Schwann Cell Caveolin-1 Expression Increases During Myelination and Decreases After Axotomy

Glia. May, 2002  |  Pubmed ID: 11968057

The caveolins are a family of related proteins that form the structural framework of caveolae. They have been implicated in the regulation of signal transduction, cell cycle control, and cellular transport processes, particularly cholesterol trafficking. Caveolin-1 is expressed by a variety of cell types, including Schwann cells, although its expression is greatest in differentiated cell types, such as endothelial cells and adipocytes. In the present work, we characterize caveolin-1 expression both during rat sciatic nerve development and after axotomy. Schwann cells express little caveolin-1 on postnatal days 1 and 6. By P30, myelinating Schwann cells express caveolin-1, which is localized in the outer/abaxonal myelin membranes as well as intracellularly. After axotomy, Schwann cell caveolin-1 expression in the distal nerve stump decreases as Schwann cells revert to a premyelinating (p75-positive) phenotype; residual caveolin-1 within the nerve largely localizes to myelin debris and infiltrating macrophages. We speculate that caveolin-1 plays a role in the biology of myelinating Schwann cells.

Insulin-like Growth Factor I Prevents Mannitol-induced Degradation of Focal Adhesion Kinase and Akt

The Journal of Biological Chemistry. Jul, 2002  |  Pubmed ID: 12011046

In our laboratory, we are interested in hyperosmolarity-induced apoptosis in neuronal cells. We have shown that high concentrations of glucose or mannitol induce apoptotic cell death in dorsal root ganglia in culture and in SH-SY5Y and SH-EP human neuroblastoma cells. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that has a critical role for transmitting integrin-mediated-signals. In this study, we report that hyperosmolar treatment mediates FAK dephosphorylation and cleavage, which is prevented by insulin-like growth factor I (IGF-I) treatment. Mannitol treatment of SH-EP cells transfected with vector (SH-EP/pSFFV) results in concentration- and time-dependent dephosphorylation and degradation of FAK. Dephosphorylation and degradation of FAK are tightly correlated with apoptotic morphological changes, including the disruption of actin stress fibers, the loss of focal adhesion sites, membrane blebbing, and cell detachment. Treatment of SH-EP/pSFFV cells with IGF-I or transfection of IGF-I receptor prevents these changes. Treatment of cells with pharmacologic inhibitors of the mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathways does not affect mannitol-induced FAK dephosphorylation and degradation. However, phosphatidylinositol 3-kinase is necessary for IGF-I-mediated protection against FAK alteration. Mannitol treatment also results in the degradation of Akt. Mannitol induces the activation of caspases-3 and -9 in a time course similar to the dephosphorylation and degradation of FAK. Treatment of the cells with ZVAD, a general caspase inhibitor, blocks the mannitol-induced FAK and Akt degradation as well as cell detachment and apoptosis. These results suggest that one of the pathways of mannitol-mediated apoptosis is through the degradation of FAK and Akt and that IGF-I protects the cells from apoptosis by blocking the activation of caspases, which may be responsible for the loss of FAK and Akt.

N-Myc and Bcl-2 Coexpression Induces MMP-2 Secretion and Activation in Human Neuroblastoma Cells

Oncogene. Jul, 2002  |  Pubmed ID: 12085233

Neuroblastoma is a peripheral nervous system tumor that accounts for 8-10% of all solid childhood tumors. N-Myc is the most reliable prognostic indicator for neuroblastoma. Bcl-2 is detected in 40-60% of primary neuroblastoma tumors and demonstrates anti-apoptotic action by conferring resistance to chemotherapy and radiation treatment. In neuroblastoma cell lines, the coexpression of N-Myc and Bcl-2 leads to increased tumorigenic properties. Matrix metalloproteinases (MMPs) are endopeptidases that degrade a wide range of basement membrane components, a process important for tumor invasion. This study investigates the effect of N-Myc and Bcl-2 on MMP expression and activation. MMP-2 expression and secretion are increased in SHEP neuroblastoma cells expressing Bcl-2 alone (SHEP/Bcl-2 cells) or both N-Myc and Bcl-2 (SHEP/N-Myc/Bcl-2 cells). MMP-2 activity is increased in the SHEP/N-Myc/Bcl-2 cells yet remains unchanged in SHEP/Bcl-2 cells. TIMP-2 expression is high in SHEP/Bcl-2 cells, which likely inhibits MMP-2 activity, and absent in SHEP/N-Myc/Bcl-2 cells, allowing MMP-2 activity. Invasion is increased in SHEP/N-Myc/Bcl-2 cells and prevented by the use of a pharmacologic MMP-2 inhibitor. These data imply that N-Myc and Bcl-2 cooperate to increase the expression, secretion, and activation of MMP-2, which likely leads to a more tumorigenic phenotype due to increased MMP-2 mediated invasion.

Control of Cell Survival by IGF Signaling Pathways

Growth Hormone & IGF Research : Official Journal of the Growth Hormone Research Society and the International IGF Research Society. Aug, 2002  |  Pubmed ID: 12175651

The insulin-like growth factor system efficiently signals to cells to grow, differentiate, and survive. One central player in the prevention of cell death is the IGF-I receptor. Transduction of signals through this receptor leads to multiple series of intracellular phosphorylation events and the activation of several signaling pathways. Mechanisms of IGF system signaling that prevent cell death continue to be identified, suggesting that cells have alternative ways to avert death signals in addition to primary protective pathways. This review describes current knowledge of the mechanisms utilized by the IGF system to promote cell survival.

Update on Diabetic Neuropathy

Current Opinion in Neurology. Oct, 2002  |  Pubmed ID: 12352003

This review will focus on recent advances in the field of diabetic neuropathy, with an emphasis on distal symmetric sensory and sensorimotor polyneuropathy. Some new information in the areas of diabetic amyotrophy and diabetic autonomic neuropathy will also be reviewed.

Signaling Mechanisms That Regulate Actin-based Motility Processes in the Nervous System

Journal of Neurochemistry. Nov, 2002  |  Pubmed ID: 12390511

Actin-based motility is critical for nervous system development. Both the migration of neurons and the extension of neurites require organized actin polymerization to push the cell membrane forward. Numerous extracellular stimulants of motility and axon guidance cues regulate actin-based motility through the rho GTPases (rho, rac, and cdc42). The rho GTPases reorganize the actin cytoskeleton, leading to stress fiber, filopodium, or lamellipodium formation. The activity of the rho GTPases is regulated by a variety of proteins that either stimulate GTP uptake (activation) or hydrolysis (inactivation). These proteins potentially link extracellular signals to the activation state of rho GTPases. Effectors downstream of the rho GTPases that directly influence actin polymerization have been identified and are involved in neurite development. The Arp2/3 complex nucleates the formation of new actin branches that extend the membrane forward. Ena/VASP proteins can cause the formation of longer actin filaments, characteristic of growth cone actin morphology, by preventing the capping of barbed ends. Actin-depolymerizing factor (ADF)/cofilin depolymerizes and severs actin branches in older parts of the actin meshwork, freeing monomers to be re-incorporated into actively growing filaments. The signaling mechanisms by which extracellular cues that guide axons to their targets lead to direct effects on actin filament dynamics are becoming better understood.

High Glucose-induced Oxidative Stress and Mitochondrial Dysfunction in Neurons

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Nov, 2002  |  Pubmed ID: 12409316

The current study examines the association between glucose induction of reactive oxygen species (ROS), mitochondrial (Mt) depolarization, and programmed cell death in primary neurons. In primary dorsal root ganglion (DRG) neurons, 45 mM glucose rapidly induces a peak rise in ROS corresponding to a 50% increase in mean Mt size at 6 h (P<0.001). This is coupled with loss of regulation of the Mt membrane potential (Mt membrane hyperpolarization, followed by depolarization, MMD), partial depletion of ATP, and activation of caspase-3 and -9. Glucose-induced activation of ROS, MMD, and caspase-3 and -9 activation is inhibited by myxothiazole and thenoyltrifluoroacetone (P<0.001), which inhibit specific components of the Mt electron transfer chain. Similarly, MMD and caspase-3 activation are inhibited by 100 microM bongkrekic acid (an inhibitor of the adenosine nucleotide translocase ANT). These results indicate that mild increases in glucose induce ROS and Mt swelling that precedes neuronal apoptosis. Glucotoxicity is blocked by inhibiting ROS induction, MMD, or caspase cleavage by specific inhibitors of electron transfer, or by stabilizing the ANT.

Neuronal Survival Following Remote Adenovirus Gene Delivery

Journal of Neurosurgery. Mar, 2002  |  Pubmed ID: 12450285

Virus-mediated central nervous system gene delivery is a promising means of treating traumatized tissue or degenerative diseases. In the present study, the authors examined gene expression and neuronal survival in the spinal cord after sciatic nerve administration of an adenovirus vector expressing a LacZ reporter gene.

Intraneural Colchicine Inhibition of Adenoviral and Adeno-associated Viral Vector Remote Spinal Cord Gene Delivery

Neurosurgery. Feb, 2003  |  Pubmed ID: 12535368

The mechanism of remote viral gene delivery to the spinal cord is unknown. The present experiment demonstrates that intraneural injection of colchicine is capable of inhibiting remote delivery of both adenoviral and adeno-associated viral (AAV) vectors, implicating axonal transport in this process.

Oxidative Stress and Diabetic Neuropathy: a New Understanding of an Old Problem

The Journal of Clinical Investigation. Feb, 2003  |  Pubmed ID: 12588877

N-Myc Overexpression Leads to Decreased Beta1 Integrin Expression and Increased Apoptosis in Human Neuroblastoma Cells

Oncogene. May, 2003  |  Pubmed ID: 12730680

Neuroblastoma is a childhood tumor thought to arise through improper differentiation of neural crest cells. Increased N-Myc expression in neuroblastoma indicates highly malignant disease and poor patient prognosis. N-myc enhances cell growth, insulin-like growth factor type I receptor (IGF-IR) expression, and tumorigenicity in combination with Bcl-2. Despite these effects, N-Myc overexpression in SHEP neuroblastoma cells (SHEP/N-Myc cells) increases serum-withdrawal and mannitol-induced apoptosis. Although we have previously shown a protective effect of IGF-I in SHEP cells, in SHEP/N-Myc cells IGF-I rescue from mannitol-induced apoptosis is prevented. N-Myc overexpression has little effect on IGF-IR signaling pathways, but results in increased Akt phosphorylation when Bcl-2 is coexpressed. A loss of integrin-mediated adhesion promotes apoptosis in many systems. SHEP/N-Myc cells have dramatically less beta1 integrin expression than control cells, consistent with previous reports. beta1 integrin expression is decreased in more tumorigenic neuroblastoma cells lines, including IMR32 and SH-SY5Y cells. Reintroduction of beta1 integrin into the N-Myc-overexpressing cells prevents mannitol-mediated apoptosis. We speculate that N-Myc repression of beta1 integrin expression leads to a less differentiated phenotype, resulting in increased growth and tumorigenesis if properly supported or apoptosis if deprived of growth sustaining molecules.

Substrate Patterning: an Emerging Technology for the Study of Neuronal Behavior

Experimental Neurology. Nov, 2003  |  Pubmed ID: 14597331

Extracellular matrix (ECM) proteins and cell-cell adhesion molecules (CAM) play important roles in neuronal development and differentiation. In the investigation of these roles, patterned substrates have proven to be a notably useful tool. Photolithographic and microprinting techniques can be used to make patterns of ECMs, CAMs, amino acids, and organofunctional groups for culturing neurons and other cell types. Experiments performed using these substrates have provided unique insights into the roles of cell-substratum adhesion, cell shape, and ECM composition on important cell functions, including survival, migration, neurite outgrowth, and development of polarity. Patterns may also be designed to localize cell bodies and confine their processes to predetermined areas of a substrate. Finally, the behavior of neurons on patterned substrates may prove helpful in the design of scaffoldings and nerve guides tailored for regeneration and repair of the nervous system.

Microvascular Complications of Impaired Glucose Tolerance

Diabetes. Dec, 2003  |  Pubmed ID: 14633845

Impaired glucose tolerance (IGT) serves as a marker for the state of insulin resistance and predicts both large- and small-vessel vascular complications, independent of a patient's progression to diabetes. Patients with IGT are at significantly increased risk for death and morbidity due to myocardial infarction, stroke, and large-vessel occlusive disease. IGT is more predictive of cardiovascular morbidity than impaired fasting glucose, probably because it is a better surrogate for the state of insulin resistance. IGT is also independently associated with traditional microvascular complications of diabetes, including retinopathy, renal disease, and polyneuropathy, which are the topics of this review. Inhibition of nitric oxide-mediated vasodilation, endothelial injury due to increased release of free fatty acids and adipocytokines from adipocytes, and direct metabolic injury of endothelial and end-organ cells contribute to vascular complications. Early detection of IGT allows intensive diet and exercise modification, which has proven significantly more effective than drug therapy in normalizing postprandial glucose and inhibiting progression to diabetes. To what degree intervention will limit recognized complications is not known.

Guidelines for the Diagnosis and Treatment of Chronic Inflammatory Demyelinating Polyneuropathy

Journal of the Peripheral Nervous System : JPNS. Dec, 2003  |  Pubmed ID: 14641652

Degradation and Dephosphorylation of Focal Adhesion Kinase During Okadaic Acid-induced Apoptosis in Human Neuroblastoma Cells

Neoplasia (New York, N.Y.). Sep-Oct, 2003  |  Pubmed ID: 14670178

Focal adhesion kinase (FAK) prevents apoptosis in many cell types. We have reported that tyrosine residues in FAK are dephosphorylated and FAK is degraded during mannitol-induced apoptosis in human neuroblastoma cells. Several studies suggest that FAK dephosphorylation and degradation are separate events. The current study defines the relationship between FAK dephosphorylation and degradation in neuroblastoma cells using okadaic acid (OA). OA, a serine phosphatase inhibitor, promotes serine/threonine phosphorylation, which in turn blocks tyrosine phosphorylation. OA induced focal adhesion loss, actin cytoskeleton disorganization, and cellular detachment, which corresponded to a loss of FAK Tyr397 phosphorylation. These changes preceded caspase-3 activation, Akt and MAP kinase activity loss, protein ubiquitination, and cellular apoptosis. Insulin-like growth factor-I prevented mannitol-induced, but not OA-induced, substrate detachment and FAK Tyr397 dephosphorylation, and the effects of OA on FAK Tyr397 phosphorylation were irreversible. The proteolytic degradation of FAK is temporally distinct from its tyrosine dephosphorylation, occurring when apoptotic pathways are already initiated and during a generalized destruction of signaling proteins. Therefore, agents resulting in the dephosphorylation of FAK may be beneficial for therapeutic treatment, irrespective of FAK protein levels, as this may result in apoptosis, which cannot be prevented by growth factor signaling.

Adeno-associated Viral Vector Gene Expression in the Adult Rat Spinal Cord Following Remote Vector Delivery

Neurobiology of Disease. Dec, 2003  |  Pubmed ID: 14678769

The current investigation tests whether adeno-associated viral vectors (rAAV) undergo remote delivery to the spinal cord via peripheral nerve injection as previously demonstrated with adenoviral vectors. The sciatic nerves of adult rats (n = 10) were injected with either an rAAV (rAAVCMV-lacZ) or adenoviral (AdCMV-lacZ) vector (1.4 x 10(7) particles/ml). After 21 days, the rAAV group demonstrated significantly higher spinal cord viral expression than the adenoviral group (P < 0.024). A second group of rats was injected with rAAV expressing the green fluorescence protein (GFP) reporter gene. GFP was detected 21 days after unilateral sciatic nerve injection in the neurons of the dorsal root ganglion and spinal cord. The codistribution of the viral genome and transgene in CNS neurons was confirmed with in situ hybridization. In summary, rAAV genes are expressed in CNS neurons following peripheral nerve injection at levels exceeding those seen following remote adenovirus injection.

Applying Database Technology to Clinical and Basic Research Bioinformatics Projects

Journal of Integrative Neuroscience. Dec, 2003  |  Pubmed ID: 15011271

This paper describes the application of database technology to medical information with the goal of providing medical and clinical researchers with the tools necessary to plan bioinformatics projects. Commercial database management systems were utilized, standard database design practices were applied, a user interface was created, data entered, and the development of analysis tools, including data mining technologies is underway. Databases were constructed based on animal and cell culture models of diabetes and clinical data. Bioinformatics is a useful tool in both basic research and clinical settings. The advantages of relational databases and an approach to managing bioinformatics projects are discussed.

Neuroinflammation, COX-2, and ALS--a Dual Role?

Experimental Neurology. May, 2004  |  Pubmed ID: 15081582

Although the root cause of many neurodegenerative diseases is unknown, neuroinflammation may play a key role in these types of disease, including amyotrophic lateral sclerosis (ALS). In the context of neurodegeneration, it is unclear if the disease is propagated through inflammation, or whether in contrast, evidence of inflammation reflects an attempt to protect against further cellular injury. Inflammatory pathways involving the cyclooxygenase (COX) enzymes and subsequent generation of prostaglandins are potential target sites for treatments to halt the progression of ALS. In the CNS, COX enzymes are localized to neurons, astrocytes, and microglia and can be induced under various conditions. In addition, there appears to be a dual role for the prostaglandin products of COX enzymes in the nervous system. Some prostaglandins promote the survival of neurons, while others promote apoptosis. In this review, the pathways of COX activity and prostaglandin production form the center of the debate regarding the dual nature of neuroinflammation. We will also discuss how this duality may affect future treatments for neurodegenerative diseases such as ALS.

IGF-I Prevents Glutamate-induced Motor Neuron Programmed Cell Death

Neurobiology of Disease. Jul, 2004  |  Pubmed ID: 15193297

Insulin-like growth factor I (IGF-I) is currently in clinical trials for treatment of amyotrophic lateral sclerosis (ALS), but little is known about how it promotes the survival of motor neurons. In the current study, we examined IGF-I-mediated neuroprotection in an in vitro model of ALS utilizing enriched cultures of embryonic rat spinal cord motor neurons. IGF-I binds to the IGF-I receptor (IGF-IR) in motor neurons and activates MAPK and the downstream effector of phosphatidylinositol 3-kinase (PI-3K) signaling, Akt. IGF-I:IGF-IR signaling involves phosphorylation of IRS-1 and Shc, but not IRS-2. Glutamate, which is elevated in the cerebrospinal fluid of ALS patients, induced DNA fragmentation and caspase-3 cleavage in the spinal cord motor neurons. These effects of glutamate were blocked by co-treatment with IGF-I. However, a delay of IGF-I treatment for as little as 30 min eliminated its neuroprotective effect. Finally, alone, neither the MAPK pathway inhibitor PD98059 nor the PI-3K inhibitor LY294002 blocked the neuroprotective effect of IGF-I, but both inhibitors together were effective in this regard. These results suggest that the dose and timing of IGF-I administration are critical for producing a neuroprotective effect, and also suggest that both the MAPK and PI-3K/Akt pathways can promote the survival of motor neurons. We discuss our results in terms of novel strategies for ALS therapy.

New Insights into the Mechanisms of Diabetic Neuropathy

Reviews in Endocrine & Metabolic Disorders. Aug, 2004  |  Pubmed ID: 15211094

Integrin Expression Regulates Neuroblastoma Attachment and Migration

Neoplasia (New York, N.Y.). Jul-Aug, 2004  |  Pubmed ID: 15256055

Neuroblastoma (NBL) is the most common malignant disease of infancy, and children with bone metastasis have a mortality rate greater than 90%. Two major classes of proteins, integrins and growth factors, regulate the metastatic process. We have previously shown that tumorigenic NBL cells express higher levels of the type I insulin-like growth factor receptor (IGF-IR) and that beta1 integrin expression is inversely proportional to tumorigenic potential in NBL. In the current study, we analyze the effect of beta1 integrin and IGF-IR on NBL cell attachment and migration. Nontumorigenic S-cells express high levels of beta1 integrin, whereas tumorigenic N-cells express little beta1 integrin. Alterations in beta1 integrin are due to regulation at the protein level, as translation is decreased in N-type cells. Moreover, inhibition of protein synthesis shows that beta1 integrin is degraded more slowly in S-type cells (SHEP) than in N-type cells (SH-SY5Y and IMR32). Inhibition of alpha5beta1 integrin prevents SHEP (but not SH-SY5Y or IMR32) cell attachment to fibronectin and increases SHEP cell migration. Increases in IGF-IR decrease beta1 integrin expression, and enhance SHEP cell migration, potentially through increased expression of alphavbeta3. These data suggest that specific classes of integrins in concert with IGF-IR regulate NBL attachment and migration.

Oxidative Stress in the Pathogenesis of Diabetic Neuropathy

Endocrine Reviews. Aug, 2004  |  Pubmed ID: 15294884

Oxidative stress results from a cell or tissue failing to detoxify the free radicals that are produced during metabolic activity. Diabetes is characterized by chronic hyperglycemia that produces dysregulation of cellular metabolism. This review explores the concept that diabetes overloads glucose metabolic pathways, resulting in excess free radical production and oxidative stress. Evidence is presented to support the idea that both chronic and acute hyperglycemia cause oxidative stress in the peripheral nervous system that can promote the development of diabetic neuropathy. Proteins that are damaged by oxidative stress have decreased biological activity leading to loss of energy metabolism, cell signaling, transport, and, ultimately, to cell death. Examination of the data from animal and cell culture models of diabetes, as well as clinical trials of antioxidants, strongly implicates hyperglycemia-induced oxidative stress in diabetic neuropathy. We conclude that striving for superior antioxidative therapies remains essential for the prevention of neuropathy in diabetic patients.

Phosphatidylinositol 3-kinase and Akt Effectors Mediate Insulin-like Growth Factor-I Neuroprotection in Dorsal Root Ganglia Neurons

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Oct, 2004  |  Pubmed ID: 15319368

Insulin-like growth factor-I (IGF-I) protects neurons of the peripheral nervous system from apoptosis, but the underlying signaling pathways are not well understood. We studied IGF-I mediated signaling in embryonic dorsal root ganglia (DRG) neurons. DRG neurons express IGF-I receptors (IGF-IR), and IGF-I activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. High glucose exposure induces apoptosis, which is inhibited by IGF-I through the PI3K/Akt pathway. IGF-I stimulation of the PI3K/Akt pathway phosphorylates three known Akt effectors: the survival transcription factor cyclic AMP response element binding protein (CREB) and the pro-apoptotic effector proteins glycogen synthase kinase-3beta (GSK-3beta) and forkhead (FKHR). IGF-I regulates survival at the nuclear level through accumulation of phospho-Akt in DRG neuronal nuclei, increased CREB-mediated transcription, and nuclear exclusion of FKHR. High glucose increases expression of the pro-apoptotic Bcl protein Bim (a transcriptional target of FKHR). However, IGF-I does not regulate Bim or anti-apoptotic Bcl-xL protein expression levels, which suggests that IGF-I neuroprotection is not through regulation of their expression. High glucose also induces loss of the initiator caspase-9 and increases caspase-3 cleavage, effects blocked by IGF-I. These data suggest that IGF-I prevents apoptosis in DRG neurons by regulating PI3K/Akt pathway effectors, including GSK-3beta, CREB, and FKHR, and by blocking caspase activation.

Adeno-associated Viral-mediated Insulin-like Growth Factor Delivery Protects Motor Neurons in Vitro

Neuromolecular Medicine. 2004  |  Pubmed ID: 15970625

Recent work has demonstrated that adeno-associated viral (AAV) vector-mediated delivery of the insulin-like growth factor (IGF-I) gene through retrograde axonal transport can prolong survival and delay disease onset in the superoxide dismutase mutant mouse model of motor neuron (MN) disease. The present experiment examines IGF-I gene transfer in vitro. Adenoviral and AAV vectors for IGF-I infect neurons triggering expression and secretion of biologically active IGF-I. AAV-mediated IGF-I expression in SH-SY5Y neurons protects both cells expressing the transgene, and bystanders without transgene expression from glutamate-induced apoptosis. Similarly, AAV-mediated IGF-I delivery in primary E15 MN culture provides a titer-dependent neuroprotection from glutamate-induced DNA fragmentation. Both infected and noninfected neurons are equally protected. These observations argue that vector-mediated IGF-I gene transfer induces secretion of active IGF-I that acts through direct effects on spinal cord MNs. This mechanism may explain the therapeutic effects observed in vivo despite relatively low affinity AAV spinal cord uptake.

Insulin-like Growth Factor-I Signaling in Human Neuroblastoma Cells

Oncogene. Jan, 2004  |  Pubmed ID: 14712218

Neuroblastoma is a heterogeneous tumor consisting of N (neuronal) and S (stromal) cells. We report that more tumorigenic and motile N cells express higher levels of IGF-I receptor (IGF-IR) than less tumorigenic, more adherent S cells. Shc, one of the two major docking partners of IGF-IR, is equally expressed in N and S cell lines. IGF-I treatment phosphorylates Shc in N cells, but only weakly activates Shc in S cells. Expression of the second partner, insulin receptor substrate (IRS), is cell type specific. S cells exclusively express IRS-1 that undergoes sustained phosphorylation by IGF-I. In contrast, N cells express IRS-2 that is transiently phosphorylated by IGF-I. Downstream of IRS-2 and Shc, IGF-I treatment results in strong activation of Akt and MAPK in N cells and activation of both pathways is required for IGF-I-mediated differentiation. Only IGF-IR activation of phosphatidylinositol-3 kinase is required for tumor edge ruffling in N and S cells, with stimulation of focal adhesion kinase (FAK) and paxillin. This detailed understanding of the 'biochemical signature' of N and S cells provides the background needed to target and disrupt specific IGF signaling pathways in an attempt to develop more effective therapies.

The Role of Growth Factors in Diabetic Peripheral Neuropathy

Journal of the Peripheral Nervous System : JPNS. Mar, 2004  |  Pubmed ID: 14871451

Peripheral neuropathy afflicts 60% of all diabetic patients. Underlying the clinical disorder is the loss or degeneration of neurons, Schwann cells, and neuronal fibers. This degenerative pathology has prompted interest in the potential of growth factors as a therapy in diabetic neuropathy. Three lines of evidence support the theory that growth factors may be important in this disorder: (1) endogenous growth factors promote survival and health of neurons, (2) expression levels of growth factors are altered in diabetic neuropathy and peripheral neuron injury, and (3) growth factors induce neuronal regeneration in in vitro and in vivo models of diabetic injury. This review surveys the roles of several growth factors in diabetic neuropathy, including the neurotrophins, insulin-like growth factors, cytokine-like growth factors, and vascular endothelial growth factor. These growth factors are examined in terms of their expression during peripheral nerve injury and their protective and regenerative effects on peripheral neurons. Growth factor-mediated neuroprotective signaling is discussed, particularly in relation to the recent research, suggesting that diabetic neuropathy-induced degeneration stems from oxidative stress. Finally, the potential of growth factors as therapeutic agents is addressed, including an assessment of past growth factor clinical trials and other potential avenues of growth factor therapy.

In Situ Imaging of Mitochondrial Outer-membrane Pores Using Atomic Force Microscopy

BioTechniques. Oct, 2004  |  Pubmed ID: 15517968

Here we describe a technique for imaging of the outer contours of the mitochondrial membrane using atomic force microscopy, subsequent to or during a toxic or metabolic challenge. Pore formation in both glucose-challenged and 1,3-dinitrobenzene (DNB)-challenged mitochondria was observed using this technique. Our approach enables quantification of individual mitochondrial membrane pore formations. With this work, we have produced some of the highest resolution images of the outer contours of the in situ mitochondrial membrane published to date. These are potentially the first images of the component protein clusters at the time of formation of the mitochondrial membrane transition pore in situ. With the current work, we have extended the application of atomic force microscopy of mitochondrial membranes to fluid imaging. We have also begun to correlate 3-D surface features of mitochondria dotted with open membrane pores with features previously viewed with electron microscopy (EM) of fixed sections.

Polyneuropathy with Impaired Glucose Tolerance: Implications for Diagnosis and Therapy

Current Treatment Options in Neurology. Jan, 2005  |  Pubmed ID: 15610705

Prediabetes is associated with a length-dependent polyneuropathy that typically is sensory predominant and painful. A diagnosis of prediabetes should be sought in patients with otherwise idiopathic sensory-predominant neuropathy by doing a 2-hour oral glucose tolerance test. Fasting plasma glucose of 100 to 125 mg/dL or 2-hour glucose 140 to 199 mg/dL (impaired glucose tolerance) constitutes prediabetes. Most patients with neuropathy associated with prediabetes (NAP) are obese and show metabolic manifestations of insulin resistance, including hyperlipidemia and hypertension. Appropriate treatment addresses hyperglycemia, insulin resistance, and neuropathic pain. Professionally administered individualized diet and exercise counseling (modeled on the Diabetes Prevention Program) has been shown to be more effective than glucose-lowering medications in preventing progression from impaired glucose tolerance to diabetes, and is the mainstay of treatment for all patients with NAP. The goals of this therapy should be a 5% to 7% reduction in weight and an increase to 30 minutes of moderate exercise five times weekly. Patients with prediabetes are at increased risk for myocardial infarction, stroke, and peripheral vascular disease. Therefore, risk reduction with control of hypertension and hyperlipidemia is essential. Neuropathic pain troubles nearly every patient with NAP, and often limits aerobic exercise. No trials have specifically addressed the patient population with NAP, and neuropathic pain treatment closely follows recommendations for diabetic neuropathy. Gabapentin, lamotrigine, and tricyclic antidepressants are well-validated first-line therapies. Adjunctive therapy with opioids, nonsteroidal anti-inflammatory drugs often are necessary. Diet and exercise seem to reduce neuropathic pain in patients with NAP.

Integrin-linked Kinase Complexes with Caveolin-1 in Human Neuroblastoma Cells

Biochemistry. Jan, 2005  |  Pubmed ID: 15654749

Integrin-linked kinase (ILK) and caveolin-1 (cav-1) are implicated in the pathogenesis of cancer. Overexpression of ILK leads to altered expression of cell cycle regulators, a decreased level of cell adhesion to the extracellular matrix, a decreased level of apoptosis, in vitro phosphorylation of Akt, and tumor formation in nude mice. Conversely, cav-1 expression is frequently downregulated in many forms of cancer. We examined whether ILK and cav-1 interact in SHEP human neuroblastoma cells because ILK is present in caveolae-enriched membranes and contains a putative cav-binding domain. SHEP cells were stably transfected with vector, wild-type ILK (ILK-wt), kinase-deficient ILK (ILK-kd), or mutant cav-binding domain ILK (ILK-mutCavbd). Control SHEP cells and ILK transfectants express high levels of ILK and cav-1. Immunoprecipitation with anti-cav-1 co-immunoprecipitates a 59 kDa protein that is immunoreactive with the anti-ILK antibody, and this interaction is partially prevented in cells expressing ILK-mutCavbd. Cav-1 and ILK partially colocalize in SHEP cells, also supporting these data. Last, affinity chromatography with a biotinylated cav-scaffolding domain peptide precipitates ILK-wt but not ILK-mutCavbd. These data suggest that the cav-binding domain of ILK and the cav-scaffolding domain of cav-1 mediate complex formation in human neuroblastoma cells.

Short-term Hyperglycemia Produces Oxidative Damage and Apoptosis in Neurons

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Apr, 2005  |  Pubmed ID: 15677696

Dorsal root ganglia neurons in culture die through programmed cell death when exposed to elevated glucose, providing an in vitro model system for the investigation of the mechanisms leading to diabetic neuropathy. This study examines the time course of programmed cell death induction, regulation of cellular antioxidant capacity, and the protective effects of antioxidants in neurons exposed to hyperglycemia. We demonstrate that the first 2 h of hyperglycemia are sufficient to induce oxidative stress and programmed cell death. Using fluorimetric analysis of reactive oxygen species (ROS) production, in vitro assays of antioxidant enzymes, and immunocytochemical assays of cell death, we demonstrate superoxide formation, inhibition of aconitase, and lipid peroxidation within 1 h of hyperglycemia. These are followed by caspase-3 activation and DNA fragmentation. Antioxidant potential increases by 3-6 h but is insufficient to protect these neurons. Application of the antioxidant alpha-lipoic acid potently prevents glucose-induced oxidative stress and cell death. This study identifies cellular therapeutic targets to prevent diabetic neuropathy. Since oxidative stress is a common feature of the micro- and macrovascular complications of diabetes, the present findings have broad application to the treatment of diabetic patients.

Differential Regulation of Insulin Receptor Substrate-1 Degradation During Mannitol and Okadaic Acid Induced Apoptosis in Human Neuroblastoma Cells

Cellular Signalling. Jun, 2005  |  Pubmed ID: 15722201

Insulin receptor substrate (IRS) proteins are major docking molecules for the type I insulin like growth factor (IGF) receptor (IGF-IR) and mediate their effects on downstream signaling molecules. In this report, we investigated IRS-1 regulation during apoptosis in human neuroblastoma SH-EP cells. Treatment of SH-EP cells with mannitol or okadaic acid (OA) induces apoptosis with the typical characteristics of anoikis. Mannitol treatment results in IRS-1 degradation with concomitant appearance of smaller fragments, likely representing caspase cleavage products. In contrast OA-induced IRS-1 degradation is accompanied by a mobility shift in IRS-1, suggesting IRS-1 serine/threonine phosphorylation. Mannitol-induced, but not OA-induced, degradation is blocked by IGF-I. Pretreatment of the cells with caspase or proteasome inhibitors also partially blocks mannitol-induced IRS-1 degradation. These results suggest two independent pathways are involved in IRS-1 degradation; one pathway is dependent on caspase activation and is blocked by IGF-I, while a second pathway is caspase-independent and IGF-I-insensitive.

Diabetic Neuropathies: a Statement by the American Diabetes Association

Diabetes Care. Apr, 2005  |  Pubmed ID: 15793206

Insulin-like Growth Factors in the Treatment of Neurological Disease

Endocrine Development. 2005  |  Pubmed ID: 15879695

Although the functional deficits of neurological diseases vary, they are all pathologically marked by neuronal degeneration. The ability of insulin-like growth factor-I (IGF-I) to support both sensory and motor neuron regeneration has suggested its potential in treatment of neurological diseases. IGF-I is pleiotrophic, stimulating survival, neurite outgrowth and motility in neurons, as well as myelination of neurons by glia. Understanding the intracellular signaling pathways that mediate these pleiotrophic responses to IGF-I is important for tailoring IGF-I treatment to the appropriate neurological deficit. This review surveys the current understanding of IGF-I pleiotrophism, the underlying signaling conferring these effects, and the status of IGF-I in treatment of human neurological disorders.

Insulin-like Growth Factor I Induces Preferential Degradation of Insulin Receptor Substrate-2 Through the Phosphatidylinositol 3-kinase Pathway in Human Neuroblastoma Cells

Endocrinology. Dec, 2005  |  Pubmed ID: 16150916

Insulin receptor substrate (IRS) signaling is regulated through serine/threonine phosphorylation, with subsequent IRS degradation. This study examines the differences in IRS-1 and IRS-2 degradation in human neuroblastoma cells. SH-EP cells are glial-like, express low levels of the type I IGF-I receptor (IGF-IR) and IRS-2 and high levels of IRS-1. SH-SY5Y cells are neuroblast-like, with high levels of IGF-IR and IRS-2 but virtually no IRS-1. When stimulated with IGF-I, IRS-1 expression remains constant in SH-EP cells; however, IRS-2 in SH-SY5Y cells shows time- and concentration-dependent degradation, which requires IGF-IR activation. SH-EP cells transfected with IRS-2 and SH-SY5Y cells transfected with IRS-1 show that only IRS-2 is degraded by IGF-I treatment. When SH-EP cells are transfected with IGF-IR or suppressor of cytokine signaling, IRS-1 is degraded by IGF-I treatment. IRS-1 and -2 degradation are almost completely blocked by phosphatidylinositol 3-kinase inhibitors and partially by proteasome inhibitors. In summary, 1) IRS-2 is more sensitive to IGF-I-mediated degradation; 2) IRS degradation is mediated by phosphatidylinositol 3-kinase and proteasome sensitive pathways; and 3) high levels of IGF-IR, and possibly the subsequent increase in Akt phosphorylation, are required for efficient IRS degradation.

New Developments in Diabetic Neuropathy

Current Opinion in Neurology. Oct, 2005  |  Pubmed ID: 16155445

Diabetic neuropathy is a debilitating consequence of type 1 and 2 diabetes. Hyperglycemia disrupts the normal function of neurons and their supporting glia at multiple levels. The complexity of this complication, combined with difficulties of delivering therapy to sensory, sympathetic and parasympathetic neurons, contributes to the intractability of this serious diabetic complication. This review summarizes recent reviews examining the state of research on and treatment of diabetic neuropathy and highlights areas of clinical and basic research that may yield new diagnostic and treatment options.

Cell Culture Modeling to Test Therapies Against Hyperglycemia-mediated Oxidative Stress and Injury

Antioxidants & Redox Signaling. Nov-Dec, 2005  |  Pubmed ID: 16356113

The concept that oxidative stress is a key mediator of nerve injury in diabetes has led us to design therapies that target oxidative stress mechanisms. Using an in vitro model of glucose-treated dorsal root ganglion (DRG) neurons in culture, we can examine both free radical generation, using fluorimetric probes for reactive oxygen species, and cell death via the TUNEL assay. The cell culture system is scaled down to a 96-well plate format, and so is well suited to high-throughput screening. In the present study, we test the ability of three drugs, nicotinamide, allopurinol, and alpha-lipoic acid, alone and in combination to prevent DRG neuron oxidative stress and cell death. This combination of drugs is currently in clinical trial in type 1 diabetic patients. We demonstrate independent effects on oxidative stress and neuronal survival for the three drugs, and neuronal protection using the three drugs in combination. The data strengthen the rationale for the current clinical trial. In addition, we describe an effective tool for rapid preclinical testing of novel therapies against diabetic neuropathy.

Establishment, Maintenance, and Transfection of in Vitro Cultures of Human Retinal Pigment Epithelium

Methods in Molecular Medicine. 2005  |  Pubmed ID: 15492384

Biomedical Research: a Culture in Crisis?

Nature Clinical Practice. Neurology. Dec, 2005  |  Pubmed ID: 16932493

Mechanisms of Disease: Mitochondria As New Therapeutic Targets in Diabetic Neuropathy

Nature Clinical Practice. Neurology. Nov, 2006  |  Pubmed ID: 17057749

Diabetic neuropathy (DN) is the most common complication of diabetes mellitus, and it imposes a considerable burden on a patient's quality of life and the health-care system. Despite the prevalence and severity of DN, there are no effective treatments. Pathogenetic evidence suggests that DN is marked by degeneration of dorsal root ganglion (DRG) neurons in peripheral nerves, and that DRG mitochondria are particularly affected. DRG mitochondria are especially vulnerable because they are the origin of reactive oxygen species production in the hyperglycemic neuron. Accumulating evidence indicates that neuronal mitochondria are subject to damage at the level of their DNA, and their outer and inner membranes, and also via deregulation of mitochondrial fission and fusion proteins that control mitochondrial shape and number. This Review will survey the mechanisms of mitochondrial degeneration in the pathogenesis of DN, highlighting potential mitochondrial sites for therapeutic intervention.

Neuropathy Among the Diabetes Control and Complications Trial Cohort 8 Years After Trial Completion

Diabetes Care. Feb, 2006  |  Pubmed ID: 16443884

To evaluate the impact of prior intensive diabetes therapy on neuropathy among former Diabetes Control and Complications Trial (DCCT) participants.

Longitudinal Assessment of Noninvasive Positive Pressure Ventilation Adjustments in ALS Patients

Journal of the Neurological Sciences. Aug, 2006  |  Pubmed ID: 16631799

The absence of data guiding optimal titration of noninvasive positive pressure ventilation (NIPPV) over time in ALS patients may contribute to the under-prescribing of NIPPV. We conducted a retrospective, single-center, chart review assessment of NIPPV pressure settings used for symptomatic treatment of ALS patients to determine NIPPV adjustments, and to compare survival between those who were tolerant and intolerant to NIPPV. All subjects were started on nocturnal NIPPV at 8 and 3 cm H2O inspiratory and expiratory pressure, respectively. Of the 18 tolerant subjects identified, 4 (22%) had no NIPPV pressure changes before death; 8 (44%), 1 change; 4 (22%), 2 changes; 1 (6%), 3 changes; and 1 (6%), 5 changes. Most pressure changes occurred during the first year of NIPPV initiation. The maximum pressure needed for comfort by any patient in this study was 19/5 cm H2O, while 4 (22%) found the original 8/3 cm H2O settings to be sufficient until death. Subjects in the tolerant group had better survival, when adjusting for age and site of symptom onset (bulbar versus limb), with a hazard ratio of 0.23 [95% confidence interval: 0.10, 0.54]. The current data suggest that ALS patients who are tolerant to NIPPV typically need at least one upward change in pressure settings. Tolerance to relatively low NIPPV inspiratory pressures is associated with improved survival.

Mitochondria in DRG Neurons Undergo Hyperglycemic Mediated Injury Through Bim, Bax and the Fission Protein Drp1

Neurobiology of Disease. Jul, 2006  |  Pubmed ID: 16684605

Dorsal root ganglia (DRG) neurons degenerate in diabetic neuropathy (DN) and exhibit mitochondrial damage. We studied mitochondria of cultured DRG neurons exposed to high glucose as an in vitro model of DN. High glucose sequentially increases the expression, activation and localization of the pro-apoptotic proteins Bim and Bax and the mitochondrial fission protein dynamin-regulated protein 1 (Drp1). High glucose causes association of Drp1/Bax, similar to other apoptotic stimuli. Collectively, these events promote mitochondrial fragmentation and reduce mitochondrial number, suggestive of apoptotic mitochondrial fission. Drp1 is also upregulated in DRG from experimentally diabetic rats, suggesting a role for mitochondrial fission in DN. Insulin-like growth factor-I (IGF-I) protects high glucose-treated DRG neurons by preventing mitochondrial accumulation of Bim and Bax but does not modulate Drp1 expression or localization. We propose that mitochondria are compromised by convergence of Bim/Bax proteins with Drp1, which contributes to high glucose-induced injury in DRG neurons.

Lifestyle Intervention for Pre-diabetic Neuropathy

Diabetes Care. Jun, 2006  |  Pubmed ID: 16732011

The purpose of this study was to evaluate intraepidermal nerve fiber density (IENFD) as a sensitive measure of neuropathy change in patients with neuropathy associated with impaired glucose tolerance (IGT) receiving lifestyle intervention based on that used in the Diabetes Prevention Program.

Evaluation of Sham Non-invasive Ventilation for Randomized, Controlled Trials in ALS

Amyotrophic Lateral Sclerosis : Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. Jun, 2006  |  Pubmed ID: 16753973

Non-invasive positive pressure ventilation (NIV) treatment of advanced respiratory insufficiency prolongs survival in ALS. To investigate the critical question of whether earlier initiation of NIV might provide additional benefit, a randomized trial with an appropriate placebo is needed. This study evaluated sub-therapeutic (sham) continuous positive airway pressure as a potential placebo. In a single-blind design, 40 ALS patients with forced vital capacity>50% were randomized to receive 30 seconds (s) of either active NIV, with 8 cm H2O inspiratory and 4 cm H2O expiratory pressure, or sham NIV with<1 cm of H2O continuous positive airway pressure at the mask. A questionnaire was then used to assess whether subjects thought that they had received a "real" or "pretend" treatment trial. The subjects' median age was 60.5 years, and 38% were female. Twelve of 20 subjects (60%) who received active NIV and 7 (35%) of the 20 subjects who received sham thought that they had tried the active treatment (p = 0.11). Only 8 (20%) of all subjects were confident about their determination that they had received "real" or "pretend" NIV. Thus, sub-therapeutic (sham) continuous positive airway pressure is a promising placebo control for NIV trials in ALS.

Insulin-like Growth Factor-I Receptor Expression Regulates Neuroblastoma Metastasis to Bone

Cancer Research. Jul, 2006  |  Pubmed ID: 16818629

Neuroblastoma is a pediatric tumor that preferentially metastasizes to bone. Patients with bone metastases have a mortality rate >93%, indicating a need for novel treatment targets. Our laboratory has shown that type I insulin-like growth factor receptor (IGF-IR) expression and activation regulate neuroblastoma cell proliferation, motility, invasion, and survival, and that expression of the IGF-IR correlates with neuroblastoma tumorigenicity. Bone expresses large amounts of IGF ligands, and the IGF system is required for normal bone physiology. The current study addresses the role of the IGF system in neuroblastoma metastasis to bone. Upon reaching the bone marrow through the circulation, neuroblastoma cells must dock at the bone marrow endothelium, extravasate into the bone microenvironment, and destroy bone tissue to allow for tumor growth. This report examines the effects of high IGF-IR expression on neuroblastoma cell interaction with bone. The current data show that neuroblastoma cells with high IGF-IR expression, either endogenously or through transfection, adhere to human bone marrow endothelial cells and subsequently migrate toward both IGF-I and human bone stromal cells. High IGF-IR-expressing neuroblastoma cells adhere tightly to bone stromal cells, flatten, and extend processes. When neuroblastoma cells are injected directly into the tibiae of mice, those cells with increased IGF-IR form both osteolytic lesions within the tibiae and secondary tumors within other sites. These results support the hypothesis that IGF-IR expression in neuroblastoma cells increases tumor cell interaction with the bone microenvironment, resulting in greater formation of metastases.

Receptor for Advanced Glycation End Products Activation Injures Primary Sensory Neurons Via Oxidative Stress

Endocrinology. Feb, 2007  |  Pubmed ID: 17095586

The receptor for advanced glycation end products (RAGE) may promote diabetic vascular and renal disease through the activation of intracellular signaling pathways that promote oxidative stress. Oxidative stress is a mediator of hyperglycemia-induced cell injury and a unifying theme for all mechanisms of diabetic complications, but there are few studies on the expression and potential contribution of RAGE in diabetic neuropathy. The current study demonstrates that dorsal root ganglia neurons express functional RAGE and respond to the RAGE ligand S100 with similar downstream signaling, oxidative stress, and cellular injury as other diabetic complication-prone tissues. RAGE-induced phosphatidylinositol-3 kinase activity is associated with formation of reactive oxygen species, caspase-3 activation, and nuclear DNA degradation. These events are prevented by treatment with the antioxidant alpha-lipoic acid. Our data indicate that therapies aimed at decreasing RAGE ligands, blocking RAGE signaling, or preventing oxidative stress could significantly decrease the development of neuropathy in diabetic patients.

Diabetic Neuropathies

Practical Neurology. Apr, 2007  |  Pubmed ID: 17430872

Aligned Electrospun Nanofibers Specify the Direction of Dorsal Root Ganglia Neurite Growth

Journal of Biomedical Materials Research. Part A. Dec, 2007  |  Pubmed ID: 17508416

Nerve injury, a significant cause of disability, may be treated more effectively using nerve guidance channels containing longitudinally aligned fibers. Aligned, electrospun nanofibers direct the neurite growth of immortalized neural stem cells, demonstrating potential for directing regenerating neurites. However, no study of neurite guidance on these fibers has yet been performed with primary neurons. Here, we examined neurites from dorsal root ganglia explants on electrospun poly-L-lactate nanofibers of high, intermediate, and random alignment. On aligned fibers, neurites grew radially outward from the ganglia and turned to follow the fibers upon contact. Neurite guidance was robust, with neurites never leaving the fibers to grow on the surrounding cover slip. To compare the alignment of neurites to that of the nanofiber substrates, Fourier methods were used to quantify the alignment. Neurite alignment, however striking, was inferior to fiber alignment on all but the randomly aligned fibers. Neurites on highly aligned substrates were 20 and 16% longer than neurites on random and intermediate fibers, respectively. Schwann cells on fibers assumed a very narrow morphology compared to those on the surrounding coverslip. The robust neurite guidance demonstrated here is a significant step toward the use of aligned, electrospun nanofibers for nerve regeneration. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.

Dysfunction of the Recurrent Laryngeal Nerve and the Potential of Gene Therapy

The Annals of Otology, Rhinology, and Laryngology. Jun, 2007  |  Pubmed ID: 17672247

Injury to the recurrent laryngeal nerve causes vocal fold paresis or paralysis resulting in poor voice quality, and possibly swallowing dysfunction and/or airway compromise. Injury can occur as part of a neurodegenerative disease process or can be due to direct nerve trauma or tumor invasion. Management depends upon symptoms, the cause and severity of injury, and the prognosis for recovery of nerve function. Surgical treatment techniques can improve symptoms, but do not restore physiologic motion. Gene therapy may be a useful adjunct to enhance nerve regeneration in the setting of neurodegenerative disease or trauma. Remote injection of viral vectors into the recurrent laryngeal nerve is the least invasive way to deliver neurotrophic factors to the nerve's cell bodies within the nucleus ambiguus, and in turn to promote nerve regeneration and enhance both nuclear and nerve survival. The purpose of this review is to discuss the potential role for gene therapy in treatment of the unsolved problem of vocal fold paralysis.

Mouse Models of Diabetic Neuropathy

Neurobiology of Disease. Dec, 2007  |  Pubmed ID: 17804249

Diabetic neuropathy (DN) is a debilitating complication of type 1 and type 2 diabetes. Rodent models of DN do not fully replicate the pathology observed in human patients. We examined DN in streptozotocin (STZ)-induced [B6] and spontaneous type 1 diabetes [B6Ins2(Akita)] and spontaneous type 2 diabetes [B6-db/db, BKS-db/db]. Despite persistent hyperglycemia, the STZ-treated B6 and B6Ins2(Akita) mice were resistant to the development of DN. In contrast, DN developed in both type 2 diabetes models: the B6-db/db and BKS-db/db mice. The persistence of hyperglycemia and development of DN in the B6-db/db mice required an increased fat diet while the BKS-db/db mice developed severe DN and remained hyperglycemic on standard mouse chow. Our data support the hypothesis that genetic background and diet influence the development of DN and should be considered when developing new models of DN.

SOD2 Protects Neurons from Injury in Cell Culture and Animal Models of Diabetic Neuropathy

Experimental Neurology. Dec, 2007  |  Pubmed ID: 17927981

Hyperglycemia-induced oxidative stress is an inciting event in the development of diabetic complications including diabetic neuropathy. Our observations of significant oxidative stress and morphological abnormalities in mitochondria led us to examine manganese superoxide dismutase (SOD2), the enzyme responsible for mitochondrial detoxification of oxygen radicals. We demonstrate that overexpression of SOD2 decreases superoxide (O(2)(-)) in cultured primary dorsal root ganglion (DRG) neurons and subsequently blocks caspase-3 activation and cellular injury. Underexpression of SOD2 in dissociated DRG cultures from adult SOD2(+/-) mice results in increased levels of O2-, activation of caspase-3 cleavage and decreased neurite outgrowth under basal conditions that are exacerbated by hyperglycemia. These profound changes in sensory neurons led us to explore the effects of decreased SOD2 on the development of diabetic neuropathy (DN) in mice. DN was assessed in SOD2(+/-) C57BL/6J mice and their SOD2(+/+) littermates following streptozotocin (STZ) treatment. These animals, while hyperglycemic, do not display any signs of DN. DN was observed in the C57BL/6Jdb/db mouse, and decreased expression of SOD2 in these animals increased DN. Our data suggest that SOD2 activity is an important cellular modifier of neuronal oxidative defense against hyperglycemic injury.

Can Drug Screening Lead to Candidate Therapies for Testing in Diabetic Neuropathy?

Antioxidants & Redox Signaling. Feb, 2008  |  Pubmed ID: 17961065

A key mechanism of dorsal root ganglia (DRG) neuron injury in high glucose is mitochondrial overload leading to oxidative stress. We screened selected compounds for the ability to prevent hyperglycemia-induced mitochondrial superoxide in primary sensory DRG neurons. Twenty five out of 1,040 compounds decreased both mitochondrial superoxide and subsequent neuronal injury. These data both validate our screening strategy and indicate further mechanistic evaluation of drug hits and related compounds. Such studies may lead to the design of rational therapeutic approaches for this severe complication of diabetes.

Kindlin-2 is an Essential Component of Intercalated Discs and is Required for Vertebrate Cardiac Structure and Function

Circulation Research. Feb, 2008  |  Pubmed ID: 18174465

Integrins and proteins that associate with integrins are implicated in normal cardiac muscle function and development. Unc-112 is a cytoplasmic adaptor protein required for the proper establishment of integrin junctions in Caenorhabditis elegans muscle. A vertebrate homolog of unc-112, kindlin-2, is an integrin-binding protein that is expressed in cardiac muscle, but its function is unknown. We sought to understand the role of kindlin-2 in the development and function of the mouse and zebrafish heart. In the mouse, we found that kindlin-2 is highly expressed in the heart and is enriched at intercalated discs and costameres. Targeted disruption of the murine kindlin-2 gene resulted in embryonic lethality before cardiogenesis. To better assess the role of kindlin-2 in cardiac muscle development, we used morpholinos to knockdown the kindlin-2 homolog in zebrafish (z-kindlin-2), which resulted in severe abnormalities of heart development. Morphant hearts were hypoplastic and dysmorphic and exhibited significantly reduced ventricular contractility. Ultrastructural analysis of these hearts revealed disrupted intercalated disc formation and a failure in the attachment of myofibrils to membrane complexes. We conclude that kindlin-2 is an essential component of the intercalated disc, is necessary for cytoskeletal organization at sites of membrane attachment, and is required for vertebrate myocardial formation and function. These findings provide the first characterization of the in vivo functions of this novel and critical regulator of cardiogenesis.

Strategic Approaches to Developing Drug Treatments for ALS

Drug Discovery Today. Jan, 2008  |  Pubmed ID: 18190866

Significant progress in understanding the cellular mechanisms of motor neuron degeneration in amyotrophic lateral sclerosis (ALS) has not been matched with the development of therapeutic strategies to prevent disease progression. The multiple potential causes and relative rarity of the disease are two significant factors that make drug development and assessment in clinical trials extremely difficult. We review recent progress in promoting therapeutics into clinical trials and highlight the value of moderate throughput screening for the acceleration and improvement of drug design.

Diabetic Neuropathy

Current Drug Targets. Jan, 2008  |  Pubmed ID: 18220708

Criteria for Creating and Assessing Mouse Models of Diabetic Neuropathy

Current Drug Targets. Jan, 2008  |  Pubmed ID: 18220709

Diabetic neuropathy (DN) is a serious and debilitating complication of both type 1 and type 2 diabetes. Despite intense research efforts into multiple aspects of this complication, including both vascular and neuronal metabolic derangements, the only treatment remains maintenance of euglycemia. Basic research into the mechanisms responsible for DN relies on using the most appropriate animal model. The advent of genetic manipulation has moved mouse models of human disease to the forefront. The ability to insert or delete genes affected in human patients offers unique insight into disease processes; however, mice are still not humans and difficulties remain in interpreting data derived from these animals. A number of studies have investigated and described DN in mice but it is difficult to compare these studies with each other or with human DN due to experimental differences including background strain, type of diabetes, method of induction and duration of diabetes, animal age and gender. This review describes currently used DN animal models. We followed a standardized diabetes induction protocol and designed and implemented a set of phenotyping parameters to classify the development and severity of DN. By applying standard protocols, we hope to facilitate the comparison and characterization of DN across different background strains in the hope of discovering the most human like model in which to test potential therapies.

The Antioxidant Response As a Drug Target in Diabetic Neuropathy

Current Drug Targets. Jan, 2008  |  Pubmed ID: 18220717

While increasing antioxidant potential is an attractive treatment strategy for diabetic neuropathy, many years of trials using high-dose oral antioxidants have not produced therapeutic results. An increasing understanding of the innate antioxidant response and the pharmacological agents that can regulate this mechanism may open new avenue for drug development. This review describes the current state of antioxidant trials and the potential for targeting the antioxidant response. In combination with antihyperglycemic agents, agents that regulate the antioxidant response may afford superior protection against cellular oxidative injury in diabetes.

Membrane Traffic and Muscle: Lessons from Human Disease

Traffic (Copenhagen, Denmark). Jul, 2008  |  Pubmed ID: 18266915

Like all mammalian tissues, skeletal muscle is dependent on membrane traffic for proper development and homeostasis. This fact is underscored by the observation that several human diseases of the skeletal muscle are caused by mutations in gene products of the membrane trafficking machinery. An examination of these diseases and the proteins that underlie them is instructive both in terms of determining disease pathogenesis and of understanding the normal aspects of muscle biology regulated by membrane traffic. This review highlights our current understanding of the trafficking genes responsible for human myopathies.

National Institute of Neurological Disorders and Stroke (NINDS): Advances in Understanding and Treating Neuropathy, 24-25 October 2006; Bethesda, Maryland

Journal of the Peripheral Nervous System : JPNS. Mar, 2008  |  Pubmed ID: 18346227

National Institute of Neurological Disorders and Stroke sponsored a meeting to explore the current status of basic and clinical research in peripheral neurobiology and clinical neuropathy. The goal of the workshop was to identify areas where additional research could lead to the development of new therapeutics in the next 5 years. Participants discussed the current understanding of disease mechanisms of axonal and demyelinating neuropathies, existing techniques in research, disease biomarkers, and assessment of neuropathy. Painful neuropathies were discussed at the basic scientific and clinical levels in relation to new insights into etiology and treatment. The meeting concluded with a discussion on therapeutic development in neuropathy and the need for a unified approach to multicenter trials. Short-term goals of the workshop were to form a working group for neuropathy, the Peripheral Neuropathy Study Group, and to translate new scientific findings into therapies and complete clinical trials.

Abnormal Muscle Spindle Innervation and Large-fiber Neuropathy in Diabetic Mice

Diabetes. Jun, 2008  |  Pubmed ID: 18362211

Large-fiber diabetic polyneuropathy (DPN) leads to balance and gait abnormalities, placing patients at risk for falls. Large sensory axons innervating muscle spindles provide feedback for balance and gait and, when damaged, can cause altered sensorimotor function. This study aimed to determine whether symptoms of large-fiber DPN in type 1 and type 2 diabetic mouse models are related to alterations in muscle spindle innervation. In addition, diabetic mice were treated with insulin to assess whether sensorimotor and spindle deficits were reversible.

The Design of Electrospun PLLA Nanofiber Scaffolds Compatible with Serum-free Growth of Primary Motor and Sensory Neurons

Acta Biomaterialia. Jul, 2008  |  Pubmed ID: 18396117

Aligned electrospun nanofibers direct neurite growth and may prove effective for repair throughout the nervous system. Applying nanofiber scaffolds to different nervous system regions will require prior in vitro testing of scaffold designs with specific neuronal and glial cell types. This would be best accomplished using primary neurons in serum-free media; however, such growth on nanofiber substrates has not yet been achieved. Here we report the development of poly(L-lactic acid) (PLLA) nanofiber substrates that support serum-free growth of primary motor and sensory neurons at low plating densities. In our study, we first compared materials used to anchor fibers to glass to keep cells submerged and maintain fiber alignment. We found that poly(lactic-co-glycolic acid) (PLGA) anchors fibers to glass and is less toxic to primary neurons than bandage and glue used in other studies. We then designed a substrate produced by electrospinning PLLA nanofibers directly on cover slips pre-coated with PLGA. This substrate retains fiber alignment even when the fiber bundle detaches from the cover slip and keeps cells in the same focal plane. To see if increasing wettability improves motor neuron survival, some fibers were plasma etched before cell plating. Survival on etched fibers was reduced at the lower plating density. Finally, the alignment of neurons grown on this substrate was equal to nanofiber alignment and surpassed the alignment of neurites from explants tested in a previous study. This substrate should facilitate investigating the behavior of many neuronal types on electrospun fibers in serum-free conditions.

Kindlin-2 is Required for Myocyte Elongation and is Essential for Myogenesis

BMC Cell Biology. 2008  |  Pubmed ID: 18611274

Integrins are required for normal muscle differentiation and disruptions in integrin signaling result in human muscle disease. The intracellular components that regulate integrin function during myogenesis are poorly understood. Unc-112 is an integrin-associated protein required for muscle development in C. elegans. To better understand the intracellular effectors of integrin signaling in muscle, we examined the mammalian homolog of Unc-112, kindlin-2.

Diabetic Neuropathy: Mechanisms to Management

Pharmacology & Therapeutics. Oct, 2008  |  Pubmed ID: 18616962

Neuropathy is the most common and debilitating complication of diabetes and results in pain, decreased motility, and amputation. Diabetic neuropathy encompasses a variety of forms whose impact ranges from discomfort to death. Hyperglycemia induces oxidative stress in diabetic neurons and results in activation of multiple biochemical pathways. These activated pathways are a major source of damage and are potential therapeutic targets in diabetic neuropathy. Though therapies are available to alleviate the symptoms of diabetic neuropathy, few options are available to eliminate the root causes. The immense physical, psychological, and economic cost of diabetic neuropathy underscore the need for causally targeted therapies. This review covers the pathology, epidemiology, biochemical pathways, and prevention of diabetic neuropathy, as well as discusses current symptomatic and causal therapies and novel approaches to identify therapeutic targets.

Rosiglitazone Reduces Renal and Plasma Markers of Oxidative Injury and Reverses Urinary Metabolite Abnormalities in the Amelioration of Diabetic Nephropathy

American Journal of Physiology. Renal Physiology. Oct, 2008  |  Pubmed ID: 18667486

Recent studies suggest that thiazolidinediones ameliorate diabetic nephropathy (DN) independently of their effect on hyperglycemia. In the current study, we confirm and extend these findings by showing that rosiglitazone treatment prevented the development of DN and reversed multiple markers of oxidative injury in DBA/2J mice made diabetic by low-dose streptozotocin. These diabetic mice developed a 14.2-fold increase in albuminuria and a 53% expansion of renal glomerular extracellular matrix after 12 wk of diabetes. These changes were largely abrogated by administration of rosiglitazone beginning 2 wk after the completion of streptozotocin injections. Rosiglitazone had no effect on glycemic control. Rosiglitazone had similar effects on insulin-treated diabetic mice after 24 wk of diabetes. Podocyte loss and glomerular fibronectin accumulation, other markers of early DN, were prevented by rosiglitazone in both 12- and 24-wk diabetic models. Surprisingly, glomerular GLUT1 levels did not increase and nephrin levels did not decrease in the diabetic animals; neither changed with rosiglitazone. Plasma and kidney markers of protein oxidation and lipid peroxidation were significantly elevated in the 24-wk diabetic animals despite insulin treatment and were reduced to near-normal levels by rosiglitazone. Finally, urinary metabolites were markedly altered by diabetes. Of 1,988 metabolite features identified by electrospray ionization time of flight mass spectrometry, levels of 56 were altered more than twofold in the urine of diabetic mice. Of these, 21 were returned to normal by rosiglitazone. Thus rosiglitazone has direct effects on the renal glomerulus to reduce reactive oxygen species accumulation to prevent type 1 diabetic mice from development of DN.

Mechanisms of Disease: the Oxidative Stress Theory of Diabetic Neuropathy

Reviews in Endocrine & Metabolic Disorders. Dec, 2008  |  Pubmed ID: 18709457

Diabetic neuropathy is the most common complication of diabetes, affecting 50% of diabetic patients. Currently, the only treatment for diabetic neuropathy is glucose control and careful foot care. In this review, we discuss the idea that excess glucose overloads the electron transport chain, leading to the production of superoxides and subsequent mitochondrial and cytosolic oxidative stress. Defects in metabolic and vascular pathways intersect with oxidative stress to produce the onset and progression of nerve injury present in diabetic neuropathy. These pathways include the production of advanced glycation end products, alterations in the sorbitol, hexosamine and protein kinase C pathways and activation of poly-ADP ribose polymerase. New bioinformatics approaches can augment current research and lead to new discoveries to understand the pathogenesis of diabetic neuropathy and to identify more effective molecular therapeutic targets.

Insulin-like Growth Factors in the Peripheral Nervous System

Endocrinology. Dec, 2008  |  Pubmed ID: 18719018

IGF-I and -II are potent neuronal mitogens and survival factors. The actions of IGF-I and -II are mediated via the type I IGF receptor (IGF-IR) and IGF binding proteins regulate the bioavailability of the IGFs. Cell viability correlates with IGF-IR expression and intact IGF-I/IGF-IR signaling pathways, including activation of MAPK/phosphatidylinositol-3 kinase. The expression of IGF-I and -II, IGF-IR, and IGF binding proteins are developmentally regulated in the central and peripheral nervous system. IGF-I therapy demonstrates mixed therapeutic results in the treatment of peripheral nerve injury, neuropathy, and motor neuron diseases such as amyotrophic lateral sclerosis. In this review we discuss the role of IGFs during peripheral nervous system development and the IGF signaling system as the potential therapeutic target for the treatment of nerve injury and motor neuron diseases.

The Utah Early Neuropathy Scale: a Sensitive Clinical Scale for Early Sensory Predominant Neuropathy

Journal of the Peripheral Nervous System : JPNS. Sep, 2008  |  Pubmed ID: 18844788

Early neuropathy is often sensory predominant and prominently involves small-diameter nerve fibers. Established neuropathy examination scales such as the Michigan Diabetic Neuropathy Scale (MDNS) and the Neuropathy Impairment Score-Lower Leg (NIS-LL) focus primarily on large-fiber sensory and motor function. Here, we validate the Utah Early Neuropathy Scale (UENS), a physical examination scale specific to early sensory predominant polyneuropathy. Compared with other scales, the UENS emphasizes severity and spatial distribution of pin (sharp) sensation loss in the foot and leg and focuses less on motor weakness. UENS, MDNS, and NIS-LL were compared in 215 diabetic or prediabetic subjects, with (129) or without neuropathy (86), and repeated in 114 neuropathy subjects after 1 year of follow-up. Neuropathy severity was also evaluated with nerve conduction studies, quantitative sensory testing, quantitative sudomotor axonal reflex testing, and intraepidermal nerve fiber density determination. The UENS had a high degree of interrater reliability (interclass correlation of 94%). UENS correlated significantly to MDNS and NIS-LL (p < 0.01), and more significantly than MDNS or NIS-LL to confirmatory tests. In this cohort, UENS had a superior profile to receiver operating characteristic analysis across a range of scores, with a sensitivity (92%) higher than MDNS (67%) or NIS-LL (81%), without sacrificing specificity. UENS more closely correlated with change in ancillary and small-fiber neuropathy measures over 1 year follow-up than did MDNS or NIS-LL. UENS is a sensitive and reproducible clinical measure of sensory and small-fiber nerve injury and may be useful in trials of early neuropathy.

Oxidative Injury and Neuropathy in Diabetes and Impaired Glucose Tolerance

Neurobiology of Disease. Jun, 2008  |  Pubmed ID: 18424057

Clinical studies suggest that impaired glucose tolerance (IGT) is associated with the development of neuropathy. The aim of the current study was to determine if neuropathy developed in the female Zucker Diabetic Fatty (ZDF) rat, an animal model of IGT and type 2 diabetes. The ZDF rat develops impaired glucose tolerance (IGT) when fed a control diet, and frank diabetes when fed a high fat diet. Following 10 weeks of hyperglycemia, sensory nerve action potentials (SNAP) and compound motor action potentials (CMAP) were reduced and sensory conduction velocities were slowed (distal>proximal) in the tail and hind limb in ZDF animals with IGT and frank diabetes (p<0.01). Neuropathy was coupled with evidence of increased reactive oxygen species (ROS) and cellular injury in dorsal root ganglion (DRG) neurons from IGT animals. Our study supports the hypothesis that neuropathy develops in an animal model of IGT and is associated with evidence of oxidative injury in DRG and peripheral nerves.

Rosiglitazone Treatment Reduces Diabetic Neuropathy in Streptozotocin-treated DBA/2J Mice

Endocrinology. Oct, 2008  |  Pubmed ID: 18583417

Diabetic neuropathy (DN) is a common complication of diabetes. Currently, there is no drug treatment to prevent or slow the development of DN. Rosiglitazone (Rosi) is a potent insulin sensitizer and may also slow the development of DN by a mechanism independent of its effect on hyperglycemia. A two by two design was used to test the effect of Rosi treatment on the development of DN. Streptozotocin-induced diabetic DBA/2J mice were treated with Rosi. DN and oxidative stress were quantified, and gene expression was profiled using the Affymetrix Mouse Genome 430 2.0 microarray platform. An informatics approach identified key regulatory elements activated by Rosi. Diabetic DBA/2J mice developed severe hyperglycemia, DN, and elevated oxidative stress. Rosi treatment did not affect hyperglycemia but did reduce oxidative stress and prevented the development of thermal hypoalgesia. Two novel transcription factor binding modules were identified that may control genes correlated to changes in DN after Rosi treatment: SP1F_ZBPF and EGRF_EGRF. These targets may be useful in designing drugs with the same efficacy as Rosi in treating DN but with fewer undesirable effects.

Insulin-like Growth Factor-I for the Treatment of Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis : Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. Apr, 2009  |  Pubmed ID: 18608100

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects both upper and lower motorneurons (MN) resulting in weakness, paralysis and subsequent death. Insulin-like growth factor-I (IGF-I) is a potent neurotrophic factor that has neuroprotective properties in the central and peripheral nervous systems. Due to the efficacy of IGF-I in the treatment of other diseases and its ability to promote neuronal survival, IGF-I is being extensively studied in ALS therapeutic trials. This review covers in vitro and in vivo studies examining the efficacy of IGF-I in ALS model systems and also addresses the mechanisms by which IGF-I asserts its effects in these models, the status of the IGF-I system in ALS patients, results of clinical trials, and the need for the development of better delivery mechanisms to maximize IGF-I efficacy. The knowledge obtained from these studies suggests that IGF-I has the potential to be a safe and efficacious therapy for ALS.

SUMOylation of the Mitochondrial Fission Protein Drp1 Occurs at Multiple Nonconsensus Sites Within the B Domain and is Linked to Its Activity Cycle

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Nov, 2009  |  Pubmed ID: 19638400

Dynamin-related protein (Drp) 1 is a key regulator of mitochondrial fission and is composed of GTP-binding, Middle, insert B, and C-terminal GTPase effector (GED) domains. Drp1 associates with mitochondrial fission sites and promotes membrane constriction through its intrinsic GTPase activity. The mechanisms that regulate Drp1 activity remain poorly understood but are likely to involve reversible post-translational modifications, such as conjugation of small ubiquitin-like modifier (SUMO) proteins. Through a detailed analysis, we find that Drp1 interacts with the SUMO-conjugating enzyme Ubc9 via multiple regions and demonstrate that Drp1 is a direct target of SUMO modification by all three SUMO isoforms. While Drp1 does not harbor consensus SUMOylation sequences, our analysis identified2 clusters of lysine residues within the B domain that serve as noncanonical conjugation sites. Although initial analysis indicates that mitochondrial recruitment of ectopically expressed Drp1 in response to staurosporine is unaffected by loss of SUMOylation, we find that Drp1 SUMOylation is enhanced in the context of the K38A mutation. This dominant-negative mutant, which is deficient in GTP binding and hydrolysis, does not associate with mitochondria and prevents normal mitochondrial fission. This finding suggests that SUMOylation of Drp1 is linked to its activity cycle and is influenced by Drp1 localization.

Vascular Endothelial Growth Factor Prevents G93A-SOD1-induced Motor Neuron Degeneration

Developmental Neurobiology. Nov, 2009  |  Pubmed ID: 19672955

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by selective loss of motor neurons (MNs). Twenty percent of familial ALS cases are associated with mutations in Cu(2+)/Zn(2+) superoxide dismutase (SOD1). To specifically understand the cellular mechanisms underlying mutant SOD1 toxicity, we have established an in vitro model of ALS using rat primary MN cultures transfected with an adenoviral vector encoding a mutant SOD1, G93A-SOD1. Transfected cells undergo axonal degeneration and alterations in biochemical responses characteristic of cell death such as activation of caspase-3. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can increase axonal outgrowth, block neuronal apoptosis, and promote neurogenesis. Decreased VEGF gene expression in mice results in a phenotype similar to that seen in patients with ALS, thus linking loss of VEGF to the pathogenesis of MN degeneration. Decreased neurotrophic signals prior to and during disease progression may increase MN susceptibility to mutant SOD1-induced toxicity. In this study, we demonstrate a decrease in VEGF and VEGFR2 levels in the spinal cord of G93A-SOD1 ALS mice. Furthermore, in isolated MN cultures, VEGF alleviates the effects of G93A-SOD1 toxicity and neuroprotection involves phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling. Overall, these studies validate the usefulness of VEGF as a potential therapeutic factor for the treatment of ALS and give valuable insight into the responsible signaling pathways and mechanisms involved.

Nerve Growth Factor Mediates Mechanical Allodynia in a Mouse Model of Type 2 Diabetes

Journal of Neuropathology and Experimental Neurology. Nov, 2009  |  Pubmed ID: 19816194

C57BLKS db/db (db/db) mice develop a neuropathy with features of human type 2 diabetic neuropathy. Here, we demonstrate that these mice develop transient mechanical allodynia at the early stage of diabetes. We hypothesized that nerve growth factor (NGF), which enhances the expression of key mediators of nociception (i.e. substance P [SP] and calcitonin gene-related peptide), contributes to the development of mechanical allodynia in these mice. We found that NGF, SP, and calcitonin gene-related peptide gene expression is upregulated in the dorsal root ganglion (DRG) of db/db mice before or during the period that they develop mechanical allodynia. There were more small- to medium-sized NGF-immunopositive DRG neurons in db/db mice than in control db+ mice; these neurons also expressed SP, consistent with its role in nociception. Nerve growth factor expression in the hind paw skin was also increased in a variety of dermal cell types and nerve fibers, suggesting the contribution of a peripheral source of NGF to mechanical allodynia. The upregulation of NGF coincided with enhanced tropomyosin-related kinase A receptor phosphorylation in the DRG. Finally, an antibody against NGF inhibited mechanical allodynia and decreased the numbers of SP-positive DRG neurons in db/db mice. These results suggest that inhibition of NGF action is a potential strategy for treating painful diabetic neuropathy.

Increased Tau Phosphorylation and Cleavage in Mouse Models of Type 1 and Type 2 Diabetes

Endocrinology. Dec, 2009  |  Pubmed ID: 19819959

As the population of the United States ages, the incidence of age-related neurodegenerative and systemic diseases including Alzheimer's disease (AD) and diabetes is increasing rapidly. Multiple studies report that patients with diabetes have a 50-75% increased risk of developing AD compared with age- and gender-matched patients without diabetes. Abnormally phosphorylated tau is a major building block of neurofibrillary tangles, a classic neuropathological characteristic of AD. In addition, proteolytic tau cleavage promotes AD progression due to cleaved tau serving as a nucleation center for the pathological assembly of tau filaments. The current study examines tau modification in type 1 (streptozotocin-injected) and type 2 (db/db) mouse models of diabetes. Tau phosphorylation is increased in the cortex and hippocampus of db/db mice compared with db+ control mouse brain. Interestingly, there is an age-dependent increase in tau cleavage that is not observed in age-matched control db+ animals. Streptozotocin injection also increased tau phosphorylation; however, the increase was less significant compared with the type 2 mouse model, and more importantly, no tau cleavage was detected. Our results suggest tau modification caused by insulin dysfunction and hyperglycemia may contribute to the increased incidence of AD in diabetes. We hypothesize that type 1 and type 2 diabetes may contribute to AD through different mechanisms; in type 2 diabetes, hyperglycemia-mediated tau cleavage may be the key feature, whereas insulin deficiency may be the major contributing factor in type 1 diabetes.

Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized MtDNA In Vitro

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. Nov, 2009  |  Pubmed ID: 19875847

Mitochondria are key regulators of cellular energy and are the focus of a large number of studies examining the regulation of mitochondrial dynamics and biogenesis in healthy and diseased conditions. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to visualize newly synthesized mtDNA in individual cells in order to study mtDNA replication within subcellular compartments of neurons. The technique combines the incorporation of BrdU and/or EdU into mtDNA together with a tyramide signal amplification protocol. Employing this technique, we visualized and measured mtDNA biogenesis in individual cells. The labeling procedure for EdU allows for more comprehensive results by allowing the comparison of its incorporation with other intracellular markers because it does not require harsh acid or enzyme digests necessary to recover the BrdU epitope. In addition, the utilization of both BrdU and EdU permits sequential pulse-chase experiments to follow the intracellular localization of mtDNA replication. The ability to quantify mitochondrial biogenesis provides an essential tool for investigating alterations in mitochondrial dynamics involved in the pathogenesis of multiple cellular disorders including neuropathies and neurodegenerative diseases.

Hyperlipidemia: a New Therapeutic Target for Diabetic Neuropathy

Journal of the Peripheral Nervous System : JPNS. Dec, 2009  |  Pubmed ID: 20021567

Emerging data establish dyslipidemia as a significant contributor to the development of diabetic neuropathy. In this review, we discuss how separate metabolic imbalances, including hyperglycemia and hyperlipidemia, converge on mechanisms leading to oxidative stress in dorsal root ganglia (DRG) sensory neurons. We conclude with suggestions for novel therapeutic strategies to prevent or reverse diabetes-induced nerve degeneration.

Sensory Neurons and Schwann Cells Respond to Oxidative Stress by Increasing Antioxidant Defense Mechanisms

Antioxidants & Redox Signaling. Mar, 2009  |  Pubmed ID: 19072199

Elevated blood glucose is a key initiator of mechanisms leading to diabetic neuropathy. Increases in glucose induce acute mitochondrial oxidative stress in dorsal root ganglion (DRG) neurons, the sensory neurons normally affected in diabetic neuropathy, whereas Schwann cells are largely unaffected. We propose that activation of an antioxidant response in DRG neurons would prevent glucose-induced injury. In this study, mild oxidative stress (1 microM H2O2) leads to the activation of the transcription factor Nrf2 and expression of antioxidant (phase II) enzymes. DRG neurons are thus protected from subsequent hyperglycemia-induced injury, as determined by activation of caspase 3 and the TUNEL assay. Schwann cells display high basal antioxidant enzyme expression and respond to hyperglycemia and mild oxidative stress via further increases in these enzymes. The botanical compounds resveratrol and sulforaphane activate the antioxidant response in DRG neurons. Other drugs that protect DRG neurons and block mitochondrial superoxide, identified in a compound screen, have differential ability to activate the antioxidant response. Multiple cellular targets exist for the prevention of hyperglycemic oxidative stress in DRG neurons, and these form the basis for new therapeutic strategies against diabetic neuropathy.

Intraspinal Cord Delivery of IGF-I Mediated by Adeno-associated Virus 2 is Neuroprotective in a Rat Model of Familial ALS

Neurobiology of Disease. Mar, 2009  |  Pubmed ID: 19135533

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating disease that is characterized by the progressive loss of motor neurons. Patients with ALS usually die from respiratory failure due to respiratory muscle paralysis. Consequently, therapies aimed at preserving segmental function of the respiratory motor neurons could extend life for these patients. Insulin-like growth factor-I (IGF-I) is known to be a potent survival factor for motor neurons. In this study we induced high levels of IGF-I expression in the cervical spinal cord of hSOD1(G93A) rats with intraspinal cord (ISC) injections of an adeno-associated virus serotype 2 vector (CERE-130). This approach reduced the extent of motor neuron loss in the treated segments of the spinal cord. However, a corresponding preservation of motor function was observed in male, but not female, hSOD1(G93A) rats. We conclude that ISC injection of CERE-130 has the potential to protect motor neurons and preserve neuromuscular function in ALS.

SciMiner: Web-based Literature Mining Tool for Target Identification and Functional Enrichment Analysis

Bioinformatics (Oxford, England). Mar, 2009  |  Pubmed ID: 19188191

SciMiner is a web-based literature mining and functional analysis tool that identifies genes and proteins using a context specific analysis of MEDLINE abstracts and full texts. SciMiner accepts a free text query (PubMed Entrez search) or a list of PubMed identifiers as input. SciMiner uses both regular expression patterns and dictionaries of gene symbols and names compiled from multiple sources. Ambiguous acronyms are resolved by a scoring scheme based on the co-occurrence of acronyms and corresponding description terms, which incorporates optional user-defined filters. Functional enrichment analyses are used to identify highly relevant targets (genes and proteins), GO (Gene Ontology) terms, MeSH (Medical Subject Headings) terms, pathways and protein-protein interaction networks by comparing identified targets from one search result with those from other searches or to the full HGNC [HUGO (Human Genome Organization) Gene Nomenclature Committee] gene set. The performance of gene/protein name identification was evaluated using the BioCreAtIvE (Critical Assessment of Information Extraction systems in Biology) version 2 (Year 2006) Gene Normalization Task as a gold standard. SciMiner achieved 87.1% recall, 71.3% precision and 75.8% F-measure. SciMiner's literature mining performance coupled with functional enrichment analyses provides an efficient platform for retrieval and summary of rich biological information from corpora of users' interests. AVAILABILITY: http://jdrf.neurology.med.umich.edu/SciMiner/. A server version of the SciMiner is also available for download and enables users to utilize their institution's journal subscriptions. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Loss of Myotubularin Function Results in T-tubule Disorganization in Zebrafish and Human Myotubular Myopathy

PLoS Genetics. Feb, 2009  |  Pubmed ID: 19197364

Myotubularin is a lipid phosphatase implicated in endosomal trafficking in vitro, but with an unknown function in vivo. Mutations in myotubularin cause myotubular myopathy, a devastating congenital myopathy with unclear pathogenesis and no current therapies. Myotubular myopathy was the first described of a growing list of conditions caused by mutations in proteins implicated in membrane trafficking. To advance the understanding of myotubularin function and disease pathogenesis, we have created a zebrafish model of myotubular myopathy using morpholino antisense technology. Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle. These changes include abnormally shaped and positioned nuclei and myofiber hypotrophy. These findings are consistent with those observed in the human disease. We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin. Finally, we identify abnormalities in the tubulo-reticular network in muscle from myotubularin zebrafish morphants and correlate these changes with abnormalities in T-tubule organization in biopsies from patients with myotubular myopathy. In all, we have generated a new model of myotubular myopathy and employed this model to uncover a novel function for myotubularin and a new pathomechanism for the human disease that may explain the weakness associated with the condition (defective excitation-contraction coupling). In addition, our findings of tubuloreticular abnormalities and defective excitation-contraction coupling mechanistically link myotubular myopathy with several other inherited muscle diseases, most notably those due to ryanodine receptor mutations. Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis.

Disorders of Cranial Nerves IX and X

Seminars in Neurology. Feb, 2009  |  Pubmed ID: 19214937

The glossopharyngeal and vagus nerves mediate the complex interplay between the many functions of the upper aerodigestive tract. Defects may occur anywhere from the brainstem to the peripheral nerve and can result in significant impairment in speech, swallowing, and breathing. Multiple etiologies can produce symptoms. In this review, the authors broadly examine the normal functions, clinical examination, and various pathologies of cranial nerves IX and X.

Insulin Receptor Substrate (IRS)-2, Not IRS-1, Protects Human Neuroblastoma Cells Against Apoptosis

Apoptosis : an International Journal on Programmed Cell Death. May, 2009  |  Pubmed ID: 19259821

Insulin receptor substrates (IRS)-1 and -2 are major substrates of insulin and type I insulin-like growth factor (IGF-I) receptor (IGF-IR) signaling. In this study, SH-EP human neuroblastoma cells are used as a model system to examine the differential roles of IRS-1 and IRS-2 on glucose-mediated apoptosis. In the presence of high glucose, IRS-1 underwent caspase-mediated degradation, followed by focal adhesion kinase (FAK) and Akt degradation and apoptosis. IRS-2 expression blocked all these changes whereas IRS-1 overexpression had no effect. In parallel, IRS-2, but not IRS-1, overexpression enhanced IGF-I-mediated Akt activation without affecting extracellular regulated kinase signaling. While IRS-1 was readily degraded by caspases, hyperglycemia-mediated IRS-2 degradation was unaffected by caspase inhibitors but blocked by proteasome and calpain inhibitors. Our data suggest that the differential degradation of IRS-1 and IRS-2 contributes to their distinct modes of action and the increased neuroprotective effects of IRS-2 in this report are due, in part, to its resistance to caspase-mediated degradation.

Reliability of Quantitative Sudomotor Axon Reflex Testing and Quantitative Sensory Testing in Neuropathy of Impaired Glucose Regulation

Muscle & Nerve. Apr, 2009  |  Pubmed ID: 19260066

Reproducible neurophysiologic testing paradigms are critical for multicenter studies of neuropathy associated with impaired glucose regulation (IGR), yet the best methodologies and endpoints remain to be established. This study evaluates the reproducibility of neurophysiologic tests within a multicenter research setting. Twenty-three participants with neuropathy and IGR were recruited from two study sites. The reproducibility of quantitative sudomotor axon reflex test (QSART) and quantitative sensory test (QST) (using the CASE IV system) was determined in a subset of patients at two sessions, and it was calculated from intraclass correlation coefficients (ICCs). QST (cold detection threshold: ICC=0.80; vibration detection threshold: ICC=0.75) was more reproducible than QSART (ICC foot=0.52). The performance of multiple tests in one setting did not improve reproducibility of QST. QST reproducibility in our IGR patients was similar to reports of other studies. QSART reproducibility was significantly lower than QST. In this group of patients, the reproducibility of QSART was unacceptable for use as a secondary endpoint measure in clinical research trials.

Hydrogen Peroxide-induced Akt Phosphorylation Regulates Bax Activation

Biochimie. May, 2009  |  Pubmed ID: 19278624

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) are involved in many cellular processes that positively and negatively regulate cell fate. H(2)O(2), acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H(2)O(2) was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H(2)O(2)-induced activation of PI3K/Akt influences post-translational modification of Bax and inactivates a key component of the cell death machinery.

Stem Cells: Comprehensive Treatments for Amyotrophic Lateral Sclerosis in Conjunction with Growth Factor Delivery

Growth Factors (Chur, Switzerland). Jun, 2009  |  Pubmed ID: 19294549

Amyotrophic lateral sclerosis (ALS) is characterized by loss of both upper and lower motor neurons. ALS progression is complex and likely due to cellular dysfunction at multiple levels, including mitochondrial dysfunction, glutamate excitotoxicity, oxidative stress, axonal dysfunction, reactive astrocytosis, and mutant superoxide dismutase expression, therefore, treatment must provide neuronal protection from multiple insults. A significant amount of ALS research focuses on growth factor-based therapies. Growth factors including insulin-like growth factor-I, vascular endothelial growth factor, brain-derived neurotrophic factor, and glial-derived neurotrophic factor exhibit robust neuroprotective effects on motor neurons in ALS models. Issues concerning growth factor delivery, stability and unwanted side effects slow the transfer of these treatments to human ALS patients. Stem cells represent a new therapeutic approach offering both cellular replacement and trophic support for the existing population. Combination therapy consisting of stem cells expressing beneficial growth factors may provide a comprehensive treatment for ALS.

Computational Methods for Predicting Sites of Functionally Important Dynamics

The Journal of Physical Chemistry. B. May, 2009  |  Pubmed ID: 19378962

Understanding and controlling biological function of proteins at the atomic level is of great importance; allosteric mechanisms provide such an interface. Experimental and computational methods have been developed to search for residue mutations that produce changes in function by altering sites of correlated motion. These methods are often observational in that altered motions are achieved by random sampling without revealing the underlying mechanism(s). We present two deterministic methods founded on structure-function relationships that predict dynamic control sites (i.e., locations that experience correlated motions as a result of altered dynamics). The first method ("static") is based on a single structure conformation (e.g., the wild type (WT)) and utilizes a graph description of atomic connectivity. The local atomic interactions are used to compute the propagation of contact paths. This description of structure connectivity reveals flexible locations that are susceptible to altered dynamics. The second method ("dynamic") is a comparative analysis between the normal modes of a WT structure and a mutant structure. A mapping function is defined that quantifies the significance of the motions in one structure projected onto the motions of the other. Each mode is considered up- or down-regulated according to its change in relative significance. This description of altered dynamics is the basis for a motion correlation analysis, from which the dynamic control sites are readily identified. The methods are theoretically derived and applied using the canonical system dihydrofolate reductase (DHFR). Both methods demonstrate a very high predictive value (p<0.005) in identifying known dynamic control sites. The dynamic method also produces a new hypothesis regarding the mechanism by which the DHFR mutant achieves hyperactivity. These tools are suitable for allosteric investigations and may greatly enhance the speed and effectiveness of other computational and experimental methods.

Elevated Triglycerides Correlate with Progression of Diabetic Neuropathy

Diabetes. Jul, 2009  |  Pubmed ID: 19411614

To evaluate mechanisms underlying diabetic neuropathy progression using indexes of sural nerve morphometry obtained from two identical randomized, placebo-controlled clinical trials.

Effects of Prior Intensive Insulin Therapy on Cardiac Autonomic Nervous System Function in Type 1 Diabetes Mellitus: the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Study (DCCT/EDIC)

Circulation. Jun, 2009  |  Pubmed ID: 19470886

The Epidemiology of Diabetes Interventions and Complications (EDIC) study, a prospective observational follow-up of the Diabetes Control and Complications Trial (DCCT) cohort, reported persistent benefit of prior intensive therapy on retinopathy and nephropathy in type 1 diabetes mellitus. We evaluated the effects of prior intensive insulin therapy on the prevalence and incidence of cardiac autonomic neuropathy (CAN) in former DCCT intensive and conventional therapy subjects 13 to 14 years after DCCT closeout.

Dyslipidemia-induced Neuropathy in Mice: the Role of OxLDL/LOX-1

Diabetes. Oct, 2009  |  Pubmed ID: 19592619

Neuropathy is a frequent and severe complication of diabetes. Multiple metabolic defects in type 2 diabetic patients result in oxidative injury of dorsal root ganglia (DRG) neurons. Our previous work focused on hyperglycemia clearly demonstrates induction of mitochondrial oxidative stress and acute injury in DRG neurons; however, this mechanism is not the only factor that produces neuropathy in vivo. Dyslipidemia also correlates with the development of neuropathy, even in pre-diabetic patients. This study was designed to explore the contribution of dyslipidemia in neuropathy.

National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Meeting Summary: Advances Toward Measuring Diabetic Retinopathy and Neuropathy: from the Bench to the Clinic and Back Again (April 4-5, 2007, Baltimore, Maryland)

Journal of Diabetes and Its Complications. May-Jun, 2009  |  Pubmed ID: 18413183

The National Institute of Diabetes and Digestive and Kidney Diseases sponsored a meeting recently to explore new ways to assess diabetic retinopathy and neuropathy, both in diabetic patients and in diabetic mice. The workshop compared current gold standards for assessment of retinopathy and neuropathy, new improvements of existing techniques, and new functional biomarkers measured with nontraditional technologies. Since the anatomical changes that comprise diabetic retinopathy and neuropathy take long to develop and have proven difficult to arrest once initiated, some talks highlighted the value of methods that are based on the pathophysiology that precedes, and might contribute to, the histopathology. In addition, a goal of the workshop was to produce a set of working criteria on phenotyping diabetic retinopathy and neuropathy that could be reviewed by the scientific community.

Patterning N-type and S-type Neuroblastoma Cells with Pluronic F108 and ECM Proteins

Journal of Biomedical Materials Research. Part A. May, 2010  |  Pubmed ID: 19609877

Influencing cell shape using micropatterned substrates affects cell behaviors, such as proliferation and apoptosis. Cell shape may also affect these behaviors in human neuroblastoma (NBL) cancer, but to date, no substrate design has effectively patterned multiple clinically important human NBL lines. In this study, we investigated whether Pluronic F108 was an effective antiadhesive coating for human NBL cells and whether it would localize three NBL lines to adhesive regions of tissue culture plastic or collagen I on substrate patterns. The adhesion and patterning of an S-type line, SH-EP, and two N-type lines, SH-SY5Y and IMR-32, were tested. In adhesion assays, F108 deterred NBL adhesion equally as well as two antiadhesive organofunctional silanes and far better than bovine serum albumin. Patterned stripes of F108 restricted all three human NBL lines to adhesive stripes of tissue culture plastic. We then investigated four schemes of applying collagen and F108 to different regions of a substrate. Contact with collagen obliterates the ability of F108 to deter NBL adhesion, limiting how both materials can be applied to substrates to produce high fidelity NBL patterning. This patterned substrate design should facilitate investigations of the role of cell shape in NBL cell behavior.

The Management of Diabetic Neuropathy in CKD

American Journal of Kidney Diseases : the Official Journal of the National Kidney Foundation. Feb, 2010  |  Pubmed ID: 20042258

Effect of Prior Intensive Insulin Treatment During the Diabetes Control and Complications Trial (DCCT) on Peripheral Neuropathy in Type 1 Diabetes During the Epidemiology of Diabetes Interventions and Complications (EDIC) Study

Diabetes Care. May, 2010  |  Pubmed ID: 20150297

To evaluate the impact of former intensive versus conventional insulin treatment on neuropathy in Diabetes Control and Complications Trial (DCCT) intensive and conventional treatment subjects with type 1 diabetes 13-14 years after DCCT closeout, during which time the two groups had achieved similar A1C levels.

Accelerated Neuritogenesis and Maturation of Primary Spinal Motor Neurons in Response to Nanofibers

Developmental Neurobiology. Jul, 2010  |  Pubmed ID: 20213755

Neuritogenesis, neuronal polarity formation, and maturation of axons and dendrites are strongly influenced by both biochemical and topographical extracellular components. The aim of this study was to elucidate the effects of polylactic acid electrospun fiber topography on primary motor neuron development, because regeneration of motor axons is extremely limited in the central nervous system and could potentially benefit from the implementation of a synthetic scaffold to encourage regrowth. In this analysis, we found that both aligned and randomly oriented submicron fibers significantly accelerated the processes of neuritogenesis and polarity formation of individual cultured motor neurons compared to flat polymer films and glass controls, likely due to restricted lamellipodia formation observed on fibers. In contrast, dendritic maturation and soma spreading were inhibited on fiber substrates after 2 days in vitro. This study is the first to examine the effects of electrospun fiber topography on motor neuron neuritogenesis and polarity formation. Aligned nanofibers were shown to affect the directionality and timing of motor neuron development, providing further evidence for the effective use of electrospun scaffolds in neural regeneration applications.

The Pleotrophic Effects of Insulin-like Growth Factor-I on Human Spinal Cord Neural Progenitor Cells

Stem Cells and Development. Dec, 2010  |  Pubmed ID: 20406098

Most stem cell therapies involve direct, intraparachymal placement of neural progenitor cells. These cells provide physical support to the endogenous neuronal population and may be engineered to provide in situ growth factor support. Insulin-like growth factor-I (IGF-I) has potent neurotrophic and neuroprotective properties and is expressed by human neural stem cells (hNSCs). IGF-I is implicated in multiple aspects of cell behavior, including proliferation, differentiation, and survival. Enhancing hNSC function through IGF-I overexpression may increase the benefits of stem cell therapy. As a first step to that goal, we examined the direct effects of IGF-I on hNSC behavior in vitro. We demonstrate that IGF-I treatment enhances both the number and length of hNSC neurites. This is correlated with a decrease in proliferation, suggesting that IGF-I promotes neurite outgrowth but not proliferation. While IGF-I activates both AKT and MAPK signaling in hNSCs, we demonstrate that IGF-I-mediated neurite outgrowth is dependent only on AKT signaling. Finally, we demonstrate that IGF-I is neuroprotective after glutamate exposure in a model of excitotoxic cell death.

DCCT and EDIC Studies in Type 1 Diabetes: Lessons for Diabetic Neuropathy Regarding Metabolic Memory and Natural History

Current Diabetes Reports. Aug, 2010  |  Pubmed ID: 20464532

The DCCT/EDIC (Diabetes Control and Complications Trial/ Epidemiology of Diabetes Interventions and Complications) provides a comprehensive characterization of the natural history of diabetic neuropathy in patients with type 1 diabetes and provides insight into the impact of intensive insulin therapy in disease progression. The lessons learned about the natural history of distal symmetrical polyneuropathy and cardiovascular autonomic neuropathy and the impact of glycemic control on neuropathy are discussed in this review.

Mitochondrial Biogenesis and Fission in Axons in Cell Culture and Animal Models of Diabetic Neuropathy

Acta Neuropathologica. Oct, 2010  |  Pubmed ID: 20473509

Mitochondrial-mediated oxidative stress in response to high glucose is proposed as a primary cause of dorsal root ganglia (DRG) neuron injury in the pathogenesis of diabetic neuropathy. In the present study, we report a greater number of mitochondria in both myelinated and unmyelinated dorsal root axons in a well-established model of murine diabetic neuropathy. No similar changes were seen in younger diabetic animals without neuropathy or in the ventral motor roots of any diabetic animals. These findings led us to examine mitochondrial biogenesis and fission in response to hyperglycemia in the neurites of cultured DRG neurons. We demonstrate overall mitochondrial biogenesis via increases in mitochondrial transcription factors and increases in mitochondrial DNA in both DRG neurons and axons. However, this process occurs over a longer time period than a rapidly observed increase in the number of mitochondria in DRG neurites that appears to result, at least in part, from mitochondrial fission. We conclude that during acute hyperglycemia, mitochondrial fission is a prominent response, and excessive mitochondrial fission may result in dysregulation of energy production, activation of caspase 3, and subsequent DRG neuron injury. During more prolonged hyperglycemia, there is evidence of compensatory mitochondrial biogenesis in axons. Our data suggest that an imbalance between mitochondrial biogenesis and fission may play a role in the pathogenesis of diabetic neuropathy.

Lack of Both Bradykinin B1 and B2 Receptors Enhances Nephropathy, Neuropathy, and Bone Mineral Loss in Akita Diabetic Mice

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2010  |  Pubmed ID: 20479236

An insertion polymorphism of the angiotensin-I converting enzyme gene (ACE) is common in humans and the higher expressing allele is associated with an increased risk of diabetic complications. The ACE polymorphism does not significantly affect blood pressure or angiotensin II levels, suggesting that the kallikrein-kinin system partly mediates the effects of the polymorphism. We have therefore explored the influence of lack of both bradykinin receptors (B1R and B2R) on diabetic nephropathy, neuropathy, and osteopathy in male mice heterozygous for the Akita diabetogenic mutation in the insulin 2 gene (Ins2). We find that all of the detrimental phenotypes observed in Akita diabetes are enhanced by lack of both B1R and B2R, including urinary albumin excretion, glomerulosclerosis, glomerular basement membrane thickening, mitochondrial DNA deletions, reduction of nerve conduction velocities and of heat sensation, and bone mineral loss. Absence of the bradykinin receptors also enhances the diabetes-associated increases in plasma thiobarbituric acid-reactive substances, mitochondrial DNA deletions, and renal expression of fibrogenic genes, including transforming growth factor beta1, connective tissue growth factor, and endothelin-1. Thus, lack of B1R and B2R exacerbates diabetic complications. The enhanced renal injury in diabetic mice caused by lack of B1R and B2R may be mediated by a combination of increases in oxidative stress, mitochondrial DNA damage and over expression of fibrogenic genes.

P38 Mediates Mechanical Allodynia in a Mouse Model of Type 2 Diabetes

Molecular Pain. 2010  |  Pubmed ID: 20482876

Painful Diabetic Neuropathy (PDN) affects more than 25% of patients with type 2 diabetes; however, the pathogenesis remains unclear due to lack of knowledge of the molecular mechanisms leading to PDN. In our current study, we use an animal model of type 2 diabetes in order to understand the roles of p38 in PDN. Previously, we have demonstrated that the C57BLK db/db (db/db) mouse, a model of type 2 diabetes that carries the loss-of-function leptin receptor mutant, develops mechanical allodynia in the hind paws during the early stage (6-12 wk of age) of diabetes. Using this timeline of PDN, we can investigate the signaling mechanisms underlying mechanical allodynia in the db/db mouse.

The Role of MTMR14 in Autophagy and in Muscle Disease

Autophagy. Aug, 2010  |  Pubmed ID: 20595810

Phosphoinositides (PIs) are a group of low-abundance phospholipids that play a critical role in the control of organelle and membrane traffic. There is strong evidence that specific PIs are also important for the regulation of autophagy. PIs are modified by a complex network of lipid kinases and phosphatases. A recent study from our laboratory focused on two PI phosphatases from the myotubularin related protein (MTMR) family, myotubularin (MTM1) and MTMR14. Using zebrafish as a model system, we found that dual knockdown of MTM1 and MTMR14 leads to an unexpectedly severe developmental motor phenotype. We found that this severe phenotype was mediated, at least in part, by dysregulation of autophagy, as demonstrated by the accumulation of autophagic vacuoles and increased levels of LC3-II. Our study provides the first in vivo evidence for a role of myotubularins, and in particular MTMR14, in the regulation of autophagy.

The Effects of Anesthesia on Measures of Nerve Conduction Velocity in Male C57Bl6/J Mice

Neuroscience Letters. Oct, 2010  |  Pubmed ID: 20691755

Animal models, particularly mice, are used extensively to investigate neurological diseases. Basic research regarding animal models of human neurological disease requires that the animals exhibit hall mark characteristics of the disease. These include disease specific anatomical, metabolic and behavioral changes. Nerve conduction velocity (NCV) is the predominant method used to assess peripheral nerve health. Normative data adjusted for age, gender and height is available for human patients; however, these data are not available for most rodents including mice. NCV may be affected by animal age and size, body temperature, stimulus strength and anesthesia. While the effects of temperature, age and size are documented, the direct and indirect effects of anesthesia on NCV are not well reported. Our laboratory is primarily concerned with animal models of diabetic neuropathy (DN) and uses NCV to confirm the presence of neuropathy. To ensure that subtle changes in NCV are reliably assayed and not directly or indirectly affected by anesthesia, we compared the effects of 4 commonly used anesthetics, isoflurane, ketamine/xylazine, sodium pentobarbital and 2-2-2 tribromoethanol on NCV in a commonly used rodent model, the C57Bl6/J mouse. Our results indicate that of the anesthetics tested, isoflurane has minimal impact on NCV and is the safest, most effective method of anesthesia. Our data strongly suggest that isoflurane should become the anesthetic of choice when performing NCV on murine models of neurological disease.

Vibration Perception Threshold As a Measure of Distal Symmetrical Peripheral Neuropathy in Type 1 Diabetes: Results from the DCCT/EDIC Study

Diabetes Care. Dec, 2010  |  Pubmed ID: 20833868

To describe the sensitivity, specificity, positive predictive value, and negative predictive value of vibration perception threshold (VPT) testing in subjects with type 1 diabetes relative to gold standard assessments of peripheral neuropathy.

How Does Diabetes Accelerate Alzheimer Disease Pathology?

Nature Reviews. Neurology. Oct, 2010  |  Pubmed ID: 20842183

Diabetes and Alzheimer disease (AD)-two age-related diseases-are both increasing in prevalence, and numerous studies have demonstrated that patients with diabetes have an increased risk of developing AD compared with healthy individuals. The underlying biological mechanisms that link the development of diabetes with AD are not fully understood. Abnormal protein processing, abnormalities in insulin signaling, dysregulated glucose metabolism, oxidative stress, the formation of advanced glycation end products, and the activation of inflammatory pathways are features common to both diseases. Hypercholesterolemia is another factor that has received attention, owing to its potential association with diabetes and AD. This Review summarizes the mechanistic pathways that might link diabetes and AD. An understanding of this complex interaction is necessary for the development of novel drug therapies and lifestyle guidelines aimed at the treatment and/or prevention of these diseases.

Literature-based Discovery of Diabetes- and ROS-related Targets

BMC Medical Genomics. 2010  |  Pubmed ID: 20979611

Reactive oxygen species (ROS) are known mediators of cellular damage in multiple diseases including diabetic complications. Despite its importance, no comprehensive database is currently available for the genes associated with ROS.

Cervical Multilevel Intraspinal Stem Cell Therapy: Assessment of Surgical Risks in Gottingen Minipigs

Spine. Feb, 2011  |  Pubmed ID: 21099736

Assessment of long-term surgical risks from multiple intraspinal cell injections.

Computational Methods for Identifying a Layered Allosteric Regulatory Mechanism for ALS-causing Mutations of Cu-Zn Superoxide Dismutase 1

Proteins. Feb, 2011  |  Pubmed ID: 21104697

The most prominent form of familial amyotrophic lateral sclerosis (fALS, Lou Gehrig's Disease) is caused by mutations of Cu-Zn superoxide dismutase 1 (SOD1). SOD1 maintains antioxidant activity under fALS causing mutations, suggesting that the mutations introduce a new, toxic, function. There are 100+ such known mutations that are chemically diverse and spatially distributed across the structure. The common phenotype leads us to propose an allosteric regulatory mechanism hypothesis: SOD1 mutants alter the correlated dynamics of the structure and differentially signal across an inherent allosteric network, thereby driving the disease mechanism at varying rates of efficiency. Two recently developed computational methods for identifying allosteric control sites are applied to the wild type crystal structure, 4 fALS mutant crystal structures, 20 computationally generated fALS mutants and 1 computationally generated non-fALS mutant. The ensemble of mutant structures is used to generate an ensemble of dynamics, from which two allosteric control networks are identified. One network is connected to the catalytic site and thus may be involved in the natural antioxidant function. The second allosteric control network has a locus bordering the dimer interface and thus may serve as a mechanism to modulate dimer stability. Though the toxic function of mutated SOD1 is unknown and likely due to several contributing factors, this study explains how diverse mutations give rise to a common function. This new paradigm for allostery controlled function has broad implications across allosteric systems and may lead to the identification of the key chemical activity of SOD1-linked ALS.

Cortical Neurons Develop Insulin Resistance and Blunted Akt Signaling: a Potential Mechanism Contributing to Enhanced Ischemic Injury in Diabetes

Antioxidants & Redox Signaling. May, 2011  |  Pubmed ID: 21194385

Patients with diabetes are at higher risk of stroke and experience increased morbidity and mortality after stroke. We hypothesized that cortical neurons develop insulin resistance, which decreases neuroprotection via circulating insulin and insulin-like growth factor-I (IGF-I). Acute insulin treatment of primary embryonic cortical neurons activated insulin signaling including phosphorylation of the insulin receptor, extracellular signal-regulated kinase (ERK), Akt, p70S6K, and glycogen synthase kinase-3β (GSK-3β). To mimic insulin resistance, cortical neurons were chronically treated with 25 mM glucose, 0.2 mM palmitic acid (PA), or 20 nM insulin before acute exposure to 20 nM insulin. Cortical neurons pretreated with insulin, but not glucose or PA, exhibited blunted phosphorylation of Akt, p70S6K, and GSK-3β with no change detected in ERK. Inhibition of the phosphatidylinositol 3-kinase (PI3-K) pathway during insulin pretreatment restored acute insulin-mediated Akt phosphorylation. Cortical neurons in adult BKS-db/db mice exhibited higher basal Akt phosphorylation than BKS-db(+) mice and did not respond to insulin. Our results indicate that prolonged hyperinsulinemia leads to insulin resistance in cortical neurons. Decreased sensitivity to neuroprotective ligands may explain the increased neuronal damage reported in both experimental models of diabetes and diabetic patients after ischemia-reperfusion injury.

Stem Cell Technology for the Study and Treatment of Motor Neuron Diseases

Regenerative Medicine. Mar, 2011  |  Pubmed ID: 21391854

Amyotrophic lateral sclerosis and spinal muscular atrophy are devastating neurodegenerative diseases that lead to the specific loss of motor neurons. Recently, stem cell technologies have been developed for the investigation and treatment of both diseases. Here we discuss the different stem cells currently being studied for mechanistic discovery and therapeutic development, including embryonic, adult and induced pluripotent stem cells. We also present supporting evidence for the utilization of stem cell technology in the treatment of amyotrophic lateral sclerosis and spinal muscular atrophy, and describe key issues that must be considered for the transition of stem cell therapies for motor neuron diseases from bench to bedside. Finally, we discuss the first-in-human Phase I trial currently underway examining the safety and feasibility of intraspinal stem cell injections in amyotrophic lateral sclerosis patients as a foundation for translating stem cell therapies for various neurological diseases.

Evidence-based Guideline: Treatment of Painful Diabetic Neuropathy--report of the American Association of Neuromuscular and Electrodiagnostic Medicine, the American Academy of Neurology, and the American Academy of Physical Medicine & Rehabilitation

Muscle & Nerve. Jun, 2011  |  Pubmed ID: 21484835

The objective of this report was to develop a scientifically sound and clinically relevant evidence-based guideline for the treatment of painful diabetic neuropathy (PDN). The basic question that was asked was: "What is the efficacy of a given treatment (pharmacological: anticonvulsants, antidepressants, opioids, others; non-pharmacological: electrical stimulation, magnetic field treatment, low-intensity laser treatment, Reiki massage, others) to reduce pain and improve physical function and quality of life (QOL) in patients with PDN?" A systematic review of literature from 1960 to August 2008 was performed, and studies were classified according to the American Academy of Neurology classification of evidence scheme for a therapeutic article. Recommendations were linked to the strength of the evidence. The results indicate that pregabalin is established as effective and should be offered for relief of PDN (Level A). Venlafaxine, duloxetine, amitriptyline, gabapentin, valproate, opioids (morphine sulfate, tramadol, and oxycodone controlled-release), and capsaicin are probably effective and should be considered for treatment of PDN (Level B). Other treatments have less robust evidence, or the evidence is negative. Effective treatments for PDN are available, but many have side effects that limit their usefulness. Few studies have sufficient information on their effects on function and QOL.

Transcriptional Profiling of Diabetic Neuropathy in the BKS Db/db Mouse: a Model of Type 2 Diabetes

Diabetes. Jul, 2011  |  Pubmed ID: 21617178

A better understanding of the molecular mechanisms underlying the development and progression of diabetic neuropathy (DN) is essential for the design of mechanism-based therapies. We examined changes in global gene expression to define pathways regulated by diabetes in peripheral nerve.

Optimization of Immunosuppressive Therapy for Spinal Grafting of Human Spinal Stem Cells in a Rat Model of ALS

Cell Transplantation. 2011  |  Pubmed ID: 21669047

Previous rodent studies employing monotherapy or combined immunosuppressive regimens have demonstrated a variable degree of spinal xenograft survival in several spinal neurodegenerative models including spinal ischemia, trauma, or amyotrophic lateral sclerosis (ALS). Accordingly, the characterization of optimal immunosuppressive protocols for the specific neurodegenerative model is critical to ensure reliable assessment of potential long-term therapeutic effects associated with cell replacement. In the present study we characterized the survival of human spinal stem cells when grafted into the lumbar spinal cords of a rodent model of ALS, SOD1 (G93A) male and female rats (60-67 days old). Four different immunosuppressive protocols were studied: i) FK506 (q12h); ii) FK506 (qd) + mycophenolate (PO; q12h, up to 7 days postop); iii) FK506 (qd) + mycophenolate (IP; q12h, up to 7 days postop); and iv) FK506 (qd) + mycophenolate (IP; qd, up to 7 days postop). Three weeks after cell grafting the number of surviving human cells was then systematically assessed. The highest density of grafted cells was seen in animals treated with FK506 (qd) and mycophenolate (IP; qd; an average 915 ± 95 grafted cells per spinal cord section). The majority of hNUMA-positive cells colocalized with doublecortin (DCX) immunoreactivity. DCX-positive neurons showed extensive axodendritic sprouting toward surrounding host neurons. In addition, migrating grafted cells were identified up to 500 μm from the graft. In animals treated with FK506 (q12h), FK506 (qd) + mycophenolate (PO; q12h) or FK506 (qd) + mycophenolate (IP; q12h), 11.8 ± 3.4%, 61.2 ± 7.8%, and 99.4 ± 8.9% [expressed as percent of the FK506 (qd) and mycophenolate (IP; qd)] cell survival was seen, respectively. In contrast to animals treated with a combination of FK506 + mycophenolate, robust CD4/8 immunoreactivity was identified in the vicinity of the injection tract in animals treated with FK506 only. These data suggest that a combined, systemically delivered immunosuppression regimen including FK506 and mycophenolate can significantly improve survival of human spinal stem cells after intraspinal transplantation in SOD1 (G93A) rats.

Diabetic Polyneuropathies: Update on Research Definition, Diagnostic Criteria and Estimation of Severity

Diabetes/metabolism Research and Reviews. Jun, 2011  |  Pubmed ID: 21695763

Prior to a joint meeting of the Neurodiab Association and International Symposium on Diabetic Neuropathy held in Toronto, Ontario, Canada, October 13-18, 2009, Solomon Tesfaye, Sheffield, UK convened a panel of neuromuscular experts to provide an update on polyneuropathies associated with diabetes (Toronto Consensus Panels on DPNs, 2009). Herein we provide definitions of typical and atypical DPN, diagnostic criteria, and approaches to diagnose sensorimotor polyneuropathy and to estimate severity. Typical DPN is a sensorimotor polyneuropathy (DSPN) usually developing on long standing hyperglycemia, consequent metabolic derangements and microvessel alterations, and frequently associated with microvessel retinal and kidney disease - but other causes must be excluded. By contrast atypical DPNs are intercurrent painful and autonomic small fiber polyneuropathies. Recognizing that there is a need to detect and estimate severity of DSPN validly and reproducibly, we define subclinical DSPN using nerve conduction criteria and define possible, probable and confirmed clinical levels of DSPN. For conduct of epidemiologic surveys and randomized controlled trials, it is necessary to pre-specify which attributes of nerve conduction are to be used, the criterion for diagnosis, reference values, correction for applicable variables, and the specific criterion for DSPN. Herein we provide the performance characteristics of several criteria for the diagnosis of sensorimotor polyneuropathy in healthy subject and diabetic subject cohorts. Also outlined here are staged and continuous approaches to estimate severity of DSPN. Copyright © 2011 John Wiley & Sons, Ltd.

Comment: Career Guidelines Promote Neurologist Satisfaction

Neurology. Aug, 2011  |  Pubmed ID: 21795654

Hyperinsulinemia Induces Insulin Resistance in Dorsal Root Ganglion Neurons

Endocrinology. Oct, 2011  |  Pubmed ID: 21810948

Insulin resistance (IR) is the major feature of metabolic syndrome, including type 2 diabetes. IR studies are mainly focused on peripheral tissues, such as muscle and liver. There is, however, little knowledge about IR in neurons. In this study, we examined whether neurons develop IR in response to hyperinsulinemia. We first examined insulin signaling using adult dorsal root ganglion neurons as a model system. Acute insulin treatment resulted in time- and concentration-dependent activation of the signaling cascade, including phosphorylation of the insulin receptor, Akt, p70S6K, and glycogen synthase kinase-3β. To mimic hyperinsulinemia, cells were pretreated with 20 nM insulin for 24 h and then stimulated with 20 nM insulin for 15 min. Chronic insulin treatment resulted in increased basal Akt phosphorylation. More importantly, acute insulin stimulation after chronic insulin treatment resulted in blunted phosphorylation of Akt, p70S6K, and glycogen synthase kinase-3β. Interestingly, when the cells were treated with phosphatidylinositol 3-kinase pathway inhibitor, but not MAPK pathway inhibitor, chronic insulin treatment did not block acute insulin treatment-induced Akt phosphorylation. Insulin-induced Akt phosphorylation was lower in dorsal root ganglion neurons from BKS-db/db compared with control BKS-db+ mice. This effect was age dependent. Our results suggest that hyperinsulinemia cause IR by disrupting the Akt-mediated pathway. We also demonstrate that hyperinsulinemia increases the mitochondrial fission protein dynamin-related protein 1. Our results suggest a new theory for the etiology of diabetic neuropathy, i.e. that, similar to insulin dependent tissues, neurons develop IR and, in turn, cannot respond to the neurotrophic properties of insulin, resulting in neuronal injury and the development of neuropathy.

Ontology-based Brucella Vaccine Literature Indexing and Systematic Analysis of Gene-vaccine Association Network

BMC Immunology. 2011  |  Pubmed ID: 21871085

Vaccine literature indexing is poorly performed in PubMed due to limited hierarchy of Medical Subject Headings (MeSH) annotation in the vaccine field. Vaccine Ontology (VO) is a community-based biomedical ontology that represents various vaccines and their relations. SciMiner is an in-house literature mining system that supports literature indexing and gene name tagging. We hypothesize that application of VO in SciMiner will aid vaccine literature indexing and mining of vaccine-gene interaction networks. As a test case, we have examined vaccines for Brucella, the causative agent of brucellosis in humans and animals.

Skeletal Muscle Weakness Due to Deficiency of CuZn-superoxide Dismutase is Associated with Loss of Functional Innervation

American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. Nov, 2011  |  Pubmed ID: 21900648

An association between oxidative stress and muscle atrophy and weakness in vivo is supported by elevated oxidative damage and accelerated loss of muscle mass and force with aging in CuZn-superoxide dismutase-deficient (Sod1(-/-)) mice. The purpose was to determine the basis for low specific force (N/cm(2)) of gastrocnemius muscles in Sod1(-/-) mice and establish the extent to which structural and functional changes in muscles of Sod1(-/-) mice resemble those associated with normal aging. We tested the hypothesis that muscle weakness in Sod1(-/-) mice is due to functionally denervated fibers by comparing forces during nerve and direct muscle stimulation. No differences were observed for wild-type mice at any age in the forces generated in response to nerve and muscle stimulation. Nerve- and muscle-stimulated forces were also not different for 4-wk-old Sod1(-/-) mice, whereas, for 8- and 20-mo-old mice, forces during muscle stimulation were 16 and 30% greater, respectively, than those obtained using nerve stimulation. In addition to functional evidence of denervation with aging, fiber number was not different for Sod1(-/-) and wild-type mice at 4 wk, but 50% lower for Sod1(-/-) mice by 20 mo, and denervated motor end plates were prevalent in Sod1(-/-) mice at both 8 and 20 mo and in WT mice by 28 mo. The data suggest ongoing denervation in muscles of Sod1(-/-) mice that results in fiber loss and muscle atrophy. Moreover, the findings support using Sod1(-/-) mice to explore mechanistic links between oxidative stress and the progression of deficits in muscle structure and function.

Stem Cell Technology for Neurodegenerative Diseases

Annals of Neurology. Sep, 2011  |  Pubmed ID: 21905078

Over the past 20 years, stem cell technologies have become an increasingly attractive option to investigate and treat neurodegenerative diseases. In the current review, we discuss the process of extending basic stem cell research into translational therapies for patients suffering from neurodegenerative diseases. We begin with a discussion of the burden of these diseases on society, emphasizing the need for increased attention toward advancing stem cell therapies. We then explain the various types of stem cells utilized in neurodegenerative disease research, and outline important issues to consider in the transition of stem cell therapy from bench to bedside. Finally, we detail the current progress regarding the applications of stem cell therapies to specific neurodegenerative diseases, focusing on Parkinson disease, Huntington disease, Alzheimer disease, amyotrophic lateral sclerosis, and spinal muscular atrophy. With a greater understanding of the capacity of stem cell technologies, there is growing public hope that stem cell therapies will continue to progress into realistic and efficacious treatments for neurodegenerative diseases.

Diabetic Neuropathy: Cellular Mechanisms As Therapeutic Targets

Nature Reviews. Neurology. Oct, 2011  |  Pubmed ID: 21912405

In patients with diabetes, nerve injury is a common complication that leads to chronic pain, numbness and substantial loss of quality of life. Good glycemic control can decrease the incidence of diabetic neuropathy, but more than half of all patients with diabetes still develop this complication. There is no approved treatment to prevent or halt diabetic neuropathy, and only symptomatic pain therapies, with variable efficacy, are available. New insights into the mechanisms leading to the development of diabetic neuropathy continue to point to systemic and cellular imbalances in metabolites of glucose and lipids. In the PNS, sensory neurons, Schwann cells and the microvascular endothelium are vulnerable to oxidative and inflammatory stress in the presence of these altered metabolic substrates. This Review discusses the emerging cellular mechanisms that are activated in the diabetic milieu of hyperglycemia, dyslipidemia and impaired insulin signaling. We highlight the pathways to cellular injury, thereby identifying promising therapeutic targets, including mitochondrial function and inflammation.

The Identification of Gene Expression Profiles Associated with Progression of Human Diabetic Neuropathy

Brain : a Journal of Neurology. Nov, 2011  |  Pubmed ID: 21926103

Diabetic neuropathy is a common complication of diabetes. While multiple pathways are implicated in the pathophysiology of diabetic neuropathy, there are no specific treatments and no means to predict diabetic neuropathy onset or progression. Here, we identify gene expression signatures related to diabetic neuropathy and develop computational classification models of diabetic neuropathy progression. Microarray experiments were performed on 50 samples of human sural nerves collected during a 52-week clinical trial. A series of bioinformatics analyses identified differentially expressed genes and their networks and biological pathways potentially responsible for the progression of diabetic neuropathy. We identified 532 differentially expressed genes between patient samples with progressing or non-progressing diabetic neuropathy, and found these were functionally enriched in pathways involving inflammatory responses and lipid metabolism. A literature-derived co-citation network of the differentially expressed genes revealed gene subnetworks centred on apolipoprotein E, jun, leptin, serpin peptidase inhibitor E type 1 and peroxisome proliferator-activated receptor gamma. The differentially expressed genes were used to classify a test set of patients with regard to diabetic neuropathy progression. Ridge regression models containing 14 differentially expressed genes correctly classified the progression status of 92% of patients (P < 0.001). To our knowledge, this is the first study to identify transcriptional changes associated with diabetic neuropathy progression in human sural nerve biopsies and describe their potential utility in classifying diabetic neuropathy. Our results identifying the unique gene signature of patients with progressive diabetic neuropathy will facilitate the development of new mechanism-based diagnostics and therapies.

The Evaluation of Distal Symmetric Polyneuropathy: A Physician Survey of Clinical Practice

Archives of Neurology. Nov, 2011  |  Pubmed ID: 22083798

OBJECTIVE: To define current clinical practice for evaluating distal symmetric polyneuropathy. DESIGN: Using a modified Dillman method, we sent surveys to 600 internists, 600 neurologists, and 45 neuromuscular specialists selected from the American Medical Association Physician Masterfile. Survey questions pertained to which tests providers would order in the following 3 scenarios: (1) the initial evaluation of distal symmetric polyneuropathy, (2) the use of additional tests if the initial evaluation was unrevealing, and (3) patients with diabetes. The t test was used to compare the number of tests ordered by physician type, and the χ(2) test was used to compare proportions of tests ordered. SETTING: National survey of physicians. PARTICIPANTS: Internists, neurologists, and neuromuscular specialists. RESULTS: The response rate was 35%. Overall, many tests were ordered for the full evaluation of distal symmetric polyneuropathy (mean [SD], 16.5 [7.2] tests), and there was substantial variation within and between provider types. Internists ordered fewer tests (mean [SD], 14.5 [6.1] tests) than did neurologists (mean [SD], 17.5 [7.9] tests) (P < .001). Regarding the glucose tolerance test, substantial differences were found between physician types, with neurologists and neuromuscular specialists ordering this test more frequently (28.6% and 72.3%, respectively) and internists ordering it less frequently (4.1%). A brain and/or spine magnetic resonance imaging scan was ordered by 19.8% of internists and 12.9% of neurologists. CONCLUSIONS: From the supporting evidence, current practice intent on evaluating distal symmetric polyneuropathy is highly variable and differs widely. For this disorder of the peripheral nerves, a high-yield test such as the glucose tolerance test is rarely used, whereas magnetic resonance imaging is likely overused. Research that defines the optimal evaluation of distal symmetric polyneuropathy has the potential to result in more efficient care.

Translational Stem Cell Therapy for Amyotrophic Lateral Sclerosis

Nature Reviews. Neurology. Dec, 2011  |  Pubmed ID: 22158518

Effective treatments are urgently needed for amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the loss of motor neurons. In 2009, the FDA approved the first phase I safety trial of direct intraspinal transplantation of neural stem cells into patients with ALS, which is currently in progress. Stem cell technologies represent a promising approach for treating ALS, but several issues must be addressed when translating promising experimental ALS therapies to patients. This article highlights the key research that supports the use of stem cells as a therapy for ALS, and discusses the rationale behind and approach to the phase I trial. Completion of the trial could pave the way for continued advances in stem cell therapy for ALS and other neurodegenerative diseases.

Potential for Promoting Recurrent Laryngeal Nerve Regeneration by Remote Delivery of Viral Gene Therapy

The Laryngoscope. Feb, 2012  |  Pubmed ID: 22241608

The aims of this study were to demonstrate the ability to enhance nerve regeneration by remote delivery of a viral vector to the crushed recurrent laryngeal nerve (RLN), to demonstrate the usefulness of a crushed RLN model to test the efficacy of viral gene therapy, and to discuss future potential applications of this approach.

Insulin Resistance in the Nervous System

Trends in Endocrinology and Metabolism: TEM. Jan, 2012  |  Pubmed ID: 22245457

Metabolic syndrome is a cluster of cardiovascular risk factors including obesity, diabetes and dyslipidemia. Insulin resistance (IR) is at the core of metabolic syndrome. In adipose tissue and muscle, IR results in decreased insulin signaling, primarily affecting downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling. It was recently proposed that neurons can develop hyperinsulinemia-induced IR, which in turn results in injury to the peripheral and central nervous systems and is probably pathogenic in common neurological disorders such as diabetic neuropathy and Alzheimer's disease (AD). This review presents evidence indicating that, similarly to insulin-dependent metabolically active tissues such as fat and muscle, neurons also develop IR and thus cannot respond to the neurotrophic properties of insulin, resulting in neuronal injury, subsequent dysfunction and disease states.

Intraspinal Transplantation of Neurogenin-expressing Stem Cells Generates Spinal Cord Neural Progenitors

Neurobiology of Disease. Jan, 2012  |  Pubmed ID: 22245661

Embryonic stem (ES) cells and their derivatives are an important resource for developing novel cellular therapies for disease. Controlling proliferation and lineage selection, however, are essential to circumvent the possibility of tumor formation and facilitate the safe translation of ES-based therapies to man. Expression of appropriate transcription factors is one approach to direct the differentiation of ES cells towards a specific lineage and stop proliferation. Neural differentiation can be initiated in ES cells by expression of Neurogenin1 (Ngn1). In this study we investigate the effects of controlled Ngn1 expression on mouse ES (mES) cell differentiation in vitro and following grafting into the rat spinal cord. In vitro, Ngn1 expression in mES cells leads to rapid and specific neural differentiation, and a concurrent decrease in proliferation. Similarly transplantation of Ngn1-expressing mES cells into the spinal cord lead to in situ differentiation and spinal precursor formation. These data demonstrate that Ngn1 expression in mES cells is sufficient promote neural differentiation and inhibit proliferation, thus establishing an approach to safely graft ES cells into the spinal cord.

A Novel Approach to Study Motor Neurons from Zebrafish Embryos and Larvae in Culture

Journal of Neuroscience Methods. Jan, 2012  |  Pubmed ID: 22285259

Zebrafish are becoming increasingly popular models for examining the mechanisms of and treatments for neurological diseases. The available methods and technology to examine disease processes in vivo are increasing, however, detailed observations of subcellular structures and processes are complex in whole organisms. To address this need, we developed a primary motor neuron (MN) culture technique for utilization with zebrafish neurological disease models. Our protocol enables the culturing of cells from embryos older than 24h post-fertilization, at points after MN axonal development and outgrowth begins, which enables MN axons to develop in vivo in the context of the normal endogenous cues of the model organism, while also providing the accessibility of an in vitro system. When utilized with the increasing number of genetically modified or transgenic models of neurological diseases, this approach provides a novel tool for the examination of cellular and subcellular disease mechanisms, and offers a new platform for therapeutic discoveries in zebrafish.

A New Member of the Multidisciplinary ALS Team: The Otolaryngologist

Amyotrophic Lateral Sclerosis : Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. Feb, 2012  |  Pubmed ID: 22292845

Abstract The multidisciplinary approach to treatment of amyotrophic lateral sclerosis (ALS) has improved the overall care of patients suffering from this disease ( 1 , 2 ). This approach typically has included neurologists, physiatrists, occupational therapists, respiratory therapists and speech therapists. Dysphonia, dysarthria, and dysphagia are three of the most common bulbar manifestations of ALS, and are often the presenting symptoms in bulbar-onset patients. Despite this, otolaryngologists are often not included in ALS management until a tracheostomy is considered. The otolaryngologist can play an important role in early diagnosis and subsequent management of bulbar manifestations of ALS, and would be a valuable member of the multidisciplinary team.

Nerve Growth Factor/p38 Signaling Increases Intraepidermal Nerve Fiber Densities in Painful Neuropathy of Type 2 Diabetes

Neurobiology of Disease. Jan, 2012  |  Pubmed ID: 21872660

Painful diabetic neuropathy (PDN) is a common, yet devastating complication of type 2 diabetes. At this time, there is no objective test for diagnosing PDN. In the current study, we measured the peptidergic intraepidermal nerve fiber densities (IENFD) from hind paws of the db/db mouse, an animal model for type 2 diabetes, during the period of mechanical allodynia from 6 to 12 weeks of age. Intraepidermal nerve fibers (IENF) of the hind footpads were identified by protein gene product (PGP) 9.5 immunohistochemistry. The peptidergic IENF were determined by double immunofluorescence using anti-PGP9.5 and antibodies against tropomyosin-receptor-kinase (Trk) A. We observed a significant increase in PGP9.5-positive IENFD at 8 and 10 weeks of age. Similarly, Trk A-positive peptidergic IENF, which also express substance P and calcitonin gene related peptide in db/db mice, were observed to be elevated from 1.5 to 2 fold over controls. This upregulation ended at 16 weeks of age, in accordance with the reduction of mechanical allodynia. Anti-NGF treatment significantly inhibited the upregulation of peptidergic IENFD during the period of mechanical allodynia, suggesting that increased neurotrophism may mediate this phenomenon. In addition, SB203580, an inhibitor of p38, blocked the increase in peptidergic IENFD in db/db mice. The current results suggest that peptidergic IENFD could be a potential diagnostic indicator for PDN in type 2 diabetes. Furthermore, the inhibition of NGF-p38 signaling could be a potential therapeutic strategy for treating this painful condition.

Effect of Mild Hyperglycemia on Autonomic Function in Obstructive Sleep Apnea

Clinical Autonomic Research : Official Journal of the Clinical Autonomic Research Society. Feb, 2012  |  Pubmed ID: 21796355

Obstructive sleep apnea (OSA) has been hypothesized to cause a hypersympathetic state, which may be the mechanism for the increased incidence of cardiovascular disease in OSA. However, there is a high prevalence of hyperglycemia in OSA patients, which may also contribute to autonomic dysfunction.