The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A novel split hand index (SI) was developed to quantify the split hand phenomenon, and its diagnostic utility was assessed in ALS patients. The split hand index was derived by dividing the product of the compound muscle action potential (CMAP) amplitude recorded over the abductor pollicis brevis and first dorsal interosseous muscles by the CMAP amplitude recorded over the abductor digiti minimi muscle. In order to assess the diagnostic utility of the split hand index, ALS patients were prospectively assessed and their results were compared to neuromuscular disorder patients. The split hand index was significantly reduced in ALS when compared to neuromuscular disorder patients (P<0.0001). Limb-onset ALS patients exhibited the greatest reduction in the split hand index, and a value of 5.2 or less reliably differentiated ALS from other neuromuscular disorders. Consequently, the split hand index appears to be a novel diagnostic biomarker for ALS, perhaps facilitating an earlier diagnosis.
25 Related JoVE Articles!
Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain
Institutions: University of Texas Medical Branch , University of Texas Medical Branch .
Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed α, β, and γ-secretases1
. Sequential proteolytic cleavage of APP with β and γ-secretases leads to the production of a small proteolytic peptide, termed Aβ, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3
. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro
, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14
. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6
, however it obtains stable secondary structure when bound to Fe657
. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro
and in intact cells4
. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8
, to modulate signaling pathways9
, and to regulate calcium signaling10
. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11
, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12
. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro
thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.
Medicine, Issue 66, Neuroscience, Cellular Biology, Molecular Biology, Amyloid precursor protein, APP, AICD, Alzheimer's Disease, Atomic Force Microscopy, Aggregation, Ubiquilin-1, Molecular Chaperone
Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures
Institutions: Max Plank Institute of Molecular Plant Physiology, University of Hohenheim.
Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1
. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2
. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana
We use full metabolic labeling of Arabidopsis thaliana
suspension cell cultures with K15
as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3
. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4
Empty Value, Issue 79, Cellular Structures, Plants, Genetically Modified, Arabidopsis, Membrane Lipids, Intracellular Signaling Peptides and Proteins, Membrane Proteins, Isotope Labeling, Proteomics, plants, Arabidopsis thaliana, metabolic labeling, stable isotope labeling, suspension cell cultures, plasma membrane fractionation, two phase system, detergent resistant membranes (DRM), mass spectrometry, membrane microdomains, quantitative proteomics
4D Imaging of Protein Aggregation in Live Cells
Institutions: Hebrew University of Jerusalem .
One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1
. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2
, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1
. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4
. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1
, target them for degradation by the ubiquitin-proteasome system5
, or direct them to protective aggregation inclusions6-9
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10
: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1
- model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12
) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts
is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 °C, or when the cell faces misfolding stress, Ubc9ts
misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.
Cellular Biology, Issue 74, Molecular Biology, Genetics, Proteins, Aggregation quality control, protein folding quality control, GFP, JUNQ (juxtanuclear quality control compartment), IPOD (insoluble protein deposit), proteostasis sensor, 4D live cell imaging, live cells, laser, cell biology, protein folding, Ubc9ts, yeast, assay, cell, imaging
Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli
Institutions: Duke University, Duke University.
Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli
. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.
Molecular Biology, Issue 88, elastin-like polypeptides, lower critical solution temperature, phase separation, inverse transition cycling, protein purification, batch purification
Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Institutions: University of Texas Southwestern Medical Center at Dallas.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
Molecular Biology, Issue 77, Biochemistry, Genetics, Cellular Biology, Structural Biology, Biophysics, Membrane Lipids, Phospholipids, Carrier Proteins, Membrane Proteins, Micelles, Molecular Motor Proteins, life sciences, biochemistry, Amino Acids, Peptides, and Proteins, lipid-protein interaction, channel reconstitution, lipid-dependent gating, voltage-gated ion channel, conformation-specific ligands, lipids
Designing Silk-silk Protein Alloy Materials for Biomedical Applications
Institutions: Rowan University, Rowan University, Cooper Medical School of Rowan University, Rowan University.
Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi
) and domestic mulberry silk (Bombyx mori
) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.
Bioengineering, Issue 90, protein alloys, biomaterials, biomedical, silk blends, computational simulation, implantable electronic devices
Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
Institutions: University of Akron.
Recombinant protein engineering has utilized Escherichia coli (E. coli)
expression systems for nearly 4 decades, and today E. coli
is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing a T7 lac
inducible vector and E. coli
expression hosts, starting from transformation to scale-up and purification.
Bioengineering, Issue 83, protein engineering, recombinant protein production, AviTag, BirA, biotinylation, pET vector system, E. coli, inclusion bodies, Ni-NTA, size exclusion chromatography
Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments
Institutions: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU).
The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis
oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (ΔIami
) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of γENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a γENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in γENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.
Biochemistry, Issue 89, two-electrode voltage-clamp, electrophysiology, biotinylation, Xenopus laevis oocytes, epithelial sodium channel, ENaC, proteases, proteolytic channel activation, ion channel, cleavage sites, cleavage fragments
Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae
Institutions: University of Manchester.
Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to <40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae
. Like prokaryotic systems, S. cerevisiae
can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-β-D-maltoside (DDM) and 1-tetradecanoyl-sn
-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.
Biochemistry, Issue 87, Membrane protein, cystic fibrosis, CFTR, ABCC7, protein purification, Cystic Fibrosis Foundation, green fluorescent protein
Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
Institutions: Ben-Gurion University of the Negev.
The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro
, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans
. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo
, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.
Biochemistry, Issue 82, aging, Caenorhabditis elegans, heat shock response, neurodegenerative diseases, protein folding homeostasis, proteostasis, stress, temperature-sensitive
A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells
Institutions: University of Basel.
Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1
. These limitations hamper the in vitro
study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2
. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo
neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3
, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.
Neuroscience, Issue 62, Human primary muscle cells, embryonic spinal cord explants, neurites, innervation, contraction, cell culture
Rapid Generation of Amyloid from Native Proteins In vitro
Institutions: The University of Texas MD Anderson Cancer Center.
Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous
components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro
. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.
Biochemistry, Issue 82, amyloid, soluble protein oligomer, amyloid precursor, protein misfolding, amyloid fibril, protein aggregate
Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)
Institutions: University Medicine Göttingen, Göttingen, Germany.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.
Medicine, Issue 61, neuroscience, amyotrophic lateral sclerosis, ALS, spinal cord, mouse, rotarod, hanging wire
Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models
Institutions: Perelman School of Medicine at the University of Pennsylvania.
Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae
. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and α-synuclein, implicated in Parkinson’s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and α-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.
Microbiology, Issue 93, Protein-misfolding disorders, yeast proteinopathy models, Hsp104, proteotoxicity, amyloid, disaggregation
Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules
Institutions: Ohio State University.
Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.
Bioengineering, Issue 92, adhesion, aggregation, Fc-fusion, cadherin, protocadherin
Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases
Institutions: University of Ulm.
Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo
. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls.
DTI data analysis is performed in a variate fashion, i.e.
voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e.
differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels.
In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.
Medicine, Issue 77, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Anatomy, Physiology, Neurodegenerative Diseases, nuclear magnetic resonance, NMR, MR, MRI, diffusion tensor imaging, fiber tracking, group level comparison, neurodegenerative diseases, brain, imaging, clinical techniques
Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury
Institutions: Thomas Jefferson University Medical College.
Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1
and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3
. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4
. Transgenic mice 4,5
and rats 6
carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R)
have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7
. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1
. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11
. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A
mice and rats, as well as spinal cord injured rats and mice 11
Medicine, Issue 55, cell transplantation, engraftment, graft, spinal cord, stem cells, precursors, ALS, amyotrophic lateral sclerosis, motor neuron, SCI, spinal cord injury
Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System
Institutions: Washington University in St. Louis School of Medicine.
Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1
as well as shift isoform ratios of specific genes 2
. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.
Neurobiology, Issue 75, Neuroscience, Medicine, Biomedical Engineering, Genetics, Anatomy, Physiology, Surgery, Pharmacology, Cerebrospinal Fluid, Rodentia, Oligonucleotides, Antisense, Drug Administration Routes, Injections, Intraventricular, Drug Delivery Systems, mouse, rat, brain, antisense oligonucleotide, osmotic pump, Bolus, Ventricle, Neurosciences, Translational, Cerebrospinal fluid, CNS, cannula, catheter, animal model, surgical techniques
Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection
Institutions: Yale School of Medicine, Howard Hughes Medical Institute.
Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments. In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent <10% of the total cell population. Laser capture microdissection (LMD) has been developed to address this problem. Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited. LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity. Standard techniques are used to recover the total RNA and measure its integrity. This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.
Neuroscience, Issue 83, Laser capture microdissection, Motor neuron, Spinal cord, Azure B, RNA, RNA-seq, qRT-PCR
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
Institutions: Ohio University.
Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1
, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2
(Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3
. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5
. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8
. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9
, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8
. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.
Medicine, Issue 59, neuroscience, muscle, electromyography, physiology, TMS, strength, motor control. sarcopenia, dynapenia, lumbar
High-throughput Yeast Plasmid Overexpression Screen
Institutions: University of Pennsylvania School of Medicine , University of Pennsylvania School of Medicine .
The budding yeast, Saccharomyces cerevisiae
, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5
. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10
. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16
. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.
Cell Biology, Issue 53, Yeast, plasmid, transformation, PEG/LioAc, high-throughput screen
ALS - Motor Neuron Disease: Mechanism and Development of New Therapies
Institutions: Johns Hopkins University.
Medicine, Issue 6, Translational Research, Neuroscience, ALS, stem cells, brain, neuron, upper motor neuron, transplantation
Interview: Protein Folding and Studies of Neurodegenerative Diseases
Institutions: MIT - Massachusetts Institute of Technology.
In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.
Neuroscience, issue 17, protein folding, brain, neuron, prion, neurodegenerative disease, yeast, screen, Translational Research
Ole Isacson: Development of New Therapies for Parkinson's Disease
Institutions: Harvard Medical School.
Medicine, Issue 3, Parkinson' disease, Neuroscience, dopamine, neuron, L-DOPA, stem cell, transplantation