imaging-metals-brain-tissue-laser-ablation-inductively-coupled-plasma

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

The University of Melbourne

A growing amount of evidence indicates that neuronal trauma can induce a recapitulation of developmental-like mechanisms for neuronal survival and regeneration. Concurrently, ontogenic dependency of central neurons for brain-derived neurotrophic factor (BDNF) is lost during maturation but is re-acquired after injury. Here we show in organotypic hippocampal slices that thyroxin, the thyroid hormone essential for normal CNS development, induces up-regulation of BDNF upon injury. This change in the effect of thyroxin is crucial to promote survival and regeneration of damaged central neurons. In addition, the effect of thyroxin on the expression of the K-Cl cotransporter (KCC2), a marker of neuronal maturation, is changed from down to up-regulation. Notably, previous results in humans have shown that during the first few days after traumatic brain injury or spinal cord injury, thyroid hormone levels are often diminished. Our data suggest that maintaining normal levels of thyroxin during the early post-traumatic phase of CNS injury could have a therapeutically positive effect.

Thyroxin regulates BDNF expression to promote survival of injured neurons.

Abnormal phosphorylation and aggregation of the microtubule-associated protein Tau are hallmarks of various neurodegenerative diseases, such as Alzheimer disease. Molecular mechanisms that regulate Tau phosphorylation are complex and currently incompletely understood. We have developed a novel live cell reporter system based on protein-fragment complementation assay to study dynamic changes in Tau phosphorylation status. In this assay, fusion proteins of Tau and Pin1 (peptidyl-prolyl cis-trans-isomerase 1) carrying complementary fragments of a luciferase protein serve as a sensor of altered protein-protein interaction between Tau and Pin1, a critical regulator of Tau dephosphorylation at several disease-associated proline-directed phosphorylation sites. Using this system, we identified several structurally distinct GABA(A) receptor modulators as novel regulators of Tau phosphorylation in a chemical library screen. GABA(A) receptor activation promoted specific phosphorylation of Tau at the AT8 epitope (Ser-199/Ser-202/Thr-205) in cultures of mature cortical neurons. Increased Tau phosphorylation by GABA(A) receptor activity was associated with reduced Tau binding to protein phosphatase 2A and was dependent on Cdk5 but not GSK3β kinase activity.

γ-Aminobutyric acid type A (GABAA) receptor activation modulates tau phosphorylation.

The secreted protease proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipid (LDL) receptor family members LDLR, very low density lipoprotein receptor (VLDLR) and apolipoprotein receptor 2 (ApoER2), and promotes their degradation in intracellular acidic compartments. In the liver, LDLR is a major controller of blood LDL levels, whereas VLDLR and ApoER2 in the brain mediate Reelin signaling, a critical pathway for proper development of the nervous system. Expression level of PCSK9 in the brain is highest in the cerebellum during perinatal development, but is also increased in the adult brain after ischemia. The mechanism of PCSK9 function and its involvement in neuronal apoptosis is poorly understood. We show here that RNAi-mediated knockdown of PCSK9 significantly reduced the death of potassium-deprived cerebellar granule neurons (CGN), as shown by reduced levels of nuclear phosphorylated c-Jun and activated caspase-3, as well as condensed apoptotic nuclei. ApoER2 protein levels were increased in PCSK9 RNAi cells. Knockdown of ApoER2 but not of VLDLR was sufficient to reverse the protection provided by PCSK9 RNAi, suggesting that proapoptotic signaling of PCSK9 is mediated by altered ApoER2 function. Pharmacological inhibition of signaling pathways associated with lipoprotein receptors suggested that PCSK9 regulates neuronal apoptosis independently of NMDA receptor function but in concert with ERK and JNK signaling pathways. PCSK9 RNAi also reduced staurosporine-induced CGN apoptosis and axonal degeneration in the nerve growth factor-deprived dorsal root ganglion neurons. We conclude that PCSK9 potentiates neuronal apoptosis via modulation of ApoER2 levels and related anti-apoptotic signaling pathways.

PCSK9 regulates neuronal apoptosis by adjusting ApoER2 levels and signaling.

Neuronal LRRTM3 (leucine-rich repeat transmembrane 3) protein has been reported to promote amyloid-β protein precursor (AβPP) processing and LRRTM3 is a candidate gene in late-onset Alzheimer's disease. To address the role of LRRTM3 in AβPP processing and amyloid-β (Aβ) production in vivo, we analyzed amyloidogenic processing of AβPP in the brains of LRRTM3-deficient mice and transgenic AβPP/PS1 mice with or without LRRTM3. We did not find differences between the genotypes in the levels of Aβ or AβPP C-terminal fragments indicating that LRRTM3 is not an essential regulator of Aβ production in adult mice. Moreover, Aβ levels in primary cortical neurons were similar between the genotypes, indicating that LRRTM3 is not required for Aβ generation in developing mice.

LRRTM3 is dispensable for amyloid-β production in mice.

The global incidence of metabolic and age-related diseases, including type 2 diabetes and Alzheimer's disease, is on the rise. In addition to traditional pharmacotherapy, drug candidates from complementary and alternative medicine are actively being pursued for further drug development. Berberine, a nutraceutical traditionally used as an antibiotic, has recently been proposed to act as a multi-target protective agent against type 2 diabetes, dyslipidemias, ischemic brain injury and neurodegenerative diseases, such as Parkinson's and Alzheimer's disease. However, the safety profile of berberine remains controversial, as isolated reports suggest risks with acute toxicity, bradycardia and exacerbation of neurodegeneration. We report that low micromolar berberine causes rapid mitochondria-dependent toxicity in primary neurons characterized by mitochondrial swelling, increased oxidative stress, decreased mitochondrial membrane potential and depletion of ATP content. Berberine does not induce caspase-3 activation and the resulting neurotoxicity remains unaffected by pan-caspase inhibitor treatment. Interestingly, inhibition of NMDA receptors by memantine and MK-801 completely blocked berberine-induced neurotoxicity. Additionally, subtoxic nanomolar concentrations of berberine were sufficient to sensitize neurons to glutamate excitotoxicity and rotenone injury. Our study highlights the need for further safety assessment of berberine, especially due to its tendency to accumulate in the CNS and the risk of potential neurotoxicity as a consequence of increasing bioavailability of berberine.

Mitochondria and NMDA receptor-dependent toxicity of berberine sensitizes neurons to glutamate and rotenone injury.

Lipoprotein receptor family members hold multiple roles in the brain, and alterations in lipoprotein receptor expression and function are implicated in neuronal stress, developmental disorders and neurodegenerative diseases, such as Alzheimer's disease. Berberine (BBR), a nutraceutical shown to have both neuroprotective and neurotoxic properties, is suggested to regulate lipoprotein receptor expression. We show that subtoxic concentration of BBR regulates neuronal lipoprotein receptor expression in a receptor- and time-dependent fashion in cerebellar granule neurons (CGN). Similarly to BBR, subtoxic concentrations of neuronal stressors cobalt chloride, thapsigargin and rotenone increased very-low-density lipoprotein receptor (VLDLR) mRNA and protein expression in CGN suggesting a conserved pathway for stress-induced upregulation of VLDLR in neurons. We also show that VLDLR upregulation is accompanied by transiently increased stabilization of hypoxia-induced factor 1 alpha (HIF-1α) and decreased β-catenin levels affecting the Wnt pathway through GSK3β phosphorylation, a crucial player in neurodegenerative processes. Our results indicate that neuronal stress differentially regulates lipoprotein receptor expression in neurons, with VLDLR upregulation as a common element as a modulator of neuronal Wnt signaling.

Stress-induced upregulation of VLDL receptor alters Wnt-signaling in neurons.

Synaptic adhesion molecules are key components in development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of chemical synapses. Several families of neuronal adhesion molecules have been identified such as the neuronal cell adhesion molecules, neurexins and neuroligins, and in particular recently several leucine-rich repeat proteins, e.g., Netrin G-ligands, SLITRKs, and LRRTMs. The LRRTMs form a family of four proteins. They have been implicated in excitatory glutamatergic synapse function and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. In addition, LRRTM3 and LRRTM4 have been found to be ligands for heparan sulfate proteoglycans, including glypican. We report here the crystal structure of a thermostabilized mouse LRRTM2, with a Tm 30 °C higher than that of the wild-type protein. We localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation, and prior information about the interaction of the ligand with neurexins, which allowed us to propose a tentative model for the LRRTM-neurexin interaction complex. We also determined affinities of the thermostabilized LRRTM2 and wild-type LRRTM1 and LRRTM2 for neurexin-β1 with and without Ca(2+). Cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapselike contacts. The structural and functional data presented here provide the first structure of an LRRTM protein and allow us to propose a model for the molecular mechanism of LRRTM function in the synaptic adhesion.

Article	Year
Thyroxin regulates BDNF expression to promote survival of injured neurons. Molecular and cellular neurosciences\| PubMed ID: 19765661	2009
γ-Aminobutyric acid type A (GABAA) receptor activation modulates tau phosphorylation. The Journal of biological chemistry\| PubMed ID: 22235112	2012
PCSK9 regulates neuronal apoptosis by adjusting ApoER2 levels and signaling. Cellular and molecular life sciences : CMLS\| PubMed ID: 22481440	2012
LRRTM3 is dispensable for amyloid-β production in mice. Journal of Alzheimer's disease : JAD\| PubMed ID: 22710909	2012
Mitochondria and NMDA receptor-dependent toxicity of berberine sensitizes neurons to glutamate and rotenone injury. PloS one\| PubMed ID: 25192195	2014
Stress-induced upregulation of VLDL receptor alters Wnt-signaling in neurons. Experimental cell research\| PubMed ID: 26751967	2016
Crystal Structure of an Engineered LRRTM2 Synaptic Adhesion Molecule and a Model for Neurexin Binding. Biochemistry\| PubMed ID: 26785044	2016

Kai Kysenius

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