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- Pluripotent stem cell-based models of spinal muscular atrophy. [REVIEW]
- Mol Cell Neurosci 2014 Dec 12.
Motor neuron diseases, as the vast majority of neurodegenerative disorders in humans, are incurable conditions that are challenging to study in vitro, owing to the obstacles in obtaining the cell types majorly involved in the pathogenesis. Recent advances in stem cell research, especially in the development of induced pluripotent stem cell (iPSC) technology, have opened up the possibility of generating a substantial amount of disease-specific neuronal cells, including motor neurons and glial cells. The present review analyzes the practical implications of iPSCs, generated from fibroblasts of patients affected by spinal muscular atrophy (SMA), and discusses the challenges in the development and optimization of in vitro disease models. Research on patient-derived disease-specific cells may shed light on the pathological processes behind neuronal dysfunction and death in SMA, thus providing new insights for the development of novel effective therapies.
- The L-type voltage-gated calcium channel modulates microglial pro-inflammatory activity. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Dec 12.
Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-d-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca(2+) entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.
- Cathepsin D deficiency induces oxidative damage in brain pericytes and impairs the blood-brain barrier. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Dec 10.
Recent evidence suggests that peripheral blood mononuclear cells (PBMCs) contribute to the pathogenesis of neuropathological changes in patients with neuronal ceroid lipofuscinosis (NCL) and lysosomal storage diseases. In order to examine the possible increase in the permeability of the blood-brain-barrier (BBB) and resultant infiltration of PBMCs due to cathepsin D (CatD) deficiency, a process underlying the onset of congenital NCL, we examined structural changes in brain vessels in CatD(-/-) mice. Consequently, the mean diameter of the brain vessels in the cerebral cortex on postnatal day 24 (P24) was significantly larger in CatD(-/-) mice than in wild-type mice. Furthermore, the mean number of brain pericytes in CatD(-/-) mice began to decline significantly on P16 and almost disappeared on P24, and oxidative DNA damage was first detected in brain pericytes on P12. Examinations with electron microscopy revealed that brain pericytes were laden with dense granular bodies, cytoplasmic vacuoles and lipid droplets. The infiltration of PBMCs characterized by segmented nucleus laden with dense granular bodies was also noted in the cerebral cortex of CatD(-/-) mice. When primary cultured microglia prepared from enhanced green fluorescent protein (GFP)-expressing transgenic rats were injected into the common carotid artery, GFP-positive microglia were detected in the brain parenchyma of CatD(-/-), but not wild-type, mice. Moreover, pepstatin A, a specific aspartic protease inhibitor, induced mitochondria-derived reactive oxygen species (ROS) production in the isolated brain pericytes, which decreased the cell viability. These observations suggest that increased lysosomal storage due to CatD deficiency causes oxidative damage in brain pericytes, subsequently resulting in an increased vessel diameter, enhanced permeability of the BBB and the infiltration of PBMCs. Therefore, protecting brain pericytes against lysosomal storage-induced oxidative stress may represent an alternative treatment strategy for congenital NCL.
- Memory and PTPIP51 - A new protein in hippocampus and cerebellum. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Dec 10.
Previously the expression of Protein Tyrosine Phosphatase Interacting Protein 51 (PTPIP51) in mouse brain was reported. Here, we investigated PTPIP51 mRNA and protein in two of the brain regions namely the hippocampus and the cerebellum of mouse brains. On a cellular level both the protein and the mRNA were related to the pyramidal cells of the hippocampal formation, the granular cells of the dentate gyrus and the cells of the adjacent strata. In the cerebellum PTPIP51 was traced in Purkinje cells, the cells of the molecular layer and the granular layer. On a subcellular level only partial co-localization was seen for the endoplasmic reticulum, but not with mitochondria. In addition the interactome of PTPIP51 was analysed. In hippocampal cells a strong interaction with PTP1B and vesicle-associated membrane protein-associated protein B (VAPB) was detected. A somewhat differing interaction profile was found in the cerebellum, where high interaction levels were found for 14-3-3, diacylglycerol kinase α (DGKα), NFκB and PTP1B. These interaction partners represent specific signalling pathways linked to building memory. PTPIP51 can be associated with nerve growth factor signalling, dendritic and axonal growth, synaptogenesis, and all processes needed for memory formation. Moreover, in HT-22 mouse hippocampal cells PTPIP51 expression was induced by administrating the fibroblast growth factor 1 (FGF-1), which is known to take part in learning/memory processes. Knocking down p38-MAPK also led to an up-regulation of PTPIP51 probably resembling a compensative mechanism. Thus, a possible connection to the processing of memories can be anticipated. Differences in the interaction profile in both regions may be attributed to the actual/local differences in memory formation.
- Regional effects of endocannabinoid, BDNF and FGF receptor signalling on neuroblast motility and guidance along the rostral migratory stream. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Dec 3.:32-43.
During development and after birth neural stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to populate the olfactory bulb (OB) with neurons. Multiple factors promote neuroblast migration, but the contribution that many of these make to guidance within the intact RMS is not known. In the present study we have characterised in detail how endocannabinoid (eCB), BDNF and FGF receptor (FGFR) signalling regulates motility and guidance, and also determined whether any of these receptors operate in a regionally restricted manner. We used in vivo electroporation in postnatal mice to fluorescently label neuroblasts, and live cell imaging to detail their migratory properties. Cannabinoid receptor antagonists rendered neuroblasts less mobile, and when they did move guidance was lost. Similar results were obtained when eCB synthesis was blocked with diacylglycerol lipase (DAGL) inhibitors, and importantly eCB function is required for directed migration at both ends of the RMS. Likewise, inhibition of BDNF signalling disrupted motility and guidance in a similar manner along the entire RMS. In contrast, altering FGFR signalling inhibits motility and perturbs guidance, but only at the beginning of the stream. Inhibition of FGFR signalling in vivo also reduces the length of the leading process on migratory neuroblasts in a graded manner along the RMS. These results provide evidence for a guidance function for all three of the above receptor systems in the intact RMS, but show that FGFR signalling is unique as it is required in a regionally specific manner.
- Benzodiazepine-dependent stabilization of GABAA receptors at synapses. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Nov 4.:101-113.
GABAA receptors constitutively enter and exit synapses by lateral diffusion in the plane of the neuronal membrane. They are trapped at synapses through their interactions with gephyrin, the main scaffolding protein at inhibitory post-synaptic densities. Previous work has shown that the synaptic accumulation and diffusion dynamics of GABAARs are controlled via excitatory synaptic activity. However, it remains unknown whether GABAAR activity can itself impact the surface trafficking of the receptors. Here we report the effects of GABAAR agonists, antagonists and allosteric modulators on the receptor's surface dynamics. Using immunocytochemistry and single particle tracking experiments on mouse hippocampal neurons, we show that the agonist muscimol decreases GABAAR and gephyrin levels at synapses and accelerates the receptor's lateral diffusion within 30-120min of treatment. In contrast, the GABAAR antagonist gabazine increased GABAAR amounts and slowed down GABAAR diffusion at synapses. The response to GABAAR activation or inhibition appears to be an adaptative regulation of GABAergic synapses. Surprisingly, the positive allosteric modulator diazepam abolished the regulation induced by muscimol, and this effect was observed on α1, α2, α5 and γ2 GABAAR subunits. Altogether these results indicate that diazepam stabilizes synaptic GABAARs and thus prevents the agonist-induced regulation of GABAAR levels at synapses. This occurred independently of neuronal activity and intracellular calcium and involved GABAAR-gephyrin interactions, suggesting that the changes in GABAAR diffusion depend on conformational changes of the receptor. Our study provides a new molecular mechanism involved in the adaptative response to changes in GABAAR activity and benzodiazepine treatments.
- Distinct patterns of compartmentalization and proteolytic stability of PDE6C mutants linked to achromatopsia. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Nov 11.:1-8.
Phosphodiesterase-6 (PDE6) is an essential effector enzyme in vertebrate photoreceptor cells. Mutations in rod and cone PDE6 cause recessive retinitis pigmentosa and achromatopsia, respectively. The mechanisms of missense PDE6 mutations underlying severe visual disorders are poorly understood. To probe these mechanisms, we expressed seven known missense mutants of cone PDE6C in rods of transgenic Xenopus laevis and examined their stability and compartmentalization. PDE6C proteins with mutations in the catalytic domain, H602L and E790K, displayed modestly reduced proteolytic stability, but they were properly targeted to the outer segment of photoreceptor cells. Mutations in the regulatory GAF domains, R104W, Y323N, and P391L led to a proteolytic degradation of the proteins involving a cleavage in the GAFb domain. Lastly, the R29W and M455V mutations residing outside the conserved PDE6 domains produced a pattern of subcellular compartmentalization different from that of PDE6C. Thus, our results suggest a spectrum of mechanisms of missense PDE6C mutations in achromatopsia including catalytic defects, protein mislocalization, or a specific sequence of proteolytic degradation.
- TDP6, a brain-derived neurotrophic factor-based trkB peptide mimetic, promotes oligodendrocyte myelination. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Oct 16.:132-140.
Brain-derived neurotrophic factor (BDNF) plays critical roles in the development and maintenance of the central (CNS) and peripheral nervous systems (PNS). BDNF exerts its biological effects via tropomyosin-related kinase B (TrkB) and the p75 neurotrophin receptor (p75(NTR)). We have recently identified that BDNF promotes CNS myelination via oligodendroglial TrkB receptors. In order to selectively target TrkB to promote CNS myelination, we have used a putative TrkB agonist, a small multicyclic peptide (tricyclic dimeric peptide 6, TDP6) previously described by us that structurally mimics a region of BDNF that binds TrkB. We confirmed that TDP6 acts as a TrkB agonist as it provoked autophosphorylation of TrkB and its downstream signalling effector extracellular related-kinase 1 and 2 (Erk1/2) in primary oligodendrocytes. Using an in vitro myelination assay, we show that TDP6 significantly promotes myelination by oligodendrocytes in vitro, as evidenced by enhanced myelin protein expression and an increased number of myelinated axonal segments. In contrast, a second, structurally distinct BDNF mimetic (cyclo-dPAKKR) that targets p75(NTR) had no effect upon oligodendrocyte myelination in vitro, despite the fact that cyclo-dPAKKR is a very effective promoter of peripheral (Schwann cell) myelination. The selectivity of TDP6 was further verified by using TrkB-deficient oligodendrocytes, in which TDP6 failed to promote myelination, indicating that the pro-myelinating effect of TDP6 is oligodendroglial TrkB-dependent. Together, our results demonstrate that TDP6 is a novel BDNF mimetic that promotes oligodendrocyte myelination in vitro via targeting TrkB.
- Huntingtin is associated with cytomatrix proteins at the presynaptic terminal. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Nov 4.:96-100.
Huntington's disease (HD) is a single gene disorder produced by expansion of the gene encoding huntingtin (htt), a large protein with features of a multi-functional scaffold. Expansion of htt's polyglutamine domain induces novel, toxic interactions and likely also disrupts normal htt function. Because of its predicted role as a scaffold, pursuit of huntingtin function and HD pathogenesis has focused on identifying htt-interacting proteins. Here we present a focused screen designed to identify htt-interacting proteins in the presynaptic terminal. To identify interactions that occur in situ, synaptosomes (isolated nerve terminals) from cerebral cortices, striata and hippocampi were subjected to chemical crosslinking followed by denaturation, immunoprecipitation using an anti-htt antibody, and nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) analyses. The presynaptic cytomatrix proteins Bassoon, Piccolo/Aczonin and Ahnak were among the most consistently identified binding partners. Co-immunoprecipitation and co-fractionation studies support the conclusion that huntingtin is a component of the presynaptic cytomatrix, a complicated network of proteins that regulates the positioning and priming of synaptic vesicles. These findings implicate htt in presynaptic functioning, and suggest that aberrant organization of presynaptic components may contribute to the neurological pathology associated with HD.
- Modulation of CaV2.1 channels by neuronal calcium sensor-1 induces short-term synaptic facilitation. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Nov 5.:124-131.
Facilitation and inactivation of P/Q-type Ca(2+) currents mediated by Ca(2+)/calmodulin binding to CaV2.1 channels contribute to facilitation and rapid depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin from its binding site and differentially modulate P/Q-type Ca(2+) currents, resulting in diverse patterns of short-term synaptic plasticity. Neuronal calcium sensor-1 (NCS-1, frequenin) has been shown to enhance synaptic facilitation, but the underlying mechanism is unclear. We report here that NCS-1 directly interacts with IQ-like motif and calmodulin-binding domain in the C-terminal domain of CaV2.1 channel. NCS-1 reduces Ca(2+)-dependent inactivation of P/Q-type Ca(2+) current through interaction with the IQ-like motif and calmodulin-binding domain without affecting peak current or activation kinetics. Expression of NCS-1 in presynaptic superior cervical ganglion neurons has no effect on synaptic transmission, eliminating effects of this calcium sensor protein on endogenous N-type Ca(2+) currents and the endogenous neurotransmitter release machinery. However, in superior cervical ganglion neurons expressing wild-type CaV2.1 channels, co-expression of NCS-1 induces facilitation of synaptic transmission in response to paired pulses and trains of depolarizing stimuli, and this effect is lost in CaV2.1 channels with mutations in the IQ-like motif and calmodulin-binding domain. These results reveal that NCS-1 directly modulates CaV2.1 channels to induce short-term synaptic facilitation and further demonstrate that CaS proteins are crucial in fine-tuning short-term synaptic plasticity.