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Papers by Bernard Schneider

Stem Cells, Sep 18, 2008

During development of the central nervous system, stem and progenitor cell proliferation and diff... more During development of the central nervous system, stem and progenitor cell proliferation and differentiation are controlled by complex inter-and intracellular interactions that orchestrate the precise spatiotemporal production of particular cell types. Within the embryonic retina, progenitor cells are located adjacent to the retinal pigment epithelium (RPE), which differentiates prior to the neurosensory retina and has the capacity to secrete a multitude of growth factors. We found that secreted proteinaceous factors in human prenatal RPE conditioned medium (RPE CM) prolonged and enhanced the growth of human prenatal retinal neurospheres. The growth-promoting activity of RPE CM was mitogen-dependent and associated with an acute increase in transcription factor phosphorylation. Expanded populations of RPE CM-treated retinal neurospheres expressed numerous neurodevelopmental and eye specification genes and markers characteristic of neural and retinal progenitor cells, but gradually lost the potential to generate neurons upon differentiation. Misexpression of Mash1 restored the neurogenic potential of long-term cultures, yielding neurons with phenotypic characteristics of multiple inner retinal cell types. Thus, a novel combination of extrinsic and intrinsic factors was required to promote both progenitor cell proliferation and neuronal multipotency in human retinal neurosphere cultures. These results support a pro-proliferative and antiapoptotic role for RPE in human retinal development, reveal potential limitations of human retinal progenitor culture systems, and suggest a means for overcoming cell fate restriction in vitro.

Nature, Jun 23, 2013

In the mammalian neocortex, segregated processing streams are thought to be important for forming... more In the mammalian neocortex, segregated processing streams are thought to be important for forming sensory representations of the environment, but how local information in primary sensory cortex is transmitted to other distant cortical areas during behaviour is unclear. Here we show task-dependent activation of distinct, largely non-overlapping long-range projection neurons in the whisker region of primary somatosensory cortex (S1) in awake, behaving mice. Using two-photon calcium imaging, we monitored neuronal activity in anatomically identified S1 neurons projecting to secondary somatosensory (S2) or primary motor (M1) cortex in mice using their whiskers to perform a texture-discrimination task or a task that required them to detect the presence of an object at a certain location. Whisking-related cells were found among S2-projecting (S2P) but not M1-projecting (M1P) neurons. A higher fraction of S2P than M1P neurons showed touch-related responses during texture discrimination, whereas a higher fraction of M1P than S2P neurons showed touch-related responses during the detection task. In both tasks, S2P and M1P neurons could discriminate similarly between trials producing different behavioural decisions. However, in trials producing the same decision, S2P neurons performed better at discriminating texture, whereas M1P neurons were better at discriminating location. Sensory stimulus features alone were not sufficient to elicit these differences, suggesting that selective transmission of S1 information to S2 and M1 is driven by behaviour.

Cellular and Molecular Gastroenterology and Hepatology, 2021

The Smn 2B/mice, a mouse model with reduced level of SMN protein, represent a good model of micro... more The Smn 2B/mice, a mouse model with reduced level of SMN protein, represent a good model of microvesicular steatohepatitis. They offer a reliable, low-cost, early-onset model to identify molecular players in the pathogenesis of NAFLD in both the adult and pediatric populations. BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is considered a health epidemic with potential devastating effects on the patients and the healthcare systems. Current preclinical models of NAFLD are invariably imperfect and generally take a long time to develop. A mouse model of survival motor neuron (SMN) depletion (Smn 2B/mice) was recently shown to develop significant hepatic steatosis in less than 2 weeks from birth. The rapid onset of fatty liver in Smn 2B/mice provides an opportunity to identify molecular markers of NAFLD. Here, we investigated whether Smn 2B/mice display typical features of NAFLD/nonalcoholic steatohepatitis (NASH). METHODS: Biochemical, histologic, electron microscopy, proteomic, and high-resolution respirometry were used. RESULTS: The Smn 2B/mice develop microvesicular steatohepatitis within 2 weeks, a feature prevented by AAV9-SMN gene therapy. Although fibrosis is not overtly apparent in histologic sections of the liver, there is molecular evidence of fibrogenesis and presence of stellate cell activation. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in hepatic dysfunction in protein output, complement, coagulation, iron homeostasis, and insulin-like growth factor-1 metabolism. The NAFLD phenotype is likely due to non-esterified fatty acid overload from peripheral lipolysis subsequent to hyperglucagonemia compounded by reduced muscle use and insulin resistance. Despite the low hepatic mitochondrial content, isolated mitochondria show enhanced b-oxidation, likely as a compensatory response, resulting in the production of reactive oxygen species. In contrast to typical NAFLD/NASH, the Smn 2B/mice lose weight because of their associated neurological condition (spinal muscular atrophy) and develop hypoglycemia. CONCLUSIONS: The Smn 2B/mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early-onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.

Background & aimsSpinal muscular atrophy (SMA) is an inherited neuromuscular disorder leading to ... more Background & aimsSpinal muscular atrophy (SMA) is an inherited neuromuscular disorder leading to paralysis and death in children. SMA patients are more susceptible to dyslipidemia as well as liver steatosis, features reproduced in SMA mouse models. As current pre-clinical models of NAFLD are invariably imperfect and generally take a long time to develop, the rapid development of liver steatosis in SMA mice provides a means to identify molecular markers of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whetherSmn2B/-mice, a model of severe SMA, display typical features of NAFLD/non-alcoholic steatohepatitis (NASH).MethodsBiochemical, histological, electron microscopy, proteomic, and high-resolution respirometry were used.ResultsTheSmn2B/-mice develop steatohepatitis early in life. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in impaired hepatic function including alterations in protein output, complement, coagu...

Astrocytes play an important role in the onset and progression of amyotrophic lateral sclerosis (... more Astrocytes play an important role in the onset and progression of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the relentless degeneration of motor neurons (MNs) in the central nervous system. Despite evidence showing that ALS astrocytes are toxic to MNs, little is understood about the earliest pathological changes that lead to their neurotoxic phenotype. In this study, we generated human astrocytes from induced pluripotent stem cells (iPSCs) harboring the ALS-associated A4V mutation in superoxide dismutase 1 (SOD1), to examine cellular pathways and network changes similar to early stages of the disease. By using proteomics as a molecular indicator, we observed significant alterations in the levels of proteins linked to ALS pathology and the cGAS-STING pathway-induced innate immunity. Interestingly, we found that the protein profile of reactive ALS astrocytes differed from that of wildtype astrocytes treated with the pro-inflammatory cytokine TNFa. Notably,...

Background: The oligomeric state of ␣-syn in vivo remains unknown. Results: ␣-syn in the CNS and ... more Background: The oligomeric state of ␣-syn in vivo remains unknown. Results: ␣-syn in the CNS and produced by erythrocytes, mammalian cells, and Escherichia coli exists predominantly as a disordered monomer. Conclusion: Native ␣-syn from various sources behaves as unstructured and monomeric. Significance: Stabilizing monomeric ␣-syn, lowering its levels, and/or inhibiting its fibrillization remain viable therapeutic strategies for Parkinson disease. Since the discovery and isolation of ␣-synuclein (␣-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that ␣-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in ␣-helical structure (Bartels, T.

Acta neuropathologica communications, Nov 23, 2020

The brain pathology of Alzheimer's disease (AD) is characterized by the misfolding and aggregatio... more The brain pathology of Alzheimer's disease (AD) is characterized by the misfolding and aggregation of both the amyloid beta (Aβ) peptide and hyperphosphorylated forms of the tau protein. Initial Aβ deposition is considered to trigger a sequence of deleterious events contributing to tau pathology, neuroinflammation and ultimately causing the loss of synapses and neurons. To assess the effect of anti-Aβ immunization in this context, we generated a mouse model by overexpressing the human tau protein in the hippocampus of 5xFAD mice. Aβ plaque deposition combined with human tau overexpression leads to an array of pathological manifestations including the formation of tau-positive dystrophic neurites and accumulation of hyperphosphorylated tau at the level of neuritic plaques. Remarkably, the presence of human tau reduces microglial clustering in proximity to the Aβ plaques, which may affect the barrier role of microglia. In this mouse model, continuous administration of anti-Aβ antibodies enhances the clustering of microglial cells even in the presence of tau. Anti-Aβ immunization increases plaque compaction, reduces the spread of tau in the hippocampal formation and prevents the formation of tau-positive dystrophic neurites. However, the treatment does not significantly reduce tau-induced neurodegeneration in the dentate gyrus. These results highlight that anti-Aβ immunization is able to enhance microglial activity around neuritic plaques, mitigating part of the tauinduced pathological manifestations.

Movement Disorders

BackgroundParkinson's disease (PD) is characterized by a severe loss of the dopaminergic (DA)... more BackgroundParkinson's disease (PD) is characterized by a severe loss of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Perturbation of protein thiol redox homeostasis has been shown to play a role in the dysregulation of cell death and cell survival signaling pathways in these neurons. Glutaredoxin 1 (Grx1) is a thiol/disulfide oxidoreductase that catalyzes the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis.ObjectivesWe evaluated if the downregulation of Grx1 could lead to dopaminergic degeneration and PD‐relevant motor deficits in mice.MethodsGrx1 was downregulated unilaterally through viral vector‐mediated transduction of short hairpin RNA against Grx1 into the SNpc. Behavioral assessment was performed through rotarod and elevated body swing test. Stereological analysis of tyrosine hydroxylase–positive and Nissl‐positive neurons was carried out to evaluate neurodegeneration.ResultsDownreg...

Antioxidants & Redox Signaling, 2019

Aims: Reactive oxygen species (ROS) generated during Alzheimer's disease (AD) pathogenesis throug... more Aims: Reactive oxygen species (ROS) generated during Alzheimer's disease (AD) pathogenesis through multiple sources are implicated in synaptic pathology observed in the disease. We have previously shown Factin disassembly in dendritic spines in early AD (34). The actin cytoskeleton can be oxidatively modified resulting in altered F-actin dynamics. Therefore, we investigated whether disruption of redox signaling could contribute to actin network disassembly and downstream effects in the amyloid precursor protein/presenilin-1 double transgenic (APP/PS1) mouse model of AD. Results: Synaptosomal preparations from 1-month-old APP/PS1 mice showed an increase in ROS levels, coupled with a decrease in the reduced form of F-actin and increase in glutathionylated synaptosomal actin. Furthermore, synaptic glutaredoxin 1 (Grx1) and thioredoxin levels were found to be lowered. Overexpressing Grx1 in the brains of these mice not only reversed F-actin loss seen in APP/PS1 mice but also restored memory recall after contextual fear conditioning. F-actin levels and F-actin nanoarchitecture in spines were also stabilized by Grx1 overexpression in APP/PS1 primary cortical neurons, indicating that glutathionylation of Factin is a critical event in early pathogenesis of AD, which leads to spine loss. Innovation: Loss of thiol/disulfide oxidoreductases in the synapse along with increase in ROS can render Factin nanoarchitecture susceptible to oxidative modifications in AD. Conclusions: Our findings provide novel evidence that altered redox signaling in the form of S-glutathionylation and reduced Grx1 levels can lead to synaptic dysfunction during AD pathogenesis by directly disrupting the Factin nanoarchitecture in spines. Increasing Grx1 levels is a potential target for novel disease-modifying therapies for AD.

The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates... more The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrPC (GDL) can also initiate neurotoxicity by inducing an intramolecular R208-H140 hydrogen bond (“H-latch”) between the α2-α3 and β2-α2 loops of PrPC. Importantly, GDL that suppress the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced nineteen individual PrPC variants to PrPC-deficient cerebellar organotypic cultured slices using adenovirus-associated viral vectors (AAV). We report that GDL toxicity requires a single N-proximal cationic residue (K27 or R27) within PrPC. Alanine substitution of K27 also prevented the toxicity of PrPC mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co-expression of wild-t...

International Journal of Molecular Sciences, 2021

Methamphetamine (METH) is a highly abused psychostimulant that is neurotoxic to dopaminergic (DAe... more Methamphetamine (METH) is a highly abused psychostimulant that is neurotoxic to dopaminergic (DAergic) nerve terminals in the striatum and increases the risk of developing Parkinson’s disease (PD). In vivo, METH-mediated DA release, followed by DA-mediated oxidative stress and mitochondrial dysfunction in pre- and postsynaptic neurons, mediates METH neurotoxicity. METH-triggered oxidative stress damages parkin, a neuroprotective protein involved in PD etiology via its involvement in the maintenance of mitochondria. It is not known whether METH itself contributes to mitochondrial dysfunction and whether parkin regulates complex I, an enzymatic complex downregulated in PD. To determine this, we separately assessed the effects of METH or DA alone on electron transport chain (ETC) complexes and the protein parkin in isolated striatal mitochondria. We show that METH decreases the levels of selected complex I, II, and III subunits (NDUFS3, SDHA, and UQCRC2, respectively), whereas DA decre...

Frontiers in Immunology, 2021

Dendritic cells (DCs) are professional antigen-presenting cells involved in the initiation of imm... more Dendritic cells (DCs) are professional antigen-presenting cells involved in the initiation of immune responses. We generated a tolerogenic DC (tolDC) line that constitutively secretes interleukin-10 (IL10-DCs), expressed lower levels of co-stimulatory and MHCII molecules upon stimulation, and induced antigen-specific proliferation of T cells. Vaccination with IL10-DCs combined with another tolDC line that secretes IL-35, reduced antigen-specific local inflammation in a delayed-type hypersensitivity assay independently on regulatory T cell differentiation. In an autoimmune model of rheumatoid arthritis, vaccination with the combined tolDCs after the onset of the disease impaired disease development and promoted recovery of mice. After stable memory was established, the tolDCs promoted CD4 downregulation and induced lymphocyte activation gene 3 (LAG-3) expression in reactivated memory T cells, reducing T cell activation. Taken together, our findings indicate the benefits of combining ...

Nature communications, Nov 1, 2017

Direct collection of extracellular fluid (ECF) plays a central role in the monitoring of neurolog... more Direct collection of extracellular fluid (ECF) plays a central role in the monitoring of neurological disorders. Current approaches using microdialysis catheters are however drastically limited in term of temporal resolution. Here we show a functional in vivo validation of a droplet collection system included at the tip of a neural probe. The system comprises an advanced droplet formation mechanism which enables the collection of neurochemicals present in the brain ECF at high-temporal resolution. The probe was implanted in a rat brain and could successfully collect fluid samples organized in a train of droplets. A microfabricated target plate compatible with most of the surface-based detection methods was specifically developed for sample analysis. The time-resolved brain-fluid samples are analyzed using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The results provide a time evolution picture of the cerebral tissues neurochemical composition for selected...

Cell Death & Disease, 2013

The Journal of Neuroscience, 2014

Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we us... more Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate significantly reduced secondary injury pathology in adult rats following spinal contusion injury and LV-ChABC treatment, with reduced cavitation and enhanced preservation of spinal neurons and axons at 12 weeks postinjury, compared with control (LV-GFP)-treated animals. To understand these neuroprotective effects, we investigated early inflammatory changes following LV-ChABC treatment. Increased expression of the phagocytic macrophage marker CD68 at 3 d postinjury was followed by increased CD206 expression at 2 weeks, indicating that large-scale CSPG digestion can alter macrophage phenotype to favor alternatively activated M2 macrophages. Accordingly, ChABC treatmentin vitroinduced a signif...

NMR in Biomedicine, 1990

We have obtained localized, water‐suppressed proton magnetic resonance spectra from eleven astroc... more We have obtained localized, water‐suppressed proton magnetic resonance spectra from eleven astrocytomas in vivo. Localized phosphorus spectra were also obtained from three of these tumors. All tumors were examined prior to surgery, radiotherapy or chemotherapy. Examinations were performed with a commercially available 1.5 Tesla combined imaging and spectroscopy system using a stimulated echo pulse sequence for protons and an ISIS pulse sequence for phosphorus. A relatively high lactate resonance intensity correlated with a more malignant histological tumor grade and more aggressive behaviour. The resonance intensity of N‐acetylaspartate/creatine was decreased and choline/creatine was increased, but these did not reliably discriminate between tumor grades. Other unidentified resonances not present in spectra of normal brain were sometimes seen. Proton magnetic resonance spectroscopy provides a new method for determining the metabolic behaviour of astrocytomas that may be useful in th...

Journal of Clinical Investigation, 2013

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is character... more Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Na v s) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na v s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Na v s, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na v 1.7-specific inhibitor, ProTxII, allowed the isolation of Na v 1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na v 1.7 and Na v 1.8 currents. The redistribution of Na v 1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L-/-). SNS-Nedd4L-/mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na v 1.7 and Na v 1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na v s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Nature neuroscience, Jan 22, 2015

In the mammalian brain, sensory cortices exhibit plasticity during task learning, but how this al... more In the mammalian brain, sensory cortices exhibit plasticity during task learning, but how this alters information transferred between connected cortical areas remains unknown. We found that divergent subpopulations of cortico-cortical neurons in mouse whisker primary somatosensory cortex (S1) undergo functional changes reflecting learned behavior. We chronically imaged activity of S1 neurons projecting to secondary somatosensory (S2) or primary motor (M1) cortex in mice learning a texture discrimination task. Mice adopted an active whisking strategy that enhanced texture-related whisker kinematics, correlating with task performance. M1-projecting neurons reliably encoded basic kinematics features, and an additional subset of touch-related neurons was recruited that persisted past training. The number of S2-projecting touch neurons remained constant, but improved their discrimination of trial types through reorganization while developing activity patterns capable of discriminating th...

Acta Neuropathologica Communications

In Parkinson’s disease, pathogenic factors such as the intraneuronal accumulation of the protein ... more In Parkinson’s disease, pathogenic factors such as the intraneuronal accumulation of the protein α-synuclein affect key metabolic processes. New approaches are required to understand how metabolic dysregulations cause degeneration of vulnerable subtypes of neurons in the brain. Here, we apply correlative electron microscopy and NanoSIMS isotopic imaging to map and quantify 13C enrichments in dopaminergic neurons at the subcellular level after pulse-chase administration of 13C-labeled glucose. To model a condition leading to neurodegeneration in Parkinson’s disease, human α-synuclein was unilaterally overexpressed in the substantia nigra of one brain hemisphere in rats. When comparing neurons overexpressing α-synuclein to those located in the control hemisphere, the carbon anabolism and turnover rates revealed metabolic anomalies in specific neuronal compartments and organelles. Overexpression of α-synuclein enhanced the overall carbon turnover in nigral neurons, despite a lower rela...

Stem Cells, Sep 18, 2008

During development of the central nervous system, stem and progenitor cell proliferation and diff... more During development of the central nervous system, stem and progenitor cell proliferation and differentiation are controlled by complex inter-and intracellular interactions that orchestrate the precise spatiotemporal production of particular cell types. Within the embryonic retina, progenitor cells are located adjacent to the retinal pigment epithelium (RPE), which differentiates prior to the neurosensory retina and has the capacity to secrete a multitude of growth factors. We found that secreted proteinaceous factors in human prenatal RPE conditioned medium (RPE CM) prolonged and enhanced the growth of human prenatal retinal neurospheres. The growth-promoting activity of RPE CM was mitogen-dependent and associated with an acute increase in transcription factor phosphorylation. Expanded populations of RPE CM-treated retinal neurospheres expressed numerous neurodevelopmental and eye specification genes and markers characteristic of neural and retinal progenitor cells, but gradually lost the potential to generate neurons upon differentiation. Misexpression of Mash1 restored the neurogenic potential of long-term cultures, yielding neurons with phenotypic characteristics of multiple inner retinal cell types. Thus, a novel combination of extrinsic and intrinsic factors was required to promote both progenitor cell proliferation and neuronal multipotency in human retinal neurosphere cultures. These results support a pro-proliferative and antiapoptotic role for RPE in human retinal development, reveal potential limitations of human retinal progenitor culture systems, and suggest a means for overcoming cell fate restriction in vitro.

Nature, Jun 23, 2013

In the mammalian neocortex, segregated processing streams are thought to be important for forming... more In the mammalian neocortex, segregated processing streams are thought to be important for forming sensory representations of the environment, but how local information in primary sensory cortex is transmitted to other distant cortical areas during behaviour is unclear. Here we show task-dependent activation of distinct, largely non-overlapping long-range projection neurons in the whisker region of primary somatosensory cortex (S1) in awake, behaving mice. Using two-photon calcium imaging, we monitored neuronal activity in anatomically identified S1 neurons projecting to secondary somatosensory (S2) or primary motor (M1) cortex in mice using their whiskers to perform a texture-discrimination task or a task that required them to detect the presence of an object at a certain location. Whisking-related cells were found among S2-projecting (S2P) but not M1-projecting (M1P) neurons. A higher fraction of S2P than M1P neurons showed touch-related responses during texture discrimination, whereas a higher fraction of M1P than S2P neurons showed touch-related responses during the detection task. In both tasks, S2P and M1P neurons could discriminate similarly between trials producing different behavioural decisions. However, in trials producing the same decision, S2P neurons performed better at discriminating texture, whereas M1P neurons were better at discriminating location. Sensory stimulus features alone were not sufficient to elicit these differences, suggesting that selective transmission of S1 information to S2 and M1 is driven by behaviour.

Cellular and Molecular Gastroenterology and Hepatology, 2021

The Smn 2B/mice, a mouse model with reduced level of SMN protein, represent a good model of micro... more The Smn 2B/mice, a mouse model with reduced level of SMN protein, represent a good model of microvesicular steatohepatitis. They offer a reliable, low-cost, early-onset model to identify molecular players in the pathogenesis of NAFLD in both the adult and pediatric populations. BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is considered a health epidemic with potential devastating effects on the patients and the healthcare systems. Current preclinical models of NAFLD are invariably imperfect and generally take a long time to develop. A mouse model of survival motor neuron (SMN) depletion (Smn 2B/mice) was recently shown to develop significant hepatic steatosis in less than 2 weeks from birth. The rapid onset of fatty liver in Smn 2B/mice provides an opportunity to identify molecular markers of NAFLD. Here, we investigated whether Smn 2B/mice display typical features of NAFLD/nonalcoholic steatohepatitis (NASH). METHODS: Biochemical, histologic, electron microscopy, proteomic, and high-resolution respirometry were used. RESULTS: The Smn 2B/mice develop microvesicular steatohepatitis within 2 weeks, a feature prevented by AAV9-SMN gene therapy. Although fibrosis is not overtly apparent in histologic sections of the liver, there is molecular evidence of fibrogenesis and presence of stellate cell activation. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in hepatic dysfunction in protein output, complement, coagulation, iron homeostasis, and insulin-like growth factor-1 metabolism. The NAFLD phenotype is likely due to non-esterified fatty acid overload from peripheral lipolysis subsequent to hyperglucagonemia compounded by reduced muscle use and insulin resistance. Despite the low hepatic mitochondrial content, isolated mitochondria show enhanced b-oxidation, likely as a compensatory response, resulting in the production of reactive oxygen species. In contrast to typical NAFLD/NASH, the Smn 2B/mice lose weight because of their associated neurological condition (spinal muscular atrophy) and develop hypoglycemia. CONCLUSIONS: The Smn 2B/mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early-onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.

Background & aimsSpinal muscular atrophy (SMA) is an inherited neuromuscular disorder leading to ... more Background & aimsSpinal muscular atrophy (SMA) is an inherited neuromuscular disorder leading to paralysis and death in children. SMA patients are more susceptible to dyslipidemia as well as liver steatosis, features reproduced in SMA mouse models. As current pre-clinical models of NAFLD are invariably imperfect and generally take a long time to develop, the rapid development of liver steatosis in SMA mice provides a means to identify molecular markers of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whetherSmn2B/-mice, a model of severe SMA, display typical features of NAFLD/non-alcoholic steatohepatitis (NASH).MethodsBiochemical, histological, electron microscopy, proteomic, and high-resolution respirometry were used.ResultsTheSmn2B/-mice develop steatohepatitis early in life. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in impaired hepatic function including alterations in protein output, complement, coagu...

Astrocytes play an important role in the onset and progression of amyotrophic lateral sclerosis (... more Astrocytes play an important role in the onset and progression of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the relentless degeneration of motor neurons (MNs) in the central nervous system. Despite evidence showing that ALS astrocytes are toxic to MNs, little is understood about the earliest pathological changes that lead to their neurotoxic phenotype. In this study, we generated human astrocytes from induced pluripotent stem cells (iPSCs) harboring the ALS-associated A4V mutation in superoxide dismutase 1 (SOD1), to examine cellular pathways and network changes similar to early stages of the disease. By using proteomics as a molecular indicator, we observed significant alterations in the levels of proteins linked to ALS pathology and the cGAS-STING pathway-induced innate immunity. Interestingly, we found that the protein profile of reactive ALS astrocytes differed from that of wildtype astrocytes treated with the pro-inflammatory cytokine TNFa. Notably,...

Background: The oligomeric state of ␣-syn in vivo remains unknown. Results: ␣-syn in the CNS and ... more Background: The oligomeric state of ␣-syn in vivo remains unknown. Results: ␣-syn in the CNS and produced by erythrocytes, mammalian cells, and Escherichia coli exists predominantly as a disordered monomer. Conclusion: Native ␣-syn from various sources behaves as unstructured and monomeric. Significance: Stabilizing monomeric ␣-syn, lowering its levels, and/or inhibiting its fibrillization remain viable therapeutic strategies for Parkinson disease. Since the discovery and isolation of ␣-synuclein (␣-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that ␣-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in ␣-helical structure (Bartels, T.

Acta neuropathologica communications, Nov 23, 2020

The brain pathology of Alzheimer's disease (AD) is characterized by the misfolding and aggregatio... more The brain pathology of Alzheimer's disease (AD) is characterized by the misfolding and aggregation of both the amyloid beta (Aβ) peptide and hyperphosphorylated forms of the tau protein. Initial Aβ deposition is considered to trigger a sequence of deleterious events contributing to tau pathology, neuroinflammation and ultimately causing the loss of synapses and neurons. To assess the effect of anti-Aβ immunization in this context, we generated a mouse model by overexpressing the human tau protein in the hippocampus of 5xFAD mice. Aβ plaque deposition combined with human tau overexpression leads to an array of pathological manifestations including the formation of tau-positive dystrophic neurites and accumulation of hyperphosphorylated tau at the level of neuritic plaques. Remarkably, the presence of human tau reduces microglial clustering in proximity to the Aβ plaques, which may affect the barrier role of microglia. In this mouse model, continuous administration of anti-Aβ antibodies enhances the clustering of microglial cells even in the presence of tau. Anti-Aβ immunization increases plaque compaction, reduces the spread of tau in the hippocampal formation and prevents the formation of tau-positive dystrophic neurites. However, the treatment does not significantly reduce tau-induced neurodegeneration in the dentate gyrus. These results highlight that anti-Aβ immunization is able to enhance microglial activity around neuritic plaques, mitigating part of the tauinduced pathological manifestations.

Movement Disorders

BackgroundParkinson's disease (PD) is characterized by a severe loss of the dopaminergic (DA)... more BackgroundParkinson's disease (PD) is characterized by a severe loss of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Perturbation of protein thiol redox homeostasis has been shown to play a role in the dysregulation of cell death and cell survival signaling pathways in these neurons. Glutaredoxin 1 (Grx1) is a thiol/disulfide oxidoreductase that catalyzes the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis.ObjectivesWe evaluated if the downregulation of Grx1 could lead to dopaminergic degeneration and PD‐relevant motor deficits in mice.MethodsGrx1 was downregulated unilaterally through viral vector‐mediated transduction of short hairpin RNA against Grx1 into the SNpc. Behavioral assessment was performed through rotarod and elevated body swing test. Stereological analysis of tyrosine hydroxylase–positive and Nissl‐positive neurons was carried out to evaluate neurodegeneration.ResultsDownreg...

Antioxidants & Redox Signaling, 2019

Aims: Reactive oxygen species (ROS) generated during Alzheimer's disease (AD) pathogenesis throug... more Aims: Reactive oxygen species (ROS) generated during Alzheimer's disease (AD) pathogenesis through multiple sources are implicated in synaptic pathology observed in the disease. We have previously shown Factin disassembly in dendritic spines in early AD (34). The actin cytoskeleton can be oxidatively modified resulting in altered F-actin dynamics. Therefore, we investigated whether disruption of redox signaling could contribute to actin network disassembly and downstream effects in the amyloid precursor protein/presenilin-1 double transgenic (APP/PS1) mouse model of AD. Results: Synaptosomal preparations from 1-month-old APP/PS1 mice showed an increase in ROS levels, coupled with a decrease in the reduced form of F-actin and increase in glutathionylated synaptosomal actin. Furthermore, synaptic glutaredoxin 1 (Grx1) and thioredoxin levels were found to be lowered. Overexpressing Grx1 in the brains of these mice not only reversed F-actin loss seen in APP/PS1 mice but also restored memory recall after contextual fear conditioning. F-actin levels and F-actin nanoarchitecture in spines were also stabilized by Grx1 overexpression in APP/PS1 primary cortical neurons, indicating that glutathionylation of Factin is a critical event in early pathogenesis of AD, which leads to spine loss. Innovation: Loss of thiol/disulfide oxidoreductases in the synapse along with increase in ROS can render Factin nanoarchitecture susceptible to oxidative modifications in AD. Conclusions: Our findings provide novel evidence that altered redox signaling in the form of S-glutathionylation and reduced Grx1 levels can lead to synaptic dysfunction during AD pathogenesis by directly disrupting the Factin nanoarchitecture in spines. Increasing Grx1 levels is a potential target for novel disease-modifying therapies for AD.

The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates... more The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrPC (GDL) can also initiate neurotoxicity by inducing an intramolecular R208-H140 hydrogen bond (“H-latch”) between the α2-α3 and β2-α2 loops of PrPC. Importantly, GDL that suppress the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced nineteen individual PrPC variants to PrPC-deficient cerebellar organotypic cultured slices using adenovirus-associated viral vectors (AAV). We report that GDL toxicity requires a single N-proximal cationic residue (K27 or R27) within PrPC. Alanine substitution of K27 also prevented the toxicity of PrPC mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co-expression of wild-t...

International Journal of Molecular Sciences, 2021

Methamphetamine (METH) is a highly abused psychostimulant that is neurotoxic to dopaminergic (DAe... more Methamphetamine (METH) is a highly abused psychostimulant that is neurotoxic to dopaminergic (DAergic) nerve terminals in the striatum and increases the risk of developing Parkinson’s disease (PD). In vivo, METH-mediated DA release, followed by DA-mediated oxidative stress and mitochondrial dysfunction in pre- and postsynaptic neurons, mediates METH neurotoxicity. METH-triggered oxidative stress damages parkin, a neuroprotective protein involved in PD etiology via its involvement in the maintenance of mitochondria. It is not known whether METH itself contributes to mitochondrial dysfunction and whether parkin regulates complex I, an enzymatic complex downregulated in PD. To determine this, we separately assessed the effects of METH or DA alone on electron transport chain (ETC) complexes and the protein parkin in isolated striatal mitochondria. We show that METH decreases the levels of selected complex I, II, and III subunits (NDUFS3, SDHA, and UQCRC2, respectively), whereas DA decre...

Frontiers in Immunology, 2021

Dendritic cells (DCs) are professional antigen-presenting cells involved in the initiation of imm... more Dendritic cells (DCs) are professional antigen-presenting cells involved in the initiation of immune responses. We generated a tolerogenic DC (tolDC) line that constitutively secretes interleukin-10 (IL10-DCs), expressed lower levels of co-stimulatory and MHCII molecules upon stimulation, and induced antigen-specific proliferation of T cells. Vaccination with IL10-DCs combined with another tolDC line that secretes IL-35, reduced antigen-specific local inflammation in a delayed-type hypersensitivity assay independently on regulatory T cell differentiation. In an autoimmune model of rheumatoid arthritis, vaccination with the combined tolDCs after the onset of the disease impaired disease development and promoted recovery of mice. After stable memory was established, the tolDCs promoted CD4 downregulation and induced lymphocyte activation gene 3 (LAG-3) expression in reactivated memory T cells, reducing T cell activation. Taken together, our findings indicate the benefits of combining ...

Nature communications, Nov 1, 2017

Direct collection of extracellular fluid (ECF) plays a central role in the monitoring of neurolog... more Direct collection of extracellular fluid (ECF) plays a central role in the monitoring of neurological disorders. Current approaches using microdialysis catheters are however drastically limited in term of temporal resolution. Here we show a functional in vivo validation of a droplet collection system included at the tip of a neural probe. The system comprises an advanced droplet formation mechanism which enables the collection of neurochemicals present in the brain ECF at high-temporal resolution. The probe was implanted in a rat brain and could successfully collect fluid samples organized in a train of droplets. A microfabricated target plate compatible with most of the surface-based detection methods was specifically developed for sample analysis. The time-resolved brain-fluid samples are analyzed using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The results provide a time evolution picture of the cerebral tissues neurochemical composition for selected...

Cell Death & Disease, 2013

The Journal of Neuroscience, 2014

Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we us... more Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate significantly reduced secondary injury pathology in adult rats following spinal contusion injury and LV-ChABC treatment, with reduced cavitation and enhanced preservation of spinal neurons and axons at 12 weeks postinjury, compared with control (LV-GFP)-treated animals. To understand these neuroprotective effects, we investigated early inflammatory changes following LV-ChABC treatment. Increased expression of the phagocytic macrophage marker CD68 at 3 d postinjury was followed by increased CD206 expression at 2 weeks, indicating that large-scale CSPG digestion can alter macrophage phenotype to favor alternatively activated M2 macrophages. Accordingly, ChABC treatmentin vitroinduced a signif...

NMR in Biomedicine, 1990

We have obtained localized, water‐suppressed proton magnetic resonance spectra from eleven astroc... more We have obtained localized, water‐suppressed proton magnetic resonance spectra from eleven astrocytomas in vivo. Localized phosphorus spectra were also obtained from three of these tumors. All tumors were examined prior to surgery, radiotherapy or chemotherapy. Examinations were performed with a commercially available 1.5 Tesla combined imaging and spectroscopy system using a stimulated echo pulse sequence for protons and an ISIS pulse sequence for phosphorus. A relatively high lactate resonance intensity correlated with a more malignant histological tumor grade and more aggressive behaviour. The resonance intensity of N‐acetylaspartate/creatine was decreased and choline/creatine was increased, but these did not reliably discriminate between tumor grades. Other unidentified resonances not present in spectra of normal brain were sometimes seen. Proton magnetic resonance spectroscopy provides a new method for determining the metabolic behaviour of astrocytomas that may be useful in th...

Journal of Clinical Investigation, 2013

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is character... more Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Na v s) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na v s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Na v s, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na v 1.7-specific inhibitor, ProTxII, allowed the isolation of Na v 1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na v 1.7 and Na v 1.8 currents. The redistribution of Na v 1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L-/-). SNS-Nedd4L-/mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na v 1.7 and Na v 1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na v s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Nature neuroscience, Jan 22, 2015

In the mammalian brain, sensory cortices exhibit plasticity during task learning, but how this al... more In the mammalian brain, sensory cortices exhibit plasticity during task learning, but how this alters information transferred between connected cortical areas remains unknown. We found that divergent subpopulations of cortico-cortical neurons in mouse whisker primary somatosensory cortex (S1) undergo functional changes reflecting learned behavior. We chronically imaged activity of S1 neurons projecting to secondary somatosensory (S2) or primary motor (M1) cortex in mice learning a texture discrimination task. Mice adopted an active whisking strategy that enhanced texture-related whisker kinematics, correlating with task performance. M1-projecting neurons reliably encoded basic kinematics features, and an additional subset of touch-related neurons was recruited that persisted past training. The number of S2-projecting touch neurons remained constant, but improved their discrimination of trial types through reorganization while developing activity patterns capable of discriminating th...

Acta Neuropathologica Communications

In Parkinson’s disease, pathogenic factors such as the intraneuronal accumulation of the protein ... more In Parkinson’s disease, pathogenic factors such as the intraneuronal accumulation of the protein α-synuclein affect key metabolic processes. New approaches are required to understand how metabolic dysregulations cause degeneration of vulnerable subtypes of neurons in the brain. Here, we apply correlative electron microscopy and NanoSIMS isotopic imaging to map and quantify 13C enrichments in dopaminergic neurons at the subcellular level after pulse-chase administration of 13C-labeled glucose. To model a condition leading to neurodegeneration in Parkinson’s disease, human α-synuclein was unilaterally overexpressed in the substantia nigra of one brain hemisphere in rats. When comparing neurons overexpressing α-synuclein to those located in the control hemisphere, the carbon anabolism and turnover rates revealed metabolic anomalies in specific neuronal compartments and organelles. Overexpression of α-synuclein enhanced the overall carbon turnover in nigral neurons, despite a lower rela...

Mol. Ther Meth Clin. Dev., 2019

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo ge... more Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.