-
BioRxiv : the Preprint Server For... Apr 2024The heterotrimeric G-protein α subunit, Gα , acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase...
The heterotrimeric G-protein α subunit, Gα , acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase mediated production of the second messenger cyclic adenosine monophosphate. Numerous mutations in the gene, which encodes Gα , have been identified as causative for an adult-onset dystonia. These mutations disrupt GPCR signaling cascades in assays through several mechanisms, and this disrupted signaling is hypothesized to lead to dystonic motor symptoms in patients. However, the cells and circuits that mutations in corrupt are not well understood. Published patterns of Gα expression outside the context of the striatum are sparse, conflicting, often lack cell type specificity, and may be confounded by expression of the close homolog of . Here, we use RNAScope in-situ hybridization to quantitatively characterize mRNA expression in brain tissue from wildtype C57BL/6J adult mice. We observed widespread expression of puncta throughout the brain, suggesting Gα is expressed in more brain structures and neuron types than previously accounted for. We quantify transcripts at a single cell level, and use neuron type specific markers to further classify and understand patterns of expression. Our data suggests that brain regions classically associated with motor control, initiation, and regulation show the highest expression of , with Purkinje Cells of the cerebellum showing the highest expression of any neuron type examined. Subsequent conditional knockout in Purkinje cells led to markedly decreased intracellular cAMP levels and downstream cAMP-dependent enzyme activation. Our work provides a detailed characterization of expression throughout the brain and the biochemical consequences of loss of Gα signaling in neurons that highly express .
PubMed: 38617339
DOI: 10.1101/2024.04.03.587766 -
Molecular Therapy. Methods & Clinical... Jun 2024Viral vector gene therapy has immense promise for treating central nervous system (CNS) disorders. Although adeno-associated virus vectors (AAVs) have had success, their...
Viral vector gene therapy has immense promise for treating central nervous system (CNS) disorders. Although adeno-associated virus vectors (AAVs) have had success, their small packaging capacity limits their utility to treat the root cause of many CNS disorders. Adenoviral vectors (Ad) have tremendous potential for CNS gene therapy approaches. Currently, the most common vectors utilize the Group C Ad5 serotype capsid proteins, which rely on the Coxsackievirus-Adenovirus receptor (CAR) to infect cells. However, these Ad5 vectors are unable to transduce many neuronal cell types that are dysfunctional in many CNS disorders. The human CD46 (hCD46) receptor is widely expressed throughout the human CNS and is the primary attachment receptor for many Ad serotypes. Therefore, to overcome the current limitations of Ad vectors to treat CNS disorders, we created chimeric first generation Ad vectors that utilize the hCD46 receptor. Using a "humanized" hCD46 mouse model, we demonstrate these Ad vectors transduce cerebellar cell types, including Purkinje cells, that are refractory to Ad5 transduction. Since Ad vector transduction properties are dependent on their capsid proteins, these chimeric first generation Ad vectors open new avenues for high-capacity helper-dependent adenovirus (HdAd) gene therapy approaches for cerebellar disorders and multiple neurological disorders.
PubMed: 38605812
DOI: 10.1016/j.omtm.2024.101243 -
Redox Biology Jun 2024Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction...
Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1 mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1 mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1 mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1 mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.
Topics: Animals; Mice; Membrane Fluidity; S-Adenosylmethionine; Mitochondrial Membranes; Niemann-Pick Disease, Type C; Glutathione; Brain; Mitochondria; Niemann-Pick C1 Protein; Disease Models, Animal; Mice, Knockout; Phosphatidylcholines
PubMed: 38599016
DOI: 10.1016/j.redox.2024.103150 -
Communications Biology Apr 2024Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest...
Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1), a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However, the regulatory network of SCA1 pathology, especially central regulators of the earliest developmental stages and inflammatory events, remains incompletely understood. Here, we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development, and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients.
Topics: Animals; Humans; Mice; Cytokines; Induced Pluripotent Stem Cells; Mice, Transgenic; Purkinje Cells; Spinocerebellar Ataxias; Ubiquitins
PubMed: 38594382
DOI: 10.1038/s42003-024-06066-z -
Acta Neurobiologiae Experimentalis Mar 2024Neuroinflammation is a process associated with degeneration and loss of neurons in different parts of the brain. The most important damage mechanisms in its formation...
Neuroinflammation is a process associated with degeneration and loss of neurons in different parts of the brain. The most important damage mechanisms in its formation are oxidative stress and inflammation. This study aimed to investigate the protective effects of cannabidiol (CBD) against neuroinflammation through various mechanisms. Thirty‑two female rats were randomly divided into 4 groups as control, lipopolysaccharide (LPS), LPS + CBD and CBD groups. After six hours following LPS administration, rats were sacrificed, brain and cerebellum tissues were obtained. Tissues were stained with hematoxylin‑eosin for histopathological analysis. Apelin and tyrosine hydroxylase synthesis were determined immunohistochemically. Total oxidant status and total antioxidant status levels were measured, and an oxidative stress index was calculated. Protein kinase B (AKT), brain-derived neurotrophic factor (BDNF), cyclic‑AMP response element‑binding protein (CREB) and nuclear factor erythroid 2‑related factor 2 (NRF2) mRNA expression levels were also determined. In the LPS group, hyperemia, degeneration, loss of neurons and gliosis were seen in all three tissues. Additionally, Purkinje cell loss in the cerebellum, as well as neuronal loss in the cerebral cortex and hippocampus, were found throughout the LPS group. The expressions of AKT, BDNF, CREB and NRF2, apelin and tyrosine hydroxylase synthesis all decreased significantly. CBD treatment reversed these changes and ameliorated oxidative stress parameters. CBD showed protective effects against neuroinflammation via regulating AKT, CREB, BDNF expressions, NRF2 signaling, apelin and tyrosine hydroxylase synthesis.
Topics: Female; Rats; Animals; Proto-Oncogene Proteins c-akt; Cannabidiol; Neuroprotective Agents; NF-E2-Related Factor 2; Dopamine; Apelin; Cyclic AMP Response Element-Binding Protein; Brain-Derived Neurotrophic Factor; Neuroinflammatory Diseases; Lipopolysaccharides; Tyrosine 3-Monooxygenase; Hippocampus; Gene Expression
PubMed: 38587319
DOI: 10.55782/ane-2024-2546 -
BioRxiv : the Preprint Server For... Mar 2024The Cre-lox system is an indispensable tool in neuroscience research for targeting gene deletions to specific cellular populations. Here we assess the utility of several...
The Cre-lox system is an indispensable tool in neuroscience research for targeting gene deletions to specific cellular populations. Here we assess the utility of several transgenic lines, along with a viral approach, for targeting cerebellar Purkinje cells. Using a combination of a fluorescent reporter line () to indicate -mediated recombination and a floxed line () we show that reporter expression does not always align precisely with loss of protein. The commonly used line exhibits a gradual mosaic pattern of recombination in Purkinje cells from P7-P14, while loss of Dag1 protein is not complete until P30. drives recombination in precursor cells that give rise to GABAergic neurons in the embryonic cerebellum, including Purkinje cells and molecular layer interneurons. However, due to its transient expression in precursors, results in stochastic loss of Dag1 protein in these neurons. , which is often described as a "pan-neuronal" line for the central nervous system, does not drive -mediated recombination in Purkinje cells. We identify a line that drives efficient and complete recombination in embryonic Purkinje cells, resulting in loss of Dag1 protein before the period of synaptogenesis. -mediated delivery of at P0 results in gradual transduction of Purkinje cells during the second postnatal week, with loss of Dag1 protein not reaching appreciable levels until P35. These results characterize several tools for targeting conditional deletions in cerebellar Purkinje cells at different developmental stages and illustrate the importance of validating the loss of protein following recombination.
PubMed: 38585758
DOI: 10.1101/2024.03.28.587263 -
Neurobiology of Disease Jun 2024We performed a comprehensive study of the morphological, functional, and genetic features of moonwalker (MWK) mice, a mouse model of spinocerebellar ataxia caused by a...
We performed a comprehensive study of the morphological, functional, and genetic features of moonwalker (MWK) mice, a mouse model of spinocerebellar ataxia caused by a gain of function of the TRPC3 channel. These mice show numerous behavioral symptoms including tremor, altered gait, circling behavior, impaired motor coordination, impaired motor learning and decreased limb strength. Cerebellar pathology is characterized by early and almost complete loss of unipolar brush cells as well as slowly progressive, moderate loss of Purkinje cell (PCs). Structural damage also includes loss of synaptic contacts from parallel fibers, swollen ER structures, and degenerating axons. Interestingly, no obvious correlation was observed between PC loss and severity of the symptoms, as the phenotype stabilizes around 2 months of age, while the cerebellar pathology is progressive. This is probably due to the fact that PC function is severely impaired much earlier than the appearance of PC loss. Indeed, PC firing is already impaired in 3 weeks old mice. An interesting feature of the MWK pathology that still remains to be explained consists in a strong lobule selectivity of the PC loss, which is puzzling considering that TRPC is expressed in every PC. Intriguingly, genetic analysis of MWK cerebella shows, among other alterations, changes in the expression of both apoptosis inducing and resistance factors possibly suggesting that damaged PCs initiate specific cellular pathways that protect them from overt cell loss.
Topics: Animals; Mice; Disease Models, Animal; Phenotype; Cerebellum; Purkinje Cells; TRPC Cation Channels; Genotype; Spinocerebellar Ataxias; Mice, Neurologic Mutants; Mice, Inbred C57BL; Mice, Transgenic
PubMed: 38575093
DOI: 10.1016/j.nbd.2024.106492 -
Memantine suppresses the excitotoxicity but fails to rescue the ataxic phenotype in SCA1 model mice.Biomedicine & Pharmacotherapy =... May 2024Spinocerebellar ataxia type 1 (SCA1) is a debilitating neurodegenerative disorder of the cerebellum and brainstem. Memantine has been proposed as a potential treatment...
Spinocerebellar ataxia type 1 (SCA1) is a debilitating neurodegenerative disorder of the cerebellum and brainstem. Memantine has been proposed as a potential treatment for SCA1. It blocks N-methyl-D-aspartate (NMDA) receptors on neurons, reduces excitotoxicity and decreases neurodegeneration in Alzheimer models. However, in cerebellar neurodegenerative diseases, the potential value of memantine is still unclear. We investigated the effects of memantine on motor performance and synaptic transmission in the cerebellum in a mouse model where mutant ataxin 1 is specifically targeted to glia. Lentiviral vectors (LVV) were used to express mutant ataxin 1 selectively in Bergmann glia (BG). In mice transduced with the mutant ataxin 1, chronic treatment with memantine improved motor activity during initial tests, presumably due to preserved BG and Purkinje cell (PC) morphology and numbers. However, mice were unable to improve their rota rod scores during next days of training. Memantine also compromised improvement in the rota rod scores in control mice upon repetitive training. These effects may be due to the effects of memantine on plasticity (LTD suppression) and NMDA receptor modulation. Some effects of chronically administered memantine persisted even after its wash-out from brain slices. Chronic memantine reduced morphological signs of neurodegeneration in the cerebellum of SCA1 model mice. This resulted in an apparent initial reduction of ataxic phenotype, but memantine also affected cerebellar plasticity and ultimately compromised motor learning. We speculate that that clinical application of memantine in SCA1 might be hampered by its ability to suppress NMDA-dependent plasticity in cerebellar cortex.
Topics: Animals; Memantine; Disease Models, Animal; Spinocerebellar Ataxias; Phenotype; Mice; Ataxin-1; Motor Activity; Cerebellum; Purkinje Cells; Receptors, N-Methyl-D-Aspartate; Mice, Transgenic; Mice, Inbred C57BL; Neuroglia; Male; Neuronal Plasticity
PubMed: 38574621
DOI: 10.1016/j.biopha.2024.116526 -
PLoS Computational Biology Apr 2024According to the motor learning theory by Albus and Ito, synaptic depression at the parallel fibre to Purkinje cells synapse (pf-PC) is the main substrate responsible...
According to the motor learning theory by Albus and Ito, synaptic depression at the parallel fibre to Purkinje cells synapse (pf-PC) is the main substrate responsible for learning sensorimotor contingencies under climbing fibre control. However, recent experimental evidence challenges this relatively monopolistic view of cerebellar learning. Bidirectional plasticity appears crucial for learning, in which different microzones can undergo opposite changes of synaptic strength (e.g. downbound microzones-more likely depression, upbound microzones-more likely potentiation), and multiple forms of plasticity have been identified, distributed over different cerebellar circuit synapses. Here, we have simulated classical eyeblink conditioning (CEBC) using an advanced spiking cerebellar model embedding downbound and upbound modules that are subject to multiple plasticity rules. Simulations indicate that synaptic plasticity regulates the cascade of precise spiking patterns spreading throughout the cerebellar cortex and cerebellar nuclei. CEBC was supported by plasticity at the pf-PC synapses as well as at the synapses of the molecular layer interneurons (MLIs), but only the combined switch-off of both sites of plasticity compromised learning significantly. By differentially engaging climbing fibre information and related forms of synaptic plasticity, both microzones contributed to generate a well-timed conditioned response, but it was the downbound module that played the major role in this process. The outcomes of our simulations closely align with the behavioural and electrophysiological phenotypes of mutant mice suffering from cell-specific mutations that affect processing of their PC and/or MLI synapses. Our data highlight that a synergy of bidirectional plasticity rules distributed across the cerebellum can facilitate finetuning of adaptive associative behaviours at a high spatiotemporal resolution.
Topics: Neuronal Plasticity; Animals; Models, Neurological; Cerebellum; Computer Simulation; Conditioning, Eyelid; Purkinje Cells; Blinking; Conditioning, Classical; Synapses; Computational Biology; Mice; Cerebellar Cortex
PubMed: 38574161
DOI: 10.1371/journal.pcbi.1011277 -
Nature Neuroscience May 2024Supervised learning depends on instructive signals that shape the output of neural circuits to support learned changes in behavior. Climbing fiber (CF) inputs to the...
Supervised learning depends on instructive signals that shape the output of neural circuits to support learned changes in behavior. Climbing fiber (CF) inputs to the cerebellar cortex represent one of the strongest candidates in the vertebrate brain for conveying neural instructive signals. However, recent studies have shown that Purkinje cell stimulation can also drive cerebellar learning and the relative importance of these two neuron types in providing instructive signals for cerebellum-dependent behaviors remains unresolved. In the present study we used cell-type-specific perturbations of various cerebellar circuit elements to systematically evaluate their contributions to delay eyeblink conditioning in mice. Our findings reveal that, although optogenetic stimulation of either CFs or Purkinje cells can drive learning under some conditions, even subtle reductions in CF signaling completely block learning to natural stimuli. We conclude that CFs and corresponding Purkinje cell complex spike events provide essential instructive signals for associative cerebellar learning.
Topics: Animals; Purkinje Cells; Mice; Optogenetics; Association Learning; Conditioning, Eyelid; Male; Mice, Inbred C57BL; Cerebellum; Nerve Fibers; Mice, Transgenic; Cerebellar Cortex; Female
PubMed: 38565684
DOI: 10.1038/s41593-024-01594-7