Cerebellar Neurons Research Articles

Temporal Silencing of Purkinje Cell Neurotransmission Reveals a Dynamic Role for Cerebellar Function in Neurodevelopment (P10-9.006)

<h3>Objective:</h3> To determine whether differential timing of disrupted cerebellar function influences the nature and severity of associated neurodevelopmental outcomes. <h3>Background:</h3> Disruptions of cerebellar function have been increasingly implicated in the pathogenesis of a wide array of neurodevelopmental disorders. While there has been increasing focus on genetically mediated etiologies of disrupted cerebellar function, less attention has been given to acquired etiologies although they underlie diseases with high incidence and prevalence, such as the neurodevelopmental disability associated with prematurity and autism spectrum disorders. The present study uses mouse transgenics to develop a synthetic model of cerebellar developmental dysfunction and interrogates the role of cerebellar neurotransmission in early brain development. <h3>Design/Methods:</h3> Using inducible tissue specific Cre-mediated recombination the present model allows for spatial and temporal induction of cerebellar dysfunction through timed excision of the vesicular GABA transporter (VGAT) specifically in Purkinje cells. Using this line, Purkinje cell neurotransmission is silenced <i>in utero</i> (EarlyOff) or at time of weaning (LateOff), corresponding developmentally to the immature and mature cerebellum. Mouse behavior and the electrophysiologic profile of cerebellar nuclear neurons was then performed comparing EarlyOff, LateOff, and control animals at 8–10 weeks of life. <h3>Results:</h3> Compared to controls EarlyOFF showed significantly altered gross motor behavior (rotarod assay, footprinting), social behavior (3-chamber assay), and hyperactivity (open field assay). Compared to controls, LateOff had equivalent performance on gross motor assays but showed significant hyperactivity. Social tests are ongoing. Cerebellar nuclear electrophysiology from awake behaving animals revealed significantly different and divergent spike profiles between EarlyOff, LateOff, and controls. <h3>Conclusions:</h3> The developmental timing of cerebellar dysfunction leads to discrepant behavioral and electrophysiologic profiles. These findings underline that the disparate phenotypes of neurodevelopmental disability associated with prematurity and autism may nonetheless have shared cerebellar etiology, emphasize the necessity of understanding how cerebellar nuclear signaling influences broad cortical function, and reveal a potential target for neuromodulatory intervention. <b>Disclosure:</b> Dr. Gill has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for Elite Medical Experts. The institution of Dr. Gill has received research support from NIH-NINDS. Miss Nguyen has nothing to disclose. Roy Sillitoe has nothing to disclose.

Dexmedetomidine Protects Cerebellar Neurons against Hyperoxia-Induced Oxidative Stress and Apoptosis in the Juvenile Rat.

The risk of oxidative stress is unavoidable in preterm infants and increases the risk of neonatal morbidities. Premature infants often require sedation and analgesia, and the commonly used opioids and benzodiazepines are associated with adverse effects. Impairment of cerebellar functions during cognitive development could be a crucial factor in neurodevelopmental disorders of prematurity. Recent studies have focused on dexmedetomidine (DEX), which has been associated with potential neuroprotective properties and is used as an off-label application in neonatal units. Wistar rats (P6) were exposed to 80% hyperoxia for 24 h and received as pretreatment a single dose of DEX (5µg/kg, i.p.). Analyses in the immature rat cerebellum immediately after hyperoxia (P7) and after recovery to room air (P9, P11, and P14) included examinations for cell death and inflammatory and oxidative responses. Acute exposure to high oxygen concentrations caused a significant oxidative stress response, with a return to normal levels by P14. A marked reduction of hyperoxia-mediated damage was demonstrated after DEX pretreatment. DEX produced a much earlier recovery than in controls, confirming a neuroprotective effect of DEX on alterations elicited by oxygen stress on the developing cerebellum.

Extracellular vesicle-based delivery of silencing sequences for the treatment of Machado-Joseph disease/spinocerebellar ataxia type 3

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominantly inherited ataxia worldwide. It is caused by an over-repetition of the trinucleotide CAG within the ATXN3 gene, which confers toxic properties to ataxin-3 (ATXN3) species. RNA interference technology has shown promising therapeutic outcomes but still lacks a non-invasive delivery method to the brain. Extracellular vesicles (EVs) emerged as promising delivery vehicles due to their capacity to deliver small nucleic acids, such as microRNAs (miRNAs). miRNAs were found to be enriched into EVs due to specific signal motifs designated as ExoMotifs. In this study, we aimed at investigating whether ExoMotifs would promote the packaging of artificial miRNAs into EVs to be used as non-invasive therapeutic delivery vehicles to treat MJD/SCA3. We found that miRNA-based silencing sequences, associated with ExoMotif GGAG and ribonucleoprotein A2B1 (hnRNPA2B1), retained the capacity to silence mutant ATXN3 (mutATXN3) and were 3-fold enriched into EVs. Bioengineered EVs containing the neuronal targeting peptide RVG on the surface significantly decreased mutATXN3 mRNA in primary cerebellar neurons from MJD YAC 84.2 and in a novel dual-luciferase MJD mouse model upon daily intranasal administration. Altogether, these findings indicate that bioengineered EVs carrying miRNA-based silencing sequences are a promising delivery vehicle for brain therapy.

Generation of three induced Pluripotent Stem Cell lines from individuals with Hypomyelination with Atrophy of Basal Ganglia and Cerebellum caused by a c.745G>A (p.D249N) autosomal dominant mutation in TUBB4A

Mutations in tubulin alpha 4a (TUBB4A) result in a spectrum of leukodystrophies, including Hypomyelination with atrophy of basal ganglia and cerebellum (H-ABC), resulting from a recurring mutation p.Asp249Asn (TUBB4AD249N). H-ABC presents with dystonia, motor and cognitive impairment and pathological features of hypomyelination and loss of cerebellar and striatal neurons. We have generated three induced pluripotent stem cell (iPSC) lines from fibroblast and peripheral blood mononuclear cells (PBMCs) of individuals with TUBB4AD249N mutation. The iPSCs were assessed to confirm a normal karyotype, pluripotency, and trilineage differentiation potential. The iPSCs will allow for disease modeling, understanding mechanisms and testing of therapeutic targets.

Patients' IgLON5 autoantibodies interfere with IgLON5-protein interactions.

Anti-IgLON5 disease is a rare neurological disorder characterized by autoantibodies against IgLON5, and pathological evidence of neurodegeneration. IgLON5 is a cell adhesion molecule but its physiological function is unknown. Our aim was to investigate the IgLON5 interactome and to determine if IgLON5 antibodies (IgLON5-abs) affect these protein interactions. IgLON5 interactome was investigated by mass spectrometry sequencing of proteins immunoprecipitated by IgLON5 autoantibodies using cultures of rat cerebellar granular neurons (CGNs). Shedding of IgLON5 was explored using HEK cells transfected with human IgLON5 plasmid and in CGNs. Interactions of IgLON5 with identified binding partners and IgLON5-abs effects were confirmed by immunofluorescence in transfected HEK cells and rat hippocampal neurons. Patients' IgLON5 antibodies co-precipitated all members of the IgLON family and three 3 additional surface proteins. IgLON5 predominantly establishes homomeric and heteromeric cis (within the cell) and trans (between cells)-interactions with other IgLON family members and undergoes spontaneous ectodomain shedding. Antibodies from patients with anti-IgLON5 disease prevent trans-interactions in hippocampal neurons independently of the IgLON5 IgG subclass distribution. We show a potentially novel pathogenic mechanism of IgLON5-abs that consists in blocking IgLON5 interactions with its binding partners. These findings extend our knowledge about the physiological role of IgLON5 and pave the way to future understanding of the pathological mechanisms of anti-IgLON5 disease.

Chronic suppression of STIM1-mediated calcium signaling in Purkinje cells rescues the cerebellar pathology in spinocerebellar ataxia type 2

Distorted neuronal calcium signaling has been reported in many neurodegenerative disorders, including different types of spinocerebellar ataxias (SCAs). Cerebellar Purkinje cells (PCs) are primarily affected in SCAs and the disturbances in the calcium homeostasis were observed in SCA PCs. Our previous results have revealed that 3,5-dihydroxyphenylglycine (DHPG) induced greater calcium responses in SCA2-58Q PC cultures than in wild type (WT) PC cultures. Here we observed that glutamate-induced calcium release in PCs cells bodies is significantly higher in SCA2-58Q PCs from acute cerebellar slices compared to WT PCs of the same age. Recent studies have demonstrated that the stromal interaction molecule 1 (STIM1) plays an important role in the regulation of the neuronal calcium signaling in cerebellar PCs in mice. The main function of STIM1 is to regulate store-operated calcium entry through the TRPC/Orai channels formation to refill the calcium stores in the ER when it is empty. Here we demonstrated that the chronic viral-mediated expression of the small interfering RNA (siRNA) targeting STIM1 specifically in cerebellar PCs alleviates the deranged calcium signaling in SCA2-58Q PCs, rescues the spine loss in these cerebellar neurons, and also improves the motor decline in SCA2-58Q mice. Thus, our preliminary results support the important role of the altered neuronal calcium signaling in SCA2 pathology and also suggest the STIM1-mediated signaling pathway as a potential therapeutic target for treatment of SCA2 patients.

Neuroprotective effects of Apis dorsata forest honey on the neurons count in cerebrum and cerebellum of mice (Mus musculus) exposed to monosodium glutamate

Introduction: Monosodium glutamate or MSG can have an effect on increasing the production of reactive oxygen species (ROS) in the brain. Apis dorsata forest honey is known to have higher antioxidant activity than Apis mellifera and Apis cerana honey. This study aims to determine the preventive effect of Apis dorsata honey on the number of pyramidal cerebrum neurons and purkinje cerebellum neurons of mice (Mus musculus) exposed to monosodium glutamate (MSG). Material and Methods: This study used 25 mice which were divided into 5 groups. In the K- group only aquadest was given. The K+ group was given 4mg/gBB of MSG. Groups P1, P2, and P3 given Apis dorsata honey at a dose of 53.82mg/20gBW, 107.64mg/20gBW, and 161.46mg/20gBW. All treatments were carried out orally for 52 days. Results: The results showed that the average number of cerebral pyramidal neurons in the K-, K+, P1, P2, and P3 groups was 12.24±0.607; 7.24±2.875; 12.48±1.513; 15.72±0.944; and 19.28±2.827. The mean number of purkinje cerebellar neurons was 4.8±1.456; 2.08±0.807; 3.08±1.035; 3.56±0.434; and 4.68±1.390. Discussion: The average number of pyramidal neurons and purkinje neurons in the group given preventive dose of Apis dorsata honey and exposed to MSG was higher and there is a significant difference compared to the group that was only exposed to MSG. It can be concluded that the administration of Apis dorsata honey can maintain the number of pyramidal cerebrum neurons and purkinje cerebellar neurons in mice exposed to MSG.

Raw Data of Immunofluorescence experiments related to the paper entitled "Potential Neurotoxic Effects of Glioblastoma-Derived Exosomes in Primary Cultures of Cerebellar Neurons via Oxidant Stress and Glutathione Depletion" (Data not published).

Raw Data of Immunofluorescence experiments related to the paper entitled "Potential Neurotoxic Effects of Glioblastoma-Derived Exosomes in Primary Cultures of Cerebellar Neurons via Oxidant Stress and Glutathione Depletion" (Data not published).

Meconic acid is a possible neuroprotector: justification on &lt;i&gt;in vitro &lt;/i&gt;experiments and its physico-chemical properties

Main representatives of gamma pyronic acid are meconic, comenic, chelidonic and kojic acid. It was found that comenic acid exerts a neuroprotective effect, and chelidonic acid has a pronounced anti-inflammatory effect. It was not studied whether meconic acid exhibits neuroprotective effects. The aim of this work was to assess the neuroprotective potential of meconic acid, taking into accout its physicochemical properties, using an in vitro model of ischemic stroke. Primary neuroglial culture was obtained from the cerebellum of 7-8-day-old Wistar rat pups by mechanical tissue dissociation. The protective effect of meconic acid on the culture of cerebellar neurons was studied using the model of glutamate toxicity and oxygen-glucose deprivation. Quantum mechanical calculations were used and experiments in the model system citrate-phosphate-luminol were conducted by the method of chemiluminescent analysis to investigate the antioxidant activity of meconic acid. The chelating properties of meconic acid with respect to Fe3+ in solutions were studied using Job's method. Meconic acid has been found to have a protective effect in in vitro models of ischemia. Its action leads to a decrease in the level of intracellular calcium and the restoration of the membrane potential of mitochondria in a culture of cerebellar neurons under glutamate exposure, resulting in an increase in the percentage of living cells under oxygen-glucose deprivation. Meconic acid has a high calculated antioxidant potential, confirmed experimentally. With an increase in the pH of the medium, stepwise binding of meconic acid with Fe3+ occurs with the formation of complexes with different ligand/metal ratios. At physiological pH, the composition of the resulting complex is 1:3. The obtained antioxidant, chelating, and cytoprotective action of meconic acid provides a basis for further study of the possible neuroprotective properties of this compound in in vivo experiments, and the data obtained in the work on its physicochemical properties can be useful for the synthesis and study of new coordination compounds based on meconic acid.

Histological and Molecular Characterization of the Inferior Olivary Nucleus and Climbing Fibers in the Goldfish, Carassius auratus.

The cerebellum receives inputs via the climbing fibers originating from the inferior olivary nucleus in the ventral medulla. In mammals, the climbing fibers entwine and terminate onto both major and peripheral branches of dendrites of the Purkinje cells. In this study, the inferior olivary nucleus and climbing fiber in the goldfish were investigated with several histological techniques. By neural tracer application to the hemisphere of the cerebellum, labeled inferior olivary neurons were found in the ventral edge of the contralateral medulla. Kainate stimulated Co + + uptake and gephyrin immunoreactivities were found in inferior olivary neurons, indicating, respectively, that they receive both excitatory (glutamatergic) and inhibitory (GABAergic or glycinergic) inputs. Inferior olivary neurons express vglut2.1 transcripts, suggesting they are glutamatergic. Around 85% of inferior olivary neurons were labeled with anti-calretinin antiserum. Calretinin immunoreactive (ir) climbing fiber terminal-like structures were distributed near the Purkinje cells and in the molecular layer. Double labeling immunofluorescence with anti-calretinin and zebrin II antisera revealed that the calretinin-ir climbing fibers run along and made synaptic-like contacts on the major dendrites of the zebrin II-ir Purkinje cells. In teleost fish, cerebellar efferent neurons, eurydendroid cells, also lie near the Purkinje cells and extend dendrites outward to intermingle with dendrites of the Purkinje cells within the molecular layer. Here we found no contacts between the climbing fiber terminals and the eurydendroid cell dendrites. These results support the idea that Purkinje cells, but not eurydendroid cells, receive strong inputs via the climbing fibers, similar to the mammalian situation.

Toward a cerebello-thalamo-cortical computational model of spinocerebellar ataxia

Computational neural network modelling is an emerging approach for optimization of drug treatment of neurological disorders and fine-tuning of rehabilitation strategies. In the current study, we constructed a cerebello-thalamo-cortical computational neural network model to simulate a mouse model of cerebellar ataxia (pcd5J mice) by manipulating cerebellar bursts through reduction of GABAergic inhibitory input. Cerebellar output neurons were projected to the thalamus and bidirectionally connected with the cortical network. Our results showed that reduction of inhibitory input in the cerebellum orchestrated the cortical local field potential (LFP) dynamics to generate specific motor outputs of oscillations of the theta, alpha, and beta bands in the computational model as well as in mouse motor cortical neurons. The therapeutic potential of deep brain stimulation (DBS) was tested in the computational model by increasing the sensory input to restore cortical output. Ataxia mice showed normalization of the motor cortex LFP after cerebellum DBS. We provide a novel approach to computational modelling to investigate the effect of DBS by mimicking cerebellar ataxia involving degeneration of Purkinje cells. Simulated neural activity coincides with findings from neural recordings of ataxia mice. Our computational model could thus represent cerebellar pathologies and provide insight into how to improve disease symptoms by restoring neuronal electrophysiological properties using DBS.

Cerebellar granule neurons induce Cyclin D1 before the onset of motor symptoms in Huntington’s disease mice

Although Huntington’s disease (HD) is classically defined by the selective vulnerability of striatal projection neurons, there is increasing evidence that cerebellar degeneration modulates clinical symptoms. However, little is known about cell type-specific responses of cerebellar neurons in HD. To dissect early disease mechanisms in the cerebellum and cerebrum, we analyzed translatomes of neuronal cell types from both regions in a new HD mouse model. For this, HdhQ200 knock-in mice were backcrossed with the calm 129S4 strain, to constrain experimental noise caused by variable hyperactivity of mice in a C57BL/6 background. Behavioral and neuropathological characterization showed that these S4-HdhQ200 mice had very mild behavioral abnormalities starting around 12 months of age that remained mild up to 18 months. By 9 months, we observed abundant Huntingtin-positive neuronal intranuclear inclusions (NIIs) in the striatum and cerebellum. The translatome analysis of GABAergic cells of the cerebrum further confirmed changes typical of HD-induced striatal pathology. Surprisingly, we observed the strongest response with 626 differentially expressed genes in glutamatergic neurons of the cerebellum, a population consisting primarily of granule cells, commonly considered disease resistant. Our findings suggest vesicular fusion and exocytosis, as well as differentiation-related pathways are affected in these neurons. Furthermore, increased expression of cyclin D1 (Ccnd1) in the granular layer and upregulated expression of polycomb group complex protein genes and cell cycle regulators Cbx2, Cbx4 and Cbx8 point to a putative role of aberrant cell cycle regulation in cerebellar granule cells in early disease.

Hypothyroidism induces motor deficit via altered cerebellar HB-EGF/EGFR and autophagy.

Thyroid hormones (TH) are vital for brain functions, while TH deficiency, i.e. hypothyroidism, induces neurological impairment in children and adults. Cerebellar neuronal apoptosis and motor deficits are crucial events in hypothyroidism; however, the underlying mechanism is less-known. Using a methimazole-treated hypothyroidism rat model, we investigated cerebellar autophagy, growth factor, and apoptotic mechanisms that participate in motor functions. We first identified that methimazole up-regulated cerebellar autophagy, marked by enhanced LC3B-II, Beclin-1, ATG7, ATG5-12, p-AMPKα/AMPKα, and p62 degradation as well as reduced p-AKT/AKT, p-mTOR/mTOR, and p-ULK1/ULK1 in developing and young adult rats. We probed upstream effectors of this abnormal autophagy and detected a methimazole-induced reduction in cerebellar phospho-epidermal growth factor receptor (p-EGFR)/EGFR and heparin-binding EGF-like growth factor (HB-EGF). Here, while a thyroxine-induced TH replenishment alleviated autophagy process and restored HB-EGF/EGFR, HB-EGF treatment regulated AKT-mTOR and autophagy signaling in the cerebellum. Moreover, neurons of the rat cerebellum demonstrated this reduced HB-EGF-dependent increased autophagy in hypothyroidism. We further checked whether the above events were related to cerebellar neuronal apoptosis and motor functions. We detected that comparable to thyroxine, treatment with HB-EGF or autophagy inhibitor, 3-MA, reduced methimazole-induced decrease in Nissl staining and increase in c-Caspase-3 and TUNEL-+ve apoptotic count of cerebellar neurons. Additionally, 3-MA, HB-EGF, and thyroxine attenuated the methimazole-induced diminution in riding time on rota-rod and grip strength for the motor performance of rats. Overall, our study enlightens HB-EGF/EGFR-dependent autophagy mechanism as a key to cerebellar neuronal loss and functional impairments in developmental hypothyroidism, which may be inhibited by HB-EGF and 3-MA treatments, like thyroxine.

Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders.

Cav2.1 channels are expressed throughout the brain and are the predominant Ca2+ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations (p.A405T in I-II loop and p.R1359W, p.R1667W and p.S1799L in IIIS4, IVS4, and IVS6 helices, respectively) identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1-e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells.

Virus-Like Cytosolic and Cell-Free Oxidatively Damaged Nucleic Acids Likely Drive Inflammation, Synapse Degeneration, and Neuron Death in Alzheimer's Disease.

Oxidative stress, inflammation, and amyloid-β are Alzheimer's disease (AD) hallmarks that cause each other and other AD hallmarks. Most amyloid-β-lowering, antioxidant, anti-inflammatory, and antimicrobial AD clinical trials failed; none stopped or reversed AD. Although signs suggest an infectious etiology, no pathogen accumulated consistently in AD patients. Neuropathology, neuronal cell culture, rodent, genome-wide association, epidemiological, biomarker, and clinical studies, plus analysis using Hill causality criteria and revised Koch's postulates, indicate that the virus-like oxidative damage-associated molecular-pattern (DAMP) cytosolic and cell-free nucleic acids accumulated in AD patients' brains likely drive neuroinflammation, synaptotoxicity, and neurotoxicity. Cytosolic oxidatively-damaged mitochondrial DNA accumulated outside mitochondria dose-dependently in preclinical AD and AD patients' hippocampal neurons, and in AD patients' neocortical neurons but not cerebellar neurons or glia. In oxidatively-stressed neural cells and rodents' brains, cytosolic oxidatively-damaged mitochondrial DNA accumulated and increased antiviral and inflammatory proteins, including cleaved caspase-1, interleukin-1β, and interferon-β. Cytosolic double-stranded RNA and DNA are DAMPs that induce antiviral interferons and/or inflammatory proteins by oligomerizing with various innate-immune pattern-recognition receptors, e.g., cyclic GMP-AMP synthase and the nucleotide-binding-oligomerization-domain-like-receptor-pyrin-domain-containing-3 inflammasome. In oxidatively-stressed neural cells, cytosolic oxidatively-damaged mitochondrial DNA caused synaptotoxicity and neurotoxicity. Depleting mitochondrial DNA prevented these effects. Additionally, cell-free nucleic acids accumulated in AD patients' blood, extracellular vesicles, and senile plaques. Injecting cell-free nucleic acids bound to albumin oligomers into wild-type mice's hippocampi triggered antiviral interferon-β secretion; interferon-β injection caused synapse degeneration. Deoxyribonuclease-I treatment appeared to improve a severe-AD patient's Mini-Mental Status Exam by 15 points. Preclinical and clinical studies of deoxyribonuclease-I and a ribonuclease for AD should be prioritized.

Loss of small GTPase Rab7 activation in prion infection negatively affects a feedback loop regulating neuronal cholesterol metabolism

Prion diseases are fatal and infectious neurodegenerative diseases that occur in humans and animals. They are caused by the misfolding of the cellular prion protein PrPc into the infectious isoform PrPSc. PrPSc accumulates mostly in endolysosomal vesicles of prion-infected cells, eventually causing neurodegeneration. In response to prion infection, elevated cholesterol levels and a reduction in membrane-attached small GTPase Rab7 have been observed in neuronal cells. Here, we investigated the molecular events causing an impaired Rab7 membrane attachment and the potential mechanistic link with elevated cholesterol levels in prion infection. We demonstrate that prion infection is associated with reduced levels of active Rab7 (Rab7.GTP) in persistently prion-infected neuronal cell lines, primary cerebellar granular neurons, and neurons in the brain of mice with terminal prion disease. In primary cerebellar granular neurons, levels of active Rab7 were increased during the very early stages of the prion infection prior to a significant decrease concomitant with PrPSc accumulation. The reduced activation of Rab7 in prion-infected neuronal cell lines is also associated with its reduced ubiquitination status, decreased interaction with its effector RILP, and altered lysosomal positioning. Consequently, the Rab7-mediated trafficking of low-density lipoprotein to lysosomes is delayed. This results in an impaired feedback regulation of cholesterol synthesis leading to an increase in cholesterol levels. Notably, transient overexpression of the constitutively active mutant of Rab7 rescues the delay in the low-density lipoprotein trafficking, hence reducing cholesterol levels and attenuating PrPSc propagation, demonstrating a mechanistic link between the loss of Rab7.GTP and elevated cholesterol levels.

Co-Administration of Vitamins B12 and D During Pregnancy Have Strong Neuroprotective Effects in Parkinson Disease.

Parkinson's disease (PD) is a common disease whose pathophysiological mechanism is not well understood. Recent research studies have shown that PD patients have low serum levels of vitamins B12 and D. Therefore, in this study, the effects of supplementation with vitamins B12 and D on PD female mice as well as their fetuses were studied. After preparation of female mice and induction of Parkinson's by rotenone administration for 19days, rotarod test was used to confirm PD induction. During this time, supplementations with vitamins B12 and D were performed. On day 19, after confirmation of PD induction, half of the mice were killed and the other half were allowed to mate with males. Viability was measured by the MTT method, and apoptosis and necrosis of cerebellar neurons were measured by flow cytometry. The RT-PCR technique was used to evaluate the relative expressions of the bax, bcl-2, miR-211, and circRNA 0,001,518 genes. Data analysis was performed by the GraphPad Prism V.8 software. Co-administration of vitamins B12 and D resulted in highest viability percentage and greatest reduction in apoptosis and necrosis of cerebellar neurons in the female mice as well as their fetuses compared to the PD females. A decrease in the relative expression of the bax and miR-211 genes and an increase in bcl-2 expression were observed in the cerebellar tissue of PD mice receiving both vitamins. Vitamins B12 and D have neuroprotective effects on PD conditions. Therefore, co-administration of these two vitamins is recommended in PD patients during pregnancy.

Therapeutic Administration of Oxcarbazepine Saves Cerebellar Purkinje Cells from Ischemia and Reperfusion Injury Induced by Cardiac Arrest through Attenuation of Oxidative Stress.

Research reports using animal models of ischemic insults have demonstrated that oxcarbazepine (a carbamazepine analog: one of the anticonvulsant compounds) extends neuroprotective effects against cerebral or forebrain injury induced by ischemia and reperfusion. However, research on protective effects against ischemia and reperfusion cerebellar injury induced by cardiac arrest (CA) and the return of spontaneous circulation has been poor. Rats were assigned to four groups as follows: (Groups 1 and 2) sham asphyxial CA and vehicle- or oxcarbazepine-treated, and (Groups 3 and 4) CA and vehicle- or oxcarbazepine-treated. Vehicle (0.3% dimethyl sulfoxide/saline) or oxcarbazepine (200 mg/kg) was administered intravenously ten minutes after the return of spontaneous circulation. In this study, CA was induced by asphyxia using vecuronium bromide (2 mg/kg). We conducted immunohistochemistry for calbindin D-28kDa and Fluoro-Jade B histofluorescence to examine Purkinje cell death induced by CA. In addition, immunohistochemistry for 4-hydroxy-2-nonenal (4HNE) was carried out to investigate CA-induced oxidative stress, and immunohistochemistry for Cu, Zn-superoxide dismutase (SOD1) and Mn-superoxide dismutase (SOD2) was performed to examine changes in endogenous antioxidant enzymes. Oxcarbazepine treatment after CA significantly increased the survival rate and improved neurological deficit when compared with vehicle-treated rats with CA (survival rates ≥ 63.6 versus 6.5%), showing that oxcarbazepine treatment dramatically protected cerebellar Purkinje cells from ischemia and reperfusion injury induced by CA. The salvation of the Purkinje cells from ischemic injury by oxcarbazepine treatment paralleled a dramatic reduction in 4HNE (an end-product of lipid peroxidation) and increased or maintained the endogenous antioxidant enzymes (SOD1 and SOD2). In brief, this study shows that therapeutic treatment with oxcarbazepine after CA apparently saved cerebellar neurons (Purkinje cells) from CA-induced neuronal death by attenuating oxidative stress and suggests that oxcarbazepine can be utilized as a therapeutic medicine for ischemia and reperfusion brain (cerebellar) injury induced by CA.

Propranolol Modulates Cerebellar Circuit Activity and Reduces Tremor.

Tremor is the most common movement disorder. Several drugs reduce tremor severity, but no cures are available. Propranolol, a β-adrenergic receptor blocker, is the leading treatment for tremor. However, the in vivo circuit mechanisms by which propranolol decreases tremor remain unclear. Here, we test whether propranolol modulates activity in the cerebellum, a key node in the tremor network. We investigated the effects of propranolol in healthy control mice and Car8wdl/wdl mice, which exhibit pathophysiological tremor and ataxia due to cerebellar dysfunction. Propranolol reduced physiological tremor in control mice and reduced pathophysiological tremor in Car8wdl/wdl mice to control levels. Open field and footprinting assays showed that propranolol did not correct ataxia in Car8wdl/wdl mice. In vivo recordings in awake mice revealed that propranolol modulates the spiking activity of control and Car8wdl/wdl Purkinje cells. Recordings in cerebellar nuclei neurons, the targets of Purkinje cells, also revealed altered activity in propranolol-treated control and Car8wdl/wdl mice. Next, we tested whether propranolol reduces tremor through β1 and β2 adrenergic receptors. Propranolol did not change tremor amplitude or cerebellar nuclei activity in β1 and β2 null mice or Car8wdl/wdl mice lacking β1 and β2 receptor function. These data show that propranolol can modulate cerebellar circuit activity through β-adrenergic receptors and may contribute to tremor therapeutics.

CTCF loss induces giant lamellar bodies in Purkinje cell dendrites

CCCTC-binding factor (CTCF) has a key role in higher-order chromatin architecture that is important for establishing and maintaining cell identity by controlling gene expression. In the mature cerebellum, CTCF is highly expressed in Purkinje cells (PCs) as compared with other cerebellar neurons. The cerebellum plays an important role in motor function by regulating PCs, which are the sole output neurons, and defects in PCs cause motor dysfunction. However, the role of CTCF in PCs has not yet been explored. Here we found that the absence of CTCF in mouse PCs led to progressive motor dysfunction and abnormal dendritic morphology in those cells, which included dendritic self-avoidance defects and a proximal shift in the climbing fibre innervation territory on PC dendrites. Furthermore, we found the peculiar lamellar structures known as “giant lamellar bodies” (GLBs), which have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe disease. GLBs are localized to PC dendrites and are assumed to be associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model in which CTCF was deleted. GLBs were not noted in PC dendrites at infancy but instead developed over time. In conjunction with GLB development in PC dendrites, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Our results revealed the important role of CTCF during normal development and in maintaining PCs and provide new insights into the molecular mechanism of GLB formation during neurodegenerative disease.