Cerebellar Sections Research Articles

The Spinocerebellar Ataxia 34-Causing W246G ELOVL4 Mutation Does Not Alter Cerebellar Neuron Populations in a Rat Model.

Spinocerebellar ataxia 34 (SCA34) is an autosomal dominant disease that arises from point mutations in the fatty acid elongase, Elongation of Very Long Chain Fatty Acids 4 (ELOVL4), which is essential for the synthesis of Very Long Chain-Saturated Fatty Acids (VLC-SFA) and Very Long Chain-Polyunsaturated Fatty Acids (VLC-PUFA) (28-34 carbons long). SCA34 is considered a neurodegenerative disease. However, a novel rat model of SCA34 (SCA34-KI rat) with knock-in of the W246G ELOVL4 mutation that causes human SCA34 shows early motor impairment and aberrant synaptic transmission and plasticity without overt neurodegeneration. ELOVL4 is expressed in neurogenic regions of the developing brain, is implicated in cell cycle regulation, and ELOVL4 mutations that cause neuroichthyosis lead to developmental brain malformation, suggesting that aberrant neuron generation due to ELOVL4 mutations might contribute to SCA34. To test whether W246G ELOVL4 altered neuronal generation or survival in the cerebellum, we compared the numbers of Purkinje cells, unipolar brush cells, molecular layer interneurons, granule and displaced granule cells in the cerebellum of wildtype, heterozygous, and homozygous SCA34-KI rats at four months of age, when motor impairment is already present. An unbiased, semi-automated method based on Cellpose 2.0 and ImageJ was used to quantify neuronal populations in cerebellar sections immunolabeled for known neuron-specific markers. Neuronal populations and cortical structure were unaffected by the W246G ELOVL4 mutation by four months of age, a time when synaptic and motor dysfunction are already present, suggesting that SCA34 pathology originates from synaptic dysfunction due to VLC-SFA deficiency, rather than aberrant neuronal production or neurodegeneration.

Progressive encephalomyelitis with rigidity and myoclonus: a pediatric case report and literature review

BackgroundProgressive encephalomyelitis with rigidity and myoclonus (PERM) is a rare and life-threatening autoimmune disease of the central nervous system. So far, only ten cases of PERM have been reported in children worldwide, including the one in this study.Case presentationWe report a case of an 11-year-old boy with PERM with an initial presentation of abdominal pain, skin itching, dysuria, urinary retention, truncal and limb rigidity, spasms of the trunk and limbs during sleep, deep and peripheral sensory disturbances, and dysphagia. A tissue-based assay using peripheral blood was positive, demonstrated by fluorescent staining of mouse cerebellar sections. He showed gradual and persistent clinical improvement after immunotherapy with intravenous immunoglobulin, steroids, plasmapheresis and rituximab.ConclusionsWe summarized the diagnosis and treatment of a patient with PERM and performed a literature review of pediatric PERM to raise awareness among pediatric neurologists. A better comprehension of this disease is required to improve its early diagnosis, treatment, and prognosis.

Zonisamide Ameliorated the Apoptosis and Inflammation in Cerebellar Tissue of Induced Alcohol Addiction Animal Model.

This study investigated the effects of zonisamide treatment on cerebellar tissues in an experimental alcohol addiction (AA) model and its potential mechanisms of action, particularly regarding apoptotic protease activating factor-1 (APAF-1) and tumor necrosis factor-alpha (TNF-α) expression. Thirty rats were divided into three groups: sham, ethanol (EtOH), and EtOH + zonisamide. AA was induced by administering 6 cc of EtOH orally every 8 h for 4 days. Zonisamide (100 mg/kg) was given to rats once daily before EtOH administration. Motor defects were evaluated using an open field maze. Serum TNF-α levels were measured from blood samples. Cerebellar sections were processed for histological examination and immunostained for APAF-1 and TNF-α. Protein interaction networks were constructed using Cytoscape, and functional annotations were performed with ShinyGO (version 0.80) software. The traveled area in the EtOH group was significantly reduced compared to the sham group (p = 0.0005). Rats in the EtOH + zonisamide group covered a larger area, with zonisamide treatment significantly improving locomotor ability compared to the EtOH group (p = 0.0463). Serum TNF-α levels were significantly elevated in the EtOH group compared to the sham group (p < 0.0001) and were significantly decreased in the EtOH + zonisamide group compared to the EtOH group (p = 0.0309). Regular cerebellar histological layers were observed in the sham group, while EtOH induction caused loss of cerebellar tissue integrity, neuronal degeneration, vascular dilatation and congestion, reduced myelin density, and neuropils in the EtOH group. Zonisamide treatment improved these pathologies, enhancing myelination and neuropil formation. Negative APAF-1 and TNF-α expressions were observed across cerebellar layers in the sham group. Due to EtOH toxicity, APAF-1 and TNF-α expression were upregulated in the EtOH group compared to the sham group (p < 0.001 for both). Zonisamide treatment downregulated these protein expressions in the EtOH + zonisamide group compared to the EtOH group (p < 0.001 and p = 0.0087, respectively). APAF-1 was primarily associated with AA through antifolate resistance, endopeptidases, and the interleukin-1 pathway, while TNF-α was predominantly enriched in infections and choline-binding, indicating zonisamide's impact on immune and inflammatory pathways. In conclusion, zonisamide treatment significantly mitigated ethanol-induced cerebellar damage and inflammation in an AA model. Zonisamide improved locomotor function and reduced serum TNF-α levels, as well as APAF-1 and TNF-α expression in cerebellar tissues. These findings suggest that zonisamide exerts its protective effects by modulating immune and inflammatory pathways, thereby preserving cerebellar integrity and function.

Calcium-Dependent Regulation of Neuronal Excitability Is Rescued in Fragile X Syndrome by a Tat-Conjugated N-Terminal Fragment of FMRP.

Fragile X syndrome (FXS) arises from the loss of fragile X messenger ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber long-term potentiation by adjusting the Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knock-out (KO) tsA-201 cells and cerebellar sections from male Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate the A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual-specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signaling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices, cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signaling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of FXS.

FXTAS Neuropathology Includes Widespread Reactive Astrogliosis and White Matter Specific Astrocyte Degeneration.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset progressive genetic neurodegenerative disorder that occurs in FMR1 premutation carriers. The temporal, spatial, and cell-type specific patterns of neurodegeneration in the FXTAS brain remain incompletely characterized. Intranuclear inclusion bodies are the neuropathological hallmark of FXTAS, which are largest and occur most frequently in astrocytes, glial cells that maintain brain homeostasis. Here, we characterized neuropathological alterations in astrocytes in multiple regions of the FXTAS brain. Striatal and cerebellar sections from FXTAS cases (n = 12) and controls (n = 12) were stained for the astrocyte markers glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1L1 (ALDH1L1) using immunohistochemistry. Reactive astrogliosis severity, the prevalence of GFAP+ fragments, and astrocyte density were scored. Double label immunofluorescence was utilized to detect co-localization of GFAP and cleaved caspase-3. FXTAS cases showed widespread reactive gliosis in both grey and white matter. GFAP staining also revealed remarkably severe astrocyte pathology in FXTAS white matter - characterized by a significant and visible reduction in astrocyte density (-38.7% in striatum and - 32.2% in cerebellum) and the widespread presence of GFAP+ fragments reminiscent of apoptotic bodies. White matter specific reductions in astrocyte density were confirmed with ALDH1L1 staining. GFAP+ astrocytes and fragments in white matter were positive for cleaved caspase-3, suggesting that apoptosis-mediated degeneration is responsible for reduced astrocyte counts. We have established that FXTAS neuropathology includes robust degeneration of astrocytes, which is specific to white matter. Because astrocytes are essential for maintaining homeostasis within the central nervous system, a loss of astrocytes likely further exacerbates neuropathological progression of other cell types in the FXTAS brain. ANN NEUROL 2024;95:558-575.

Two patients of immunotherapy-responsive autoimmune cerebellar ataxia fulfilled with criteria of multiple system atrophy

We report two patients with autoimmune cerebellar ataxia who fulfilled the diagnostic criteria of multiple system atrophy (MSA) and responded to immunotherapies. Patient 1 was a 72-year-old man who was diagnosed with clinically probable MSA according to Movement Disorder Society criteria. Patient 2 was a 68-year-old man who was diagnosed with clinically established MSA according to Movement Disorder Society criteria. Both patients showed cerebellar ataxia, autonomic dysfunction, and pyramidal tract signs; however, they also had atypical clinical features. Patient 1 exhibited self-‍limiting mild improvement of clinical symptoms and had inflammatory findings in his cerebrospinal fluid. Patient 2 showed a rapidly progressive clinical course. We therefore examined anti-neuronal antibodies using tissue-based immunohistochemical assays with frozen rat cerebellum sections. We detected autoantibodies that mainly reacted with the cytoplasm of Purkinje cells. The two patients then underwent immunotherapies, which led to substantial improvements in their clinical symptoms. Our findings indicate that some patients with autoimmune cerebella ataxia have clinical features that resemble MSA, and respond well to immunotherapies.

Examining the microglial TSPO phenotype in Alzheimer’s disease

AbstractBackgroundNeuroinflammation is a key pathological feature of Alzheimer’s disease (AD), with microglia being the main inflammatory cell type in the CNS. In order to examine these cells in vivo, PET ligands targeting the microglial translocator protein (TSPO) have been developed. However, it remains unclear the specific target of these ligands, whether this is all microglia or a subset of cells. We aim to elucidate this by identifying the microglial phenotype most associated with TSPO in humans.MethodHuman post‐mortem sections of temporal lobe (TL) and cerebellum (Cb) from cases classified by Braak stage (0‐II, III‐IV, V‐VI; each n = 10) were immunostained for pan‐Aß, pTau, and microglial markers TSPO, Iba1 and HLA‐DR with protein load quantified. Double immunofluorescent staining was performed with TSPO and microglial markers HLA‐DR and CD68 with positive cells counted.ResultTL showed an increase in Aβ (P&lt;0.0001), pTau (P&lt;0.0001) and TSPO (P&lt;0.0001) protein load, but not Iba1 or HLA‐DR, with progressing Braak stage. Aβ (P = 0.0008) and Iba1 (P = 0.012) load increased in the Cb, but not pTau, TSPO or HLA‐DR. Of note, TSPO load in TL was associated with pTau. In both areas, no difference was found in number of HLA+ or CD68+ cells, though TL TSPO+ cell number increased significantly (P = 0.0347). There was no difference in the number of HLA‐DR+/TSPO+ or CD68+/TSPO+ cells with progressing Braak stage. However, significantly more CD68+/TSPO+ than HLA‐DR+/TSPO+ cells were detected in both the TL (P&lt;0.0001) and Cb (P = 0.009) regions.ConclusionThe results suggest that TSPO expression is related to late stage disease (pTau) and microglial phagocytosis (CD68), a function we have previously identified associated with the presence of dementia, cognitive decline and tau pathology. Further additional analysis with other microglial markers is currently ongoing.

Evaluation of prenatal administration of valproic acid on the cerebellum of albino rat offspring

Introduction: Autism is a severe neurodevelopmental disorder of poorly understood etiology; which may be genetic, epigenetic or environmental. Valproic acid (VPA), the most widely used antiepileptic drug, has been reported to increase the risk of autism among the offspring of human mothers who are medicated with it during early pregnancy. Aim: The current work aimed to study the biochemical and histological changes in the cerebellum of the offspring of prenatally VPA treated rats. Materials and Methods: Twelve pregnant female albino rats were divided into two groups; control and VPA treated (50 mg/kg/day orally). The cerebellar sections of male offspring rats were subjected to biochemical tests (brain tissue Malondialdehyde (MDA), Superoxide dismutase (SOD), Tumor necrosis factor-alpha (TNF-α) and glutamate), histological examination, immunohistochemical analyses for the expression of glial fibrillary acidic protein (GFAP) and Bcl-2 associated X protein (Bax), and electro microscopical studies. Results: VPA caused significant elevation in the brain levels of oxidative stress marker MDA, proinflammatory cytokine TNF-α and excitatory neurotransmitter glutamate, with significant reduction in the level of brain antioxidant marker SOD, compared to the control group.

P04.07.B CHARACTERIZATION OF ARHGAP36/MYC-INDUCED GROUP 3 MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is one of the most common pediatric brain cancers of the central nervous system. It is stratified into 4 molecular subtypes (WNT, SHH, group 3, and group 4), which differ from one to the other for various features, such as cellular morphology, age of onset, set of mutations, and percentage of survival. A common point between the four MB subgroups is the absence of treatments for this disease. The current gold standard treatment has deleterious side effects for the patients, which spans from growth defects to low hormone levels and problems with learning. Analyzing transcriptomic data of group 3 MB patients we found ARHGAP36 as a possible gene involved in tumor formation or propagation. Rho GTPase-Activating Protein 36 (ARHGAP36) is a gene that plays a role in regulating PKA activity. Specifically, the ARHGAP36 protein can inhibit PKA function through two distinct mechanisms. Firstly, it acts as a pseudosubstrate for the catalytic subunit of the PKA protein. Secondly, it can drive protein degradation via a lysosome-mediated process. MATERIAL AND METHODS We took advantage of the Piggybac/transposon technology to induce a strong and stable overexpression of our genes of interest to generate two different models, one in-vivo and one in-vitro with induced pluripotent stem cells-derived human cerebellar organoids. We also exploited the cre/LoxP system in transgenic mice to overexpress ARHGAP36 under the control of specific promoters. The analysis was conducted using standard techniques such as RT-qPCR and Western Blot, together with immunofluorescence of the cerebellar section and live imaging of the modified cerebellar organoids. The gene expression profile of the tumors obtained in mice was analyzed by RNA sequencing and compared to known group 3 MB mouse models. RESULTS We analyzed RNA-sequencing data from medulloblastoma patients and found that high expression of ARHGAP36 correlated with poor prognosis in patients. Through PKA inhibition, ARHAP36 induces ectopic activation of SHH signaling and cell proliferation leading to abnormal development of the cerebellum in mouse embryos. Notably, despite its inductive activity on the SHH pathway, ARHGAP36 expression is higher in group 3 and group 4 MB, rather than in the SHH subgroup. In this study, we demonstrate that intracranial transfection of P0 mouse pups with plasmids overexpressing MYC and ARHGAP36 can generate group 3 medulloblastoma in-vivo. We studied and then confirmed the involvement of the binding with PKA as a tumorigenic mechanism in the MYC/ARHGAP36 co-overexpression using several mutated forms of the gene. We studied the obtained tumors for the presence of group 3 MB immunophenotypical markers, as well as their gene expression profile by RNA-seq. CONCLUSION These findings can become of key interest as possible druggable targets to efficiently treat group 3 medulloblastoma patients in the future.

Ablation of Projection Glutamatergic Neurons in the Lateral Cerebellar Nuclei Alters Motor Coordination in Vglut2-Cre+ Mice.

Cerebellar nuclei (CN) constitute the sole cerebellar output to the rest of the central nervous system and play a central role in cerebellar circuits. Accumulating evidence from both human genetics and animal studies point to a crucial role for CN connectivity in neurological diseases, including several types of ataxia. However, because of the compact and restricted topography and close functional connection between the CN and the cerebellar cortex, identifying cerebellar deficits exclusively linked to CN is challenging. In this study, we have experimentally ablated large projection glutamatergic neurons of the lateral CN and evaluated the impact of this selective manipulation on motor coordination in mice. To this end, through stereotaxic surgery, we injected the lateral CN of Vglut2-Cre+ mice with an adeno-associated virus (AAV) encoding a Cre-dependent diphtheria toxin receptor (DTR), followed by an intraperitoneal injection of diphtheria toxin (DT) to ablate the glutamatergic neurons of the lateral nucleus. Double immunostaining of cerebellar sections with anti-SMI32 and -GFP antibodies revealed GFP expression and provided evidence of SMI32+ neuron degeneration at the site of AAV injection in the lateral nucleus of Vglut2-Cre+ mice. No changes were observed in Vglut2-Cre negative mice. Analysis of motor coordination by rotarod test indicated that the latency to fall was significantly different before and after AAV/DT injection in the Vglut2-Cre+ group. Elapsed time and number of steps in the beam walking test were significantly higher in AAV/DT injected Vglut2-Cre+ AAV/DT mice compared to controls. We demonstrate for the first time that partial degeneration of glutamatergic neurons in the lateral CN is sufficient to induce an ataxic phenotype.

Septin-3 autoimmunity in patients with paraneoplastic cerebellar ataxia

BackgroundSeptins are cytoskeletal proteins with filament forming capabilities, which have multiple roles during cell division, cellular polarization, morphogenesis, and membrane trafficking. Autoantibodies against septin-5 are associated with non-paraneoplastic cerebellar ataxia, and autoantibodies against septin-7 with encephalopathy with prominent neuropsychiatric features. Here, we report on newly identified autoantibodies against septin-3 in patients with paraneoplastic cerebellar ataxia. We also propose a strategy for anti-septin autoantibody determination.MethodsSera from three patients producing similar immunofluorescence staining patterns on cerebellar and hippocampal sections were subjected to immunoprecipitation followed by mass spectrometry. The identified candidate antigens, all of which were septins, were expressed recombinantly in HEK293 cells either individually, as complexes, or combinations missing individual septins, for use in recombinant cell-based indirect immunofluorescence assays (RC-IIFA). Specificity for septin-3 was further confirmed by tissue IIFA neutralization experiments. Finally, tumor tissue sections were analyzed immunohistochemically for septin-3 expression.ResultsImmunoprecipitation with rat cerebellum lysate revealed septin-3, -5, -6, -7, and -11 as candidate target antigens. Sera of all three patients reacted with recombinant cells co-expressing septin-3/5/6/7/11, while none of 149 healthy control sera was similarly reactive. In RC-IIFAs the patient sera recognized only cells expressing septin-3, individually and in complexes. Incubation of patient sera with five different septin combinations, each missing one of the five septins, confirmed the autoantibodies’ specificity for septin-3. The tissue IIFA reactivity of patient serum was abolished by pre-incubation with HEK293 cell lysates overexpressing the septin-3/5/6/7/11 complex or septin-3 alone, but not with HEK293 cell lysates overexpressing septin-5 as control. All three patients had cancers (2 × melanoma, 1 × small cell lung cancer), presented with progressive cerebellar syndromes, and responded poorly to immunotherapy. Expression of septin-3 was demonstrated in resected tumor tissue available from one patient.ConclusionsSeptin-3 is a novel autoantibody target in patients with paraneoplastic cerebellar syndromes. Based on our findings, RC-IIFA with HEK293 cells expressing the septin-3/5/6/7/11 complex may serve as a screening tool to investigate anti-septin autoantibodies in serological samples with a characteristic staining pattern on neuronal tissue sections. Autoantibodies against individual septins can then be confirmed by RC-IIFA expressing single septins.

Alteration of Aquaporins 1 and 4 immunohistochemical and gene expression in the cerebellum of diabetic albino rat

Aquaporins (AQPs) are a family of transmembrane channel proteins. AQP1 and AQP4 are expressed in cerebellum amongst others. This study was designed to assess the effect of diabetes on AQP1 and AQP4 expression in cerebellum of rats. Diabetes was induced by a single intraperitoneal injection of Streptozotocin 45 mg/kg in 24 adult male Sprague Dawley rats. Six rats from control and diabetic groups were sacrificed at one, four, and eight weeks post diabetic confirmation. After eight weeks, measurement of malondialdehyde (MDA), reduced glutathione (GSH) concentrations, and cerebellar mRNA expression for AQP1 and AQP4 genes were performed. Immunohistochemical evaluation of AQP1, AQP4, and glial fibrillary acidic protein (GFAP) for cerebellar sections was performed for all groups. Diabetes caused degenerative changes in Purkinje cells with a significant increase in the cerebellar level of MDA and AQP1 immunoreactivity and a significant decrease in GSH level and AQP4 expression levels. However, the alteration in the AQP1 mRNA level was not statistically significant. GFAP immunoreactivity was increased in 8 W diabetic rats following its decrease in 1 W diabetic rats. Diabetes caused some alteration in the AQPs 1 and 4 expression in the cerebellum of diabetic rats which may contribute to diabetes-induced cerebellar complications.

STLV-1 Commonly Targets Neurons in the Brain of Asymptomatic Non-Human Primates.

The human T-cell leukemia virus (HTLV)-1 is responsible for an aggressive neurodegenerative disease (HAM/TSP) and multiple neurological alterations. The capacity of HTLV-1 to infect central nervous system (CNS) resident cells, together with the neuroimmune-driven response, has not been well-established. Here, we combined the use of human induced pluripotent stem cells (hiPSC) and of naturally STLV-1-infected nonhuman primates (NHP) as models with which to investigate HTLV-1 neurotropism. Hence, neuronal cells obtained after hiPSC differentiation in neural polycultures were the main cell population infected by HTLV-1. Further, we report the infection of neurons with STLV-1 in spinal cord regions as well as in brain cortical and cerebellar sections of postmortem NHP. Additionally, reactive microglial cells were found in infected areas, suggesting an immune antiviral response. These results emphasize the need to develop new efficient models by which to understand HTLV-1 neuroinfection and suggest an alternative mechanism that leads to HAM/TSP.

Localization and Characterization of Major Neurogenic Niches in the Brain of the Lesser-Spotted Dogfish Scyliorhinus canicula.

Adult neurogenesis is defined as the ability of specialized cells in the postnatal brain to produce new functional neurons and to integrate them into the already-established neuronal network. This phenomenon is common in all vertebrates and has been found to be extremely relevant for numerous processes, such as long-term memory, learning, and anxiety responses, and it has been also found to be involved in neurodegenerative and psychiatric disorders. Adult neurogenesis has been studied extensively in many vertebrate models, from fish to human, and observed also in the more basal cartilaginous fish, such as the lesser-spotted dogfish, Scyliorhinus canicula, but a detailed description of neurogenic niches in this animal is, to date, limited to the telencephalic areas. With this article, we aim to extend the characterization of the neurogenic niches of S. canicula in other main areas of the brain: we analyzed via double immunofluorescence sections of telencephalon, optic tectum, and cerebellum with markers of proliferation (PCNA) and mitosis (pH3) in conjunction with glial cell (S100β) and stem cell (Msi1) markers, to identify the actively proliferating cells inside the neurogenic niches. We also labeled adult postmitotic neurons (NeuN) to exclude double labeling with actively proliferating cells (PCNA). Lastly, we observed the presence of the autofluorescent aging marker, lipofuscin, contained inside lysosomes in neurogenic areas.

Does Withania somnifera mitigate the structural alterations of the rat brain associated with propylthiouracil?

The repercussion of propylthiouracil (PTU) use postnatally on brain histology have not yet been intensely scrutinized. To examine whether Withania somnifera mitigate the structural effects of propylthiouracil on rat brain, rats were distributed into group A enrolled ten pups that were received -orally- distilled water (D.W) daily from postnatal day (PND) 3 to PND 43. Group B: enrolled pups (n=8) subjected to oral doses of PTU (1 mg/kg/day) from PND 3 to PND 25. Then they were gavaged with D.W till PND 43. Group C: included pups (n=8) that were subjected orally to PTU (1mg/kg/day) PND 3 to PND 25 with receiving levothyroxine (four microgram /100g/day) from PND 25 to PND 43. Group D included pups (n=8) that were treated with oral PTU 1 mg/kg/day PND 3 to PND 25 with receiving Withania somnifera extract (200 mg/kg/day) from PND 25 to PND 43. Cerebellar sections of rats of group B exhibited disorganization of the cerebellar cortex with a falling off in the Purkinje cells' count and the appearance of degenerated cells. Hippocampal sections (of rats of group B) proclaimed a falling off in the breadth of the pyramidal zone of cornu Amonis. Sections of the cerebral cortex of rats in group B exhibited the presence of large degenerated neurons. Sections of rats' brains belonging to groups C and D showed improved cerebellar and cerebral cortex and hippocampal and cerebral cortical segments. Levothyroxine and Withania somnifera mitigates the structural changes in the peripubertal rat brain induced by postnatal PTU administration.

Automated Immunofluorescence Staining for Analysis of Mitotic Stages and Division Orientation in Brain Sections.

Microcephaly often results from mitotic defects in neuronal progenitors, frequently by decreasing proliferation rates or shifting cell fates. During neurogenesis, oriented cell division-the molecular control of mitotic spindle positioning to control the axis of division-represents an important mechanism to balance expansion of the progenitor pool with generating cellular diversity. While mostly studied in the context of cortical development, more recently, spindle orientation has emerged as a key player in the formation of other brain regions such as the cerebellum. Here we describe methods to perform automated dual-color fluorescent immunohistochemistry on murine cerebellar sections using the mitotic markers phospho-Histone H3 and Survivin, and detail analytical and statistical approaches to display and compare division orientation datasets.

Mechanisms underpinning Carpolobia lutea G. Don ethanol extract's neurorestorative and antipsychotic-like activities in an NMDA receptor antagonist model of schizophrenia

Ethnopharmacological relevancePersistent ketamine insults to the central nervous system block NMDA receptors and disrupt putative neurotransmission, oxido–nitrosative, and inflammatory pathways, resulting in schizophrenia-like symptoms in animals. Previously, the ethnomedicinal benefits of Carpolobia lutea against insomnia, migraine headache, and insanity has been documented, but the mechanisms of action remain incomplete. Aim of the studyPresently, we explored the neuro-therapeutic role of Carpolobia lutea ethanol extract (C. lutea) in ketamine-induced schizophrenia-like symptoms in mice. Materials and methodsSixty-four male Swiss (22 ± 2 g) mice were randomly assigned into eight groups (n = 8/group) and exposed to a reversal ketamine model of schizophrenia. For 14 days, either distilled water (10 mL/kg; p.o.) or ketamine (20 mg/kg; i.p.) was administered, following possible reversal treatments with C. lutea (100, 200, 400, and 800 mg/kg; p.o.), haloperidol (1 mg/kg, p.o.), or clozapine (5 mg/kg; p.o.) beginning on days 8–14. During the experiment, a battery of behavioral characterizations defining schizophrenia-like symptoms were obtained using ANY-maze software, followed by neurochemical, oxido-inflammatory and histological assessments in the mice brains. ResultsA 7-day reversal treatment with C. lutea reversed predictors of positive, negative and cognitive symptoms of schizophrenia. C. lutea also mitigated ketamine-induced neurochemical derangements as evidenced by modulations of dopamine, glutamate, norepinephrine and serotonin neurotransmission. Also, the increased acetylcholinesterase activity, malondialdehyde nitrite, interleukin-6 and tumor necrosis-factor-α concentrations were reversed by C. lutea accompanied with elevated levels of catalase, superoxide dismutase and reduced glutathione. Furthermore, C. lutea reversed ketamine-induced neuronal alterations in the prefrontal cortex, hippocampus and cerebellum sections of the brain. ConclusionThese findings suggest that C. lutea reverses the cardinal symptoms of ketamine-induced schizophrenia in a dose-dependent fashion by modulating the oxido-inflammatory and neurotransmitter-related mechanisms.

High quality mapping of chromatin at or near the nuclear lamina from small numbers of cells reveals cell cycle and developmental changes of chromatin at the nuclear periphery.

The chromatin associated with the nuclear lamina (NL) is referred to as lamina-associated domains (LADs). Here, we present an adaptation of the tyramide-signal amplification sequencing (TSA-seq) protocol, which we call chromatin pull down-based TSA-seq (cTSA-seq), that can be used to map chromatin regions at or near the NL from as little as 50 000 cells. The cTSA-seq mapped regions are composed of previously defined LADs and smaller chromatin regions that fall within the Hi-C defined B-compartment containing nuclear peripheral heterochromatin. We used cTSA-seq to map chromatin at or near the assembling NL in cultured cells progressing through early G1. cTSA-seq revealed that the distal ends of chromosomes are near or at the reassembling NL during early G1, a feature similar to those found in senescent cells. We expand the use of cTSA-seq to the mapping of chromatin at or near the NL from fixed-frozen mouse cerebellar tissue sections. This mapping reveals a general conservation of NL-associated chromatin and identifies global and local changes during cerebellar development. The cTSA-seq method reported here is useful for analyzing chromatin at or near the NL from small numbers of cells derived from both in vitro and in vivo sources.

Neuroprotective effects of quercetin on the cerebellum of zinc oxide nanoparticles (ZnoNps)-exposed rats

ABSTRACT Engineered nanomaterials induce hazardous effects at the cellular and molecular levels. We investigated different mechanisms underlying the neurotoxic potential of zinc oxide nanoparticles (ZnONPs) on cerebellar tissue and clarified the ameliorative role of Quercetin supplementation. Forty adult male albino rats were divided into control group (I), ZnONPs-exposed group (II), and ZnONPs and Quercetin group (III). Oxidative stress biomarkers (MDA & TOS), antioxidant biomarkers (SOD, GSH, GR, and TAC), serum interleukins (IL-1β, IL-6, IL-8), and tumor necrosis factor alpha (TNF-α) were measured. Serum micro-RNA (miRNA): miRNA-21-5p, miRNA-122-5p, miRNA-125b-5p, and miRNA-155-3p expression levels were quantified by real-time quantitative polymerase-chain reaction (RT-QPCR). Cerebellar tissue sections were stained with Hematoxylin & Eosin and Silver stains and examined microscopically. Expression levels of Calbindin D28k, GFAP, and BAX proteins in cerebellar tissue were detected by immunohistochemistry. Quercetin supplementation lowered oxidative stress biomarkers levels and ameliorated the antioxidant parameters that were decreased by ZnONPs. No significant differences in GR activity were detected between the study groups. ZnONPs significantly increased serum IL-1β, IL-6, IL-8, and TNF-α which were improved with Quercetin. Serum miRNA-21-5p, miRNA-122-5p, miRNA-125b-5p, and miRNA-155-p expression levels showed significant increase in ZnONPs group, while no significant difference was observed between Quercetin-treated group and control group. ZnONPs markedly impaired cerebellar tissue structure with decreased levels of calbindin D28k, increased BAX and GFAP expression. Quercetin supplementation ameliorated cerebellar tissue apoptosis, gliosis and improved calbindin levels. In conclusion: Quercetin supplementation ameliorated cerebellar neurotoxicity induced by ZnONPs at cellular and molecular basis by different studied mechanisms.Abbreviations: NPs: Nanoparticles, ROS: reactive oxygen species, ZnONPs: Zinc oxide nanoparticles, AgNPs: silver nanoparticles, BBB: blood–brain barrier, ncRNAs: Non-coding RNAs, miRNA: Micro RNA, DMSO: Dimethyl sulfoxide, LPO: lipid peroxidation, MDA: malondialdehyde, TBA: thiobarbituric acid, TOS: total oxidative status, ELISA: enzyme-linked immunosorbent assay, H2O2: hydrogen peroxide, SOD: superoxide dismutase, GR: glutathione reductase, TAC: total antioxidant capacity, IL-1: interleukin-1, TNF: tumor necrosis factor alpha, cDNA: complementary DNA, RT-QPCR: Real-time quantitative polymerase-chain reaction, ABC: Avidin biotin complex technique, DAB: 3′, 3-diaminobenzidine, SPSS: Statistical Package for Social Sciences, ANOVA: One way analysis of variance, Tukey’s HSD: Tukey’s Honestly Significant Difference, GFAP: glial fiberillar acitic protein, iNOS: Inducible nitric oxide synthase, NO: nitric oxide, HO-1: heme oxygenase-1, Nrf2: nuclear factor erythroid 2-related factor 2, NF-B: nuclear factor-B, SCI: spinal cord injury, CB: Calbindin

The Discovery of Anti-Yo (Anti-PCA1) Antibody in Patients with Paraneoplastic Cerebellar Degeneration: Opening a Window into Autoimmune Neurological Disease.

Prior to 1982, ovarian and certain other cancers were known to have a rare complication of progressive cerebellar ataxia, a disorder characterized pathologically by severe-often total-obliteration of cerebellar Purkinje cells. However, the cause of cerebellar injury in these patients was unknown. In that year, we began studies in which sera from individuals with this disorder were reacted with frozen sections of human cerebellum. These studies revealed that patients with ovarian cancer and cerebellar degeneration had high titers of antibodies directed against cytoplasmic antigens of Purkinje cells and deep cerebellar nuclei-a previously undescribed pattern of antibody response which was subsequently found not to be present in ovarian cancer patients who remained neurologically normal. This antibody, now known as "anti-Yo" or "anti-PCA1" provides a marker for rapidly progressive cerebellar ataxia and is heavily associated with gynecological and breast malignancies. Although the role of anti-Yo antibody in cerebellar injury has not been established in living animals, in vitro studies have demonstrated that anti-Yo antibody causes Purkinje cell death in the absence of T lymphocytes. In this commentary, we describe our studies leading to initial discovery of anti-Yo antibody, discuss the relationship of this discovery to current knowledge of paraneoplastic neurological disease, and outline some important questions which remain to be resolved before we fully understand the pathogenesis and optimal treatment of this disorder.