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BioRxiv : the Preprint Server For... Feb 2024Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje...
Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. We recently reported a family with a paternally inherited intragenic duplication with a range of neurodevelopmental disorders, including autism spectrum disorder (ASD), learning difficulties, and speech and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional knockout (KO and cKO, respectively) mouse lines. KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity and repetitive behaviors, altered social behaviors, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors were also prominent in cKO animals. By Golgi staining, KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrates a significant increase in Bergmann glia volume in the molecular layer of KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents (IPSCs) in the KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.
PubMed: 38405978
DOI: 10.1101/2024.02.18.580354 -
BioRxiv : the Preprint Server For... Feb 2024We identified a novel variant, E171Q, in a neonate with very frequent ectopy and reduced ejection fraction which normalized after arrhythmia suppression by flecainide....
BACKGROUND
We identified a novel variant, E171Q, in a neonate with very frequent ectopy and reduced ejection fraction which normalized after arrhythmia suppression by flecainide. This clinical picture is consistent with multifocal ectopic Purkinje-related premature contractions (MEPPC). Most previous reports of MEPPC have implicated variants such as R222Q that neutralize positive charges in the S4 voltage sensor helix of the channel protein Na1.5 and generate a gating pore current.
METHODS AND RESULTS
E171 is a highly conserved negatively-charged residue located in the S2 transmembrane helix of Na1.5 domain I. E171 is a key component of the Gating Charge Transfer Center, a region thought to be critical for normal movement of the S4 voltage sensor helix. We used heterologous expression, CRISPR-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and molecular dynamics simulations to demonstrate that E171Q generates a gating pore current, which was suppressed by a low concentration of flecainide (IC50 = 0.71±0.07 µM). R222Q shifts voltage dependence of activation and inactivation in a negative direction but we observed positive shifts with E171Q. E171Q iPSC-CMs demonstrated abnormal spontaneous activity and prolonged action potentials. Molecular dynamics simulations revealed that both R222Q and E171Q proteins generate a water-filled permeation pathway that underlies generation of the gating pore current.
CONCLUSION
Previously identified MEPPC-associated variants that create gating pore currents are located in positively-charged residues in the S4 voltage sensor and generate negative shifts in the voltage dependence of activation and inactivation. We demonstrate that neutralizing a negatively charged S2 helix residue in the Gating Charge Transfer Center generates positive shifts but also create a gating pore pathway. These findings implicate the gating pore pathway as the primary functional and structural determinant of MEPPC and widen the spectrum of variants that are associated with gating pore-related disease in voltage-gated ion channels.
PubMed: 38405820
DOI: 10.1101/2024.02.13.580021 -
Frontiers in Physiology 2024Severe acute global cerebral hypoxia can lead to significant disability in humans. Although different animal models have been described to study hypoxia, there is no...
Severe acute global cerebral hypoxia can lead to significant disability in humans. Although different animal models have been described to study hypoxia, there is no endogenous model that considers hypoxia and its effect on the brain as an independent factor. Thus, we developed a minimally invasive rat model, which is based on the non-depolarizing muscle blocking agent rocuronium in anesthetized animals. This drug causes respiratory insufficiency by paralysis of the striated muscles. In this study, 14 rats underwent 12 min of hypoxemia with an oxygen saturation of approximately 60% measured by pulse oximetry; thereafter, animals obtained sugammadex to antagonize rocuronium immediately. Compared to controls (14 rats, anesthesia only), hypoxic animals demonstrated significant morphological alterations in the hippocampus (cell decrease in the CA 1 region) and the cerebellum (Purkinje cell decrease), as well as significant changes in hypoxia markers in blood (Hif2α, Il1β, Tgf1β, Tnfα, S100b, cspg2, neuron-specific enolase), hippocampus (Il1β, Tnfα, S100b, cspg2, NSE), and cerebellum (Hif1α, Tnfα, S100b, cspg2, NSE). Effects were more pronounced in females than in males. Consequently, this model is suitable to induce hypoxemia with consecutive global cerebral hypoxia. As significant morphological and biochemical changes were proven, it can be used to investigate therapeutic and preventive drugs for global cerebral hypoxia.
PubMed: 38405120
DOI: 10.3389/fphys.2024.1293247 -
International Journal of Molecular... Feb 2024Although more than 30 different types of neuropeptides have been identified in various cell types and circuits of the cerebellum, their unique functions in the... (Review)
Review
Although more than 30 different types of neuropeptides have been identified in various cell types and circuits of the cerebellum, their unique functions in the cerebellum remain poorly understood. Given the nature of their diffuse distribution, peptidergic systems are generally assumed to exert a modulatory effect on the cerebellum via adaptively tuning neuronal excitability, synaptic transmission, and synaptic plasticity within cerebellar circuits. Moreover, cerebellar neuropeptides have also been revealed to be involved in the neurogenetic and developmental regulation of the developing cerebellum, including survival, migration, differentiation, and maturation of the Purkinje cells and granule cells in the cerebellar cortex. On the other hand, cerebellar neuropeptides hold a critical position in the pathophysiology and pathogenesis of many cerebellar-related motor and psychiatric disorders, such as cerebellar ataxias and autism. Over the past two decades, a growing body of evidence has indicated neuropeptides as potential therapeutic targets to ameliorate these diseases effectively. Therefore, this review focuses on eight cerebellar neuropeptides that have attracted more attention in recent years and have significant potential for clinical application associated with neurodegenerative and/or neuropsychiatric disorders, including brain-derived neurotrophic factor, corticotropin-releasing factor, angiotensin II, neuropeptide Y, orexin, thyrotropin-releasing hormone, oxytocin, and secretin, which may provide novel insights and a framework for our understanding of cerebellar-related disorders and have implications for novel treatments targeting neuropeptide systems.
Topics: Humans; Cerebellum; Purkinje Cells; Neurons; Cerebellar Cortex; Neuropeptides; Cerebellar Diseases
PubMed: 38397008
DOI: 10.3390/ijms25042332 -
Biomedicine & Pharmacotherapy =... Apr 2024Ethanol is one of the psychoactive substances most used by young individuals, usually in an intermittent and episodic manner, also called binge drinking. In the...
Ethanol binge drinking exposure during adolescence displays long-lasting motor dysfunction related to cerebellar neurostructural damage even after long-term withdrawal in female Wistar rats.
Ethanol is one of the psychoactive substances most used by young individuals, usually in an intermittent and episodic manner, also called binge drinking. In the adolescent period, brain structures undergo neuromaturation, which increases the vulnerability to psychotropic substances. Our previous studies have revealed that ethanol binge drinking during adolescence elicits neurobehavioral alterations associated with brain damage. Thus, we explored the persistence of motor function impairment and cerebellum damage in the context of ethanol withdrawal periods (emerging adulthood and adult life) in adolescent female rats. Female Wistar rats (35 days old) received orally 4 cycles of ethanol (3.0 g/kg/day) or distilled water in 3 days on-4 days off paradigm (35th until 58th day of life). Motor behavioral tests (open field, grip strength, beam walking, and rotarod tests) and histological assays (Purkinje's cell density and NeuN-positive cells) were assessed on the 1-, 30-, and 60-days of binge alcohol exposure withdrawal. Our findings demonstrate that the adolescent binge drinking exposure paradigm induced cerebellar cell loss in all stages evaluated, measured through the reduction of Purkinje's cell density and granular layer neurons. The cerebellar tissue alterations were accompanied by behavioral impairments. In the early withdrawal, the reduction of spontaneous movement, incoordination, and unbalance was seen. However, the grip strength reduction was found at long-term withdrawal (60 days of abstinence). The cerebellum morphological changes and the motor alterations persisted until adulthood. These data suggest that binge drinking exposure during adolescence causes motor function impairment associated with cerebellum damage, even following a prolonged withdrawal, in adult life.
Topics: Rats; Animals; Female; Rats, Wistar; Ethanol; Binge Drinking; Alcohol Drinking; Cerebellum; Alcoholism; Substance Withdrawal Syndrome; Age Factors
PubMed: 38394853
DOI: 10.1016/j.biopha.2024.116316 -
PLoS Computational Biology Feb 2024Investigating and modelling the functionality of human neurons remains challenging due to the technical limitations, resulting in scarce and incomplete 3D anatomical...
Investigating and modelling the functionality of human neurons remains challenging due to the technical limitations, resulting in scarce and incomplete 3D anatomical reconstructions. Here we used a morphological modelling approach based on optimal wiring to repair the parts of a dendritic morphology that were lost due to incomplete tissue samples. In Drosophila, where dendritic regrowth has been studied experimentally using laser ablation, we found that modelling the regrowth reproduced a bimodal distribution between regeneration of cut branches and invasion by neighbouring branches. Interestingly, our repair model followed growth rules similar to those for the generation of a new dendritic tree. To generalise the repair algorithm from Drosophila to mammalian neurons, we artificially sectioned reconstructed dendrites from mouse and human hippocampal pyramidal cell morphologies, and showed that the regrown dendrites were morphologically similar to the original ones. Furthermore, we were able to restore their electrophysiological functionality, as evidenced by the recovery of their firing behaviour. Importantly, we show that such repairs also apply to other neuron types including hippocampal granule cells and cerebellar Purkinje cells. We then extrapolated the repair to incomplete human CA1 pyramidal neurons, where the anatomical boundaries of the particular brain areas innervated by the neurons in question were known. Interestingly, the repair of incomplete human dendrites helped to simulate the recently observed increased synaptic thresholds for dendritic NMDA spikes in human versus mouse dendrites. To make the repair tool available to the neuroscience community, we have developed an intuitive and simple graphical user interface (GUI), which is available in the TREES toolbox (www.treestoolbox.org).
Topics: Humans; Mice; Animals; Dendrites; Neurons; Pyramidal Cells; Hippocampus; Drosophila; Mammals
PubMed: 38394339
DOI: 10.1371/journal.pcbi.1011267 -
Cells Feb 2024Internal granular progenitors (IGPs) in the developing cerebellar cortex of ferrets differentiate towards neural and glial lineages. The present study tracked IGPs that...
Internal granular progenitors (IGPs) in the developing cerebellar cortex of ferrets differentiate towards neural and glial lineages. The present study tracked IGPs that proliferated in response to valproic acid (VPA) to determine their fate during cerebellar cortical histogenesis. Ferret kits were used to administer VPA (200 μg/g body weight) on postnatal days 6 and 7. EdU and BrdU were injected on postnatal days 5 and 7, respectively, to label the post-proliferative and proliferating cells when exposed to VPA. At postnatal day 20, when the external granule layer was most expanded, EdU- and BrdU-single-labeled cells were significantly denser in the inner granular layer of VPA-exposed ferrets than in controls. No EdU- or BrdU-labeling was found in Purkinje cells and molecular layer interneurons. Significantly higher percentages of NeuN and Pax6 immunostaining in VPA-exposed ferrets revealed VPA-induced differentiation of IGPs towards granular neurons in BrdU-single-labeled cells. In contrast, both EdU- and BrdU-single-labeled cells exhibited significantly greater percentages of PCNA immunostaining, which appeared in immature Bergman glia, in the internal granular layer of VPA-exposed ferrets. These findings suggest that VPA affects the proliferation of IGPs to induce differentiative division towards granular neurons as well as post-proliferative IGPs toward differentiation into Bergmann glia.
Topics: Humans; Animals; Ferrets; Valproic Acid; Bromodeoxyuridine; Cerebellar Cortex; Purkinje Cells
PubMed: 38391920
DOI: 10.3390/cells13040308 -
Journal of Advanced Veterinary and... Dec 2023Andaliman () is a potent medicinal plant in Asia. This present study aimed to reveal the effectivity of Andaliman fruit extract in alleviating hyperglycemia, sensory and...
OBJECTIVE
Andaliman () is a potent medicinal plant in Asia. This present study aimed to reveal the effectivity of Andaliman fruit extract in alleviating hyperglycemia, sensory and motoric balance disorders, histopathology of the cerebellum, and tissue oxidative stress in diabetic mice induced by alloxan.
MATERIALS AND METHODS
Diabetes induction was performed by intraperitoneally injecting alloxan monohydrate [200 mg/kg body weight (BW)]. Subsequently, the mice were treated daily with an ethanolic extract of Andaliman fruit (0, 150, 300, 450 mg/kg BW per oral) for 28 days, followed by measurements of blood glucose, paw sensitivity, motoric balance, histopathology of the cerebellum, and malondialdehyde (MDA) levels. Moreover, the phytochemical constituents of the extract were elucidated by liquid chromatography.
RESULTS
Higher doses of Andaliman fruit extract could significantly attenuate the elevation of random and fasting blood glucose ( < 0.05) and improve paw sensitivity responses ( < 0.05) and motoric balances ( < 0.05) in diabetic mice. Moreover, Andaliman fruit extract could significantly attenuate the degeneration of cerebellar Purkinje cells ( < 0.05) and suppress MDA levels in the blood ( < 0.05) while blunting the MDA in the brain tissue ( < 0.05). Phytochemical screening revealed 39 compounds in the Andaliman extract belonging to the groups of alkaloids (26 compounds), flavonoids (12 compounds), and terpenoids (1 compound).
CONCLUSION
The ethanolic extract of Andaliman fruit is capable of ameliorating diabetic neuropathy, motor balance disorders, and Purkinje cell degeneration while also reducing oxidative stress in the peripheral system. Hence, Andaliman extract is a promising candidate for formulation as an herbal remedy against the detrimental outcomes of diabetes mellitus.
PubMed: 38370902
DOI: 10.5455/javar.2023.j716 -
Journal of Neuroinflammation Feb 2024Myeloid-derived suppressor cells (MDSCs) constitute a recently discovered bone-marrow-derived cell type useful for dealing with neuroinflammatory disorders. However,...
BACKGROUND
Myeloid-derived suppressor cells (MDSCs) constitute a recently discovered bone-marrow-derived cell type useful for dealing with neuroinflammatory disorders. However, these cells are only formed during inflammatory conditions from immature myeloid cells (IMCs) that acquire immunosuppressive activity, thus being commonly gathered from diseased animals. Then, to obtain a more clinically feasible source, we characterized IMCs directly derived from healthy bone marrow and proved their potential immunosuppressive activity under pathological conditions in vitro. We then explored their neuroprotective potential in a model of human cerebellar ataxia, the Purkinje Cell Degeneration (PCD) mouse, as it displays a well-defined neurodegenerative and neuroinflammatory process that can be also aggravated by invasive surgeries.
METHODS
IMCs were obtained from healthy bone marrow and co-cultured with activated T cells. The proliferation and apoptotic rate of the later were analyzed with Tag-it Violet. For in vivo studies, IMCs were transplanted by stereotactic surgery into the cerebellum of PCD mice. We also used sham-operated animals as controls of the surgical effects, as well as their untreated counterparts. Motor behavior of mice was assessed by rotarod test. The Purkinje cell density was measured by immunohistochemistry and cell death assessed with the TUNEL technique. We also analyzed the microglial phenotype by immunofluorescence and the expression pattern of inflammation-related genes by qPCR. Parametric tests were applied depending on the specific experiment: one or two way ANOVA and Student's T test.
RESULTS
IMCs were proven to effectively acquire immunosuppressive activity under pathological conditions in vitro, thus acting as MDSCs. Concerning in vivo studios, sham-operated PCD mice suffered detrimental effects in motor coordination, Purkinje cell survival and microglial activation. After intracranial administration of IMCs into the cerebellum of PCD mice, no special benefits were detected in the transplanted animals when compared to untreated mice. Nonetheless, this transplant almost completely prevented the impairments caused by the surgery in PCD mice, probably by the modulation of the inflammatory patterns.
CONCLUSIONS
Our work comprise two main translational findings: (1) IMCs can be directly used as they behave as MDSCs under pathological conditions, thus avoiding their gathering from diseased subjects; (2) IMCs are promising adjuvants when performing neurosurgery.
Topics: Mice; Humans; Animals; Myeloid Cells; Cerebellum; Purkinje Cells; Monocytes; Immunosuppressive Agents
PubMed: 38355633
DOI: 10.1186/s12974-023-03000-8 -
BioRxiv : the Preprint Server For... May 2024High-density probes allow electrophysiological recordings from many neurons simultaneously across entire brain circuits but don't reveal cell type. Here, we develop a...
High-density probes allow electrophysiological recordings from many neurons simultaneously across entire brain circuits but don't reveal cell type. Here, we develop a strategy to identify cell types from extracellular recordings in awake animals, revealing the computational roles of neurons with distinct functional, molecular, and anatomical properties. We combine optogenetic activation and pharmacology using the cerebellum as a testbed to generate a curated ground-truth library of electrophysiological properties for Purkinje cells, molecular layer interneurons, Golgi cells, and mossy fibers. We train a semi-supervised deep-learning classifier that predicts cell types with greater than 95% accuracy based on waveform, discharge statistics, and layer of the recorded neuron. The classifier's predictions agree with expert classification on recordings using different probes, in different laboratories, from functionally distinct cerebellar regions, and across animal species. Our classifier extends the power of modern dynamical systems analyses by revealing the unique contributions of simultaneously-recorded cell types during behavior.
PubMed: 38352514
DOI: 10.1101/2024.01.30.577845