3-Acetylpyridine-induced ataxic-like motor impairments are associated with plastic changes in the Purkinje cells of the rat cerebellum.

Ataxias are characterized by aberrant movement patterns closely related to cerebellar dysfunction. Purkinje cell axons are the sole outputs from the cerebellar cortex, and dysfunctional activity of Purkinje cells has been associated with ataxic movements. However, the synaptic characteristics of Purkinje cells in cases of ataxia are not yet well understood. The nicotinamide antagonist 3-acethylpyridine (3-AP) selectively destroys inferior olivary nucleus neurons so it is widely used to induce cerebellar ataxia. Five days after 3-AP treatment (65mg/kg) in adult male Sprague-Dawley rats, motor incoordination was revealed through BBB and Rotarod testing. In addition, in Purkinje cells from lobules V-VII of the cerebellar vermis studied by the Golgi method, the density of dendritic spines decreased, especially the thin and mushroom types. Western blot analysis showed a decrease in AMPA and PSD-95 content with an increase of the α-catenin protein, while GAD-67 and synaptophysin were unchanged. Findings suggest a limited capacity of Purkinje cells to acquire and consolidate afferent excitatory inputs and an aberrant, rigid profile in the movement-related output patterns of Purkinje neurons that likely contributes to the motor-related impairments characteristic of cerebellar ataxias.

Clinical, imaging and histopathological characterization of a series of three cats with cerebellar cortical degeneration.

BACKGROUND: Neurological inherited disorders are rare in domestic animals. Cerebellar cortical degeneration remains amongst the most common of these disorders. The condition is defined as the premature loss of fully differentiated cerebellar components due to genetic or metabolic defects. It has been studied in dogs and cats, and various genetic defects and diagnostic tests (including magnetic resonance imaging (MRI)) have been refined in these species. Cases in cats remain rare and mostly individual, and few diagnostic criteria, other than post-mortem exam, have been evaluated in reports with multiple cases. Here, we report three feline cases of cerebellar cortical degeneration with detailed clinical, diagnostic imaging and post-mortem findings. CASE PRESENTATION: The three cases were directly (siblings, case #1 and #2) or indirectly related (same farm, case #3) and showed early-onset of the disease, with clinical signs including cerebellar ataxia and tremors. Brain MRI was highly suggestive of cerebellar cortical degeneration on all three cases. The relative cerebrospinal fluid (CSF) space, relative cerebellum size, brainstem: cerebellum area ratio, and cerebellum: total brain area ratio, were measured and compared to a control group of cats and reference cut-offs for dogs in the literature. For the relative cerebellum size and cerebellum: total brain area ratio, all affected cases had a lower value than the control group. For the relative CSF space and brainstem: cerebellum area ratio, the more affected cases (#2 and #3) had higher values than the control group, while the least affected case (#3) had values within the ranges of the control group, but a progression was visible over time. Post-mortem examination confirmed the diagnosis of cerebellar cortical degeneration, with marked to complete loss of Purkinje cells and associated granular layer depletion and proliferation of Bergmann glia. One case also had Wallerian-like degeneration in the spinal cord, suggestive of spinocerebellar degeneration. CONCLUSION: Our report further supports a potential genetic component for the disease in cats. For the MRI examination, the relative cerebellum size and cerebellum: total brain area ratio seem promising, but further studies are needed to establish specific feline cut-offs. Post-mortem evaluation of the cerebellum remains the gold standard for the final diagnosis.

Harlequin mice exhibit cognitive impairment, severe loss of Purkinje cells and a compromised bioenergetic status due to the absence of Apoptosis Inducing Factor.

The functional integrity of the central nervous system relies on complex mechanisms in which the mitochondria are crucial actors because of their involvement in a multitude of bioenergetics and biosynthetic pathways. Mitochondrial diseases are among the most prevalent groups of inherited neurological disorders, affecting up to 1 in 5000 adults and despite considerable efforts around the world there is still limited curative treatments. Harlequin mice correspond to a relevant model of recessive X-linked mitochondrial disease due to a proviral insertion in the first intron of the Apoptosis-inducing factor gene, resulting in an almost complete depletion of the corresponding protein. These mice exhibit progressive degeneration of the retina, optic nerve, cerebellum, and cortical regions leading to irremediable blindness and ataxia, reminiscent of what is observed in patients suffering from mitochondrial diseases. We evaluated the progression of cerebellar degeneration in Harlequin mice, especially for Purkinje cells and its relationship with bioenergetics failure and behavioral damage. For the first time to our knowledge, we demonstrated that Harlequin mice display cognitive and emotional impairments at early stage of the disease with further deteriorations as ataxia aggravates. These functions, corresponding to higher-order cognitive processing, have been assigned to a complex network of reciprocal connections between the cerebellum and many cortical areas which could be dysfunctional in these mice. Consequently, Harlequin mice become a suitable experimental model to test innovative therapeutics, via the targeting of mitochondria which can become available to a large spectrum of neurological diseases.

Neuroanatomy of autism: what is the role of the cerebellum?

Autism (or autism spectrum disorder) was initially defined as a psychiatric disorder, with the likely cause maternal behavior (the very destructive "refrigerator mother" theory). It took several decades for research into brain mechanisms to become established. Both neuropathological and imaging studies found differences in the cerebellum in autism spectrum disorder, the most widely documented being a decreased density of Purkinje cells in the cerebellar cortex. The popular interpretation of these results is that cerebellar neuropathology is a critical cause of autism spectrum disorder. We challenge that view by arguing that if fewer Purkinje cells are critical for autism spectrum disorder, then any condition that causes the loss of Purkinje cells should also cause autism spectrum disorder. We will review data on damage to the cerebellum from cerebellar lesions, tumors, and several syndromes (Joubert syndrome, Fragile X, and tuberous sclerosis). Collectively, these studies raise the question of whether the cerebellum really has a role in autism spectrum disorder. Autism spectrum disorder is now recognized as a genetically caused developmental disorder. A better understanding of the genes that underlie the differences in brain development that result in autism spectrum disorder is likely to show that these genes affect the development of the cerebellum in parallel with the development of the structures that do underlie autism spectrum disorder.

The effects of anaesthesia on cell death in a porcine model of neonatal hypoxic-ischaemic brain injury.

Background: Hypothermia is neuroprotective after neonatal hypoxic-ischaemic brain injury. However, systemic cooling to hypothermic temperatures is a stressor and may reduce neuroprotection in awake pigs. We compared two experiments of global hypoxic-ischaemic injury in newborn pigs, in which one group received propofol-remifentanil and the other remained awake during post-insult hypothermia treatment. Methods: In both studies, newborn pigs were anaesthetised using halothane during a 45-min global hypoxic-ischaemic insult induced by reducing Fio2 and graded hypotension until a low-voltage <7 µV electroencephalogram was achieved. On reoxygenation, the pigs were randomly allocated to receive 24 h of normothermia or hypothermia. In the first study (n=18) anaesthesia was discontinued and the pigs' tracheas were extubated. In the second study (n=14) anaesthesia was continued using propofol and remifentanil. Brain injury was assessed after 72 h by classical global histopathology, Purkinje cell count, and apoptotic cell counts in the hippocampus and cerebellum. Results: Global injury was nearly 10-fold greater in the awake group compared with the anaesthetised group (P=0.021). Hypothermia was neuroprotective in the anaesthetised pigs but not the awake pigs. In the hippocampus, the density of cleaved caspase-3-positive cells was increased in awake compared with anaesthetised pigs in normothermia. In the cerebellum, Purkinje cell density was reduced in the awake pigs irrespective of treatment, and the number of cleaved caspase-3-positive Purkinje cells was greatly increased in hypothermic awake pigs. We detected no difference in cleaved caspase-3 in the granular cell layer or microglial reactivity across the groups. Conclusions: Our study provides novel insights into the significance of anaesthesia/sedation during hypothermia for achieving optimal neuroprotection.

A combination of chlorzoxazone and folic acid improves recognition memory, anxiety and depression in SCA3-84Q mice.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is reported to be the most common type of autosomal dominant cerebellar ataxia (ADCA). SCA3 patients suffer from a progressive decline in motor coordination and other disease-associated symptoms. Moreover, recent studies have reported that SCA3 patients also exhibit symptoms of cerebellar cognitive affective syndrome (CCAS). We previously observed signs of CCAS in mouse model of SCA3. Particularly, SCA3-84Q mice suffer from anxiety, recognition memory decline, and also exhibit signs of low mood and aversion to activity. Here we studied the effect of long-term injections of SK channels activator chlorzoxazone (CHZ) together and separately with the folic acid (FA) on the cerebellar Purkinje cell (PC) firing and histology, and also on the motor and cognitive functions as well as mood alterations in SCA3-84Q hemizygous transgenic mice. We realized that both CHZ and CHZ-FA combination had similar positive effect on pure cerebellum impairments including PC firing precision, PC histology, and motor performance in SCA3-84Q mice. However, only the CHZ-FA combination, but not CHZ, had significantly ameliorated the signs of anxiety and depression, and also noticeably improved recognition memory in SCA3-84Q mice. Our results suggest that the combination therapy for both ataxia and non-motor symptoms is required for the complex treatment of ADCA.

Phenotypical, genotypical and pathological characterization of the moonwalker mouse, a model of ataxia.

We performed a comprehensive study of the morphological, functional, and genetic features of moonwalker (MWK) mice, a mouse model of spinocerebellar ataxia caused by a gain of function of the TRPC3 channel. These mice show numerous behavioral symptoms including tremor, altered gait, circling behavior, impaired motor coordination, impaired motor learning and decreased limb strength. Cerebellar pathology is characterized by early and almost complete loss of unipolar brush cells as well as slowly progressive, moderate loss of Purkinje cell (PCs). Structural damage also includes loss of synaptic contacts from parallel fibers, swollen ER structures, and degenerating axons. Interestingly, no obvious correlation was observed between PC loss and severity of the symptoms, as the phenotype stabilizes around 2 months of age, while the cerebellar pathology is progressive. This is probably due to the fact that PC function is severely impaired much earlier than the appearance of PC loss. Indeed, PC firing is already impaired in 3 weeks old mice. An interesting feature of the MWK pathology that still remains to be explained consists in a strong lobule selectivity of the PC loss, which is puzzling considering that TRPC is expressed in every PC. Intriguingly, genetic analysis of MWK cerebella shows, among other alterations, changes in the expression of both apoptosis inducing and resistance factors possibly suggesting that damaged PCs initiate specific cellular pathways that protect them from overt cell loss.

Purkinje cell dysfunction causes disrupted sleep in ataxic mice.

Purkinje cell dysfunction disrupts movement and causes disorders such as ataxia. Recent evidence suggests that Purkinje cell dysfunction may also alter sleep regulation. Here, we used an ataxic mouse model generated by silencing Purkinje cell neurotransmission (L7Cre;Vgatfx/fx) to better understand how cerebellar dysfunction impacts sleep physiology. We focused our analysis on sleep architecture and electrocorticography (ECoG) patterns based on their relevance to extracting physiological measurements during sleep. We found that circadian activity was unaltered in the mutant mice, although their sleep parameters and ECoG patterns were modified. The L7Cre;Vgatfx/fx mutant mice had decreased wakefulness and rapid eye movement (REM) sleep, whereas non-REM sleep was increased. The mutants had an extended latency to REM sleep, which is also observed in human patients with ataxia. Spectral analysis of ECoG signals revealed alterations in the power distribution across different frequency bands defining sleep. Therefore, Purkinje cell dysfunction may influence wakefulness and equilibrium of distinct sleep stages in ataxia. Our findings posit a connection between cerebellar dysfunction and disrupted sleep and underscore the importance of examining cerebellar circuit function in sleep disorders.

Foxp and Skor family proteins control differentiation of Purkinje cells from Ptf1a- and Neurog1-expressing progenitors in zebrafish.

Cerebellar neurons, such as GABAergic Purkinje cells (PCs), interneurons (INs) and glutamatergic granule cells (GCs) are differentiated from neural progenitors expressing proneural genes, including ptf1a, neurog1 and atoh1a/b/c. Studies in mammals previously suggested that these genes determine cerebellar neuron cell fate. However, our studies on ptf1a;neurog1 zebrafish mutants and lineage tracing of ptf1a-expressing progenitors have revealed that the ptf1a/neurog1-expressing progenitors can generate diverse cerebellar neurons, including PCs, INs and a subset of GCs in zebrafish. The precise mechanisms of how each cerebellar neuron type is specified remains elusive. We found that genes encoding the transcriptional regulators Foxp1b, Foxp4, Skor1b and Skor2, which are reportedly expressed in PCs, were absent in ptf1a;neurog1 mutants. foxp1b;foxp4 mutants showed a strong reduction in PCs, whereas skor1b;skor2 mutants completely lacked PCs, and displayed an increase in immature GCs. Misexpression of skor2 in GC progenitors expressing atoh1c suppressed GC fate. These data indicate that Foxp1b/4 and Skor1b/2 function as key transcriptional regulators in the initial step of PC differentiation from ptf1a/neurog1-expressing neural progenitors, and that Skor1b and Skor2 control PC differentiation by suppressing their differentiation into GCs.

The Effect of Nucleo-Olivary Stimulation on Climbing Fiber EPSPs in Purkinje Cells.

Climbing fibers, connecting the inferior olive and Purkinje cells, form the nervous system's strongest neural connection. These fibers activate after critical events like motor errors or anticipation of rewards, leading to bursts of excitatory postsynaptic potentials (EPSPs) in Purkinje cells. The number of EPSPs is a crucial variable when the brain is learning a new motor skill. Yet, we do not know what determines the number of EPSPs. Here, we measured the effect of nucleo-olivary stimulation on periorbital elicited climbing fiber responses through in-vivo intracellular Purkinje cell recordings in decerebrated ferrets. The results show that while nucleo-olivary stimulation decreased the probability of a response occurring at all, it did not reduce the number of EPSPs. The results suggest that nucleo-olivary stimulation does not influence the number of EPSPs in climbing fiber bursts.

Protein kinase Cγ negatively regulates the intrinsic excitability in zebrin-negative cerebellar Purkinje cells.

Protein kinase C γ (PKCγ), a neuronal isoform present exclusively in the central nervous system, is most abundantly expressed in cerebellar Purkinje cells (PCs). Targeted deletion of PKCγ causes a climbing fiber synapse elimination in developing PCs and motor deficit. However, physiological roles of PKCγ in adult mouse PCs are little understood. In this study, we aimed to unravel the roles of PKCγ in mature mouse PCs by deleting PKCγ from adult mouse PCs of PKCγfl/fl mice via cerebellar injection of adeno-associated virus (AAV) vectors expressing Cre recombinase under the control of the PC-specific L7-6 promoter. Whole cell patch-clamp recording of PCs showed higher intrinsic excitability in PCs virally lacking PKCγ [PKCγ-conditional knockout (PKCγ-cKO) PCs] than in wild-type (WT) mouse PCs in the zebrin-negative module, but not in the zebrin-positive module. AAV-mediated PKCγ re-expression in PKCγ-deficient mouse PCs in the zebrin-negative module restored the enhanced intrinsic excitability to a level comparable to that of wild-type mouse PCs. In parallel with higher intrinsic excitability, we found larger hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents in PKCγ-cKO PCs located in the zebrin-negative module, compared with those in WT mouse PCs in the same region. However, pharmacological inhibition of the HCN currents did not restore the enhanced intrinsic excitability in PKCγ-cKO PCs in the zebrin-negative module. These results suggested that PKCγ suppresses the intrinsic excitability in zebrin-negative PCs, which is likely independent of the HCN current inhibition.

Ligustilide inhibits Purkinje cell ferritinophagy via the ULK1/NCOA4 pathway to attenuate valproic acid-induced autistic features.

BACKGROUND: Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder in which social impairment is the core symptom. Presently, there are no definitive medications to cure core symptoms of ASD, and most therapeutic strategies ameliorate ASD symptoms. Treatments with proven efficacy in autism are imminent. Ligustilide (LIG), an herbal monomer extracted from Angelica Sinensis and Chuanxiong, is mainly distributed in the cerebellum and widely used in treating neurological disorders. However, there are no studies on its effect on autistic-like phenotypes and its mechanism of action. PURPOSE: Investigate the efficacy and mechanism of LIG in treating ASD using two Valproic acid(VPA)-exposed and BTBR T + Itpr3tf/J (BTBR) mouse models of autism. METHODS: VPA-exposed mice and BTBR mice were given LIG for treatment, and its effect on autistic-like phenotype was detected by behavioral experiments, which included a three-chamber social test. Subsequently, RNA-Sequence(RNA-Seq) of the cerebellum was performed to observe the biological changes to search target pathways. The autophagy and ferroptosis pathways screened were verified by WB(Western Blot) assay, and the cerebellum was stained by immunofluorescence and examined by electron microscopy. To further explore the therapeutic mechanism, ULK1 agonist BL-918 was used to block the therapeutic effect of LIG to verify its target effect. RESULTS: Our work demonstrates that LIG administration from P12-P14 improved autism-related behaviors and motor dysfunction in VPA-exposed mice. Similarly, BTBR mice showed the same improvement. RNA-Seq data identified ULK1 as the target of LIG in regulating ferritinophagy in the cerebellum of VPA-exposed mice, as evidenced by activated autophagy, increased ferritin degradation, iron overload, and lipid peroxidation. We found that VPA exposure-induced ferritinophagy occurred in the Purkinje cells, with enhanced NCOA4 and Lc3B expressions. Notably, the therapeutic effect of LIG disappeared when ULK1 was activated. CONCLUSION: LIG treatment inhibits ferritinophagy in Purkinje cells via the ULK1/NCOA4-dependent pathway. Our study reveals for the first time that LIG treatment ameliorates autism symptoms in VPA-exposed mice by reducing aberrant Purkinje ferritinophagy. At the same time, our study complements the pathogenic mechanisms of autism and introduces new possibilities for its therapeutic options.

Activating Lobule VI PCTH+-Med Pathway in Cerebellum Blocks the Acquisition of Methamphetamine Conditioned Place Preference in Mice.

Cerebellum has been implicated in drug addiction; however, its underlying cellular populations and neuronal circuitry remain largely unknown. In the current study, we identified a neural pathway from tyrosine hydroxylase (TH)-positive Purkinje cells (PCTH+) in cerebellar lobule VI to calcium/calmodulin-dependent protein kinase II (CaMKII)-positive glutamatergic neurons in the medial cerebellar nucleus (MedCaMKII), forming the lobule VI PCTH+-MedCaMKII pathway in male mice. In naive male mice, inhibition of PCTH+ neurons activated Med neurons. During conditioned place preference (CPP) training, exposure to methamphetamine (METH) inhibited lobule VI PCTH+ neurons while excited MedCaMKII neurons in mice. Silencing MedCaMKII using a tetanus toxin light chain (tettox) suppressed the acquisition of METH CPP in mice but resulted in motor coordination deficits in naive mice. In contrast, activating lobule VI PCTH+ terminals within Med inhibited the activity of Med neurons and subsequently blocked the acquisition of METH CPP in mice without affecting motor coordination, locomotor activity, and sucrose reinforcements in naive mice. Our findings identified a novel lobule VI PCTH+-MedCaMKII pathway within the cerebellum and explored its role in mediating the acquisition of METH-preferred behaviors.

Innovative in vivo rat model for global cerebral hypoxia: a new approach to investigate therapeutic and preventive drugs.

Introduction: 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. Methods: 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. Results: 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, Il1ß, Tgf1ß, Tnfα, S100b, cspg2, neuron-specific enolase), hippocampus (Il1ß, Tnfα, S100b, cspg2, NSE), and cerebellum (Hif1α, Tnfα, S100b, cspg2, NSE). Effects were more pronounced in females than in males. Discussion: 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.

Investigating the role of Purkinje fibers and synaptic connectivity in balance regulation through comprehensive ultrastructural and immunohistochemical analysis of the donkey's (Equus asinus) cerebellum.

The donkey's extraordinary capacity to endure substantial loads over long distances while maintaining equilibrium suggests a distinctive cerebellar architecture specialized in balance regulation. Consequently, our study aims to investigate the intricate histophysiology of the donkey's cerebellum using advanced ultrastructural and immunohistochemical methodologies to comprehend the mechanisms that govern this exceptional ability. This study represents the pioneering investigation to comprehensively describe the ultrastructure and immunohistochemistry within the donkey cerebellum. Five adult donkeys' cerebella were utilized for the study, employing stains such as hematoxylin, eosin, and toluidine blue to facilitate a comprehensive histological examination. For immunohistochemical investigation, synaptophysin (SP), calretinin, and glial fibrillary acidic protein were used and evaluated by the Image J software. Furthermore, a double immunofluorescence staining of SP and neuron-specific enolase (NSE) was performed to highlight the co-localization of these markers and explore their potential contribution to synaptic function within the donkey cerebellum. This investigation aims to understand their possible roles in regulating neuronal activity and synaptic connectivity. We observed co-expression of SP and NSE in the donkey cerebellum, which emphasizes the crucial role of efficient energy utilization for motor coordination and balance, highlighting the interdependence of synaptic function and energy metabolism. The Purkinje cells were situated in the intermediate zone of the cerebellum cortex, known as the Purkinje cell layer. Characteristically, the Purkinje cell's bodies exhibited a distinct pear-like shape. The cross-section area of the Purkinje cells was 107.7 ± 0.2 µm2 , and the Purkinje cell nucleus was 95.7 ± 0.1 µm2 . The length and diameter of the Purkinje cells were 36.4 × 23.4 µm. By scanning electron microscopy, the body of the Purkinje cell looked like a triangular or oval with a meandrous outer surface. The dendrites appeared to have small spines. The Purkinje cells' cytoplasm was rich with mitochondria, rough endoplasmic reticulum, ribosomes, Golgi apparatus, multivesicular bodies, and lysosomes. Purkinje cell dendrites were discovered in the molecular layer, resembling trees. This study sheds light on the anatomical and cellular characteristics underlying the donkey's exceptional balance-maintaining abilities.

Knockdown of the Non-canonical Wnt Gene Prickle2 Leads to Cerebellar Purkinje Cell Abnormalities While Cerebellar-Mediated Behaviors Remain Intact.

Autism spectrum disorders (ASD) involve brain wide abnormalities that contribute to a constellation of symptoms including behavioral inflexibility, cognitive dysfunction, learning impairments, altered social interactions, and perceptive time difficulties. Although a single genetic variation does not cause ASD, genetic variations such as one involving a non-canonical Wnt signaling gene, Prickle2, has been found in individuals with ASD. Previous work looking into phenotypes of Prickle2 knock-out (Prickle2-/-) and heterozygous mice (Prickle2-/+) suggest patterns of behavior similar to individuals with ASD including altered social interaction and behavioral inflexibility. Growing evidence implicates the cerebellum in ASD. As Prickle2 is expressed in the cerebellum, this animal model presents a unique opportunity to investigate the cerebellar contribution to autism-like phenotypes. Here, we explore cerebellar structural and physiological abnormalities in animals with Prickle2 knockdown using immunohistochemistry, whole-cell patch clamp electrophysiology, and several cerebellar-associated motor and timing tasks, including interval timing and eyeblink conditioning. Histologically, Prickle2-/- mice have significantly more empty spaces or gaps between Purkinje cells in the posterior lobules and a decreased propensity for Purkinje cells to fire action potentials. These structural cerebellar abnormalities did not impair cerebellar-associated behaviors as eyeblink conditioning and interval timing remained intact. Therefore, although Prickle-/- mice show classic phenotypes of ASD, they do not recapitulate the involvement of the adult cerebellum and may not represent the pathophysiological heterogeneity of the disorder.

Human cerebellar organoids with functional Purkinje cells.

Research on human cerebellar development and disease has been hampered by the need for a human cell-based system that recapitulates the human cerebellum's cellular diversity and functional features. Here, we report a human organoid model (human cerebellar organoids [hCerOs]) capable of developing the complex cellular diversity of the fetal cerebellum, including a human-specific rhombic lip progenitor population that have never been generated in vitro prior to this study. 2-month-old hCerOs form distinct cytoarchitectural features, including laminar organized layering, and create functional connections between inhibitory and excitatory neurons that display coordinated network activity. Long-term culture of hCerOs allows healthy survival and maturation of Purkinje cells that display molecular and electrophysiological hallmarks of their in vivo counterparts, addressing a long-standing challenge in the field. This study therefore provides a physiologically relevant, all-human model system to elucidate the cell-type-specific mechanisms governing cerebellar development and disease.

Aging, Neurodegenerative Disorders, and Cerebellum.

An important part of the central nervous system (CNS), the cerebellum is involved in motor control, learning, reflex adaptation, and cognition. Diminished cerebellar function results in the motor and cognitive impairment observed in patients with neurodegenerative disorders such as Alzheimer's disease (AD), vascular dementia (VD), Parkinson's disease (PD), Huntington's disease (HD), spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Friedreich's ataxia (FRDA), and multiple sclerosis (MS), and even during the normal aging process. In most neurodegenerative disorders, impairment mainly occurs as a result of morphological changes over time, although during the early stages of some disorders such as AD, the cerebellum also serves a compensatory function. Biological aging is accompanied by changes in cerebellar circuits, which are predominantly involved in motor control. Despite decades of research, the functional contributions of the cerebellum and the underlying molecular mechanisms in aging and neurodegenerative disorders remain largely unknown. Therefore, this review will highlight the molecular and cellular events in the cerebellum that are disrupted during the process of aging and the development of neurodegenerative disorders. We believe that deeper insights into the pathophysiological mechanisms of the cerebellum during aging and the development of neurodegenerative disorders will be essential for the design of new effective strategies for neuroprotection and the alleviation of some neurodegenerative disorders.

Ucp4 Knockdown of Cerebellar Purkinje Cells Induces Bradykinesia.

Although uncoupling protein 4 (UCP4) is the most abundant protein reported in the brain, the biological function of UCP4 in cerebellum and pathological outcome of UCP4 deficiency in cerebellum remain obscure. To evaluate the role of Ucp4 in the cerebellar Purkinje cells (PCs), we generated the conditional knockdown of Ucp4 in PCs (Pcp2cre;Ucp4fl/fl mice) by breeding Ucp4fl/fl mice with Pcp2cre mice. Series results by Western blot, immunofluorescent staining, and triple RNAscope in situ hybridization confirmed the specific ablation of Ucp4 in PCs in Pcp2cre;Ucp4fl/fl mice, but did not affect the expression of Ucp2, the analog of Ucp4. Combined behavioral tests showed that Pcp2cre;Ucp4fl/fl mice displayed a characteristic bradykinesia in the spontaneous movements. The electromyogram recordings detection excluded the possibility of hypotonia in Pcp2cre;Ucp4fl/fl mice. And the electrical patch clamp recordings showed the altered properties of PCs in Pcp2cre;Ucp4fl/fl mice. Moreover, transmission electron microscope (TEM) results showed the increased mitochondrial circularity in PCs; ROS probe imaging showed the increased ROS generation in molecular layer; and finally, microplate reader assay showed the significant changes of mitochondrial functions, including ROS, ATP, and MMP in the isolated cerebellum tissue. The results suggested that the specific knockdown of mitochondrial protein Ucp4 could damage PCs possibly by attacking their mitochondrial function. The present study is the first to report a close relationship between UCP4 deletion with PCs impairment, and suggests the importance of UCP4 in the substantial support of mitochondrial function homeostasis in bradykinesia. UCP4 might be a therapeutic target for the cerebellar-related movement disorder.

Cerebellar contribution to autism-relevant behaviors in fragile X syndrome models.

Cerebellar dysfunction has been linked to autism spectrum disorders (ASDs). Although cerebellar pathology has been observed in individuals with fragile X syndrome (FXS) and in mouse models of the disorder, a cerebellar functional contribution to ASD-relevant behaviors in FXS has yet to be fully characterized. In this study, we demonstrate a critical cerebellar role for Fmr1 (fragile X messenger ribonucleoprotein 1) in ASD-relevant behaviors. First, we identify reduced social behaviors, sensory hypersensitivity, and cerebellar dysfunction, with loss of cerebellar Fmr1. We then demonstrate that cerebellar-specific expression of Fmr1 is sufficient to impact social, sensory, cerebellar dysfunction, and cerebro-cortical hyperexcitability phenotypes observed in global Fmr1 mutants. Moreover, we demonstrate that targeting the ASD-implicated cerebellar region Crus1 ameliorates behaviors in both cerebellar-specific and global Fmr1 mutants. Together, these results demonstrate a critical role for the cerebellar contribution to FXS-related behaviors, with implications for future therapeutic strategies.