Numb positively regulates Hedgehog signaling at the ciliary pocket.

Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.

Insights into the genetic architecture of cerebellar lobules derived from the UK Biobank.

In this work we endeavor to further understand the genetic architecture of the cerebellum by examining the genetic underpinnings of the different cerebellar lob(ul)es, identifying their genetic relation to cortical and subcortical regions, as well as to psychiatric disorders, as well as traces of their evolutionary trajectories. We confirm the moderate heritability of cerebellar volumes, and reveal genetic clustering and variability across their different substructures, which warranted a detailed analysis using this higher structural resolution. We replicated known genetic correlations with several subcortical volumes, and report new cortico-cerebellar genetic correlations, including negative genetic correlations between anterior cerebellar lobules and cingulate, and positive ones between lateral Crus I and lobule VI with cortical measures in the fusiform region. Heritability partitioning for evolutionary annotations highlighted that the vermis of Crus II has depleted heritability in genomic regions of "archaic introgression deserts", but no enrichment/depletion of heritability in any other cerebellar regions. Taken together, these findings reveal novel insights into the genetic underpinnings of the different cerebellar lobules.

Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion.

The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients.

Cerebellum Purkinje cell vulnerability in aged rats with memory impairment.

The cerebellum is involved in higher order cognitive function and is susceptible to age-related atrophy. However, limited evidence has directly examined the cerebellum's role in cognitive aging. To interrogate potential substrates of the relationship between cerebellar structure and memory in aging, here we target the Purkinje cells (PCs). The sole output neurons of the cerebellum, PC loss and/or degeneration underlie a variety of behavioral abnormalities. Using a rat model of normal cognitive aging, we immunostained sections through the cerebellum for the PC-specific protein, calbindin-D28k. Although morphometric quantification revealed no significant difference in total PC number as a function of age or cognitive status, regional cell number was a more robust correlate of memory performance in the young cerebellum than in aged animals. Parallel biochemical analysis of PC-specific protein levels in whole cerebellum additionally revealed that calbindin-D28k and Purkinje cell protein-2 (pcp-2) levels were lower selectively in aged rats with spatial memory impairment compared to both young animals and aged rats with intact memory. These results suggest that cognitive aging is associated with cerebellum vulnerability, potentially reflecting disruption of the cerebellum-medial temporal lobe network.

Neuroprotective Effect of Sterculia setigera Leaves Hydroethanolic Extract.

Plants are a valuable source of information for pharmacological research and new drug discovery. The present study aimed to evaluate the neuroprotective potential of the leaves of the medicinal plant Sterculia setigera. In vitro, the effect of Sterculia setigera leaves dry hydroethanolic extract (SSE) was tested on cultured cerebellar granule neurons (CGN) survival when exposed to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA), using the viability probe fluorescein diacetate (FDA), a lactate dehydrogenase (LDH) activity assay, an immunocytochemical staining against Gap 43, and the quantification of the expression of genes involved in apoptosis, necrosis, or oxidative stress. In vivo, the effect of intraperitoneal (ip) injection of SSE was assessed on the developing brain of 8-day-old Wistar rats exposed to ethanol neurotoxicity by measuring caspase-3 activity on cerebellum homogenates, the expression of some genes in tissue extracts, the thickness of cerebellar cortical layers and motor coordination. In vitro, SSE protected CGN against H2O2 and 6-OHDA-induced cell death at a dose of 10 µg/mL, inhibited the expression of genes Casp3 and Bad, and upregulated the expression of Cat and Gpx7. In vivo, SSE significantly blocked the deleterious effect of ethanol by reducing the activity of caspase-3, inhibiting the expression of Bax and Tp53, preventing the reduction of the thickness of the internal granule cell layer of the cerebellar cortex, and restoring motor functions. Sterculia setigera exerts neuroactive functions as claimed by traditional medicine and should be a good candidate for the development of a neuroprotective treatment against neurodegenerative diseases.

Folding of the cerebellar cortex is clade-specific in form but universal in degree.

Like the cerebralcortex, the surface of the cerebellum is repeatedly folded. Unlike the cerebralcortex, however, cerebellar folds are much thinner and more numerous; repeatthemselves largely along a single direction, forming accordion-like folds transverseto the mid-sagittal plane; and occur in all but the smallest cerebella. We haveshown previously that while the location of folds in mammalian cerebral cortex isclade-specific, the overall degree of folding strictly follows a universalpower law relating cortical thickness and the exposed and total surface areas predictedfrom the minimization of the effective free energy of an expanding, self-avoidingsurface of a certain thickness. Here we show that this scaling law extends tothe folding of the mid-sagittal sections of the cerebellum of 53 speciesbelonging to six mammalian clades. Simultaneously, we show that each clade hasa previously unsuspected distinctive spatial pattern of folding evident at themid-sagittal surface of the cerebellum. We note, however, that the mammaliancerebellum folds as a multi-fractal object, because of the difference betweenthe outside-in development of the cerebellar cortex around a preexisting coreof already connected white matter, compared to the inside-out development ofthe cerebral cortex with a white matter volume that develops as the cerebralcortex itself gains neurons. We conclude that repeated folding, one of the mostrecognizable features of biology, can arise simply from the interplay betweenthe universal applicability of the physics of self-organization and biological,phylogenetical clade-specific contingency, without the need for invokingselective pressures in evolution.

ADNP dysregulates methylation and mitochondrial gene expression in the cerebellum of a Helsmoortel-Van der Aa syndrome autopsy case.

BACKGROUND: Helsmoortel-Van der Aa syndrome is a neurodevelopmental disorder in which patients present with autism, intellectual disability, and frequent extra-neurological features such as feeding and gastrointestinal problems, visual impairments, and cardiac abnormalities. All patients exhibit heterozygous de novo nonsense or frameshift stop mutations in the Activity-Dependent Neuroprotective Protein (ADNP) gene, accounting for a prevalence of 0.2% of all autism cases worldwide. ADNP fulfills an essential chromatin remodeling function during brain development. In this study, we investigated the cerebellum of a died 6-year-old male patient with the c.1676dupA/p.His559Glnfs*3 ADNP mutation. RESULTS: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain. CONCLUSION: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target.

Re-examining the Mysterious Role of the Cerebellum in Pain.

Pain is considered a multidimensional experience that embodies not merely sensation, but also emotion and perception. As is appropriate for this complexity, pain is represented and processed by an extensive matrix of cortical and subcortical structures. Of these structures, the cerebellum is gaining increasing attention. Although association between the cerebellum and both acute and chronic pain have been extensively detailed in electrophysiological and neuroimaging studies, a deep understanding of what functions are mediated by these associations is lacking. Nevertheless, the available evidence implies that lobules IV-VI and Crus I are especially pertinent to pain processing, and anatomical studies reveal that these regions connect with higher-order structures of sensorimotor, emotional, and cognitive function. Therefore, we speculate that the cerebellum exerts a modulatory role in pain via its communication with sites of sensorimotor, executive, reward, and limbic function. On this basis, in this review, we propose numerous ways in which the cerebellum might contribute to both acute and chronic pain, drawing particular attention to emotional and cognitive elements of pain. In addition, we emphasise the importance of advancing our knowledge about the relationship between the cerebellum and pain by discussing novel therapeutic opportunities that capitalize on this association.

The olivary input to the cerebellum dissociates sensory events from movement plans.

Neurons in the inferior olive are thought to anatomically organize the Purkinje cells (P-cells) of the cerebellum into computational modules, but what is computed by each module? Here, we designed a saccade task in marmosets that dissociated sensory events from motor events and then recorded the complex and simple spikes of hundreds of P-cells. We found that when a visual target was presented at a random location, the olive reported the direction of that sensory event to one group of P-cells, but not to a second group. However, just before movement onset, it reported the direction of the planned movement to both groups, even if that movement was not toward the target. At the end of the movement if the subject experienced an error but chose to withhold the corrective movement, only the first group received information about the sensory prediction error. We organized the P-cells based on the information content of their olivary input and found that in the group that received sensory information, the simple spikes were suppressed during fixation, then produced a burst before saccade onset in a direction consistent with assisting the movement. In the second group, the simple spikes were not suppressed during fixation but burst near saccade deceleration in a direction consistent with stopping the movement. Thus, the olive differentiated the P-cells based on whether they would receive sensory or motor information, and this defined their contributions to control of movements as well as holding still.

Resection of the quadrangular lobule of the cerebellum to increase exposure of the cerebellomesencephalic fissure: an anatomical study with clinical correlation.

OBJECTIVE: The lateral aspect of the cerebellomesencephalic fissure frequently harbors vascular pathology and is a common surgical corridor used to access the pons tegmentum, as well as the cerebellum and its superior and middle peduncles. The quadrangular lobule of the cerebellum (QLC) represents an obstacle to reach these structures. The authors sought to analyze and compare exposure of the cerebellar interpeduncular region (CIPR) before and after QLC resection and provide a case series to evaluate its clinical applicability. METHODS: Forty-two sides of human brainstems were prepared with Klingler's method and dissected. The exposure area before and after resection of the QLC was measured and statistically studied. A case series of 59 patients who underwent QLC resection for the treatment of CIPR lesions was presented and clinical outcomes were evaluated at 1-year follow-up. RESULTS: The anteroposterior surgical corridor of the CIPR increased by 10.3 mm after resection of the QLC. The mean exposure areas were 42 mm2 before resection of the QLC and 159.6 mm2 after resection. In this series, ataxia, extrapyramidal syndrome, and akinetic mutism were found after surgery. However, all these cases resolved within 1 year of follow-up. Modified Rankin Scale score improved by 1 grade, on average. CONCLUSIONS: QLC resection significantly increased the exposure area, mainly in the anteroposterior axis. This surgical strategy appears to be safe and may help the neurosurgeon when operating on the lateral aspect of the cerebellomesencephalic fissure.

Behaviour-correlated profiles of cerebellar-cerebral functional connectivity observed in independent neurodevelopmental disorder cohorts.

The cerebellum, through its connectivity with the cerebral cortex, plays an integral role in regulating cognitive and affective processes, and its dysregulation can result in neurodevelopmental disorder (NDD)-related behavioural deficits. Identifying cerebellar-cerebral functional connectivity (FC) profiles in children with NDDs can provide insight into common connectivity profiles and their correlation to NDD-related behaviours. 479 participants from the Province of Ontario Neurodevelopmental Disorders (POND) network (typically developing = 93, Autism Spectrum Disorder = 172, Attention Deficit/Hyperactivity Disorder = 161, Obsessive-Compulsive Disorder = 53, mean age = 12.2) underwent resting-state functional magnetic resonance imaging and behaviour testing (Social Communication Questionnaire, Toronto Obsessive-Compulsive Scale, and Child Behaviour Checklist - Attentional Problems Subscale). FC components maximally correlated to behaviour were identified using canonical correlation analysis. Results were then validated by repeating the investigation in 556 participants from an independent NDD cohort provided from a separate consortium (Healthy Brain Network (HBN)). Replication of canonical components was quantified by correlating the feature vectors between the two cohorts. The two cerebellar-cerebral FC components that replicated to the greatest extent were correlated to, respectively, obsessive-compulsive behaviour (behaviour feature vectors, rPOND-HBN = -0.97; FC feature vectors, rPOND-HBN = -0.68) and social communication deficit contrasted against attention deficit behaviour (behaviour feature vectors, rPOND-HBN = -0.99; FC feature vectors, rPOND-HBN = -0.78). The statistically stable (|z| > 1.96) features of the FC feature vectors, measured via bootstrap re-sampling, predominantly comprised of correlations between cerebellar attentional and control network regions and cerebral attentional, default mode, and control network regions. In both cohorts, spectral clustering on FC loading values resulted in subject clusters mixed across diagnostic categories, but no cluster was significantly enriched for any given diagnosis as measured via chi-squared test (p > 0.05). Overall, two behaviour-correlated components of cerebellar-cerebral functional connectivity were observed in two independent cohorts. This suggests the existence of generalizable cerebellar network differences that span across NDD diagnostic boundaries.

Behind the wheel: exploring gray matter variations in experienced drivers.

Background: Driving is a complex skill involving various cognitive activities. Previous research has explored differences in the brain structures related to the navigational abilities of drivers compared to non-drivers. However, it remains unclear whether changes occur in the structures associated with low-level sensory and higher-order cognitive abilities in drivers. Methods: Gray matter volume, assessed via voxel-based morphometry analysis of T1-weighted images, is considered a reliable indicator of structural changes in the brain. This study employs voxel-based morphological analysis to investigate structural differences between drivers (n = 22) and non-drivers (n = 20). Results: The results indicate that, in comparison to non-drivers, drivers exhibit significantly reduced gray matter volume in the middle occipital gyrus, middle temporal gyrus, supramarginal gyrus, and cerebellum, suggesting a relationship with driving-related experience. Furthermore, the volume of the middle occipital gyrus, and middle temporal gyrus, is found to be marginally negative related to the years of driving experience, suggesting a potential impact of driving experience on gray matter volume. However, no significant correlations were observed between driving experiences and frontal gray matter volume. Conclusion: These findings suggest that driving skills and experience have a pronounced impact on the cortical areas responsible for low-level sensory and motor processing. Meanwhile, the influence on cortical areas associated with higher-order cognitive function appears to be minimal.

Cortico-cerebellar coordination facilitates neuroprosthetic control.

Temporally coordinated neural activity is central to nervous system function and purposeful behavior. Still, there is a paucity of evidence demonstrating how this coordinated activity within cortical and subcortical regions governs behavior. We investigated this between the primary motor (M1) and contralateral cerebellar cortex as rats learned a neuroprosthetic/brain-machine interface (BMI) task. In neuroprosthetic task, actuator movements are causally linked to M1 "direct" neurons that drive the decoder for successful task execution. However, it is unknown how task-related M1 activity interacts with the cerebellum. We observed a notable 3 to 6 hertz coherence that emerged between these regions' local field potentials (LFPs) with learning that also modulated task-related spiking. We identified robust task-related indirect modulation in the cerebellum, which developed a preferential relationship with M1 task-related activity. Inhibiting cerebellar cortical and deep nuclei activity through optogenetics led to performance impairments in M1-driven neuroprosthetic control. Together, these results demonstrate that cerebellar influence is necessary for M1-driven neuroprosthetic control.

Plastic vasomotion entrainment.

The presence of global synchronization of vasomotion induced by oscillating visual stimuli was identified in the mouse brain. Endogenous autofluorescence was used and the vessel 'shadow' was quantified to evaluate the magnitude of the frequency-locked vasomotion. This method allows vasomotion to be easily quantified in non-transgenic wild-type mice using either the wide-field macro-zoom microscopy or the deep-brain fiber photometry methods. Vertical stripes horizontally oscillating at a low temporal frequency (0.25 Hz) were presented to the awake mouse, and oscillatory vasomotion locked to the temporal frequency of the visual stimulation was induced not only in the primary visual cortex but across a wide surface area of the cortex and the cerebellum. The visually induced vasomotion adapted to a wide range of stimulation parameters. Repeated trials of the visual stimulus presentations resulted in the plastic entrainment of vasomotion. Horizontally oscillating visual stimulus is known to induce horizontal optokinetic response (HOKR). The amplitude of the eye movement is known to increase with repeated training sessions, and the flocculus region of the cerebellum is known to be essential for this learning to occur. Here, we show a strong correlation between the average HOKR performance gain and the vasomotion entrainment magnitude in the cerebellar flocculus. Therefore, the plasticity of vasomotion and neuronal circuits appeared to occur in parallel. Efficient energy delivery by the entrained vasomotion may contribute to meeting the energy demand for increased coordinated neuronal activity and the subsequent neuronal circuit reorganization.

Cortical-cerebellar circuits changes in preschool ASD children by multimodal MRI.

OBJECTIVE: To investigate the alterations in cortical-cerebellar circuits and assess their diagnostic potential in preschool children with autism spectrum disorder using multimodal magnetic resonance imaging. METHODS: We utilized diffusion basis spectrum imaging approaches, namely DBSI_20 and DBSI_combine, alongside 3D structural imaging to examine 31 autism spectrum disorder diagnosed patients and 30 healthy controls. The participants' brains were segmented into 120 anatomical regions for this analysis, and a multimodal strategy was adopted to assess the brain networks using a multi-kernel support vector machine for classification. RESULTS: The results revealed consensus connections in the cortical-cerebellar and subcortical-cerebellar circuits, notably in the thalamus and basal ganglia. These connections were predominantly positive in the frontoparietal and subcortical pathways, whereas negative consensus connections were mainly observed in frontotemporal and subcortical pathways. Among the models tested, DBSI_20 showed the highest accuracy rate of 86.88%. In addition, further analysis indicated that combining the 3 models resulted in the most effective performance. CONCLUSION: The connectivity network analysis of the multimodal brain data identified significant abnormalities in the cortical-cerebellar circuits in autism spectrum disorder patients. The DBSI_20 model not only provided the highest accuracy but also demonstrated efficiency, suggesting its potential for clinical application in autism spectrum disorder diagnosis.

The role of cerebellum in learned vocal communication in adult songbirds.

Injury, tumors, ischemia, and lesions in the cerebellum show the involvement of this region in human speech. The association of the cerebellum with learned birdsong has only been identified recently. Cerebellar dysfunction in young songbirds causes learning disabilities, but its role in adult songbirds has not been established. The aim of this study was to investigate the role of the deep cerebellar nuclei (DCN) in adult birdsong. We created bilateral excitotoxic lesions in the DCN of adult male zebra finches (Taeniopygia guttata) and recorded their songs for up to 4 months. Using magnetic resonance imaging (MRI) and immunohistochemistry, we validated the lesion efficacy. We found that the song duration significantly increased from 14 weeks post-op; the increase in duration was caused by a greater number of introductory notes as well as a greater number of syllables sung after the introductory notes. On the other hand, the motif duration decreased from 8 weeks after DCN lesions were induced, which was due to faster singing of syllables, not changes in inter-syllable interval length. DCN lesions also caused a decrease in the fundamental frequency of syllables. In summary, we showed that DCN lesions influence the temporal and acoustic features of birdsong. These results suggest that the cerebellum influences singing in adult songbirds.

Ruptured aneurysm in collateral arteries with blood-flow impairment of posterior inferior cerebellar artery: a report of three cases.

Herein, we report three cases of cerebellar hemorrhage due to a ruptured small aneurysm located on a collateral artery compensating for one or more stenotic or occluded major cerebellar arteries. In each case, endovascular distant parent artery occlusion of both the collateral artery and aneurysm was performed to prevent rebleeding. A ruptured small aneurysm in a collateral artery may be observed in patients with hemorrhage in an atypical cerebellar region, especially in cases of stenosis or occlusion of the vertebral artery or posterior inferior cerebellar artery. Thus, cerebral angiography is recommended to rule out collateral artery aneurysm.

Large-scale cranial window for in vivo mouse brain imaging utilizing fluoropolymer nanosheet and light-curable resin.

Two-photon microscopy enables in vivo imaging of neuronal activity in mammalian brains at high resolution. However, two-photon imaging tools for stable, long-term, and simultaneous study of multiple brain regions in same mice are lacking. Here, we propose a method to create large cranial windows covering such as the whole parietal cortex and cerebellum in mice using fluoropolymer nanosheets covered with light-curable resin (termed the 'Nanosheet Incorporated into light-curable REsin' or NIRE method). NIRE method can produce cranial windows conforming the curved cortical and cerebellar surfaces, without motion artifacts in awake mice, and maintain transparency for >5 months. In addition, we demonstrate that NIRE method can be used for in vivo two-photon imaging of neuronal ensembles, individual neurons and subcellular structures such as dendritic spines. The NIRE method can facilitate in vivo large-scale analysis of heretofore inaccessible neural processes, such as the neuroplastic changes associated with maturation, learning and neural pathogenesis.