Pumilio1 haploinsufficiency leads to SCA1-like neurodegeneration by increasing wild-type Ataxin1 levels.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1(+/-) mice to SCA1 mice (Atxn1(154Q/+)) exacerbated disease progression, whereas breeding them to Atxn1(+/-) mice normalized Ataxin1 levels and largely rescued the Pum1(+/-) phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.

Whisker kinematics in the cerebellum.

The whisker system is widely used as a model system for understanding sensorimotor integration. Purkinje cells in the crus regions of the cerebellum have been reported to linearly encode whisker midpoint, but it is unknown whether the paramedian and simplex lobules as well as their target neurons in the cerebellar nuclei also encode whisker kinematics and if so which ones. Elucidating how these kinematics are represented throughout the cerebellar hemisphere is essential for understanding how the cerebellum coordinates multiple sensorimotor modalities. Exploring the cerebellar hemisphere of mice using optogenetic stimulation, we found that whisker movements can be elicited by stimulation of Purkinje cells in not only crus1 and crus2, but also in the paramedian lobule and lobule simplex; activation of cells in the medial paramedian lobule had on average the shortest latency, whereas that of cells in lobule simplex elicited similar kinematics as those in crus1 and crus2. During spontaneous whisking behaviour, simple spike activity correlated in general better with velocity than position of the whiskers, but it varied between protraction and retraction as well as per lobule. The cerebellar nuclei neurons targeted by the Purkinje cells showed similar activity patterns characterized by a wide variety of kinematic signals, yet with a dominance for velocity. Taken together, our data indicate that whisker movements are much more prominently and diversely represented in the cerebellar cortex and nuclei than assumed, highlighting the rich repertoire of cerebellar control in the kinematics of movements that can be engaged during coordination. KEY POINTS: Excitation of Purkinje cells throughout the cerebellar hemispheres induces whisker movement, with the shortest latency and longest duration within the paramedian lobe. Purkinje cells have differential encoding for the fast and slow components of whisking. Purkinje cells encode not only the position but also the velocity of whiskers. Purkinje cells with high sensitivity for whisker velocity are preferentially located in the medial part of lobule simplex, crus1 and lateral paramedian. In the downstream cerebellar nuclei, neurons with high sensitivity for whisker velocity are located at the intersection between the medial and interposed nucleus.

Pre-ataxic loss of intrinsic plasticity and motor learning in a mouse model of SCA1.

Spinocerebellar ataxias are neurodegenerative diseases, the hallmark symptom of which is the development of ataxia due to cerebellar dysfunction. Purkinje cells, the principal neurons of the cerebellar cortex, are the main cells affected in these disorders, but the sequence of pathological events leading to their dysfunction is poorly understood. Understanding the origins of Purkinje cells dysfunction before it manifests is imperative to interpret the functional and behavioural consequences of cerebellar-related disorders, providing an optimal timeline for therapeutic interventions. Here, we report the cascade of events leading to Purkinje cells dysfunction before the onset of ataxia in a mouse model of spinocerebellar ataxia 1 (SCA1). Spatiotemporal characterization of the ATXN1[82Q] SCA1 mouse model revealed high levels of the mutant ATXN1[82Q] weeks before the onset of ataxia. The expression of the toxic protein first caused a reduction of Purkinje cells intrinsic excitability, which was followed by atrophy of Purkinje cells dendrite arborization and aberrant glutamatergic signalling, finally leading to disruption of Purkinje cells innervation of climbing fibres and loss of intrinsic plasticity of Purkinje cells. Functionally, we found that deficits in eyeblink conditioning, a form of cerebellum-dependent motor learning, precede the onset of ataxia, matching the timeline of climbing fibre degeneration and reduced intrinsic plasticity. Together, our results suggest that abnormal synaptic signalling and intrinsic plasticity during the pre-ataxia stage of spinocerebellar ataxias underlie an aberrant cerebellar circuitry that anticipates the full extent of the disease severity. Furthermore, our work indicates the potential for eyeblink conditioning to be used as a sensitive tool to detect early cerebellar dysfunction as a sign of future disease.

Sphingolipid metabolism governs Purkinje cell patterned degeneration in Atxn1[82Q]/+ mice.

Patterned degeneration of Purkinje cells (PCs) can be observed in a wide range of neuropathologies, but mechanisms behind nonrandom cerebellar neurodegeneration remain unclear. Sphingolipid metabolism dyshomeostasis typically leads to PC neurodegeneration; hence, we questioned whether local sphingolipid balance underlies regional sensitivity to pathological insults. Here, we investigated the regional compartmentalization of sphingolipids and their related enzymes in the cerebellar cortex in healthy and pathological conditions. Analysis in wild-type animals revealed higher sphingosine kinase 1 (Sphk1) levels in the flocculonodular cerebellum, while sphingosine-1-phosphate (S1P) levels were higher in the anterior cerebellum. Next, we investigated a model for spinocerebellar ataxia type 1 (SCA1) driven by the transgenic expression of the expanded Ataxin 1 protein with 82 glutamine (82Q), exhibiting severe PC degeneration in the anterior cerebellum while the flocculonodular region is preserved. In Atxn1[82Q]/+ mice, we found that levels of Sphk1 and Sphk2 were region-specific decreased and S1P levels increased, particularly in the anterior cerebellum. To determine if there is a causal link between sphingolipid levels and neurodegeneration, we deleted the Sphk1 gene in Atxn1[82Q]/+ mice. Analysis of Atxn1[82Q]/+; Sphk1-/- mice confirmed a neuroprotective effect, rescuing a subset of PCs in the anterior cerebellum, in domains reminiscent of the modules defined by AldolaseC expression. Finally, we showed that Sphk1 deletion acts on the ATXN1[82Q] protein expression and prevents PC degeneration. Taken together, our results demonstrate that there are regional differences in sphingolipid metabolism and that this metabolism is directly involved in PC degeneration in Atxn1[82Q]/+ mice.

Evaluation and Management of Airway Foreign Bodies in the Emergency Department Setting.

BACKGROUND: Airway foreign body can be a life-threatening issue in pediatric and adult patients, and the majority of these patients will first present to the emergency department. OBJECTIVE: This article provides a narrative review of the diagnosis and management of airway foreign bodies for the emergency clinician. DISCUSSION: Foreign bodies in the upper and lower airways are potentially life threatening. This affects all age groups but is more common in pediatric patients. A history of a witnessed ingestion or aspiration event should raise the clinical suspicion for an aspirated foreign body. Patients with upper-airway foreign bodies are more likely to present in respiratory distress when compared with lower-airway foreign bodies, which often present with more subtle signs. Stridor, drooling, and wheezing suggest respiratory distress, but the presenting clinical picture is often unclear and may only include a cough. Immediate intervention is required in the patient with hemodynamic instability or respiratory distress. Airway management including laryngoscopy, fiberoptic bronchoscopy, and cricothyrotomy may be needed in these patients, with the emphasis on removing the obstructing foreign body and securing the airway. Specialist consultation can assist in retrieving the foreign body and managing the airway. If the patient is stable, imaging and specialist consultation for potential operating room intervention should be considered. CONCLUSIONS: An understanding of the presentation, evaluation, and management of the patient with an airway foreign body is essential for emergency clinicians.

Extensive cryptic splicing upon loss of RBM17 and TDP43 in neurodegeneration models.

Splicing regulation is an important step of post-transcriptional gene regulation. It is a highly dynamic process orchestrated by RNA-binding proteins (RBPs). RBP dysfunction and global splicing dysregulation have been implicated in many human diseases, but the in vivo functions of most RBPs and the splicing outcome upon their loss remain largely unexplored. Here we report that constitutive deletion of Rbm17, which encodes an RBP with a putative role in splicing, causes early embryonic lethality in mice and that its loss in Purkinje neurons leads to rapid degeneration. Transcriptome profiling of Rbm17-deficient and control neurons and subsequent splicing analyses using CrypSplice, a new computational method that we developed, revealed that more than half of RBM17-dependent splicing changes are cryptic. Importantly, RBM17 represses cryptic splicing of genes that likely contribute to motor coordination and cell survival. This finding prompted us to re-analyze published datasets from a recent report on TDP-43, an RBP implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as it was demonstrated that TDP-43 represses cryptic exon splicing to promote cell survival. We uncovered a large number of TDP-43-dependent splicing defects that were not previously discovered, revealing that TDP-43 extensively regulates cryptic splicing. Moreover, we found a significant overlap in genes that undergo both RBM17- and TDP-43-dependent cryptic splicing repression, many of which are associated with survival. We propose that repression of cryptic splicing by RBPs is critical for neuronal health and survival. CrypSplice is available at www.liuzlab.org/CrypSplice.

Pathogenesis of severe ataxia and tremor without the typical signs of neurodegeneration.

Neurological diseases are especially devastating when they involve neurodegeneration. Neuronal destruction is widespread in cognitive disorders such as Alzheimer's and regionally localized in motor disorders such as Parkinson's, Huntington's, and ataxia. But, surprisingly, the onset and progression of these diseases can occur without neurodegeneration. To understand the origins of diseases that do not have an obvious neuropathology, we tested how loss of CAR8, a regulator of IP3R1-mediated Ca(2+)-signaling, influences cerebellar circuit formation and neural function as movement deteriorates. We found that faulty molecular patterning, which shapes functional circuits called zones, leads to alterations in cerebellar wiring and Purkinje cell activity, but not to degeneration. Rescuing Purkinje cell function improved movement and reducing their Ca(2+) influx eliminated ectopic zones. Our findings in Car8(wdl) mutant mice unveil a pathophysiological mechanism that may operate broadly to impact motor and non-motor conditions that do not involve degeneration.

Cerebellar zonal patterning relies on Purkinje cell neurotransmission.

Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. The proper connectivity of zones is critical for motor coordination and motor learning, and in several neurological diseases cerebellar circuits degenerate in zonal patterns. Despite recent advances in understanding zone function, we still have a limited understanding of how zones are formed. Here, we focused our attention on Purkinje cells to gain a better understanding of their specific role in establishing zonal circuits. We used conditional mouse genetics to test the hypothesis that Purkinje cell neurotransmission is essential for refining prefunctional developmental zones into sharp functional zones. Our results show that inhibitory synaptic transmission in Purkinje cells is necessary for the precise patterning of Purkinje cell zones and the topographic targeting of mossy fiber afferents. As expected, blocking Purkinje cell neurotransmission caused ataxia. Using in vivo electrophysiology, we demonstrate that loss of Purkinje cell communication altered the firing rate and pattern of their target cerebellar nuclear neurons. Analysis of Purkinje cell complex spike firing revealed that feedback in the cerebellar nuclei to inferior olive to Purkinje cell loop is obstructed. Loss of Purkinje neurotransmission also caused ectopic zonal expression of tyrosine hydroxylase, which is only expressed in adult Purkinje cells when calcium is dysregulated and if excitability is altered. Our results suggest that Purkinje cell inhibitory neurotransmission establishes the functional circuitry of the cerebellum by patterning the molecular zones, fine-tuning afferent circuitry, and shaping neuronal activity.

In vivo analysis of Purkinje cell firing properties during postnatal mouse development.

Purkinje cell activity is essential for controlling motor behavior. During motor behavior Purkinje cells fire two types of action potentials: simple spikes that are generated intrinsically and complex spikes that are induced by climbing fiber inputs. Although the functions of these spikes are becoming clear, how they are established is still poorly understood. Here, we used in vivo electrophysiology approaches conducted in anesthetized and awake mice to record Purkinje cell activity starting from the second postnatal week of development through to adulthood. We found that the rate of complex spike firing increases sharply at 3 wk of age whereas the rate of simple spike firing gradually increases until 4 wk of age. We also found that compared with adult, the pattern of simple spike firing during development is more irregular as the cells tend to fire in bursts that are interrupted by long pauses. The regularity in simple spike firing only reached maturity at 4 wk of age. In contrast, the adult complex spike pattern was already evident by the second week of life, remaining consistent across all ages. Analyses of Purkinje cells in alert behaving mice suggested that the adult patterns are attained more than a week after the completion of key morphogenetic processes such as migration, lamination, and foliation. Purkinje cell activity is therefore dynamically sculpted throughout postnatal development, traversing several critical events that are required for circuit formation. Overall, we show that simple spike and complex spike firing develop with unique developmental trajectories.

Deep Brain Stimulation of the Interposed Cerebellar Nuclei in a Conditional Genetic Mouse Model with Dystonia.

Dystonia is a neurological disease that is currently ranked as the third most common motor disorder. Patients exhibit repetitive and sometimes sustained muscle contractions that cause limb and body twisting and abnormal postures that impair movement. Deep brain stimulation (DBS) of the basal ganglia and thalamus can be used to improve motor function when other treatment options fail. Recently, the cerebellum has garnered interest as a DBS target for treating dystonia and other motor disorders. Here, we describe a procedure for targeting DBS electrodes to the interposed cerebellar nuclei to correct motor dysfunction in a mouse model with dystonia. Targeting cerebellar outflow pathways with neuromodulation opens new possibilities for using the expansive connectivity of the cerebellum to treat motor and non-motor diseases.

Purkinje cell microzones mediate distinct kinematics of a single movement.

The classification of neuronal subpopulations has significantly advanced, yet its relevance for behavior remains unclear. The highly organized flocculus of the cerebellum, known to fine-tune multi-axial eye movements, is an ideal substrate for the study of potential functions of neuronal subpopulations. Here, we demonstrate that its recently identified subpopulations of 9+ and 9- Purkinje cells exhibit an intermediate Aldolase C expression and electrophysiological profile, providing evidence for a graded continuum of intrinsic properties among PC subpopulations. By identifying and utilizing two Cre-lines that genetically target these floccular domains, we show with high spatial specificity that these subpopulations of Purkinje cells participate in separate micromodules with topographically organized connections. Finally, optogenetic excitation of the respective subpopulations results in movements around the same axis in space, yet with distinct kinematic profiles. These results indicate that Purkinje cell subpopulations integrate in discrete circuits and mediate particular parameters of single movements.

Postsynaptic plasticity of Purkinje cells in mice is determined by molecular identity.

Cerebellar learning is expressed as upbound or downbound changes in simple spike activity of Purkinje cell subpopulations, but the underlying mechanism remains enigmatic. By visualizing murine Purkinje cells with different molecular identities, we demonstrate that the potential for induction of long-term depression is prominent in downbound and minimal in the upbound subpopulation. These differential propensities depend on the expression profile, but not on the synaptic inputs, of the individual Purkinje cell involved, highlighting the functional relevance of intrinsic properties for memory formation.

Olivocerebellar control of movement symmetry.

Coordination of bilateral movements is essential for a large variety of animal behaviors. The olivocerebellar system is critical for the control of movement, but its role in bilateral coordination has yet to be elucidated. Here, we examined whether Purkinje cells encode and influence synchronicity of left-right whisker movements. We found that complex spike activity is correlated with a prominent left-right symmetry of spontaneous whisker movements within parts, but not all, of Crus1 and Crus2. Optogenetic stimulation of climbing fibers in the areas with high and low correlations resulted in symmetric and asymmetric whisker movements, respectively. Moreover, when simple spike frequency prior to the complex spike was higher, the complex spike-related symmetric whisker protractions were larger. This finding alludes to a role for rebound activity in the cerebellar nuclei, which indeed turned out to be enhanced during symmetric protractions. Tracer injections suggest that regions associated with symmetric whisker movements are anatomically connected to the contralateral cerebellar hemisphere. Together, these data point toward the existence of modules on both sides of the cerebellar cortex that can differentially promote or reduce the symmetry of left and right movements in a context-dependent fashion.

Purkinje Cell Activity Resonation Generates Rhythmic Behaviors at the Preferred Frequency of 8 Hz.

Neural activity exhibits oscillations, bursts, and resonance, enhancing responsiveness at preferential frequencies. For example, theta-frequency bursting and resonance in granule cells facilitate synaptic transmission and plasticity mechanisms at the input stage of the cerebellar cortex. However, whether theta-frequency bursting of Purkinje cells is involved in generating rhythmic behavior has remained neglected. We recorded and optogenetically modulated the simple and complex spike activity of Purkinje cells while monitoring whisker movements with a high-speed camera of awake, head-fixed mice. During spontaneous whisking, both simple spike activity and whisker movement exhibit peaks within the theta band. Eliciting either simple or complex spikes at frequencies ranging from 0.5 to 28 Hz, we found that 8 Hz is the preferred frequency around which the largest movement is induced. Interestingly, oscillatory whisker movements at 8 Hz were also generated when simple spike bursting was induced at 2 and 4 Hz, but never via climbing fiber stimulation. These results indicate that 8 Hz is the resonant frequency at which the cerebellar-whisker circuitry produces rhythmic whisking.

In vivo 5-ethynyluridine (EU) labelling detects reduced transcription in Purkinje cell degeneration mouse mutants, but can itself induce neurodegeneration.

Fluorescent staining of newly transcribed RNA via metabolic labelling with 5-ethynyluridine (EU) and click chemistry enables visualisation of changes in transcription, such as in conditions of cellular stress. Here, we tested whether EU labelling can be used to examine transcription in vivo in mouse models of nervous system disorders. We show that injection of EU directly into the cerebellum results in reproducible labelling of newly transcribed RNA in cerebellar neurons and glia, with cell type-specific differences in relative labelling intensities, such as Purkinje cells exhibiting the highest levels. We also observed EU-labelling accumulating into cytoplasmic inclusions, indicating that EU, like other modified uridines, may introduce non-physiological properties in labelled RNAs. Additionally, we found that EU induces Purkinje cell degeneration nine days after EU injection, suggesting that EU incorporation not only results in abnormal RNA transcripts, but also eventually becomes neurotoxic in highly transcriptionally-active neurons. However, short post-injection intervals of EU labelling in both a Purkinje cell-specific DNA repair-deficient mouse model and a mouse model of spinocerebellar ataxia 1 revealed reduced transcription in Purkinje cells compared to controls. We combined EU labelling with immunohistology to correlate altered EU staining with pathological markers, such as genotoxic signalling factors. These data indicate that the EU-labelling method provided here can be used to identify changes in transcription in vivo in nervous system disease models.

Neuromodulation of the cerebellum rescues movement in a mouse model of ataxia.

Deep brain stimulation (DBS) relieves motor dysfunction in Parkinson's disease, and other movement disorders. Here, we demonstrate the potential benefits of DBS in a model of ataxia by targeting the cerebellum, a major motor center in the brain. We use the Car8 mouse model of hereditary ataxia to test the potential of using cerebellar nuclei DBS plus physical activity to restore movement. While low-frequency cerebellar DBS alone improves Car8 mobility and muscle function, adding skilled exercise to the treatment regimen additionally rescues limb coordination and stepping. Importantly, the gains persist in the absence of further stimulation. Because DBS promotes the most dramatic improvements in mice with early-stage ataxia, we postulated that cerebellar circuit function affects stimulation efficacy. Indeed, genetically eliminating Purkinje cell neurotransmission blocked the ability of DBS to reduce ataxia. These findings may be valuable in devising future DBS strategies.

Region-specific preservation of Purkinje cell morphology and motor behavior in the ATXN1[82Q] mouse model of spinocerebellar ataxia 1.

Purkinje cells are the primary processing units of the cerebellar cortex and display molecular heterogeneity that aligns with differences in physiological properties, projection patterns, and susceptibility to disease. In particular, multiple mouse models that feature Purkinje cell degeneration are characterized by incomplete and patterned Purkinje cell degeneration, suggestive of relative sparing of Purkinje cell subpopulations, such as those expressing Aldolase C/zebrinII (AldoC) or residing in the vestibulo-cerebellum. Here, we investigated a well-characterized Purkinje cell-specific mouse model for spinocerebellar ataxia type 1 (SCA1) that expresses human ATXN1 with a polyQ expansion (82Q). Our pathological analysis confirms previous findings that Purkinje cells of the vestibulo-cerebellum, i.e., the flocculonodular lobes, and crus I are relatively spared from key pathological hallmarks: somatodendritic atrophy, and the appearance of p62/SQSTM1-positive inclusions. However, immunohistological analysis of transgene expression revealed that spared Purkinje cells do not express mutant ATXN1 protein, indicating the sparing of Purkinje cells can be explained by an absence of transgene expression. Additionally, we found that Purkinje cells in other cerebellar lobules that typically express AldoC, not only display severe pathology but also show loss of AldoC expression. The relatively preserved flocculonodular lobes and crus I showed a substantial fraction of Purkinje cells that expressed the mutant protein and displayed pathology as well as loss of AldoC expression. Despite considerable pathology in these lobules, behavioral analyses demonstrated a relative sparing of related functions, suggestive of sufficient functional cerebellar reserve. Together, the data indicate that mutant ATXN1 affects both AldoC-positive and AldoC-negative Purkinje cells and disrupts normal parasagittal AldoC expression in Purkinje cells. Our results show that, in a mouse model otherwise characterized by widespread Purkinje cell degeneration, sparing of specific subpopulations is sufficient to maintain normal performance of specific behaviors within the context of the functional, modular map of the cerebellum.

Differential spatiotemporal development of Purkinje cell populations and cerebellum-dependent sensorimotor behaviors.

Distinct populations of Purkinje cells (PCs) with unique molecular and connectivity features are at the core of the modular organization of the cerebellum. Previously, we showed that firing activity of PCs differs between ZebrinII-positive and ZebrinII-negative cerebellar modules (Zhou et al., 2014; Wu et al., 2019). Here, we investigate the timing and extent of PC differentiation during development in mice. We found that several features of PCs, including activity levels, dendritic arborization, axonal shape and climbing fiber input, develop differentially between nodular and anterior PC populations. Although all PCs show a particularly rapid development in the second postnatal week, anterior PCs typically have a prolonged physiological and dendritic maturation. In line herewith, younger mice exhibit attenuated anterior-dependent eyeblink conditioning, but faster nodular-dependent compensatory eye movement adaptation. Our results indicate that specific cerebellar regions have unique developmental timelines which match with their related, specific forms of cerebellum-dependent behaviors.

Purkinje cell misfiring generates high-amplitude action tremors that are corrected by cerebellar deep brain stimulation.

Tremor is currently ranked as the most common movement disorder. The brain regions and neural signals that initiate the debilitating shakiness of different body parts remain unclear. Here, we found that genetically silencing cerebellar Purkinje cell output blocked tremor in mice that were given the tremorgenic drug harmaline. We show in awake behaving mice that the onset of tremor is coincident with rhythmic Purkinje cell firing, which alters the activity of their target cerebellar nuclei cells. We mimic the tremorgenic action of the drug with optogenetics and present evidence that highly patterned Purkinje cell activity drives a powerful tremor in otherwise normal mice. Modulating the altered activity with deep brain stimulation directed to the Purkinje cell output in the cerebellar nuclei reduced tremor in freely moving mice. Together, the data implicate Purkinje cell connectivity as a neural substrate for tremor and a gateway for signals that mediate the disease.

Persistent motor dysfunction despite homeostatic rescue of cerebellar morphogenesis in the Car8 waddles mutant mouse.

BACKGROUND: Purkinje cells play a central role in establishing the cerebellar circuit. Accordingly, disrupting Purkinje cell development impairs cerebellar morphogenesis and motor function. In the Car8wdl mouse model of hereditary ataxia, severe motor deficits arise despite the cerebellum overcoming initial defects in size and morphology. METHODS: To resolve how this compensation occurs, we asked how the loss of carbonic anhydrase 8 (CAR8), a regulator of IP3R1 Ca2+ signaling in Purkinje cells, alters cerebellar development in Car8wdl mice. Using a combination of histological, physiological, and behavioral analyses, we determined the extent to which the loss of CAR8 affects cerebellar anatomy, neuronal firing, and motor coordination during development. RESULTS: Our results reveal that granule cell proliferation is reduced in early postnatal mutants, although by the third postnatal week there is enhanced and prolonged proliferation, plus an upregulation of Sox2 expression in the inner EGL. Modified circuit patterning of Purkinje cells and Bergmann glia accompany these granule cell adjustments. We also find that although anatomy eventually normalizes, the abnormal activity of neurons and muscles persists. CONCLUSIONS: Our data show that losing CAR8 only transiently restricts cerebellar growth, but permanently damages its function. These data support two current hypotheses about cerebellar development and disease: (1) Sox2 expression may be upregulated at sites of injury and contribute to the rescue of cerebellar structure and (2) transient delays to developmental processes may precede permanent motor dysfunction. Furthermore, we characterize waddles mutant mouse morphology and behavior during development and propose a Sox2-positive, cell-mediated role for rescue in a mouse model of human motor diseases.