Mitochondrial damage and impaired mitophagy contribute to disease progression in SCA6.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease that manifests in midlife and progressively worsens with age. SCA6 is rare, and many patients are not diagnosed until long after disease onset. Whether disease-causing cellular alterations differ at different disease stages is currently unknown, but it is important to answer this question in order to identify appropriate therapeutic targets across disease duration. We used transcriptomics to identify changes in gene expression at disease onset in a well-established mouse model of SCA6 that recapitulates key disease features. We observed both up- and down-regulated genes with the major down-regulated gene ontology terms suggesting mitochondrial dysfunction. We explored mitochondrial function and structure and observed that changes in mitochondrial structure preceded changes in function, and that mitochondrial function was not significantly altered at disease onset but was impaired later during disease progression. We also detected elevated oxidative stress in cells at the same disease stage. In addition, we observed impairment in mitophagy that exacerbates mitochondrial dysfunction at late disease stages. In post-mortem SCA6 patient cerebellar tissue, we observed metabolic changes that are consistent with mitochondrial impairments, supporting our results from animal models being translatable to human disease. Our study reveals that mitochondrial dysfunction and impaired mitochondrial degradation likely contribute to disease progression in SCA6 and suggests that these could be promising targets for therapeutic interventions in particular for patients diagnosed after disease onset.

Synapse-specific burst coding sustained by local axonal translation.

Neurotransmission in the brain is unreliable, suggesting that high-frequency spike bursts rather than individual spikes carry the neural code. For instance, cortical pyramidal neurons rely on bursts in memory formation. Protein synthesis is another key factor in long-term synaptic plasticity and learning but is widely considered unnecessary for synaptic transmission. Here, however, we show that burst neurotransmission at synapses between neocortical layer 5 pyramidal cells depends on axonal protein synthesis linked to presynaptic NMDA receptors and mTOR. We localized protein synthesis to axons with laser axotomy and puromycylation live imaging. We whole-cell recorded connected neurons to reveal how translation sustained readily releasable vesicle pool size and replenishment rate. We live imaged axons and found sparsely docked RNA granules, suggesting synapse-specific regulation. In agreement, translation boosted neurotransmission onto excitatory but not inhibitory basket or Martinotti cells. Local axonal mRNA translation is thus a hitherto unappreciated principle for sustaining burst coding at specific synapse types.

A mitochondrial-targeted antioxidant (MitoQ) improves motor coordination and reduces Purkinje cell death in a mouse model of ARSACS.

Mitochondrial deficits have been observed in animal models of Autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) and in patient-derived fibroblasts. We investigated whether mitochondrial function could be restored in Sacs-/- mice, a mouse model of ARSACS, using the mitochondrial-targeted antioxidant ubiquinone MitoQ. After 10weeks of chronic MitoQ administration in drinking water, we partially reversed motor coordination deficits in Sacs-/- mice but did not affect litter-matched wild-type control mice. MitoQ administration led to a restoration of superoxide dismutase 2 (SOD2) in cerebellar Purkinje cell somata without altering Purkinje cell firing deficits. Purkinje cells in anterior vermis of Sacs-/- mice normally undergo cell death in ARSACS; however, Purkinje cells numbers were elevated after chronic MitoQ treatment. Furthermore, Purkinje cell innervation of target neurons in the cerebellar nuclei of Sacs-/- mice was also partially restored with MitoQ treatment. Our data suggest that MitoQ is a potential therapeutic treatment for ARSACS and that it improves motor coordination via increasing cerebellar Purkinje cell mitochondria function and reducing Purkinje cell death.

Activation of TrkB-Akt signaling rescues deficits in a mouse model of SCA6.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease resulting in motor coordination deficits and cerebellar pathology. Expression of brain-derived neurotrophic factor (BDNF) is reduced in postmortem tissue from SCA6 patients. Here, we show that levels of cerebellar BDNF and its receptor, tropomyosin receptor kinase B (TrkB), are reduced at an early disease stage in a mouse model of SCA6 (SCA684Q/84Q). One month of exercise elevated cerebellar BDNF expression and improved ataxia and cerebellar Purkinje cell firing rate deficits. A TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), likewise improved motor coordination and Purkinje cell firing rate and elevated downstream Akt signaling. Prolonged 7,8-DHF administration persistently improved ataxia when treatment commenced near disease onset but was ineffective when treatment was started late. These data suggest that 7,8-DHF, which is orally bioavailable and crosses the blood-brain barrier, is a promising therapeutic for SCA6 and argue for the importance of early intervention for SCA6.

Loss of Flocculus Purkinje Cell Firing Precision Leads to Impaired Gaze Stabilization in a Mouse Model of Spinocerebellar Ataxia Type 6 (SCA6).

Spinocerebellar Ataxia Type 6 (SCA6) is a mid-life onset neurodegenerative disease characterized by progressive ataxia, dysarthria, and eye movement impairment. This autosomal dominant disease is caused by the expansion of a CAG repeat tract in the CACNA1A gene that encodes the α1A subunit of the P/Q type voltage-gated Ca2+ channel. Mouse models of SCA6 demonstrate impaired locomotive function and reduced firing precision of cerebellar Purkinje in the anterior vermis. Here, to further assess deficits in other cerebellar-dependent behaviors, we characterized the oculomotor phenotype of a knock-in mouse model with hyper-expanded polyQ repeats (SCA684Q). We found a reduction in the efficacy of the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) in SCA6 mutant mice, without a change in phase, compared to their litter-matched controls. Additionally, VOR motor learning was significantly impaired in SCA684Q mice. Given that the floccular lobe of the cerebellum plays a vital role in the generation of OKR and VOR calibration and motor learning, we investigated the firing behavior and morphology of floccular cerebellar Purkinje cells. Overall, we found a reduction in the firing precision of floccular lobe Purkinje cells but no morphological difference between SCA684Q and wild-type mice. Taken together, our findings establish that gaze stabilization and motor learning are impaired in SCA684Q mice and suggest that altered cerebellar output contributes to these deficits.

Molecular Identity and Location Influence Purkinje Cell Vulnerability in Autosomal-Recessive Spastic Ataxia of Charlevoix-Saguenay Mice.

Patterned cell death is a common feature of many neurodegenerative diseases. In patients with autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) and mouse models of ARSACS, it has been observed that Purkinje cells in anterior cerebellar vermis are vulnerable to degeneration while those in posterior vermis are resilient. Purkinje cells are known to express certain molecules in a highly stereotyped, patterned manner across the cerebellum. One patterned molecule is zebrin, which is expressed in distinctive stripes across the cerebellar cortex. The different zones delineated by the expression pattern of zebrin and other patterned molecules have been implicated in the patterning of Purkinje cell death, raising the question of whether they contribute to cell death in ARSACS. We found that zebrin patterning appears normal prior to disease onset in Sacs-/- mice, suggesting that zebrin-positive and -negative Purkinje cell zones develop normally. We next observed that zebrin-negative Purkinje cells in anterior lobule III were preferentially susceptible to cell death, while anterior zebrin-positive cells and posterior zebrin-negative and -positive cells remained resilient even at late disease stages. The patterning of Purkinje cell innervation to the target neurons in the cerebellar nuclei (CN) showed a similar pattern of loss: neurons in the anterior CN, where inputs are predominantly zebrin-negative, displayed a loss of Purkinje cell innervation. In contrast, neurons in the posterior CN, which is innervated by both zebrin-negative and -positive puncta, had normal innervation. These results suggest that the location and the molecular identity of Purkinje cells determine their susceptibility to cell death in ARSACS.

Purkinje cell axonal swellings enhance action potential fidelity and cerebellar function.

Axonal plasticity allows neurons to control their output, which critically determines the flow of information in the brain. Axon diameter can be regulated by activity, yet how morphological changes in an axon impact its function remains poorly understood. Axonal swellings have been found on Purkinje cell axons in the cerebellum both in healthy development and in neurodegenerative diseases, and computational models predicts that axonal swellings impair axonal function. Here we report that in young Purkinje cells, axons with swellings propagated action potentials with higher fidelity than those without, and that axonal swellings form when axonal failures are high. Furthermore, we observed that healthy young adult mice with more axonal swellings learn better on cerebellar-related tasks than mice with fewer swellings. Our findings suggest that axonal swellings underlie a form of axonal plasticity that optimizes the fidelity of action potential propagation in axons, resulting in enhanced learning.

4E-BP2-dependent translation in cerebellar Purkinje cells controls spatial memory but not autism-like behaviors.

Recent studies have demonstrated that selective activation of mammalian target of rapamycin complex 1 (mTORC1) in the cerebellum by deletion of the mTORC1 upstream repressors TSC1 or phosphatase and tensin homolog (PTEN) in Purkinje cells (PCs) causes autism-like features and cognitive deficits. However, the molecular mechanisms by which overactivated mTORC1 in the cerebellum engenders these behaviors remain unknown. The eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) is a central translational repressor downstream of mTORC1. Here, we show that mice with selective ablation of 4E-BP2 in PCs display a reduced number of PCs, increased regularity of PC action potential firing, and deficits in motor learning. Surprisingly, although spatial memory is impaired in these mice, they exhibit normal social interaction and show no deficits in repetitive behavior. Our data suggest that, downstream of mTORC1/4E-BP2, there are distinct cerebellar mechanisms independently controlling social behavior and memory formation.

Losing the Beat: Contribution of Purkinje Cell Firing Dysfunction to Disease, and Its Reversal.

The cerebellum is a brain structure that is highly interconnected with other brain regions. There are many contributing factors to cerebellar-related brain disease, such as altered afferent input, local connectivity, and/or cerebellar output. Purkinje cells (PC) are the principle cells of the cerebellar cortex, and fire intrinsically; that is, they fire spontaneous action potentials at high frequencies. This review paper focuses on PC intrinsic firing activity, which is altered in multiple neurological diseases, including ataxia, Huntington Disease (HD) and autism spectrum disorder (ASD). Notably, there are several cases where interventions that restore or rescue PC intrinsic activity also improve impaired behavior in these mouse models of disease. These findings suggest that rescuing PC firing deficits themselves may be sufficient to improve impairment in cerebellar-related behavior in disease. We propose that restoring PC intrinsic firing represents a good target for drug development that might be of therapeutic use for several disorders.

Optimizing Optogenetic Activation of Purkinje Cell Axons to Investigate the Purkinje Cell - DCN Synapse.

Optogenetics is a state-of-the-art tool for interrogating neural circuits. In the cerebellum, Purkinje cells serve as the sole output of the cerebellar cortex where they synapse on neurons in the deep cerebellar nuclei (DCN). To investigate the properties of this synaptic connection, we sought to elicit time-locked single action potentials from Purkinje cell axons. Using optical stimulation of channelrhodopsin-2 (ChR2)-expressing Purkinje cells combined with patch-clamp recordings of Purkinje cells and DCN neurons in acute cerebellar slices, we determine the photostimulation parameters required to elicit single time-locked action potentials from Purkinje cell axons. We show that axons require longer light pulses than somata do to elicit single action potentials and that Purkinje cell axons are also more susceptible to light perturbations. We then demonstrate that these empirically determined photostimulation parameters elicit time-locked synaptic currents from postsynaptic cells in the DCN. Our results highlight the importance of optimizing optogenetic stimulation conditions to interrogate synaptic connections.

The impact of light during the night.

Exposing chicks to one hour of light during the night disrupts the release of a hormone that is needed by cells in the developing brain to survive.

ATAT1 regulates forebrain development and stress-induced tubulin hyperacetylation.

α-Tubulin acetyltransferase 1 (ATAT1) catalyzes acetylation of α-tubulin at lysine 40 in various organisms ranging from Tetrahymena to humans. Despite the importance in mammals suggested by studies of cultured cells, the mouse Atat1 gene is non-essential for survival, raising an intriguing question about its real functions in vivo. To address this question, we systematically analyzed a mouse strain lacking the gene. The analyses revealed that starting at postnatal day 5, the mutant mice display enlarged lateral ventricles in the forebrain, resembling ventricular dilation in human patients with ventriculomegaly. In the mice, ventricular dilation is due to hypoplasia in the septum and striatum. Behavioral tests of the mice uncovered deficits in motor coordination. Birth-dating experiments revealed that neuronal migration to the mutant septum and striatum is impaired during brain development. In the mutant embryonic fibroblasts, we found mild defects in cell proliferation and primary cilium formation. Notably, in these cells, ATAT1 is indispensable for tubulin hyperacetylation in response to high salt, high glucose, and hydrogen peroxide-induced oxidative stress. We investigated the role of ATAT1 in the hematopoietic system using multicolor flow cytometry and found that this system remains normal in the mutant mice. Although tubulin acetylation was undetectable in a majority of mutant tissues, residual levels were detected in the heart, skeletal muscle, trachea, oviduct, thymus and spleen. This study thus not only establishes the importance of ATAT1 in regulating mouse forebrain development and governing tubulin hyperacetylation during stress responses, but also suggests the existence of an additional α-tubulin acetyltransferase.

Sacs R272C missense homozygous mice develop an ataxia phenotype.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS [MIM 270550]) is an early-onset neurodegenerative disorder caused by mutations in the SACS gene. Over 200 SACS mutations have been identified. Most mutations lead to a complete loss of a sacsin, a large 520 kD protein, although some missense mutations are associated with low levels of sacsin expression. We previously showed that Sacs knock-out mice demonstrate early-onset ataxic phenotype with neurofilament bundling in many neuronal populations. To determine if the preservation of some mutated sacsin protein resulted in the same cellular and behavioral alterations, we generated mice expressing an R272C missense mutation, a homozygote mutation found in some affected patients. Though SacsR272C mice express 21% of wild type brain sacsin and sacsin is found in many neurons, they display similar abnormalities to Sacs knock-out mice, including the development of an ataxic phenotype, reduced Purkinje cell firing rates, and somatodendritic neurofilament bundles in Purkinje cells and other neurons. Together our results support that Sacs missense mutation largely lead to loss of sacsin function.

Altered synaptic and firing properties of cerebellar Purkinje cells in a mouse model of ARSACS.

KEY POINTS: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative human disease characterized in part by ataxia and Purkinje cell loss in anterior cerebellar lobules. A knock-out mouse model has been developed that recapitulates several features of ARSACS. Using this ARSACS mouse model, we report changes in synaptic input and intrinsic firing in cerebellar Purkinje cells, as well as in their synaptic output in the deep cerebellar nuclei. Changes in firing are observed in anterior lobules that later exhibit Purkinje cell death, but not in posterior lobules that do not. Our results show that both synaptic and intrinsic alterations in Purkinje cell properties likely contribute to disease manifestation in ARSACS; these findings resemble pathophysiological changes reported in several other ataxias. ABSTRACT: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disease that includes a pronounced and progressive cerebellar dysfunction. ARSACS is caused by an autosomal recessive loss-of-function mutation in the Sacs gene that encodes the protein sacsin. To better understand the cerebellar pathophysiology in ARSACS, we studied synaptic and firing properties of Purkinje cells from a mouse model of ARSACS, Sacs-/- mice. We found that excitatory synaptic drive was reduced onto Sacs-/- Purkinje cells, and that Purkinje cell firing rate, but not regularity, was reduced at postnatal day (P)40, an age when ataxia symptoms were first reported. Firing rate deficits were limited to anterior lobules that later display Purkinje cell death, and were not observed in posterior lobules where Purkinje cells are not lost. Mild firing deficits were observed as early as P20, prior to the manifestation of motor deficits, suggesting that a critical level of cerebellar dysfunction is required for motor coordination to emerge. Finally, we observed a reduction in Purkinje cell innervation onto target neurons in the deep cerebellar nuclei (DCN) in Sacs-/- mice. Together, these findings suggest that multiple alterations in the cerebellar circuit including Purkinje cell input and output contribute to cerebellar-related disease onset in ARSACS.

Transient cerebellar alterations during development prior to obvious motor phenotype in a mouse model of spinocerebellar ataxia type 6.

KEY POINTS: Spinocerebellar ataxia type 6 (SCA6) is a midlife-onset neurodegenerative disease caused by a CACNA1A mutation; CACNA1A is also implicated in cerebellar development. We have previously shown that when disease symptoms are present in midlife in SCA684Q/84Q mice, cerebellar Purkinje cells spike with reduced rate and precision. In contrast, we find that during postnatal development (P10-13), SCA684Q/84Q Purkinje cells spike with elevated rate and precision. Although surplus climbing fibres are linked to ataxia in other mouse models, we found surplus climbing fibre inputs on developing (P10-13) SCA684Q/84Q Purkinje cells when motor deficits were not detected. Developmental alterations were transient and were no longer observed in weanling (P21-24) SCA684Q/84Q Purkinje cells. Our results suggest that changes in the developing cerebellar circuit can occur without detectable motor abnormalities, and that changes in cerebellar development may not necessarily persist into adulthood. ABSTRACT: Although some neurodegenerative diseases are caused by mutations in genes that are known to regulate neuronal development, surprisingly, patients may not present disease symptoms until adulthood. Spinocerebellar ataxia type 6 (SCA6) is one such midlife-onset disorder in which the mutated gene, CACNA1A, is implicated in cerebellar development. We wondered whether changes were observed in the developing cerebellum in SCA6 prior to the detection of motor deficits. To address this question, we used a transgenic mouse with a hyper-expanded triplet repeat (SCA684Q/84Q ) that displays late-onset motor deficits at 7 months, and measured cerebellar Purkinje cell synaptic and intrinsic properties during postnatal development. We found that firing rate and precision were enhanced during postnatal development in P10-13 SCA684Q/84Q Purkinje cells, and observed surplus multiple climbing fibre innervation without changes in inhibitory input or dendritic structure during development. Although excess multiple climbing fibre innervation has been associated with ataxic symptoms in several adult transgenic mice, we observed no detectable changes in cerebellar-related motor behaviour in developing SCA684Q/84Q mice. Interestingly, we found that developmental alterations were transient, as both Purkinje cell firing properties and climbing fibre innervation from weanling-aged (P21-24) SCA684Q/84Q mice were indistinguishable from litter-matched control mice. Our results demonstrate that significant alterations in neuronal circuit development may be observed without any detectable behavioural read-out, and that early changes in brain development may not necessarily persist into adulthood in midlife-onset diseases.

Transient Developmental Purkinje Cell Axonal Torpedoes in Healthy and Ataxic Mouse Cerebellum.

Information is carried out of the cerebellar cortical microcircuit via action potentials propagated along Purkinje cell axons. In several human neurodegenerative diseases, focal axonal swellings on Purkinje cells - known as torpedoes - have been associated with Purkinje cell loss. Interestingly, torpedoes are also reported to appear transiently during development in rat cerebellum. The function of Purkinje cell axonal torpedoes in health as well as in disease is poorly understood. We investigated the properties of developmental torpedoes in the postnatal mouse cerebellum of wild-type and transgenic mice. We found that Purkinje cell axonal torpedoes transiently appeared on axons of Purkinje neurons, with the largest number of torpedoes observed at postnatal day 11 (P11). This was after peak developmental apoptosis had occurred, when Purkinje cell counts in a lobule were static, suggesting that most developmental torpedoes appear on axons of neurons that persist into adulthood. We found that developmental torpedoes were not associated with a presynaptic GABAergic marker, indicating that they are not synapses. They were seldom found at axonal collateral branch points, and lacked microglia enrichment, suggesting that they are unlikely to be involved in axonal refinement. Interestingly, we found several differences between developmental torpedoes and disease-related torpedoes: developmental torpedoes occurred largely on myelinated axons, and were not associated with changes in basket cell innervation on their parent soma. Disease-related torpedoes are typically reported to contain neurofilament; while the majority of developmental torpedoes did as well, a fraction of smaller developmental torpedoes did not. These differences indicate that developmental torpedoes may not be functionally identical to disease-related torpedoes. To study this further, we used a mouse model of spinocerebellar ataxia type 6 (SCA6), and found elevated disease-related torpedo number at 2 years. However, we found normal levels of developmental torpedoes in these mice. Our findings suggest that the transient emergence of Purkinje cell axonal torpedoes during the second postnatal week in mice represents a normal morphological feature in the developing cerebellar microcircuit.

4-aminopyridine reverses ataxia and cerebellar firing deficiency in a mouse model of spinocerebellar ataxia type 6.

Spinocerebellar ataxia type 6 (SCA6) is a devastating midlife-onset autosomal dominant motor control disease with no known treatment. Using a hyper-expanded polyglutamine (84Q) knock-in mouse, we found that cerebellar Purkinje cell firing precision was degraded in heterozygous (SCA6(84Q/+)) mice at 19 months when motor deficits are observed. Similar alterations in firing precision and motor control were observed at disease onset at 7 months in homozygous (SCA6(84Q/84Q)) mice, as well as a reduction in firing rate. We further found that chronic administration of the FDA-approved drug 4-aminopyridine (4-AP), which targets potassium channels, alleviated motor coordination deficits and restored cerebellar Purkinje cell firing precision to wildtype (WT) levels in SCA6(84Q/84Q) mice both in acute slices and in vivo. These results provide a novel therapeutic approach for treating ataxic symptoms associated with SCA6.

Long-Term Potentiation by Theta-Burst Stimulation Using Extracellular Field Potential Recordings in Acute Hippocampal Slices.

This protocol describes how to carry out theta-burst long-term potentiation (LTP) with extracellular field recordings in acute rodent hippocampal slices. This method is relatively simple and noninvasive and provides a way to sample many neurons simultaneously, making it suitable for applications requiring higher throughput than whole-cell recording.