Purkinje Cell Soma Research Articles

Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells

As cerebellar granule cells (GCs) coordinate the formation of regular cerebellar networks during postnatal development, molecules in GCs are expected to be involved. Here, we test the effects of the knockdown (KD) of multiple epidermal growth factor-like domains protein 11 (MEGF11), which is a homolog of proteins mediating astrocytic phagocytosis but is substantially increased at the later developmental stages of GCs on cerebellar development. MEGF11-KD in GCs of developing mice results in abnormal cerebellar structures, including extensively ectopic Purkinje cell (PC) somas, and in impaired motor functions. MEGF11-KD also causes abnormally asynchronous synaptic release from GC axons, parallel fibers, before the appearance of abnormal cerebellar structures. Interestingly, blockade of this abnormal synaptic release restores most of the cerebellar structures. Thus, apart from phagocytic functions of its related homologs in astrocytes, MEGF11 in GCs promotes proper PC development and cerebellar network formation by regulating immature synaptic transmission.

Cerebellar axonopathy in Shivers horses identified by spatial transcriptomic and proteomic analyses.

Shivers in horses is characterized by abnormal hindlimb movement when walking backward and is proposed to be caused by a Purkinje cell (PC) axonopathy based on histopathology. Define region-specific differences in gene expression within the lateral cerebellar hemisphere and compare cerebellar protein expression between Shivers horses and controls. Case-control study of 5 Shivers and 4 control geldings ≥16.2 hands in height. Using spatial transcriptomics, gene expression was compared between Shivers and control horses in PC soma and lateral cerebellar hemisphere white matter, consisting primarily of axons. Tandem-mass-tag (TMT-11) proteomic analysis was performed on lateral cerebellar hemisphere homogenates. Differences in gene expression between Shivers and control horses were evident in principal component analysis of axon-containing white matter but not PC soma. In white matter, there were 455/1846 differentially expressed genes (DEG; 350 ↓DEG, 105 ↑DEG) between Shivers and controls, with significant gene set enrichment of the Toll-Like Receptor 4 (TLR4) cascade, highlighting neuroinflammation. There were 50/936 differentially expressed proteins (DEP). The 27 ↓DEP highlighted loss of axonal proteins including intermediate filaments (5), myelin (3), cytoskeleton (2), neurite outgrowth (2), and Na/K ATPase (1). The 23 ↑DEP were involved in the extracellular matrix (7), cytoskeleton (7), redox balance (2), neurite outgrowth (1), signal transduction (1), and others. Our findings support axonal degeneration as a characteristic feature of Shivers. Combined with histopathology, these findings are consistent with the known distinctive response of PC to injury where axonal changes occur without a substantial impact on PC soma.

Electrophysiological and Imaging Analysis of GFP-Tagged Protein Kinase C γ Translocation in Cerebellar Purkinje Cells.

The cerebellum contains the highest density of protein kinase C (PKC) in the central nervous system. PKCγ, the major isotype accounting for over half of the PKCs in the cerebellum, is expressed exclusively in Purkinje cells (PCs). Inactivated PKCγ, which is localized in the cytoplasm of PC dendrites and soma, begins to translocate to the cell membrane upon activation. However, the physiological conditions that induce PKCγ translocation in PC remain largely unknown. Here, we virally expressed PKCγ-GFP in PCs and examined the conditions that induced its translocation to PC dendrites by whole-cell patch clamp analysis combined with confocal GFP fluorescence imaging. A single or repetitive (150 pulses at 5Hz for 30s) electrical stimulation to a climbing fiber (CF), which produced a complex spike(s) in PC, failed to induce translocation of PKCγ-GFP to the dendritic shaft of PCs. Direct current injection (+ 2 nA for 3s) to PC also did not induce the translocation, although PCs generated simple spikes continuously at high rates. In contrast, high-frequency parallel fiber (PF) stimulation (50 pulses at 50Hz for 1s), which triggered action potentials followed by sustained depolarization (known as mGluR1-mediated slow depolarization), caused translocation of cytoplasmic PKCγ-GFP to the membrane. Low-frequency PF stimulation (150 pulses at 5Hz for 30s) induced continuous simple spike firing but did not induce translocation. These results suggest that CF-triggered depolarization, which causes Ca2+ influx through voltage-gated Ca2+ channels throughout PC dendrites and somas, is insufficient to induce the translocation of PKCγ, instead requiring high-frequency PF stimulation that activates mGluR1.

Cerebellar Calcium-Binding Protein and Neurotrophin Receptor Defects in Down Syndrome and Alzheimer's Disease.

Cerebellar hypoplasia is a major characteristic of the Down syndrome (DS) brain. However, the consequences of trisomy upon cerebellar Purkinje cells (PC) and interneurons in DS are unclear. The present study performed a quantitative and qualitative analysis of cerebellar neurons immunostained with antibodies against calbindin D-28k (Calb), parvalbumin (Parv), and calretinin (Calr), phosphorylated and non-phosphorylated intermediate neurofilaments (SMI-34 and SMI-32), and high (TrkA) and low (p75NTR) affinity nerve growth factor (NGF) receptors as well as tau and amyloid in DS (n = 12), Alzheimer's disease (AD) (n = 10), and healthy non-dementia control (HC) (n = 8) cases. Our findings revealed higher Aβ42 plaque load in DS compared to AD and HC but no differences in APP/Aβ plaque load between HC, AD, and DS. The cerebellar cortex neither displayed Aβ40 containing plaques nor pathologic phosphorylated tau in any of the cases examined. The number and optical density (OD) measurements of Calb immunoreactive (-ir) PC soma and dendrites were similar between groups, while the number of PCs positive for Parv and SMI-32 were significantly reduced in AD and DS compared to HC. By contrast, the number of SMI-34-ir PC dystrophic axonal swellings, termed torpedoes, was significantly greater in AD compared to DS. No differences in SMI-32- and Parv-ir PC OD measurements were observed between groups. Conversely, total number of Parv- (stellate/basket) and Calr (Lugaro, brush, and Golgi)-positive interneurons were significantly reduced in DS compared to AD and HC. A strong negative correlation was found between counts for Parv-ir interneurons, Calr-ir Golgi and brush cells, and Aβ42 plaque load. Number of TrkA and p75NTR positive PCs were reduced in AD compared to HC. These findings suggest that disturbances in calcium binding proteins play a critical role in cerebellar neuronal dysfunction in adults with DS.

Cerebellar developmental deficits underlie neurodegenerative disorder spinocerebellar ataxia type 23.

Spinocerebellar ataxia type 23 (SCA23) is a late‐onset neurodegenerative disorder characterized by slowly progressive gait and limb ataxia, for which there is no therapy available. It is caused by pathogenic variants in PDYN, which encodes prodynorphin (PDYN). PDYN is processed into the opioid peptides α‐neoendorphin and dynorphins (Dyn) A and B; inhibitory neurotransmitters that function in pain signaling, stress‐induced responses and addiction. Variants causing SCA23 mostly affect Dyn A, leading to loss of secondary structure and increased peptide stability. PDYN R212W mice express human PDYN containing the SCA23 variant p.R212W. These mice show progressive motor deficits from 3 months of age, climbing fiber (CF) deficits from 3 months of age, and Purkinje cell (PC) loss from 12 months of age. A mouse model for SCA1 showed similar CF deficits, and a recent study found additional developmental abnormalities, namely increased GABAergic interneuron connectivity and non‐cell autonomous disruption of PC function. As SCA23 mice show a similar pathology to SCA1 mice in adulthood, we hypothesized that SCA23 may also follow SCA1 pathology during development. Examining PDYN R212W cerebella during development, we uncovered developmental deficits from 2 weeks of age, namely a reduced number of GABAergic synapses on PC soma, possibly leading to the observed delay in early phase CF elimination between 2 and 3 weeks of age. Furthermore, CFs did not reach terminal height, leaving proximal PC dendrites open to be occupied by parallel fibers (PFs). The observed increase in vGlut1 protein—a marker for PF‐PC synapses—indicates that PFs indeed take over CF territory and have increased connectivity with PCs. Additionally, we detected altered expression of several critical Ca2+ channel subunits, potentially contributing to altered Ca2+ transients in PDYN R212W cerebella. These findings indicate that developmental abnormalities contribute to the SCA23 pathology and uncover a developmental role for PDYN in the cerebellum.

Role of β3-adrenergic receptor in the modulation of synaptic transmission and plasticity in mouse cerebellar cortex.

Convergent lines of evidence have recently highlighted β3-adrenoreceptors (ARs) as a potentially critical target in the regulation of nervous and behavioral functions, including memory consolidation, anxiety, and depression. Nevertheless, the role of β3-ARs in the cerebellum has been never investigated. To address this issue, we first examined the effects of pharmacological manipulation of β3-ARs on motor learning in mice. We found that blockade of β3-ARs by SR 59230A impaired the acquisition of the rotarod task with no effect on general locomotion. Since the parallel fiber-Purkinje cell (PF-PC) synapse is considered to be the main cerebellar locus of motor learning, we assessed β3-AR modulatory action on this synapse as well as its expression in cerebellar slices. We demonstrate, for the first time, a strong expression of β3-ARs on Purkinje cell soma and dendrites. In addition, whole-cell patch-clamp recordings revealed that bath application of β3-AR agonist CL316,243 depressed the PF-PC excitatory postsynaptic currents via a postsynaptic mechanism mediated by the PI3K signaling pathway. Application of CL316,243 also interfered with the expression of PF long-term potentiation, whereas SR 59230A prevented the induction of LTD at PF-PC synapse. These results underline the critical role of β3-AR on cerebellar synaptic transmission and plasticity and provide a new mechanism for adrenergic modulation of motor learning.

The TMEM240 Protein, Mutated in SCA21, Is Expressed in Purkinje Cells and Synaptic Terminals

A variety of missense mutations and a stop mutation in the gene coding for transmembrane protein 240 (TMEM240) have been reported to be the causative mutations of spinocerebellar ataxia 21 (SCA21). We aimed to investigate the expression of TMEM240 protein in mouse brain at the tissue, cellular, and subcellular levels. Immunofluorescence labeling showed TMEM240 to be expressed in various areas of the brain, with the highest levels in the hippocampus, isocortex, and cerebellum. In the cerebellum, TMEM240 was detected in the deep nuclei and the cerebellar cortex. The protein was expressed in all three layers of the cortex and various cerebellar neurons. TMEM240 was localized to climbing, mossy, and parallel fiber afferents projecting to Purkinje cells, as shown by co-immunostaining with VGLUT1 and VGLUT2. Co-immunostaining with synaptophysin, post-synaptic fractionation, and confirmatory electron microscopy showed TMEM240 to be localized to the post-synaptic side of synapses near the Purkinje-cell soma. Similar results were obtained in human cerebellar sections. These data suggest that TMEM240 may be involved in the organization of the cerebellar network, particularly in synaptic inputs converging on Purkinje cells. This study is the first to describe TMEM240 expression in the normal mouse brain.

Phenylbutyrate administration reduces changes in the cerebellar Purkinje cells population in PDC-deficient mice

In humans, pyruvate dehydrogenase complex (PDC) deficiency impairs brain energy metabolism by reducing the availability of the functional acetyl‑CoA pool. This "hypometabolic defect" results in congenital lactic acidosis and abnormalities of brain morphology and function, ranging from mild ataxia to profound psychomotor retardation. Our previous study showed reduction in total cell number and dendritic arbors in the cerebellar Purkinje cells in systemic PDC‑deficient mice. Phenylbutyrate has been shown to increase PDC activity in cultured fibroblasts from PDC‑deficient patients. Hence, we investigated the effects of postnatal (days 2‑35) phenylbutyrate administration on the cerebellar Purkinje cell population in PDC‑deficient female mice. Histological analyses of different regions of cerebellar cortex from the brain‑specific PDC‑deficient saline‑injected mice revealed statistically significant reduction in the Purkinje cell density and increased cell size of the individual Purkinje cell soma compared to control PDC‑normal, saline‑injected group. Administration of phenylbutyrate to control mice did not cause significant changes in the Purkinje cell density and cell size in the studied regions. In contrast, administration of phenylbutyrate variably lessened the ill effects of PDC deficiency on Purkinje cell populations in different areas of the cerebellum. Our results lend further support for the possible use of phenylbutyrate as a potential treatment for PDC deficiency.

Cerebellar Neurodegeneration and Neuronal Circuit Remodeling in Golgi pH Regulator-Deficient Mice.

The Golgi apparatus plays an indispensable role in posttranslational modification and transport of proteins to their target destinations. Although it is well established that the Golgi apparatus requires an acidic luminal pH for optimal activity, morphological and functional abnormalities at the neuronal circuit level because of perturbations in Golgi pH are not fully understood. In addition, morphological alteration of the Golgi apparatus is associated with several neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. Here, we used anatomical and electrophysiological approaches to characterize morphological and functional abnormalities of neuronal circuits in Golgi pH regulator (GPHR) conditional knock-out mice. Purkinje cells (PCs) from the mutant mice exhibited vesiculation and fragmentation of the Golgi apparatus, followed by axonal degeneration and progressive cell loss. Morphological analysis provided evidence for the disruption of basket cell (BC) terminals around PC soma, and electrophysiological recordings showed selective loss of large amplitude responses, suggesting BC terminal disassembly. In addition, the innervation of mutant PCs was altered such that climbing fiber (CF) terminals abnormally synapsed on the somatic spines of mutant PCs in the mature cerebellum. The combined results describe an essential role for luminal acidification of the Golgi apparatus in maintaining proper neuronal morphology and neuronal circuitry.

A Quantitative Study of Empty Baskets in Essential Tremor and Other Motor Neurodegenerative Diseases.

The underlying biology of essential tremor (ET) is poorly understood. Purkinje cell (PC) loss has been observed in some studies, although this finding remains somewhat controversial. Basket cells are interneurons whose axonal collaterals form a plexus around PC soma. When there is PC loss, this basket plexus appears empty. We used dual immunohistochemical staining for calbindin D28k and glutamic acid decarboxylase to quantify "empty baskets" as an indirect and alternative method of detecting PC loss. Microscopic analyses on 127 brains included ET and a spectrum of motor neurodegenerative diseases (50 ET, 27 spinocerebellar ataxias [SCAs], 25 Parkinson disease, 25 controls). The median percentage of empty baskets in ET patients was 1.5 times higher than controls (48.8% vs 33.5%, p < 0.001) but lower in ET than in SCA1 (59.7%, p = 0.011), SCA2 (77.5%, p = 0.003), and SCA6 (87.0%, p < 0.001). PC loss is not a feature of SCA3, and the median percentage of empty baskets (30.1%) was similar to controls (p = 0.303). These data provide support for PC loss in ET and are consistent with the notion that ET could represent a mild form of cerebellar degeneration with an intermediate degree of PC loss.

The Functional Organization and Integration of Purkinje Cells in the Cerebellum

The overall object of this project is to create a 3D model of the cerebellum in order to illustrate the anatomical organization of Purkinje neurons, and to provide a better understanding of how they are integrated into other locations of the brain. Because the communication between the cerebrum and cerebellum is a closed system of polysynaptic circuits, understanding the underlying anatomy and physiology is a significant challenge. Classic staining techniques do not have the resolution nor the sophistication to capture the complete organization of the cerebellar cortex, let alone the entire cerebrocerebellar circuit. To overcome this challenge, a more precise staining technique has been developed to understand the complex functional organization of the cerebellum. Using a genetically modified variant of the rabies virus, neural connectivity can be imaged with extreme detail. The rabies is modified so that the glycoprotein required for the trans‐synaptic jump is deleted and replaced with a GFP protein, thus creating the ability to accurately label the connections to a single neuron. As a result of this staining technique, the Purkinje neurons can be traced using the cutting edge tracing software Neurolucida. The traced Purkinje cell somas and their axons can then be rendered in 3D and used for analysis and quantification. Having the ability to visualize the origin of the axons of specific cell types that communicate with the deep cerebellar nuclei in high 3D resolution may begin to provide an understanding of how the input of Purkinje cells is integrated with other excitatory inputs from elsewhere in the brain. Moreover, a 3D model of the cerebellum could also provide a more complete explanation of how the cerebellum quickly and accurately coordinates the inputs and outputs into movement, and is able to predict body movements in space.Support or Funding Informationhttps://www.sciencedirect.com/science/article/pii/0306452284901933This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function.

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ataxias and other movement disorders marked by progressive degeneration of the cerebellum. The goal of this protocol is to assess mitochondrial dysfunction in Spinocerebellar ataxia type 1 (SCA1) and assess the efficacy of pharmacological targeting of metabolic respiration via the water-soluble compound succinic acid to slow disease progression. This approach is applicable to other cerebellar diseases and can be adapted to a host of water-soluble therapies. Ex vivo analysis of mitochondrial respiration is used to detect and quantify disease-related changes in mitochondrial function. With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic booster succinic acid by dissolving this compound directly into the home cage drinking water. The ability of the drug to pass the blood brain barrier can be deduced using high performance liquid chromatography (HPLC). The efficacy of these compounds can then be tested using multiple behavioral paradigms including the accelerating rotarod, balance beam test and footprint analysis. Cytoarchitectural integrity of the cerebellum can be assessed using immunofluorescence assays that detect Purkinje cell nuclei and Purkinje cell dendrites and soma. These methods are robust techniques for determining mitochondrial dysfunction and the efficacy of treatment with water-soluble compounds in cerebellar neurodegenerative disease.

Ca2+ current facilitation determines short‐term facilitation at inhibitory synapses between cerebellar Purkinje cells

Short-term facilitation takes place at GABAergic synapses between cerebellar Purkinje cells (PCs). By directly patch clamp recording from a PC axon terminal, we studied the mechanism of short-term facilitation. We show that the Ca(2+) currents elicited by high-frequency action potentials were augmented in a [Ca(2+) ]i -dependent manner. The facilitation of synaptic transmission showed 4-5th power dependence on the Ca(2+) current facilitation, and was abolished when the Ca(2+) current amplitude was adjusted to be identical. Short-term facilitation of Ca(2+) currents predominantly mediates short-term facilitation at synapses between PCs. Short-term synaptic facilitation is critical for information processing of neuronal circuits. Several Ca(2+) -dependent positive regulations of transmitter release have been suggested as candidate mechanisms underlying facilitation. However, the small sizes of presynaptic terminals have hindered the biophysical study of short-term facilitation. In the present study, by directly recording from the axon terminal of a rat cerebellar Purkinje cell (PC) in culture, we demonstrate a crucial role of [Ca(2+) ]i -dependent facilitation of Ca(2+) currents in short-term facilitation at inhibitory PC-PC synapses. Voltage clamp recording was performed from a PC axon terminal visualized by enhanced green fluorescent protein, and the Ca(2+) currents elicited by the voltage command consisting of action potential waveforms were recorded. The amplitude of presynaptic Ca(2+) current was augmented upon high-frequency paired-pulse stimulation in a [Ca(2+) ]i -dependent manner, leading to paired-pulse facilitation of Ca(2+) currents. Paired recordings from a presynaptic PC axon terminal and a postsynaptic PC soma demonstrated that the paired-pulse facilitation of inhibitory synaptic transmission between PCs showed 4-5th power dependence on that of Ca(2+) currents, and was completely abolished when the Ca(2+) current amplitude was adjusted to be identical. Thus, short-term facilitation of Ca(2+) currents predominantly mediates short-term synaptic facilitation at synapses between PCs.

Linking Essential Tremor to the Cerebellum: Neuropathological Evidence.

A fundamental question about essential tremor (ET) is whether its associated pathological changes and disease mechanisms are linkable to a specific brain region. To that end, recent tissue-based studies have made significant strides in elucidating changes in the ET brain. Emerging from these studies is increasing neuropathological evidence linking ET to the cerebellum. These studies have systematically identified a broad range of structural, degenerative changes in the ET cerebellum, spanning across all Purkinje cell compartments. These include the dendritic compartment (where there is an increase in number of Purkinje cell dendritic swellings, a pruning of the dendritic arbor, and a reduction in spine density), the cell body (where, aside from reductions in Purkinje cell linear density in some studies, there is an increase in the number of heterotopic Purkinje cell soma), and the axonal compartment (where a plethora of changes in axonal morphology have been observed, including an increase in the number of thickened axonal profiles, torpedoes, axonal recurrent collaterals, axonal branching, and terminal axonal sprouting). Additional changes, possibly due to secondary remodeling, have been observed in neighboring neuronal populations. These include a hypertrophy of basket cell axonal processes and changes in the distribution of climbing fiber-Purkinje cell synapses. These changes all distinguish ET from normal control brains. Initial studies further indicate that the profile (i.e., constellation) of these changes may separate ET from other diseases of the cerebellum, thereby serving as a disease signature. With the discovery of these changes, a new model of ET has arisen, which posits that it may be a neurodegenerative disorder centered in the cerebellar cortex. These newly emerging neuropathological studies pave the way for anatomically focused, hypothesis-driven, molecular mechanistic studies of disease pathogenesis.

Control of Inhibitory Synaptic Outputs by Low Excitability of Axon Terminals Revealed by Direct Recording

An axon is thought to faithfully conduct action potentials to its terminals. However, many features of the axon and axon terminals, especially at inhibitory synapses, remain unknown. By directly recording from the axon and terminal of a cultured cerebellar Purkinje cell (PC), we demonstrate that low membrane excitability of axon terminals shapes synaptic output. Simultaneous measurements of presynaptic capacitance and evoked IPSCs revealed PC axon terminals contained large readily releasable synaptic vesicles that exhibited a low release probability. Nevertheless, IPSCs evoked by stimulating a PC soma underwent frequency-dependent depression. Direct axonal recordings showed that high-frequency action potentials were faithfully conducted over axonal bifurcations but were attenuated around terminals. Sparse Na(+) channels relative to enriched voltage-gated K(+) channels in terminals caused short-term depression of IPSCs by reducing Ca(2+) influx. Together with confirmation in slice recordings, our findings reveal a presynaptic mechanism that shapes short-term synaptic depression without depleting releasable vesicles.

Cerebellum neurotransmission during postnatal development: [Pt(O,O′-acac)(γ-acac)(DMS)] vs cisplatin and neurotoxicity

Several chemotherapeutic drugs are known to cause neurotoxicity. Platinum-based agents in use or in clinical trials display neurotoxic potential accompanied by neurological complications; recent studies have identified a large number of behavioural issues in paediatric oncology patients.To understand the toxicity of platinum drugs at the molecular and cellular levels, this study compares the possible cytotoxic effects of an older platinum compound, cisplatin and a new platinum compound, [Pt(O,O′-acac)(γ-acac)(DMS)], on the CNS of postnatally developing rats, which is much more vulnerable to injury than the CNS of adult rats. Since several drugs interact with neurotransmitters during neuronal maturation, we performed immunostainings with antibodies raised against markers of glutamate and GABA, the major neurotransmitters in the cerebellum. After a single injection of cisplatin at postnatal day 10 (PD10), the labelling of Purkinje cells with the neurotransmitter markers evidenced alterations between PD11 and PD30, i.e. atrophy of the dendrite tree, changes in the distribution of synaptic contacts of parallel and climbing fibres, delay in the elimination of transient synapses on cell soma and severely impaired pinceau formation at the axon hillock. After treatment with [Pt(O,O′-acac)(γ-acac)(DMS)], the sole relevant change concerned the timing of climbing fibres elimination; the transient synapses disappearance on the Purkinje cell soma was delayed in some cells; instead, the growth of Purkinje cell dendrite tree was normal as was the formation of inhibitory synaptic contacts on these neurons.These findings add new evidence not only on the lower neurotoxicity of [Pt(O,O′-acac)(γ-acac)(DMS)] vs cisplatin but also on the involvement of neurotransmitters and relative synaptic connections in the maturation of central nerve tissue.

Tissue-type plasminogen activator is an extracellular mediator of Purkinje cell damage and altered gait

Purkinje neurons are a sensitive and specialised cell type important for fine motor movement and coordination. Purkinje cell damage manifests as motor incoordination and ataxia — a prominent feature of many human disorders including spinocerebellar ataxia and Huntington's disease. A correlation between Purkinje degeneration and excess cerebellar levels of tissue-type plasminogen activator (tPA) has been observed in multiple genetically-distinct models of ataxia. Here we show that Purkinje loss in a mouse model of Huntington's disease also correlates with a 200% increase in cerebellar tPA activity. That elevated tPA levels arise in a variety of ataxia models suggests that tPA is a common mediator of Purkinje damage. To address the specific contribution of tPA to cerebellar dysfunction we studied the T4 mice line that overexpresses murine tPA in postnatal neurons through the Thy1.2 gene promoter, which directs preferential expression to Purkinje cells within the cerebellum. Here we show that T4 mice develop signs of cerebellar damage within 10weeks of birth including atrophy of Purkinje cell soma and dendrites, astrogliosis, reduced molecular layer volume and altered gait. In contrast, T4 mice displayed no evidence of microgliosis, nor any changes in interneuron density, nor alteration in the cerebellar granular neuron layer. Thus, excess tPA levels may be sufficient to cause targeted Purkinje cell degeneration and ataxia. We propose that elevated cerebellar tPA levels exert a common pathway of Purkinje cell damage. Therapeutically lowering cerebellar tPA levels may represent a novel means of preserving Purkinje cell integrity and motor coordination across a wide range of neurodegenerative diseases.

Enhanced inhibitory neurotransmission in the cerebellar cortex of Atp1a3‐deficient heterozygous mice

Dystonia is characterized by excessive involuntary and prolonged simultaneous contractions of both agonist and antagonist muscles. Although the basal ganglia have long been proposed as the primary region, recent studies indicated that the cerebellum also plays a key role in the expression of dystonia. One hereditary form of dystonia, rapid-onset dystonia with parkinsonism (RDP), is caused by loss of function mutations of the gene for the Na pump α3 subunit (ATP1A3). Little information is available on the affected brain regions and mechanism for dystonia by the mutations in RDP. The Na pump is composed of α and β subunits and maintains ionic gradients of Na(+) and K(+) across the cell membrane. The gradients are utilized for neurotransmitter reuptake and their alteration modulates neural excitability. To provide insight into the molecular aetiology of RDP, we generated and analysed knockout heterozygous mice (Atp1a3(+/-)). Atp1a3(+/-) showed increased symptoms of dystonia that is induced by kainate injection into the cerebellar vermis. Atp1a3 mRNA was highly expressed in Purkinje cells and molecular-layer interneurons, and its product was concentrated at Purkinje cell soma, the site of abundant vesicular γ-aminobutyric acid transporter (VGAT) signal, suggesting the presynaptic localization of the α3 subunit in the inhibitory synapse. Electrophysiological studies showed that the inhibitory neurotransmission at molecular-layer interneuron-Purkinje cell synapses was enhanced in Atp1a3(+/-) cerebellar cortex, and that the enhancement originated via a presynaptic mechanism. Our results shed light on the role of Atp1a3 in the inhibitory synapse, and potential involvement of inhibitory synaptic dysfunction for the pathophysiology of dystonia.

Mature Purkinje Cells Require the Retinoic Acid-Related Orphan Receptor-α (RORα) to Maintain Climbing Fiber Mono-Innervation and Other Adult Characteristics

Neuronal maturation during development is a multistep process regulated by transcription factors. The transcription factor RORα (retinoic acid-related orphan receptor α) is necessary for early Purkinje cell (PC) maturation but is also expressed throughout adulthood. To identify the role of RORα in mature PCs, we used Cre-lox mouse genetic tools in vivo that delete it specifically from PCs between postnatal days 10-21. Up to 14 d of age, differences between mutant and control PCs were not detectable: both were mono-innervated by climbing fibers (CFs) extending along their well-developed dendrites with spiny branchlets. By week 4, mutant mice were ataxic, some PCs had died, and remaining PC soma and dendrites were atrophic, with almost complete disappearance of spiny branchlets. The innervation pattern of surviving RORα-deleted PCs was abnormal with several immature characteristics. Notably, multiple functional CF innervation was reestablished on these mature PCs, simultaneously with the relocation of CF contacts to the PC soma and their stem dendrite. This morphological modification of CF contacts could be induced even later, using lentivirus-mediated depletion of rora from adult PCs. These data show that the late postnatal expression of RORα cell-autonomously regulates the maintenance of PC dendritic complexity, and the CF innervation status of the PC (dendritic vs somatic contacts, and mono-innervation vs multi-innervation). Thus, the differentiation state of adult neurons is under the control of transcription factors; and in their absence, adult neurons lose their mature characteristics and acquire some characteristics of an earlier developmental stage.

Purkinje Cell Ataxin-1 Modulates Climbing Fiber Synaptic Input in Developing and Adult Mouse Cerebellum

Previous studies indicate that while transgenic mice with ATXN1[30Q]-D776-induced disease share pathological features caused by ATXN1[82Q] having an expanded polyglutamine tract, they fail to manifest the age-related progressive neurodegeneration seen in spinocerebellar ataxia type 1. The shared features include morphological alterations in climbing fiber (CF) innervation of Purkinje cells (PCs). To further investigate the ability of ataxin-1 (ATXN1) to impact CF/PC innervation, this study used morphological and functional approaches to examine CF/PC innervation during postnatal development in ATXN1[30Q]-D776 and ATXN1[82Q] cerebella. Notably, ATXN1[30Q]-D776 induced morphological alterations consistent with the development of the innervation of PCs by CFs being compromised, including a reduction of CF translocation along the PC dendritic tree, and decreased pruning of CF terminals from the PC soma. As previously shown for ATXN1[82Q], ATXN1[30Q]-D776 must enter the nucleus of PCs to induce these alterations. Experiments using conditional ATXN1[30Q]-D776 mice demonstrate that both the levels and specific timing of mutant ATXN1 expression are critical for alteration of the CF-PC synapse. Together these observations suggest that ATXN1, expressed exclusively in PCs, alters expression of a gene(s) in the postsynaptic PC that are critical for its innervation by CFs. To investigate whether ATXN1[30Q]-D776 curbs the progressive disease in ATXN1[82Q]-S776 mice, we crossed ATXN1[30Q]-D776 and ATXN1[82Q]-S776 mice and found that double transgenic mice developed progressive PC atrophy. Thus, the results also show that to develop progressive cerebellar degeneration requires expressing ATXN1 with an expanded polyglutamine tract.