Ataxic Mice Research Articles

Frataxin deficiency unveils cell-context dependent actions of insulin-like growth factor I on neurons

BackgroundFriedreich’s ataxia (FRDA) is a neurodegenerative disease caused by deficiency of the mitochondrial iron chaperone frataxin (Fxn). FRDA has no cure, but disease-modifying strategies to increase frataxin are under study. Because insulin-like growth factor I (IGF-I) has therapeutic effects in various types of cerebellar ataxia and exerts protective actions on mitochondrial function, we explored the potential Fxn-stimulating activity of this growth factor on brain cells.ResultsIGF-I normalized frataxin levels in frataxin-deficient neurons and astrocytes through its canonical Akt/mTOR signaling pathway. IGF-I also stimulated frataxin in normal astrocytes but not in normal neurons, whereas IGF-I stimulated the Akt/mTOR pathway in both types of cells. This cell context-dependent action of IGF-I on neurons suggested that the intrinsic regulation of Fxn in neurons is different than in astrocytes. Indeed, neurons express much higher levels of frataxin and are much more sensitive to Fxn deficiency than astrocytes; i.e.: only neurons die in the absence of frataxin. In addition, the half-life of frataxin is shorter in neurons than in astrocytes, while after blockade of the proteasome only neurons responded to IGF-I with an increase in frataxin levels. We also explore a potential therapeutic utility of IGF-I in FRDA-like transgenic mice (YG8R mice) and found that treatment with IGF-I normalized motor coordination in these moderately ataxic mice.ConclusionExposure to IGF-I unveiled a cell-specific regulation of frataxin in neurons as compared to astrocytes. Collectively, these results indicate that IGF-I exerts cell-context neuroprotection in frataxin deficiency that maybe therapeutically effective.

ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice

Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)31. This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissue-specific effects, which may be partially alleviated by the induction of FBXW8.

Kv1.1 knock-in ataxic mice exhibit spontaneous myokymic activity exacerbated by fatigue, ischemia and low temperature

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by myokymia and attacks of ataxic gait often precipitated by stress. Several genetic mutations have been identified in the Shaker-like K+ channel Kv1.1 (KCNA1) of EA1 individuals, including V408A, which result in remarkable channel dysfunction. By inserting the heterozygous V408A, mutation in one Kv1.1 allele, a mouse model of EA1 has been generated (Kv1.1V408A/+). Here, we investigated the neuromuscular transmission of Kv1.1V408A/+ ataxic mice and their susceptibility to physiologically relevant stressors. By using in vivo preparations of lateral gastrocnemius (LG) nerve–muscle from Kv1.1+/+ and Kv1.1V408A/+ mice, we show that the mutant animals exhibit spontaneous myokymic discharges consisting of repeated singlets, duplets or multiplets, despite motor nerve axotomy. Two-photon laser scanning microscopy from the motor nerve, ex vivo, revealed spontaneous Ca2+ signals that occurred abnormally only in preparations dissected from Kv1.1V408A/+ mice. Spontaneous bursting activity, as well as that evoked by sciatic nerve stimulation, was exacerbated by muscle fatigue, ischemia and low temperatures. These stressors also increased the amplitude of compound muscle action potential. Such abnormal neuromuscular transmission did not alter fiber type composition, neuromuscular junction and vascularization of LG muscle, analyzed by light and electron microscopy. Taken together these findings provide direct evidence that identifies the motor nerve as an important generator of myokymic activity, that dysfunction of Kv1.1 channels alters Ca2+ homeostasis in motor axons, and also strongly suggest that muscle fatigue contributes more than PNS fatigue to exacerbate the myokymia/neuromyotonia phenotype. More broadly, this study points out that juxtaparanodal K+ channels composed of Kv1.1 subunits exert an important role in dampening the excitability of motor nerve axons during fatigue or ischemic insult.

A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease

A type of lower motor neuron (LMN) disease inherited as autosomal recessive in Romney sheep was characterized with normal appearance at birth, but with progressive weakness and tetraparesis after the first week of life. Here, we carried out genome-wide homozygosity mapping using Illumina Ovine SNP50 BeadChips on lambs descended from one carrier ram, including 19 sheep diagnosed as affected and 11 of their parents that were therefore known carriers. A homozygous region of 136 consecutive single-nucleotide polymorphism (SNP) loci on chromosome 2 was common to all affected sheep and it was the basis for searching for the positional candidate genes. Other homozygous regions shared by all affected sheep spanned eight or fewer SNP loci. The 136-SNP region contained the sheep ATP/GTP-binding protein 1 (AGTPBP1) gene. Mutations in this gene have been shown to be related to Purkinje cell degeneration (pcd) phenotypes including ataxia in mice. One missense mutation c.2909G>C on exon 21 of AGTPBP1 was discovered, which induces an Arg to Pro substitution (p.Arg970Pro) at amino-acid 970, a conserved residue for the catalytic activity of AGTPBP1. Genotyping of this mutation showed 100% concordant rate with the recessive pattern of inheritance in affected, carrier, phenotypically normal and unrelated normal individuals. This is the first report showing a mutant AGTPBP1 is associated with a LMN disease in a large mammal animal model. Our finding raises the possibility of human patients with the same etiology caused by this gene or other genes in the same pathway of neuronal development.

Abnormal grooming activity in Dab1scm (scrambler) mutant mice

Dab1scm mutant mice, characterized by cell ectopias and degeneration in cerebellum, hippocampus, and neocortex, were compared to non-ataxic controls for different facets of grooming caused by brief water immersions, as well as some non-grooming behaviors. Dab1scm mutants were strongly affected in their quantitative functional parameters, exhibiting higher starting latencies before grooming relative to non-ataxic littermates of the A/A strain, fewer grooming bouts, and grooming components of shorter duration, with an unequal regional distribution targeting almost totally the rostral part (head washing and forelimb licking) of the animal. Only bouts of a single grooming element were preserved. The cephalocaudal order of grooming elements appeared less disorganized, mutant and control mice initiating the grooming with head washing and forelimb licking prior to licking posterior parts. However, mutants differed from controls in that all their bouts were incomplete but uninterrupted, although intergroup difference for percentage of the incorrect transitions was not significant. In contrast to grooming, Dab1scm mice ambulated for a longer time. During walking episodes, they exhibited more body scratching than controls, possibly to compensate for the lack of licking different body parts. In conjunction with studies with other ataxic mice, these results indicate that the cerebellar cortex affects grooming activity and is consequently involved in executing various components, but not in its sequential organization, which requires other brain regions such as cerebral cortices or basal ganglia.

Altered neurotransmitter release machinery in mice deficient for the deubiquitinating enzyme Usp14

Homozygous ataxic mice (ax(J)) express reduced levels of the deubiquitinating enzyme Usp14. They develop severe tremors by 2-3 wk of age, followed by hindlimb paralysis, and death by 6-8 wk. While changes in the ubiquitin proteasome system often result in the accumulation of ubiquitin protein aggregates and neuronal loss, these pathological markers are not observed in the ax(J) mice. Instead, defects in neurotransmission were observed in both the central and peripheral nervous systems of ax(J) mice. We have now identified several new alterations in peripheral neurotransmission in the ax(J) mice. Using the two-microelectrode voltage clamp technique on diaphragm muscles of ax(J) mice, we observed that under normal neurotransmitter release conditions ax(J) mice lacked paired-pulse facilitation and exhibited a frequency-dependent increase in rundown of the end plate current at high-frequency stimulation (HFS). Combined electrophysiology and styryl dye staining revealed a significant reduction in quantal content during the initial and plateau portions of the HFS train. In addition, uptake of styryl dyes (FM dye) during HFS demonstrated that the size of the readily releasable vesicle pool was significantly reduced. Destaining rates for styryl dyes suggested that ax(J) neuromuscular junctions are unable to mobilize a sufficient number of vesicles during times of intense activity. These results imply that ax(J) nerve terminals are unable to recruit a sufficient number of vesicles to keep pace with physiological rates of transmitter release. Therefore, ubiquitination of synaptic proteins appears to play an important role in the normal operation of the neurotransmitter release machinery and in regulating the size of pools of synaptic vesicles.

Selective loss of AMPA receptor subunits at inhibitory neuron synapses in the cerebellum of the ataxic stargazer mouse

AMPA receptor subunits (GluA1–4) are trafficked to membrane synaptic sites by transmembrane AMPA receptor regulatory proteins (TARPs). In the stargazer mutant mouse, expression of TARP-γ2 (stargazin) is severely reduced, resulting in cerebellar ataxia. Stargazer granule cells (GCs) have a complete loss of functional AMPARs, as γ2 is their main TARP; hence mossy fiber (MF)–GC synapses are silent. The aim of the current study was to investigate how the stargazin deficit affects expression levels of AMPAR subunits at output synapses from GC parallel fibers (PF) onto inhibitory neurons in the molecular layer. Cerebella from male litter-pairs of stargazer and control mice were analyzed by post-embedding immunogold-microscopy. Levels of GluA2/3 and GluA4 were evaluated by measuring relative density of immunogold at PF–Purkinje cell (PF–PC) and PF–interneuron (PF–In) synapses respectively. In total, 100 synapses were analyzed in each pair of stargazer and control littermates. GluA2/3 and GluA4 expression was significantly reduced throughout the stargazer cerebellar cortex. GluA2/3 levels were reduced by 52% (p < 0.001) at PF–PC synapses, and GluA4 levels by 31% (p < 0.001) at PF–In synapses in stargazers. In neither case, however, was there a total loss of synaptic AMPAR subunits as occurs at MF–GC synapses. As the inhibitory neurons of the molecular layer express other TARPs in addition to stargazin, TARP compensation may limit the loss of GluA subunits at these synapses and explain why they are not silent like the MF–GC synapses. These data suggest that the ataxic phenotype in stargazers is primarily due to absence of AMPARs at cerebellar MF–GC synapses.

Purkinje cell-specific ablation of Cav2.1 channels is sufficient to cause cerebellar ataxia in mice.

The Cacna1a gene encodes the α1A subunit of voltage-gated CaV2.1 Ca2+ channels that are involved in neurotransmission at central synapses. CaV2.1-α1-knockout (α1KO) mice, which lack CaV2.1 channels in all neurons, have a very severe phenotype of cerebellar ataxia and dystonia, and usually die around postnatal day 20. This early lethality, combined with the wide expression of CaV2.1 channels throughout the cerebellar cortex and nuclei, prohibited determination of the contribution of particular cerebellar cell types to the development of the severe neurobiological phenotype in Cacna1a mutant mice. Here, we crossed conditional Cacna1a mice with transgenic mice expressing Cre recombinase, driven by the Purkinje cell-specific Pcp2 promoter, to specifically ablate the CaV2.1-α1A subunit and thereby CaV2.1 channels in Purkinje cells. Purkinje cell CaV2.1-α1A-knockout (PCα1KO) mice aged without difficulties, rescuing the lethal phenotype seen in α1KO mice. PCα1KO mice exhibited cerebellar ataxia starting around P12, much earlier than the first signs of progressive Purkinje cell loss, which appears in these mice between P30 and P45. Secondary cell loss was observed in the granular and molecular layers of the cerebellum and the volume of all individual cerebellar nuclei was reduced. In this mouse model with a cell type-specific ablation of CaV2.1 channels, we show that ablation of CaV2.1 channels restricted to Purkinje cells is sufficient to cause cerebellar ataxia. We demonstrate that spatial ablation of CaV2.1 channels may help in unraveling mechanisms of human disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s12311-011-0302-1) contains supplementary material, which is available to authorized users.

Rpl27a mutation in the sooty foot ataxia mouse phenocopies high p53 mouse models

Ribosomal stress is an important, yet poorly understood, mechanism that results in activation of the p53 tumour suppressor. We present a mutation in the ribosomal protein Rpl27a gene (sooty foot ataxia mice), isolated through a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for p53 pathway defects, that shares striking phenotypic similarities with high p53 mouse models, including cerebellar ataxia, pancytopenia and epidermal hyperpigmentation. This phenocopy is rescued in a haploinsufficient p53 background. A detailed examination of the bone marrow in these mice identified reduced numbers of haematopoietic stem cells and a p53-dependent c-Kit down-regulation. These studies suggest that reduced Rpl27a increases p53 activity in vivo, further evident with a delay in tumorigenesis in mutant mice. Taken together, these data demonstrate that Rpl27a plays a crucial role in multiple tissues and that disruption of this ribosomal protein affects both development and transformation.

Patterned Purkinje cell loss in the ataxic sticky mouse

The ataxic sticky (sti/sti) mouse is a spontaneous autosomal recessive mutant resulting from a disruption in the editing domain of the alanyl-tRNA synthetase (Aars) gene. The sticky phenotype is characterized by a small body size, a characteristic unkempt coat and neurological manifestations including marked tremor and ataxia starting at 6 weeks of age. The present study was undertaken to examine the spatiotemporal features of Purkinje cell degeneration in the sticky mouse. Purkinje cell loss was found to be both progressive and patterned, with vermal lobules VI, IX and X, crus 1 of the hemisphere, and the flocculus and paraflocculus being differentially resistant to degeneration. The pattern of Purkinje cell degeneration in sticky is not random - in general, the sphingosine kinase 1a-immunonegative Purkinje cell subset is preferentially susceptible to early cell death. In addition, zebrin II/aldolase C expression in the sticky cerebellum is profoundly downregulated, whereas the heat-shock protein 25 is both ectopically expressed in some scattered Purkinje cells and downregulated in other Purkinje cells in which it is normally expressed constitutively. Compared with many mouse mutants with patterned Purkinje cell death, in which successive stripes of cell loss are very clear, Purkinje cell loss in sticky shows a less clear-cut pattern between different Purkinje cell subtypes, with the result that preferential survival is less dramatic. This may represent a secondary consequence of the downregulation of zebrin II expression.

Human umbilical mesenchymal stem cells enhance the expression of neurotrophic factors and protect ataxic mice

Cerebellar ataxias, which comprise a wide spectrum of progressive disorders, are incurable at present. It has been reported that human umbilical mesenchymal stem cell (HU-MSC) transplantation has a protective effect on neurodegenerative diseases. In this study, we investigated the effect of HU-MSCs on ataxic mice induced by cytosine beta-D-arabinofuranoside (Ara-C). The ataxic mouse received an intravenous injection of 2 × 10 6 HU-MSCs once a week for three consecutive weeks. Neurological function was scored weekly by rotarod test and open field test. The mouse cerebellar volume and weight were also measured. The apoptotic cells, pathological alternations and distribution of HU-MSCs were determined by TUNEL assay and immunohistochemistry staining respectively. Double immunostaining was carried out to investigate the dynamics of HU-MSCs in the host animals. Neurotrophic factors in cerebellar tissue and serum were measured by Q-PCR and ELISA. Our results showed that HU-MSCs implantation significantly improved the motor skills of ataxic mice 8 weeks after application. HU-MSCs also alleviated cerebellar atrophy and decreased the number of apoptotic cells in the therapeutic group. Implanted HU-MSCs stayed in cerebellum for at least three months with no obvious differentiation. HU-MSC treated mice had enhanced expression of insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) in cerebellum extraction and blood serum. Double immunostaining revealed that a few MAB1287 positive cells co-localized with IGF-1 or VEGF express cells. Our results suggest that HU-MSC treatment is capable of alleviating the motor impairments and cerebellar atrophy in the ataxic mouse model, probably via promoting particular neurotrophic factors.

Smad2 Protein Disruption in the Central Nervous System Leads to Aberrant Cerebellar Development and Early Postnatal Ataxia in Mice

Smad2 is a critical mediator of TGF-β signals that are known to play an important role in a wide range of biological processes in various cell types. Its role in the development of the CNS, however, is largely unknown. Mice lacking Smad2 in the CNS (Smad2-CNS-KO) were generated by a Cre-loxP approach. These mice exhibited behavioral abnormalities in motor coordination from an early postnatal stage and mortality at approximately 3 weeks of age, suggestive of severe cerebellar dysfunction. Gross observation of Smad2-CNS-KO cerebella demonstrated aberrant foliations in lobule IX and X. Further analyses revealed increased apoptotic cell death, delayed migration and maturation of granule cells, and retardation of dendritic arborization of Purkinje cells. These findings indicate that Smad2 plays a key role in cerebellar development and motor function control.

Altered Purkinje cell responses and calmodulin expression in the spontaneously ataxic mouse, Pogo

Ataxia is often associated with altered cerebellar motor control, a process in which Purkinje cells (PCs) play a principal role. Pogo mice display severe motor deficits characterized by an ataxic gait accompanying hindlimb hyperextension. Here, using whole-cell patch-clamp recordings, we show that parallel fiber (PF)-excitatory post-synaptic currents (PF-EPSCs) are reduced, paired-pulse facilitation (PPF) is increased and PF-PC long-term depression (LTD) is impaired in Pogo mice; in contrast, climbing-fiber EPSCs are preserved. In control mice, treatment with the calmodulin antagonist calmidazolium (5 μm) impaired PPF and LTD. Notably, cerebellar calmodulin expression was significantly reduced in Pogo mice compared with control mice. Control PCs predominantly exhibited a tonic firing pattern, whereas the firing pattern in Pogo PCs was mainly a complex burst type. These results implicate alterations in PC responses and calmodulin content in the abnormal cerebellar function of Pogo mice.

Nucleolar Disruption and Cajal Body Disassembly are Nuclear Hallmarks of DNA Damage‐Induced Neurodegeneration in Purkinje Cells

The Purkinje cell (PC) degeneration (pcd) phenotype results from mutation in nna1 gene and is associated with the degeneration and death of PCs during the postnatal life. Although the pcd mutation is a model of the ataxic mouse, it shares clinical and pathological characteristics of inherited human spinocerebellar ataxias. PC degeneration in pcd mice provides a useful neuronal system to study nuclear mechanisms involved in DNA damage-dependent neurodegeneration, particularly the contribution of nucleoli and Cajal bodies (CBs). Both nuclear structures are engaged in housekeeping functions for neuronal survival, the biogenesis of ribosomes and the maturation of snRNPs and snoRNPs required for pre-mRNA and pre-rRNA processing, respectively. In this study, we use ultrastructural analysis, in situ transcription assay and molecular markers for DNA damage, nucleoli and CB components to demonstrate that PC degeneration involves the progressive accumulation of nuclear DNA damage associated with disruption of nucleoli and CBs, disassembly of polyribosomes into monoribosomes, ribophagy and shut down of nucleolar and extranucleolar transcription. Microarray analysis reveals that four genes encoding repressors of nucleolar rRNA synthesis (p53, Rb, PTEN and SNF2) are upregulated in the cerebellum of pcd mice. Collectively, these data support that nucleolar and CB alterations are hallmarks of DNA damage-induced neurodegeneration.

Stargazin Modulates Neuronal Voltage-dependent Ca2+ Channel Cav2.2 by a Gβγ-dependent Mechanism

Loss of neuronal protein stargazin (gamma(2)) is associated with recurrent epileptic seizures and ataxia in mice. Initially, due to homology to the skeletal muscle calcium channel gamma(1) subunit, stargazin and other family members (gamma(3-8)) were classified as gamma subunits of neuronal voltage-gated calcium channels (such as Ca(V)2.1-Ca(V)2.3). Here, we report that stargazin interferes with G protein modulation of Ca(V)2.2 (N-type) channels expressed in Xenopus oocytes. Stargazin counteracted the Gbetagamma-induced inhibition of Ca(V)2.2 channel currents, caused either by coexpression of the Gbetagamma dimer or by activation of a G protein-coupled receptor. Expression of high doses of Gbetagamma overcame the effects of stargazin. High affinity Gbetagamma scavenger proteins m-cbetaARK and m-phosducin produced effects similar to stargazin. The effects of stargazin and m-cbetaARK were not additive, suggesting a common mechanism of action, and generally independent of the presence of the Ca(V)beta(3) subunit. However, in some cases, coexpression of Ca(V)beta(3) blunted the modulation by stargazin. Finally, the Gbetagamma-opposing action of stargazin was not unique to Ca(V)2.2, as stargazin also inhibited the Gbetagamma-mediated activation of the G protein-activated K(+) channel. Purified cytosolic C-terminal part of stargazin bound Gbetagamma in vitro. Our results suggest that the regulation by stargazin of biophysical properties of Ca(V)2.2 are not exerted by direct modulation of the channel but via a Gbetagamma-dependent mechanism.

CaV2.1 channelopathies

Mutations in the CACNA1A gene that encodes the pore-forming alpha1 subunit of human voltage-gated CaV2.1 (P/Q-type) Ca2+ channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2, and spinocerebellar ataxia type 6 (SCA6). For each channelopathy, the review describes the disease phenotype as well as the functional consequences of the disease-causing mutations on recombinant human CaV2.1 channels and, in the case of FHM1 and SCA6, on neuronal CaV2.1 channels expressed at the endogenous physiological level in knockin mouse models. The effects of FHM1 mutations on cortical spreading depression, the phenomenon underlying migraine aura, and on cortical excitatory and inhibitory synaptic transmission in FHM1 knockin mice are also described, and their implications for the disease mechanism discussed. Moreover, the review describes different ataxic spontaneous cacna1a mouse mutants and the important insights into the cerebellar mechanisms underlying motor dysfunction caused by mutant CaV2.1 channels that were obtained from their functional characterization.

Zebrin II Expressing Purkinje Cell Phenotype—Related and—Unrelated Cerebellar Abnormalities in Ca˅2.1 Mutant, Rolling Mouse Nagoya

Rolling mouse Nagoya is an ataxic mutant mouse that carries a mutation in a gene encoding for the alpha 1A subunit of the voltage-gated P/Q-type Ca2+ channel (Ca˅2.1). This report summarizes our studies and others concerning cerebellar abnormalities in rolling mice based on chemical neuroanatomy. While there are no obvious cerebellar deformations in this mutant mouse, the altered functions of Purkinje cells can be revealed as a reduced expression of type 1 ryanodine receptor (RyR1) in all Purkinje cells uniformly throughout the cerebellum, and as an ectopic expression of tyrosine hydroxylase (TH) in the Purkinje cell subsets with the zebrin II—immunopositive phenotype. As the mutated Ca˅2.1 channel is expressed at uniform levels in all Purkinje cells, its copresence with RyR1 staining suggests that a Ca˅2.1 channel dysfunction links with the expression of RyR1 in Purkinje cells of rolling mice. However, an ectopic expression of TH in the Purkinje cells is topologically related to the projection of corticotrophin-releasing factor—immunopositive climbing fibers rather than expression of the mutated Ca˅2.1 channel. On the other hand, increased levels of serotonin (5-HT) in 5-HTergic fibers were revealed immunohistochemically in Purkinje cells of the vermis of rolling cerebellum. Thus, to determine whether or not cerebellar abnormalities are related to Purkinje cell populations revealed by zebrin II expression is essential for enhancing our understanding of the pathogenesis of hereditary cerebellar ataxic mutants such as rolling mice.

AF4 Is a Critical Regulator of the IGF-1 Signaling Pathway during Purkinje Cell Development

Deregulation of the insulin-like growth factor 1 (IGF-1) signaling pathway is a recurrent finding in mouse models and human patients with cerebellar ataxia and thus represents a common pathological cascade in neuronal cell death that may be targeted for therapy. We have previously identified a point mutation in AF4, a transcription cofactor of RNA polymerase II elongation and chromatin remodeling, that causes progressive and highly specific Purkinje cell (PC) death in the ataxic mouse mutant robotic, leading to the accumulation of AF4 in PCs. Here we confirm that the spatiotemporal pattern of PC degeneration in the robotic cerebellum correlates with the specific profile of AF4 upregulation. To identify the underlying molecular pathways, we performed microarray gene expression analysis of PCs obtained by laser capture microdissection (LCM) at the onset of degeneration. Igf-1 was significantly downregulated in robotic PCs compared with wild-type controls before and throughout the degenerative process. Consistently, we observed a decrease in the activation of downstream signaling molecules including type 1 IGF receptor (IGF-1R) and the extracellular signal-regulated kinase (ERK) 1 and ERK2. Chromatin immunoprecipitation confirmed that Igf-1 is a direct and the first validated target of the AF4 transcriptional regulatory complex, and treatment of presymptomatic robotic mice with IGF-1 indeed markedly delayed the progression of PC death. This study demonstrates that small changes in the levels of a single transcriptional cofactor can deleteriously affect normal cerebellum function and opens new avenues of research for the manipulation of the IGF-1 pathway in the treatment of cerebellar ataxia in humans.

Lentiviral vector-mediated rescue of motor behavior in spontaneously occurring hereditary ataxic mice

Hotfoot5J mice are spontaneously occurring ataxic mice that lack δ2 glutamate receptor (GluRδ2) protein in cerebellar Purkinje cells. Here we aimed to rescue the ataxic phenotype of hotfoot5J mice by lentiviral vector-mediated expression of recombinant GluRδ2 in Purkinje cells. Lentiviral vectors expressing GluRδ2 were injected into the cerebellar cortex of hotfoot5J mice 6 or 7 days after birth, and the effects were studied on postnatal day 30. The motor behavior of hotfoot5J mice treated with vectors expressing GluRδ2 was markedly rescued, whereas the ataxia of hotfoot5J mice treated with vectors expressing GFP was comparable to that of non-injected hotfoot5J littermates. Furthermore, the impaired release probability of glutamate from parallel fiber terminals and the failure of developmental elimination of surplus climbing fibers from Purkinje cells in hotfoot5J mice were completely rescued by GluRδ2 expression. These results indicate the therapeutic potential of viral vector-based gene therapy for hereditary cerebellar ataxia and other neuronal disorders.

Loss of calcium channels in the cerebellum of the ataxic and epileptic stargazer mutant mouse

The stargazer mouse displays cerebellar ataxia and absence epilepsy as a result of a single, recessive mutation on chromosome 15 which silences the expression of the voltage-dependent calcium channel (VDCC) subunit γ2, termed stargazin. Stargazin is the predominant γ-subunit expressed in the cerebellum and is essential for correct assembly and trafficking of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-subtype of glutamate receptors (AMPARs) to postsynaptic membranes. As a functional association between AMPARs and VDCCs has been reported, and loss of stargazin results in a loss of AMPA receptors at cerebellar synapses, we investigated whether the loss of stargazin might also change the expression levels of calcium channels at cerebellar synapses. We present data showing that the stargazin mutation affects the expression of postsynaptic L-type Cav1.2 (α1C-class) but not presynaptic P/Q-type Cav2.1 (α1A-class) calcium channel proteins at cerebellar synapses. Both Western blot and immunogold analyses demonstrated a significant reduction in the levels of L-type calcium channel Cav1.2 at stargazer cerebellar synapses compared to their non-ataxic littermates. This is in contrast to stargazer hippocampal synapses where no differences were detected in Cav1.2 and 2.1 levels compared to controls, likely due to compensation by subunit γ8. The loss of L-type calcium channel Cav1.2 at stargazer cerebellar synapses suggests that stargazin mutation may contribute to the loss of VDCCs at postsynaptic sites. It is therefore possible that stargazin is involved in the trafficking of both AMPARs and VDCCs or in the formation of a functional AMPA receptor–calcium channel complex in the postsynaptic membrane.