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1.  Recessive Mutations in SPTBN2 Implicate β-III Spectrin in Both Cognitive and Motor Development 
PLoS Genetics  2012;8(12):e1003074.
β-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding β-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as “Lincoln ataxia,” because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of β-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that β-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.
Author Summary
β-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Mutations in β-III spectrin cause spinocerebellar ataxia type 5 (SCA5), sometimes called Lincoln ataxia because it was first described in the relatives of United States President Abraham Lincoln. This is generally an adult-onset progressive cerebellar disorder. Recessive mutations have not previously been described in any of the brain spectrins. We identified a homozygous mutation in SPTBN2, which causes a more severe disorder than SCA5, with a developmental cerebellar ataxia, which is present from childhood; in addition there is marked cognitive impairment. We call this novel condition SPARCA1 (Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1). This condition could be caused by two separate gene mutations; but we show, using a combination of genome-wide mapping, whole-genome sequencing, and detailed behavioural and neuropathological analysis of a β-III spectrin mouse knockout, that both the ataxia and cognitive impairment are caused by the recessive mutations in β-III spectrin. SPARCA1 is one of a family of neuronal spectrinopathies and illustrates the importance of spectrins in brain development and function.
doi:10.1371/journal.pgen.1003074
PMCID: PMC3516553  PMID: 23236289
2.  β-III spectrin is critical for development of Purkinje cell dendritic tree and spine morphogenesis 
The Journal of Neuroscience  2011;31(46):16581-16590.
Mutations in the gene encoding β-III spectrin give rise to spinocerebellar ataxia type 5 (SCA5), a neurodegenerative disease characterized by progressive thinning of the molecular layer, loss of Purkinje cells and increasing motor deficits. A mouse lacking full-length β-III spectrin (β-III−/−) displays a similar phenotype. In vitro and in vivo analyses of Purkinje cells lacking β-III spectrin, reveal a critical role for β-III spectrin in Purkinje cell morphological development. Disruption of the normally well-ordered dendritic arborization occurs in Purkinje cells from β-III−/− mice, specifically showing a loss of monoplanar organization, smaller average dendritic diameter and reduced densities of Purkinje cell spines and synapses. Early morphological defects appear to affect distribution of dendritic, but not axonal, proteins. This study confirms that thinning of the molecular layer associated with disease pathogenesis is a consequence of Purkinje cell dendritic degeneration, as Purkinje cells from 8-month old β-III−/− mice have drastically reduced dendritic volumes, surface areas and total dendritic lengths compared to 5–6 week old β-III−/− mice. These findings highlight a critical role of β-III spectrin in dendritic biology and are consistent with an early developmental defect in β-III−/− mice, with abnormal Purkinje cell dendritic morphology potentially underlying disease pathogenesis.
doi:10.1523/JNEUROSCI.3332-11.2011
PMCID: PMC3374928  PMID: 22090485
3.  Spectrin mutations that cause spinocerebellar ataxia type 5 impair axonal transport and induce neurodegeneration in Drosophila 
The Journal of Cell Biology  2010;189(1):143-158.
How spectrin mutations caused Purkinje cell death becomes clearer following studies that examined the effect of expressing mutant SCA5 in the fly eye. Mutant spectrin causes deficits in synapse formation at the neuromuscular junction and disrupts vesicular trafficking.
Spinocerebellar ataxia type 5 (SCA5) is an autosomal dominant neurodegenerative disorder caused by mutations in the SPBTN2 gene encoding β-III–spectrin. To investigate the molecular basis of SCA5, we established a series of transgenic Drosophila models that express human β-III–spectrin or fly β-spectrin proteins containing SCA5 mutations. Expression of the SCA5 mutant spectrin in the eye causes a progressive neurodegenerative phenotype, and expression in larval neurons results in posterior paralysis, reduced synaptic terminal growth, and axonal transport deficits. These phenotypes are genetically enhanced by both dynein and dynactin loss-of-function mutations. In summary, we demonstrate that SCA5 mutant spectrin causes adult-onset neurodegeneration in the fly eye and disrupts fundamental intracellular transport processes that are likely to contribute to this progressive neurodegenerative disease.
doi:10.1083/jcb.200905158
PMCID: PMC2854382  PMID: 20368622
4.  Genome-wide mRNA sequencing of a single canine cerebellar cortical degeneration case leads to the identification of a disease associated SPTBN2 mutation 
BMC Genetics  2012;13:55.
Background
Neonatal cerebellar cortical degeneration is a neurodegenerative disease described in several canine breeds including the Beagle. Affected Beagles are unable to ambulate normally from the onset of walking and the main pathological findings include Purkinje cell loss with swollen dendritic processes. Previous reports suggest an autosomal recessive mode of inheritance. The development of massively parallel sequencing techniques has presented the opportunity to investigate individual clinical cases using genome-wide sequencing approaches. We used genome-wide mRNA sequencing (mRNA-seq) of cerebellum tissue from a single Beagle with neonatal cerebellar cortical degeneration as a method of candidate gene sequencing, with the aim of identifying the causal mutation.
Results
A four-week old Beagle dog presented with progressive signs of cerebellar ataxia and the owner elected euthanasia. Histopathology revealed findings consistent with cerebellar cortical degeneration. Genome-wide mRNA sequencing (mRNA-seq) of RNA from cerebellum tissue was used as a method of candidate gene sequencing. After analysis of the canine orthologues of human spinocerebellar ataxia associated genes, we identified a homozygous 8 bp deletion in the β-III spectrin gene, SPTBN2, associated with spinocerebellar type 5 in humans. Genotype analysis of the sire, dam, ten clinically unaffected siblings, and an affected sibling from a previous litter, showed the mutation to fully segregate with the disorder. Previous studies have shown that β-III spectrin is critical for Purkinje cell development, and the absence of this protein can lead to cell damage through excitotoxicity, consistent with the observed Purkinje cell loss, degeneration of dendritic processes and associated neurological dysfunction in this Beagle.
Conclusions
An 8 bp deletion in the SPTBN2 gene encoding β-III spectrin is associated with neonatal cerebellar cortical degeneration in Beagle dogs. This study shows that mRNA-seq is a feasible method of screening candidate genes for mutations associated with rare diseases when a suitable tissue resource is available.
doi:10.1186/1471-2156-13-55
PMCID: PMC3413603  PMID: 22781464
Beta-III spectrin; Beagle dogs; Cerebellar cortical degeneration; Spinocerebellar ataxia type 5; Genome-wide mRNA sequencing; Cerebellum; mRNA-seq; SPTBN2; Canine; Next generation sequencing
5.  Lithium Therapy Improves Neurological Function and Hippocampal Dendritic Arborization in a Spinocerebellar Ataxia Type 1 Mouse Model 
PLoS Medicine  2007;4(5):e182.
Background
Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disorder characterized by progressive motor and cognitive dysfunction. Caused by an expanded polyglutamine tract in ataxin 1 (ATXN1), SCA1 pathogenesis involves a multifactorial process that likely begins with misfolding of ATXN1, which has functional consequences on its interactions, leading to transcriptional dysregulation. Because lithium has been shown to exert neuroprotective effects in a variety of conditions, possibly by affecting gene expression, we tested the efficacy of lithium treatment in a knock-in mouse model of SCA1 (Sca1154Q/2Q mice) that replicates many features of the human disease.
Methods and Findings
Sca1154Q/2Q mice and their wild-type littermates were fed either regular chow or chow that contained 0.2% lithium carbonate. Dietary lithium carbonate supplementation resulted in improvement of motor coordination, learning, and memory in Sca1154Q/2Q mice. Importantly, motor improvement was seen when treatment was initiated both presymptomatically and after symptom onset. Neuropathologically, lithium treatment attenuated the reduction of dendritic branching in mutant hippocampal pyramidal neurons. We also report that lithium treatment restored the levels of isoprenylcysteine carboxyl methyltransferase (Icmt; alternatively, Pccmt), down-regulation of which is an early marker of mutant ATXN1 toxicity.
Conclusions
The effect of lithium on a marker altered early in the course of SCA1 pathogenesis, coupled with its positive effect on multiple behavioral measures and hippocampal neuropathology in an authentic disease model, make it an excellent candidate treatment for human SCA1 patients.
Huda Zoghbi and colleagues show that lithium treatment initiated before or after disease onset improves multiple symptoms of neurodegeneration in a mouse model of spinocerebellar ataxia.
Editors' Summary
Background.
Spinocerebellar ataxia type 1 (SCA1) is an inherited, incurable neurodegenerative disease in which the neurons (cells that transmit information between the brain and body) in the cerebellum (the brain region that coordinates movement) gradually die. Symptoms of the disease, which usually begins in early adult life, include poor coordination of movement (ataxia), slurred speech, and cognitive (learning and memory) problems. As more neurons die, these symptoms get worse until breathing difficulties eventually cause death. SCA1 is a “triplet repeat disease.” Information for making proteins is stored in DNA as groups of three nucleotides (codons), each specifying a different amino acid (the building blocks of proteins). In triplet repeat diseases, patients inherit a mutant gene containing abnormally long stretches of repeated codons. In SCA1, the repeated codon is CAG, which specifies glutamine. Consequently, SCA1 is a “polyglutamine disease,” a group of neurodegenerative disorders in which an abnormal protein (a different one for each disease) containing a long stretch of glutamine forms nuclear inclusions (insoluble lumps of protein) in neurons that, possibly by trapping essential proteins, cause neuronal death. In SCA1, the abnormal protein is ataxin 1, which is made in many neurons including the cerebellar neurons (Purkinje cells) that coordinate movement.
Why Was This Study Done?
Early in SCA1, the production of several messenger RNAs (the templates for protein production) decreases, possibly because transcription factors (proteins that control gene expression) interact with the mutant protein. Could the progression of SCA1 be slowed, therefore, by using an agent that affects gene expression? In this study, the researchers have investigated whether lithium can slow disease progression in an animal model of SCA1. They chose lithium for their study because this drug (best known for stabilizing mood in people with bipolar [manic] depression) affects gene expression, is neuroprotective, and has beneficial effects in animal models of Huntington disease, another polyglutamine disease.
What Did the Researchers Do and Find?
The researchers bred mice carrying one mutant gene for ataxin 1 containing a very long CAG repeat and one normal gene (Sca1154Q/2Q mice; genes come in pairs). These mice develop symptoms similar to those seen in people with SCA1. After weaning, the mice and their normal littermates were fed normal food or food supplemented with lithium for several weeks before assessing their ability to coordinate their movements and testing their cognitive skills. Dietary lithium (given before or after symptoms appeared) improved both coordination and learning and memory in the Sca1154Q/2Q mice but had little effect in the normal mice. Lithium did not change the overall appearance of the cerebellum in the Sca1154Q/2Q mice nor reduce the occurrence of nuclear inclusions, but it did partly reverse hippocampal neuron degeneration in these animals. The researchers discovered this effect by examining the shape of the hippocampal neurons in detail. These neurons normally have extensive dendrites—branch-like projections that make contact with other cells—but these gradually disappear in Sca1154Q/2Q mice; lithium partly reversed this loss. Finally, lithium also restored the level of Icmt/Pccmt mRNA in the cerebellum to near normal in the Sca1154Q/2Q mice—this mRNA is one of the first to be reduced by ataxin 1 toxicity.
What Do These Findings Mean?
These findings show that treatment with lithium slows neurodegeneration in a mouse model of SCA1, even when it is given only after the first symptoms of the disease have appeared. Unfortunately, lithium did not improve the life span of the Sca1154Q/2Q mice (although this could be because the mutant SCA1 protein has some deleterious effects outside the brain). Thus, lithium is unlikely to cure SCA1, but it could provide some help to people with this devastating disease, even if (as is usual), their condition is not diagnosed until the disease is quite advanced. However, because drugs that work in animal models of diseases do not necessarily work in people, patients with SCA1 (or other polyglutamine diseases, which might also benefit from lithium supplementation) should not be treated with lithium until human trials of this approach have been completed.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040182.
The US National Ataxia Foundation provides information for patients
International Network of Ataxia Friends has information for patients and carergivers on ataxias, including SCA1
GeneTests provides information for health care providers and researchers about SCA1
Online Mendelian Inheritance in Man (OMIM) has detailed scientific information on SCA1
Huntington's Outreach Project for Education offers information for lay people from Stanford University on trinucleotide repeat disorders including SCA1
doi:10.1371/journal.pmed.0040182
PMCID: PMC1880853  PMID: 17535104
6.  Progress in pathogenesis studies of spinocerebellar ataxia type 1. 
Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited disorder characterized by progressive loss of coordination, motor impairment and the degeneration of cerebellar Purkinje cells, spinocerebellar tracts and brainstem nuclei. Many dominantly inherited neurodegenerative diseases share the mutational basis of SCA1: the expansion of a translated CAG repeat coding for glutamine. Mice lacking ataxin-1 display learning deficits and altered hippocampal synaptic plasticity but none of the abnormalities seen in human SCA1; mice expressing ataxin-1 with an expanded CAG tract (82 glutamine residues), however, develop Purkinje cell pathology and ataxia. These results suggest that mutant ataxin-1 gains a novel function that leads to neuronal degeneration. This novel function might involve aberrant interaction(s) with cell-specific protein(s), which in turn might explain the selective neuronal pathology. Mutant ataxin-1 interacts preferentially with a leucine-rich acidic nuclear protein that is abundantly expressed in cerebellar Purkinje cells and other brain regions affected in SCA1. Immunolocalization studies in affected neurons of patients and SCA1 transgenic mice showed that mutant ataxin-1 localizes to a single, ubiquitin-positive nuclear inclusion (NI) that alters the distribution of the proteasome and certain chaperones. Further analysis of NIs in transfected HeLa cells established that the proteasome and chaperone proteins co-localize with ataxin-1 aggregates. Moreover, overexpression of the chaperone HDJ-2/HSDJ in HeLa cells decreased ataxin-1 aggregation, suggesting that protein misfolding might underlie NI formation. To assess the importance of the nuclear localization of ataxin-1 and its role in SCA1 pathogenesis, two lines of transgenic mice were generated. In the first line, the nuclear localization signal was mutated so that full-length mutant ataxin-1 would remain in the cytoplasm; mice from this line did not develop any ataxia or pathology. This suggests that mutant ataxin-1 is pathogenic only in the nucleus. To assess the role of the aggregates, transgenic mice were generated with mutant ataxin-1 without the self-association domain (SAD) essential for aggregate formation. These mice developed ataxia and Purkinje cell abnormalities similar to those seen in SCA1 transgenic mice carrying full-length mutant ataxin-1, but lacked NIs. The nuclear milieu is thus a critical factor in SCA1 pathogenesis, but large NIs are not needed to initiate pathogenesis. They might instead be downstream of the primary pathogenic steps. Given the accumulated evidence, we propose the following model for SCA1 pathogenesis: expansion of the polyglutamine tract alters the conformation of ataxin-1, causing it to misfold. This in turn leads to aberrant protein interactions. Cell specificity is determined by the cell-specific proteins interacting with ataxin-1. Submicroscopic protein aggregation might occur because of protein misfolding, and those aggregates become detectable as NIs as the disease advances. Proteasome redistribution to the NI might contribute to disease progression by disturbing proteolysis and subsequent vital cellular functions.
PMCID: PMC1692607  PMID: 10434309
7.  FGF14 Regulates the Intrinsic Excitability of Cerebellar Purkinje Neurons 
Neurobiology of disease  2008;33(1):81-88.
A missense mutation in the fibroblast growth factor 14 (FGF14) gene underlies SCA27, an autosomal dominant spinocerebellar ataxia in humans. Mice with a targeted disruption of the Fgf14 locus (Fgf14−/−) develop ataxia resembling human SCA27. We tested the hypothesis that loss of FGF14 affects the firing properties of Purkinje neurons, which play an important role in motor control and coordination. Current clamp recordings from Purkinje neurons in cerebellar slices revealed attenuated spontaneous firing in Fgf14−/− neurons. Unlike in the wild type animals, more than 80% of Fgf14−/− Purkinje neurons were quiescent and failed to fire repetitively in response to depolarizing current injections. Immunohistochemical examination revealed reduced expression of Nav1.6 protein in Fgf14−/− Purkinje neurons. Together, these observations suggest that FGF14 is required for normal Nav1.6 expression in Purkinje neurons, and that the loss of FGF14 impairs spontaneous and repetitive firing in Purkinje neurons by altering the expression of Nav1.6 channels.
doi:10.1016/j.nbd.2008.09.019
PMCID: PMC2652849  PMID: 18930825
Spinocerebellar ataxia; Intracellular fibroblast growth factor 14 (iFGF14); Purkinje neurons; Nav 1.6; SCA27
8.  Early changes in cerebellar physiology accompany motor dysfunction in the polyglutamine disease, Spinocerebellar Ataxia type 3 
The relationship between cerebellar dysfunction, motor symptoms and neuronal loss in the inherited ataxias, including the polyglutamine disease Spinocerebellar Ataxia type 3 (SCA3), remains poorly understood. We demonstrate that prior to neurodegeneration, Purkinje neurons in a mouse model of SCA3 exhibit increased intrinsic excitability resulting in depolarization block and the loss of the ability to sustain spontaneous repetitive firing. These alterations in intrinsic firing are associated with increased inactivation of voltage-activated potassium currents. Administration of an activator of calcium-activated potassium channels, SKA-31, partially corrects abnormal Purkinje cell firing and improves motor function in SCA3 mice. Finally, expression of the disease protein, ataxin-3, in transfected cells increases the inactivation of Kv3.1 channels and shifts the activation of Kv1.2 channels to more depolarized potentials. Our results suggest that in SCA3, early Purkinje neuron dysfunction is associated with altered physiology of voltage-activated potassium channels. We further suggest that the observed changes in Purkinje neuron physiology contribute to disease pathogenesis, underlie at least some motor symptoms, and represent a promising therapeutic target in SCA3.
doi:10.1523/JNEUROSCI.2789-11.2011
PMCID: PMC3170039  PMID: 21900579
9.  Computational analysis of calcium signaling and membrane electrophysiology in cerebellar Purkinje neurons associated with ataxia 
BMC Systems Biology  2012;6:70.
Background
Mutations in the smooth endoplasmic reticulum (sER) calcium channel Inositol Trisphosphate Receptor type 1 (IP3R1) in humans with the motor function coordination disorders Spinocerebellar Ataxia Types 15 and 16 (SCA15/16) and in a corresponding mouse model, the IP3R1delta18/delta18 mice, lead to reduced IP3R1 levels. We posit that increasing IP3R1 sensitivity to IP3 in ataxias with reduced IP3R1 could restore normal calcium response. On the other hand, in mouse models of the human polyglutamine (polyQ) ataxias, SCA2, and SCA3, the primary finding appears to be hyperactive IP3R1-mediated calcium release. It has been suggested that the polyQ SCA1 mice may also show hyperactive IP3R1. Yet, SCA1 mice show downregulated gene expression of IP3R1, Homer, metabotropic glutamate receptor (mGluR), smooth endoplasmic reticulum Ca-ATP-ase (SERCA), calbindin, parvalbumin, and other calcium signaling proteins.
Results
We create a computational model of pathological alterations in calcium signaling in cerebellar Purkinje neurons to investigate several forms of spinocerebellar ataxia associated with changes in the abundance, sensitivity, or activity of the calcium channel IP3R1. We find that increasing IP3R1 sensitivity to IP3 in computational models of SCA15/16 can restore normal calcium response if IP3R1 abundance is not too low. The studied range in IP3R1 levels reflects variability found in human and mouse ataxic models. Further, the required fold increases in sensitivity are within experimental ranges from experiments that use IP3R1 phosphorylation status to adjust its sensitivity to IP3. Results from our simulations of polyglutamine SCAs suggest that downregulation of some calcium signaling proteins may be partially compensatory. However, the downregulation of calcium buffer proteins observed in the SCA1 mice may contribute to pathology. Finally, our model suggests that the calcium-activated voltage-gated potassium channels may provide an important link between calcium metabolism and membrane potential in Purkinje cell function.
Conclusion
Thus, we have established an initial platform for computational evaluation and prediction of ataxia pathophysiology. Specifically, the model has been used to investigate SCA15/16, SCA1, SCA2, and SCA3. Results suggest that experimental studies treating mouse models of any of these ataxias with appropriately chosen peptides resembling the C-terminal of IP3R1 could adjust receptor sensitivity, and thereby modulate calcium release and normalize IP3 response. In addition, the model supports the hypothesis of IP3R1 supersensitivity in SCA1.
doi:10.1186/1752-0509-6-70
PMCID: PMC3468360  PMID: 22703638
Model; IP3R; Homer; CAG repeat instability; KCa channels; Ataxia; Computational; BK channel; Spinocerebellar; Virtual cell
10.  A Family with Spinocerebellar Ataxia Type 5 Found to Have a Novel Missense Mutation Within a SPTBN2 Spectrin Repeat 
Cerebellum (London, England)  2013;12(2):162-164.
OBJECTIVE
Identification of a novel missense mutation in the SPTBN2 gene of a family with a clinical diagnosis of spinocerebellar ataxia type 5 (SCA5).
METHODS
A family with late-onset autosomal dominant pure cerebellar ataxia, consistent with SCA5 but lacking previously reported SPTBN2 mutations, was identified. DNA was collected from seven individuals across two generations and the SPTBN2 gene on chromosome 11 was sequenced.
RESULTS
A nonsynonymous heterozygous substitution in exon 12 was detected in individuals diagnosed with SCA5 while unaffected family members did not possess this variant. The identified c.1415C>T variant results in a p.T472M substitution in the second SPEC domain of the beta-III spectrin protein. The threonine at position 472 is not in close proximity to the characteristic residues that define the SPEC domain and is variable across diverse SPEC domains, yet is highly conserved in SPTBN2. Consistent with these observations, bioinformatic analysis of the p.T472M variant suggests it to be pathological.
CONCLUSION
Two deletions within the SPTBN2 SPEC domains (E532_M544del and L629_R634delinsW) have been previously reported to cause SCA5, but this is the first missense mutation in this region of the protein shown to likely be pathogenic.
doi:10.1007/s12311-012-0408-0
PMCID: PMC3574192  PMID: 22843192
ATAXIA; SCA5; SPTBN2
11.  Requirement for Zebrafish Ataxin-7 in Differentiation of Photoreceptors and Cerebellar Neurons 
PLoS ONE  2012;7(11):e50705.
The expansion of a polyglutamine (polyQ) tract in the N-terminal region of ataxin-7 (atxn7) is the causative event in spinocerebellar ataxia type 7 (SCA7), an autosomal dominant neurodegenerative disorder mainly characterized by progressive, selective loss of rod-cone photoreceptors and cerebellar Purkinje and granule cells. The molecular and cellular processes underlying this restricted neuronal vulnerability, which contrasts with the broad expression pattern of atxn7, remains one of the most enigmatic features of SCA7, and more generally of all polyQ disorders. To gain insight into this specific neuronal vulnerability and achieve a better understanding of atxn7 function, we carried out a functional analysis of this protein in the teleost fish Danio rerio. We characterized the zebrafish atxn7 gene and its transcription pattern, and by making use of morpholino-oligonucleotide-mediated gene inactivation, we analysed the phenotypes induced following mild or severe zebrafish atxn7 depletion. Severe or nearly complete zebrafish atxn7 loss-of-function markedly impaired embryonic development, leading to both early embryonic lethality and severely deformed embryos. More importantly, in relation to SCA7, moderate depletion of the protein specifically, albeit partially, prevented the differentiation of both retina photoreceptors and cerebellar Purkinje and granule cells. In addition, [1–232] human atxn7 fragment rescued these phenotypes showing strong function conservation of this protein through evolution. The specific requirement for zebrafish atxn7 in the proper differentiation of cerebellar neurons provides, to our knowledge, the first in vivo evidence of a direct functional relationship between atxn7 and the differentiation of Purkinje and granule cells, the most crucial neurons affected in SCA7 and most other polyQ-mediated SCAs. These findings further suggest that altered protein function may play a role in the pathophysiology of the disease, an important step toward the development of future therapeutic strategies.
doi:10.1371/journal.pone.0050705
PMCID: PMC3511343  PMID: 23226359
12.  Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 2 
Spinocerebellar ataxia type 2 (SCA2), is an autosomal dominantly inherited, neurodegenerative disease caused by an expansion of polyglutamine (polyQ) tracts in the cytosolic protein ataxin-2 (Atx2). Cerebellar Purkinje cells (PCs) are predominantly affected in SCA2. The cause of PC degeneration in SCA2 is unknown. Here we demonstrate that mutant Atx2-58Q, but not wild type Atx2-22Q, specifically associates with the cytosolic carboxy-terminal region of type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1), an intracellular calcium (Ca2+) release channel. Association with Atx2-58Q increased the sensitivity of InsP3R1 to activation by InsP3 in planar lipid bilayer reconstitution experiments. To validate physiological significance of these findings, we performed a series of experiments with an SCA2-58Q transgenic mouse model that expresses human full-length Atx2-58Q protein under the control of a PC-specific promoter. In Ca2+ imaging experiments, we demonstrated that stimulation with DHPG resulted in higher Ca2+ responses in 58Q PC cultures than in wild type (WT) PC cultures. DHPG-induced Ca2+ responses in 58Q PC cultures were blocked by the addition of ryanodine, an inhibitor of the ryanodine receptor (RyanR). We further demonstrated that application of glutamate induced more pronounced cell death in 58Q PC cultures than in WT PC cultures. Glutamate-induced cell death of 58Q PC cultures was attenuated by dantrolene, a clinically relevant RyanR inhibitor and Ca2+ stabilizer. In whole animal experiments, we demonstrated that long-term feeding of 58Q mice with dantrolene alleviated age-dependent motor deficits (quantified in beamwalk and rotarod assays) and reduced PC loss observed in untreated SCA2-58Q mice by 12 months of age (quantified by stereology). Results of our studies indicate that disturbed neuronal Ca2+ signaling may play an important role in SCA2 pathology and also suggest the RyanR as a potential therapeutic target for treatment of SCA2 patients.
doi:10.1523/JNEUROSCI.0660-09.2009
PMCID: PMC2749883  PMID: 19625506
calcium signaling; neurodegeneration; ataxin-2; spinocerebellar ataxia type 2; SCA2; Purkinje cells; apoptosis; transgenic mouse; stereology; dantrolene
13.  Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments 
The Journal of Cell Biology  2001;155(5):739-746.
The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, βIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and βIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. βIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.
doi:10.1083/jcb.200109026
PMCID: PMC2150881  PMID: 11724816
βIV spectrin; sodium channel Nav1.6; neurofascin; NrCAM; axon hillock
14.  Loss of Intrinsic Organization of Cerebellar Networks in Spinocerebellar Ataxia Type 1: Correlates with Disease Severity and Duration 
Cerebellum (London, England)  2011;10(2):218-232.
The spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of cerebellar degenerative disorders, characterized by progressive gait unsteadiness, hand incoordination, and dysarthria. The mutational mechanism in SCA1, a dominantly inherited form of SCA, consists of an expanded trinucleotide CAG repeat. In SCA1, there is loss of Purkinje cells, neuronal loss in dentate nucleus, olives, and pontine nuclei. In the present study, we sought to apply intrinsic functional connectivity analysis combined with diffusion tensor imaging to define the state of cerebellar connectivity in SCA1. Our results on the intrinsic functional connectivity in lateral cerebellum and thalamus showed progressive organizational changes in SCA1 noted as a progressive increase in the absolute value of the correlation coefficients. In the lateral cerebellum, the anatomical organization of functional clusters seen as parasagittal bands in controls is lost, changing to a patchy appearance in SCA1. Lastly, only fractional anisotropy in the superior peduncle and changes in functional organization in thalamus showed a linear dependence to duration and severity of disease. The present pilot work represents an initial effort describing connectivity biomarkers of disease progression in SCA1. The functional changes detected with intrinsic functional analysis and diffusion tensor imaging suggest that disease progression can be analyzed as a disconnection syndrome.
doi:10.1007/s12311-010-0214-5
PMCID: PMC3091958  PMID: 20886327
Networks; MRI; Biomarkers; Ataxia
15.  Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2 
Human Molecular Genetics  2012;22(2):271-283.
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited disorder, which is caused by a pathological expansion of a polyglutamine (polyQ) tract in the coding region of the ATXN2 gene. Like other ataxias, SCA2 most overtly affects Purkinje cells (PCs) in the cerebellum. Using a transgenic mouse model expressing a full-length ATXN2Q127-complementary DNA under control of the Pcp2 promoter (a PC-specific promoter), we examined the time course of behavioral, morphologic, biochemical and physiological changes with particular attention to PC firing in the cerebellar slice. Although motor performance began to deteriorate at 8 weeks of age, reductions in PC number were not seen until after 12 weeks. Decreases in the PC firing frequency first showed at 6 weeks and paralleled deterioration of motor performance with progression of disease. Transcription changes in several PC-specific genes such as Calb1 and Pcp2 mirrored the time course of changes in PC physiology with calbindin-28 K changes showing the first small, but significant decreases at 4 weeks. These results emphasize that in this model of SCA2, physiological and behavioral phenotypes precede morphological changes by several weeks and provide a rationale for future studies examining the effects of restoration of firing frequency on motor function and prevention of future loss of PCs.
doi:10.1093/hmg/dds427
PMCID: PMC3526159  PMID: 23087021
16.  ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice 
PLoS Genetics  2012;8(8):e1002920.
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.
Author Summary
Frequent age-associated neurodegenerative disorders like Alzheimer's, Parkinson's, and Lou Gehrig's disease are being elucidated molecularly by studying rare heritable variants. Various hereditary neurodegenerative disorders are caused by polyglutamine expansions in different proteins. In spite of this common pathogenesis and the pathological aggregation of most affected proteins, investigators were puzzled that the pattern of affected neuron population varies and that molecular mechanisms seem different between such disorders. The polyglutamine expansions in the Ataxin-2 (ATXN2) protein are exceptional in view of the lack of aggregate clumps in nuclei of affected Purkinje neurons and well documented alterations of RNA processing in the resulting disorders SCA2 and ALS. Here, as a faithful disease model and to overcome the unavailability of autopsied patient brain tissues, we generated and characterized an ATXN2-CAG42-knock-in mouse mutant. Our data show that the unspecific, chronically present mutation leads to progressive insolubility and to reduced soluble levels of the disease protein and of an interactor protein, which modulates RNA processing. Compensatory efforts are particularly weak in vulnerable tissue. They appear to include the increased degradation of the toxic disease protein by FBXW8. Thus the link between protein and RNA pathology becomes clear, and crucial molecular targets for preventive therapy are identified.
doi:10.1371/journal.pgen.1002920
PMCID: PMC3431311  PMID: 22956915
17.  Sequential Degradation of αII and βII Spectrin by Calpain in Glutamate or Maitotoxin-Stimulated Cells 
Biochemistry  2007;46(2):502-513.
Calpain-catalyzed proteolysis of αII-spectrin is a regulated event associated with neuronal long-term potentiation, platelet and leukocyte activation, and other processes. Calpain proteolysis is also linked to apoptotic and non-apoptotic cell death following excessive glutamate exposure, hypoxia, HIV-gp120/160 exposure, or toxic injury. The molecular basis for these divergent consequences of calpain action, and their relationship to spectrin proteolysis, is unclear. Calpain preferentially cleaves αII spectrin in vitro in repeat 11 between residues Y1176 and G1177. Unless stimulated by Ca++ and calmodulin (CaM), βII spectrin proteolysis in vitro is much slower. We identify additional unrecognized sites in spectrin targeted by calpain in vitro and in vivo. Bound CaM induces a second αII spectrin cleavage at G1230*S1231. βII spectrin is cleaved at four sites. One cleavage only occurs in the absence of CaM at high enzyme-to-substrate ratios near the βII spectrin COOH-terminus. CaM promotes βII spectrin cleavages at Q1440*S1441, S1447*Q1448, and L1482*A1483. These sites are also cleaved in the absence of CaM in recombinant βII spectrin fusion peptides, indicating that they are probably shielded in the spectrin heterotetramer and become exposed only after CaM binds αII spectrin. Using epitope-specific antibodies prepared to the calpain cleavage sites in both αII and βII spectrin, we find in cultured rat cortical neurons that brief glutamate exposure (a physiologic ligand) rapidly stimulates αII spectrin cleavage only at Y1176*G1177, while βII spectrin remains intact. In cultured SH-SY5Y cells that lack an NMDA receptor, glutamate is without effect. Conversely, when stimulated by calcium influx (via maitotoxin), there is rapid and sequential cleavage of αII and then βII spectrin, coinciding with the onset of non-apoptotic cell death. These results identify: i) novel calpain target sites in both αII and βII spectrin; ii) trans-regulation of proteolytic susceptibility between the spectrin subunits in vivo; and iii) the preferential cleavage of αII spectrin vs. βII spectrin when responsive cells are stimulated by engagement of the NMDA receptor. We postulate that calpain proteolysis of spectrin can activate two physiologically distinct responses: one that enhances skeletal plasticity without destroying the spectrin-actin skeleton, characterized by preservation of βII spectrin; or an alternative response closely correlated with non-apoptotic cell death and characterized by proteolysis of βII spectrin and complete dissolution of the spectrin skeleton.
doi:10.1021/bi061504y
PMCID: PMC2825692  PMID: 17209560
cytoskeleton; proteolysis; necrosis; cell death; glutamate; fodrin
18.  Transgene Rescue Identifies an Essential Function for Drosophila β Spectrin in the Nervous System and a Selective Requirement for Ankyrin-2–binding Activity 
Molecular Biology of the Cell  2010;21(16):2860-2868.
The Gal4-UAS system was used to overexpress or knock down β spectrin with dsRNA in a variety of Drosophila tissues. Unexpectedly, overexpression in most tissues tested was lethal, whereas knockdown failed to produce a detectable phenotype in the same tissues. The lethality of a β spectrin mutation was rescued by expression of β spectrin in neurons.
The protein spectrin is ubiquitous in animal cells and is believed to play important roles in cell shape and membrane stability, cell polarity, and endomembrane traffic. Experiments here were undertaken to identify sites of essential β spectrin function in Drosophila and to determine whether spectrin and ankyrin function are strictly linked to one another. The Gal4-UAS system was used to drive tissue-specific overexpression of a β spectrin transgene or to knock down β spectrin expression with dsRNA. The results show that 1) overexpression of β spectrin in most of the cell types studied was lethal; 2) knockdown of β spectrin in most tissues had no detectable effect on growth or viability of the organism; and 3) nervous system-specific expression of a UAS-β spectrin transgene was sufficient to overcome the lethality of a loss-of-function β spectrin mutation. Thus β spectrin expression in other cells was not required for development of fertile adult males, although females lacking nonneuronal spectrin were sterile. Previous data indicated that binding of the DAnk1 isoform of ankyrin to spectrin was partially dispensable for viability. Domain swap experiments here uncovered a different requirement for neuronal DAnk2 binding to spectrin and establish that DAnk2-binding is critical for β spectrin function in vivo.
doi:10.1091/mbc.E10-03-0180
PMCID: PMC2921109  PMID: 20573981
19.  RNA Gain-of-Function in Spinocerebellar Ataxia Type 8 
PLoS Genetics  2009;5(8):e1000600.
Microsatellite expansions cause a number of dominantly-inherited neurological diseases. Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins. Bi-directional expression of the spinocerebellar ataxia type 8 (SCA8) CTG CAG expansion produces CUG expansion RNAs (CUGexp) from the ATXN8OS gene and a nearly pure polyglutamine expansion protein encoded by ATXN8 CAGexp transcripts expressed in the opposite direction. Here, we present three lines of evidence that RNA gain-of-function plays a significant role in SCA8: 1) CUGexp transcripts accumulate as ribonuclear inclusions that co-localize with MBNL1 in selected neurons in the brain; 2) loss of Mbnl1 enhances motor deficits in SCA8 mice; 3) SCA8 CUGexp transcripts trigger splicing changes and increased expression of the CUGBP1-MBNL1 regulated CNS target, GABA-A transporter 4 (GAT4/Gabt4). In vivo optical imaging studies in SCA8 mice confirm that Gabt4 upregulation is associated with the predicted loss of GABAergic inhibition within the granular cell layer. These data demonstrate that CUGexp transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUGexp disorders. Moreover, our demonstration that relatively short CUGexp transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.
Author Summary
We describe several lines of evidence that RNA gain-of-function effects play a significant role in spinocerebellar ataxia type 8 (SCA8) and has broader implications for understanding the CNS effects of other trinucleotide expansion disorders including myotonic dystrophy type 1, Huntington disease like-2, and spinocerebellar ataxia type 7. The SCA8 mutation is bidirectionally transcribed resulting in the expression of CUGexp transcripts from ATXN8OS and CAGexp transcripts and polyglutamine protein from the overlapping ATXN8 gene. These data suggest that SCA8 pathogenesis involves toxic gain-of-function effects at the RNA (CUGexp) and/or protein (PolyQ) levels. We present three lines of evidence that CUGexp transcripts play a significant role in SCA8: 1) CUGexp transcripts accumulate as ribonuclear inclusions that co-localize with MBNL1 in selected neurons; 2) loss of Mbnl1 enhances motor deficits in SCA8 mice; 3) SCA8 CUGexp transcripts trigger alternative splicing changes and increased expression of the CUGBP1-MBNL1 regulated CNS target, GABA-A transporter 4 (GAT4/Gabt4) which is associated with the predicted loss of GABAergic inhibition within the granular cell layer in SCA8 mice. Additionally, alternative splicing changes and GAT4 upregulation are induced by CUGexp but not CAGexp transcripts. From a therapeutic viewpoint, it is promising that this change is reversed in cells overexpressing MBNL1.
doi:10.1371/journal.pgen.1000600
PMCID: PMC2719092  PMID: 19680539
20.  Autosomal dominant cerebellar ataxia type III: a review of the phenotypic and genotypic characteristics 
Autosomal Dominant Cerebellar Ataxia (ADCA) Type III is a type of spinocerebellar ataxia (SCA) classically characterized by pure cerebellar ataxia and occasionally by non-cerebellar signs such as pyramidal signs, ophthalmoplegia, and tremor. The onset of symptoms typically occurs in adulthood; however, a minority of patients develop clinical features in adolescence. The incidence of ADCA Type III is unknown. ADCA Type III consists of six subtypes, SCA5, SCA6, SCA11, SCA26, SCA30, and SCA31. The subtype SCA6 is the most common. These subtypes are associated with four causative genes and two loci. The severity of symptoms and age of onset can vary between each SCA subtype and even between families with the same subtype. SCA5 and SCA11 are caused by specific gene mutations such as missense, inframe deletions, and frameshift insertions or deletions. SCA6 is caused by trinucleotide CAG repeat expansions encoding large uninterrupted glutamine tracts. SCA31 is caused by repeat expansions that fall outside of the protein-coding region of the disease gene. Currently, there are no specific gene mutations associated with SCA26 or SCA30, though there is a confirmed locus for each subtype. This disease is mainly diagnosed via genetic testing; however, differential diagnoses include pure cerebellar ataxia and non-cerebellar features in addition to ataxia. Although not fatal, ADCA Type III may cause dysphagia and falls, which reduce the quality of life of the patients and may in turn shorten the lifespan. The therapy for ADCA Type III is supportive and includes occupational and speech modalities. There is no cure for ADCA Type III, but a number of recent studies have highlighted novel therapies, which bring hope for future curative treatments.
doi:10.1186/1750-1172-8-14
PMCID: PMC3558377  PMID: 23331413
SCA5; SCA6; SCA11; SCA26; SCA30; SCA31; SPTBN2; CACNA1A; TTBK2; BEAN
21.  Cellular Fusion for Gene Delivery to SCA1 Affected Purkinje Neurons 
Cerebellar Purkinje neurons (PNs) possess a well characterized propensity to fuse with bone marrow-derived cells (BMDCs), producing heterokaryons with Purkinje cell identities. This offers the potential to rescue/repair at risk or degenerating PNs in the inherited ataxias, including Spinocerebellar Ataxia 1 (SCA1), by introducing therapeutic factors through BMDCs to potentially halt or reverse disease progression. In this study, we combined gene therapy and a stem cell-based treatment to attempt repair of at-risk PNs through cell-cell fusion in a Sca1154Q/2Q knock-in mouse model. BMDCs enriched for the hematopoietic stem cell (HSC) population were genetically modified using adeno-associated viral vector 7 (AAV7) to carry SCA1 modifier genes and transplanted into irradiated Sca1154Q/2Q mice. Binucleated Purkinje heterokaryons with sex-mismatched donor Y chromosomes were detected and successfully expressed the modifier genes in vivo. Potential effects of the new genome within Purkinje heterokaryons were evaluated using nuclear inclusions (NIs) as a biological marker to reflect possible modifications of the SCA1 disease process. An overall decrease in number of NIs and an increase in the number of surviving PNs were observed in treated Sca1154Q/2Q. Furthermore, Bergmann glia were found to have fusogenic potential with the donor population and reveal another potential route of therapeutic entry into at-risk cells of the SCA1 cerebellum. This study presents a first step towards a proof of principle that combines somatic cellular fusion events with a neuroprotective gene therapy approach for providing potential neuronal protection/repair in a variety of neurodegenerative disorders.
doi:10.1016/j.mcn.2011.03.003
PMCID: PMC3100720  PMID: 21420496
Spinocerebellar Ataxia 1; Bone marrow derived cells; Hematopoietic stem cells; Gene therapy; AAV; Stem cell fusion
22.  A SEL1L Mutation Links a Canine Progressive Early-Onset Cerebellar Ataxia to the Endoplasmic Reticulum–Associated Protein Degradation (ERAD) Machinery 
PLoS Genetics  2012;8(6):e1002759.
Inherited ataxias are characterized by degeneration of the cerebellar structures, which results in progressive motor incoordination. Hereditary ataxias occur in many species, including humans and dogs. Several mutations have been found in humans, but the genetic background has remained elusive in dogs. The Finnish Hound suffers from an early-onset progressive cerebellar ataxia. We have performed clinical, pathological, and genetic studies to describe the disease phenotype and to identify its genetic cause. Neurological examinations on ten affected dogs revealed rapidly progressing generalized cerebellar ataxia, tremors, and failure to thrive. Clinical signs were present by the age of 3 months, and cerebellar shrinkage was detectable through MRI. Pathological and histological examinations indicated cerebellum-restricted neurodegeneration. Marked loss of Purkinje cells was detected in the cerebellar cortex with secondary changes in other cortical layers. A genome-wide association study in a cohort of 31 dogs mapped the ataxia gene to a 1.5 Mb locus on canine chromosome 8 (praw = 1.1×10−7, pgenome = 7.5×10−4). Sequencing of a functional candidate gene, sel-1 suppressor of lin-12-like (SEL1L), revealed a homozygous missense mutation, c.1972T>C; p.Ser658Pro, in a highly conserved protein domain. The mutation segregated fully in the recessive pedigree, and a 10% carrier frequency was indicated in a population cohort. SEL1L is a component of the endoplasmic reticulum (ER)–associated protein degradation (ERAD) machinery and has not been previously associated to inherited ataxias. Dysfunctional protein degradation is known to cause ER stress, and we found a significant increase in expression of nine ER stress responsive genes in the cerebellar cortex of affected dogs, supporting the pathogenicity of the mutation. Our study describes the first early-onset neurodegenerative ataxia mutation in dogs, establishes an ERAD–mediated neurodegenerative disease model, and proposes SEL1L as a new candidate gene in progressive childhood ataxias. Furthermore, our results have enabled the development of a genetic test for breeders.
Author Summary
Hereditary ataxias are a heterogeneous group of rare disorders characterized by progressive cerebellar neurodegeneration. Several causative mutations have been identified in various forms of human ataxias. In addition to humans, inherited ataxias have been described in several other species, including the domestic dog. In this study, we have studied the clinical and genetic properties of cerebellar ataxia in the Finnish Hound dog breed. The breed suffers from a progressive ataxia that has an early onset before the age of 3 months. Affected puppies have difficulties in coordinating their movements and balance, and have to be euthanized due to rapidly worsening symptoms. Our pedigree analysis suggested an autosomal recessive mode of inheritance, which was confirmed by identifying a homozygous mutation in the SEL1L gene through genome-wide association and linkage analyses. The SEL1L protein functions in a protein quality control pathway that targets misfolded proteins to degradation in the endoplasmic reticulum. Mutations in the SEL1L gene have not been previously found in ataxias. Our study indicates SEL1L as a novel candidate gene for human childhood ataxias, establishes a large animal model to investigate mechanisms of cerebellar neurodegeneration, and enables carrier screening for breeding purposes.
doi:10.1371/journal.pgen.1002759
PMCID: PMC3375262  PMID: 22719266
23.  Clinical, neuropathological, and molecular study in two families with spinocerebellar ataxia type 6 (SCA6) 
To clarify the clinical, neuropathological, and molecular characteristics of spinocerebellar ataxia type 6 (SCA6), two unrelated Japanese families with SCA6 were studied. A clinical feature of the two families was late onset "pure" cerebellar ataxia. Pathologically, three SCA6 brains consistently showed Purkinje cell dominant cortical cerebellar degeneration. Morphometric analysis showed that loss of the cerebellar granule cells and inferior olivary neurons were very mild compared with the severity of Purkinje cell loss. There was no obvious ubiquitin immunoreactive nuclear inclusions. All affected patients had identical expanded alleles, and the expansion was also homogeneously distributed throughout the brain without mosaicism. The present study showed that SCA6 is characterised by Purkinje cell dominant cortical cerebellar degeneration, highly stable transmission of the CAG repeat expansion, and lack of ubiquitin immunoreactive nuclear inclusions.


PMCID: PMC1736420  PMID: 10369828
24.  Role of Inositol 1,4,5-Trishosphate Receptors in Pathogenesis of Huntington's Disease and Spinocerebellar Ataxias 
Neurochemical research  2011;36(7):1186-1197.
Huntington's disease (HD) and spinocerebellar ataxias (SCAs) are autosomal-dominant neurodegenerative disorders. HD is caused by polyglutamine (polyQ) expansion in the amino-terminal region of a protein huntingtin (Htt) and primarily affects medium spiny striatal neurons (MSN). Many SCAs are caused by polyQ-expansion in ataxin proteins and primarily affect cerebellar Purkinje cells. The reasons for neuronal dysfunction and death in HD and SCAs remain poorly understood and no cure is available for the patients. Our laboratory discovered that mutant huntingtin, ataxin-2 and ataxin-3 proteins specifically bind to the carboxy-terminal region of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an intracellular Ca2+ release channel. Moreover, we found that association of mutant huntingtin or ataxins with IP3R1 causes sensitization of IP3R1 to activation by IP3 in planar lipid bilayers and in neuronal cells. These results suggested that deranged neuronal Ca2+ signaling might play an important role in pathogenesis of HD, SCA2 and SCA3. In support of this idea, we demonstrated a connection between abnormal Ca2+ signaling and neuronal cell death in experiments with HD, SCA2 and SCA3 transgenic mouse models. Additional data in the literature indicate that abnormal neuronal Ca2+ signaling may also play an important role in pathogenesis of SCAl, SCA5, SCA6, SCA14 and SCA15/16. Based on these results I propose that IP3R and other Ca2+ signaling proteins should be considered as potential therapeutic targets for treatment of HD and SCAs.
doi:10.1007/s11064-010-0393-y
PMCID: PMC3094593  PMID: 21210219
Calcium signaling; Huntingtin; Neurodegeneration; Polyglutamine expansion; Inositol 1,4,,5-trisphosphate; Ataxin-2; Ataxin-3; Spinocerebellar ataxias; Transgenic mice; NMDA receptor; Apoptosis; Mitochondria; Memantine
25.  Caenorhabditis elegans β-G Spectrin Is Dispensable for Establishment of Epithelial Polarity, but Essential for Muscular and Neuronal Function 
The Journal of Cell Biology  2000;149(4):915-930.
The Caenorhabditis elegans genome encodes one α spectrin subunit, a β spectrin subunit (β-G), and a β-H spectrin subunit. Our experiments show that the phenotype resulting from the loss of the C. elegans α spectrin is reproduced by tandem depletion of both β-G and β-H spectrins. We propose that α spectrin combines with the β-G and β-H subunits to form α/β-G and α/β-H heteromers that perform the entire repertoire of spectrin function in the nematode. The expression patterns of nematode β-G spectrin and vertebrate β spectrins exhibit three striking parallels including: (1) β spectrins are associated with the sites of cell–cell contact in epithelial tissues; (2) the highest levels of β-G spectrin occur in the nervous system; and (3) β spec-trin-G in striated muscle is associated with points of attachment of the myofilament apparatus to adjacent cells. Nematode β-G spectrin associates with plasma membranes at sites of cell–cell contact, beginning at the two-cell stage, and with a dramatic increase in intensity after gastrulation when most cell proliferation has been completed. Strikingly, depletion of nematode β-G spectrin by RNA-mediated interference to undetectable levels does not affect the establishment of structural and functional polarity in epidermis and intestine. Contrary to recent speculation, β-G spectrin is not associated with internal membranes and depletion of β-G spectrin was not associated with any detectable defects in secretion. Instead β-G spectrin-deficient nematodes arrest as early larvae with progressive defects in the musculature and nervous system. Therefore, C. elegans β-G spectrin is required for normal muscle and neuron function, but is dispensable for embryonic elongation and establishment of early epithelial polarity. We hypothesize that heteromeric spectrin evolved in metazoans in response to the needs of cells in the context of mechanically integrated tissues that can withstand the rigors imposed by an active organism.
PMCID: PMC2174577  PMID: 10811831
membrane skeleton; unc-70; RNAi; cell–cell contact

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