[show abstract][hide abstract] ABSTRACT: All spinocerebellar ataxias (SCAs) are rare diseases. SCA1, 2, 3 and 6 are the four most common SCAs, all caused by expanded polyglutamine-coding CAG repeats. Their pathomechanisms are becoming increasingly clear and well-designed clinical trials will be needed.
To characterize the clinical manifestations of spinocerebellar ataxia (SCA) 1, 2, 3 and 6 and their natural histories in the United States (US), we conducted a prospective multicenter study utilized a protocol identical to the European consortium study, using the Scale for the Assessment and Rating of Ataxia (SARA) score as the primary outcome, with follow-ups every 6 months up to 2 years.
We enrolled 345 patients (60 SCA1, 75 SCA2, 138 SCA3 and 72 SCA6) at 12 US centers. SCA6 patients had a significantly later onset, and SCA2 patients showed greater upper-body ataxia than patients with the remaining SCAs. The annual increase of SARA score was greater in SCA1 patients (mean +/- SE: 1.61 +/- 0.41) than in SCA2 (0.71 +/- 0.31), SCA3 (0.65 +/- 0.24) and SCA6 (0.87 +/- 0.28) patients (p = 0.049). The functional stage also worsened faster in SCA1 than in SCA2, 3 and 6 (p = 0.002).
The proportions of different SCA patients in US differ from those in the European consortium study, but as in the European patients, SCA1 progress faster than those with SCA2, 3 and 6. Later onset in SCA6 and greater upper body ataxia in SCA2 were noted. We conclude that progression rates of these SCAs were comparable between US and Europe cohorts, suggesting the feasibility of international collaborative clinical studies.
[show abstract][hide abstract] ABSTRACT: The autosomal dominant spinocerebellar ataxias are a diverse and clinically heterogeneous group of disorders characterized by degeneration and dysfunction of the cerebellum and its associated pathways. Clinical and diagnostic evaluation can be challenging because of phenotypic overlap among causes, and a stratified and systematic approach is essential. Recent advances include the identification of additional genes causing dominant genetic ataxia, a better understanding of cellular pathogenesis in several disorders, the generation of new disease models that may stimulate development of new therapies, and the use of new DNA sequencing technologies, including whole-exome sequencing, to improve diagnosis.
[show abstract][hide abstract] ABSTRACT: Machado-Joseph disease (MJD) is a dominantly inherited ataxia caused by a polyglutamine-coding expansion in the ATXN3 gene. Suppressing expression of the toxic gene product represents a promising approach to therapy for MJD and other polyglutamine diseases. We performed an extended therapeutic trial of RNA interference (RNAi) targeting ATXN3 in a mouse model expressing the full human disease gene and recapitulating key disease features. Adeno-associated virus encoding a microRNA-like molecule, miRATXN3, was delivered bilaterally into the cerebellum of 6-8 week old MJD mice, which were then followed to end-stage disease to assess the safety and efficacy of anti-ATXN3 RNAi. Despite effective, lifelong suppression of ATXN3 in the cerebellum and the apparent safety of miRATXN3, motor impairment was not ameliorated in treated MJD mice and survival was not prolonged. These results with an otherwise effective RNAi agent suggest that targeting a large extent of the cerebellum alone may not be sufficient for effective human therapy. Artificial miRNAs or other nucleotide-based suppression strategies targeting ATXN3 more widely in the brain should be considered in future preclinical tests.Molecular Therapy (2013); doi:10.1038/mt.2013.144.
[show abstract][hide abstract] ABSTRACT: 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 before 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.
Journal of Neuroscience 09/2011; 31(36):13002-14. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder caused by mutations in the NPC1 or NPC2 genes. Loss of function mutations in either gene disrupt intracellular lipid trafficking and lead to a clinically heterogeneous phenotype that invariably includes neurological dysfunction and early death. The mechanism by which impaired lipid transport leads to neurodegeneration is poorly understood. Here we used mice with a conditional null allele to establish the timing and cell type that underlie neurodegeneration due to Npc1 deficiency. We show that global deletion of Npc1 in adult mice leads to progressive weight loss, impaired motor function and early death in a time course similar to that resulting from germline deletion. These phenotypes are associated with the occurrence of characteristic neuropathology including patterned Purkinje cell loss, axonal spheroids and reactive gliosis, demonstrating that there is not a significant developmental component to NPC neurodegeneration. Furthermore, we show that these same changes occur when Npc1 is specifically deleted only in neurons, establishing that neuronal deficiency is sufficient to mediate central nervous system (CNS) disease. In contrast, astrocyte-specific deletion does not impact behavioral phenotypes, CNS histopathology or synaptic function. We conclude that defects arising in neurons, but not in astrocytes, are the determining factor in the development of NPC neuropathology.
Human Molecular Genetics 08/2011; 20(22):4440-51. · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pathways regulating neuronal vulnerability are poorly understood, yet are central to identifying therapeutic targets for degenerative neurological diseases. Here, we characterize mechanisms underlying neurodegeneration in Niemann-Pick type C (NPC) disease, a lysosomal storage disorder characterized by impaired cholesterol trafficking. To date, the relative contributions of neuronal and glial defects to neuron loss are poorly defined. Using gene targeting, we generate Npc1 conditional null mutant mice. Deletion of Npc1 in mature cerebellar Purkinje cells leads to an age-dependent impairment in motor tasks, including rotarod and balance beam performance. Surprisingly, these mice did not show the early death or weight loss that are characteristic of global Npc1 null mice, suggesting that Purkinje cell degeneration does not underlie these phenotypes. Histological examination revealed the progressive loss of Purkinje cells in an anterior-to-posterior gradient. This cell autonomous neurodegeneration occurs in a spatiotemporal pattern similar to that of global knockout mice. A subpopulation of Purkinje cells in the posterior cerebellum exhibits marked resistance to cell death despite Npc1 deletion. To explore this selective response, we investigated the electrophysiological properties of vulnerable and susceptible Purkinje cell subpopulations. Unexpectedly, Purkinje cells in both subpopulations displayed no electrophysiological abnormalities prior to degeneration. Our data establish that Npc1 deficiency leads to cell autonomous, selective neurodegeneration and suggest that the ataxic symptoms of NPC disease arise from Purkinje cell death rather than cellular dysfunction.
Human Molecular Genetics 12/2009; 19(5):837-47. · 7.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ataxias constitute a heterogeneous group of diseases in which cerebellar dysfunction typically underlies the major neurologic manifestations. It is increasingly clear that ataxia can result directly from mutations in ion channels or from perturbations in ion channel physiology in the absence of a primary channel defect. Neuronal dysfunction stemming from perturbed channel activity likely explains some motor deficits in episodic and degenerative ataxias. Understanding these pathophysiologic changes may reveal novel therapeutic targets for symptomatic treatment of ataxia.
Archives of neurology 10/2009; 66(10):1196-201. · 6.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Neurobiology of Disease 11/2008; 33(1):81-8. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: Calcium-activated potassium channels modulate calcium signaling cascades and membrane potential in both excitable and non-excitable cells. In this article we will review the physiological properties, the structure activity relationships of the existing peptide and small molecule modulators and the therapeutic importance of the three small-conductance channels KCa2.1-KCa2.3 (a.k.a. SK1-SK3) and the intermediate-conductance channel KCa3.1 (a.k.a. IKCa1). The apamin-sensitive KCa2 channels contribute to the medium afterhyperpolarization and are crucial regulators of neuronal excitability. Based on behavioral studies with apamin and on observations made in several transgenic mouse models, KCa2 channels have been proposed as targets for the treatment of ataxia, epilepsy, memory disorders and possibly schizophrenia and Parkinson's disease. In contrast, KCa3.1 channels are found in lymphocytes, erythrocytes, fibroblasts, proliferating vascular smooth muscle cells, vascular endothelium and intestinal and airway epithelia and are therefore regarded as targets for various diseases involving these tissues. Since two classes of potent and selective small molecule KCa3.1 blocker, triarylmethanes and cyclohexadienes, have been identified, several of these postulates have already been validated in animal models. The triarylmethane ICA-17043 is currently in phase III clinical trials for sickle cell anemia while another triarylmethane, TRAM-34, has been shown to prevent vascular restenosis in rats and experimental autoimmune encephalomyelitis in mice. Experiments showing that a cyclohexadiene KCa3.1 blocker reduces infarct volume in a rat subdural hematoma model further suggest KCa3.1 as a target for the treatment of traumatic and possibly ischemic brain injury. Taken together KCa2 and KCa3.1 channels constitute attractive new targets for several diseases that currently have no effective therapies.
Current Medicinal Chemistry 02/2007; 14(13):1437-57. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many neurons, including pyramidal cells of the cortex, express a slow afterhyperpolarization (sAHP) that regulates their firing. Although initial findings suggested that the current underlying the sAHP could be carried through SK(Ca) channels, recent work has uncovered anomalies that are not congruent with this idea. Here, we used overexpression and dominant-negative strategies to assess the involvement of SK(Ca) channels in mediating the current underlying the sAHP in pyramidal cells of the cerebral cortex. Pyramidal cells of layer V exhibit robust AHP currents composed of two kinetically and pharmacologically distinguishable currents known as the medium AHP current (I(mAHP)) and the slow AHP current (I(sAHP)). I(mAHP) is blocked by the SK(Ca) channel blockers apamin and bicuculline, whereas I(sAHP) is resistant to these agents but is inhibited by activation of muscarinic receptors. To test for a role for SK(Ca) channels, we overexpressed K(Ca)2.1 (SK1) and K(Ca)2.2 (SK2), the predominant SK(Ca) subunits expressed in the cortex, in pyramidal cells of cultured brain slices. Overexpression of K(Ca)2.1 and K(Ca)2.2 resulted in a fourfold to fivefold increase in the amplitude of I(mAHP) but had no detectable effect on I(sAHP). As an additional test, we examined I(sAHP) in a transgenic mouse expressing a truncated SK(Ca) subunit (SK3-1B) capable of acting as a dominant negative for the entire family of SK(Ca)-IK(Ca) channels. Expression of SK3-1B profoundly inhibited I(mAHP) but again had no discernable effect on I(sAHP). These results are inconsistent with the proposal that SK(Ca) channels mediate I(sAHP) in pyramidal cells and indicate that a different potassium channel mediates this current.
Journal of Neuroscience 05/2004; 24(14):3537-42. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cerebellar ataxia, a devastating neurological disease, may be initiated by hyperexcitability of deep cerebellar nuclei (DCN) secondary to loss of inhibitory input from Purkinje neurons that frequently degenerate in this disease. This mechanism predicts that intrinsic DCN hyperexcitability would cause ataxia in the absence of upstream Purkinje degeneration. We report the generation of a transgenic (Tg) model that supports this mechanism of disease initiation. Small-conductance calcium-activated potassium (SK) channels, regulators of firing frequency, were silenced in the CNS of Tg mice with the dominant-inhibitory construct SK3-1B-GFP. Transgene expression was restricted to the DCN within the cerebellum and was detectable beginning on postnatal day 10, concomitant with the onset of cerebellar ataxia. Neurodegeneration was not evident up to the sixth month of age. Recordings from Tg DCN neurons revealed loss of the apamin-sensitive after-hyperpolarization current (IAHP) and increased spontaneous firing through SK channel suppression, indicative of DCN hyperexcitability. Spike duration and other electrogenic conductance were unaffected. Thus, a purely electrical alteration is sufficient to cause cerebellar ataxia, and SK openers such as the neuroprotective agent riluzole may reduce neuronal hyperexcitability and have therapeutic value. This dominant-inhibitory strategy may help define the in vivo role of SK channels in other neuronal pathways.
Journal of Clinical Investigation 03/2004; 113(4):582-90. · 12.81 Impact Factor
[show abstract][hide abstract] ABSTRACT: Small conductance Ca2+-activated K+ channels, products of the SK1-SK3 genes, regulate membrane excitability both within and outside the nervous system. We report the characterization of a SK3 variant (SK3-1C) that differs from SK3 by utilizing an alternative first exon (exon 1C) in place of exon 1A used by SK3, but is otherwise identical to SK3. Quantitative RT-PCR detected abundant expression of SK3-1C transcripts in human lymphoid tissues, skeletal muscle, trachea, and salivary gland but not the nervous system. SK3-1C did not produce functional channels when expressed alone in mammalian cells, but suppressed SK1, SK2, SK3, and IKCa1 channels, but not BKCa or KV channels. Confocal microscopy revealed that SK3-1C sequestered SK3 protein intracellularly. Dominant-inhibitory activity of SK3-1C was not due to a nonspecific calmodulin sponge effect since overexpression of calmodulin did not reverse SK3-1C-mediated intracellular trapping of SK3 protein, and calmodulin-Ca2+-dependent inactivation of CaV channels was not affected by SK3-1C overexpression. Deletion analysis identified a dominant-inhibitory segment in the SK3-1C C terminus that resembles tetramerization-coiled-coiled domains reported to enhance tetramer stability and selectivity of multimerization of many K+ channels. SK3-1C may therefore suppress calmodulin-gated SKCa/IKCa channels by trapping these channel proteins intracellularly via subunit interactions mediated by the dominant-inhibitory segment and thereby reduce functional channel expression on the cell surface. Such family-wide dominant-negative suppression by SK3-1C provides a powerful mechanism to titrate membrane excitability and is a useful approach to define the functional in vivo role of these channels in diverse tissues by their targeted silencing.
Journal of Biological Chemistry 03/2004; 279(8):6893-904. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The small-conductance calcium-activated K(+) channel SK3 (SKCa3/KCNN3) regulates electrical excitability and neurotransmitter release in monoaminergic neurons, and has been implicated in schizophrenia, ataxia and anorexia nervosa. We have identified a novel SK3 transcript, SK3-1B that utilizes an alternative first exon (exon 1B), but is otherwise identical to SK3. SK3-1B, mRNA is widely distributed in human tissues and is present at 20-60% of SK3 in the brain. The SK3-1B protein lacks the N-terminus and first transmembrane segment, and begins eight residues upstream of the second transmembrane segment. When expressed alone, SK3-1B did not produce functional channels, but selectively suppressed endogenous SK3 currents in the pheochromocytoma cell line, PC12, in a dominant-negative fashion. This dominant inhibitory effect extended to other members of the SK subfamily, but not to voltage-gated K(+) channels, and appears to be due to intracellular trapping of endogenous SK channels. The effect of SK3-1B expression is very similar to that produced by expression of the rare SK3 truncation allele, SK3-Delta, found in a patient with schizophrenia. Regulation of SK3 and SK3-1B levels may provide a potent mechanism to titrate neuronal firing rates and neurotransmitter release in monoaminergic neurons, and alterations in the relative abundance of these proteins could contribute to abnormal neuronal excitability, and to the pathogenesis of schizophrenia.
[show abstract][hide abstract] ABSTRACT: Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.
Journal of Biological Chemistry 12/2001; 276(46):43145-51. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1–3) mediate the slow afterhyperpolarization
in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here
we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that
share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tsκ, and PO1 were ineffective. Lei
blocked these channels more potently than PO5 because of the presence of Ala1, Phe2, and Met7. By replacing Met7 in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab7, a selective SKCa2 inhibitor (K
d = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab7 by replacing His521 with the corresponding SKCa2 residue (Asn367). Intracerebroventricular injection of Lei-Dab7 into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers.
Lei-Dab7 will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.
Journal of Biological Chemistry 11/2001; 276(46):43145-43151. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: KCNN3 is a member of the gene family, KCNN1-4, encoding the small and intermediate conductance calcium-activated potassium channels. Long CAG-repeat alleles of this gene have been found to be over-represented in patients with schizophrenia in a number of population-based association studies, and this gene maps to human chromosome 1q21, a region recently implicated in schizophrenia by linkage. To set the stage for a further functional evaluation of KCNN3, we defined the nature of the genomic locus in the size, structure, and sequence of its introns and exons and the function of potential upstream regulatory regions. We isolated P1-derived artificial chromosome (PAC) clones from a genomic library and identified an overlapping available bacterial artificial chromosome (BAC) clone. Cosmids subcloned from the PAC and BAC clones were then sequenced and merged with the sequence in the public database. The KCNN3 gene spans over 163.1 kb and is composed of eight exons and seven introns. All of the exon-intron junctions conform closely to consensus splice sites. The proximal 2.5 kb of the 5'-flanking sequence was obtained and analyzed for potential transcription factor binding sites. In the proximal 2.5 kb upstream region, potential sites for the Ikaros factor (IK2), homeodomain factor Nkx-2.5/Csx (NKX25), nuclear factor of activated T-cells (NFAT), upstream stimulating factor (USF), c-AMP responsive element binding protein (CREB), POU factor Brn2 (BRN-2), myeloid zinc finger protein (MZF1), vitellogenin binding protein (VBP), HNF3 forkhead homologue 2 (HFH2), and transcription initiation were identified, as well as several potential AP-1 and AP-4 sites. Finally, a 2261-bp fragment of this upstream region was cloned into a promoterless pGL3-luciferase vector, where it produced orientation-dependent expression of the reporter gene in transiently transfected PC12 cells, cells which natively express functional KCNN3 channels, suggesting that this cloned fragment includes competent promoter elements of this gene.
Journal of Human Genetics 02/2001; 46(8):463-70. · 2.37 Impact Factor