Identification of a calmodulin-regulated soybean Ca(2+)-ATPase (SCA1) that is located in the plasma membrane

Ca(2)+-ATPases are key regulators of Ca(2+) ion efflux in all eukaryotes. Animal cells have two distinct families of Ca(2+) pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane. In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non-plasma membrane locations. Here, we identify a plant cDNA, designated SCA1 (for soybean Ca(2+)-ATPase 1), that encodes Ca(2+)-ATPase and is located at the plasma membrane. The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein. The Ca(2+)-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K(1/2) approximately 10 nM). Two calmodulin binding sequences were identified in the N-terminal domain. An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca(2+) pumps. Our results indicate that SCA1p is structurally distinct from the plasma membrane-localized Ca(2+) pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations. In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress.     

Nonmuscle myosin IIA and IIB have distinct functions in the exocytosis-dependent process of cell membrane repair

Vesicle generation, recruitment, and exocytosis are essential for repairing disruptions of cell membranes. The functions of nonmuscle myosin IIA and IIB in this exocytotic process of membrane repair were studied by the antisense technique. Knockdown of myosin IIB suppressed wound-induced exocytosis and the membrane resealing process. Knockdown of myosin IIA did not suppress exocytosis at an initial wound and had no inhibitory effect on the resealing at initial wounds but did inhibit the facilitated rate of resealing normally found at repeated wounds made at the same site. COS-7 cells, which lack myosin IIA, did not show the facilitated response of membrane resealing to a repeated wound. S91 melanoma cells, a mutant cell line lacking myosin Va, showed normal membrane resealing and normal facilitated responses. We concluded that myosin IIB was required for exocytosis and therefore cell membrane repair itself and that myosin IIA was required in facilitation of cell membrane repair at repeated wounds. Myosin IIB was primarily at the subplasmalemma cortex and myosin IIA was concentrated at the trans-Golgi network consistent with their distinct roles in vesicle trafficking in cell membrane repair.     

CHIP protects from the neurotoxicity of expanded and wild-type ataxin-1 and promotes their ubiquitination and degradation

CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemagglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration.     

Hsp105alpha suppresses the aggregation of truncated androgen receptor with expanded CAG repeats and cell toxicity

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The N-terminal fragment of AR containing the expanded polyglutamine tract aggregates in cytoplasm and/or in nucleus and induces cell death. Some chaperones such as Hsp40 and Hsp70 have been identified as important regulators of polyglutamine aggregation and/or cell death in neuronal cells. Recently, Hsp105alpha, expressed at especially high levels in mammalian brain, has been shown to suppress apoptosis in neuronal cells and prevent the aggregation of protein caused by heat shock in vitro. However, its role in polyglutamine-mediated cell death and toxicity has not been studied. In the present study, we examined the effects of Hsp105alpha on the aggregation and cell toxicity caused by expansion of the polyglutamine tract using a cellular model of SBMA. The transient expression of truncated ARs (tARs) containing an expanded polyglutamine tract caused aggregates to form in COS-7 and SK-N-SH cells and concomitantly apoptosis in the cells with the nuclear aggregates. When Hsp105alpha was overexpressed with tAR97 in the cells, Hsp105alpha was colocalized to aggregates of tAR97, and the aggregation and cell toxicity caused by expansion of the polyglutamine tract were markedly reduced. Both beta-sheet and alpha-helix domains, but not the ATPase domain, of Hsp105alpha were necessary to suppress the formation of aggregates in vivo and in vitro. Furthermore, Hsp105alpha was found to localize in nuclear inclusions formed by ARs containing an expanded polyglutamine tract in tissues of patients and transgenic mice with SBMA. These findings suggest that overexpression of Hsp105alpha suppresses cell death caused by expansion of the polyglutamine tract without chaperone activity, and the enhanced expression of the essential domains of Hsp105alpha in brain may provide an effective therapeutic approach for CAG repeat diseases.     

Cytoplasmic Location of α1A Voltage-Gated Calcium Channel C-Terminal Fragment (Cav2.1-CTF) Aggregate Is Sufficient to Cause Cell Death

The human α_1A voltage-dependent calcium channel (Ca_v2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Ca_v2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.     

Proteolytic processing regulates pathological accumulation in dentatorubral-pallidoluysian atrophy

Dentatorubral-pallidoluysian atrophy is caused by polyglutamine (polyQ) expansion in atrophin-1 (ATN1). Recent studies have shown that nuclear accumulation of ATN1 and cleaved fragments with expanded polyQ is the pathological process underlying neurodegeneration in dentatorubral-pallidoluysian atrophy. However, the mechanism underlying the proteolytic processing of ATN1 remains unclear. In the present study, we examined the proteolytic processing of ATN1 aiming to understand the mechanisms of ATN1 accumulation with polyQ expansion. Using COS-7 and Neuro2a cells that express the ATN1 gene, in which ATN1 was accumulated by increasing the number of polyQs, we identified a novel C-terminal fragment containing a polyQ tract. The mutant C-terminal fragment with expanded polyQ selectively accumulated in the cells, and this was also demonstrated in the brain tissues of patients with dentatorubral-pallidoluysian atrophy. Immunocytochemical and biochemical studies revealed that full-length ATN1 and C-terminal fragments displayed individual localization. The mutant C-terminal fragment was preferentially found in the cytoplasmic membrane/organelle and insoluble fractions. Accordingly, it is assumed that the proteolytic processing of ATN1 regulates the localization of C-terminal fragments. Accumulation of the C-terminal fragment was enhanced by inhibition of caspases in the cytoplasm of COS-7 cells. Collectively, these results suggest that the C-terminal fragment plays a principal role in the pathological accumulation of ATN1 in dentatorubral-pallidoluysian atrophy.     

Two regions of the tail are necessary for the isoform-specific functions of nonmuscle myosin IIB

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB(-/-) fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament.     

Reduced Levels of Proteasome Products in a Mouse Striatal Cell Model of Huntington’s Disease

Huntington’s disease is the result of a long polyglutamine tract in the gene encoding huntingtin protein, which in turn causes a large number of cellular changes and ultimately results in neurodegeneration of striatal neurons. Although many theories have been proposed, the precise mechanism by which the polyglutamine expansion causes cellular changes is not certain. Some evidence supports the hypothesis that the long polyglutamine tract inhibits the proteasome, a multiprotein complex involved in protein degradation. However, other studies report normal proteasome function in cells expressing long polyglutamine tracts. The controversy may be due to the methods used to examine proteasome activity in each of the previous studies. In the present study, we measured proteasome function by examining levels of endogenous peptides that are products of proteasome cleavage. Peptide levels were compared among mouse striatal cell lines expressing either 7 glutamines (STHdh ^Q7/Q7) or 111 glutamines in the huntingtin protein, either heterozygous (STHdh ^Q7/Q111) or homozygous (STHdh ^Q111/Q111). Both of the cell lines expressing huntingtin with 111 glutamines showed a large reduction in nearly all of the peptides detected in the cells, relative to levels of these peptides in cells homozygous for 7 glutamines. Treatment of STHdh ^Q7/Q7 cells with proteasome inhibitors epoxomicin or bortezomib also caused a large reduction in most of these peptides, suggesting that they are products of proteasome-mediated cleavage of cellular proteins. Taken together, these results support the hypothesis that proteasome function is impaired by the expression of huntingtin protein containing long polyglutamine tracts.     

Molecular aspects of higher plant P-type Ca(2+)-ATPases

Recent genomic data in the model plant Arabidopsis thaliana reveal the existence of at least 11 Ca(2+)-ATPase genes, and an analysis of expressed sequence tags suggests that the number of calcium pumps in this organism might be even higher. A phylogenetic analysis shows that 11 Ca(2+)-ATPases clearly form distinct groups, type IIA (or ECA for ER-type Ca(2+)-ATPase) and type IIB (ACA for autoinhibited Ca(2+)-ATPase). While plant IIB calcium pumps characterized so far are localized to internal membranes, their animal homologues are exclusively found in the plasma membrane. However, Arabidopsis type IIB calcium pump isoforms ACA8, ACA9 and ACA10 form a separate outgroup and, based on the high molecular masses of the encoded proteins, are good candidates for plasma membrane bound Ca(2+)-ATPases. All known plant type IIB calcium ATPases seem to employ an N-terminal calmodulin-binding autoinhibitor. Therefore it appears that the activity of type IIB Ca(2+)-ATPases in plants and animals is controlled by N-terminal and C-terminal autoinhibitory domains, respectively. Possible functions of plant calcium pumps are described and - beside second messenger functions directly linked to calcium homeostasis - new data on a putative involvement in secretory and salt stress functions are discussed.     

The MAL proteolipid restricts detergent-mediated membrane pore expansion and percolation

Differential solubilization of membrane components by cold 1% Triton X-100 extraction is common practice in cell biology and membrane research, used to define components of, or localization within membrane domains called lipid rafts. In this study, extraction of biological membranes was continuously monitored in single cells by confocal microscopy. The distributions of fluorescently-tagged proteins that label raft and non-raft membranes, cytosolic and cytoskeletal proteins were continuously monitored upon addition of the detergent. Membranes containing the non-raft membrane protein VSVG-GFP were immediately extracted from the plasma membrane, whereas raft-membrane proteins were predominantly resistant to the detergent. The morphological characteristics of differential membrane solubilization consisted of the formation of pores that expand and percolate as the detergent-mediated solubilization proceeds. Pore expansion and percolation was much slower and more restricted in non-polarized MDCK cells than in COS-7 cells. Heterologous overexpression in COS-7 cells of the fluorescently-tagged human MAL, a tetra-spanning, lipid-raft-associated protein, significantly slowed and limited membrane pore expansion and percolation. Extensive percolation resulting in large holes in the membrane was observed for the raft-associated, GPI-GFP-labeled membranes in COS-7 cells. Quantitative analysis carried out using pixel intensity variance as an indicator of membrane pore expansion demonstrated that the MAL protein is capable of modifying the plasma membrane, thereby increasing its resistance to detergent-induced pore formation.     

Insights into the molecular basis of polyglutamine neurodegeneration from studies of a spinocerebellar ataxia type 7 mouse model

Spinocerebellar ataxia type 7 (SCA7) is one member of a growing list of neurodegenerative disorders that are all caused by CAG repeat expansions that produce disease by encoding elongated polyglutamine tracts in a variety of apparently unrelated proteins. In this review, we provide an overview of our efforts to determine the molecular basis of polyglutamine neurotoxicity in SCA7 by modeling this polyglutamine repeat disorder in mice. We discuss how our SCA7 mouse model develops a phenotype that is reminiscent of the retinal and cerebellar disease pathology seen in human patients. All of these findings are considered in the context of numerous other models of polyglutamine disease pathology in mice and other organisms, together with various other in vitro and biochemical studies. We present the competing hypotheses of polyglutamine disease pathogenesis, and explain how our studies of SCA7 brainstem and retinal degeneration using this mouse model have yielded insights into possible mechanisms and pathways of polyglutamine disease pathology. In addition to illustrating how our SCA7 mouse model has allowed us to develop and advance notions of disease pathogenesis, we propose a model of polyglutamine molecular pathology that attempts to integrate the key observations in the field. We close by describing why our SCA7 mouse model should be useful for the next phase of polyglutamine disease research--the development of therapies, and predict that this stage of experimentation will continue to rely heavily on the mouse.     

Poly(Q) Expansions in ATXN7 Affect Solubility but Not Activity of the SAGA Deubiquitinating Module

Spinocerebellar ataxia type 7 (SCA7) is a debilitating neurodegenerative disease caused by expansion of a polyglutamine [poly(Q)] tract in ATXN7, a subunit of the deubiquitinase (DUB) module (DUBm) in the SAGA complex. The effects of ATXN7-poly(Q) on DUB activity are not known. To address this important question, we reconstituted the DUBm in vitro with either wild-type ATXN7 or a pathogenic form, ATXN7-92Q NT, with 92 Q residues at the N terminus (NT). We found that both forms of ATXN7 greatly enhance DUB activity but that ATXN7-92Q NT is largely insoluble unless it is incorporated into the DUBm. Cooverexpression of DUBm components in human astrocytes also promoted the solubility of ATXN7-92Q, inhibiting its aggregation into nuclear inclusions that sequester DUBm components, leading to global increases in ubiquitinated H2B (H2Bub) levels. Global H2Bub levels were also increased in the cerebellums of mice in a SCA7 mouse model. Our findings indicate that although ATXN7 poly(Q) expansions do not change the enzymatic activity of the DUBm, they likely contribute to SCA7 by initiating aggregates that sequester the DUBm away from its substrates.     

CAG repeat instability, cryptic sequence variation and pathogeneticity: evidence from different loci.

Different aspects of expanded polyglutamine tracts and of their pathogenetic role are taken into consideration here. (i) The (CAG)n length of wild-type alleles of the Huntington disease gene was analysed in instability-prone tumour tissue from colon cancer patients to test whether the process leading to the elongation of alleles towards the expansion range involves single-unit stepwise mutations or larger jumps. The analysis showed that length changes of a single unit had a relatively low frequency. (ii) The observation of an expanded spinocerebellar ataxia (SCA)1 allele with an unusual pattern of multiple CAT interruptions showed that cryptic sequence variations are critical not only for sequence length stability but also for the expression of the disease phenotype. (iii) Small expansions of the (CAG)n sequence at the CACNA1A gene have been reported as causing SCA6. The analysis of families with SCA6 and episodic ataxia type 2 showed that these phenotypes are, in fact, expressions of the same disorder caused either by point mutations or by small (CAG)n expansions. A gain of function has been hypothesized for all proteins containing an expanded polyglutamine stretch, including the alpha 1A subunit of the voltage-gated calcium channel type P/Q coded by the CACNA1A gene. Because point mutations at the same gene with similar phenotypic consequences are highly unlikely to have this effect, an alternative common pathogenetic mechanism for all these mutations, including small expansions, can be hypothesized.     

Polyglutamine genes interact to modulate the severity and progression of neurodegeneration in Drosophila

The expansion of polyglutamine tracts in a variety of proteins causes devastating, dominantly inherited neurodegenerative diseases, including six forms of spinal cerebellar ataxia (SCA). Although a polyglutamine expansion encoded in a single allele of each of the responsible genes is sufficient for the onset of each disease, clinical observations suggest that interactions between these genes may affect disease progression. In a screen for modifiers of neurodegeneration due to SCA3 in Drosophila, we isolated atx2, the fly ortholog of the human gene that causes a related ataxia, SCA2. We show that the normal activity of Ataxin-2 (Atx2) is critical for SCA3 degeneration and that Atx2 activity hastens the onset of nuclear inclusions associated with SCA3. These activities depend on a conserved protein interaction domain of Atx2, the PAM2 motif, which mediates binding of cytoplasmic poly(A)-binding protein (PABP). We show here that PABP also influences SCA3-associated neurodegeneration. These studies indicate that the toxicity of one polyglutamine disease protein can be dramatically modulated by the normal activity of another. We propose that functional links between these genes are critical to disease severity and progression, such that therapeutics for one disease may be applicable to others.     

Proteomic analyses of K(v)2.1 channel phosphorylation sites determining cell background specific differences in function

The K(v)2.1 potassium channel plays an important role in regulating membrane excitability and is highly phosphorylated in mammalian neurons. Our previous results showed that variable phosphorylation of K(v)2.1 at multiple sites allows graded activity-dependent regulation of channel gating. Our previous studies also found functional differences between recombinant K(v)2.1 channels expressed in HEK293 cells and COS-1 cells that were eliminated upon complete dephosphorylation of K(v)2.1. To better understand how phosphorylation affects K(v)2.1 gating in HEK293 and COS-1 cells we used stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry to determine the level of phosphorylation at one newly and thirteen previously identified sites on K(v)2.1 purified from HEK293 and COS-1 cells. We identified seven phosphorylation sites on the K(v)2.1 C-terminus that exhibit different levels of phosphorylation in HEK293 and COS-1 cells. Six sites have enhanced phosphorylation in HEK293 compared to COS-1, while one site exhibits enhanced phosphorylation in COS-1 cells. No sites were found phosphorylated in one cell type and not the other. Interestingly, the sites exhibiting differential phosphorylation in HEK293 and COS-1 cells under basal conditions are similar to the subset targeted by calcineurin-mediated signaling pathways. The data presented here suggests that differential phosphorylation at a specific subset of sites, as opposed to utilization of novel cell-specific phosphorylation sites, can explain differences in the gating properties of K(v)2.1 in different cell types under basal conditions, and in the same cell type under basal versus stimulated conditions.     

Extended polyglutamine tracts cause aggregation and structural perturbation of an adjacent beta barrel protein

Formation of fibrillar intranuclear inclusions and related neuropathologies of the CAG-repeat disorders are linked to the expansion of a polyglutamine tract. Despite considerable effort, the etiology of these devastating diseases remains unclear. Although polypeptides with glutamine tracts recapitulate many of the observed characteristics of the gene products with CAG repeats, such as in vitro and in vivo aggregation and toxicity in model organisms, extended polyglutamine segments have also been reported to structurally perturb proteins into which they are inserted. Additionally, the sequence context of a polyglutamine tract has recently been shown to modulate its propensity to aggregate. These findings raise the possibility that indirect influences of the repeat tract on adjacent protein domains are contributory to pathologies. Destabilization of an adjacent domain may lead to loss of function, as well as favoring non-native structures in the neighboring domain causing them to be prone to intermolecular association and consequent aggregation. To explore these phenomena, we have used chimeras of a well studied globular protein and exon 1 of huntingtin. We find that expansion of the polyglutamine segment beyond the pathological threshold (>35 glutamines) results in structural perturbation of the neighboring protein whether the huntingtin exon is N- or C-terminal. Elongation of the polyglutamine region also substantially increases the propensity of the chimera to aggregate, both in vitro and in vivo, and in vitro aggregation kinetics of a chimera with a 53-glutamine repeat follow a nucleation polymerization mechanism with a monomeric nucleus.     

Polyglutamine Genes Interact to Modulate the Severity and Progression of Neurodegeneration in Drosophila

The expansion of polyglutamine tracts in a variety of proteins causes devastating, dominantly inherited neurodegenerative diseases, including six forms of spinal cerebellar ataxia (SCA). Although a polyglutamine expansion encoded in a single allele of each of the responsible genes is sufficient for the onset of each disease, clinical observations suggest that interactions between these genes may affect disease progression. In a screen for modifiers of neurodegeneration due to SCA3 in Drosophila, we isolated atx2, the fly ortholog of the human gene that causes a related ataxia, SCA2. We show that the normal activity of Ataxin-2 (Atx2) is critical for SCA3 degeneration and that Atx2 activity hastens the onset of nuclear inclusions associated with SCA3. These activities depend on a conserved protein interaction domain of Atx2, the PAM2 motif, which mediates binding of cytoplasmic poly(A)-binding protein (PABP). We show here that PABP also influences SCA3-associated neurodegeneration. These studies indicate that the toxicity of one polyglutamine disease protein can be dramatically modulated by the normal activity of another. We propose that functional links between these genes are critical to disease severity and progression, such that therapeutics for one disease may be applicable to others.     

Hsp70 and Hsp40 chaperones do not modulate retinal phenotype in SCA7 mice

Nine neurodegenerative diseases, including spinocerebellar ataxia type 7 (SCA7), are caused by the expansion of polyglutamine stretches in the respective disease-causing proteins. A hallmark of these diseases is the aggregation of expanded polyglutamine-containing proteins in nuclear inclusions that also accumulate molecular chaperones and components of the ubiquitin-proteasome system. Manipulation of HSP70 and HSP40 chaperone levels has been shown to suppress aggregates in cellular models, prevent neuronal death in Drosophila, and improve to some extent neurological symptoms in mouse models. An important issue in mammals is the relative expression levels of toxic and putative rescuing proteins. Furthermore, overexpression of both HSP70 and its co-factor HSP40/HDJ2 has never been investigated in mice. We decided to address this question in a SCA7 transgenic mouse model that progressively develops retinopathy, similar to SCA7 patients. To co-express HSP70 and HDJ2 with the polyglutamine protein, in the same cell type, at comparable levels and with the same time course, we generated transgenic mice that express the heat shock proteins specifically in rod photoreceptors. While co-expression of HSP70 with its co-factor HDJ2 efficiently suppressed mutant ataxin-7 aggregation in transfected cells, they did not prevent either neuronal toxicity or aggregate formation in SCA7 mice. Furthermore, nuclear inclusions in SCA7 mice were composed of a cleaved mutant ataxin-7 fragment, whereas they contained the full-length protein in transfected cells. We propose that differences in the aggregation process might account for the different effects of chaperone overexpression in cellular and animal models of polyglutamine diseases.     

Pathogenic mechanisms of a polyglutamine-mediated neurodegenerative disease, spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited neurodegenerative diseases caused by the expansion of a CAG trinucleotide repeat encoding a polyglutamine tract. SCA1 patients lose motor coordination and develop slurred speech, spasticity, and cognitive impairments. Difficulty with coordinating swallowing and breathing eventually causes death. Genetic evidence indicates that the disease mutation induces a toxic gain of function in the SCA1 encoded protein ATXN1. The discovery that residues in ATXN1 outside of the polyglutamine tract are crucial for pathogenesis hinted that alterations in the normal function of this protein are linked to its toxicity. Biochemical and genetic studies provide evidence that the polyglutamine expansion enhances interactions that are normally regulated by phosphorylation at Ser(776) and a subsequent alteration in its interaction with other cellular proteins. Moreover, the finding that other ATXN1 interactions are decreased in disease suggests that the polyglutamine expansion contributes to disease by both a gain-of-function mechanism and partial loss of function.