PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins

The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, beta-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders.     

SUMO-1 interacts with mutant ataxin-1 and colocalizes to its aggregates in Purkinje cells of SCA1 transgenic mice

Spinocerebellar ataxia type 1 (SCA1) is one of several progressive neurodegenerative diseases caused by the expanded polyglutamine tract in ataxin-1, the SCA1 gene product. In SCA1 patients and transgenic mice, the affected neuronal cells contain a large ubiquitin-positive aggregate which is derived from the mutant ataxin-1. Small ubiquitin-like modifier-1 (SUMO-1) is one of the most intriguing ubiquitin-like modifiers being conjugated to target proteins and modulating a number of cellular pathways. Recent findings that the aggregates from several neurodegenerative diseases are SUMO-1-positive prompted us to examine the implication of SUMO-1 in SCA1 pathogenesis. In our yeast two-hybrid experiments using mutant ataxin-1 as bait, we identified a SUMO-1 protein that directly binds to ataxin-1 protein. Interestingly, we found that most of the mutant ataxin-1-derived aggregates were SUMO-1-positive both in Purkinje cells of SCA1 transgenic mice and in HeLa cells, but not wild-type ataxin-1 in HeLa cells. In addition, the aggregates in Purkinje cells of SCA1 transgenic mice were positive against both anti-SUMO-1 and anti-ubiquitin antibodies. These results show that the SUMO-1 protein interacts with mutant ataxin-1 and colocalizes with its aggregates which suggests the involvement of the SUMO-1 system in the pathogenesis of SCA1 disease.     

Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice

Polyglutamine-induced neurodegeneration in transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene is modulated by subcellular distribution of ataxin-1 and by components of the protein folding/degradation machinery. Since phosphorylation is a prominent mechanism by which these processes are regulated, we examined phosphorylation of ataxin-1 and found that serine 776 (S776) was phosphorylated. Residue 776 appeared to affect cellular deposition of ataxin-1[82Q] in that ataxin-1[82Q]-A776 failed to form nuclear inclusions in tissue culture cells. The importance of S776 for polyglutamine-induced pathogenesis was examined by generating ataxin-1[82Q]-A776 transgenic mice. These mice expressed ataxin-1[82Q]-A776 within Purkinje cell nuclei, yet the ability of ataxin-1[82Q]-A776 to induce disease was substantially reduced. These studies demonstrate that polyglutamine tract expansion and localization of ataxin-1 to the nucleus of Purkinje cells are not sufficient to induce disease. We suggest that S776 of ataxin-1 also has a critical role in SCA1 pathogenesis.     

Capacitative calcium entry deficits and elevated luminal calcium content in mutant presenilin-1 knockin mice

Dysregulation of calcium signaling has been causally implicated in brain aging and Alzheimer's disease. Mutations in the presenilin genes (PS1, PS2), the leading cause of autosomal dominant familial Alzheimer's disease (FAD), cause highly specific alterations in intracellular calcium signaling pathways that may contribute to the neurodegenerative and pathological lesions of the disease. To elucidate the cellular mechanisms underlying these disturbances, we studied calcium signaling in fibroblasts isolated from mutant PS1 knockin mice. Mutant PS1 knockin cells exhibited a marked potentiation in the amplitude of calcium transients evoked by agonist stimulation. These cells also showed significant impairments in capacitative calcium entry (CCE, also known as store-operated calcium entry), an important cellular signaling pathway wherein depletion of intracellular calcium stores triggers influx of extracellular calcium into the cytosol. Notably, deficits in CCE were evident after agonist stimulation, but not if intracellular calcium stores were completely depleted with thapsigargin. Treatment with ionomycin and thapsigargin revealed that calcium levels within the ER were significantly increased in mutant PS1 knockin cells. Collectively, our findings suggest that the overfilling of calcium stores represents the fundamental cellular defect underlying the alterations in calcium signaling conferred by presenilin mutations.     

Comparative characterization of GPRC5B and GPRC5CLacZ knockin mice; behavioral abnormalities in GPRC5B-deficient mice

Although GPRC5B and GPRC5C are categorized into the G protein-coupled receptor family C, including glutamate receptors, GABA receptors, and taste receptors, their physiological functions remain unknown. Since both receptors are expressed in the brain and evolutionarily conserved from fly to human, it is conceivable that they have significant biological roles particularly in the central nervous system (CNS). We generated GPRC5B- and GPRC5C-deficient mice to examine their roles in the CNS. Both homozygous mice were viable, fertile, and showed no apparent histological abnormalities, though GPRC5B-deficient mice resulted in partial perinatal lethality. We demonstrated that the expressions of GPRC5B and GPRC5C are developmentally regulated and differentially distributed in the brain. GPRC5B-deficient mice exhibited altered spontaneous activity pattern and decreased response to a new environment, while GPRC5C-deficient mice have no apparent behavioral deficits. Thus, GPRC5B has important roles for animal behavior controlled by the CNS. In contrast, GPRC5C does not affect behavior, though it has a high sequence similarity to GPRC5B. These findings suggest that family C, group 5 (GPRC5) receptors in mammals are functionally segregated from their common ancestor.     

Keratin 5 knockout mice reveal plasticity of keratin expression in the corneal epithelium

We have recently demonstrated that the keratin K3 gene, which is active in the suprabasal human corneal epithelium, is missing in the genome of the mouse. We show that a normal K3 gene exists in a wide variety of mammals while in rodents the gene is converted to a pseudogene with a very strong sequence drift. The availability of K5-/- mice provides a unique opportunity to investigate type-specific keratin function during corneal differentiation in the absence of both K5 and K3. Here, we report that the deletion of K5, which in wild-type mice forms a cytoskeleton with K12, does neither cause keratin aggregation nor cytolysis in the cornea. This is due to the induction of K4 in corneal epithelial cells, normally restricted to corneal stem stem cells residing in the limbus. Using a combination of antibodies and RT-PCR, we identified additional keratins expressed in the mouse cornea including K23 which was previously thought to be specific for pancreatic carcinomas. This reflects an unexpected complexity of keratin expression in the cornea. Our data suggest that in the absence of mechanical stress, corneal differentiation does not depend on distinct keratin pairs, supporting a concept of functional redundancy, at least for certain keratins.     

Histone deacetylase-3 interacts with ataxin-7 and is altered in a spinocerebellar ataxia type 7 mouse model

Spinocerebellar ataxia type 7 (SCA7) is caused by a toxic polyglutamine (polyQ) expansion in the N-terminus of the protein ataxin-7. Ataxin-7 has a known function in the histone acetylase complex, Spt/Ada/Gcn5 acetylase (STAGA) chromatin-remodeling complex. We hypothesized that some histone deacetylase (HDAC) family members would impact the posttranslational modification of normal and expanded ataxin-7 and possibly modulate ataxin-7 function or neurotoxicity associated with the polyQ expansion. Interestingly, when we coexpressed each HDAC family member in the presence of ataxin-7 we found that HDAC3 increased the posttranslational modification of normal and expanded ataxin-7. Specifically, HDAC3 stabilized ataxin-7 and increased modification of the protein. Further, HDAC3 physically interacts with ataxin-7. The physical interaction of HDAC3 with normal and polyQ-expanded ataxin-7 affects the toxicity in a polyQ-dependent manner. We detect robust HDAC3 expression in neurons and glia in the cerebellum and an increase in the levels of HDAC3 in SCA7 mice. Consistent with this we found altered lysine acetylation levels and deacetylase activity in the brains of SCA7 transgenic mice. This study implicates HDAC3 and ataxin-7 interaction as a target for therapeutic intervention in SCA7, adding to a growing list of neurodegenerative diseases that may be treated by HDAC inhibitors.     

Aminergic neurons: State control and plasticity in three model systems

Aminergic neurons have particular functions in many systems, and in this review their role is discussed and compared in three systems: those parts of the central nervous system controlling sleep and waking in the cat; the superior cervical ganglion; and the isolated nervous system of Aplysia.In the cat the aminergic neurons are most important in a waking state during which time external information is received, processed, and can be retrieved, and during which time habituation and sensitization occur. Aminergic neurons appear to have similar roles in state control in plasticity in both the Aplysianervous system and the superior cervical ganglion. The striking similarities in the role of aminergic neurons in these three systems support the speculation that aminergic neurons have uniquely important roles in regulation of the plastic properties of neurons.     

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.     

Developmental abnormalities of the thymus in hea/hea mutant mice.

This study found that the thymus of hea/hea mutant mice (hea mice) became atrophic in early phase of life and that the differentiation of CD4-CD8- (Double Negative; DN) into CD4+CD8+ (Double Positive; DP) cells during the development of T cells in the thymus was abnormal. The thymus development of hea mice was different from that of normal littermates. After 6 days of age, the numbers of thymocytes in hea mice decreased. The total numbers of DP cells in the thymus of hea mice reached the maximum at 6-9 days of age and then decreased after 10 days. The total numbers of DN cells in the thymus were almost constant in hea mice and normal littermates. These results indicate abnormalities in the process of differentiation from DN to DP cells in the thymus of hea mice. Flow cytometoric analysis indicated the presence of a large number of apoptotic and necrotic cells in the thymus of 13-15-day-old hea mice. However, there were no significant differences in the amount of mRNAs of Fas, Fas ligand and IL-7 between hea mice and normal littermates. Splenocytes from hea mice produced the same amount of cytokine mRNAs as normal littermate mice and the hea mutation (Ttc7(fsn-hea)) did not affect serum levels of IgM immunoglobulin. However, activated T cells from hea mice showed more secretion of cytokines derived from Th2 cells than from Th1 cells, so they might be affected by abnormalities of the immune system.     

Molecular analysis of Spinocerebellar ataxias in Koreans: frequencies and reference ranges of SCA1, SCA2, SCA3, SCA6, and SCA7.

Spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders. CAG repeat expansions in the causative genes have been identified as the basic cause of several types of SCAs, and have been used for the diagnoses and classifications of patients with ataxia. In order to assess the frequency and CAG repeat size ranges of SCAs, and to establish an effective strategy for molecular diagnosis, we performed a molecular analysis of SCA1, SCA2, SCA3, SCA6, and SCA7 in 76 patients. These patients were as follows: 32 with dominant inheritance, 39 sporadic cases, and 5 with unknown family histories. The normal and affected CAG repeat size ranges were established at five SCA loci in Koreans, which was consistent with previous reports. The total prevalence of the five types of SCAs was 39.5% in the 76 patients with ataxia, regardless of their family history. It was 75.0% in the 32 families with a dominant inheritance. The most frequent type was SCA3 (15.8%), followed by SCA2 (14.5%). Both types combined formed 76.7% of the 30 patients with CAG expansions. SCA1, SCA6, and SCA7 were less frequent, affecting 3.9%, 2.6%, and 2.6% of the cases, respectively. This mutation spectrum is quite different from a previous report concerning Koreans, but is similar to the distributions that are seen in several ethnic populations worldwide. For a correct and effective diagnosis of SCAs, we suggest that a molecular diagnosis be undertaken, even in patients without a family history, as well as those with a family history. A stepwise approach is also recommended. Patients with ataxia should be tested for SCA2 and SCA3. Individuals testing negative should be tested for SCA1, SCA6, and SCA7.     

Crystal structures of Drosophila mutant translin and characterization of translin variants reveal the structural plasticity of translin proteins

Translin protein is highly conserved in eukaryotes. Human translin binds both ssDNA and RNA. Its nucleic acid binding site results from a combination of basic regions in the octameric structure. We report here the first biochemical characterization of wild-type Drosophila melanogaster (drosophila) translin and a chimeric translin, and present 3.5 A resolution crystal structures of drosophila P168S mutant translin from two crystal forms. The wild-type drosophila translin most likely exists as an octamer/decamer, and binds to the ssDNA Bcl-CL1 sequence. In contrast, ssDNA binding-incompetent drosophila P168S mutant translin exists as a tetramer. The structures of the mutant translin are identical in both crystal forms, and their C-terminal residues are disordered. The chimeric protein, possessing two nucleic acid binding motifs of drosophila translin, the C-terminal residues of human translin, and serine at position 168, attains the octameric state and binds to ssDNA. The present studies suggest that the oligomeric status of translin critically influences the DNA binding properties of translin proteins.     

Accelerated plaque accumulation, associative learning deficits, and up-regulation of alpha 7 nicotinic receptor protein in transgenic mice co-expressing mutant human presenilin 1 and amyloid precursor proteins

Familial Alzheimer's disease-associated mutations in presenilin 1 or 2 or amyloid precursor protein result in elevated beta-amyloid, beta-amyloid accumulation, and plaque formation in the brains of affected individuals. By crossing presenilin 1 transgenic mice carrying the A246E mutation with plaque-producing amyloid precursor protein K670N/M671L transgenic mice (Tg2576), we show that co-expression of both mutant transgenes results in acceleration of amyloid accumulation and associative learning deficits. At 5 months of age with no detectable plaque pathology, amyloid precursor protein transgenic animals are impaired in contextual fear learning following two pairings of conditioned and unconditioned stimuli but appear normal following a more robust five-pairing training. At 9 months of age when beta-amyloid deposition is evident, these mice are impaired following both two-pairing and five-pairing protocols. Mice carrying both transgenes are impaired in contextual fear conditioning at either age. All transgenic animal groups performed as well as controls in cued fear conditioning, indicating that the contextual fear learning deficits are hippocampus-specific. The associative learning impairments are coincident with elevated alpha 7 nicotinic acetylcholine receptor protein in the dentate gyrus. These findings provide two robust and rapid assays for beta-amyloid-associated effects that can be performed on young animals: impaired contextual fear learning and up-regulation of alpha 7 nicotinic receptors.