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Machado–Joseph disease is an autosomal dominant spinocerebellar degeneration caused by the expansion of a polyglutamine tract within the gene product, ataxin-3. We have previously shown that increased oxidative stress and decreased expression of Hsp27 may be contributory factors to the disease progression. In this study, we utilized neuroblastoma SK-N-SH cells stably transfected with full-length expanded ataxin-3 to further investigate the mechanism(s) resulting in the decreased expression of Hsp27. Results from 35 S-methionine pulse-chase labeling and protein degradation assays revealed that decreased Hsp27 in mutant MJD cells is due to defects in protein synthesis. Our results further demonstrated that Hsp27 degradation is independent of the proteasome degradation pathway. In addition, we showed that overexpression of Hsp27 desensitizes mutant MJD cells to apoptotic stress. Taken together, these findings provide the first evidence that expanded ataxin-3 interferes with Hsp27 synthesis, which may contribute to the impairment of the cells’ ability to respond to stresses and trigger the progression of this late-onset disease.

Huntington's disease (HD) is characterised by the proteolytic production of N-terminal fragments of huntingtin containing polyglutamine repeats that forms intracellular ubiquitinated aggregates in the affected neurons. Using cellular and transgenic mice model of HD, we report here that BAG-1 co-immunoprecipitates with the polyglutamine-expanded truncated N-terminal huntingtin (tNhtt) and associates with their aggregates through its interaction with the chaperones Hsc70/Hsp70. We further demonstrate that the over expression of BAG-1 protects polyglutamine-expanded tNhtt induced cell death. Since, BAG-1 is essential for cell survival, its association with tNhtt aggregates might disrupt its normal function and thereby promote polyglutamine-expanded tNhtt-induced cell death.

The genetic defect dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a CAG trinucleotide repeat. The mutant gene is translated into protein whose electrophoretic mobility correlates to the number of expanded CAG trinucleotide repeats, indicating that the protein carries an expanded glutamine repeat. Using two polyclonal antibodies raised against the DRPLA gene product in immunoblotting, we determined the untruncated DRPLA proteins, and showed that the amounts of mutant and wild-type DRPLA proteins were similar in DRPLA brain tissues and lymphoblastoid cells, suggesting that regulation of the level of translation of the DRPLA gene is not central to the development of the disease.

The expansion of a polyQ repeat within the ataxin-2 protein causes spinocerebellar ataxia type 2 (SCA2). However, neither the precise pathological mechanism nor the physiological functions of ataxin-2 are known. Ataxin-2 contains 47 (S/T)P sequences, which are targeted by proline-directed protein kinases such as the cyclin-dependent kinase 5 (Cdk5). We hypothesized that ataxin-2 is phosphorylated by Cdk5. In fact, phosphorylation of ataxin-2 by Cdk5-p25 was shown using two methods: in vitro(32)P labeling and electrophoretic mobility shift on Phos-tag SDS-PAGE. The fractionation of ataxin-2 into three portions, the N-terminal fragment (NF, amino acids 1-507), the middle fragment (MF, amino acids 508-905), and the C-terminal fragment (CF, amino acids 906-1313) showed that NF and MF were phosphorylated slightly and highly, respectively, by Cdk5-p25 when expressed in COS-7 cells. Cdk5-mediated phosphorylation induced the degradation of NF remarkably and MF moderately. Furthermore, toxic ataxin-2-41Q underwent proteasomal degradation after phosphorylation by Cdk5. These results suggest that Cdk5 controls the abundance of both normal and polyQ-expanded ataxin-2 protein in neurons, which implies that Cdk5 activity is a therapeutic approach for SCA2.

Nurr1 is an orphan nuclear receptor essential for development and survival of dopaminergic neurons. Mutations in Nurr1 are associated with Parkinson's disease (PD) and there is a correlation between Nurr1 and tyrosine hydroxylase (TH) expression in PD brain. Two domains, activation function 1 (AF1) at the N-terminus and AF2 at the C-terminus of Nurr1, are important for Nurr1 activation. AF1 domain is conserved in NGFI-B/Nurr1/Nor-1 family members and MAPK signal pathway is involved in AF1 activity. Using in vitro phoshorylation assays, we have shown that ERK2 is a kinase to phosphorylate Nurr1 on multiple sites. S126 and T132, which are located near AF1 core of Nurr1, are dominant sites phosphorylated by ERK2. Moreover, using GST pull-down and co-IP assays, we identified that both the N-terminus of Nurr1 containing three ERK docking domains and another ERK docking domain in Nurr1 DNA binding domain are able to bind to ERK2. Furthermore, overexpression of a constitutively active form of MEK1, together with Nurr1 and mouse ERK2, greatly increases the tyrosine hydroxylase expression in SH-SY5Y cells. Reporter gene assays show that Nurr1Delta124-133/T185A, an ERK2 phospho-site mutant form, could not further increase its transcriptional activity on TH promoter, suggesting that Nurr1 phosphorylation by ERK2 may regulate its transcriptional activity on TH promoter. Thus, our results indicate that Nurr1 phosphorylation by ERK2 may play a role in regulating the TH expression.

Human apolipoprotein E (apoE) comprises three isoforms, apoE2, apoE3 and apoE4, and apoE4 has been reported as a risk factor of Alzheimer's disease (AD). One of the clinical symptoms of AD is disorder of memory that has been suggested to be related with synaptic plasticity such as long-term potentiation (LTP). Here, we show the enhancement of hippocampal LTP at younger age in knock-in mice lacking mouse apoE, but instead expressing human apoE4. The enhancement of LTP in apoE4 knock-in mice is age-dependent, and it disappears in adult apoE4 knock-in mice. In apoE3 knock-in mice LTP is unaltered, thus human apoE4, but not apoE3, specifically modulates synaptic plasticity at younger age. Since basal synaptic transmission and distribution of glutamate receptors, as well as presynaptic functions, are intact in apoE4 knock-in mice, postsynaptic functional modification of LTP through lipid homeostasis is suggested. ApoE4 knock-in mice would be a useful animal model of human apoE4 carriers, and our finding that LTP is enhanced in younger apoE4 knock-in mice is in accord with the previous report showing higher intelligence in young human apoE4 carriers.

To date, eleven independent FAD pedigrees with the 717 Val→Ile mutation have been identified. Interestingly, five pedigrees were of Japanese origin and four were of British origin. The apparent ethnic prediction of this mutation raises the possibility that there is a founder effect in these two island nations. We did not observe any significant linkage disequilibrium in any locus of APP and GT12 loci in the five Japanese FAD pedigrees with the 717 Val→Ile mutation. A founder effect would probably not be present in Japanese FAD pedgrees with the 717 Val→Ile mutation.

The monoclonal antibody, Alz 50, is known to label many dystrophic neurites and neurofibrillary tangles in Alzheimer's disease (AD) brain. Using immunoprecipitation and immunoblotting, we have compared Alz 50 to monoclonal antibodies directed at two known components of neurofibrillary tangles, tau and ubiquitin, in order to characterize further the antigens recognized by Alz 50 in AD brain. Alz 50 labeled purified tau proteins in a highly similar fashion to two well-characterized tau monoclonal antibodies. Alz 50 precipitated proteins at the molecular weight of 50–70 k Da from AD but not normal brain; these proteins were reactive with the tau antibodies. In addition, Alz 50 precipitated proteins migrating principally around 160–180 kDa from AD but not normal brain; the relationship of the latter proteins to tau remains unclear. None of these proteins reacted with a ubiquitin antibody. We hypothesize that the proteins recognized by Alz 50 at much higher levers in AD than normal brain include modified and aggregated forms of tau.