Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.

Two mutations in the DJ-1 gene on chromosome1p36 have been identified recently to cause early-onset, autosomal recessive Parkinson's disease. As no information is available regarding the distribution of DJ-1 protein in the human brain, in this study we used a monoclonal antibody for DJ-1 to map its distribution in frontal cortex and substantia nigra, regions invariably involved in Parkinson's disease. Western blotting of human frontal cortex showed DJ-1 to be an abundant protein in control, idiopathic Parkinson's disease, cases with clinical and pathological phenotypes of Parkinson's disease with R98Q polymorphism for DJ-1, and in progressive supranuclear palsy (PSP) brains. We also showed that DJ-1 immunoreactivity (IR) was particularly prominent in astrocytes and astrocytic processes in both control and Parkinson's disease frontal cortex, whereas neurons showed light or no DJ-1 IR. Only occasional Lewy bodies (LBs), the pathological hallmarks of Parkinson's disease, showed faint DJ-1 IR, localized to the outer halo. In preclinical studies we showed that DJ-1 is expressed in primary hippocampal and astrocyte cultures of mouse brain. By 2D gel analysis we also showed multiple pI isoforms for DJ-1 ranging between 5.5-6.6 in both control and Parkinson's disease brains, whilst exposure of M17 cells to the oxidizing agent paraquat was manifested as a shift in pI of endogenous DJ-1 towards more acidic isoforms. We conclude that DJ-1 is not an essential component of LBs and Lewy neurites, is expressed mainly by astrocytes in human brain tissue and is sensitive to oxidative stress conditions. These results are consistent with the hypothesis that neuronal-glial interactions are important in the pathophysiology of Parkinson's disease.

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Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.

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In March 2013, the California Institute for Regenerative Medicine, in collaboration with the NIH Center for Regenerative Medicine, held a 2-day workshop on cell therapies for Parkinson's disease (PD), with the goals of reviewing the state of stem cell research for the treatment of PD and discussing and refining the approach and the appropriate patient populations in which to plan and conduct new clinical trials using stem cell-based therapies for PD. Workshop participants identified priorities for research, development, and funding; discussed existing resources and initiatives; and outlined a path to the clinic for a stem cell-based therapy for PD. A consensus emerged among participants that the development of cell replacement therapies for PD using stem cell-derived products could potentially offer substantial benefits to patients. As with all stem cell-based therapeutic approaches, however, there are many issues yet to be resolved regarding the safety, efficacy, and methodology of transplanting cell therapies into patients. Workshop participants agreed that designing an effective stem cell-based therapy for PD will require further research and development in several key areas. This paper summarizes the meeting. STEM CELLS TRANSLATIONAL MEDICINE 2014;3:979-991

Antibodies to a-synuclein label Lewy bodies and Lewy neurites in Parkinson's disease, dementia with Lewy bodies and in cases of Alzheimer's disease (AD) with Lewy bodies. These antibodies also label glial cytoplasmic inclusions in multiple system atrophy (MSA). The aim of the current study is to investigate the prevalence and biochemical nature of a-synuclein in AD and MSA brains, compared to control brain, using antibodies raised against different regions of a-synuclein.In AD, the immunoreactivity of a-synuclein on western blots suggests the presence of agpgated protein, as well as a concomitant reduction in the amount of monomeric a-synuclein compared to that in control. The aggregated a-synuclein in AD appears as a smear of immunoreactivity on wcstem blots, and is absent from the equivalent hction of control brain. As this smear is not labelled by antibodies to the C-terminus of a-synuclein, we suggest that this aggrrgated a-synuclcin in AD ni.~ be either cleaved or may become inaccessible to the antibody. prior to or following the aggregation.We arc also investigating the nature of a-synuclein found in MSA brain. Current results show that the pattern of a-synuclein deposited in MSA brain differs both from control and from the smear detected in AD.Calcium storage in Ihc ER serves several functions. Cell sumulation typically leads to cytosolic signals based on uansient Ca" release from ER. Within the ER lumen, the processes of folding, subunit assembly and other postlranslational protein modifications. as well as transport and quality control of protein synthesis seem to be calcium dependent (I). These processes are mediated hy the ER-resident chaperone proteins, some of which (e.g. GRWS. GRP94) are known a q strcss proteins because their expression is enhanced by treatments that interfere with the ER protein synthesis machinery.We have investigated the expression of two ER calcium pump isoforms (Ca"-ATF'ases). SERCAZb and SERCA3. in E l 2 cells. Treatments with calcium ionophore. thapsigargin or EGTA resulted in a high induction of the SERCA2b mRNA. but surprisingly not of the SERCA3 -A.The common feature of rhcsc treatments is that thcy deplete the ER for calcium. We further showed that SERCAZb mRNA is also higbly induced in the presence of DlT. tunicamycin. azctidine (a proline analog) and brefcldin A, agents known to upregulale the classical ER-resident stress proteins. The expression of SERCA2b. but not of SERCA3. was also increased at the protein level. Since all cells depend on thc ER for protein synthesis. maturation and transport, these results may explain why SERCA2b. rather than SERCA3. is the ubiquitously disuihuted ER Ca"-ATPax isofom.Our results suggest that SERCA2b is a new member of the ER stress protein family. These results raise a number of further questions. including: is SERCA2b gene regulated by the unfolded protein response pathway similar to the classical ER stress proteins (2)? Is SERCA2b induced in neurons in vivo under pathological conditions (degeneration. ischemia)? Does such induc...

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