TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.

2 (1). The most dramatic PD-associated mutation L166P impairs DJ-1 dimer formation and dramatically destabilizes the protein (2-7). Other mutations such as M26I (8) and E64D (9) have more subtle defects with unclear cellular consequences (4, 7, 10, 11). In addition to this genetic association, DJ-1 is neuropathologically linked to PD. DJ-1 is up-regulated in reactive astrocytes, and it is oxidatively modified in brains of sporadic PD patients (12)(13)(14).DJ-1 protects against oxidative stress and mitochondrial toxins in cell culture (15-17) as well as in diverse animal models (18 -21). The cytoprotective effects of DJ-1 may be stimulated by oxidation and mediated by molecular chaperoning (22, 23), and/or facilitation of the pro-survival Akt and suppression of apoptosis signal-regulating kinase 1 (ASK1) pathways (6,24,25). The cytoprotective activity of DJ-1 against oxidative stress depends on its cysteine residues (15,17,26). Among the three cysteine residues of DJ-1, the most prominent one is the easiest oxidizable that is in a constrained conformation (28), but the other cysteine residues Cys-46 and Cys-53 have been implicated with DJ-1 activity as well (22). However, the molecular basis of oxidation-mediated cytoprotective activity of DJ-1 is not clear. Moreover, the roles of PD-mutated and in vivo oxidized methionines are not known.Here we have mutagenized all oxidizable residues within DJ-1 and studied the effects on protein stability and function. The PD-associated mutation M26I within the DJ-1 dimer interface selectively reduced protein expression as well as ASK1 suppression and cytoprotective activity in oxidatively stressed cells. These cell culture results support a pathogenic effect of the clinical M26I mutation (8). Furthermore, oxidation-defective C106A mutation abolished binding to ASK1 and cytoprotective activity of DJ-1, whereas the designed higher order oxidation

The Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulates proinflammatory responses in mouse astrocyte-rich primary cultures. When treated with a Toll-like receptor 4 agonist, the bacterial endotoxin lipopolysaccharide (LPS), Dj-1-knockout astrocytes generated >10 times more nitric oxide (NO) than littermate controls. Lentiviral reintroduction of DJ-1 restored the NO response to LPS. The enhanced NO production in Dj-1(-/-) astrocytes was mediated by a signaling pathway involving reactive oxygen species leading to specific hyperinduction of type II NO synthase [inducible NO synthase (iNOS)]. These effects coincided with significantly increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and p38(MAPK) inhibition suppressed NO production and iNOS mRNA and protein induction. Dj-1(-/-) astrocytes also induced the proinflammatory mediators cyclooxygenase-2 and interleukin-6 significantly more strongly, but not nerve growth factor. Finally, primary neuron cultures grown on Dj-1(-/-) astrocytes became apoptotic in response to LPS in an iNOS-dependent manner, directly demonstrating the neurotoxic potential of astrocytic DJ-1 deficiency. These findings identify DJ-1 as a regulator of proinflammatory responses and suggest that loss of DJ-1 contributes to PD pathogenesis by deregulation of astrocytic neuroinflammatory damage.

Mutations in the PARK7 gene encoding DJ-1 cause autosomal recessive Parkinson disease. The most deleterious point mutation is the L166P substitution, which resides in a structure motif comprising two ␣-helices (G and H) separated by a kink. Here we subjected the C-terminal helix-kink-helix motif to systematic site-directed mutagenesis, introducing helix-incompatible proline residues as well as conservative substitutions into the helical interface. Furthermore, we generated deletion mutants lacking the H-helix, the kink, and the entire C terminus. When transfected into neural and nonneural cell lines, steady-state levels of G-helix breaking and kink deletion mutants were dramatically lower than wildtype DJ-1. The effects of H-helix breakers were comparably smaller, and the non-helix breaking mutants only slightly destabilized DJ-1. The decreased steady-state levels were due to accelerated protein degradation involving in part the proteasome. G-helix breaking DJ-1 mutations abolished dimer formation. These structural perturbations had functional consequences on the cytoprotective activities of DJ-1. The destabilizing mutations conferred reduced cytoprotection against H 2 O 2 in transiently retransfected DJ-1 knock-out mouse embryonic fibroblasts. The loss of survival promoting activity of the DJ-1 mutants with destabilizing C-terminal mutations correlated with impaired anti-apoptotic signaling. We found that wild-type, but not mutant DJ-1 facilitated the Akt pathway and simultaneously blocked the apoptosis signal-regulating kinase 1, with which DJ-1 interacted in a redoxdependent manner. Thus, the G-helix and kink are critical determinants of the C-terminal helix-kink-helix motif, which is absolutely required for stability and the regulation of survival-promoting redox signaling of the Parkinson disease-associated protein DJ-1. DJ-1 is the gene mutated in the PARK7 locus associated with autosomal-recessive Parkinson disease (PD)4 (1). It is believed that loss of function accounts for the symptoms in DJ-1 mutation bearers, but it remains to be shown exactly what physiological role of DJ-1 is depleted in PD. Post-mortem studies on sporadic PD patients showed that DJ-1 did not accumulate in Lewy bodies, the neuropathological hallmark lesions of PD and related diseases. Rather, DJ-1 was prominently expressed in reactive astrocytes under neurodegenerative conditions, including PD (2-4), as well as in a transgenic mouse model of Lewy pathology (3). Astrocytes have a high antioxidative capacity and support adjacent neurons suffering from oxidative stress (5). Oxidative modifications of DJ-1 were found in brains of patients with PD and Alzheimer disease (2, 6). Thus, DJ-1 upregulation appears to be associated with oxidative stress in neurodegenerative brain.Overexpression of DJ-1 conferred resistance against H 2 O 2 , 1-methyl-4-phenylpyridinium, bisphenol A, and other oxidative stressors in neuroblastoma cells (7-9). Conversely, RNA silencing (9) or targeted disruption of the DJ-1 gene (10, 11) enhanced cytotoxicity medi...

Mutations in the PARK7 gene DJ-1 are associated with recessive hereditary Parkinson's disease (PD). Fibrillar inclusions of alpha-synuclein comprise the neuropathological hallmarks of PD and related Lewy body diseases as well as multiple system atrophy (MSA). Moreover, neuronal and glial inclusions containing tau have been observed in alpha-synucleinopathy patients. Using a collection of antibodies against DJ-1, we have performed a comprehensive investigation of DJ-1 in alpha-synucleinopathies and tauopathies. DJ-1 was abundantly expressed in reactive astrocytes of patients with neurodegenerative diseases. Likewise, DJ-1 antiserum immunostained reactive astrocytes that became abundant with disease progression in the brain stem of transgenic mice expressing mutant [A30P]alpha-synuclein. Human Lewy bodies as well as Lewy body-like inclusions in the alpha-synuclein transgenic mice were DJ-1 negative. Neuronal tau inclusions were DJ-1 immunopositive in Pick's disease (PiD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and Alzheimer's disease. In addition, we found DJ-1-immunopositive glial inclusions in CBD, PSP and MSA. Biochemical extraction experiments revealed the specific presence of insoluble, modified DJ-1 in PiD and MSA. Our results suggest that DJ-1 is up-regulated in reactive astrocytes as well as in neuronal and glial cells with specific alpha-synucleinopathy and tauopathy.

Although hereditary parkinsonism is very rare compared with sporadic Parkinson's disease (PD), 1 the identification of PARK genes has greatly expanded the molecular understanding of the most common neurodegenerative movement disorders (1). The discovery of PARK1/␣-SYNUCLEIN has led to the identification of ␣-synuclein fibrils as the neuropathological hallmark of PD, and the investigation of ␣-synuclein has provided important clues to the molecular mechanisms of PD (2, 3). Moreover, two enzymes involved in ubiquitin metabolism were found to be encoded by PARK2 (the ubiquitin ligase parkin) and PARK5 (ubiquitin C-terminal hydrolase-L1), suggesting that failure of the ubiquitin-proteasome system generally contributes to PD (4).More recently, DJ-1 was identified to be the gene mutated in the PARK7 locus (5). Loss-of-function mutations of DJ-1 are compatible with the recessive inheritance of PARK7 (6). In the Dutch kindred, protein-coding sequences are deleted altogether (5). Complete loss of functional DJ-1 was also predicted for a young-onset PD patient with compound frameshift and splice mutations (7). It remains to be shown what physiological function of DJ-1 is depleted. Previous studies have implicated DJ-1 with tumor progression, RNA binding, male fertility, androgen receptor signaling, and cellular management of oxidative stress (8 -14). Furthermore, there is structural similarity of DJ-1 with bacterial proteases and hydroperoxidases (15-18). However, the function of DJ-1 in the brain, where it is expressed at rather moderate levels (5, 8, 9), is completely unknown.A second PARK7 family from Italy (19) was found to bear a point mutation in the DJ-1 gene, leading to replacement of leucine 166 with proline (5). The L166P mutation is predicted to break a characteristic ␣-helical fold in the DJ-1 structure. More recently, a second point mutation (E64D) was found in a small kindred of Turkish ancestry.2 The homozygous index patient had early-onset PD along with significant depletion of striatal dopamine receptors, as evidenced by the reduced [ 18 F]FP-CIT uptake. Subclinical PD was indicated by reduction of [18 F]FP-CIT positron emission in a homozygous sister, whereas a heterozygous brother had normal [18 F]FP-CIT uptake. Five more siblings as well as the heterozygous mother were unaffected.To elucidate the molecular basis of the deficits of DJ-1 point mutations, the expression, processing, and turnover of WT and mutant DJ-1 were examined. Expression levels of mutant

Loss of function of DJ-1 (PARK7) is associated with autosomal recessive early-onset Parkinson's disease (PD), one of the major age-related neurological diseases. In this study, we extended former studies on DJ-1 knockout mice by identifying subtle morphological and behavioural phenotypes. The DJ-1 gene trapinduced null mutants exhibit less dopamine-producing neurons in the ventral tegmental area (VTA). They also exhibit slight changes in behaviour, i.e. diminished rearing behaviour and impairments in object recognition. Furthermore, we detected subtle phenotypes, which suggest that these animals compensate for the loss of DJ-1. First, we found a significant upregulation of mitochondrial respiratory enzyme activities, a mechanism known to protect against oxidative stress. Second, a close to significant increase in c-Jun N-terminal kinase 1 phosphorylation in old DJ-1-deficient mice hints at a differential activation of neuronal cell survival pathways. Third, as no change in the density of tyrosine hydroxylase (TH)-positive terminals in the striatum was observed, the remaining dopamine-producing neurons likely compensate by increasing axonal sprouting. In summary, the present data suggest that DJ-1 is implicated in major non-motor symptoms of PD appearing in the early phases of the disease -such as subtle impairments in motivated behaviour and cognition -and that under basal conditions the loss of DJ-1 is compensated

Our work discloses a complete workflow to detect, count and genetically analyze individual CTCs isolated from blood samples. This method has a central impact on the early detection of metastasis development. The combination of cell quantification and genetic analysis provides the clinicians with a powerful tool not available so far.