We have previously linked families with autosomal-dominant, late-onset parkinsonism to chromosome 12p11.2-q13.1 (PARK8). By high-resolution recombination mapping and candidate gene sequencing in 46 families, we have found six disease-segregating mutations (five missense and one putative splice site mutation) in a gene encoding a large, multifunctional protein, LRRK2 (leucine-rich repeat kinase 2). It belongs to the ROCO protein family and includes a protein kinase domain of the MAPKKK class and several other major functional domains. Within affected carriers of families A and D, six post mortem diagnoses reveal brainstem dopaminergic degeneration accompanied by strikingly diverse pathologies. These include abnormalities consistent with Lewy body Parkinson's disease, diffuse Lewy body disease, nigral degeneration without distinctive histopathology, and progressive supranuclear palsy-like pathology. Clinical diagnoses of Parkinsonism with dementia or amyotrophy or both, with their associated pathologies, are also noted. Hence, LRRK2 may be central to the pathogenesis of several major neurodegenerative disorders associated with parkinsonism.

Loss of parkin function is responsible for the majority of autosomal recessive parkinsonism. Here, we show that parkin is not only a stress-protective, but also a stress-inducible protein. Both mitochondrial and endoplasmic reticulum (ER) stress induce an increase in parkin-specific mRNA and protein levels. The stress-induced upregulation of parkin is mediated by ATF4, a transcription factor of the unfolded protein response (UPR) that binds to a specific CREB/ATF site within the parkin promoter. Interestingly, c-Jun can bind to the same site, but acts as a transcriptional repressor of parkin gene expression. We also present evidence that mitochondrial damage can induce ER stress, leading to the activation of the UPR, and thereby to an upregulation of parkin expression. Vice versa, ER stress results in mitochondrial damage, which can be prevented by parkin. Notably, the activity of parkin to protect cells from stress-induced cell death is independent of the proteasome, indicating that proteasomal degradation of parkin substrates cannot explain the cytoprotective activity of parkin. Our study supports the notion that parkin has a role in the interorganellar crosstalk between the ER and mitochondria to promote cell survival under stress, suggesting that both ER and mitochondrial stress can contribute to the pathogenesis of Parkinson's disease.

The maintenance of the mitochondrial genomic integrity is a prerequisite for proper mitochondrial function. Due to the high concentration of reactive oxygen species (ROS) generated by the oxidative phosphorylation pathway, the mitochondrial genome is highly exposed to oxidative stress leading to mitochondrial DNA injury. Accordingly, mitochondrial DNA damage was shown to be associated with ageing as well as with numerous human diseases including neurodegenerative disorders and cancer. To date, several methods have been described to detect damaged mitochondrial DNA, but those techniques are semi-quantitative and often require high amounts of genomic input DNA. We developed a rapid and quantitative method to evaluate the relative levels of damage in mitochondrial DNA by using the real time-PCR amplification of mitochondrial DNA fragments of different lengths. We investigated mitochondrial DNA damage in SH-SY5Y human neuroblastoma cells exposed to hydrogen peroxide or stressed by over-expression of the tyrosinase gene. In the past, there has been speculation about a variable vulnerability to oxidative stress along the mitochondrial genome. Our results indicate the existence of at least one mitochondrial DNA hot spot, namely the D-Loop, being more prone to ROS-derived damage.

Mutations in the parkin gene are the most common cause of recessive familial Parkinson disease (PD). Parkin has been initially characterized as an ubiquitin E3 ligase, but the pathological relevance of this activity remains uncertain. Recently, an impressive amount of evidence has accumulated that parkin is involved in the maintenance of mitochondrial function and biogenesis. We used a human neuroblastoma cell line as a model to study the influence of endogenous parkin on mitochondrial genomic integrity. Using an unbiased chromatin immunoprecipitation approach, we found that parkin is associated physically with mitochondrial DNA (mtDNA) in proliferating as well as in differentiated SH-SY5Y cells. In vivo, the association of parkin with mtDNA could be confirmed in brain tissue of mouse and human origin. Replication and transcription of mtDNA were enhanced in SH-SY5Y cells over-expressing the parkin gene. The ability of parkin to support mtDNA-metabolism was impaired by pathogenic parkin point mutations. Most importantly, we show that parkin protects mtDNA from oxidative damage and stimulates mtDNA repair. Moreover, higher susceptibility of mtDNA to reactive oxygen species and reduced mtDNA repair capacity was observed in parkin-deleted fibroblasts of a PD patient. Our data indicate a novel role for parkin in directly supporting mitochondrial function and protecting mitochondrial genomic integrity from oxidative stress.

Recombination signal sequences (RSS) flank V, D, and J coding segments and serve as the sites for recognition and cleavage by the recombinase. Each RSS consists of two conserved elements, the heptamer and the nonamer, and a spacer element of either 12 or 23 bases of fixed length but variable nucleotide composition. Recombination events are limited by the "12/23 rule" to those in which a pair of RSS participate, one 12-spacer signal and one 23-spacer signal. All coding segments of a given class (V, D, or J) have the same arrangement of spacer lengths, with the arrangement of signals limiting recombination events to those that could potentially encode a functional antigen receptor (17).RAG1 and RAG2 carry out the initial stages of V(D)J recombination during which signal sequences are recognized and bound, and double-strand breaks are introduced at the border of the signal sequence and the coding segment (22). Studies with purified proteins have shown that double-strand break formation occurs in two steps (18). First, the RAG proteins introduce a single-strand nick at the 5Ј end of the heptamer, adjacent to the coding DNA. A direct transesterification reaction follows, in which the free hydroxyl at the 3Ј end of the coding sequence attacks the phosphodiester bond between the coding sequence and the RSS of the opposite strand, resulting in a blunt 5Ј phosphorylated signal end and a covalently sealed hairpin coding end (18, 31).Stable, site-specific binding and the two cleavage steps require both RAG1 and RAG2. In addition, a divalent metal ion is required for binding and cleavage. The identity of the metal ion profoundly influences the behavior of the recombinase (9, 32). Interdependent, or coupled, cleavage occurs with purified proteins when Mg 2ϩ is the divalent metal ion (32).

SummaryIn this study, a flagella-related protein gene cluster is described for Halobacterium salinarum. The fla gene cluster is located upstream of the flagellin genes flgB1 -3 and oriented in the opposite direction. It consists of nine open reading frames (ORFs

In Alcaligenes eutrophus HI6 the hyp gene complex consists of six open reading frames hypAl, B l , F I , C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NADreducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypHl and hypFl were originally considered to form a single open reading frame designated hypB (Dernedde, J., Eitinger, M. & Friedrich, B. (1 993) Arch. Microhiol. 159, 545-5531. Re-examination of the relevant sequence identified hypBl and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, BI and F l deletion mutants (cl 11) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. lmmunoblot analysis showed the presence of the H,-activating SH subunit (HoxH) at levels comparable to those observed in the wildtype strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. "Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class I1 mutants bearing deletions in hypAl, B l and F I showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.Keywords: Alculigenes eutrophus; hyp genes ; in-frame deletions; hydrogenase processing; nickel incorporation.Alculigenes eutrophus HI 6 , a facultative chemolithoautotrophic bacterium, is able to grow on molecular hydrogen as the sole energy source. Oxidation of hydrogen is mediated by two NiFe-containing hydrogenases: a heteroditneric membranebound enzyme (MBH;Schink and Schlegel, 1979) and a cytoplasmic heterotetrameric protein (SH; Schneider and Schlegel, 1976). MBH is considered to donate electrons to the respiratory chain via a membrane-bound b-type cytochroine as has been shown for Wolinella succinogenes hydrogenase (Dross et al., 1992). The FMN-containjng SH transfers electrons to NAD as the physiological acceptor. The genes (hox) encoding the two hydrogenases of A. eutrophus are located on a transmissible 450-kb megaplasmid. They are organised in two separate operons Corresporrdenw to

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson’s disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes α-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.