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Age-related neurodegenerative diseases are a severe and increasingly worrisome burden for our aging population. Most of the chronic neurodegenerative diseases (Parkinson’s disease [PD], Lewy body dementia [LBD], Alzheimer’s disease, frontotemporal dementia [FTD], amyotrophic lateral sclerosis [ALS], etc.) are characterized by intracellular protein inclusions that are specific for each of these diseases. We investigate the structural, molecular, cellular, and histopathological mechanisms underlying aggregation of the PD/DLB-associated synaptic protein α-synuclein as well as the FTD/ALS-associated nucleic acid binding proteins TDP-43 and FUS/TLS. Pathological phosphorylation pathways and oxidative modifications are modelled in cell culture and animal models. Cytotoxic mechanisms including impairments of the ubiquitin-proteasome system and mitochondrial malfunction modulated by PD-associated genes (PINK1 and parkin, DJ-1, LRRK2, and others) are studied. Novel RNA targets of TDP-43 and FUS/TLS are screened using microarray and RNAseq, and are validated structurally and functionally. We wish to understand the molecular basis of the remarkable specificity of intracellular protein aggregation killing particular neuronal subpopulations, which cause the characteristic syndromes of neurodegenerative movement disorders and dementias.
We are using transgenic mice expressing human mutant A30P α-synuclein under the control of a Thy1 promoter, which recapitulate human α-synucleinopathy down to the ultrastructural level (Neumann et al., 2002). Cognitive behavior of (Thy1)-h[A30P]αSYN mice is impaired in an age-dependent manner, most likely due to development of neuropathology within the amygdala circuitry (Freichel et al., 2007). Moreover, old transgenic mice ultimately die of locomotor deterioration, caused by brain stem and spinal motoneuron pathology. Based on our experimental evidence these transgenic mice serve as a valuable model for LB dementia. Heinrich Schell together with Cindy Boden analyses and characterizes the effects of α-synuclein modifications (with emphasis on phosphorylation) and aggregation on neuronal dysfunction and behavioural impairments. This work is supported by the Helmholtz Alliance for Mental Health in an Aging Society (HelMA) and the German Center for Neurodegenerative Diseases (DZNE).
Missense mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant hereditary PD and a considerable genetic risk factor for sporadic PD. However, little is known about the molecular mechanisms of LRRK2 activation, and the biological role of LRRK2 is still largely unknown. LRRK2 belongs to a small, distinct group of protein kinases characterized by a GTP binding ROCO domain, comprising LRRK1, LRRK2 and death-associated protein kinase. We are investigating the biochemistry of ROCO kinase regulation (Klein et al., 2009) and explore how specifically LRRK2 is embedded in cellular signaling pathways with particular emphasis on the understanding of pathophysiology of PD-causing LRRK2 mutations. We found that LRRK2 is capable of inducing a-synuclein expression via the extracellular signal-regulated kinase module (Carballo-Carbajal et al., 2010). This work is supported by the Helmholtz Alliance for Mental Health in an Aging Society.
Most of the familial PD cases are caused by recessive mutations in the PARK2/PARKIN gene, which may also be a genetic risk factor for sporadic PD. The PARKIN gene product functions as an E3 ubiquitin protein ligase for a variety of unrelated substrate proteins. Lysine-48 linked polyubiquitination leads to protein tarteting to the proteasome and subsequent degradation. In addition, additional specific linkages with ubiquitin and ubiquitin-like modifiers, further complex regulatory functions and cellular effects. To shed light into the diverse effects of parkin-mediated ubiquitin protein modifications, Dr. Sven Geisler investigates its regulation in cell-free assays and cell culture.Special emphasis is on the role of parkin in the autophagic degradation of damaged mitochondria. Parkin is recruited to experimentally depolarized mitochondria in a PINK1-dependent manner, which is differentially affected by PD mutations in both genes (Geisler et al. 2010). We discovered that parkin mediates an new kind of K27-linked ubiquitinylation of the outer mitochondrial membrane protein VDAC1. RNAi experiments provided evidence for the importance of PINK1/parkin-mediated mitophagy, identified involved autophagy mediators, and are currently expanded to further understand how parkin und PINK1 regulate mitochondrial turnover.This work is supported by the German National Genome Research Network NGFNplus and the EU FP7 Consortium MEndelian FOrms of Parkinsonism (MEFOPA).
Parkinson-associated genomic deletions as well as the dramatically destabilising L166P point mutation cause loss of the cytoprotective protein DJ-1. The effecs of other Parkinson-associated mutations is less obvious. In ongoing structure-function studies we could show that the M26I mutation reduces DJ-1 protein stability particularly in a DJ-1 null background. This causes a loss of neuroprotective activity due to dysregulation of an incorporation of DJ-1 in the apoptosis signal-regulating kinase 1 (ASK1) signalosome. Further systematic mutagenesis of all oxidizable methionine und cysteine residues of DJ-1 confirmed the importance of the central C106, but also showed a role for the peripheral cysteines C53 und C46 (Waak et al. 2009a). We suggest that this second, peripheral redox site in DJ-1 modulates the activity of this cytoprotective Parkinson gene product (Kahle et al. 2009).
The induction of DJ-1 in reactive astrocytes also indicates a glial role. We could demonstrate that DJ-1 regulates neuroinflammatory processes in astrocytes (Waak et al. 2009b). DJ-1ko astrocytes stimulated with the bacterial endotoxin lipopolysaccharide (LPS) produced excessive nitric oxide (NO) via selective activation of p38MAPK to specifically induce type II NO synthase (iNOS). In the presence of LPS, primary neurons cultured on stimulated astrocytes DJ-1ko conducted iNOS-dependent apoptosis, directly proving the neurotoxic potential of DJ-1ko astrocytes. For in vivo confirmation we generated C. elegans mutants, and found regulation of p38MAPK and several response genes by the nematode ortholog DJR-1 when innate immunity signaling in worms was induced by growth on pathogenic bacteria (Cornejo Castro et al. 2010). Thus, one of the functions of DJ-1 is evolutionary conserved regulation of innate immunity signaling, whose loss may contribute to Parkinson pathogenesis by deregulated neuroinflammatory damage.
The molecular mechanisms of intrinsic neuroprotection and neuroinflammatory regulation of DJ-1 are further elucidated in vitro and various in vivo models by Emmy Rannikko together with Stephanie Weber. This work is supported by the German National Genome Research Network NGFNplus.
TDP-43 and FUS/TLS in cytosolic and nuclear inclusions were recently identified as neuropathological hallmarks of frontotemporal dementia (FTD) and amyotrophic lateral sklerosis (ALS). It is to show now if the cytosolic aggregates are actively neurotoxic or if their cytosolic sequestration of these nuclear proteins deprives neurons of vital RNA processing factors. To identify novel target genes, we conduct expression profiling studies after RNA interference. We have identified the intracellular transport protein histone deacetylase 6 and the exon junction complex component SKAR as novel TDP-43 target mRNAs and validated them structurally and functionally in non-neuronal and neuronal cells treated with siRNA and lentiviral shRNA vectors as well as in TDP-43 mutant animal models (Fiesel et al. 2010 and 2011). Friederike Hans is currently validating novel yeast 2-hybrid interactors of TDP-43. We have just completed an RNAseq analysis of FUS/TLS-silenced cells, validations are done by Catherine Thömmes. This work is supported by the German Research Concil (DFG), the German Ministry of Education and Research (BMBF) Competence Network "Degenerative Dementias" (KNDD) and the German Center for Neurodegenerative Disease (DZNE).
Klein, C. L., Rovelli, G., Springer, W., Gasser, T. , and Kahle, P. J. (2009) Homo- and heterodimerization of leucine-rich repeat kinases via the ROC-COR domains: LRRK2 kinase inhibition by the LRRK2 ROC-COR fragment. J. Neurochem. 111, 703-715
Geisler, S., Holmström, K. M., Skujat, D., Fiesel, F. C., Rothfuss, O. C., Kahle, P. J., and Springer, W. (2010). PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat. Cell Biol. 12, 119-131
Freichel, C., Neumann, M., Ballard, T., Müller, V., Woolley, M., Ozmen, L., Borroni, E., Kretzschmar, H. A., Haass, C., Spooren, W., and Kahle, P. J. (2006) Age-dependent cognitive decline and amygdala pathology in α-synuclein transgenic mice. Neurobiol. Aging 28, 1421-1435
Fiesel, F. C., Voigt, A., Weber, S. S., Van den Heute, C., Waldenmaier, A., Görner, K., Walter, M., Anderson, M. L., Kern, J. V., Rasse, T. M., Schmidt, T., Springer, W., Kirchner, R., Bonin, M., Neumann, M., Baekelandt, V., Alunni-Fabbroni, M., Schulz, J. B., and Kahle, P. J. (2010) Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6. EMBO J. 29, 209-221
Fiesel, F. C., Weber, S. S., Supper, J., Zell, A., and Kahle, P. J. (2011) TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR. Nucleic Acids Res. in press
Neumann, M., Kahle, P. J., Giasson, B. I., Ozmen, L., Borroni, E., Spooren, W., Müller, V., Odoy, S., Fujiwara, H., Hasegawa, M., et al. (2002). Misfolded proteinase K-resistant hyperphosphorylated α-synuclein in aged transgenic mice with locomotor deterioration and in human α-synucleinopathies. J Clin Invest 110, 1429-1439.
Waak, J., Weber, S. S., Görner, K., Schall, C., Ichijo, H., Stehle, T., and Kahle, P. J. (2009) Oxidizable residues mediating protein stability and cytoprotective interaction of DJ-1 with apoptosis signal-regulating kinase-1. J. Biol. Chem. 284, 14245-14257