Functional Neurogenetics

Laboratory of Functional Neurogenetics

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 molecular, cellular, and histopathological mechanisms underlying aggregation of the PD/DLB-associated synaptic protein α-synuclein and the FTD/ALS-associated TAR DNA-binding protein. Pathological proteolytic processing, phosphorylation pathways, and oxidative modifications are modelled in cell culture and transgenic animal models such as mice and worms. Cytotoxic mechanisms including impairments of the ubiquitin-proteasome system and mitochondrial malfunction modulated by PD-associated genes (parkin, DJ-1, LRRK2, PINK1, HtrA2/Omi, and others) are studied. 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 in human patients and are recapitulated in our transgenic mouse models.

Figure 1: Aggregation/Lewy body formation of alpha-synuclein in various model systems

Lewy-like neuropathology in transgenic mouse brain (immunostaining of α-synuclein phosphorylated at serine-129)

Characterization and Behavioural Consequences of α-Synucleinopathy in Transgenic Mice

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 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.

Regulation of leucine-rich repeat kinase 2 activity and cellular signaling

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.

Cytoprotective Role of Parkin on Dopaminergic Neurodegeneration

Mutations in the PARK2/ PARKIN gene account for most cases of hereditary parkinsonism, and may genetically predispose to PD. Parkin is a broadly functional cytoprotective enzyme with E3 ubiqutin ligase activity. However, it is not understood how parkin protects the PD affected dopaminergic (DAergic) neurons. To investigate the molecular and cellular mechanisms of DA neurotoxicity and their modulation by PD-associated genes, Dr. Takafumi Hasegawa has developed novel neuronal cell lines co-expressing human PD-associated genes together with transcriptionally regulated tyrosinase. Tyrosinase, a key enzyme in the biosynthesis of melanin, catalyzes both the hydroxylation of tyrosine to L-DOPA and the subsequent conversion of L-DOPA and DA to their specific o-quinones. Massive intracellular production of reactive oxygen species occurs as a by-product, which leads to the activation of apoptotic pathways. Using this cell culture model of PD-relevant oxidative DA neurotoxicity, Hasegawa et al. (2008) could demonstrate that parkin suppresses apoptotic stress-activated protein kinase signaling. We are currently identifying the regulatory parkin effector proteins, and their exact ubiquitin modification(s). Dr. Hasegawa is recipient of a Humboldt fellowship.

Intracellular ROS formation triggered by tyrosinase

Regulation and Cellular Effects of Parkin E3 Ubiquitin Ligase Activities

Most of the familial PD cases are caused by mutations in the parkin gene, causing autosomal recessive juvenile Parkinsonism (AR-JP). 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 proteasom and subsequent degradation. In addition, dependent on the quantity of ubiquitin moieties ligated, their specific linkage, as well as the usage of various ubiquitin-like modifiers (UBLs), further complex regulatory functions, other than simple degradation, have mediated. To shed light into the diverse effects of protein modification by UBLs, Dr. Wolfdieter Springer investigates the regulation as well as the cooperative actions among ubiquitinating enzymes with respect to linkage and ligation of ubiquitin and related modifiers in cell-free assays, transfected cell lines, and C. elegans.

Recently, a new role of parkin in the autophagic degradation of depolarised mitochondria emerged. Together with Sven Geisler, Dr. Springer managed to unveil the molecular mechanism. Parkin is recruited to depolarised mitochondria in a PINK1-dependent manner, which is differentially affected by 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 VDAC. RNAi experiments proved the importance of our newly discovered mechanism of mitophagy. Thus, the recessive Parkinson gene products parkin und PINK1 are not only involved in the regulation of mitochondrial morphology, but are also functionally pivotal in the clearance of damaged mitochondria.

This work is supported by the German National Genome Research Network NGFNplus and the EU FP7 Consortium MEndelian FOrms of Parkinsonism (MEFOPA).

Molecular Mechanisms of DJ-1 Mediated Anti-Oxidative and Neuroinflammatory Responses

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. This work is supported by the German National Genome Research Network NGFNplus.

DJ-1 dimer

Cell Biology of the FTD/ALS Associated Nuclear Splice Factor TDP-43

Similar to α-synuclein, the major constituent of Lewy Bodies, proteolytic fragments of the nucleic acid binding protein TDP-43 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 the cytosolic sequestration of the nuclear protein TDP-43 deprives neurons of a vital splicing / transcription factor. To identify novel TDP-43 target genes, Fabienne Fiesel conducts expression profiling studies after RNA interference. We have identified histone deacetylase 6 as a novel TDP-43 target mRNA and validated it in non-neuronal and neuronal cells treated with siRNA and viral shRNA vectors as well as in TDP-43 mutant animal models, together with Friederike Hans and help of Stephanie Weber. This work is supported by the German Ministry of Education and Research (BMBF) Competence Network "Degenerative Dementias" and the German Center for Neurodegenerative Disease (DZNE).

Key publications

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

Carballo-Carbajal, I., Weber-Endress, S., Rovelli, G., Chan, D., Wolozin, B., Klein, C. L., Patenge, N., Gasser, T., and Kahle, P. J. (2010) Leucine-rich repreat kinase 2 induces a-synuclein expression via the extracellular signal-regulated kinase pathway. Cell. Signal., in press

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

Neumann, M., Kahle, P. J., Giasson, B. I., Ozmen, L., Borroni, E., Spooren, W., Muller, 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., Waldenmaier, A., Görner, K., Alunni-Fabbroni, M., Schell, H., Vogt-Weisenhorn, D., Pham, T.-T., Reumers, V., Baekelandt, V., Wurst, W., and Kahle, P. J. (2009) Regulation of astrocyte inflammatory responses by the Parkinson's disease-associated gene DJ-1. FASEB J. 23, 2478-2489

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