Research

Research in patient material

Alpha-synuclein

Other genes linked to PD

Autophagy

Research in patient material:
Together with Demitris Vassilatis at BRFAA, we have established a Biobank and Database of patients afflicted with Parkinson’s Disease (PD) and of controls.  This forms the basis of ongoing studies that investigate the genetic basis of PD in Greece.  The Biobank, originally comprising only of DNA, is being enriched with material such as RNA, serum, plasma and fibroblasts, in order to perform peripheral biomarker studies. There is a specific focus on rare Mendelian forms of PD, while we are also examining, in collaboration with the laboratory of Dr. Michelakakis at the Institute of Child Health, biological material from patients with Gaucher Disease, who have an increased risk of developing PD.  In such subjects, we are currently examining the levels and conformations of alpha-synuclein, but also peripheral indices of lysosomal and especially autophagic function (see below).  We have furthermore begun to perform biochemical studies of such markers in post-mortem brain samples of patients with Parkinson’s Disease.

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Alpha-synuclein:
The lab has a special interest in the study of the key molecule alpha-synuclein, a presynaptic neuronal protein of uncertain function. Genetic, neuropathological and biochemical data indicate that alpha-synuclein, and particularly its tendency to form oligomeric, aggregated structures, is intricately linked to the pathogenesis of both familial and sporadic PD. Given the evidence that the levels of alpha-synuclein are important for PD pathogenesis, we have been studying the transcriptional and post-translational mechanisms involved in their control. We have identified a novel pathway for alpha-synuclein transcriptional regulation within the first Intron of the gene and the involved transcription factors (Clough et al., 2007, 2009, 2011). Efforts are under way to examine the relevance of these findings in vivo and in our Biobank material.

We have also identified pathways within lysosomes and proteasomes that differentially degrade forms of alpha-synuclein (Cuervo et al., 2004; Vogiatzi et al., 2008; Xilouri et al., 2008; Emmanouilidou et al., 2010a). Importantly, aberrant forms of alpha-synuclein inhibit such degradation systems, creating a vicious cycle of disease propagation (Xilouri et al., 2009; Emmanouilidou et al., 2010a). We have focused particularly on the lysosomal process of Chaperone-Mediated Autophagy (CMA) and its relationship with other degradation systems, such as macroautophagy. We have shown that this is the main pathway for alpha-synuclein degradation in neurons (Vogiatzi et al., 2008), and our recent results indicate that overexpression of Lamp2a, the rate-limiting step in this pathway, leads to a decrease of alpha-synuclein levels and an amelioration of its toxic effects in cellular and animal models. The in vivo studies were performed through intranigral injection of Adeno-asssociated Virus (AAV) expressing concomitantly toxic alpha-synuclein and neuroprotective Lamp2a (collaboration with Deniz Kirik, Lund) (Xilouri et al., 2013).  It is thus possible that targeting the CMA pathway may offer therapeutic advances in Parkinson’s Disease and related synucleinopathies. 

Given the genetic association of the lysosomal protein GBA with Parkinson’s Disease, we are investigating its reciprocal interrelationship with alpha-synuclein (Argyriou et al., 2012; Dermentzaki et al., 2013).  Our findings so far suggest that a decrease of GBA activity does not necessarily lead to changes in alpha-synuclein levels or conformations.  On the other hand, Gaucher Disease patients, harboring homozygote or compound heterozygote GBA mutations, appear to have an increased tendency for alpha-synuclein oligomerization.  These findings have led us to the study of the effects of mutant forms of GBA on alpha-synuclein in cell culture and animal models, in collaboration with the lab of Dr. Vekrellis.

As a tool to understand the pathological effects of alpha-synuclein, we have created an SH-SY5Y neuronal cell line that inducibly expresses human wild type alpha-synuclein. Oligomeric alpha-synuclein accumulates with time in these cells, while there is also progressive neurodegeneration (Vekrellis et al., 2009). This cell line therefore represents a very useful tool where mechanisms of toxicity and possible therapeutic intervention can be tested (Vekrellis et al., 2009; Dulovic et al., 2014).  Currently, we are examining systematically, using in part bioinformatic tools, candidate genes that may manifest a modifying effect in this model.  

Another aspect of the toxic effect of alpha-synuclein that deserves attention is the apparent selective vulnerability of the noradrenergic system to the toxic effects of the aberrant protein (Sotiriou et al., 2010). These studies were performed in transgenic mice, available in our laboratory, overexpressing alpha-synuclein under a neuron-specific promoter.  In future studies we aim to decipher the molecular basis of this vulnerability and examine its behavioral consequences.

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Other genes linked to PD:
We are furthermore examining the interrelationship of alpha-synuclein with other proteins genetically linked in PD, such as the dopaminergic survival factor Nurr-1. In collaboration with the laboratory of Dr. Vassilatis, we are investigating the mutual interaction of these proteins in cellular and animal models.  Concerning other genes linked to the disease, we have found that a polymorphic variant of UCH-L1, S18Y, which may be protective in PD, acts as a powerful antioxidant (Kyratzi et al., 2008). This work has recently been extended to the in vivo level, in the MPTP mouse model, where we have confirmed the protective effects of S18Y UCH-L1 delivered to the nigra via striatal adenoviral transduction (Xilouri et al., 2012).  In future studies, we aim to identify the molecular basis of this effect. Furthermore, a team within our lab headed by Dr. Hardy Rideout is examining the pathogenic effects of LRRK2 mutations linked to PD, focusing on the aspects of LRRK2 oligomerization and impingement on the extrinsic apoptotic pathway (Ho et al., 2009). We are also studying the inter-relationship between LRRK2 and the CMA pathway, in analogy to our studies with alpha-synuclein.

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Autophagy:
Given the association of alpha-synuclein with CMA and Parkinson’s Disease, we are studying the more general significance of this lysosomal degradation pathway in the Nervous System within our «ARISTEIA» Programme, «CMAin NEURO» (Link).  We have, in collaboration with Panos Politis at BRFAA, focused on the function of CMA in neuronal development, and in particular in the process of neural fate specification. Furthermore, it appears that this pathway also plays an important role in neuronal homeostasis in the adult Nervous System. We are examining possible neuronal substrates of this pathway using proteomics, in collaboration with Tonia Vlahou at BRFAA.

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