Functional characterization of HtrA2 in the pathogenesis of Parkinson´s disease

Abstract

The mitochondrial protease HtrA2 is localized in the mitochondrial intermembrane space and has a pro-apoptotic function when released into the cytosol. Its role in neuroprotection was initially identified by the demonstration of neurodegeneration and mitochondrial dysfunction in the mice lacking HtrA2 expression or function (Jones et al, 2003; Martins et al, 2004). Furthermore Parkinson’s disease (PD)-associated variants of HtrA2 have been described in German, Belgian and Taiwanese patients (Strauss et al, 2005; Bogaerts et al, 2008; Lin et al, 2007). Although several studies have pointed to the importance of a protective role for HtrA2 within the mitochondria, the mechanism of neuroprotection is still not clear. It has been shown that G399S mutant HtrA2 displays a reduced serine protease activity in vitro following activating stimuli arguing in favor of a dominant negative effect of the G399S mutation in relation to PD(Strauss et al., 2005). Therefore a mouse model of human G399S HtrA2 overexpression (TG HtrA2 G399S) was chosen to investigate its pathogenic role in vivo compared to overexpression of human WT HtrA2 (TG HtrA2 WT). We assessed the effect of WT HtrA2 and G399S HtrA2 overexpression in mice using behavioral, immunohistochemical and biochemical approaches in order to further define the role of HtrA2 in PD. The TG HtrA2 WT mice showed significant motor impairments compared to Non-TG mice, while TG HtrA2 G399S mice revealed no difference compared to Non-TG mice using rotarod and beam walk test, which was not expected according to our hypothesis. As a potential cause for motor impairments, we found a significant increase of apoptotic nuclei in the cerebellum, cortex and basal ganglia of TG HtrA2 using TUNEL staining. Significant increase in apoptotic nuclei was also found in cerebellum and cortex of TG HtrA2 G399S mice. Alpha-synuclein aggregates were found in cerebellum, cortex and basal ganglia of TG HtrA2 WT mice, which were shown to be PK resistant. Increased amounts of ubiquitinated proteins were observed in cerebellum of the TG HtrA2 WT mice which represents another hint for impairment in degradation mechanisms and aggregation of insoluble alpha-synculein in this mouse line. Although no obvious change in expression pattern of mitochondrial proteins was observed in cerebellum, cortex and basal ganglia, but significant upregulation of HtrA2 substrate XIAP in basal ganglia, and cleaved caspases in cerebellum and cortex of TG HtrA2 WT and cortex of TG HtrA2 G399S mice were detected. Upregulation of XIAP in basal ganglia might be a compensatory mechanism, but also pointing to the fact that there might be another substrate involved in the cell death caused by HtrA2. Since TG G399S HtrA2 mice do not present with a gain of toxic function, this rather argues against a dominant loss of function mechanism of the G399S mutation according to the behavioral assays. But if results from the biochemical and immunohistochemical assays are taken together we see some non-significant trends in TG HtrA2 G399S mice compared to Non-TG mice, which could be a result of unequal expression of the transgene in the cerebellum in TG HtrA2 G399S mice compared to TG HtrA2 WT mice. Although, the differences in transgene expression cannot completely rule out the differences in phenotype, the conclusion remains that the G399S HtrA2 mutation represents a dominant loss of function and further experiments in a HtrA2 KO background are required.