Paquet-Durand Lab

Cell Death Mechanisms

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Methods and Techniques

We are studying cell death pathways using pharmacological and genetic manipulations on retinal degeneration models, specific knock-out animals, organotypic retinal tissue cultures and in vivo animal models. For the latter we use a variety of in vivo application techniques combined with specific drug delivery systems, as well as in vivo imaging and functional testing approaches. Experimental manipulations are evaluated using methods that allow high spatiotemporal resolution of metabolic processes in living retinal tissues.

In wild-type (wt) mice (upper panel) PDE6 beta is expressed in the outer segments of photoreceptors. In rd1 photoreceptors (lower panel) the PDE6 mutation leads to a loss of expression and a dramatic accumulation of cGMP at post-natal day 11 when compared with wt. cGMP accumulation coincides with increased activation of CaMKII and PKC theta in rd1 photoreceptor segments. In the rd1 situation a subpopulation of photoreceptor cell bodies shows strong increases in calpain activity, oxidatively damaged DNA, and PARP activity. Degenerating rd1 photoreceptors also show nuclear translocation of AIF and a positive reaction in the TUNEL assay. The images shown here are representative of at least 3 different specimens from each genotype. ONL/INL stands for outer/inner nuclear layer, respectively; figure modified after Sancho-Pelluz et al., 2008.

Processes under investigation concern regulation of cyclic nucleotide synthesis and degradation, activities and expression of protein kinases A and G (PKA, PKG), calpains, poly-ADP-ribose-polymerase (PARP), histone deacetylases (HDAC) as well as factors implicated with oxidative stress (Figure 1). Particular emphasis is put on assessing metabolic activity rather than gene/protein expression. New data generated serves as basis for the development of novel experimental treatments on in vitro retinal explants and where appropriate on in vivo animal models.

We have developed or adapted new methods to biochemically assay enzyme activity in individual retinal cells. Using such in situ biochemical methods, we have identified several novel factors that are highly relevant for photoreceptor degeneration including calpain-type proteases, PARP, PKG and HDAC. Continued studies on these and other factors involved in neurodegeneration may eventually afford a profound understanding of the underlying molecular cell death pathways. It is our hope that that this will provide novel, rational approaches for the development of treatments for inherited retinal degenerations as well as for other currently untreatable CNS disorders.