Novel In Situ Activity Assays for the Quantitative Molecular Analysis of Neurodegenerative Processes in the Retina
The mechanisms of neuronal cell death are still only poorly understood, which has hindered the advancement
of therapies for many currently untreatable neurodegenerative diseases. This calls for the development of new methods
which reveal critical molecular mechanisms of the celldeath machinery with both high sensitivity and cellular resolution.
Using animal models for hereditary neurodegeneration in the retina, we have developed or adapted different biochemical
assays to determine the enzymatic activities of calpain, poly-ADP-ribose-polymerase (PARP), and histone deacetylase
(HDAC) directly and in situ. Additionally, the enzymatic activity of cGMP-dependent protein kinase (PKG) was assessed
indirectly using in situ immunohistological techniques to detect PKG-activity-dependent products. Combining these assays
with in situ cell death markers revealed close temporospatial correlations, suggesting causal connections between the
PKG, HDAC, PARP and calpain activities and neuronal cell death. Using different pharmacological and genetic manipulations,
causality could indeed be demonstrated. Surprisingly, the often dramatic rises in metabolic activities didnot match
by corresponding increases in expression, highlighting the importance of analyses of protein activities at the cellular level.
The above mentioned studies identified a number of metabolic processes previously unknownto be involved in inherited
retinal degeneration. Comparing different animal retinal degeneration models uncovered striking similarities in enzymatic
activities, suggesting a generality of the destructive pathways. Taken together, these findings provided a number of novel
targets for neuroprotection and as such opened up new perspectives for the therapy of hereditary neurodegeneration in the
retina and possibly other parts of the central nervous system.
Keywords: calpain, cell death, cGMP, HDAC, PARP, TUNEL.
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