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Content archived on 2024-05-29

Mitochondrial pathways in neurodegeneration

Final Activity Report Summary - DEMISE (Mitochondrial pathways in neurodegeneration)

The objective of the project, as described in the proposal, was to delineate the mitochondrial pathways involved in neuronal necrotic cell death, with the ultimate aim being to decipher the biochemistry of necrosis. We initially identified a lysosomal mutant that suppressed necrotic cell death. Therefore, because of interesting preliminary results, research was initiated by analysing the role of lysosomes in neurodegeneration. We followed a molecular genetic approach towards dissection of lysosomal mechanisms involved in c. elegans necrotic death.

Double mutants of suppressors and enhancers of necrotic cell death were constructed. We found that different mutations in genes which resulted in altered lysosomal biogenesis and function markedly affected neuronal necrosis. We additionally tested the effect of alkalisation of acidic endosomal and lysosomal compartments by weak bases and found that it protected against necrotic cell death. This observation suggested that targeting intracellular pH changes could be an effective strategy towards countering neurodegeneration.

The fate of lysosomes during neurodegeneration was followed in vivo. For that, a genetically encoded fluorescent marker that targeted lysosomes was constructed. Transgenic lines specifically expressing the lysosomal marker in dying neurons were constructed. Strikingly, we found that lysosomes fused and localised exclusively around a swollen nucleus. In advanced stages of cell death, the nucleus condensed and migrated towards the periphery of the cell, while green fluorescent protein (GFP)-labeled lysosomal membranes faded, indicating lysosomal rupture. Similarly, we optically followed the fate of lysosomes in the different lysosomal mutants described by introducing the same fluorescent marker.

Our study directly implicated lysosomes and acidic cellular organelles in the execution of necrotic cell death and, to the best of our knowledge, was the first to monitor lysosomal alterations during necrosis in vivo, in any organism. Such information could be effectively utilised towards identifying candidate common intervention targets, in an effort to battle numerous pathological conditions in humans. In the meanwhile genetically encoded fluorescent markers and transgenic animals that would enable us to follow mitochondrial dynamics during neuronal necrotic cell death were constructed.

Ageing represented a high risk factor for the appearance of neurodegenerative disorders. Mitochondria play a prominent role in ageing and several neurodegenerative disorders were associated with mitochondrial dysfunction. As a following up of her PhD thesis, the contractor studied the role of the mitochondrial prohibitin complex in the process of ageing. The prohibitin complex in eukaryotes consisted of two subunits (PHB1 and PHB2) that assembled into a macromolecular structure of approximately 1 MD in the inner mitochondrial membrane. The evolutionary conservation and the ubiquitous expression of PHB genes in mammalian tissues suggested an important function for the prohibitin complex. However, the physiological role of the complex remained elusive. The PHB complex was essential for c. elegans embryonic development. The postembryonic depletion of PHB proteins resulted in gonad differentiation defects and sterility, reduced physiological rhythms and oxygen consumption rates and increased sensitivity to oxidative stress. Muscle mitochondria appeared fragmented and disorganised in phb(RNAi)-treated animals, suggesting that the prohibitin complex has an important role in establishing or maintaining the integrity of mitochondrial membranes. The contractor investigated the effect of phb-1 and phb-2 knock down on the lifespan of wild type animals as well as indifferent mutant genetic backgrounds. Prohibitin depletion had no effect on the lifespan of N2 animals at 20 degrees, although sometimes a slight reduction in lifespan could be observed. However, phb-1/2(RNAi) animals showed increased lifespan at 25 degrees as well as increased intrinsic thermotolerance. This indicated that changes in temperature had a dramatic effect on mitochondrial function and in ageing. Insulin signalling was one of the major regulating pathways.