2 Materials and Methods 2.1 Materials
CHX ActD
4.8 Implications for neurodegeneration in vivo
In the presented culture model, different programs trigger degeneration of neuronal projections and apoptotic demise of neuronal somata and caspase-independent disruption of neurites precedes neuronal apoptosis. Activation of caspases seems to be the main, but not sole execution system in neuronal apoptosis. When caspases are inhibited either by energy depletion or synthetic agents, neurons can still undergo delayed demise and this death is characterized by apoptotic features.
Such a cell culture system models a situation where caspase inhibition may save time for the recovery of neurons in stress situations. If this does not occur, then other proteases may take over the execution to prevent the persistence of damaged cells. This suggests that: (i) the finding of neurons dying in vivo with apoptotic features does not always imply that caspase activation was causally involved in neuronal demise; and (ii) that treatment of neurodegenerative disease solely with caspase inhibitors may not always be the most efficient way to rescue neurons. The combined use of caspase inhibitors with treatments aimed to foster active regeneration of sublethally injured neurons, may be the most effective therapeutic approach [410].
5 Summary
• In cerebellar granule cells, the microtubule disassembling poisons colchicine, nocodazole, vincristine, and vinblastine induced fragmentation of microtubules which resulted in the degeneration of the axodendritic network which was followed by apoptotic demise of neuronal somata. The microtubule stabilizing drug taxol prevented both fragmentation of microtubules and apoptosis.
• Colchicine-induced apoptosis was accompanied by mitochondrial cytochrome c release and damage, proteolytic activation of execution caspase-3 and -7, subsequent proteolysis of fodrin, advanced chromatin condensation and fragmentation, large scale and oligonucleosomal DNA fragmentation, and PS translocation to the outer surface of the plasma membrane. In the presence of peptide caspase inhibitors, apoptotic changes were prevented, although cytochrome c was released from mitochondria and the axodendritic network was still destroyed.
• Depletion of intracellular ATP by either mitochondrial inhibitors or inhibition of glycolysis blocked caspase activation, nuclear apoptotic features, and PS exposure, while the primary effect of microtubule disruption and neurite loss still occurred. Conversely, repletion of intracellular ATP by enhanced glycolysis restored all apoptotic features.
Thus, sufficient ATP is required for the execution of neuronal apoptosis and energy deficiency may lead to the persistence of degenerating and dysfunctional neurons.
• Neither caspase inhibition by ATP depletion or pharmacological agents nor antiapoptotic Bcl-2 prevented microtubule breakdown or neurite loss. But neuronal somata were protected from apoptosis under these conditions. However, after a lag period, caspase-inhibited as well as Bcl-2 overexpressing neurons underwent delayed cell death. This implies that death programs for neurite degeneration and apoptosis can occur independently from each other and that block of caspases may not rescue neurons from demise under degenerative conditions.
• The delayed caspase-independent death was characterized by partial chromatin condensation, large scale DNA fragmentation and PS exposure. Inhibitors of the proteasome reduced caspase-independent apoptosis, implying that noncaspase proteases can take over execution of apoptosis in neurons.
Zusammenfassung
Colchicin, Nocodazol, Vincristin und Vinblastin lösten in Kleinhirnkörnerzellen die De-polymerisierung von Microtubuli aus. Die Zerstörung dieser Strukturen führte zur Degeneration des neuronalen Netzwerks, dem der apoptotische Untergang der neuronalen Zellkörper folgte. Taxol, eine microtubulistabilisierende Substanz verhinderte sowohl die Zerstörung der Microtubuli als auch die nachfolgende Apoptose. Die durch Colchicin ausgelöste Apoptose war durch folgende morphologische und biochemische Merkmale gekennzeichnet: Schädigung der Mitochondrien und Freisetzung von Cytochrome c, proteo-lytische Aktivierung der Exekutionscaspasen 3 und 7, nachfolgende Proteolyse von Fodrin, Kondensierung und Fragmentierung des Chromatins, hochmolekulare und oligonukleosomale DNA-Fragmentierung und Umverteilung von Phosphatidylserin (PS) in die äußere Schicht der Plasmamembran. Die meisten dieser apoptotischen Veränderungen wurden durch die Gabe von Caspaseinhibitoren verhindert, obwohl Cytochrome c aus den Mitochondrien freigesetzt und das axodendritische Netzwerk zerstört wurde. Wenn der intrazelluläre ATP-Spiegel abgesenkt wurde, entweder durch Hemmstoffe, die direkt auf Mitochondrien wirkten oder durch Inhibition der Glykolyse wurden sowohl Caspasenaktivierung, als auch apoptotische Kernveränderungen und die PS-Umverteilung verhindert, während der primäre Effekt, die Zerstörung von Microtubuli und der Verlust von Neuriten weiterhin stattfand.
Umgekehrt führte die Wiederherstellung des ATP-Gehalts durch erhöhte Glykolyse zur erneuten Ausprägung der apoptotischen Merkmale. Daher kann angenommen werden, daß für die Durchführung der Apoptose genügend ATP in den Neuronen vorhanden sein muss und könnte erklären, wieso unter energiedefizienten Zuständen degenerierende Neuronen persistieren können. Die Zerstörung der Microtubuli und der Neuritenverlust wurden weder durch Caspaseninhibition noch durch das anti-apoptotische Protein Bcl-2 verhindert, dennoch waren die neuronalen Zellkörper vor Apoptose geschützt. Nach einer gewissen Zeit starben allerdings auch diese Neuronen. Dies impliziert, daß unterschiedliche Todesprogramme verantwortlich für Neuritenverlust oder für Apoptose existieren und daß die Hemmung von Caspasen nicht ausreichen könnte, um Neuronen unter neurodegenerativen Bedingungen vor dem Zelluntergang zu bewahren. Der verzögerte und unter Caspaseninhibition auftretende Zelltod war durch eine partielle Chromatinkondensierung, hochmolekulare DNA-Frag-mentierung und PS-Umverteilung gekennzeichnet. Proteasominhibitoren reduzierten diese von Caspasen unabhängig auftretende Apoptose, was daraufschliessen läßt, daß andere Proteasen wie zum Beispiel das Proteasom die Durchführung des apoptotischen Zelltods übernehmen können.
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