Optical Analysis of [Ca 2+ ]i and Mitochondrial Signaling Pathways: Implications for the Selective Vulnerability of Motoneurons in Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective loss of a defined motoneurons (MNs) population in the brain stem and spinal cord of both human ALS and corresponding mouse models of this neurodegenerative disease. While disruptions of [Ca2+]i and a strong interaction between metabolic mechanisms and mitochondria have been associated with selective MNs degeneration, the underlying mechanisms are only little understood. Although the complex etiologies of ALS are not yet fully understood, a variety of studies indicate that mitochondrial dysfunction and disruption of the Ca2+ homeostasis represent critical neuropathological characteristics during the disease progression. On the basis of the studies done so far, little is known about the involvement of mitochondria in Ca2+ regulation of HMNs in 8-9 and 14-15 weeks adult WT and corresponding SOD1G93A mice. Therefore, the first aim of this dissertation was to define and evaluate the contribution of mitochondria in Ca2+ signaling and clearance of physiological Ca2+ loads in adult brain stem slice preparations from SOD1G93A mice containing selectively vulnerable HMNs and to compare the results to the corresponding wild-type (WT) littermates. Second, the work aimed at characterizing the cellular and morphological consequences of mitochondrial dysfunction in SH-SY5Y cells transfected with WT-SOD1 and SOD1G93A gene, with special attention to alteration in Ca2+ homeostasis. Furthermore, in order to check if calbindin-D28K (CB-D28K) protects cells from dysfunction and degeneration via buffering of [Ca2+]i, Ca2+ involvement as a risk factor was studied in primary cultured neuronal cells at days E18 from cortex of mouse that expresses different levels of (low and high) CB-D28K , as was previously shown that Ca2+-binding proteins such as CB-D28K and PV are absent in MN populations (HMNs/SMNs) lost early in ALS. Finally, we intend to study the mechanisms and possible role of glia in MNs-glia signaling, leading to MNs and glial cell death as a consequence of alterations in glutamate signaling, hypoxia induced cell death, mitochondrial depolarization and their clinical relevance in ALS.To elucidate the underlying factors as well as consequences of mitochondrial dysfunction in ALS on MN membrane properties and intracellular Ca2+ homeostasis, we utilized rapid camera based CCD imaging techniques in 8-9 and 14-15 weeks adult brain stem slice preparations from mouse containing selectively vulnerable HMNs obtained from mSOD1G93A and corresponding healthy MNs in WT littermates. In the case of HMNs, which are vulnerable to cell death in ALS, disruption of the ΔΨm by bath application of the mitochondrial uncoupler FCCP(2µM) provoked a substantial Ca2+ release from mitochondrial stores which was significantly diminished in HMNs of SOD1G93A mice at symptomatic stage (14-15 weeks adult mice) of motor dysfunction. Mitochondrial Ca2+ release responses after FCCP application were almost ~3 fold larger in WT as compared to SOD1G93A animals, supporting the hypothesis that mitochondrial Ca2+ homeostasis is significantly disturbed in the SOD1G93A mice. The application of the SERCA inhibitor CPA to empty the ER Ca2+ store, in the presence of FCCP, resulted in a separate defined Ca2+ release, indicating that ER stores are able to operate independently of mitochondria, but are significantly small and different from FCCP evoked Ca2+ release. Responses of ΔΨm were in agreement with a model, where the presence of the mutated gene leads to a substantial disruption of the ΔΨmm in symptomatic SOD1G93A animals, whereas in corresponding 8-9 weeks WT and pre symptomatic mice ΔΨm remains identical. To evaluate the contribution of mitochondria in buffering Ca2+ loads simultaneously in cytosol and mitochondria, we simultaneously monitor cytosolic ([Ca2+]i) and mitochondrial calcium ([Ca2+]m) concentrations in SH-SY5Y cells transfected with either WT or mSOD1G93A gene. At first, [Ca2+]i was monitored by loading SH-SY5Y cells with the fluorescent dye Fura-2 AM where bath application of 30mM KCl evoked reproducible, voltage dependent Ca2+ elevations, and responses were comparable in wtSOD1 and mSOD1G93A expressing cells. In contrast, bath application of FCCP evoked significantly smaller (~1.86 fold, n = 23 cells) intracellular Ca2+ release responses in mSOD1 expressing cells, suggesting impaired mitochondrial calcium stores. To further test this idea, CCD camera imaging system that allows us to simultaneously monitor [Ca2+]i (Fura-2) and [Ca2+]m concentrations (Rhod-2) with a temporal resolution in millisecond time domain were developed. This permits us to clearly separate dynamic profiles of [Ca2+]i and [Ca2+]m. An experimental observation during different pharmacological conditions (K+, FCCP and Caffeine) supports the concept that the presence of mSOD1 severely disrupts mitochondrial Ca2+ regulation.To explain the consequences of buffering characteristics of MNs (The MNs in ALS showed a low Ca2+ buffering capacity), Ca2+ imaging studies following depolarization induced stimulus (60mM K+) were done in primary neuronal cells expressing CB-D28K. Our data shows that indeed CB-D28K buffers [Ca2+]i where low and high CB-D28K transfected cells display a significant (~2 times) reduction in the peak amplitude of the sustained [Ca2+]i increase. Decay time constant (τ) in CB-D28K transfected cells was also relatively slower (~60s) in contrast to non-transfected cells, where baseline recovery time is ~30-35s while showing little differences in the area under the time - concentration curve (AUC). The observation is in good agreement with the Low buffering hypothesis stating that low buffers provide MNs with rapid Ca2+ dynamics during physiological activity, but represents a significant risk factor during ALS-related MN disease. Further, we made a comparative study of glia, MN s-glia signaling and relationship between [Ca2+]i, ΔΨm and MNs-glia cross-talk. We develop a protocol which allows us a parallel observation of individually identified MNs and glial cells, and thus directly compare the physiological reactions in these two cell types in rat brain stem slice network. This procedure has a main advantage that even subtle differences in reactions can be outlined, because the MNs-glia networks are exposed to identical experimental conditions. We found significant differences between glial cell and MNs Ca2+ signaling. In glutamate treated MNs, the large rise in ROS generation is in parallel with large, sustained, glutamate-mediated Ca2+ influx from the extracellular space, which dominates over the Ca2+ release from the stores. In glial cells, the capacity to down regulate [Ca2+]i clearly provides protection against increased ROS generation. These results indicate that high levels of [Ca2+]i are not necessarily toxic to glia and MNs networks, and potential-driven uptake of Ca2+ into mitochondria is required to trigger NMDA receptor- stimulated neuronal death.Taken together, the results presented here suggest that adult SOD1G93A mouse are characterized by a substantial disruption of the [Ca2+]i / mitochondrial interplay and MN degeneration caused by mSOD1 may be attributed to long term exposures to altered Ca2+ homeostasis and mitochondrial dysfunction. These observations are in good agreement with the models suggesting that an impairment of the Ca2+/ mitochondrial system is a critical element of ALS pathogenesis. A thorough understanding of these events will undoubtedly give clues to a better understanding of the processes involved in MN degeneration and thus help in designing better therapeutic strategies to treat selectively vulnerable MNs in ALS.