Ca2+ Signaling en CISD2, een Wolfram Syndroom Geassocieerd Proteïne: Van Moleculaire Mechanismes tot Neuronale Functie

Calcium (Ca²⁺) signaling is a fundamental regulator of cellular homeostasis, orchestrating processes such as metabolism, autophagy, and apoptosis. The endoplasmic reticulum (ER) and mitochondria form a dynamic communication axis at mitochondria-associated membranes (MAMs), enabling efficient Ca²⁺ transfer that sustains mitochondrial bioenergetics and influences cell fate decisions. Dysregulation of this interface is implicated in numerous pathologies, including neurodegeneration and Wolfram syndrome (WS). This thesis investigates the multifaceted role of CISD2, a redox-active protein localized at MAMs, in shaping intracellular Ca²⁺ signaling and inter-organellar communication, with particular emphasis on its interactions with IP₃Rs, RyRs, and BCL-2 family proteins. CISD2 and IP₃R-Mediated Ca²⁺ Signaling at MERCS Our work demonstrates that CISD2 directly interacts with IP₃R1, binding primarily to the ligand-binding region (LBR) and, with lower affinity, to the modulatory domain. Biophysical assays revealed sub-micromolar dissociation constants, suggesting a transient and regulatory nature of this interaction, potentially requiring additional cofactors for stabilization. Loss of CISD2 disrupted ER-mitochondrial contact sites and impaired Ca²⁺ transfer, yet functional consequences were strikingly cell-type-specific. Neurons exhibited severe mitochondrial dysfunction, diminished oxidative phosphorylation, and heightened apoptosis sensitivity, whereas proliferative HeLa cells remained largely resilient. These findings underscore the unique dependency of neurons on tightly regulated ER-mitochondrial Ca²⁺ fluxes and suggest that compensatory mechanisms in dividing cells buffer CISD2 loss. Furthermore, CISD2 deficiency profoundly suppressed cytosolic Ca²⁺ signals in neurons but not in HeLa cells, supporting the hypothesis that altered Ca²⁺-dependent cell death regulation underlies differential vulnerability. An alternative mechanism involves calpain 2, a Ca²⁺-activated protease that CISD2 inhibits; disruption of the delicate balance between calpain isoforms in neurons may amplify proteolytic cascades and contribute to neurodegeneration. CISD2, Autophagy, and Mitochondrial Quality Control Beyond Ca²⁺ signaling, CISD2 influences autophagic flux and mitophagy. CISD2 loss increased autophagy in both neurons and HeLa cells, suggesting a regulatory role independent of mitochondrial metabolism. Previous studies by other groups implicated CISD2 in BCL-2-Beclin 1 scaffolding and AMPK modulation, while emerging evidence positions CISD proteins as targets of the Pink1/Parkin pathway. These findings highlight CISD2 as a critical node linking Ca²⁺ signaling, autophagy, and mitochondrial quality control. CISD2 and BCL-2 Functional Interplay This thesis also elucidates the interaction between CISD2 and BCL-2, a well-established anti-apoptotic protein and modulator of IP₃R activity. CISD2 binds the BH4 domain of BCL-2 with sub-micromolar affinity, as confirmed by microscale thermophoresis and mutational analysis. While this interaction does not alter BCL-2's canonical inhibition of IP₃R-mediated Ca²⁺ release or its anti-apoptotic function, CISD2 selectively counteracts BCL-2's ability to reduce ER-mitochondrial tethering and Ca²⁺ transfer. These findings suggest that CISD2 fine-tunes BCL-2 activity at MAMs, potentially forming part of a larger macromolecular complex with IP₃Rs. Spatially restricted regulation of IP₃Rs at MERCS could have profound implications for mitochondrial metabolism and cell survival under stress conditions. CISD2 as a Regulator of RyR Activity In addition to IP₃Rs, CISD2 modulates RyR channel activity. Our data reveal that CISD2 interacts with RyR2 and RyR3 and acts as an inhibitory regulator of their Ca²⁺ release. Loss of CISD2 potentiated RyR-mediated Ca²⁺ flux, whereas re-expression of wild-type or Fe-S cluster-deficient CISD2 attenuated these responses, indicating that redox functionality is not essential for RyR inhibition. Interestingly, the WS2-associated CISD2N72S mutant retained RyR-binding capacity but exhibited variable inhibitory effects, suggesting potential dominant-negative behavior under certain conditions. Preliminary findings indicate an isoform-specific role for CISD2 in mitochondrial Ca²⁺ uptake: RyR3-driven transfer was abolished in CISD2-deficient cells, whereas RyR2-mediated coupling remained intact. This selective dependence on CISD2 for RyR3-mitochondria communication represents a novel aspect of organellar Ca²⁺ dynamics and warrants further investigation. Integration and Disease Relevance Collectively, this thesis positions CISD2 as a central regulator of ER-mitochondrial communication, integrating Ca²⁺ signaling with mitochondrial metabolism, autophagy, and cell survival pathways. Neurons in partical appear to be highly sensitive to loss of MERCS homeostasis and Ca2+ dysregulation by loss of CISD2. Through the work of others, absence of WFS1 has been linked to dysregulation of similar cellular processes in neurons. Collectively, these findings unite the two divergent WS-related proteins through similar dysregulated cellular processes. Finally, the diverging impacts of CISD2 on IP₃Rs and RyRs underscore the complexity of its role in intracellular Ca²⁺ dynamics and highlight the need for cell-type-specific analyses to fully understand its physiological and pathological significance.