The role of the actin-binding proteins cofilin1 and INF2 on mitochondrial dynamics and cellular resilience
Neurological diseases, such as stroke, Alzheimer’s disease and related dementias are among the most prevalent disorders leading to disability and death worldwide. Many cell death pathways, including apoptosis, necrosis and necroptosis, oxytosis or ferroptosis, relevant for these pathologies, converg...
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|Summary:||Neurological diseases, such as stroke, Alzheimer’s disease and related dementias are among the most prevalent disorders leading to disability and death worldwide. Many cell death pathways, including apoptosis, necrosis and necroptosis, oxytosis or ferroptosis, relevant for these pathologies, converge at the level of neuronal demise through oxidative stress. In the last decades, many efforts were accomplished to identify underlying pathophysiological mechanisms leading to neuronal demise and subsequent deficits in brain function. However, it still remains obscure which molecular mechanisms contribute to these pathologies and how they are interconnected. The role of mitochondria and respective dynamics dependent on the actin-binding proteins cofilin1 and INF2 contributing to mitochondrial regulation and to neuronal demise are illuminated in the present work. In particular, cofilin1 loss-of-function studies in MEF cells, demonstrated that absence of this actin-binding protein indirectly contributes to mitochondrial fission via DRP1 activation. Mitochondrial dynamics is especially substantial for the generation as well as delivery of ATP to cellular areas with high energy demand; and fission events are frequently associated with impaired mitochondrial function as a prerequisite for cell death. In the case of cofilin1 knockdown, however, mitochondrial fragmentation, was not associated with any mitochondrial impairment, substantiated by an identical bioenergetic profile of cofilin1-/- cells and control cells, unaltered ATP levels and a preserved mitochondrial integrity as assessed by TMRE measurements. Additionally, cofilin1 knockout was linked to increased basal mitochondrial Ca2+ level through elevated MCU expression, putatively contributing to mitochondrial fission. The role of cofilin1 in cell death paradigms induced by erastin or glutamate was negligible in MEF cells, and deletion of the protein had no relevant effect on cellular resilience.
In neuronal cells, however, cofilin1 was identified, for the first time, as a redox sensor in oxidative stress-induced cell death pathways, namely oxytosis and ferroptosis, thereby linking detrimental cellular ROS accumulation to mitochondrial demise through this actin-regulating protein. In particular, cofilin1 deletion in neuronal HT22 cells exerted substantial beneficial effects on mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential or bioluminescent-based measurement of ATP levels. Intriguingly, HT22 cells deficient for cofilin1 exhibited a profound glycolytic shift as a response to erastin or glutamate toxicity to meet their energy demand, whereas control cells were metabolically inactive. Surprisingly, interfering with another actin-binding protein, namely INF2, exerted similar effects on cellular resistance of neuronal HT22 cells comparable to cofilin1 knockdown. Accordingly, mitochondrial parameters were significantly preserved after oxytosis and ferroptosis induction resulting in enhanced cellular survival. Recent findings from this study and by others, suggesting that actin dynamics is directly linked to the regulation of mitochondrial fusion and fission, guided this project towards uncovering the potential of INF2 to impact mitochondrial morphology. This study unraveled an indirect role for INF2 on the regulation of mitochondrial fission by affecting actin dynamics and DRP1 activity in neuronal cells.
Notably, cofilin1 was confirmed being as a key player under pathophysiological conditions induced by glutamate in primary cortical neurons, as cofilin1 deficient cells were substantially protected against the induced excitotoxicity. Mitochondrial respiration was significantly preserved in cofilin1-/- primary neurons under excitotoxic conditions, thereby maintaining cellular survival. Additionally, decrease of cofilin-actin rod formation in cofilin1-/- deficient neurons might also contribute to the observed protective effects.
The present data on isolated mitochondria treated with the recombinant cofilin1 protein provide a further link to toxicity-related mitochondrial impairment by cofilin1 itself. Direct effects of cofilin1 were demonstrated by assessing the mitochondrial membrane potential, mitochondrial ROS accumulation and mitochondrial respiration. Interestingly, the detrimental impact of cofilin1 on mitochondria is dependent on oxidation of crucial cysteine residues at position 139 and 147, as mutations of these cysteine residues to serine abolished the noxious character of cofilin1.
Overall, the present findings reveal, for the first time, that the actin-binding proteins cofilin1 and INF2 play a crucial role in paradigms of oxidative stress and that inhibition of these proteins results in protective effects in neuronal cells that were particularly attributed to the preserved mitochondrial integrity and function. Thus, interfering with the pathological activation of actin-binding proteins, such as cofilin1 or INF2 may offer an effective therapeutic strategy in neurodegenerative diseases.|
|Physical Description:||167 Pages|