Increased Mitochondrial Calcium Uptake and Concomitant Hyperactivity by Presenilin Loss Promotes mTORC1 Signaling to Drive Neurodegeneration

Abstract

BackgroundMetabolic dysfunction and protein aggregation are common characteristics that occur in age-related neurodegenerative disease, such as Alzheimer’s disease (AD). However, the mechanisms underlying these abnormalities remain poorly understood. Mutations in the presenilin genes are the primary cause of early onset familial AD, but despite their identification over 20 years ago, their role in the disease remains unclear. MethodsThe model system Caenorhabditis elegans was utilized to study the in vivo function of the highly conserved presenilin ortholog SEL-12 in the nervous system. Cell biological and biochemical assays were employed to monitor changes to proteostasis and autophagic flux in sel-12 mutants. Immunoblotting was used to assess alterations to the activity of the mTORC1 pathway, a central inhibitor of autophagy. Genetic and pharmaceutical strategies to reduce mTORC1 activity, and fluorescent reporters and biosensors were expressed in the mechanosensory neurons to measure mTORC1’s influence on proteotoxicity, neuronal health and mitochondrial morphology. Additionally, behavioral response to touch was employed to determine the role mTORC1 activity has in neuronal function in sel-12 mutants. RNA interference by standard feeding methods was used to assess the contribution of autophagy to mTORC1-mediated sel-12 defects. ResultsLoss of SEL-12 results in the hyperactivation of the mTORC1 pathway and mTORC1-dependent reduction in autophagy. This hyperactivation is caused by elevated mitochondrial calcium signaling and concomitant mitochondrial hyperactivity. Reducing mTORC1 activity improves proteostasis defects and neurodegenerative phenotypes associated with loss of SEL-12 function. Consistent with high mTORC1 activity, we find that SEL-12 loss reduces autophagy, and this reduction is prevented by limiting mitochondrial calcium uptake or mitochondrial respiration. Moreover, the improvements in proteostasis and neuronal defects in sel-12 mutants due to mTORC1 inhibition require the induction of autophagy.ConclusionSEL-12 has a critical role in mediating mitochondrial calcium homeostasis and activity. In the absence of presenilin function mitochondrial calcium uptake and mitochondrial activity is increased. This mitochondrial hyperactivity stimulates mTORC1 signaling, which inhibits autophagy and promotes proteostasis decline and neuronal dysfunction in sel-12 mutants. These data suggest that the mTORC1 pathway is a potential therapeutic target for treating AD.

Deregulation of Mitochondrial Calcium Handling Due to Presenilin Loss Disrupts Redox Homeostasis and Promotes Neuronal Dysfunction

“…Considering that, in addition to an increase in cytosolic calcium levels, sel-12 mutants have a significant increase in basal mitochondrial calcium levels that impacts mitochondrial and neuronal function [14,26], we investigated mitochondrial calcium levels in egl-19(n2368gf) and unc-2(zf35gf) mutants. Using a mitochondrial calcium biosensor (mito-GCaMP6f::SL2::mCherry) expressed in the TRNs [14,26], we found that unlike the elevated mitochondrial calcium levels observed in sel-12 mutants, both egl-19(n2368gf) and unc-2(zf35gf) mutants were indistinguishable from wild type animals and thus do not show an increase in mitochondrial calcium (Figure 1B). These data indicate that a rise in cytosolic calcium levels does not correlate with a rise in mitochondrial calcium levels.…”

“…Among them, calcium signaling plays an important role in ROS generation, and calcium signaling dysregulation is an important contributor to neuronal pathology. Specifically, elevated intracellular calcium concentration has been associated with increased oxidative stress in AD models [26,63,64]. fAD, which is caused predominantly by mutations in the presenilin encoding genes, is also associated with altered calcium signaling (Cheung et al, 2008;Chan et al, 2000;Lee et al, 2006;Stutzmann et al, 2004) [7,8,11,12].…”

“…The dysregulation of this pathway has been implicated across multiple diseases, including Alzheimer's disease, underscoring mTORC1's importance in regulating cell behavior [48,49]. Inhibiting mTORC1 improves a range of neurodegenerative phenotypes in sel-12 mutants, including impairments to neuronal and mitochondrial morphology, protein homeostasis, neurodegeneration, and behavior, yet did not reduce mitochondrial calcium levels [26]. Since a rise in mitochondrial ROS production and oxidative stress following mitochondrial calcium uptake mediates the neuronal defects observed in sel-12 mutants [14,37], we asked if increased mTORC1 activity contributes to disrupted mitochondrial redox homeostasis.…”

“…We have previously demonstrated that mTORC1 signaling is hyperactivated in sel-12 mutants and is a critical driver of neurodegeneration in sel-12 mutants [26]. Thus, since it was previously shown that increased ROS is a critical contributor to the neurodegeneration observed in sel-12 animals [14], and we have found that mTORC1 activity also contributes to the elevated ROS and neurodegeneration in sel-12 mutants ( [26]; Figure 3A), we asked whether the ultimate cause of neurodegeneration in sel-12 mutants is the hyperactivation of the mTORC1 signaling pathway in sel-12 mutants. To test this, we examined two mutants that have loss-of-function mutations in two negative regulators of mTORC1 signaling activity.…”

“…While loss of nprl-3 function has been shown to activate mTORC1 signaling activity in C. elegans [61], the impact of sesn-1 inactivation on mTORC1 activity has not been investigated in C. elegans. Thus, to determine whether loss of sesn-1 results in the activation of mTORC1 signaling activity, we examined the levels of phosphorylated RSKS-1/s6 kinase, a downstream target of mTORC1 signaling, in these mutants [26,62]. Consistent with NPRL-3 and SESN-1 acting as negative regulators of mTORC1 signaling, we found that nprl-3 and sesn-1 mutants had significantly elevated phosphorylated RSKS-1 compared to wild type animals, indicating higher mTORC1 activity in these animals compared to wild type animals (Figure 6A,B).…”