Giovanni Manfredi - US grants

Mitochondria are the main sources of energy in the cell. They contain their own DNA (mtDNA), whose genes encode components of the respiratory chain/oxidative phosphorylation system. They are essential for the normal functioning of all cells in the body, and are absolutely critical for the function of those tissues that are highly dependent on aerobic metabolism, especially muscle and brain. Since 1988, both mtDNA point mutations and mtDNA rearrangements (i.e. large-scale deletions and duplications) have been associated with a heterogeneous group of sporadic, mendelian, and maternally-inherited mitochondrial encephalomyopathies. These mutations generally cause an impairment of the respiratory chain, with a reduction in ATP synthesis. However, very little is known about how affected cells cope with the reduced ATP production: for example, which ATP-dependent cellular functions are preserved and which are down-regulated or abolished. Understanding ATP distribution inside mutant, as well as normal, cells would be extremely important for the interpretation of the biochemical and clinical phenotype of mitochondrial disorders. This Career Development Award Application proposes to investigate the effect of mtDNA abnormalities, on the intracellular ATP pool in different cell compartments, with particular emphasis on the mitochondria, the cytoplasmic membrane, and the nucleus. We plan to study the ATP content in cytoplasmic hybrids of human mtDNA- less cyss ( rho o cells ) repopulated with mitochondria derived from patients tissues, by targeting a recombinant firefly luciferase to different cell compartments. Utilizing a similar experimental approach, we will also attempt a novel genetic strategy for treatment of point mutations in the mtDNA ATPase6 gene, that are responsible for a maternally- inherited form of Leigh syndrome (MILS): to recode the ATPase 6 gene to contain the universal genetic code by in vitro mutagenesis, to fuse a mitochondrial targeting sequence to the recoded sequence, and then to transfer this construct into the nucleus, in order to express the gene from nuclear DNA and target it back to mitochondrial ( allotopic expression). Allotopic expression of the recoded wild-type genes should partially restore the APT synthetic function in mutant cells.

DESCRIPTION (provided by applicant): As a researcher trained in clinical neurology and basic science with a primary interests in the field of mitochondrial disorders, my principal scientific goal is to better understand the pathogenic mechanisms of this group of diseases and to identify strategies to treat them. My main scientific career goals are to make novel and important contributions to the field of mitochondrial disorders and to satisfy the requirements to become a tenured faculty member in a prestigious academic institution. I believe that the Department of Neurology and Neuroscience at the Weill medical College is an ideal environment to conduct my work and to fulfill my career goals because it provides a fertile ground for scientific growth by allowing interactions with topnotch scientists within the tri-institutional organization that includes Cornell University, Memorial Sloan Kettering, and Rockefeller University. The scope of this proposal is twofold. First, to modulate mitochondrial ATP synthesis in the syndrome NARP (neuropathy ataxia and retinitis pigmentosa) caused by mutations in the mtDNA encoded ATPase 6 (A6). Second, to develop cellular and animal models recapitulating the features of NARP. Such models will serve to investigate in vivo the pathogenic mechanisms underlying mitochondrial disorders and to test therapeutic approaches. Aim 1: NARP cells generate increased free radicals. The mitochondrial respiratory chain is defective presumably due to damage or inhibition and ATP synthesis in NARP cybrid cells can be improved by antioxidants. We will define the mechanisms underlying the respiratory chain dysfunction and test the effects of antioxidants in cells directly derived from NARP patients. Aim 2: We showed that the expression of a wild type A6 protein allotopically from the nucleus improved mitochondrial ATP synthesis in a cybrid model of NARP. The goal is to assess whether allotopic expression of A6 will improve ATP synthesis in patient-derived cells and whether this approach can have a therapeutic use. Aim 3: There are no animal models of NARP. Exogenous mtDNA cannot be transferred into mitochondria to generate transgenic models of NARP. We will test two alternative strategies to generate mutants that recapitulate the biochemical and clinical defects of NARP: a) By allotopic expression from the nucleus of a mutant A6 targeted to normal mitochondria; b) By introducing mutations in a nuclear-encoded mitochondrial protein, ATPase subunit C, at crucial sites of interaction with A6.

[unreadable] DESCRIPTION (provided by applicant): The most common form of familial ALS (FALS) is due to mutations in the Cu.Zn superoxide dismutase (SOD1) gene. SOD1-FALS is caused by a "toxic gain of function". Mitochondrial dysfunction has been demonstrated in SOD1-FALS models, and a growing body of evidence suggests that it may play a role in the pathogenesis of the disease. Although the majority of SOD1 resides in the cytosol, a significant proportion of SOD1 is localized in the mitochondria. The pathological role of mitochondrial SOD1 in FALS remains to be clarified. We propose to investigate the involvement of mitochondrial SOD1 in mitochondrial dysfunction and its role in the pathogenesis of SOD1-FALS. 1) Mitochondrial dysfunction, potentially resulting in energy depletion and apoptosis, has been observed in transgenic mice expressing mutant SOD1. To determine if mutant SOD1 localized in mitochondria causes mitochondrial dysfunction we will study neuronal cells expressing mutant or wild type SOD1 selectively targeted to mitochondria by specific cleavable protein targeting sequences. 2) To determine whether mitochondrial dysfunction associated with FALS is directly caused by mutant SOD1 in mitochondria and whether the selective expression of mutant SOD1 in mitochondria results in a pathological phenotype in vivo we will generate and study transgenic mice expressing mutant or wild type SOD1 targeted to mitochondria by specific cleavable protein targeting sequences. 3) Very little is known about the mechanisms underlying import of SOD1 in mammalian mitochondria. We will investigate: A) the molecular mechanisms involved in SOD1 mitochondrial import in affected and unaffected tissues; B) which protein domains are implicated in SOD1 mitochondrial import; C) the impact of SOD1 post-translational modifications and conformation on mitochondrial import. 4) Preliminary results suggest that SOD1 interacts with other mitochondrial proteins. A) Proteins with mitochondrial isoforms have been shown to interact with mutant but not wild type SOD1 in a yeast two- hybrid system. We will study these interactions in mammalian cells. B) We will search for novel protein interactions between SOD1 and mitochondrial proteins. [unreadable] [unreadable]

Neurons depend on a finely tuned transport machinery to keep their cell bodies and extensive processes connected. Increasing evidence suggests that organelle transport is impaired in diseases of motor neurons (MN), where cellular components have to move long distances along axons, and that transport defects may contribute to why MN are specifically affected in amyotrophic lateral sclerosis (ALS). The central hypothesis of this proposal is that impaired mitochondrial dynamics (i.e., transport, fusion, fission) is a primary lesion in ALS MN: when transport is impaired, mitochondria cannot traffic normally to and from crucial sites of energy utilization, such as synaptic terminals, resulting in mitochondrial mislocalization and dysfunction, which in turn causes energy depletion, impaired calcium homeostasis, and ultimately cell degeneration. In this proposal, we will investigate mitochondrial dynamics defects in primary MN from transgenic animal models expressing mutant SOD1, which causes a familial form of ALS. We will use a novel, photo-activatable, fluorescent protein targeted to mitochondria, (mito-Dendra), and live confocal cell imaging. We will investigate the correlations between mitochondrial dynamics defects, mitochondrial structural abnormalities and bioenergetic dysfunction. Our preliminary data strongly suggest that mitochondrial dynamics is abnormal in SOD1 mutant MN and that this abnormality correlates with impaired bioenergetics. First, we will characterize how mutant SOD1 affects mitochondrial transport and determine whether mitochondrial transport defects are specific to MN or if they affect other neural cell types. Furthermore, since ALS involves other cell types besides MN, we will determine whether astrocytes and microglia, which are directly implicated in ALS pathogenesis, play a role in impairing mitochondrial dynamics and function in MN. Second, we will determine how defective mitochondrial dynamics in mutant SOD1 MN affects the interactions with muscle cells at the nuromuscular junction (NMJ), in compartmentalized innervated MN-muscle co-cultures. Third, to verify that mitochondrial dynamics impairment is a primary defect in MN degeneration we will establish the role of mitochondrial transport in maintaining MN and NMJs in normal, wild type, MN, where anterograde mitochondrial transport has been impaired by a genetic approach, independent of mutant SOD1.

DESCRIPTION (provided by applicant): Phosphorylation of mitochondrial proteins is a rapid and efficient way to regulate oxidative phosphorylation (OXPHOS) and maintain energy homeostasis. Mitochondrial PKA modulates enzymes of the electron transfer chain through protein phosphorylation, which is regulated by a signaling pathway, involving mitochondrial soluble adenylyl cyclase (sAC), generating cAMP that activates PKA. This signaling cascade serves as a metabolic sensor that modulates energy conversion in mitochondria. Cytochrome oxidase (COX) is a pacemaker of ETC fluxes, whose activity is modulated by phosphorylation of its subunits and by ATP allosteric inhibition. We showed that COX is a target of the signaling pathway and identified subunit IV of COX (COXIV) as a target for phosphorylation. We also demonstrated that phosphorylation of S58 in COXIV- 1 is responsible for modulation of COX activity. We propose that this regulatory pathway participates to metabolic adaptive responses to OXPHOS defects and could be a target for pharmacological intervention. The goal of this project is to understand the physiological implications of intramitochondrial sAC-cAMP- PKA signaling and S58 COXIV-1 phosphorylation, in vivo. We will understand how this system behaves in healthy tissues and in tissues affected by mitochondrial defects. To this end, we will generate and study mice, in which the molecular players of the regulatory pathway are genetically modified and assess the outcomes on clinical phenotype and mitochondrial biochemistry. In aim 1, we will generate transgenic mice expressing inducible sAC selectively targeted to the mitochondrial matrix (mito-sAC), which is expected produce high basal metabolism. In aim2, we will generate COXIV-1 S58A knockin mice, lacking the phosphorylated site, and thus incapable of up-regulating ETC fluxes and enhancing ATP production, resulting in exercise intolerance, decreased glucose utilization, impaired thermogenesis, and increased fat storage. In aim 3, we will investigate sAC-cAMP-PKA modulation in a mouse model of OXPHOS defect caused by conditional and inducible genetic disruption of COX. These mice recapitulate biochemical and clinical characteristics of mitochondrial diseases. We will assess how sAC-cAMP-PKA modulation and protein phosphorylation in mitochondria from affected tissues and correlate it with disease progression.

DESCRIPTION (provided by applicant): This is an application for renewal of the project entitled "the pathogenic role of mutant SOD1 in mitochondria". Mitochondrial Ca2+ overload leads to deleterious consequences, including mitochondrial de-energization, structural changes, and bioenergetic failure, which can result in cell death. The overarching hypothesis of this application is that mitochondrial Ca2+ overload plays a fundamental role in the pathogenesis of familial ALS (FALS) associated with SOD1 mutations. Mitochondrial dysfunction, one of the cardinal features of ALS, causes increased susceptibility to Ca2+ overload. Our preliminary results show impaired mitochondrial Ca2+ handling and susceptibility to overload in the CNS of mutant SOD1 transgenic mice. They also show that female mutant SOD1 mice are partially protected against mitochondrial Ca2+ overload by a pathway of Ca2+ release involving estrogen, mitochondrial estrogen receptor (ER), and cyclophilin D (CyPD), a modulator of mitochondrial Ca2+ induced damage. In this application, we provide a mechanistic hypothesis for the involvement of mitochondria and for the gender differences in FALS. The broad goals are to define the mechanisms of mitochondrial Ca2+ handling regulation and to develop approaches to prevent Ca2+-overload in FALS mitochondria. Our specific aims are to: 1) Define the role of the estrogen receptor in Ca2+ handling in FALS mitochondria by investigating, in vivo and ex vivo, ER2 KO mice crossed with G93A mutant SOD1 mice. Then, with the G85R mutant SOD1 mouse model, to determine if the mechanisms of estrogen-ER2 and CyPD dependent neuroprotection are common to different SOD1 mutants. 2) Define the biochemical and molecular basis of estrogen-ER regulation of mitochondrial Ca2+ handling and the effects of mutant SOD1. In the mouse crosses established in aim 1, we will determine: i) how estrogen-ER2 modulates mitochondrial bioenergetics and the CyPD- dependent Ca2+ release pathway, ii) the mitochondrial localization of the ER2 and the ER- CyPD interactions with SOD1, by mitochondrial fractionation, immuno-electron microscopy, and immuno-precipitation. 3) Test different complementary approaches to prevent Ca2+ overload in FALS mitochondria and improve disease in mutant SOD1 mice by: i) chronic administration of estrogen to male SOD1 mutant mice, ii) boosting mitochondrial bioenergetics with expression of a mitochondrial soluble adenylyl cyclase that enhances oxidative phosphorylation, iii) mild mitochondrial uncoupling achieved with overexpression of uncoupling protein 2 (UCP2). PUBLIC HEALTH RELEVANCE: Mitochondria play a fundamental role in regulating neuronal life and death. One of the main functions of mitochondria is to take care of intracellular calcium (Ca2+). Mitochondrial dysfunction is one of the cardinal features of ALS and causes increased susceptibility to Ca2+ overload. Mitochondrial Ca2+ overload leads to deleterious consequences, including mitochondrial failure, which can result in cell death. The leading hypothesis of this study is that mitochondrial Ca2+ overload plays a fundamental role in the pathogenesis of familial ALS (FALS) associated with SOD1 mutations. Therefore the broad goals of this project are to define the mechanisms of mitochondrial Ca2+ handling regulation and to develop approaches to prevent Ca2+-overload in FALS mitochondria to ameliorate the disease.

DESCRIPTION (provided by applicant): Mitochondrial diseases are heterogeneous genetic disorders caused by respiratory chain (RC) impairment. Attempts to treat mitochondrial diseases have been disappointing so far, mostly due to the lack of defined targets. The leading hypothesis of this application is that mitochondrial disease pathogenesis involves the blockage of crucial steps of the inter-organ amino acid metabolism. We have identified previously unrecognized defects in glutamine metabolism in cells harboring mitochondrial DNA mutations associated with human mitochondrial diseases. We found that the energetic utilization of glutamine through the glutamine-glutamate-¿-ketoglutarate pathway was impaired in these cells. We were able to rescue the metabolic defect by supplementation with compounds that bypass the enzymatic blockages. Glutamine is the most abundant and versatile circulating amino acid, mostly synthesized in skeletal muscle and released in the blood where it plays an important role as a carrier of nitrogen, carbon, and energy between organs. The various glutamine-utilization pathways in the body depend on the specialized metabolism of each tissue and play a crucial role in the inter-organs integrated metabolism that regulates metabolites homeostasis. The goal of this application is to define in vivo the altered glutamine pathways and to bypass the metabolic blockages with dietary supplementation, thus identifying new approaches to the therapy of mitochondrial diseases. To this end, we propose the following aims: 1) Metabolites imbalance in the COX10 KO mouse. We will investigate the glutamine utilization and synthesis pathways in vivo in a mouse model of RC defect caused by genetic disruption of cytochrome c oxidase (COX) assembly, resulting in a progressive mitochondriopathy. The levels of relevant metabolites will be determined in plasma and in tissues, and will be correlated with the bioenergetics, redox, acid/base and nitrogen states. The vulnerability of the affected tissues will be evaluated and correlated with disease progression. 2) Dietary supplementation in the COX10 KO mouse. In preliminary studies in vitro, metabolites that effectively bypass metabolic blocks in RC deficient cells have been identified. These metabolites will be supplemented in the diet of the COX10 KO mouse. The specialized metabolism of different tissues, the inter-organ metabolic homeostasis, and the physiological alterations in relation to disease progression will be assessed. The potential preclinical impact o this aim is that it will provide a rationale for clinical trials based on dietary supplementation, using physiological compounds.

DESCRIPTION (provided by applicant): We have recently identified a new unfolded protein response (UPR) in the mitochondrial inter-membrane space (IMS-UPR). The focus of this application is on the role of the IMS-UPR in familial amyotrophic lateral sclerosis (fALS) associated with misfolded mutant SOD1. Our data indicates that accumulation of misfolded proteins in the IMS leads to the activation of AKT, which then promotes the phosphorylation and activation of the estrogen receptor alpha (ERalpha). In turn, activated ERalpha promotes the transcription of an array of genes aimed at reducing IMS-stress. We and others have demonstrated that mutant SOD1 localizes both to the cytoplasm and the IMS. Cell lines and mouse models were generated, in which SOD1-G93A is either in both the cytoplasm and the IMS or in the IMS-only. We used these models to test the activation of the IMS-UPR. Our preliminary data in cell lines and mouse models of SOD1-G93A ALS suggests that, while the IMS-fraction of SOD1G93A activates a cyto-protective IMS-UPR, the inhibition of the proteasome by cytosolic SOD1G93A fails to limit the import of the mutant protein in the IMS. As a result, sustained and unresolved IMS-stress occurs. We hypothesize that, under these conditions, mitochondrial damage increases and the IMS-UPR switches from being cyto-protective to being pro-death. We further hypothesize that this switch does eventually happen in the IMS-only model but at a much later time. Further, while the activation of the ERalpha by AKT is independent of estrogen, it nevertheless synergizes with estrogen. Since estrogen is known to be neuro-protective, the role of estrogen in the setting of the IMS-UPR remains to be defined. To test our hypothesis we propose the following specific aims: Specific aim 1: Monitoring the IMS-UPR throughout the natural history of the disease in ALS in SOD1-G93A and IMS-only SOD1-G93A transgenic mice. Specific aim 2: Testing the effects of ERalpha ablation on the natural history of the disease in the untargeted and IMS-only SOD1-G93A models. Specific aim 3: Dissecting the role of the ligand-dependent and ligand-independent functions of the ERalpha in the activation of the IMS-UPR.

? DESCRIPTION (provided by applicant): Sporadic ALS (sALS) includes by far the largest ALS patient population, but very little is known about the causes of the disease. Patients affected by amyotrophic lateral sclerosis (ALS) have bioenergetic abnormalities, which can contribute to disease pathogenesis, and metabolic profiles could represent predisposing factors to develop ALS and affect disease course. Metabolic changes may also influence the response to therapeutics, and the lack of effective drugs for ALS may be in part attributable to insufficient understanding of metabolism as a disease modifier. To investigate energy metabolism we used a novel fluorimetric assay for mitochondrial membrane potential (MMP) and mitochondrial mass (MM), in primary sALS skin fibroblasts. We found that sALS patients have on average significantly increased membrane potential, which inversely correlates with disease onset. Furthermore, unbiased metabolomics studies identified unequivocal differences in intermediate metabolite profiles between ALS and control fibroblasts. To our knowledge, this is the first functional evidence in living cells that energy metabolism is altered in sporadic ALS. A major goal of the application is the identification of fibroblasts metabolism as a predictive factor for he evolution of ALS and for evaluating metabolic changes as disease modifiers. To this end, in aim 1, we propose to define a metabolite signature linked to energy metabolism in fibroblasts, in the extracellular medium, and in the plasma of the subjects from which skin biopsies were taken. We also propose to investigate the correlation of bioenergetic and metabolic parameters with disease status and progression. In aim 2, to understand if the changes in fibroblasts reflect a metabolic reprogramming affecting the cell types most involved in ALS, we will convert fibroblasts into induced pluripotent stem cells (iPSC) and then motor neurons, and study their bioenergetics and metabolic profiles. Importantly, to investigate if metabolic changes originate from genetic or epigenetic causes, we will determine if bioenergetic and metabolic changes persist or not after de-differentiation of iPSC followed by back-differentiation into fibroblasts.

? DESCRIPTION (provided by applicant): Neurons are highly dependent on mitochondrial metabolism, because of high energetic demands and the need to maintain intracellular calcium homeostasis. To provide energy to the extensive neuronal cytoplasm of large projections cells, such as motor neurons, the mitochondrial network has to be adequately maintained and distributed. Dynamic mitochondrial transport, fusion, and fission, ensure that healthy mitochondria are provided at sites of high-energy utilization and calcium buffering. New mitochondria are generated by regulated biogenesis and damaged mitochondria are subjected to quality control (MQC): damaged proteins are removed by proteolytic and proteosomal systems. Irreparably damaged mitochondria are eliminated by mitophagy. The balance between mitochondrial biogenesis and mitochondrial elimination ensures that adequate pools of functional mitochondria are available and that accumulated damaged mitochondria do not release toxic molecules, such as free radicals and pro-apoptotic factors, or excessive amounts of calcium. Extensive mitochondrial damage in motor neurons has been described in vitro and in vivo in mutant SOD1 models and in other forms of familial and sporadic ALS, raising two fundamental questions: 1) Why in SOD1- ALS MQC is incapable of clearing damaged mitochondria? We found an increase in the ubiquitin-binding adaptor p62 associated with mitochondria, suggesting enhanced mitophagy, but delayed mitochondrial clearance. We also found that Parkin, an ubiquitin ligase involved in both proteosomal degradation of mitochondrial proteins and mitophagy, is decreased. 2) Does the failure to clear of damaged mitochondria play a role in the pathogenesis of SOD1-ALS? This question implies that impaired MCQ may lead to accumulation of damaged mitochondria and play a pathogenic role. The overarching hypothesis of this application is that in ALS motor neurons MQC fails, either because the demand exceeds capabilities or because the MQC is dysfunctional. To test this hypothesis and study the underlying mechanisms we propose two specific aims, each with two sets of studies. Specific Aim 1 will investigate the causes of MQC impairment in SOD1-ALS. Study 1 will identify which steps of the MQC are impaired in mutant SOD1 neurons in vivo and in vitro. Study 2 will determine if MQC impairment in SOD1 motor neurons is caused by excessive mitochondrial damage. Specific Aim 2 will assess the effects of Parkin modulation in SOD1-ALS. Study 1 will assess the impact of inducible/conditional genetic deletion of the MQC component Parkin on SOD1-ALS. Study 2 will assess the impact of Parkin genetic overexpression on SOD1-ALS. The application aims at the mechanistic understanding of a pathogenic pathway that links MQC with mitochondrial dysfunction and motor neuron degeneration in SOD1-ALS. The findings will unveil novel disease mechanisms that could be addressed therapeutically by targeted approaches aimed at modulating mitochondria MQC in ALS and other neurodegenerative diseases by pharmacological intervention.

Mitochondrial permeability transition (MPT) is an inner membrane permeabilization event, which can result in irreversible de-energization and swelling of mitochondria, leading to release of pro-death factors. Mitochondrial Ca2+ overload is the best-characterized trigger of MPT and has been implicated in the pathogenesis of diverse paradigms of neuronal death, such as ischemia-reperfusion injury, where a large influx of cytosolic Ca2+ triggers mitochondrial Ca2+ overload. While uncontrolled MPT can result in mitochondrial disruption, under certain conditions, MPT could provide mitochondria with a Ca2+ release outlet, allowing Ca2+ recycling and protecting mitochondria from Ca2+ overload. Estrogen receptors (ER) have been implicated in various paradigms of neuronal injury, and MPT modulation could be one of the mechanisms whereby they exert their role. Our studies revealed an unprecedented role of the ER? in modulating MPT. In mouse brain mitochondria, estrogen decreases mitochondrial Ca2+ capacity in an ER? and cyclophilin-D (CyPD, an MPT activator) dependent manner. Mitochondria from ER? knock out (ER?KO) mice have reduced sensitivity to cyclosporine A, a potent CyPD inhibitor and CyPD genetic ablation in ER?KO does not further increase Ca2+ capacity. These results point to ER? as a novel regulator of Ca2+-dependent MPT that functionally interacts with CyPD. In this application, we will test the hypothesis that ER? localized in mitochondria (mER?) regulates MPT, independently of transcriptional effects. The goals are to investigate the mechanisms of MPT modulation by mER? and to test the effects of MPT modulation by mER? in models of neuronal injury that involve mitochondrial Ca2+ toxicity, such as oxygen glucose deprivation (OGD) and glutamatergic toxicity. To this end we propose 1) to study the mechanisms of regulation of Ca2+-mediated MPT by ER?. This regulation will be investigated using a multipronged approach, involving biochemical and molecular studies. 2) To assess the role of ER? MPT regulation in neuronal Ca2+-mediated injury. Evidence suggests that Ca2+ dependent MPT and its regulator CyPD are involved in ischemic neuronal injury. We will use neuronal OGD and exposure to glutamatergic agents, both well-known paradigms of neuronal toxicity involving mitochondrial Ca2+ overload, to test the effects of genetic and pharmacological modulation of ER?. The impact of the project will be two-fold: first, it will elucidate novel mechanisms of MPT regulation; second, it will assess if MPT modulation by mER? could be protective in neuronal injury.

Astrocytes play a fundamental role in ALS pathogenesis, participating in motor neuron (MN) degeneration in a non-cell autonomous manner. In spinal astrocytes from the G93A SOD1 mouse model of familial ALS, we found that ER oxidative stress induces post-translational changes in regulatory elements of ER calcium homeostasis (store operated calcium entry, SOCE), which leads to aberrant regulation of ER calcium levels and signaling. The resulting ER calcium signaling dysregulation participates in the toxic processes. In preparation to this application, we have studied human iPSC-derived SOD1 mutant astrocytes and found that aberrant ER calcium regulation is a common abnormal phenotype between the human cells and the mouse model of familial ALS, supporting its significance in ALS pathogenesis. Based on new preliminary studies, we hypothesize that ER calcium dysregulation in ALS astrocytes originates from enhanced oxidative protein folding process operated by the PDI-Ero1 enzyme pathway. Increased oxidative protein folding activity leads to excessive hydrogen peroxide production, glutathione consumption, and overburdening of molecular chaperones. We propose that this aberrant process causes oxidative modifications of proteins involved in calcium homeostasis. We postulate that these molecular mechanisms in ALS astrocytes are at the basis of the functional changes in intracellular calcium regulation and contribute to MN toxicity. The overarching goal of this application is to provide proof of principle that modulating ER calcium dysregulation pathways in ALS astrocytes represents a viable approach for improving ALS. To achieve this goal we propose three specific aims, which investigate different levels of regulation of ER calcium signaling and test the impact of modulating these pathways by pharmacological and genetic approaches on the toxicity of ALS astrocytes to MNs.

Selective estrogen receptors modulators (SERMs), such as tamoxifen and raloxifene, have been shown to be neuroprotective, but the mechanisms of neuroprotection are unknown. The underlying hypothesis of this application is that raloxifene is a neuroprotective in ALS by activating the estrogen receptor alpha (ER??- dependent mitochondrial unfolded protein response (UPRmt). This hypothesis stems from our recent findings in the SOD1-G93A mouse model of familial ALS. In this mouse, we found activation of the ER?-mediated axis of the UPRmt. We reported that, upon accumulation of misfolded SOD1 in the mitochondrial intermembrane space (IMS), the ER? signaling pathway is activated and promotes the expression of the IMS protease OMI, subunits of the 26S proteasome and the nuclear respiratory factor 1 (NRF1), a direct target of ER?, which in turn stimulates the transcription of several mitochondrial proteins. We recently studied this pathway in the SOD1- G93A mice and found that females activate the UPRmt more potently than males and this correlates with the longer survival in females. Further, we showed that accumulation of misfolded SOD1-G93A in ER??knockout mice failed to activate the UPRmt. Taken together, these results indicate that the neuroprotective effect of the UPRmt requires ER?-mediated activation of three major targets, OMI, NRF1 and the 26S proteasome. We tested whether estrogen, tamoxifen, and raloxifene differentially induced the UPRmt specifically in the spinal cord. We found that raloxifene is a potent inducer of NRF1 and the proteasome in the spinal cord, while tamoxifen or estrogen are not. We also performed preliminary pilot studies on the effect of chronic raloxifene administration to SOD1-G93A mice and found that it stimulates UPRmt and delays disease progression. Based on these exciting results, in this exploratory application we will test the hypothesis that raloxifene may represent a novel therapeutic approach against ALS by maintaining the activation of the UPRmt. We propose two specific aims. Aim 1: will systematically test the protective effects of different doses of raloxifene in the SOD1-G93A mouse model of ALS, starting at presymptomatic and symptomatic disease stages. Aim 2: will validate that the molecular targets have been hit upon treatment with raloxifene, by assessing the activation of the ER? axis of the UPRmt and its transcriptional effects in the spinal cord of SOD1-G93A mice.

Skeletal muscle, heart, and brain are high-energy requiring tissues that are severely affected by mitochondrial dysfunction. Recently a novel form of genetic disease affecting mitochondria has been associated with mutations in a mitochondrial protein, CHCHD10 (D10), whose function is still largely unknown. Mutant D10 causes severe autosomal dominant mitochondrial diseases, with diverse phenotypic features, ranging from myopathy to motor neuron disease and frontotemporal dementia. We previously showed that in mitochondria D10 forms a dimeric complex with its paralog protein, CHCHD2 (D2). Interestingly, mutations in D2 are also associated with familial neurodegenerative diseases. To study the manifestations and disease mechanisms of mutant D10 in vivo, we have generated a knock in mouse harboring the first pathogenic D10 mutation reported in humans (S59L, corresponding to mouse S55L). In D10S55L mouse muscle and heart mitochondria, D10 and D2 accumulate and aggregate, leading to mitochondrial dysfunction and degeneration. These abnormalities result in a profound integrated mitochondrial stress response (ISRmt), altering transcriptional profiles and metabolism, and ultimately resulting in fatal cardiomyopathy. Conversely, D10 knock out mice do not manifest mtISR and are phenotypically normal, suggesting that D10S55L causes disease through a toxic mechanism and not a loss of function. In this application, we will study the normal function of D10 and D2 and the mechanisms underlying mitochondrial alterations in D10S55L mice. Since D10 mutations cause neurodegeneration in humans, we will also investigate the involvement of the nervous system in D10S55L mice. We will then identify metabolic and molecular biomarkers to help monitor disease course. Lastly, we will test the effects of pharmacological modulation of ISRmt in D10S55L mice as a therapeutic strategy. The impact of this project will be to facilitate rationale approaches to target disease pathogenesis in patients with D10 mutations, which could be extended to other mitochondrial diseases mediated by ISRmt

Many neurodegenerative diseases, including ALS, are characterized by mitochondrial dysfunction and defects in the ubiquitin-proteasome pathway. However, why the central nervous system is more prone to these defects than other tissues is unknown. In addition, several of the CNS-associated diseases show sexual disparity but, again, the mechanistic source of this observation is unclear. The current application addresses both the increased sensitivity of the CNS to proteostasis and mitochondrial defects and sex disparity. The Germain group first described an estrogen receptor alpha (ERa) driven axis of the mitochondria unfolded protein response (UPRmt), which promotes the activity of the proteasome, as well as the transcription of mitochondrial genes. More recently, the Germain and Manfredi labs characterized this pathway in the SOD1-G93A model of familial ALS, a model in which males show earlier disease onset than females. We found that females maintain the ability to activate the ER axis of the UPRmt longer than males. These observations raise the possibility that interventions aimed at activating the ERa axis of the UPRmt early on in the disease course may delay the progression of ALS and potentially other CNS-associated diseases. Data presented in this application demonstrate that treatment with the FDA-approved selective estrogen modulator (SERM) raloxifene, but not estrogen or tamoxifen up- regulates expression of both the activity of the proteasome at multiple levels and mitochondria genes. Further, we found that raloxifene delays disease progression in this model, in females specifically, despite the fact that the serum level achieved in our trial was 10-fold lower than what is possible to achieve clinically in humans treated chronically with raloxifene. This suggests that raloxifene is unique in its remarkable ability to increase two of the key pathways associated with diseases affecting the spinal cord, such as ALS, and possibly other components of the CNS. Moreover, our findings also suggest that if levels of raloxifene closer to those achieved with human regimens can be achieve in mice, the protective effect of raloxifene could be much improved. Based on these results, we propose the three following specific aims. Aim 1: Understanding the molecular basis of the differential effect of estrogen, tamoxifen and raloxifene on the transcriptional activity of the ER in the spinal cord and expand the analysis of their effects on other parts of the CNS. Aim 2: Optimize raloxifene delivery,, alone or in combination with the proteasome activator oleuropein and extend the beneficial effect to males. Aim 3: Extending raloxifene-based therapy to a mutant Ubiquilin2 mouse model of ALS/FTD. The program proposed here is an aggressive and ambitious attempt at testing the neuroprotective effects of raloxifene in ALS. As thousands of Americans suffer from this devastating disease, which has no effective therapy, we feel that the ambitious approaches proposed are well justified.

Mitochondria play essential roles in cell biology because are central hubs of most metabolic pathways. They are not only essential for energy conversion, but also for the biosynthesis and catabolism of virtually all cell constituents. Mitochondrial dysfunction causes havoc in all cells, but especially in those cell types that are highly dependent on mitochondrial energetic and metabolic functions, such as neurons and glia. Genetic alterations of the mitochondrial proteome, which includes more than 1000 proteins, encoded by both the nuclear and the mitochondrial genomes, result in primary mitochondrial disorders. These diseases, for which there is currently no effective treatment, result in severe and often fatal neurodegeneration. Mitochondrial dysfunction also plays a role in the pathogenesis of many age-related neurodegenerative disorders, such as Alzheimer and Parkinson disease and ALS. Therefore, addressing therapeutically the consequences of mitochondrial dysfunction could have a profound impact on the treatment of many human disorders. A major challenge in devising effective treatments for mitochondrial encephalopathies is our limited understanding of the ramifications of the effects of mitochondrial dysfunction. The conventional view that these disorders are caused simply by energy failure is inadequate, as it is becoming increasingly clear that mitochondrial dysfunction affects much more than just ATP generation and leads to an extensive rewiring of cell metabolism. An exciting new development in the field is the observation that various types of mitochondrial dysfunction activate transcriptional and metabolic responses that involve multiple stress signaling pathways. We and others have identified a ?mitochondrial integrated stress response? (mtISR) in diverse genetic forms of mitochondrial disorders, suggesting that mtISR is strongly associated with mitochondrial diseases and a potential pathogenic common denominator. We postulate that, while in the short term these responses may be compensatory, if sustained and unresolved, they become maladaptive and causes imbalances of key metabolites, which may be more detrimental than the energy defect itself. While we now fully appreciate these maladaptive mechanisms in peripheral tissues, such as muscle and heart, very little is known about them in the CNS affected by mitochondrial encephalopathies. A deeper knowledge of the characteristics and the consequences of the mtISR in the CNS is needed to understand its pathogenic significance and develop targets therapeutic strategies. Our research group has a long-standing commitment to investigating the pathogenic mechanisms of mitochondrial diseases and we have accumulated over two decades of expertise in studying the mechanisms of mitochondrial encephalopathies and mitochondrial dysfunction in neurodegeneration. In this R35 application, we focus on fundamental gaps in knowledge on the mtISR in mitochondrial encephalopathies by studying disease models that recapitulate human diseases. We will use a series of approaches, both established and technologically innovative, to generate a blueprint of the metabolic rewiring in the diseased CNS and identify targets potentially responsive to therapeutic modulation.