Kartik Venkatachalam, Ph.D. - US grants

DESCRIPTION (provided by applicant): Many lysosomal storage diseases (LSDs) cause childhood-onset neurodegeneration leading to profound psychomotor retardation and ophthalmological abnormalities. In general, LSDs are notoriously difficult to treat because although these diseases are monogenic in origin, they typically affect a host of cellular signaling cascades and cell biological processes. The pleiotropy associated with LSDs prevents the development of suitable therapeutic strategies that simultaneously target the multiple disease outcomes. Moreover, it is becoming increasingly clear that LSDs are also characterized by neurodevelopmental abnormalities such as diminished axonal development in the cortex and corpus callosum. Unfortunately, the mechanistic basis for these neuronal defects associated with LSDs remain poorly understood. The overarching goal of this proposal is to address these conceptual gaps using a Drosophila model of an LSD called mucolipidosis type IV (MLIV) that arises from loss of function mutations in a lysosomal Ca2+ channel called TRPML1. We previously established that the fly TRPML1 homolog, TRPML, is a late-endosomal/amphisomal Ca2+ channel that drives the fusion of these vesicles with lysosomes. Here, we will leverage the genetic tractability of the Drosophila to address the critical mechanistic questions regarding the neuropathology of LSDs. In Aim 1, we will test the hypothesis that loss of TRPML results in alterations in the organization of cholesterol-enriched ordered membrane microdomains called lipid rafts. Because lipid rafts are critical for the functioning of a plethora of cellular signalig processes, alterations in the stability of these domains could provide a mechanistic explanation for the pleiotropy associated with lysosomal dysfunction. In Aim 2, we will test the hypothesis that TRPML promotes synaptic growth by activating developmental c-Jun Kinase (JNK) signaling in neurons. Interestingly, diminished JNK activation results in hypoplasia and agenesis of axonal tracts of the cortex and corpus callosum. Therefore, decreased JNK activation following lysosomal dysfunction signaling may be the molecular explanation for why LSDs are characterized by axonal growth defects. If successful, these studies should provide us with mechanistic insight into some of the common neurological outcomes associated with lysosomal dysfunction and also aid in the establishment of concepts for therapeutically targeting the neurological sequelae of LSDs.

PROJECT SUMMARY Mucolipidosis type IV (MLIV) is an untreatable lysosomal storage disease caused by loss-of-function mutations in MCOLN1, gene encoding an endolysosomal cation channel TRPML1. MLIV patients display severe motor and cognitive disabilities. The main difficulty with MLIV treatment is that TRPML1 is completely lost in most MLIV patients. Thus, therapies based on stimulating residual TRPML1 activity are not likely to be effective. Because TRPML1 is not an endolysosomal enzyme, enzyme replacement and/or substrate reduction are not viable options. These barriers can be overcome by identifying and attenuating toxic signaling processes triggered by TRPML1 loss. Our preliminary data show transcriptional upregulation of the gene encoding ganglioside-induced differentiation-associated protein 1 (GDAP1) in the absence of TRPML1. Forced reduction of GDAP1 levels in cells lacking TRPML1 ameliorates the mitochondrial dysfunction that we have previously shown in MLIV. Given that GDAP1 expression is triggered by gangliosides ? lipids that accumulate in MLIV cells ? and that ganglioside accumulation has been shown in MLIV, we propose that gangliosides buildup in cell lacking TRPML1 triggers GDAP1 induction. We propose that the elevated GDAP1 in MLIV cells induces mitochondrial damage, vesicular trafficking defects, and neurodegeneration. Our model redefines the current view of MLIV pathogenesis, and suggests that suppressing GDAP1 expression and/or suppressing ganglioside production by pharmacological means may be used to mitigate tissue damage in MLIV. Here we combine the expertise of our labs in cell biology, Drosophila genetics and neurophysiology to test this model. In Aim 1, we will answer whether gangliosides and GDAP1 mediate the mitochondrial and vesicle trafficking defects observed in MLIV. The outcome of these experiments could confirm that extracellular gangliosides are a novel therapeutic target for MLIV. In Aim 2, we will test whether the loss of the TRPML1 homolog in Drosophila (trpml) leads to neurodegeneration via GDAP1-dependent mitochondrial dysfunction, using flies that lack trpml and recapitulate the key neurodegenerative features of MLIV. Specifically, we will examine whether GDAP1- dependent mitochondrial dysfunction and neurodegeneration occur in the Drosophila model of MLIV. We anticipate that our findings in the Drosophila MLIV model will inform future studies using human MLIV cells and vertebrate models. We will develop novel paradigms describing the mechanisms underlying mitochondrial dysfunction in an untreatable childhood neurological disease. Our approach, which bi-directionally combines in vitro cell physiology and the utility a Drosophila model, will allow us to make unprecedented progress to develop a deeper understanding of the mitochondrial dysfunction and neurodegeneration in MLIV.

PROJECT SUMMARY The overarching goal of this proposal is to further our understanding of evolutionarily conserved mechanisms that modulate mitochondrial proliferation and energy metabolism. Although significant progress has been made in understanding the consequences of mitochondrial dysfunction and characterizing the emergent disease, it remains unclear whether manipulation of overall mitochondrial proliferation to alter mitochondrial number and/or mass would impact on disease phenotypes. This conceptual gap exists largely due to the genetic redundancy, relative intractability, and functional complexity associated with regulation of mitochondrial proliferation and function in mammals. To mitigate these limitations, mitochondria are often studied in Drosophila, which are genetically tractable organisms characterized by extensive conservation with mammals in terms of mitochondrial biology. However, transcriptional networks that regulate mitochondrial proliferation and function are unknown in Drosophila. We have now identified a master transcriptional regulator of mitochondrial proliferation in Drosophila that is both necessary and sufficient to determine mitochondrial mass. Thus, we are in a position to finally delineate the relationship between mitochondrial proliferation and function. In Aim 1, we will test the hypothesis that the transcription factor we have identified modulates mitochondrial function in addition to mitochondrial mass. Successful completion of this aim could present unprecedented opportunities for en masse modulation of mitochondrial function in Drosophila models of human diseases. In Aim 2, we will examine the interrelationship between AMPK, a known regulator of mitochondrial biogenesis and metabolism, with the identified regulatory network. These studies could reveal previously unrecognized, fundamental mechanisms by which AMPK regulates mitochondrial function. In Aim 3, we will test whether the signaling network and the human homologs of the identified transcription factor regulate mitochondrial biogenesis and/or function in the human cell. Taken together, our experimental strategies are designed to reveal novel conceptual insights into the regulation of mitochondrial proliferation and function. Furthermore, our multidisciplinary and systems-based approach will enable a deeper understanding of the pathophysiology of mitochondrial diseases. We hope that upon completion of these studies, we and other biomedical researchers can leverage the insights gleaned to inform innovative avenues for therapeutic intervention for treating human diseases involving mitochondrial dysfunction.

SUMMARY Accumulation of neurofibrillary tangles comprised of Tau, or mutations in the gene encoding this protein, are common in Alzheimer?s disease (AD) and related dementias such as frontotemporal dementia (FTD). Tau contributes to neurodegeneration via two related pathways. First, Tau forms aggregates that are toxic to neurons. Autophagy and lysosomal degradation are of particular interest when considering the removal of Tau aggregates. The idea here is that potentiation of autophagy and lysosomal degradation would rid the cells of aggregates, and thus, alleviate neurotoxicity. Second, at a cellular level, Tau perturbs several key aspects of neuronal function. Studies have shown that mutant Tau can lead to Ca2+ dyshomeostasis, perturbations in neuronal excitability and bioenergetics, and alterations in transcriptional and translational programs. The overarching goal of our exploratory grant application is to bridge these two concepts. We built the aims of this project on the basis of preliminary studies that demonstrate the onset of severe lethality upon expression of human tau or tauR406W in Drosophila glutamatergic neurons. We propose the involvement of two distinct modules of Ca2+ dyshomeostasis working in sequence to impart dysfunction. The first involves elevated inositol trisphosphate (IP3) production and IP3 receptor (IP3R)-mediated ER Ca2+ release. The Ca2+ coming out of the ER is loaded into endolysosomes, and subsequent release of vesicular Ca2+ through the TRPML endolysosomal Ca2+ release channels leads to decline in animal viability. In support of this model, knockdown of genes encoding IP3R or TRPML almost completely prevented the lethality stemming from either Tau or TauR406W. Furthermore, epistasis analyses place TRPML downstream of IP3R in the sequence of events leading to neurotoxicity. In this application, we propose three specific aims to delineate the mechanisms underlying these intriguing findings. In Aim 1, we will determine the cell biological correlates of IP3R- and TRPML-dependent decline of organismal viability. We will test the hypothesis that neuronal cell death imposed by Tau or TauR406W are either delayed or prevented by the genetic attenuation of the IP3R?TRPML axis. In Aim 2, we will test the hypothesis that Ca2+ released by IP3Rs is sequestered into endolysosomes. Thus, we will determine whether IP3R hyperactivation in neurons expressing tau variants leads to an increase in endolysosomal Ca2+ content and elevated TRPML activity. Finally, in Aim 3, we seek to elucidate the mechanisms linking TRPML-mediated endolysosomal Ca2+ release with neurotoxicity. Successful completion of these aims would point to the critical involvement of an IP3R?TRPML axis in neurotoxicity resulting from Tau variants, and motivate future studies designed to evaluate the conservation of these pathways in mouse models of AD and FTD.

Tau tangles are common features of Alzheimer?s disease related dementias (ADRDs) and induce a range of pathological perturbations in neurons including hyperexcitability and Ca2+ dyshomeostasis. In this application, we use Drosophila to demonstrate that progressive neurotoxicity elicited by Tau or hexanucleotide repeat expansion of C9ORF72 involves elevated production of the second messenger, inositol trisphosphate (IP3), and activation of IP3 receptor (IP3R)-mediated ER Ca2+ release. Thus, premature lethality stemming from expression of ADRD-causing transgenes was almost fully suppressed by attenuation of IP3 production or knockdown IP3Rs. Although IP3Rs have been previously implicated in neurodegeneration, we have uncovered a novel mechanism underlying channel hyperactivation. We show expression of ADRD-causing transgenes leads to loss of neuronal membrane potential, and that the resulting depolarization is what increases IP3R activity. Our preliminary findings are consistent with the notion that depolarization increases association of an enzyme responsible for IP3 production, phospholipase Cb (PLCb), with its phosphoinositide substrate, PIP2. Greater PLCb?PIP2 interactions in depolarized cells lead to elevated IP3 production upon stimulation of PLCb-coupled receptors. In Aim 1, we will test the aforementioned hypothesis in fly and mouse neurons expressing mutant tau. We also ask how ADRD neurons lose their ability to maintain membrane potential. Based on electrophysiological recordings and analyses of RNA-seq datasets, we hypothesize that chronic depolarization stems from diminished abundance of neuronal K+ leak channels that are needed for establishing normal resting membrane potential. In Aim 2, we seek to determine how depolarization potentiates PLCb?IP3R signaling. Our findings suggest that membrane potential dependent regulation of PLCb?PIP2 association and hydrolysis depend on the lipids? acyl side chains. Taken in conjunction with our findings that depolarization promotes IP3 production, we hypothesize that PLCb activity depends on PIP2 side chain identity, and increases in depolarized cells due to the preferential hydrolysis of species with longer and more unsaturated side chains. Successful completion of the proposed studies would demonstrate that IP3R hyperactivation in ADRD neurons is agnostic to the causal mutations ? an insight that speaks to the wide applicability of our findings. We also hope to develop strategies that can be leveraged to selectively change the population of PIP2 species at the plasma membrane in order to correct pathological Ca2+ dyshomeostasis that occurs in ADRD.

Ionic homeostasis in the somatodendritic compartment of neurons is maintained by pumps that utilize the energy of ATP hydrolysis to set the resting membrane potential and prevent toxic elevations in cytosolic [Ca2+]. The relative contributions of glycolysis and mitochondrial respiration in meeting the ATP burden associated with the activity of these pumps is poorly understood. Also unclear is how these two axes of ATP production are perturbed in neurodegenerative disease such as Alzheimer?s and related dementias (ADRDs), which exhibit bioenergetic deficits and ionic dyshomeostasis. In this proposal, we detail our plans to elucidate the relative contributions of glycolysis and mitochondrial ATP synthesis to the somatodendritic ATP burden stemming with depolarization and the release of Ca2+ from the endoplasmic reticulum (ER). By imaging of cytosolic/mitochondrial [Ca2+] and [ATP]/[ADP] ratio in live dissociated Drosophila neurons we have formulated the model that ATP burden of depolarization is so tightly coupled to ATP synthesis such that somatodendritic [ATP]/[ADP] ratio remained stable in depolarized neurons. Our preliminary data also suggest that depolarization elicits ATP production in the somatodendritic compartment without a necessity for concomitant changes in mitochondrial [Ca2+]. Given that in the absence of matrix [Ca2+] elevations mitochondrial ATP production is not potentiated, we hypothesize that glycolysis, rather than OXPHOS, is the favored bioenergetic response to depolarization. We will test this hypothesis in aim 1, and also determine whether the Na+/K+ ATPase and plasma membrane Ca2+ ATPase (PMCA) are the recipients of glycolysis-derived ATP in depolarized neurons. Aim 2 is driven by our preliminary finding that ER Ca2+ release via inositol trisphosphate receptors (IP3Rs) in depolarized neurons desynchronized ATP production from consumption. We will also probe the significance of Ca2+ transfer between the ER and mitochondria, and attendant changes in neuronal bioenergetics in a fly model of tauopathies, which stem from our preliminary findings show that expression of a toxic human Tau variant in fly glutamatergic neurons disrupted Ca2+ transfer between ER and mitochondria, and evoked toxicity that was consistent with Ca2+ dyshomeostasis. In summary, we will use a range of imaging tools and direct measures of bioenergetics to address fundamental questions of metabolic regulation in neurons, and interrogate the mechanism by which these parameters are perturbed in a model of ADRD.