Marco Sardiello - US grants

DESCRIPTION (provided by applicant): Neuronal ceroid lipofuscinoses (NCLs) are among the most devastating inherited disorders of childhood and the most common cause of neurodegeneration in children in the U.S. There is currently no cure for these disorders, and treatments remain largely supportive. NCLs are characterized by the progressive intralysosomal accumulation of ceroid lipopigment; this accumulation is thought to result from defects in lysosomal metabolism or trafficking, but could itself contribute to pathogenesis. We hypothesize that enhancing clearance processes will counteract disease progression in NCLs, even if the primary defect remains uncorrected. To test this hypothesis, we will boost lysosomal function by using genetic (Tfeb) and chemical (trehalose) enhancers of clearance processes in two mouse models of NCLs. In Aim 1 we will generate and characterize transgenic mice that conditionally express either a normal or constitutively active form of the transcription factor EB (TFEB), a master modulator of lysosomal and autophagic pathways. We will also perform molecular genetic analyses that will enable us to map the TFEB targetome, a crucial step in understanding its activities in health and disease. In Aim 2 we will cross these transgenics with Cln3 ex7/8 and Cln8mnd mice, two well-characterized models of NCLs, to investigate the effects of genetic TFEB enhancement on disease course. Importantly, we use models of NCL caused by mutations in two distinct genes in order to rigorously test whether TFEB- mediated clearance can indeed mitigate disease regardless of the underlying defect. In Aim 3 we will study the metabolism of trehalose, a putative inducer of autophagic pathways, which our data indicate activates TFEB; we will test the effect of trehalose on disease course in the two NCL models. Results from this project will show whether genetically or chemically induced lysosomal enhancement is able to counteract disease progression in NCLs and impact health and life span of affected animals. Positive results from this study will be the foundation for the development of a therapy for these devastating disorders. In addition, any knowledge gained from studies on NCLs could, in principle, be applicable to other neurodegenerative disorders caused by defects in lysosome-mediated cellular clearance.

PROJECT SUMMARY/ABSTRACT Neuronal ceroid lipofuscinoses (NCLs or Batten disease) are among the most devastating inherited disorders of childhood and the most common cause of neurodegeneration in children in the U.S. There is currently no cure for these disorders, and treatments remain largely supportive. NCLs are characterized by the progressive intralysosomal accumulation of undegraded cellular material; this accumulation is thought to result from defects in the autophagy-lysosomal pathway, but could itself contribute to pathogenesis. Our data show that deficiency of the juvenile Batten disease protein, CLN3, impairs maturation of a subset of lysosomal enzymes and that trehalose-mediated activation of TFEB, a master regulator of the autophagy-lysosomal pathway, ameliorates disease burden in a mouse model of juvenile Batten disease (JNCL). We propose to study novel mechanisms of TFEB activation that could lead to translational applications for JNCL and other neurodegenerative disorders caused by defects in lysosome-mediated cellular clearance. First, we will test the hypothesis that trehalose- induced lysosomal enhancement corrects defective maturation of lysosomal enzymes in JNCL mice (Aim 1). We will test this hypothesis by conducting experiments of protein maturation and by unbiased proteomic analyses based on the use of a knock-in Lamp1FLAG mouse line we have generated to efficiently isolate lysosomes from mouse tissues. Second, we will test the hypothesis that reduction or inhibition of Akt, a kinase inhibitor of TFEB we have identified, will decrease neuropathology of JNCL mice (Aim 2). We will reduce Akt activity by using two complementary approaches: genetically, by using Akt1-/- mice, and pharmacologically, by using an Akt drug inhibitor that is currently in clinical development. Third, we will test the hypothesis that synergistic pharmacological activation of TFEB by modulation of two orthogonal pathways will result in a greater enhancement of the autophagy-lysosomal system and better reduction of JNCL pathological hallmarks than either strategy alone (Aim 3). This hypothesis is based on our finding that the non-receptor tyrosine kinase, Src, is an essential factor for activation of mTORC1, another kinase inhibitor of TFEB. These studies will pioneer pharmacological activation of TFEB in a model of neurodegenerative disorder. If successful, this study will provide a powerful paradigm of TFEB activation that could lay the foundation for the clinical treatment of Batten disease and, potentially, additional neurodegenerative storage disorders caused by impairment of the autophagy-lysosomal pathway.

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.

        PROJECT SUMMARY/ABSTRACT    Lysosomes control a substantial part of cellular metabolism by acting as the main catabolic hub of the cell and  serving as a platform for the integration of numerous signals that modulate cell death, growth and proliferation.  Most lysosomal functions rely on a set of more than 50 acid hydrolases that degrade a wide variety of  macromolecules. Lysosomal enzymes are trafficked to the lysosome in two stages: transport of the newly  synthesized proteins from the endoplasmic reticulum (ER) to the Golgi complex, and their subsequent  receptor-­assisted transfer from the Golgi to endolysosomal compartments. How lysosomal enzymes are  transported from the ER to the Golgi complex is unknown and, to our knowledge, the simple model of a bulk,  unregulated transportation has never been questioned. We have identified two candidate ER receptors, CLN6  and CLN8, whose deficiency results in altered maturation of lysosomal enzymes and lysosomal storage  disorder-­like diseases. We propose to study how CLN6 and CLN8 function in the pathway of maturation of  lysosomal enzymes. First, we will test the hypothesis that CLN6 and CLN8 directly interact with lysosomal  enzymes and that such interaction is disrupted by disease-­associated mutations on either CLN6/CLN8 or on  the surface of lysosomal enzymes (Aim 1). Second, we will examine the trafficking and maturation of newly  synthesized lysosomal enzymes to identify the exact step that is disrupted by CLN6 and CLN8 deficiency. We  will also define CLN6 and CLN8 functions in vivo by carrying out detailed tissue-­specific analyses of lysosomal  composition in CLN6-­ and CLN8-­deficient mouse lines by LC-­MS/MS-­based proteomics. To this aim, we have  generated a knock-­in Lamp1FLAG mouse line to efficiently isolate lysosomes from the desired tissues (Aim 2).  Third, we will identify the protein domains and motifs that are involved in CLN6/CLN8 interaction and that direct  their sorting across the compartments of the early secretory pathway via COP-­coated vesicles (Aim 3). We will  accomplish our goals with a multi-­disciplinary approach that uses the tools of biochemistry, molecular biology,  cell biology and mouse engineering and we will also develop a new method of in vivo lysosome isolation from  mouse tissues. Our results are likely to have important consequences for our understanding of the  mechanisms governing lysosomal biogenesis and of the molecular pathogenesis of numerous human  diseases. Some of the regulatory mechanisms we uncover may serve in the future as targets for modulating  lysosomal biogenesis in diseases resulting from impaired lysosomal function or in conditions, such as certain  types of cancer, that are characterized by aberrant or unrestricted lysosomal activation. 

Abstract Aging is characterized by the progressive inability of cells, tissues, and organs to maintain their functional integrity and is accompanied by an increased susceptibility to morbidity?the odds to develop neurodegeneration, cancer, or cardiovascular disease increase continuously with age. There is currently no general, actionable strategy to slow down dysregulation of cell and tissue homeostasis in the aging organism. As a result, advances in healthcare in the modern era have paradoxically increased frailty and morbidity among the elderly by increasing lifespan without significantly impacting on age-related homeostasis dysregulation. Owing to a continuously aging world population, there is therefore an urgent and unmet need to identify actionable cellular and molecular targets for the development of treatments aimed at increasing healthspan along with lifespan. Hallmarks of aging are a decline in lysosome-mediated degradation pathways and chromatin dysregulation. This proposal focuses on a transcription factor EB-mediated lysosome-to-nucleus signaling pathway, the modulation of which extends mouse lifespan by 30% in males. We will systematically and mechanistically investigate the three components of this signaling pathway?namely the lysosome, the signaling transducers, and chromatin regulators?during aging by leveraging unique tools that we have developed to study lysosomal content and signaling components. Results from this study will provide the first age-associated atlas of the lysosomal content in the mammalian brain and will pioneer the investigation of lysosome-to-nucleus signaling in aging. Knowledge resulting from this study could lay the foundation for future translational investigations of clinical treatment of aging and age-related neurodegenerative disease.