Alessandra d'Azzo - US grants

Lysosomal storage diseases (LSDs) comprise a group of over 40 inherited disorders of metabolism caused by single gene mutations affecting lysosomal hydrolases. Collectively, their incidence is 1:5000 live births. While each disease has a specific clinical phenotype, they all lead to the accumulation of partially metabolized carbohydrate or lipid substrates in Lysosomes of many cells; which causes the pathologic symptoms. In general, the severity and age of onset of the clinical syndromes are determined by the level of residual enzyme activity, implying that even modes increases, if realized early in life, might affect cure. Many of the severe forms of LSDs result in marked neurologic deterioration and mental retardation for which no successful therapy is currently available. Thus, there is a compelling need to develop new approaches for treatment of LSDs with central nervous system (CNS) involvement. A key feature for therapy of these diseases is the ability of cells from both unaffected individuals and from LSD patients, to take up secreted, extracellular Lysosomal hydrolases via receptor-mediated endocytosis and to route them to Lysosomes, where they function normally. The overall goal of this grant is to use murine models for galactosialidosis and GMI-gangliosidosis to answer the fundamental question of whether a homogeneous population of overexpressing bone marrow (BM) progenitor cells can serve as a source of corrective enzyme to treat LSDs. Dr. D~Azzo will use BM from transgenic mice, overexpressing the appropriate deficient enzyme in erythroid or macrophage/monocyte lineages, for delivery of corrective protein to systemic tissues; and the CNS, of affected animals. While erythroid cells, because of their numbers, may provide a significant source of corrective enzyme to visceral organs, they hypothesize that the primary role of macrophages will be to carry the enzyme across the blood-brain barrier into the CNS. This strategy should, in principle, overcome the technical problems associated with vector-mediated gene transfer and ectopic expression. Using this approach, they will evaluate the potential of engineered BM-derived cells: 1) to serve as sources of corrective enzymes: 2) to compare the extent of correction in different organs to determine the age of recipients at which transplantation produces any benefit; 3) to confirm the idea that precursor cells of the macrophage/monocyte lineage can cross the blood-brain barrier early in life and thereby protect the CNS; and 4) to predict the longevity of efficacious treatment. The investigators postulate that results from these studies will provide crucial information for the design of expression cassettes for future use in viral or nonviral vectors, and will have direct applicability to the treatment of LSD patients. Ultimately, this approach may have general relevance to the development of gene therapy for other diseases.

DESCRIPTION (provided by applicant): The scope of this proposal is to study the function of mammalian lysosomal neuraminidase (NEU1) in normal cell metabolism, and the consequences of its loss in human diseases. NEU1 belongs to the ubiquitous superfamily of sialidases. Mammalian neuraminidases include cytosolic, lysosomal, and plasma membrane isoforms, and clues about the physiologic roles of these hydrolases, in particular NEU1, have emerged only recently. NEU1 initiates the hydrolysis of sialo-glyconjugates by removing terminal sialic acid residues. The enzyme is unique among sialidases in that it must be associated with protective protein/cathepsin A (PPCA) for intracellular routing and lysosomal activation. Finally, NEU1 is linked to 2 neurodegenerative diseases of metabolism: Sialidosis is caused by structural lesions in NEU1, and galactosialidosis (GS), a combined deficiency of NEU1 and (3-galactosidase, is caused by the absence of PPCA. The proposed studies are based on 3 Specific Aims. In Aim 1, we will investigate the structure- function relationship between NEU1 and PPCA. The 3D structure of the PPCA precursor will be used to target mutagenesis of potential contact sites between the 2 proteins and NEU1 mutations identified in patients with sialidosis. We will also use overlapping peptides that span the full-length PPCA and NEU1 to identify domains crucial for NEU1/PPCA interaction, intracellular transport, and activation. These biochemical studies will be coupled to determine the 3D structure of the Neu1/PPCA complex. In Aim 2, we will compare the characteristics of Neu1~*~ mice and PPCA^~ mice to determine the molecular bases of sialidosis and GS and to identify yet unknown Neu1 functions in normal cell physiology. In Aim 3, we will implement various enzyme replacement therapy approaches in both models to assess the correction of the systemic phenotypes in these diseases. We are in position to develop this line of investigation, because we have established appropriate genetic and biochemical systems for the proposed studies and can rely on the expertise of an outstanding structural biologist for the crystallography part of the project. Our overall goal is to gain a broader understanding of NEU1 function in normal physiology and in the pathophysiology of the neurodegenerative diseases sialidosis and GS. These diseases affect primarily infants and children. Findings from these proposed studies should increase our knowledge about NEU1 function and improve the design of future therapies for pediatric patients with these catastrophic diseases.

DESCRIPTION (provided by applicant): Lysosomal storage diseases (LSDs) comprise a large group of monogenic, metabolic disorders caused by deficiency of lysosomal hydrolases. Accumulation of intermediate metabolites in lysosomes affects systemic organs and the central nervous system (CNS). However, it is still largely unknown how an impaired lysosomal system influences cellular homeostasis and ultimately leads to organ dysfunction. The poor understanding of pathogenesis adds to the challenge in finding a treatment for LSDs that would be effective for both systemic and CNS disease. The long-term objective of the proposed research is to achieve a comprehensive evaluation of the CNS dysfunction that occurs in galactosialidosis (GS; protective protein/cathepsin A [PPCA deficiency] and GM1-gangliosidosis (GM1; beta-galactosidase [b-gal deficiency]). This knowledge will allow us to assess the feasibility, limitations, and effectiveness of specific therapies. To accomplish our goal, we have developed mouse models of cells that overexpress the correcting enzyme in a lineage specific manner effectively restore systemic organ function in GS mice, but only could partially ameliorate severely affected area of the CNS, such as the cerebellum. As a step toward developing more effective therapies for the CNS disease, we propose the following aims: Aim 1. We will complete our ongoing studies of the phenotypes of GS and GM1 mice so that the pathogenesis of neurodegeneration can be elucidated. The combined use of histological staining and immunocytochemistry with antibodies to cellular and molecular markers for neurons and glia will enable us to determine the status and characteristics of cells at disease sites, and during disease progression. Aim 2. We will generate transgenic mice in which expression of the therapeutic proteins (PPCA or beta gal) is targeted to neurons and glia by using cell-specific promoters. These transgenic models will be crossed with the respective null mice. This approach will allow us to assess the efficacy of neural cells to produce and secrete PPCA and beta-gal and of affected neighboring cells to take up the enzymes. Aim 3. We will perform ex vivo gene transfer using genetically modified, deficient BM cells for syngeneic BMT of GS and GM1. This approach will create the most realistic scenario to gene therapy in the patients. In parallel, we will determine the efficacy of in vivo administration of a recombinant adeno-associated virus (rAAV) expressing the enzymes in correcting difficult to treat CNS regions. These combined studies should provide a solid base for the development of feasible cure for these diseases, leading to clinical treatments that have a greater chance of success and a lower risk of adverse effects.

DESCRIPTION (provided by applicant): The identities of the ubiquitin-protein ligases that regulate myogenesis are largely unknown. However, we have identified a gene that encodes a novel suppressor of cytokine signaling (SOCS) protein, 077, which is expressed exclusively in striated skeletal and cardiac muscle. Ozz interacts with the Elongin B/C complex and, in turn, promotes the assembly of a RING-type ubiquitin ligase whose putative substrates are a-catenin and embryonic myosin heavy chain (MyHCemb). The expression of Ozz is regulated during myogenesis; in myoblasts, the proteasome rapidly degrades the phosphorylated form of Ozz, and in myotubes, the dephosphorylation of a tyrosine residue within the SOCS box stabilizes Ozz and enables it to bind the other components of the Ozz-E3 ligase complex. Our hypothesis is that the Ozz-E3 ligase complex plays a crucial role in the regulation of the exchange of developmental isoforms of MyHC for the adult forms and in the assembly of sarcomeres into myofibrils stabilized by a-catenin; both processes permit differentiation and remodeling of muscle fibers. The objective of this proposal is to test this hypothesis by investigating the mechanisms that regulate the expression of Ozz protein, the function of 077 during myogenesis, and the manner in which Ozz regulates the activity of the E3 ligase complex that it specifies. In the first specific aim, we will characterize the assembly and regulation of the endogenous Ozz-E3 ligase complex during the differentiation of muscle cells in vitro. Several molecular and biochemical approaches will be used to purify the endogenous Ozz-E3 ligase complex at different stages of muscle-cell differentiation and to analyze the ubiquitination activity of Ozz-E3 ligase. In the second specific aim, we will study in vivo the function of Ozz in myogenesis and muscle homeostasis. We have generated a mouse strain carrying a null mutation at the Ozz locus. These mice will facilitate the detailed analysis of the function of Ozz-mediated ubiquitination during myogenesis. We will focus specifically on the potential role of the Ozz-E3 complex in the regulation of sarcomere remodeling and maturation during development, growth, and aging of striated muscle. Understanding the role of the Ozz-E3 ligase complex will provide new insight about the mechanisms that control the early stages of muscle differentiation and regeneration; elucidation of these regulatory mechanisms may also provide new insight regarding treatment for cardiac and skeletal myopathies.

DESCRIPTION (provided by applicant): The goal of the proposed research is to achieve in-depth understanding of the molecular mechanisms underlying the CNS pathogenesis in neurodegenerative GM1-gangliosidosis (GM1). This lysosomal storage disease (LSD) is caused by deficiency of lysosomal 2-galactosidase (2-gal) that results in impaired lysosomal degradation and storage of GM1-ganglioside (GM1). Gangliosides are basic components of cell membranes, and GM1 is the primary ganglioside in the vertebrate brain. The accumulation of GM1 contributes to disease pathogenesis, but the molecular pathways involved remain largely unexplored. Our successful generation of 2- gal-/- mice, an animal model that closely resembles GM1 in humans, has facilitated studies that would be difficult or impossible to undertake in children. During the last granting period, we initiated a comprehensive analysis of neuronal cell death in the 2-gal-/- mouse model. Findings from those studies have contributed insight about the role of GM1 in normal cell metabolism and enabled us to develop the following 2 Specific Aims for this proposal. In Aim 1, we will elucidate the pathologic consequences of GM1 accumulation at the level of the ER membrane that leads to Ca2+release and, in turn, activation of the UPR and neuronal cell death in 2-gal-/- mice. We will determine whether GM1 directly influences the biochemical properties of the ER-specific Ca2+ pump and channels that control intracellular Ca2+ levels. We will apply sophisticated in vitro and in vivo assays to verify a potential physical interaction of GM1 with these ER membrane proteins. In Aim 2 we propose to investigate whether a cross-talk between the ER and the mitochondria occurs during the neuronal apoptosis mediated by the disruption of intracellular Ca2+ homeostasis in the 2-gal-/- mice. These studies are based on the hypothesis that excessive release of Ca2+ from the ER provokes Ca2+ imbalance in the cytosol, which, in turn, may impact on other organelles, in particular the mitochondria. Analyses of mitochondrial morphology and function will be paralleled by direct measurement of intracellular Ca2+ trafficking. We will also examine whether GM1 directly affects mitochondrial membrane permeability by testing if this ganglioside is incorporated in the mitochondrial membranes and, hence, perturbs mitochondrial integrity. Considering the pivotal role assigned to the Bcl-2 family of proteins in both ER- and mitochondria-mediated apoptosis, and their interplay with both ER and mitochondrial membrane components we will also determine whether GM1 may act upstream of ER Ca2+ release by activating these apoptogenic factors. RELEVANCE: GM1 is a catastrophic neurodegenerative disease that affects infants and children. We are in the position to gain full understanding of the events in GM1 that cause cell death in the brain. The proposed studies may also reveal basic biological processes controlled by the molecules that are accumulated in GM1. This knowledge is essential for designing new therapies for children with GM1 and possibly those with other LSDs.

DESCRIPTION (provided by applicant): The goal of the proposed research is to achieve in-depth understanding of the molecular mechanisms underlying the CNS pathogenesis in GM1-gangliosidosis, a catastrophic neurodegenerative lysosomal storage disease (LSD) caused by deficiency of 2-galactosidase (2-gal). In this disease massive and progressive accumulation of GM1-ganglioside (GM1) particularly in the CNS is thought to play a major role in disease pathogenesis, albeit the molecular pathways involved remain largely unexplored. Gangliosides are sialic acid- containing glycosphingolipids (GSLs) particularly abundant in the nervous system. As key modulators of intracellular Ca2+ flux, they have been implicated in cellular processes downstream of Ca2+ signaling. Changes in their chemical composition and concentration can therefore alter normal cell function and lead to cell death. Our successful generation of a 2-gal-/- mouse model that closely resembles the human disease has enabled us to initiate a comprehensive analysis of the molecular bases of neurodegeneration characteristic of this disorder in children. We have demonstrated that in these mice excessive intracellular concentration of GM1 induces apoptosis by depletion of endoplasmic reticulum (ER) Ca2+ stores and activation of an unfolded protein response (UPR). Given the spatial interplay between the ER and the mitochondria and the role of Ca2+ in conveying apoptotic signals emanating from these compartments, we now plan to investigate whether GM1 can induce both ER stress- and mitochondria-mediated apoptosis by influencing Ca2+ homeostasis in these organelles. The studies put forward in the two Aims of this proposal are designed to elucidate the following points. 1) We will examine the potential effect(s) of increased levels of GM1 on ER membrane channels and pump that regulate intracellular Ca2+ concentration. 2) We will focus on the role of GM1 on specific membrane components of the mitochondria-associated membranes or MAMs, the sites of juxtaposition between ER and mitochondrial membranes that regulate the kinetics of Ca2+ flux between these organelles. 3) We will study the downstream effects of ER Ca2+ depletion on the mitochondria. Considering the role of the Bcl-2 family of proteins in both ER- and mitochondria-mediated apoptosis, and their response to Ca2+ levels in these organelles, 4) we will explore a putative, additional involvement of these proteins in the neurodegenerative events characteristic of GM1-gangliosidosis. PUBLIC HEALTH RELEVANCE: GM1-gangliosidosis is a catastrophic neurodegenerative disease that affects infants and children for which there is no treatment. We are in the position to gain full understanding of the events in this disease that cause cell death in the brain. We are confident that these studies will enable the identification of novel mechanisms of pathogenesis and may reveal basic biological processes controlled by GM1. This knowledge is essential for designing new therapies for children with GM1-gangliosidosis and possibly those with other GSL storage diseases.

DESCRIPTION (provided by applicant): Genetic lesions affecting lysosomal metabolism alter cell and tissue homeostasis and affect a multitude of physiological processes, as documented by the complex multiorgan phenotypes of lysosomal storage diseases (LSDs). The long-term scope of this study is to gain insight into the molecular bases of the pathogenesis of sialidosis, severe neurodegenerative LSD linked to the deficiency of the lysosomal sialidase NEU1. NEU1 initiates the hydrolysis of sialo-glycoconjugates by removing their terminal sialic acids. The loss of NEU1 activity results in oversialylation of its substrates, which in turn, can change their biochemical properties and function. The focus of this application is to dissect the role of NEU1 as a newly identified negative regulator of the physiological process of lysosomal exocytosis (LEX) and to test the hypothesis that excessive LEX, resulting from NEU1 loss of function, is the common pathogenic determinant of the systemic and neurological abnormalities that are characteristic of sialidosis. Key to this study is the finding that NEU1 controls the extent of LEX by modulating the sialic acid content of one of its substrates, LAMP1. In Neu1r/r cells, lysosomes that are tagged with oversialylated Lamp1 are more prone to dock at the plasma membrane and engage in LEX upon calcium influx. We hypothesize that the excessive release of lysosomal contents into the extracellular space changes the composition of cells' plasma membranes and the extracellular matrix with deleterious consequences on the integrity and function of many organs. We propose to test this paradigm in a series of studies in mutant mice, namely Neu1r/r mice, an accurate preclinical model of sialidosis. In Aim 1, we will identify the biochemical and molecular effectors downstream of excessive LEX that cause the progressive expansion of muscle connective tissue and consequent muscle degeneration in Neu1r/r mice. In Aim 2, we will identify those factors that cause the progressive formation of amyloidogenic bodies in the hippocampal region of the Neu1r/r brain, which resembles the Alzheimer diseaserlike neurodegenerative phenotype. In Aim 3, we propose a series of biochemical approaches to determine whether LAMP1 plays a primary role in trafficking lysosomes to the plasma membrane and, if so, how this process occurs. We believe that the experiments proposed herein will give insight into previously undiscovered functions of lysosomal NEU1 beyond basic lysosomal degradation. We also expect that the results from these studies will highlight new aspects of the pathogenesis of sialidosis that have the potential to advance the development of alternative therapies for this devastating childhood disease.

DESCRIPTION (provided by applicant): This project is focused on the development of gene therapy for galactosialidosis (GS), an autosomal recessive lysosomal storage disease belonging to the glycoproteinosis subgroup. GS is caused by a primary defect in the lysosomal carboxypeptidase, protective protein/cathepsin A, that results in the secondary combined deficiency of ?- galactosidase and neuraminidase. The initial participants targeted for gene transfer will be individuals with the late, infantile phenotype in that survival into childhood and adolescence is common without accompanying neurological signs allowing reversal of the somatic phenotype to be of high potential clinical relevance. We have made the following advances which support the application of gene transfer into the liver for the treatment of GS: 1) developed a novel, rAAV self-complementary vector encoding PPCA and shown that it can correct phenotype in the GS (PPCA-/-) mouse model; 2) demonstrated that an analogous rAAV vector encoding human coagulation Factor IX restores FIX production in participants in a clinical trial with hemophilia B; 3) implemented a GMP compliant production and purification methodology for the hPPCA vector that we anticipate using in our clinical trial; and 4) defined with the FDA the remaining pre-clinical studies necessary to obtain an IND for a gene transfer trial for GS. In Specific Aim 1, we propose to produce a clinical lot of our self-complementary, rAAV hPPCA vector and in Specific Aim 2 to perform the final pre-clinical dose finding, biodistribution and toxicity studies in the PPCA mouse model. In Sub-Aim 3.1, we have begun to identify and characterize the clinical features and demography of patients with galactosialidosis with a goal of identifying a subgroup with the late infantile form for a gene transfer trial. Sub-am 3.2 is to perform the proposed gene transfer trial for participants with galactosialidosis. The tril will be monitored for evidence of gene transfer, for biochemical evidence of PPCA production and for correction of the clinical manifestations of the disorder.

ABSTRACT Changes in the sialic acid content of neural glycoproteins have been reported in neurodegenerative and neuroinflammatory diseases, including Alzheimer?s disease (AD). However, how these changes govern neuropathogenic processes remains poorly understood. The central hypothesis of these studies is that impaired de-sialylation affects the physiological and functional properties of specific sialo-glycoproteins in neurons and microglia, initiating a pathogenic cascade leading to neurodegeneration and neuroinflammation. We will test this hypothesis from the angle of the sialic acid-cleaving enzyme NEU1, a lysosomal sialidase, using a mouse model of the lysosomal storage disease, sialidosis, as an experimental tool. We have shown that Neu1?/? mice develop a hippocampal neuropathology with features of an amyloidosis, including accumulation of an oversialylated APP, a substrate of NEU1, and widespread neuroinflammation. Ablating NEU1 expression in a canonical model of AD (5xFAD) leads to exacerbation of the amyloidosis phenotype, while increasing NEU1 expression, via a gene therapy approach, reduces plaque formation. These findings implicate NEU1 as the potential underpinning cause, although the mechanisms linking the cause to the effects still need to be deciphered. The studies proposed in this application are directed to closing this knowledge gap, because of these previous observations and new compelling preliminary data. Besides APP, we have identified additional potential substrates of NEU1, including those that participate directly in the amyloidogenic process (BACE1 and nicastrin) and those that are responsible for neuroinflammatory processes, such as microglial phagocytosis (CD68 and TREM2). We will explore the idea that by controlling the levels of sialic acids on these specific AD-related glycoproteins in neurons (Aim 1) and microglia (Aim 2), NEU1 functions as a crucial regulator of key biological processes that maintain brain homeostasis. In Aim 1, we will examine the effects of altered sialylation on APP, and its sialylated proteolytic enzymes, BACE1 and nicastrin, in Neu1?/? mice and in established AD mouse models crossed into the Neu1?/? background. We will use a combination of subcellular fractionation methods, immunofluorescent microscopy, and high-resolution 3D imaging to determine alterations in intracellular distribution, processing and turnover rate of these glycoproteins. In Aim2, we will study how impaired de-sialylation of microglial receptors and signaling proteins affects their subcellular localization, turnover rate, and the cellular functions they are known to control. To focus our studies exclusively on microglia we will cross the above-mentioned mice with a mouse model that expresses GFP almost solely in microglia. Overall the experimental design of this grant application will enable us to elucidate mechanisms of pathogenesis leading to neuronal and microglial dysfunction with direct translational implications for patients with AD.