Yiannis A. Ioannou - US grants

The overall objective of the proposed research is to develop gene transfer strategies designed to overcome the three main obstacles to effective neural gene therapy for neuropathic lysosomal storage disease (LSD) with mental retardation. These obstacles are: 1) global neural delivery, 2) neuronal-specific targeting, and 3) continuous neuronal expression. Relevant to this proposal are out previous observations: 1) that the overexpression of human lysosomal enzymes results in their selective secretion, 2) that covalent coupling of neuronotropic polypeptides to lysosomal enzymes markedly enhanced their uptake and endosomal internalization in cultured neurons, and 3) that the blood brain barrier can be transiently and safely opened for neural delivery of macromolecules or enzymes in cats by infusion of hyperosmolar mannitol. The optimal neuronal-targeted overexpression constructs and neural delivery strategies will be assessed in animal models of human LSD in Project 4 of this Program Project.

The overall objective of the proposed research is to investigate the mechanisms of intracellular cholesterol transport, especially cholesterol egress from the endosome/lysosome. These studies will exploit the experiment of nature, NPC disease, in which at least two different defective genes impair cholesterol egress and result in endosomal/lysosomal cholesterol accumulation and a neurodegenerative phenotype. Recently, the NPC1 gene on chromosome 18 was isolated by positional cloning. The 4.5 kb cDNA encodes a novel 1278 residue polypeptide with several putative membrane spanning regions, which presumably is involved in cholesterol transport (e.g. transporter, pump, docking protein, etc.). Initial studies will determine the subcellular location and topology o the wild-type NPC1 protein. Immunohistochemistry and immunoelectron microscopy using monoclonal and polyclonal antibodies will be used to define the subcellular location of NPC1. The cytosolic or lumenal topology of the five NPC1-predicted hydrophilic loops will be assessed by expression and analysis of a series of NPC1 cDNAs with flag-tags in each of the loops. Our results indicate that the sterol- sensing domain (SSD) of NPC1 is in the same orientation as in HMG-CoA reductase and SCAP, whose topologies are known. The significance of the NPC1 SSD will be further evaluated. In addition, our studies indicate that loop "c" is functionally significant, as constructs containing a FLAG sequence in this loop fail to complement NPC fibroblasts. To identify structure/function relationships, putative functional domains will be expressed and their potential inhibitory effects on the endogenous protein will be assessed. These studies should enhance our understanding of subcellular cholesterol transport and metabolism and provide insights into the pathogenesis of NPC disease. We should emphasize that we have already made significant contributions in the subcellular location (defined the location of NPC1 as the late endosome and not lysosomes) and topology determination of NPC1 (solved the complete topology of this polytopic glycoprotein) and have identified a novel protein targeting motif, a functional domain of NPC1 and a potential 85 kDa protein that associates with NPC1.

DESCRIPTION (provided by applicant): Recently, we have identified a large polytopic membrane protein, NPC1L1 that shares significant homology (42% amino acid identity) with the Niemann-Pick C1 (NPC1) disease protein. NPC1 disease is a severe lysosomal lipidosis in which the egress of cholesterol and other lipids from the endosomal/lysosomal (E/L) system is defective, leading to neurodegeneration and premature demise. The proteins responsible for the two forms of NPC disease, NPC1 and NPC2, were recently identified. Preliminary characterization of these proteins suggests that NPC1 may act as a lipid permease on the membranes of late endosomes, whereas NPC2 is a small, soluble, cholesterol-binding lysosomal protein. The role of our newly identified NPC1 homologue, NPC1L1, is currently unknown and no diseases have been ascribed to its loss of function. Based on the homology between NPC1 and NPC1L1 and our preliminary data, we hypothesize that NPC1L1 has a lipid permease function similar to that of NPC1 but resides in a different subcellular location. Therefore, the overall objectives of the proposed research are to characterize the function of NPC1L1 and determine its role in subcellular lipid and/or cholesterol transport. Efforts will first be directed towards the analysis and characterization of the topology and intracellular location of the NPC1L1 protein to determine the cellular location in which it functions. Solution of its membrane topology will establish the direction of its potential pump activity and also its relationship to the newly identified resistance-nodulation-division (RND) family of eukaryotic permeases. Next, analysis and characterization of the function(s) of the NPC1L1 protein and its potential regulation by subcellular cholesterol and or lipid levels will be carried out to determine whether NPC1L1 exhibits a fatty acid, or other lipid, permease activity. These studies will be accomplished by expression of NPC1L1 in prokaryotes engineered to contain mutations in their endogenous RND permease genes relevant to our studies. Expression in yeast and mammalian cells will also be utilized, as needed, to further characterize the function(s) of this protein. Finally, generation and characterization of an NPC1L1 knockout mouse model should provide us with the necessary data to completely characterize the physiological function of NPC1L1 and its involvement in lipid/cholesterol transport or homeostasis.

[unreadable] DESCRIPTION (provided by applicant): NPC1 disease is a severe lysosomal lipidosis in which the egress of cholesterol and other lipids from the endosomal/ lysosomal (E/L) system is defective, leading to neurodegeneration and premature demise. The protein responsible for the major form of NPC disease, NPC1 is known. Preliminary characterization suggests that NPC1 may act as a lipid permease on the membranes of late endosomes. However, the exact function(s) of NPC1 and its role in NPC disease pathogenesis remain elusive and no form of treatment for this debilitating disorder is currently available. We hypothesize that many NPC1 mutations are disease causing due to the fact that the mutant proteins are unable to mature from the endoplasmic reticulum and are thus unable to be targeted to the late endosome. Pharmacologically relevant chemical chaperones have shown great promise recently in enhancing mutant protein maturation and thus partial rescue of protein activity. We will first establish an assay for detecting the maturation and ER exit of mutant NPC1 proteins. Second, we will use our in-house high throughput facility to identify chemical chaperones that can rescue mutant NPC1 proteins. These molecules will then be evaluated in cell-based assays to determine the level of NPC phenotype correction and assess their utility in future animal and NPC1 patient studies. Successful identification of small molecule chaperones will provide "Proof-of-principle" for this type of approach, and a new avenue of research for NPC1 and other devastating neurological disorders caused by membrane protein misfolding. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): During recent years, the vesicles of the endosomal/lysosomal (E/L) system have emerged as key sites for the regulation of many cellular functions. Their biological importance is exemplified by the occurrence of numerous lysosomal storage diseases (LSDs), each resulting from the deficiency of a single protein in the system, that manifest with severe phenotypes, usually leading to neurodegeneration and early death. How these single gene defects can produce such severe phenotypes is not entirely clear;dissection of the metabolic changes that occur within the E/L system should provide insights towards understanding disease pathogenesis and provide new avenues for screening, early diagnosis, and monitoring of therapeutic approaches. That disease pathogenesis of the LSDs originates in the E/L system presents unique challenges for the characterization of metabolic changes in patients, since circulating biological fluids do not offer a comprehensive view of these changes and obtaining tissue samples on a regular basis is not feasible. We will use a novel approach involving exosomes to identify and characterize the metabolic changes that occur in LSDs. Exosomes are uniquely suited for this type of study because they are secreted by many cell types and are found in biological fluids such as plasma, urine, and cerebrospinal fluid and are derived from the membranes of late endosomes. Thus, they contain a subset of proteins normally found there and can serve as useful source material to characterize the changes that occur within the E/L system as a result of disease. We hypothesize that exosomes derived from disease cells will reflect protein and lipid changes that are specific to the disease. In this respect, exosomes will provide a "fingerprint" or "barcode" unique to each LSD. Here, we propose to: 1) test the hypothesis that exosomes from human disease cells have unique protein and/or lipid identifiers that will distinguish them from those of normal cells and reveal alterations of specific metabolic pathways. We will map these pathways and validatelevaluate these changes in vitro and in vivo. 2) Test the prediction that changes in glucose metabolism correlate with NPC1 disease severity and can be used to monitor disease progression. In short, this new approach is a new paradigm in metabolic analysis and will facilitate the efficient discovery/characterization of altered LSD metabolic pathways and provide us with the next step in understanding lysosomal storage disease pathogenesis.

DESCRIPTION (provided by applicant): NPC1 disease is a severe lysosomal lipidosis in which the egress of cholesterol and other lipids from the endosomal/lysosomal (E/L) system is impeded, leading to severe neurodegeneration and premature demise. At present there are no treatment options for patients with NPC1 or other lysosomal storage disorders (LSDs) that present with neurodegenerative phenotypes, mainly due to the inability of corrective proteins to cross the blood-brain barrier. Therefore, a new paradigm is needed to address the large family of LSDs with neuropathology. We hy7pothesiae that suppressor proteins can be identified for the LSDs and that the expression of these proteins can be modulated via small chemical molecules that can gain access to the central nervous system and limit the severity of these disorders. In essence, this approach bypasses the defective lysosomal protein and therefore we have termed it "Orphan receptor bypass therapy" or "ORByT". We will first carry out a high-throughput screen to identify pharmacologically active, for ORByT-relevant small molecules for NPC1 disease. Using Rab9 as a "suppressor" candidate for NPC1, we will use the recently established high throughput screening pharmacologically active for NPC1 disease. Second, we will evaluate candidate molecules for the therapeutic potential. Small molecule compounds identified in Aim 1 will be tested for their ability to correct the NPC1 lipid transport block phenotype. Those compounds that are confirmed to restore lipid transport in NPC cells will be evaluated in vivo using an NPC mouse model to determine their therapeutic potential prior to further development to improve their potency, toxicity profile, and brain delivery. Successful identification of small molecules will provide "Proof-of-principle" for the ORByt strategy and a new approach to therapy for NPC1 and other devastating neurological LSDs. PUBLIC HEALTH RELEVANCE: Diseases that affect the brain are currently difficult if not impossible to treat. We are developing a novel approach to treat devastating genetic diseases with neurologic involvement that are currently untreatable.

A major goal in biomedicine is the identification of disease biomarkers in biological fluids that can be used in disease screening, early diagnosis, and monitoring of therapeutic approaches. The development of such biomarkers for lysosomal storage diseases (LSDs) has not been successful thus far. However, the availability of these biomarkers is needed and absolutely necessary to evaluate the safety and efficacy of disease interventions, especially for those LSDs with a neurodegenerative phenotype. Most biomarker studies have focused on comparing plasma proteins between a normal and a disease state. The major problem with this approach is the complexity of the plasma proteome, i.e. the proteins that can be found circulating in plasma. We have established a novel approach for identifying disease biomarkers in plasma and other fluids such as urine. This unique approach eliminates the difficulties inherent in analysis of the plasma proteome by first identifying potential biomarker proteins in a less complex system: the exosome. Exosomes are uniquely suited for the identification of the LSD disease biomarkers. They are small vesicles derived from the membranes of late endosomes that contain a subset of proteins normally found in endosomes. Many cells secrete exosome and thus it is likely that the proteins contained within them can be isolated and identified from cellular secretions. Because LSDs often present with abnormalities in endosomal/lysosomal compartments, we hypothesize that exosome will reflect protein and lipid changes specific to the disease defect exhibited by the cell from which they are derived/isolated. Here, we propose to: 1) test the prediction that exosomes from Neimann-Pick C1 cells will have unique protein and/or lipid identifiers (biomarkers) that will distinguish them from those of normal cells; and 2) evaluate whether these potential biomarkers are detectable in biological fluids including plasma, urine and CSF; and 3) test the prediction that changes in glucose metabolism correlate with NPC1 disease severity and can be used to monitor disease progression. In short, this new approach could facilitate the efficient discovery of LSD biomarkers and provide us with the next step in developing therapies that can be evaluated a in meaningful way in clinical trials.

Niemann Pick Type C (NPC) is a rare neurodegenerative lipidosis that is characterized by lipid storage in the endosomal/lysosomal system. Current treatment modalities for this devastating disease are currently non-existent due to the severe obstacles associated with accessing the central nervous system with proteins or genes. The majority of mutations causing NPC disease are missense mutations. Studies have shown that some of these mutations, including the most prevalent I1061T allele, result in the production of proteins that may be functional but are targeted for degradation due to misfolding. Furthermore, we and others have observed that overepression of the mutant proteins can rescue the disease phenotype, suggesting that upregulation of the endogenous NPC1 mutant protein is a new drug treatment modality for the disorder. We hypothesize that small chemical molecules that can increase the expression of NPC1 can be identified and thus propose the following specific aims: 1) High throughput screening for identification of small molecules that upregulate NPC1 expression, utilizing the MLSCN (Molecular Libraries Screening Center Network) compound collection for drug-like small chemical molecules, and 2) Characterize and confirm positive hits, using in vitro assays, for their therapeutic potential in the treatment of NPC1 disease. These studies will result in the identification of candidate compounds that will ultimately be evaluated in vivo in an NPC mouse model to determine their therapeutic potential and future drug development.

DESCRIPTION (provided by applicant): Treatment modalities for lysosomal stage diseases (LSDs) with neuropathology such as Niemann-Pick Type C Disease (NPC) are currently non-existent due to the severe obstacles associated with accessing the central nervous system with proteins or genes, Also, these rare orphan disorders do not attract the interest of pharmaceutical companies, further contributing to the lack of prospects for any form of treatment of therpy. We have developed a new paradigm, termed "Orphan Receptor Bypass Therapy" (ORByT), to address these disorders. This paradigm posits the existence of endogenous "suppressor" proteins whose expression can dramatically improve LSD pathogenesis. The goal is the identification/discovery of small chemical compounds that can modulate the expression of these proteins and provide a new treatment modality for these devastating disorders. We have previously shown that the small GTPase protein Rab9 meets this requirement and can act as suppressor of the NPC phenotype. Thus, we propose to first carry out: * High throughput screening for identification of small molecules that upregulate Rab9 expression, utilizing the MLSCN (Molecular Libraries Screening Center Network) compound collection for drug-like small chemical molecules, and * Characterize and confirm positive hits, using in vitro assay, for their therapeutic potential in the treatment of NPC1 disease, * These studies will result in the identification of candidate compounds that will ultimately be evaluated in vivo in an NPC mouse model to determine their therapeutic potential and future drug development.