Vivian Hook - US grants

9319439 Hook Cell-cell communication in neuroendocrine systems utilizes small, potent, peptide hormones and neurotransmitters that are secreted from nerve cells. These peptide hormones and peptide neurotransmitters are synthesized as large protein precursors or prohormones that require specific proteolytic processing to yield the smaller physiologically active peptides. The prohormone processing enzymes represent an important point of regulation since it is this step where the inactive prohormone is converted to the smaller active forms. Active processing enzymes are prerequisite for the formation of potent functionally relevant neuropeptides. Dr. Hook i examining the role of a 70 kDa aspartyl protease in manufacturing enkephalin and neuropeptide Y peptides from its larger precursor products. These two peptides serve very important functions. Enkephalin is an endogenous opiate that plays a critical role in controlling pain and Neuropeptide Y is shown to be involved in feeding, shifting of biological rhythms, hypotension and reproduction. Dr. Hook will use state-of- the-art technologies to purify and characterize this enzyme. She will then generate large quantities of the prohormone to establish that the newly purified protease produces enkephalin and neuropeptide Y. Finally, Dr. Hook will use a technique called directed mutagenesis to determine the specific sites of action of the 70 kDa aspartyl protease. The results from this work is fundamental for understanding the molecular basis of precursor processing in generating active neuropeptides as well as contributing to a more general understanding about the control of biologically important messengers. The greatest promise of this basic research on how peptides are normally regulated may be in ultimate treatment of drug addiction since it appears that the regulation of the levels and activities of enkephalin may be involved. ***

Proteolytic processing of inactive proenkephalin is required to produce active endogenous opioid enkephalin peptides. the goal of this proposal is to examine the relative roles of several proenkephalin (PE) processing enzymes -- (a) a novel 'prohormone thiol protease', (b) PC2 and PC3 prohormone convertases, and (c) carboxypeptidase H -- to determine which of these is the primary and rate-limiting enzyme in enkephalin biosynthesis. We have identified a novel 'prohormone thiol protease', PTP, that is involved in PE processing. We also demonstrate PC2 and PC3 (PC = prohormone convertase) activities in enkephalin-containing chromaffin granules, implicating a role for these subtilisin-like proteases in PE processing. with these findings, specific aims 1 and 2 will compare PTP, PC2, and PC3 activities with respect to the order to PE cleavages and products generated, as well as determine enzyme kinetic constants to assess substrate affinity and maximal enzyme activity. These in vitro studies of PE processing enzymes will utilize as substrate recombinant PE generated by high level expression in E. Coli. Expression of the PE cDNA provides milligram amounts of PE that allows definitive identification of PE products by peptide microsequencing and amino acid compositional analyses. Importantly, adequate levels of recombinant PE also allows analysis of the kinetic constants, Km and Vmax, near in vivo levels of PE. Subsequent to endoproteolytic processing of PE, carboxypeptidase H is required to remove COOH-terminal basic residues. The processing of the CPH precursor, pro-CPH, like other proteolytic cascades (such as in blood clotting) may be catalyzed by a protease involved in one of the first steps of processing. Therefore, specific aim 3 will assess processing of pro-CPH by purified PTP, PC2, and PC3. To obtain a more complete understanding of CPH biosynthesis, specific aim 3 will also characterize multiple CPH mRNAs by RT-PCR (reverse transcriptase-polymerase chain reaction), DNA sequence analysis of CPH cDNAs, and limited DNA sequence analysis of genomic clones to assess if CPH transcripts arise from differential transcriptional initiation sites, alternative RNA splicing, or choice of polyadenylation site(s). These studies will provide a comprehensive study of the roles of PTP, PC2, PC3, and CPH in proenkephalin processing, and will indicate the rate-limiting PE processing enzyme whose regulation should be investigated in future investigations. These studies may lead to identification of target enzyme(s) for development of therapeutic drugs that modify the opiate system through PE processing.

Peptide neurotransmitters are synthesized as protein precursors that require proteolytic processing to form the active neuropeptides. The goal of this proposal is to obtain biochemical and molecular characterization of two components -- a novel 'prohormone thiol protease' (PTP) and its endogenous alpha1-antichymotrypsin-like (ACT-like) protease inhibitor -- that may be involved in enkephalin and tachykinin precursor processing. Only the mature processed enkephalin and substance P peptides, and not the precursors, function as neurotransmitters. Therefore, investigation of PTP as a major processing enzyme and its regulation by ACT-like protease inhibitor is crucial for understanding molecular mechanisms of neurotransmission. Biochemical assessment of PTP's precursor selectivity, cleavage site specificity, and in vitro processing of recombinant proenkephalin (PE) and beta-protachykinin (beta-PT) will be conducted. High level expression of PE and beta-PT in E. coli provides adequate quantities of precursors needed for in vitro PTP kinetic studies with PE and beta-PT near their in vivo concentrations. Importantly, recombinant precursors allow identification of processing products by peptide microsequencing analyses. Molecular cloning will utilize PTP's partial NH2-terminal amino acid sequence in multiple cloning approaches: (a) RT-PCR and RACE PCR (polymerase chain reaction) with complementary degenerate oligonucleotides, combined with nested PCR, to generate a partial PTP cDNA for screening cDNA libraries, (b) use of complementary oligonucleotides for cDNA library screening, (c) expression cloning using an antibody that recognizes PTP. The PTP cDNA will allow comparison of the primary structure of PTP with other proteases. In the second part of this project, inhibition of PTP by alpha1-ACT-like protein will be investigated with respect to inhibitory potency, interactions with PTP, and microsequencing. Colocalization of PTP and ACT-like inhibitor in neuroendocrine cells will be assessed by immunofluorescence and immunoelectron microscopy. In the last part of this project, studies will assess the functional importance of PTP in cellular PE processing by inhibiting PTP with a potent cysteine protease inhibitor, and by inhibiting PTP expression with antisense oligonucleotides. These studies will provide significant advances in understanding how PTP and ACT-like inhibitor(s) are involved in peptide neurotransmitter production. Knowledge obtained will provide insight into new therapeutic strategies for normal and diseased brains.

This laboratory's research program will investigate the biochemistry and molecular biology of proenkephalin (PE) processing enzymes required to synthesize active enkephalin opiate peptides. Specifically, the research plan will determine the relative importance of the novel 'prohormone thiol protease' (PTP) that is inhibited by endogenous alpha1-antichymotrypsin (ACT), as well as the roles of PC 1/3 and PC2 (PC - prohormone convertase), in PE processing. The first part of the project will compare PTP, PC 1/3, and PC2 processing of recombinant PE, obtained by high level expression in E. coli, with respect to PE products, cleavage sites, kinetics, and theoretical activities of each enzyme in vivo. Comparison of precursor selectivity of PTP, PC 1/3 and PC2 in processing recombinant pro-neuropeptide Y and beta-protachykinin precursors into NPY and substance P neuropeptides may suggest selective PE processing enzymes. Structural characterization of PTP through molecular cloning will test the hypothesis that PTP may be a unique cysteine protease. Preliminary NH2- terminal sequencing and a partial PTP cDNA indicate a novel sequence. Cloning of a full-length PTP cDNA will be accomplished by obtaining partial clones through RT-PCR (reverse transcriptase-polymerase chain reaction), and 5'- and 3'-RACE (rapid amplification of cDNA ends); partial clones will be used to screen cDNA libraries. Secondly, the project will characterize inhibition of PTP by endogenous alpha1-antichymotrypsin (ACT) with respect to Ki, complex formation, and biochemical properties. Full- length ACT cDNA clones will be obtained by screening cDNA libraries with partial clones. Production of specific PTP and ACT antibodies will allow studies of ACT and PTP colocalization in enkephalin-containing brain regions and endocrine tissues by immunofluorescence cell staining, immunoelectron microscopy, and western blots. Thirdly, the ability of the potent PTP inhibitor Ep453 to reduce PE processing in chromaffin cells will be assessed by pulse-chase labeling of PE and by determining cellular levels of (Met)enkephalin by RIA. Overall these studies will establish the relative importance of PTP, compared to PC 1/3 and PC2, in PE processing.

tissue /cell culture; chromaffin cells; biomedical facility; PC12 cells; transfection; animal tissue;

tissue /cell culture

DESCRIPTION (provided by applicant): Elucidation of the mechanisms of action by which drugs of abuse modify brain function requires knowledge of fundamental neuroscience with respect to how individual molecular components control neurotransmission and behavior. This program for predoctoral and postdoctoral fellows will provide research training in relevant neuroscience for understanding how drugs of abuse modify brain function. This interdisciplinary training program by the excellent faculty of the Graduate Program in Neurosciences at the Univ. of Calif., San Diego (UCSD) will span protein chemistry for synthesis of opioid receptor ligands, biochemistry and macromolecular structure of protein components involved in neurotransmission, and pharmacology of receptor-mediated signal neuronal systems in pain and behavior that are related to the actions of drugs of abuse. The integration of analyzing brain function at several levels-protein biochemistry as sites of drug interactions, molecular biology, neurobiology, physiology, and neuronal circuits in pain and behavior- is required for drug abuse research to elucidate how these chemical drugs modify regulatory receptors, enzymes, and other components that regulate neurotransmission and behavior. This training program will instruct six predoctoral students and two postdoctoral trainees in multidisciplinary and integrated research areas in neuroscience for the goal of enhancing our knowledge of how drugs of abuse modify brain function. Each trainee will be mentored by a primary NIDA-funded faculty advisor of this training program. Incorporation of modern and interdisciplinary approaches will be achieved with a secondary faculty mentor with expertise in current neuroscience disciplines that will enhance drug abuse research. Multidisciplinary research projects involving the training faculty will be developed. Exchange of scientific knowledge among trainees and faculty will be achieved through coursework, seminars, formal scientific presentations and scientific discussions. The high quality faculty, and their commitment to research training, will allow this program to train young scientists who will possess the appropriate expertise and scientific knowledge for elucidation of drug actions. Furthermore, modern up-to date facilities at UCSD will be accessible to trainees, including newly established Proteomic Laboratory, bioinformatic analyses of the Protein Data Bank and protein structure at the San Diego Supercomputer Center (SDSC) that includes drug informatics, the core transgenic mouse facility, peptide synthesis and sequencing core, and molecular and cell biology facilities. Trainees of this program will acquire the knowledge required for their continued investigations on the principles of drug action to investigate mechanisms of drugs of abuse. PUBLIC HEALTH RELEVANCE: This research training is relevant for developing scientists to elucidate mechanisms of drug abuse action to maintain human health and to improve health in disease.

DESCRIPTION (provided by applicant): The goal of this NIH shared instrumentation application is to establish an integrated, state-of-the-art, DNA sequencing core system at the Buck Institute to facilitate ongoing and future scientific research on the molecular mechanisms of aging and age-related diseases. The latest technology has developed new capillary electrophoresis sequencing instrumentation to allow high quality, rapid DNA sequencing to be achieved in a completely automated format that utilizes sensitive fluorescent dye terminators. These features of the ABI 3100 Genetic Analyzer System for capillary electrophoresis system are advantageous over conventional slab gel dye terminator DNA sequencing that is used by most other DNA sequencing facilities. The automation of capillary preparation, sample loading, and electrophoresis significantly improves clarity and consistency of results; the automated format avoids operator errors that occur with the manual slab gel format of conventional DNA sequencers. The integrated DNA sequencing system will include the Expedite 8909 oligonucleotide synthesizer for synthesis of primers needed for the high volume of DNA sequencing; this unit also has the unique capability for synthesizing peptide nucleic acids (PNA) that are especially effective for antisense gene expression, as well as synthesis of modified and unmodified oligonucleotides. This integrated DNA sequencing system will enhance ongoing NIH-funded research (and research funded by other sources) for principal investigators at the Buck Institute, will complement existing cores in genomics, proteomics, and morphology and cell biology, and will foster collaborations among investigators at the Institute.

DESCRIPTION (provided by applicant): Enkephalin and Beta-endorphin opioid peptide neurotransmitters are synthesized as proenkephalin (PE) and proopiomelanocortin (POMC) precursors, respectively, which require proteolytic processing in secretory vesicles to form active neuropeptides. The extent of PE and POMC processing in chromaffin cells and pituitary, respectively, is limited and varied. These observations suggest that endogenous protease inhibitors may be involved in proneuropeptide processing. Therefore, this study has investigated the hypothesis that endogenous protease inhibitors maybe colocalized with, and inhibit, pro-neuropeptide processing enzymes. Our molecular studies have established two novel, secretory vesicle protease inhibitors, endopin 1 and 2 that posses distinct target protease specificity. These two endopins share homology with the serpin family of protease inhibitors, and possess active sites consistent with pro-neuropeptide processing. Endopins are colocalized with, and inhibit the PE processing enzyme PTP ('prohormone thiol protease'); endopins also inhibit POMC cleaving activity in pituitary secretory vesicles. In continuing studies, the goal of this proposal will be to compare the roles of endopin 1 and 2 in the proteolytic processing of the pro-neuropeptides PE and POMC. In specific aim 1, active-site mutants and chimeras of endopins will define structural features of the reactive site loop responsible for inhibition. Inactive mutants will be used as controls in endopin expression studies (aims 2, 3, 5). In specific aims 2 and 3, regulation of PE and POMC processing by endopins will be assessed in primary cultures of chromaffin and pituitary cells, respectively, by endopin sense and antisense expression. The tissue distribution of the endopins and their subcellular localization in neuropeptide containing secretory vesicles will be studied in specific aim 4 by confocal immunofluorescence and immunoelectronmicroscopy. Endopins 1 and 2 inhibit PE and POMC processing activities in secretory vesicles, suggesting target proteases of endopins in these vesicles. Because secretory vesicle endopins form complexes with proteases, endopins can be used as a tool for molecular cloning of endopin-interacting protease(s) from pituitary and chromaffin cells (aim5). Evaluation of an endopin-interacting clone for enhanced PE and POMC processing, regulated secretion, and inhibition by endopin(s) will lead to consideration of the clone as an endopin target protease. This study will define a new, endogenous serpin protease inhibitor mechanism for regulating peptide neurotransmitter production. Results will indicate the role of endopins in processing PE and POMC into active enkephalin and Beta-endorphin opioid peptides, studied here as model peptide neurotransmitters. Serpin regulation of opioid peptides that mediate analgesia and stress may lead to development of new therapeutic approaches in pain and neurologic disease.

Neuropeptide Y (NPY) and catestatin peptides are secreted from adrenomedullary chromaffin cells and sympathetic nerves for the regulation of blood pressure. NPY acts as a direct vasoconstrictor, and catestatin functions as an autocrine regulator to inhibit nicotine-stimulated catecholamine release. Elevated NPY and reduced catestatin in essential hypertension implicate their participation as neuroeffectors in regulating blood pressure. Importantly, knowledge of the major proteolytic enzymes (s) responsible for converting their respective prohormone precursors into active NPY and catestatin is crucial for understanding regulatory mechanisms that control blood pressure. The prohormone precursors of NPY and catestatin, pro-NPY and chromogranin A (CgA), respectively, undergo proteolytic processing within secretory vesicles of adrenal medulla, known as chromaffin granules. We have identified secretory vesicle cathepsin L, previously known as ?prohormone thiol protease? (PTP), as a key processing enzyme for pro-NPY and CgA. Moreover, this project discovered a novel endogenous serpin, endopin 2 that inhibits secretory vesicle cathepsin L. In addition, the subtilisin-like PC1 and PC2 proteases in secretory vesicles may also participate in pro-NPY and CgA processing. Based on these new findings, the goal of Project 3 will be to assess the roles of secretory vesicle cathepsin L and endopin 2, compared to PC1 and PC2, in the production of NPY and catestatin neuropeptides that regulate blood pressure. This project will test the hypothesis that secretory vesicle cathepsin L may be a major processing enzyme for NPY and catestatin, compared to PC1 and PC2 enzymes. Our new results support the emerging biological role of cathepsin L function in secretory vesicles for proteolysis of pro-NPY and CgA. Moreover, our recent studies of cathepsin L knockout mice suggest participation of this protease in NPY production in adrenals. These results lead to the next phase of this study that will (1) evaluate in vitro and cellular processing of pro-NPY and CgA by secretory vesicle cathepsin L, compared to PC1 and PC2, (2) assess the cellular and tissue distribution of cathepsin L and PC enzymes in secretory vesicles that contain NPY and catestatin, (3) conduct cellular antisense and gene knockout studies to examine the relative roles of cathepsin L and PC enzymes for NPY and catestatin production, and (4) evaluate endopin 2 as an endogenous serpin inhibitor of cathepsin L for neuropeptide production. Results will demonstrate the relative roles for cathepsin L and endopin 2, compared to PC1 and PC2, in the biosynthesis of active NPY and catestatin peptide regulators. Project 3 complements the program project theme of understanding the regulation of sympathetic neuroeffectors that participate in blood pressure regulation.

[unreadable] DESCRIPTION (provided by applicant): The biosynthesis of the endogenous beta-endorphin opioid peptide requires proteolytic processing of its POMC (proopiomelanocortin) precursor. Beta-endorphin is a key regulator of analgesia, behavior, and stress. It is, therefore, critical to define the proteolytic pathway(s) required to convert POMC into active beta- endorphin. Our studies have identified secretory vesicle cathepsin L as a key processing enzyme for POMC, based on exciting new results from cathepsin L knockout mice showing reduced levels of beta-endorphin. These novel results were obtained by purification from secretory vesicles, active-site affinity labeling, and peptide microsequencing to identify the processing activity as cathepsin L. Cathepsin L is localized to POMC-containing secretory vesicles, as well as neuropeptide-containing secretory vesicles. These new results implicate a significant role for secretory vesicle cathepsin L in beta-endorphin and neuropeptide production. The cleavage specificity of cathepsin L for dibasic processing sites generates peptide intermediates with NH2-terminal basic residues, indicating that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Arg/Lys aminopeptidase activity is colocalized in neurosecretory vesicles with beta-endorphin and neuropeptides. These new results indicate cathepsin L and Arg/Lys aminopeptidase as a new protease pathway for prohormone processing, in addition to the well known subtilisin-like PC1 and PC2 and carboxypeptidase E/H pathway. Thus, the goal of this proposal will be to determine the role of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, compared to PC1 and PC2, for processing POMC into beta- endorphin. This goal will be achieved in four specific aims to (1) determine the effects of reduced enzyme activities on POMC processing in (a) cathepsin L knockout mice, compared to PC1 and PC2 knockout mice, in pituitary and brain, (b) siRNA experiments to reduce enzyme levels in pituitary cells and brain neuronal cells, as well as in experiments for direct chemical inhibition of cathepsin L, (2) assess localization of cathepsin L with beta-endorphin and PC enzymes in secretory vesicles of pituitary and brain, (3) determine the role of each processing enzyme in (a) cellular POMC processing in PC12 neuroendocrine cells (and GH3 cells) by cotransfection of each enzyme with POMC, and in (b) in vitro kinetic and cleavage site studies of POMC processing, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for beta- endorphin production. Results will establish roles for secretory vesicle cathepsin L and Arg/Lys aminopeptidase processing pathway in the biosynthesis of beta-endorphin. New findings from this project will enhance our knowledge of the complexity of biosynthetic mechanisms for endogenous opioid and neuropeptide systems. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The biosynthesis of the endogenous beta-endorphin opioid peptide requires proteolytic processing of its POMC (proopiomelanocortin) precursor. Beta-endorphin is a key regulator of analgesia, behavior, and stress. It is, therefore, critical to define the proteolytic pathway(s) required to convert POMC into active beta- endorphin. Our studies have identified secretory vesicle cathepsin L as a key processing enzyme for POMC, based on exciting new results from cathepsin L knockout mice showing reduced levels of beta-endorphin. These novel results were obtained by purification from secretory vesicles, active-site affinity labeling, and peptide microsequencing to identify the processing activity as cathepsin L. Cathepsin L is localized to POMC-containing secretory vesicles, as well as neuropeptide-containing secretory vesicles. These new results implicate a significant role for secretory vesicle cathepsin L in beta-endorphin and neuropeptide production. The cleavage specificity of cathepsin L for dibasic processing sites generates peptide intermediates with NH2-terminal basic residues, indicating that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Arg/Lys aminopeptidase activity is colocalized in neurosecretory vesicles with beta-endorphin and neuropeptides. These new results indicate cathepsin L and Arg/Lys aminopeptidase as a new protease pathway for prohormone processing, in addition to the well known subtilisin-like PC1 and PC2 and carboxypeptidase E/H pathway. Thus, the goal of this proposal will be to determine the role of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, compared to PC1 and PC2, for processing POMC into beta- endorphin. This goal will be achieved in four specific aims to (1) determine the effects of reduced enzyme activities on POMC processing in (a) cathepsin L knockout mice, compared to PC1 and PC2 knockout mice, in pituitary and brain, (b) siRNA experiments to reduce enzyme levels in pituitary cells and brain neuronal cells, as well as in experiments for direct chemical inhibition of cathepsin L, (2) assess localization of cathepsin L with beta-endorphin and PC enzymes in secretory vesicles of pituitary and brain, (3) determine the role of each processing enzyme in (a) cellular POMC processing in PC12 neuroendocrine cells (and GH3 cells) by cotransfection of each enzyme with POMC, and in (b) in vitro kinetic and cleavage site studies of POMC processing, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for beta- endorphin production. Results will establish roles for secretory vesicle cathepsin L and Arg/Lys aminopeptidase processing pathway in the biosynthesis of beta-endorphin. New findings from this project will enhance our knowledge of the complexity of biosynthetic mechanisms for endogenous opioid and neuropeptide systems. [unreadable] [unreadable] [unreadable]

Neuropeptide Y (NPY) and catestatin peptides are secreted from adrenomedullary chromaffin cells and sympathetic nerves for the regulation of blood pressure. NPY acts as a direct vasoconstrictor, and catestatin functions as an autocrine regulator to inhibit nicotine-stimulated catecholamine release. Elevated NPY and reduced catestatin in essential hypertension implicate their participation as neuroeffectors in regulating blood pressure. Importantly, knowledge of the major proteolytic enzymes (s) responsible for converting their respective prohormone precursors into active NPY and catestatin is crucial for understanding regulatory mechanisms that control blood pressure. The prohormone precursors of NPY and catestatin, pro-NPY and chromogranin A (CgA), respectively, undergo proteolytic processing within secretory vesicles of adrenal medulla, known as chromaffin granules. We have identified secretory vesicle cathepsin L, previously known as ?prohormone thiol protease? (PTP), as a key processing enzyme for pro-NPY and CgA. Moreover, this project discovered a novel endogenous serpin, endopin 2 that inhibits secretory vesicle cathepsin L. In addition, the subtilisin-like PC1 and PC2 proteases in secretory vesicles may also participate in pro-NPY and CgA processing. Based on these new findings, the goal of Project 3 will be to assess the roles of secretory vesicle cathepsin L and endopin 2, compared to PC1 and PC2, in the production of NPY and catestatin neuropeptides that regulate blood pressure. This project will test the hypothesis that secretory vesicle cathepsin L may be a major processing enzyme for NPY and catestatin, compared to PC1 and PC2 enzymes. Our new results support the emerging biological role of cathepsin L function in secretory vesicles for proteolysis of pro-NPY and CgA. Moreover, our recent studies of cathepsin L knockout mice suggest participation of this protease in NPY production in adrenals. These results lead to the next phase of this study that will (1) evaluate in vitro and cellular processing of pro-NPY and CgA by secretory vesicle cathepsin L, compared to PC1 and PC2, (2) assess the cellular and tissue distribution of cathepsin L and PC enzymes in secretory vesicles that contain NPY and catestatin, (3) conduct cellular antisense and gene knockout studies to examine the relative roles of cathepsin L and PC enzymes for NPY and catestatin production, and (4) evaluate endopin 2 as an endogenous serpin inhibitor of cathepsin L for neuropeptide production. Results will demonstrate the relative roles for cathepsin L and endopin 2, compared to PC1 and PC2, in the biosynthesis of active NPY and catestatin peptide regulators. Project 3 complements the program project theme of understanding the regulation of sympathetic neuroeffectors that participate in blood pressure regulation.

DESCRIPTION (provided by applicant): The biosynthesis of the endogenous beta-endorphin opioid peptide requires proteolytic processing of its POMC (proopiomelanocortin) precursor. Beta-endorphin is a key regulator of analgesia, behavior, and stress. It is, therefore, critical to define the proteolytic pathway(s) required to convert POMC into active beta- endorphin. Our studies have identified secretory vesicle cathepsin L as a key processing enzyme for POMC, based on exciting new results from cathepsin L knockout mice showing reduced levels of beta-endorphin. These novel results were obtained by purification from secretory vesicles, active-site affinity labeling, and peptide microsequencing to identify the processing activity as cathepsin L. Cathepsin L is localized to POMC-containing secretory vesicles, as well as neuropeptide-containing secretory vesicles. These new results implicate a significant role for secretory vesicle cathepsin L in beta-endorphin and neuropeptide production. The cleavage specificity of cathepsin L for dibasic processing sites generates peptide intermediates with NH2-terminal basic residues, indicating that Arg/Lys aminopeptidase is then necessary to remove such basic residues. Arg/Lys aminopeptidase activity is colocalized in neurosecretory vesicles with beta-endorphin and neuropeptides. These new results indicate cathepsin L and Arg/Lys aminopeptidase as a new protease pathway for prohormone processing, in addition to the well known subtilisin-like PC1 and PC2 and carboxypeptidase E/H pathway. Thus, the goal of this proposal will be to determine the role of secretory vesicle cathepsin L and Arg/Lys aminopeptidase, compared to PC1 and PC2, for processing POMC into beta- endorphin. This goal will be achieved in four specific aims to (1) determine the effects of reduced enzyme activities on POMC processing in (a) cathepsin L knockout mice, compared to PC1 and PC2 knockout mice, in pituitary and brain, (b) siRNA experiments to reduce enzyme levels in pituitary cells and brain neuronal cells, as well as in experiments for direct chemical inhibition of cathepsin L, (2) assess localization of cathepsin L with beta-endorphin and PC enzymes in secretory vesicles of pituitary and brain, (3) determine the role of each processing enzyme in (a) cellular POMC processing in PC12 neuroendocrine cells (and GH3 cells) by cotransfection of each enzyme with POMC, and in (b) in vitro kinetic and cleavage site studies of POMC processing, and (4) obtain biochemical and molecular analyses of Arg/Lys aminopeptidase for beta- endorphin production. Results will establish roles for secretory vesicle cathepsin L and Arg/Lys aminopeptidase processing pathway in the biosynthesis of beta-endorphin. New findings from this project will enhance our knowledge of the complexity of biosynthetic mechanisms for endogenous opioid and neuropeptide systems.

DESCRIPTION (provided by applicant): Huntington's Disease (HD) is an inherited neurodegenerative disorder characterized by motor and cognitive impairments. The mutant huntingtin (htt) protein with expanded polyglutamine repeats are responsible for HD based on studies in human HD brains and in transgenic mice expressing mutant htt. Proteolytic fragments of htt are involved in the HD disease process. In human brain, N-terminal htt fragments in HD brains have been demonstrated. Expression of mutant N-terminal htt fragments in transgenic mice induces behavioral features and neuronal deficits that resemble HD. Notably, distinct patterns of htt N- and C-terminal fragments in human HD brains has been demonstrated. However, identification of these in vivo brain htt proteolytic fragments has not yet been determined. The critical, unsolved question is what are the primary sequences of htt proteolytic fragments in affected striatum and cortex of human HD brains? Therefore, the goal of this proposal will be to evaluate the primary sequences of N-terminal, N-domain, and C-domain htt fragments from human HD striatum and cortex, as well as from HD mouse models that express full-length htt. The first aim will evaluate the peptide sequences of in vivo htt fragments from mouse brain regions of transgenic HD mouse models that express full-length htt. Htt fragments will be purified by protein chromatographic resins that provide highly enriched htt proteins for purification;purification will include anti- htt affinity columns with well-characterized antisera that recognize the N-terminal region, N-domain, and C- domain areas of full-length htt. Purified htt fragments will be subjected to peptide sequencing by mass spectrometry, including sequencing of N- and C-terminal regions. Results will indicate peptide sequences of htt proteolytic fragments in mouse models of HD that express full-length htt. The second aim will evaluate the peptide sequences of human htt fragments purified from human HD brain regions - cortex and striatum - by mass spectrometry. The purification of htt fragments from human brain regions will utilize the purification procedure developed in aim 1 for mouse htt fragments. Mass spectrometry of purified human htt fragments will be conducted as described for htt fragments isolated from mouse brain. Results will provide key knowledge of the htt fragment sequences in human HD brain. Results of this project are essential for the next stages of HD research to define the most neurotoxic htt fragments, to elucidate the full spectrum of proteases that generate toxic htt fragments, and to move forward to drug discovery of protease inhibitors that may reduce production of htt fragments. This project will have extraordinarily high benefit for future development of effective therapeutic treatments for HD. PUBLIC HEALTH RELEVANCE: The goal of this project is to evaluate the primary peptide sequences of the in vivo mutant huntingtin (htt) protein fragments that are known to be responsible for the development of Huntington's disease (HD). These htt fragments in HD brains of patients have not yet been completely defined with respect to their primary amino acid sequences, which will be achieved in this project by their analyses by purification and current mass spectrometry approaches. The knowledge gained from this project is essential for understanding key htt mechanisms underlying HD, which will provide future strategies to improve the disease condition.

DESCRIPTION (provided by applicant): Huntington's Disease (HD) is a neurological disorder resulting from CAG triplet repeat genetic mutation of the IT15 gene that encodes the huntingtin protein. Extensive data in the field indicate that proteolytic fragment(s) of htt mediate the HD disease process, resulting in severe motor disturbances. The goal of this project will be to discover novel marine natural product compounds as potential therapeutic agents for Huntington's disease (HD). This project fulfills a major gap in the HD and neurodegenerative disease field for discovery of novel agents as protease inhibitors for future development of therapeutic agents for HD. Furthermore, this project uses novel marine natural products as a unique opportunity for drug discovery in the protease target area to reduce production of neurotoxic htt proteolytic fragments that participate in HD. This unique project will integrate expertise in protease biochemistry and neurobiology by Dr. Vivian Hook, marine natural products for discovery of therapeutic agents by Dr. William Gerwick, and model striatal neuronal cells, as well as transgenic mice, expressing mutant huntingtin (htt) protein of Huntington's disease by Dr. Albert La Spada at the Univ. of Calif., San Diego. The project plan will implement interdisciplinary efforts of three highly experienced laboratories in the required disciplines to discover novel protease inhibitors of huntingtin protein proteolysis as a means to reduce cellular mutant htt neurotoxicity. This project can progress at a rapid pace since the collaborating laboratories have the required expertise, and are located in close proximity to one another at UC San Diego. HD drug discovery of this project will be accomplished in two specific aims. The first aim will evaluate marine natural product compounds for inhibition of protease activities thought to be involved in HD, which includes caspase, calpain, lysosomal proteases (cathepsins L, B, and D), and matrix metalloprotease 10. Cyanobacteria algal marine natural product compounds are a rich source of potent drug molecules with unique structural diversity. In aim 2, cyanobacterial marine natural compounds will be tested in cellular assays for reduction of N-terminal htt fragments and protection from cell death, using striatal-like neuronal cells that express full-length mutant huntingtin (htt) protein, and in control cells expressing normal htt protein. Effective compounds identified from aims 1 and 2 will be prioritized by their inhibitory potencies. These compounds will be planned for a future R01 project that will conduct in vivo testing of compounds in HD mouse models for effectiveness to improve the motor impairment in HD transgenic mice. Results can indicate candidate marine natural drug compounds for HD drug development. PUBLIC HEALTH RELEVANCE: Huntington's Disease (HD) is a neurological disorder resulting in motor disturbances as well as dementia. The goal of the proposed new project will be to discover novel therapeutic agents for Huntington's disease (HD). This unique project will integrate the joint expertise of three experts in protease and cellular biochemistry, marine natural product drug discovery, and cellular with genetic mouse models of HD by Dr. Vivian Hook, Dr. William Gerwick, and Dr. Albert La Spada, respectively, at the Univ. of Calif., San Diego that will facilitate development of therapeutic agents for HD.

DESCRIPTION (provided by applicant): The N-terminal truncated pyroglutamate (pGlu) modified forms of beta-amyloid (A ¿), pGluA ¿ (3-40/42) (referred to as pGluA ¿), display high neurotoxicity, neurodegeneration and memory loss in Alzheimer's disease. Notably, pGluA ¿ is abundant in AD brains and is present at levels greater than A¿ (1-40/42). The pGluA ¿ accelerates formation of A¿ and pGluA ¿ oligomers that cause neurotoxic cell death and memory deficits in AD. The pGluA ¿ (3-40/42) peptides begin with N-terminal glutamate, the third amino acid of A¿ (1-40/42). pGluA¿ peptides are generated from A ¿ (1-40/42) by aminopeptidases that remove the N-terminal aspartate and alanine residues, followed by cyclization of the N-terminal glutamate. The aminopeptidases required to generate pGluA¿ peptides have not yet been elucidated. Therefore, the goal of this project will be to identify the aminopeptidase mechanisms responsible for generating neurotoxic pGluA¿ that participates with A ¿ in development of AD. Results can provide novel protease targets for Alzheimer's disease. The hypothesis of this project is that the aspartate aminopeptidase and alanine aminopeptidase enzymes generate A ¿ (3-40/42) which is converted to pGluA¿(3-40/42) by glutaminyl cyclase (QC). This hypothesis is supported by our preliminary data demonstrating the presence of aspartate and alanine aminopeptidase activities in secretory vesicles that contain pGluA¿ (3-40/42) and A¿ (1-40/42). This hypothesis will be assessed in three specific aims to (1) identify aspartate and alanine aminopeptidases in A ¿ -producing secretory vesicles that remove the N-terminal Asp and Ala residues from A ¿ (1-40/42), (2) evaluate identified aminopeptidases by gene silencing and expression for their roles in producing pGluA¿ (3-40/42), and (3) evaluate inhibitors of these aminopeptidase activities to reduce production of pGluA¿ (3- 40/42). This project will identify new aminopeptidase mechanisms for producing neurotoxic pGluA¿ peptides. Results will also yield candidate inhibitors of these aminopeptidases for future investigation of aminopeptidase regulation in Alzheimer's disease.

Project Summary This project addresses the fundamental question of ?What `human' protease mechanisms are responsible for producing active peptide neurotransmitters, neuropeptides, that are a fundamental requirement for all brain and nervous system functions?? The unique hypothesis to address this question is that human-specific cathepsin V, combined with proteases identified in non-human animal models for neuropeptide production, participates in producing active neuropeptides. Fundamental knowledge of the human protease mechanisms for producing neuropeptides are relevant to neuropeptide regulation in neurological and mental health throughout aging, and which are impacted by environmental conditions including drugs of abuse. Studies in mice have demonstrated the important role of mouse cathepsin L in secretory vesicles for producing opioid and other neuropeptides. Interestingly, human-specific cathepsin V is the closest human homologue to mouse cathepsin L, suggesting a role for cathepsin V in neuropeptide production. The human-specific cathepsin V gene is not present in mouse or other species. We investigated human cathepsin V and found that it participates as a significant protease for enkephalin neuropeptide production. These findings lead to the goal of this `human focused' project to define the human protease mechanisms for neuropeptide production by cathepsin V, combined with cathepsin L and the PC1/3 & PC2 convertases that function in non-human species for neuropeptide production. Human induced pluripotent stem cell (hiPSC) neurons produce numerous neuropeptides and will be used as an innovative model for human protease mechanisms in neuropeptide biosynthesis. Parallel studies of neuropeptides and processing proteases in human brain regions rich in neuropeptides will support findings gained from the hiPSC model. Innovative neuropeptidomics mass spectrometry will identify neuropeptide profiles in an unbiased and high throughput manner. The first aim will define the cellular role in hiPSC neurons of cathepsin V in the production of opioid neuropeptides with comparison to cathepsin L, PC1/3, and PC2 proteases by gene silencing and expression, combined with mass spectrometry peptide identifications. The second aim will conduct in vitro biochemical studies of cathepsin V processing of opioid pro-neuropeptides compared to cathepsin L, PC1/3, and PC2, to define cleavage sites, peptide products, and kinetic efficiencies, with parallel analyses in human brain regions. The third aim will investigate diverse neuropeptides by neuropeptidomics of hiPSC neurons and human brain tissues to evaluate the role of these human proteases in producing diverse neuropeptides. Results will define the basic human protease mechanisms for peptide neurotransmitter production that is fundamental for brain and nervous system functions in health and disease.

PROJECT SUMMARY/ABSTRACT This application directly responds to the objective of RFA-AG-17-051. We have recently reported a positive correlation of the levels of pathogenic phosphorylated microtubule-associated protein tau (p-tau) in plasma- derived neuronal exosomes in Alzheimer's disease (AD) patients, suggesting their potential application for biomarkers and their implication as a novel machinery of spreading pathogenic molecules in the brain. However, the exact mechanisms regulating exosome biogenesis and secretion, and their contribution to the propagation of pathogenic molecules are poorly understood. Here we propose comprehensive approaches to delineate the molecular mechanisms that regulate biogenesis, secretion and trafficking of exosomes in different brain cell types (neurons, astrocytes and microglia) in vitro and in vivo. This project will (1) analyze the roles of the ESCRT (endosomal sorting complexes required for transport)-dependent pathway, combined with the ESCRT-independent (lipid- and tetraspanin-dependent pathways), for exosome biogenesis and secretion in murine and human neuronal cell types, (2) define the molecular machinery components responsible for exosome biogenesis and secretion in human induced pluripotent stem cells (hiPSCs) derived from AD patients, (3) characterize the exosome protein interactions with target cells for propagation using AD hiPSCs, and (4) characterize the trafficking of exosomes originated from specific neuronal and glial cell types in the central nervous system to periphery. We have assembled a group of collaborative investigators with established programs in cell biology, exosome biology, proteomics and animal models of neurodegenerative disorders. The proposed research work will develop a new understanding of exosomal biology and detailed functions of exosomes in progression of AD pathology. The findings gained from this research project will have a potential to discover new molecular targets for suppression of the disease spread via exosomes, and also address the National Alzheimer's Project Act plan to accelerate basic research toward development of AD therapeutics.

Chronic pain is a long-term, debilitating condition that is typically treated with prescription opioid drugs over lengthy periods of time with repeated drug administration. The problem with long-term opioid drug use is the development of addiction that results in detrimental drug tolerance and dependence, drug abuse, and overdose death. Non-addictive drugs to treat chronic pain are an unmet need in therapeutics, so that patients will not be subjected to the detrimental consequences of long- term opioid use for chronic pain conditions of nerve injury, stroke, cancer, arthritis, and many related conditions. Development of effective, non-addicting pain medications is a public health priority, indicated by NIH. This project addresses the critical need to develop non-addictive drugs for the treatment of chronic pain. The strategy to find a non-addictive therapeutic agent for chronic pain will be to target antisense oligonucleotide (ASO) drug molecules to reduce spinal dynorphin A and alleviate chronic pain. The ASO approach to alleviate chronic pain obliterates the need for long-term opioid drug use that causes addiction. Dynorphin A has been validated as a key factor in the development and maintenance of chronic pain, since knockout of the dynorphin gene blocks chronic pain in mouse models of chronic pain, and IT (intrathecal) administration of antibodies to dynorphin A attenuates the chronic pain state. Participation of dynorphin A in chronic pain is indicated by elevation of spinal dynorphin during chronic pain, and IT administration of dynorphin enhances chronic pain. ASO targeting of spinal dynorphin is a logical and innovative approach for development of non- addictive drugs to treat chronic pain. The research plan will be achieved in two specific aims. Aim 1 will design and screen ASO molecules for reduction of dynorphin A in cultured neurons using quantitative mass spectrometry analyses of peptides. Aim 2 will assess IT ASOs for their abilities to reduce the elevation in spinal dynorphin A during chronic pain modeled by SNL (spinal nerve injury) in mice, and to alleviate the chronic pain state as assessed by behavioral assays for pain. Pilot data shows that an anti-dynorphin ASO reduces spinal dynorphin A in mice in vivo, supporting the feasibility of this project. Effective candidate ASOs will be evaluated for initial ASO pharmacokinetics, efficacy to reduce chronic pain, and dynorphin A pharmacodynamics. This project will make important contributions to the field to develop non-addictive drugs for the treatment of chronic pain.

Traumatic brain injury (TBI) and Alzheimer's disease (AD) result in long-term behavioral deficits and brain neurodegeneration. There are no effective therapeutic agents for TBI or AD; therefore, advances in mechanism- based understanding of these brain disorders can identify new drug targeting approaches. Significantly, cathepsin B has been shown as a novel mechanism participating in behavioral dysfunctions and neuropathology of TBI and AD. The premise for the cathepsin B mechanism is that (a) cathepsin B is elevated in TBI and AD patients, (b) knockout (KO) of the cathepsin B gene in TBI and AD mouse models improves deficits in behavioral dysfunctions, respectively, and (c) cathepsin B KO reduces brain neuropathology. TBI and AD results in lysosomal leakage and redistribution of cathepsin B from lysosomes to the cytosol to result in cell death and activation of inflammatory IL-1? in brain. These findings lead to the hypothesis that cytosolic cathepsin B participates in the pathogenesis of TBI and AD. To test this hypothesis, it will be ideal to inhibit the pathogenic cytosolic cathepsin, without affecting its normal lysosomal function, with pH selective inhibitors as molecular probes. Our data shows that cathepsin B displays different peptide cleavage properties at neutral cytosolic pH compared to lysosomal acidic pH. These differential cleavage properties support the development of selective substrates and peptide inhibitors of cytosolic compared to lysosomal cathepsin B. The goal of this project will be to develop pH selective inhibitors of neutral cytosolic cathepsin B, compared to acidic lysosomal cathepsin B, as molecular probes for evaluation of the hypothesized pathogenic role of cytosolic cathepsin B during cellular lysosomal leakage which leads to neurodegeneration and behavioral deficits of TBI and AD. Aim 1 will assess the selective cleavage properties of cathepsin B at neutral and acidic pHs, achieved by global 'Multiplex Substrate Profiling Mass Spectrometry (MSP-MS) and positional scanning using a synthetic combinatorial library (PSSCL), for design and testing of pH selective peptide substrates. Aim 2 will utilize pH selective assays of cathepsin B to identify natural product inhibitors, achieved by screening collections of marine and terrestrial natural products, and assessing selectivity and potency. Aim 3 will develop peptidic inhibitors of cathepsin B, achieved by modifying pH selective peptide substrates with AOMK, CMK, or VS groups; selectivity and potency will be assessed. Peptidic and natural product inhibitors will be assessed for effects on cell death and IL-1? levels during A?- and H2O2-induced lysosomal leakage in neurons and glial cells. Aim 4 will characterize in vivo lysosomal leakage of cathepsin B in TBI and AD mouse models with respect to time-course, brain regions, and neuronal and glial cells. Cathepsin B inhibitors, known and newly developed inhibitors, will be given before and during lysosomal leakage for evaluation of improvements in behavioral deficits and neuropathology. The novel inhibitors will advantageously target pathogenic cytosolic cathepsin B, rather than normal lysosomal cathepsin B, for future development of TBI and AD therapeutics.