Victor Faundez, M.D., Ph.D. - US grants

DESCRIPTION (provided by applicant): A defining feature of presynaptic terminals is the presence of synaptic vesicles (SVs), secretory organelles that store and secrete neurotransmitters. SVs functions are specified by the organelle membrane protein composition. Thus, mechanisms controlling SV formation and composition are pivotal for synapse function. In this proposal we focus on the vesicle biogenesis pathway controlled by the adaptor complex AP-3, a coat complex that sorts membrane proteins from early endosomes to SVs. SV protein composition is regulated by two isoforms of the adaptor complex AP-3, neuronal and ubiquitous, the later thought to participate exclusively in lysosome biogenesis. Genetic defects in the neuronal AP-3 isoform hinder targeting of SV membrane proteins. Surprisingly, null mouse mutants in a lysosomal sorting pathway, the ubiquitous AP-3 route, trigger accruement of SV-specific proteins in SVs. These unexpected results lead us to propose the novel concept that SV and lysosomal sorting mechanisms present on the same endosome compete for membrane proteins to be delivered into two alternative routes, SVs or lysosomes. This concept departs from the traditional view of lysosomes, which are viewed as terminal organelles involved in the disposal of normal and pathological cellular components. Furthermore, our model provides a novel way to understand the contribution of lysosome targeting mechanisms to familial and sporadic forms of neurodegeneration that affect children and adult individuals. Our central hypothesis is that: AP-3-isoform-specific mechanisms target SV membrane proteins from a common endosomal compartment to two competing pathways: either to a SV biogenesis route or to a late endosome-lysosomal path. In this proposal, we will focus on four predictions derived from our hypothesis. These predictions will be systematically explored using a combination of mouse deficient models that affect SV and endo- lysosomal targeting, high-resolution immuno-electron and in vivo imaging microscopy, as well as the molecular analysis of isolated SVs and endosomes. Information gained in this proposal will illuminate our understanding of how late endosomes-lysosome sorting processes affect synapses under physiological and pathological conditions. PUBLIC HEALTH RELEVANCE: We propose a model that will contribute knowledge to understand how lysosomes and synapses interface and contribute to neurodegenerative and psychiatric disorders.

[unreadable] DESCRIPTION (provided by applicant): Zinc plays fundamental and diverse roles in cells yet excess free zinc is associated with metal cytotoxicity. To balance these opposing effects, cells have evolved universal mechanisms controlling cytoplasmic metal concentration. Cells accomplish this goal by zinc extrusion into the extracellular space, chelation by cytosolic chaperones, or sequestration within intracellular compartments. This last mechanism is the less explored process and it constitutes the main focus of our proposal. Zinc plays fundamental roles in synaptic physiology as well as in acute and chronic pathological conditions, ranging from excitotoxicity to the formation of amyloid aggregates characteristic of neurodegenerative diseases. Despite these fundamental roles of zinc, our understanding of the contribution of intracellular compartment in metal sequestration and homoeostasis is limited. In neurons, organellar zinc is stored in synaptic vesicles (SVs) by the activity of a synaptic vesicle specific zinc transporter, ZnT3. We have isolated and characterized by proteomics a ZnT3- enriched vesicle population. In these vesicles, we have identified ~ 140 molecular targets, several of which either up- or down-regulate vesicular endosomal zinc stores. These molecules provide a unique set of tools to assess the role of intracellular organelles, en particular endosomes and SV, in normal and pathological metal homeostasis. Our studies suggest that ZnT3 transport function are regulated by the nature of the compartment in which the ZnT3 transporter resides. Consistent with this notion, we have identified three targeting mechanisms that control ZnT3 subcellular localization that have the potential to regulate ZnT3 zinc transport function. In this proposal, we will specifically explore these novel regulatory paradigms testing the hypothesis that endosome-specific zinc transporter interactions regulate zinc transporter activity and resistance to metal-induced cytotoxicity. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neuronal zinc participates in key processes such as modulation of excitatory neurotransmission. In contrast, neurons are particularly susceptible to excess of this metal. To balance these opposing effects, cells possess mechanisms to finely control free cytoplasmic metal concentration. Among these mechanisms, zinc homeostasis by organelle metal sequestration relies on ZnT/SLC30 zinc transporter family members. These mechanisms are the focus of this application. The main ZnT/SLC30 zinc transporter in neurons is ZnT3. ZnT3 is located in synaptic vesicles and its genetic deficiency modulates pathology ranging from epilepsy to Alzheimer's disease. During our previous funding period, we discovered that ZnT3 distribution and zinc transport activity are controlled by its oligomerization state. ZnT3 dimers confer cellular resistance to zinc toxicity by an inter-ZnT3 dityrosine bond whose generation is catalyzed by redox mechanisms. This is the first example of a membrane protein regulated by dityrosine bonds. We propose that compartment-specific ZnT3 transporter oligomerization by redox mechanisms regulates metal toxicity resistance. In this application, we test this hypothesis in vitro and in vivo using dimerization gain- and loss-of-function mutations i ZnT3 as well as mice carrying deficiencies or gain-of-function in the ZnT3 trafficking and transport pathways. Our studies will impact our understanding and possibly treatment of acute and chronic neurological disease processes where zinc play a role such as epilepsy and Alzheimer's disease.

DESCRIPTION (provided by applicant): Copper is a powerful toxic oxidant for neurons whose free levels must be tightly controlled. It is also an essential micronutrient necessary for neurona enzymatic reactions, such as neurotransmitter and neuropeptide synthesis. The importance of copper to neuronal cells is illustrated by Menkes disease, an X-linked genetic disorder characterized by neuronal tissue copper starvation, altered neuronal polarity, and cell survival phenotypes. The precise cellular mechanisms underlying these copper- dependent neuronal phenotypes remain unknown and constitute the focus of this application. The gene affected in Menkes disease, ATP7A, is a Golgi localized protein that loads copper into secretory proteins. This fact suggests that Menkes disease phenotypes result from alterations in the entire copper sensitive secreted proteome. We propose to test this hypothesis by comprehensively identifying the human neuronal copper-sensitive secreted proteome in human induced pluripotent stem cells (iPSCs) differentiated into neurons. We will test the participation of the copper-sensitive proteome in the progression and severity of Menkes disease neuronal pathology. Such knowledge will contribute to our understanding and development of therapeutics in Menkes disease as well as diseases affected by copper availability, such as Alzheimer's disease.

How do mutations in genes implicated in human mental/neurodevelopmental disorders progressively scale up from a single molecular defect to protein networks (interactome), the physiology of the synapse, and behavior? We seek answers to this question as they hold promising explanatory and interventional power in neurodevelopmental disorders. We have chosen to address this question using the combined power of reverse genetics in Drosophila, cell-free reconstitution, and mutant mouse experimentation on the experimentally defined dysbindin-BLOC-1 interactome. We evaluate mechanisms and phenotypic consequences of genetically manipulating the neurodevelopmental disorder pathway constituted by dysbindin-BLOC-1 and two dysbindin-BLOC-1 interactome mechanisms at the synapse. These mechanisms are NSF- and SNARE-dependent vesicle fusion and Arp2/3-dependent actin polymerization. In this application, will determine the functional consequences of genetically perturbing these two dysbindin-BLOC-1 mechanisms on the physiology of the Drosophila NMJ synapse and two forms of synaptic plasticity: presynaptic homeostatic plasticity and a simple form of learning, short-term olfactory habituation. We postulate that the dysbindin-BLOC-1 interactome is necessary for presynaptic endosome vesicle traffic to establish presynaptic homeostatic plasticity and olfactory habituation. Our application is an important contribution to the novel and emerging concept that defective actin polymerization and vesicle fusion at synapses are core mechanisms impaired in mental disease.

How  does  copper  exposure  perturb  neuronal  cells  leading  to  cell  death?  This  application  seeks  to  address  this  question  by  studying  novel  mechanisms  that  we  discovered  whose  genetic  defects  confer  susceptibility  to  or  protect  against  metal  toxicants.  We  propose  that  these  copper  homeostasis  mechanisms  are  shared  with  pathways  affected  in  common  neurodegenerative  disorders,  such  as  Parkinson?s  disease. Copper is an essential micronutrient but, in addition, copper is also a powerful  neurotoxicant  whose  free  levels  in  the  cytoplasm  must  be  tightly  controlled.  Here  we  focus on a genetic defect that renders cells susceptible to copper, Menkes disease, such  that normal environmental copper becomes toxic to cells in culture. Menkes disease, is a  progressive  childhood  neurodegeneration  caused  by  mutations  of  the  copper  pump  ATP7A.  In  this  application,  we  present  exciting  data  revealing  novel  mechanisms  associated  to  Menkes  copper  toxicity  which  are  shared  with  genetic  forms  of  neurodegeneration. We postulate that copper toxicity is modulated by membrane traffic  mechanisms  controlling  copper  transporters  expression  and  subcellular  location,  ubiquitination,  and  mitochondrial  metabolism.  This  proposal  will  test  this  hypothesis  in  mice  and  Drosophila  carrying  mutations  in  pathways  associated  to  ATP7A.  Genetic  defects  in  these  ATP7A  regulatory  pathways  also  cause  neurodegeneration.  The  completion  of  this  proposal  will  open  the  door  for  clinical  interventions  to  improve  outcomes  of  neurological  diseases  where  environmental  factors  participate  in  pathogenesis.