Karen Chang - US grants

The long-term goal ofthis research is to understand the molecular mechanisms underlying the phenotypes seen in Down syndrome (DS). DS is a complicated disorder caused by full or partial triplication of chromosome 2 l , and the genotype-phenotype correlations are riot well understood. DS patients have a number of clinical manifestations, including mental retardation, congenital heart defects;motor deficits, and early oriset Alzheimer's disease (AD). DS neuronarcultures and tissues exhibit oxidative damages such as impaired mitochondrial enzyme activities arid increased prbductibn of toxic reactive oxygen species (ROS), Using Drosophila as a model system, we have:previously demonstrated that nebula, the Droisophila homolog of human DSCR1. can regulate protein kinase A and calcineurin activities to alter learning and memory. In addition, we found that nebula is located ih the mitochondria, interacts with the adenine nucleotide ti-ansjocator (ANT), and is important for the maintenance of nriitochondrial functipn and integrity. Mitochondrial dysfunction has recently emerged as a common theme that underlies numerous neurological disorders, including DS and AD. Accordingly, ttie experiments described;in this propiosal are aimed at understanding the effects of perturbed nebula/DSCRI level on mitochohdriarfunction and cell sun/ival, factors controlling its subcellular localization and function, and its possible contribution to AD neuropathologies. The specific aims are: (1) To determine the physiological consequences of abnormal mitochondrial function due to nebula mutations. (2) To idehttfy signaling pathways regiilatihg nebula localization and fund (3) To investigate whether nebula/DSCRI overexpression contributesi to AD nieuropathologies;

Following neurotransmitter release, synaptic vesicle membrane and protein components are rapidly retrieved from the plasma membrane through endocytosis, and functional synaptic vesicles are subsequently regenerated from the endocytosed components. This fundamental process, called synaptic vesicle recycling, is crucial for sustaining neuronal communication across a wide range of neuronal activities. Impaired synaptic vesicle recycling can thus deleteriously affect neuronal survival and function, and is associated with numerous neurological disorders. There are multiple modes of synaptic vesicle endocytosis, including clathrin-mediated endocytosis and clathrin-independent bulk endocytosis. While it is widely known that dynamic phosphorylation of synaptic proteins by kinases delicately regulates clathrin-mediated endocytosis, mechanisms modulating clathrin-independent bulk endocytosis are not well understood. In this proposal, will use Drosophila melanogaster, which offers the advantage of powerful genetics and well-characterized glutamatergic synapses at the neuromuscular junction, to investigate the functions of a synaptic kinase called Minibrain (MNB), also known as DYRK1A, in regulating multiple steps of synaptic vesicle recycling. We will take a multidisciplinary approach combining genetics, biochemistry, cell biology, and electrophysiological analyses to address the role of MNB/DYRK1A in clathrin-independent bulk endocytosis and synaptic vesicle regeneration. We will address the following questions in this proposal: 1) what is the role of MNB/.DYRK1A in regulating clathrin-independent bulk endocytosis? 2) Does MNB regulate bulk endocytosis through phosphorylation of Synaptojanin, a previously identified synaptic target of MNB? 3) Is MNB required for the recovery of functional synaptic vesicles pools? 4) What is the mechanism by which MNB regulates functional recovery of synaptic vesicles? As MNB/DYRK1A is upregulated in Down syndrome and mutated in some cases of Autism, a thorough understanding of MNB functions at the synapse will provide novel insights into mechanisms underlying neurological disorders and contribute to therapeutic development in the future.

Project Summary Chemotherapy-induced peripheral neuropathy (CIPN) is a major cause of dose reduction or discontinuation of an otherwise successful anti-cancer therapy. Patients often experience debilitating chronic pain and numbness due to damage of peripheral nerves. Cellular mechanisms underlying CIPN are not well understood, but disruptions in mitochondrial functions and transport have been posited to contribute to CIPN. However, due to limitations in monitoring mitochondrial transport in vivo, whether disruptions in mitochondrial transport indeed contributes to CIPN remains a major unanswered question. Furthermore, mechanisms for how chemotherapeutics induce mitochondrial transport problems in peripheral neurons are not well understood. This proposal investigates how paclitaxel, a common therapeutic known to cause CIPN, alters mitochondrial transport and contributes to peripheral neuropathy in an intact Drosophila model system. Once established, this approach can be used to screen through other chemotherapeutics on their ability to alter mitochondrial movement and cause CIPN, as well as to identify drugs that could protect against CIPN. Specifically, we will use novel optogenetic tools to address the following questions in vivo. 1) Does paclitaxel alter mitochondrial movement in sensory neurons? 2) Does altered mitochondrial movement contribute to neuropathy? 3) Does paclitaxel elevate mitochondrial ROS and mitochondrial Ca2+ levels to influence mitochondrial movement and cause neuropathy? 4) Does restoring mitochondrial movement prevent neuropathy? Elucidating the link between chemotherapeutics treatment, changes in mitochondrial Ca2+ and ROS contents, and mitochondrial transport and neuropathy will reveal new insights into mechanisms that contribute to CIPN.

The ability of neurons to rapidly modify synaptic structure and strength in response to neuronal activity, a process called activity-induced structural and functional plasticity, is crucial for neuronal development and complex brain functions such as learning and memory. Recent evidence suggests that integrin receptors, a major class of cell adhesion molecules, play an important role in synaptic plasticity. However, how neurons dynamically couple synaptic demand to structural remodeling, and what activates integrin during neuronal activity remain elusive. My lab has recently identified a novel protein named Shriveled (Shv) that activates integrin via outside-in signaling. We now have evidence showing that Shv is selectively released during intense neuronal activity, and that shv mutant cannot undergo synaptic remodeling in response to neuronal activity at the Drosophila neuromuscular junction. In this proposal, we will: (1) investigate the role that Shv plays during development to regulate synaptic growth and function; (2) delineate how neuronal activity regulates Shv release and activity-induced structural and functional plasticity; (3) elucidate molecular and cellular mechanisms underlying activity-induced structural remodeling. As a healthy nervous system depends on the ability of neurons to dynamically adjust synaptic strength and modify synaptic structure in response to neuronal activity, elucidating Shv functions and mechanisms underlying activity-induced synaptic remodeling may lead to new therapeutic strategies to treat or prevent neurological and psychiatric disorders.

PROJECT SUMMARY While amyloid plaques and neurofibrillary tangles are Alzheimer's disease (AD) defining features, Alzheimer himself originally identified a third pathology? inflammation of the brain's glial support cells. Neuroinflammation in AD is characterized by reactive astrocytes and microglia that surround amyloid plaques and chronically secrete inflammatory innate immune cytokines. The dominant view for decades has been that all forms of inflammation damage the AD brain. Yet, non-steroidal anti-inflammatory drugs failed to produce a positive signal for AD primary prevention. This raises a fundamental question: should we be blocking or possibly even promoting inflammation as an AD therapeutic? While the focus has mainly been on pro-inflammatory cerebral innate immunity, little attention has been paid to factors that curtail peripheral innate immune responses. The unifying theme of our work is that `rebalancing' peripheral innate immunity to homeostasis by releasing immunosuppression will limit AD progression. Strikingly, our focus on innate immunity in AD has just recently been validated by genome-wide association studies. These results have taken the field by storm; identifying clusters of AD risk alleles in core peripheral macrophage pathways. As a key cytokine suppressor of innate immunity and inflammation, transforming growth factor-beta (TGF-?) mRNA abundance is increased in AD patient brains. We hypothesize that the AD brain over- compensates to pro-inflammatory signals by producing these abnormally high levels of TGF-?. Paradoxically, this sets up early, low-level and chronic neuroinflammation that fails to support amyloid-? (A?) clearance. I and my team have shown in published and preliminary data that genetic or pharmacologic blockade of TGF-?- Smad 2/3 signaling in peripheral macrophages leads to brain entry of these cells and A? phagocytosis; sparing neurons from injury and restoring learning and memory. To further explore this theme, we have now generated the TgF344-AD rat that recapitulates cognitive impairment and the full array of human AD pathological features: neuroinflammation, plaques, tangles, and frank neuronal loss. In AIM 1, we will use non-invasive longitudinal imaging approaches to determine whether early neuroinflammation preempts later cognitive impairment, A? deposition, structural connectivity changes and neuronal death in TgF344-AD rats. AIM 2 is designed to longitudinally evaluate if blocking peripheral innate immune TGF-? signaling licenses A? phagocytosis and mitigates AD-like changes by delivering cutting- edge nanoparticles containing small molecule TGF-?-Smad 2/3 signaling inhibitor payload to hematogenous macrophages. Finally, we will pharmacologically delete peripheral macrophages to definitively establish if they are responsible for the beneficial effects of TGF-? signaling inhibition. While AD animal model studies are typically limited by cross-sectional designs, this project will break this barrier by coupling the most advanced multimodal, longitudinal brain imaging with peripheral TGF-? signaling inhibition in the TgF344-AD rat.