Zachary Freyberg, M.D., Ph.D. - US grants

DESCRIPTION (provided by applicant): The overall goal of this K08 Mentored Clinical Scientist Career Development Award is to provide me with the mentored training to become an independent investigator as a physician-scientist pursuing translational neuroscience research in psychostimulant abuse. My specific career goals include application of genetic, behavioral and imaging tools towards development of an improved understanding of the basic neurobiology and synaptic signaling mechanisms underlying drug abuse and addiction. To accomplish this, I propose to use the fly Drosophila melanogaster as a model system to identify relevant molecular targets efficiently and for eventual testing and validation in rodent models. The fly provides a powerful model system to study mechanisms of psychostimulant signaling given its advantage of allowing concurrent investigation of biochemical pathways at molecular and behavioral levels. In addressing gaps in my training, my K08 training goals are: 1) to develop expertise in fly neurobiology and genetics, and 2) to develop expertise in imaging pre- and postsynaptic dopamine neuronal signaling. As proposed for this award, I will use a multidisciplinary approach combining behavioral and imaging studies to investigate the molecular pathophysiology underlying psychostimulant abuse, with a focus on amphetamine (AMPH) action. Though the primary sites of action for AMPH have been identified, the downstream signaling pathways are poorly understood. My colleagues and I have shown that fly larvae respond to AMPH by crawling faster and this is dependent both on presynaptic dopamine transporter and postsynaptic dopamine (DA) D2 receptors (D2R). While postsynaptic D2R signals may be mediated by G1i/o-dependent and/or arrestin-dependent/G1i/o-independent (Arr-dependent) downstream signaling pathways, it is not known whether AMPH action is dependent on one or both pathways. Importantly, the kinase Akt behaves as an intermediary between several signaling molecules downstream of D2R known to mediate AMPH-stimulated locomotion and understanding its regulation may shed light on molecular mechanisms of AMPH action. I will test the hypothesis that AMPH stimulation of DA postsynaptic neurons in the fly leads to D2R-dependent activation of both G1i/o-dependent and Arr-dependent signaling pathways. I will address 2 specific aims in this work: 1) to test whether AMPH-stimulated locomotion is dependent on the Arr-dependent and/or G1i/o-dependent pathways using RNAi knockdown of signaling molecules in both pathways, and 2) to determine in vivo effects on Akt activity of AMPH-mediated postsynaptic D2R activation via multiphoton imaging of the Akt biosensor within the intact living fly brain. These novel approaches and findings will facilitate further characterization of AMPH's molecular actions and move us toward critically needed treatments and to better models of stimulant pathophysiology. PUBLIC HEALTH RELEVANCE: Abuse of psychostimulant drugs such as amphetamine represents a major public health problem with a broad array of adverse health consequences, including dependence, overdose and death. Although these drugs have been some of the most used and abused substances for the last century, our knowledge of their molecular mechanisms of action in the brain remains rudimentary. This Mentored Clinical Scientist Research Career Development Award (K08) will enable Dr. Zachary Freyberg to elucidate mechanisms of action for stimulants such as amphetamine and therefore open the door to novel therapeutic interventions.

Dementia with Lewy bodies (DLB) is the second most common dementia, following Alzheimer?s disease, with prevalence rising significantly in people aged 65 years and older. The core pathology in DLB is progressive pathologic accumulation of misfolded ?-synuclein in Lewy bodies that especially targets midbrain dopamine (DA) neurons and leads to their degeneration. This neuronal loss in response to synucleinopathy is an important source of vulnerability not only in DLB, but is also found in Alzheimer?s disease. Given the shared features across disorders, identifying mechanisms of DA neurodegeneration in response to synucleinopathy may provide critical insights into neuron vulnerability and resilience common to age-related neurodegenerative disorders. However, we still lack basic understanding of the mechanisms underlying neuronal vulnerability and resilience. Significantly, we have established complementary approaches comparing Drosophila and mouse models of age-related neurodegenerative disease. We exploit the similarities and differences between the two species to provide novel insights into evolutionarily conserved cellular and molecular pathways that affect age-related neurodegeneration. Using these approaches, we have identified the vesicular glutamate transporter VGLUT (VGLUT2 in mammals and dVGLUT in Drosophila) as a new modulator of resilience to neurodegeneration in DA neurons. Indeed, DA neurons that upregulate VGLUT expression are more resilient including in response to pathologic accumulation of ?-synuclein ? a conserved response in flies and mice. To elucidate VGLUT?s roles in DA neuron vulnerability, we established an innovative suite of intersectional genetic tools to selectively distinguish and manipulate DA neurons that express VGLUT in flies and mice. With these tools and models, we can now answer key questions: 1) what are the conserved mechanisms regulating expression of DA neuron VGLUT in synucleinopathy? and 2) do regulators of VGLUT expression modulate resilience to neurodegeneration caused by synucleinopathy? To address this, we take advantage of the fly?s genetic tractability to rapidly identify new targets of vulnerability and resilience and then test these candidates in mouse models of synucleinopathy. Our central hypotheses are: i) DA neuron VGLUT expression is under tight regulatory control and the genes involved are critical for VGLUT upregulation in synucleinopathy (Aim 1). ii) Upregulation of VGLUT expression is a pro-survival coping mechanism and the genes that modulate this increased VGLUT expression in DA neurons contribute to resilience in synucleinopathy (Aim 2). Using our comparative approaches, we may identify novel interventional targets conserved across species to boost resilience in DA neurons. These new targets may ultimately be tested in other cell types as a broader intervention for prevention, mitigation and treatment of neurodegeneration in DLB, Alzheimer?s disease and Alzheimer?s disease-related dementias.

Antipsychotic drugs (APDs) treat several highly prevalent psychiatric illnesses including schizophrenia, bipolar disorder and major depressive disorder, making them among the most widely prescribed medications today. Yet, APDs also cause profound metabolic disturbances including weight gain, glucose intolerance, and insulin resistance, and increase risks of type 2 diabetes (T2D) and cardiovascular disease. Significantly, all APDs cause metabolic side effects to differing degrees, and current treatments to reduce these metabolic symptoms have only limited efficacy. The mechanisms by which APDs produce metabolic disturbances are not well understood. The single unifying property of all APDs is their blockade of dopamine D2-like receptors, including D2 (D2R) and D3 (D3R) receptors, suggesting a role for these receptors in APD-induced metabolic dysfunction. Though D2R and D3R are expressed in the central nervous system in hypothalamic regions that mediate appetite and feeding behavior, interventional studies targeting these centers have not reduced APD-induced metabolic dysfunction. This suggests that APD effects on the hypothalamus do not fully explain the metabolic effects of these drugs. Notably, we and others found D2R and D3R are also expressed in human and rodent insulin-secreting pancreatic ?-cells, and dopamine inhibits glucose-stimulated insulin secretion (GSIS). This suggests pancreatic DA signaling modulates GSIS and raises the possibility that APDs also act on pancreatic endocrine cells to drive dysglycemia. Indeed, we recently found: (1) APD blockade of ?-cell D2R/D3R disrupts dopamine?s inhibition of GSIS, leading to elevated insulin secretion ? a potential driver of insulin resistance in T2D. We similarly found that ?-cell-specific D2R knockout mice exhibit hyperinsulinemia in vivo, further supporting a role for D2-like receptors as modulators of insulin release. (2) ?-cells also express D2R and D3R, and APD blockade of ?-cell D2R/D3R profoundly elevates glucagon secretion. These data are consistent with work showing APD-induced hyperglucagonemia in vivo which drives hyperglycemia. Thus, we hypothesize that pancreatic ?- and ?-cell D2R/D3R signaling is important for glucose homeostasis and disrupting this signaling leads to dysglycemia. Using new genetic and pharmacologic tools we developed, we propose to establish how D2R and D3R signaling in ?- and ?-cells regulates islet insulin and glucagon secretion. We also propose to better understand the intracellular mechanisms by which these receptors signal, and by which APDs alter intracellular signaling pathways to induce dysglycemia (Aims 1, 2). In parallel, we will examine the therapeutic potential of peripheral D2R/D3R agonism by determining if pharmacological stimulation of specifically peripheral D2R/D3R can ameliorate or prevent APD-induced dysglycemia in vivo in mice and in human islets (Aim 3). Ultimately, our work may elucidate new pancreatic D2R/D3R signaling mechanisms that APDs disrupt to produce dysglycemia, and lead to novel drugs that prevent or significantly reduce APDs? metabolic side effects.

PROJECT SUMMARY Opioid and cocaine abuse prevalence has skyrocketed in the United States, fueling the current epidemic of overdose deaths. Despite the public health impact of opioids and cocaine, we still lack a fundamental understanding of the mechanisms by which these drugs work, particularly across cellular and circuit levels. Further understanding of the neuroanatomy of the neural circuitry underlying opioid and cocaine reward is a critical initial step in targeting and elucidating their mechanisms. However, comprehensively visualizing relevant circuits in drug reward has been limited by approaches to contextualize these circuits and their response to drugs of abuse in the whole brain. We developed an approach to rapidly image the whole brain in three-dimensional (3D) space using ultra-fast high-resolution ribbon-scanning confocal microscopy. Our ribbon-scanning confocal imaging approach can image and visualize an entire rodent brain in less than 24 hours, where more conventional approaches (e.g., light-sheet) currently require days or even weeks. Furthermore, our ribbon-scanning confocal approach reaches diffraction-limited resolutions (~200-300nm), enabling us to visualize individual cells in the brain and their ultrastructure. We can apply these unique tools to begin solving the fundamental questions: 1) What is the precise circuitry that defines drug reward? And 2) What are the differential effects of cocaine and opioids on this circuitry? Like many drugs of abuse, cocaine and opioids rely on neurotransmission from dopamine (DA) neurons in the ventral tegmental area (VTA). However, until recently, parsing the connectivity of unique subpopulations of DA neurons and their potential roles in drug reward has been difficult. We developed a suite of intersectional genetic tools to definitively dissect the anatomical and functional properties of these different subpopulations within the same brain. We will integrate our 3D ribbon-scanning confocal imaging of DA neuron subpopulations with immunolabeling of neuronal activity markers to visualize precisely which DA neurons are activated in response to cocaine and opioids. Using whole brain immunolabeling and imaging, we will also visualize and map drug-dependent neuronal activity changes in the whole brain with the potential to reveal new populations of neurons differentially response to cocaine and opioids. Our overall objectives are to: Comprehensively map the distribution of DA neuron subpopulations including DA/glutamate co-transmitting cells relative to the overall DA system within whole brain (Aim 1); and to determine how cocaine and opioids differentially affect the activity of these DA neuron subpopulations (Aim 2). We will generate a comprehensive 3D brain atlas to identify the roles of unique subpopulations of DA neurons highly relevant to cocaine and opioids, which will serve as a proof of principle for the implementation of our ultra-fast high-resolution 3D ribbon-scanning confocal microscopy. Our proposal will foster future development of the first 3D high-resolution comprehensive maps of neurotransmission within in whole brain to study addiction.