Itzhak Mano - US grants

Communication between neurons in the brain depends on secretion and removal of small signaling molecules called neuro-transmitters. Recent studies provided some insights into the structure and functional mechanisms of transporters, special proteins that remove neuro-transmitters from neuronal connections. However, we still do not understand the function of transporters at the whole-animal level. In the current project, the researchers employ an intriguing approach to this question, using a small, free-living, and transparent nematode. Numerous studies in the past have shown that basic biological mechanisms are conserved through evolution from these worms to humans. The researchers will take advantage of unique features of this tiny animal, including powerful molecular and genetic tools, and the ability to visualize neuronal activity in the whole animal. They will study how the tiny nematode, with its simple nervous system, uses neuro-transmitter transporters with special structures and unusual locations to control its neuronal activity. The study will increase understanding of the differences in molecular structure of transport proteins and their effects on neuronal signaling, whole animal physiology, and the strategies used by animals to control neuronal activity.

The study will enhance cutting edge scientific studies in a public university located in the heart of Harlem, New York City. The project is design in a way that puts special emphasis on the participation of undergraduate students from this minority-rich academic environment. Furthermore, it provides exposure to state-of-the-art neuroscience research to medical students from a unique program dedicated to the education and service of minority and underprivileged communities. Together, the current research project offers a unique and powerful perspective on neuronal function in evolution, and the translation of molecular structural differences to the whole-animal physiology. Furthermore, this study will encourage and facilitate the participation of minority and disadvantaged communities in cutting edge molecular neuroscience.

Abstract: Activation of Transcriptional Neuroprotective Programs in Nematode Excitotoxicity Excitotoxicity is a leading mechanism of necrotic neurodegeneration in stroke/brain ischemia, triggered by the exaggerated activation of Glutamate Receptors (GluRs). The complex labyrinth of signaling cascades that are suggested to lead from GluR over-activation to neurodegeneration prevents full understanding of the significance of core mechanisms. Surprisingly, GluRs also act to subdue toxic events by activating signaling cascades that lead to the transcription of pro-survival genes. Though these cascades offer an escape route from excitotoxic neurodegeneration, their identity is also shrouded in doubt and controversy. A few transcription factors (TFs) such as CREB & FoxO were suggested to be involved, but the study of their role needs to be re-examined more carefully: recent data suggests CREB?s & FoxO?s mechanisms of activation might be very different than previously proposed, and each one on its own might be too widely used to offer sufficient specificity. Accurate information on the mode of CREB and FoxO activation or their ability to mount a concerted transcriptional neuroprotective program in excitotoxic necrosis is missing, partially due to the confusion caused by the extensive crosstalk and redundancy in mammalian signaling cascades. We address these gaps by studying how both CREB and FoxO are regulated during excitotoxic necrosis and how they mitigate Glu overstimulation. We use C. elegans, a powerful, genetically accessible model where signaling cascades are simplified, but their core is conserved. We make novel observations on both cascades, suggesting that the insulin/FoxO signaling cascade is a novel conduit for GluR regulation of neuroprotective transcription, and that activation of CREB in excitotoxic necrosis is non-canonical. We aim to determine if GluRs connect through Tamalin & Cytohesin to Insulin/FoxO signaling as a novel conduit to induce neuroprotection. We will use fluorescent tags and co-IP in transgenic animals to test for colocalization & interaction. We will over-express interaction domains to disrupt exiting interactions and test the effect on excitotoxicity; We further aim to determine if CREB activation in excitotoxicity is non-canonical. We will study the involvement of candidate components by KO, express phosphorylation-site mutants in transgenic animals, and monitor the effect on neurodegeneration ; Lastly, we aim to identify transcriptional targets co-regulated by FoxO & CREB that protect from excitotoxicity. We will perform a transcriptome analysis specifically in cells at risk of degeneration, compare excitotoxic to neuroprotective conditions, and select candidate targets for future research using evaluation by a well-defined set of criteria. Put together, we will illuminate the pathways used to trigger neuroprotection in response to Glu overstimulation, and detect mediators of neuroprotection in excitotoxic necrosis. Understanding the concerted neuroprotective signaling cascades and identifying conserved mediators of neuroprotection in the nematode can provide novel insights into similar processes in mammals, ultimately inspiring new therapeutic interventions in stroke / brain ischemia.

The widespread use of Glutamate (Glu) as the major excitatory neurotransmitter (NT) in the mammalian brain is both critical for normal physiology and a source of predicaments: a) As seen in stroke and a range of neurodegenerative diseases, any disruption in Glu clearance causes its accumulation, leading to over- excitation of postsynaptic cells and excitotoxic neurodegeneration. b) The use of the same NT in so many adjacent synapses can cause signaling to ?bleed over? between neuronal circuits, and the loss of processing fidelity. An idealized view of the brain describes synapses as well insulated from each other, enveloped by glia that expresses high levels of Glu clearing transporters (GluTs). However, a more realistic examination reveals that some particularly-important brain areas (e.g., hippocampus) show severely deficient glial isolation, with estimated 2/3 of released Glu seeping out of the original synapse. How sufficient Glu clearance is achieved in glia-deficient brain areas remains unclear. To overcome the limitation of current techniques we will study Glu clearance in the glia-deficient synaptic hub of the C. elegans nerve ring. We are aided by the availability of information on the precise identification of individual neurons, the exact location of their synapses, the circuits that they participate in, and the sensory inputs and behavioral outputs of these circuits. Together with animal transparency and the wide availability of optogenetic tools, this is an ideal system to study Glu clearance without perturbing interstitial fluids. In our recent studies we have discovered that specific synapses fall into watershed territories of Glu clearance, and that synapses might be affected by the agitation of body fluids. We therefore propose a novel concept, where Glu clearance in a glia-deficient synaptic hub can be robust enough to allow functional synaptic isolation. Such robust clearance depends on division of labor between proximal and distal GluTs, and is facilitated by agitation and perfusion of interstitial fluids. To provide further support to this model we will use genetically-encoded florescent Ca2+ reporters (GCaMP) to follow synaptic activity and assign additional synapses and circuits to GluT drainage territories; we will stimulate one circuit and record responses from an adjacent one to detect spillover; we will use genetically-encoded fluorescent detectors to study the flow of Glu in the interstitial space; we will study the effect of paralysis on neuronal responses and Glu flow; We will correlated the differences between the structure of proximal and distal GluTs to potential differences transport in affinity and capacity. These studies will provide novel insights to mechanisms of robust Glu clearance in the absence of glia, and highlight the significance of agitation of interstitial fluids in synaptic areas that are deficient in glia insulation, a feature shared between nematodes and some areas of the mammalian brain. These insights will aid in the design of future therapeutic interventions to prevent excitotoxicity (seen in stroke and a range of neurodegenerative diseases), and highlight the significance of vascular pulsatility in CNS physiology.

Stroke / brain ischemia is a leading cause of death, and survivors require extensive long-term rehabilitation and care. Stroke is also a major source of medical disparity. The study of the mechanism neuronal damage in brain ischemia has seen many setbacks, since some critical events typically happen before admission to the emergency department. At the molecular level, key events include synaptic accumulation of glutamate, hyper-stimulation of postsynaptic receptors, Ca2+ influx, functional mitochondrial collapse, and cellular disintegration in a process called excitotoxicity. In spite of extensive efforts to develop intervention strategies, identified canonical events take place too early to be treated in the clinic, while subsequent proposed events remain highly controversial and scenario-specific. We now study these later steps in the C. elegans animal model system, under the premise that events that are conserved through large evolutionary distances are likely to be key steps in the essential core of the degenerative process. We take advantage of the particularly powerful set of technologies available in this model system. We hypothesize that while a number of signaling cascades converge on the mitochondria to produce excitotoxic necrosis, two novel effects are particularly important: 1) we identify a scantly studied mechanism where the Ca2+ sensitive kinase DAPK cooperates with p53 to cause necrosis by translocation of p53 into the mitochondrial matrix, interaction with CypD, and opening of the mitochondrial inner membrane?s mPTP. 2) we suggest that overstimulation of the mitochondria (following the excessive depolarization of the postsynaptic neurons) causes buildup of mitochondrial lipid peroxides, leading to membrane damage and cellular necrosis by ferroptosis. Finally, we suggest that additional novel mechanisms could be identified by an unbiased screen designed to detect new, previously unappreciated mechanisms in excitotoxic necrosis. We aim to study the DAPK/mitochondrial p53/CypD/mPTP axis by combining imaging, genetic KOs, and conditional expression. We will use similar approaches to study mitochondrial ferroptosis in excitotoxicity. Depending on progress in the previous aims, we will characterize novel mutants that show suppressed or enhanced excitotoxic necrosis. We will illuminate conserved, novel, and non-immediate mechanisms of neuronal damage in excitotoxicity. We hope these insights can later be used to develop new intervention strategies in stroke, a major cause of medical disparity. Relevance to human health: This project addressed stroke, a critical unmet need in healthcare with particular significance of minority populations. The life-threatening initial condition, and the devastating effects on quality of life for stroke survivors, call for a concerted effort to find new intervention strategies. We illuminate novel non-immediate mechanisms in stroke-related excitotoxicity and address core processes likely to be conserved across many forms of this neurodegenerative process. Stroke remains a leading cause of death and disability, pressing the need to find novel intervention strategies in a clinically feasible time-frame.