Ali P. Haghighi, Ph.D. - US grants

DESCRIPTION (provided by applicant): Appropriate regulation of synaptic strength is essential for maintaining stability in neural circuits. This regulation hinges on a balance between molecular mechanisms that promote change in synaptic function in response to extracellular and intracellular cues, and homeostatic mechanisms that seek to adjust neuronal function within a normal range, ensuring stability in neural circuits circuits1-2. This proposal is designed to unravel molecular components and mechanisms that contribute to homeostatic mechanisms at the synapse. My group has been taking advantage of the Drosophila larval neuromuscular junction (NMJ) as a model synapse. When postsynaptic function is reduced at this synapse, a robust homeostatic retrograde signal is initiated in the postsynaptic muscles, which feeds back to the presynaptic motor neuron to cause a compensatory enhancement in presynaptic neurotransmitter release1. The NMJ is a particularly well-suited model for studying this feedback or retrograde signaling mechanism, since the short life cycle of flies together with the powerful genetics available in Drosophila allow for an efficient identification and characterizatio of genes and mechanisms that participate in this coordinated process. In particular, our recently published work3 as well a wealth of unpublished preliminary findings indicate that translational mechanism that control do novo protein synthesis are essential for the ability of the NMJ to induce this retrograde compensation in neurotransmitter release. In addition, we have strong preliminary data that a Parkinson's related genes interacts with translational mechanisms and thereby influences synaptic transmission at the NMJ. We have also identified potential translational targets for postsynaptic translation that may further shed light into the nature of tis signaling. Our research plan is based on a wealth of preliminary data and unpublished observations and utilizes a multidisciplinary approach that combines Drosophila genetics with molecular biology, biochemistry, imaging and electrophysiology. We have a unique opportunity for understanding how retrograde signaling operates at synapses to induce homeostatic effects. In light of the highly conserved nature of these signaling molecules, our findings hold the promise of being translated to higher organisms and pave the way for future therapeutic approaches aimed at tackling nervous system diseases.

Summary Somatic stem cells (SCs) ensure homeostasis of high-turnover tissues by adjusting their proliferative activity in response to a wide range of damage and stress signals. To guarantee efficient regeneration while also preserving the size of the SC population during regenerative episodes, SC division modes can dynamically shift between symmetrically preserving divisions, symmetrically depleting divisions, and asymmetric divisions. Such a dynamic system allows rapid responses of the tissue to changing environmental conditions, by, for example, scaling the number of SCs according to the size of the tissue. The regulatory mechanisms that allow such dynamic responses of SCs to environmental conditions remain poorly understood. The applicant proposes a project that will explore the control of intestinal stem cells (ISCs) of the Drosophila gut, and is designed to specifically address how different ISC division modes are regulated in response to nutrient and stress signals. Based on preliminary studies, the applicant hypothesizes that oscillations in the intracellular concentration of Ca2+ are influenced by stress and nutrient signals and that the cytosolic Ca2+ concentration serves as an integrating signal to elicit dynamic responses of ISCs to changing environmental conditions. To test this hypothesis, the applicant proposes studies that take advantage of the genetic accessibility of Drosophila ISCs and will combine live imaging and transcriptome analysis to probe ISC responses to genetic and environmental perturbations. Specifically, the work will (i) explore the control of asymmetric and symmetric ISC divisions in response to nutrients and stress, (ii) test whether a signaling pathway regulating the Ca2+- responsive transcription factor CRTC integrates stress and dietary signals to control ISC activity, and (iii) assess whether ISCs are regulated by cooperative regulation of gene expression by CRTC and other signal-responsive transcription factors. The Drosophila ISC system has provided rich insight into stem cell regulation and the maintenance of tissue homeostasis. The regulatory processes in this system are evolutionarily conserved. Understanding the role of Ca2+ signaling in ISCs in the context of adaptive tissue growth, homeostatic regeneration, and epithelial stress responses is thus likely to provide significant new leads for possible therapies of human diseases, including intestinal cancers and inflammatory diseases.

PROJECT SUMMARY/ABSTRACT Alzheimer?s disease (AD) is the 6th leading cause of death in the United States, and is estimated to cost more than $200B/year. Uniquely among the top ten causes of death, we have little ability to treat or prevent the disease. Although the precise etiology of AD is still under investigation, strong evidence suggests that inflammation plays a critical role in the progression of the disease. Chronic low-grade inflammation increases systemically with age and correlates with most age-related diseases. This includes AD. One recognized driver of the age-related increase is systemic inflammation. Accumulating evidence indicates that loss of peripheral proteostasis could be a key source of systemic inflammation; in particular experimental data in model organisms show that proteostatic decline in both the intestine as well as skeletal muscle could be important sources of systemic inflammation, which in turn triggers inflammatory signaling in the central nervous system (CNS), affecting health, function and behavior, as well as A? plaque deposition and microglial activation. This supports a connection between intestinal-initiated inflammation and AD, but mechanistic insights remain poor. Here, the applicants propose to test the hypothesis that age-related proteostatic decline in the intestinal epithelium as well as in skeletal muscle are sources of inflammatory cytokines that result in elevated inflammation in the central nervous system (CNS) and thus increases AD progression and pathology. To test this hypothesis, the applicants propose studies in fruit flies, which allow tissue-specific genetic perturbations and characterization in exquisite detail. Furthermore, Drosophila neurobiology is well-described, several AD models are available, and most signaling pathways involved, including the ER unfolded protein response (UPRER) and the JAK/STAT inflammatory pathways, are conserved, but show less redundancy and complexity in flies than in vertebrates. Published work and preliminary data generated by the applicants suggest that age-related dysfunction of the intestinal microbiota is associated with strong chronic activation of the UPRER in the intestinal epithelium, and causes inflammation in the brain, influencing neuronal health and function in a Drosophila model of AD. In addition, applicants present preliminary data that links synaptic calcium influx to the regulation of muscle proteostasis as a novel mechanism to control age-related decline of muscle proteostasis. Using these novel methods, the preliminary findings by the applicants demonstrate that attenuating muscle proteostasis in aged flies can decrease the inflammatory load in the brain, improve locomotion behavior and extend lifespan. The applicants propose a mechanistic study to delineate the signaling pathways and physiological consequences of the loss of intestinal and muscle proteostasis on the brain. Based on the conserved signaling mechanisms studied, it can be anticipated that the findings of this study will pave the way for the development of novel interventions that could alleviate the symptoms of AD.

PROJECT SUMMARY Age-related neurodegenerative diseases like Alzheimer?s Disease exhibit a breakdown in neuronal protein homeostasis (proteostasis). The relationship between age-related metabolic dysfunctions and protein aggregation in such diseases remains poorly understood. Elucidating the molecular basis for the age-related loss of proteostasis is expected to inspire new therapeutic approaches, not only for diseases like Alzheimer?s, but more broadly for a wide range of age-related diseases. Increasingly, this promise of ?Geroscience? is being recognized as critical for extending our healthspan. Among the most productive experimental approaches in Geroscience is the use of genetically accessible model systems for the study of longevity. These models have allowed the identification of single gene mutations and of interventions that extend lifespan, highlighting the plasticity of the aging process and suggesting avenues to significantly alter its course. The cellular events impacted by such interventions and causing the lifespan extension, however, remain largely unclear. In many cases, the effect on longevity is associated with changes in proteostasis and metabolism. To understand the relationship between proteostasis and longevity in detail, however, requires an integrated approach that investigates the effects of lifespan extending perturbations on global protein homeostasis and metabolic flux in a well-defined genetic system. Here, the applicants propose such integration by combining the expertise of groups using genetic approaches (Jasper), proteomic and bioinformatic approaches (Schilling and Ghaemmaghami), and metabolomic approaches (Ramanathan) to develop models for protein and metabolic homeostasis in long-lived mutants of Drosophila. Recent technological advances in the field of mass spectrometry have enabled global analyses of protein turnover rates and metabolic flux in complex organisms. Combining these technologies with detailed analysis of lifespan-extending genetic perturbations is expected to provide transformative new insights into molecular changes required for longevity. The aims proposed by the applicants are to (i) assess age-related changes in global protein turnover and metabolic flux, (ii) determine if changes in energy metabolism downstream of the Jun-N-terminal Kinase and Insulin signaling pathways influence protein turnover, and (iii) perform genetic studies to explore the causes of aging and longevity. It is anticipated that combining the strengths of the Drosophila system with state-of-the-art proteomic and metabolomic approaches will significantly accelerate the discovery of fundamental mechanisms influencing physiology and cell function with age, providing new therapeutic avenues for age-related diseases.