Stephen J. Crocker - US grants

? DESCRIPTION (provided by applicant): Proteomic and Functional Analyses of Astrocyte Exosomes Astrocytes are the most abundant cell type in the central nervous system (CNS). Over the past decade increasing attention has been paid to the critical homeostatic and diverse roles astrocytes play in the CNS health and disease. The emerging influence of astrocytes in CNS physiology and pathology has been highlighted by how factors secreted by astrocytes impact CNS development, synaptic transmission, neurodegeneration, or infection. Hence, understanding how astrocytes respond to and are regulated by extracellular cues is expected to identify fundamental mechanisms that govern how the CNS adapts and responds to injury or inflammation. Exosomes are a class of very small extracellular vesicles that are known to be secreted by many cell types. Recent studies have now identified critical functions for exosomes in mediating cell signaling and inflammatory responses. However, little is known about the function of exosomes released from astrocytes in response to inflammation and no previous study has detailed what proteins are contained in astrocytic exosomes. This gap in our knowledge will be addressed in this application as follows: Specific Aim 1 will generate a proteomic map of the proteins contained within exosomes isolated from astrocytes, under basal and inflammatory conditions, using a cutting edge proteomic fractionation platform. Specific Aim 2 will determine the function of astrocyte exosomes as putative mediators of astrocytic responses to inflammatory stimuli both in vitro and in vivo. We provide preliminary data that demonstrate a qualitative and quantitative increase in the release of exosomes from cultured astrocytes in response to treatment with interleukin-1beta. Importantly, we present data to indicate that astrocyte-derived exosomes are present within systemic circulation and can be collected and quantified from blood serum. We hypothesize that exosomes represent a fundamental component of the astrocytic response to inflammatory and neuropathological conditions. Results from these studies will provide a new functional basis to understand how exosomes define astrocyte function. This work will provide the scientific community with two shared outcomes: (1) we will produce a proteomic map of astrocytic exosomes under basal and inflammatory conditions that might lead to high fidelity biomarker identification, and (2) we will determine the functional role of astrocytic exosomes in in vitro and in vivo models. Thus, these studies may stimulate widespread interest in astrocyte-derived exosomes as mediators of intercellular communication in the CNS in health and disease.

Abstract Myelination in the CNS is important for many higher order brain functions such as cognition, memory and complex motor learning behaviors. Loss of CNS myelination in diseases like multiple sclerosis (MS) represents a critical pathological event. Chronic demyelination is linked with disease progression making remyelination a therapeutic target for addressing progressive MS. Loss of bladder control is a pervasive issue among multiple sclerosis (MS) patients. It is also a leading cause of hospitalization among this patient population. It is estimated that nearly all MS patients will experience lower urinary tract dysfunction. Since symptom prevalence increases with disease duration, every MS patients will eventually experience urgency to urinate, urinary incontinence, frequency of urination, and/or retention of urine. Urinary dysfunction in MS patients remain a difficult therapeutic challenge because the etiology of bladder dysfunction among these patients is not well understood. While specific bladder dysfunction likely varies from patient-to-patient and with age, sex and history of urinary tract infections, it is generally thought that demyelination indirectly affects the bladder reflex and contributes to problems of urine storage (frequency) and emptying (retention). However, our new data presented in this proposal support bladder dysfunction as a direct corollary of CNS demyelination. Further, we hypothesize that CNS myelination may underlie the therapeutic efficacy of antimuscarinic agents which are mainstay therapy for patients with neurogenic and non-neurogenic bladder control disorders. Interestingly, an in parallel with the therapeutic effects of anti-muscarinic effects on remyelination in human MS patients, these agents take several weeks to become maximally effective among even non-MS patients with urinary disorders. Because anti-muscarinic agents have been implicated as a means to stimulate oligodendrocyte maturation, and these agents can stimulate central nervous system (CNS) remyelination in MS patients, we propose that CNS myelination underlies the development of bladder dysfunction. Therefore, bladder function could therefore serve as a surrogate marker for evaluating the efficacy of CNS remyelinating therapies. Hence, the objective of this study will be to determine whether CNS myelination underlies the therapeutic effects of anti-muscarinic treatments on bladder function in a model of CNS demyelination. We hypothesize that CNS demyelination is responsible for bladder dysfunction and the clinical benefits of muscarinic antagonists that are clinically measured as enhanced control of bladder function, are mediated through remyelination by oligodendrocytes. The results of this study will address several salient questions: (Aim 1) we will determine whether CNS demyelination impairs bladder function, (Aim 2). we will establish whether oligodendrocytes are required for the anti-muscarinic treatment effects on bladder dysfunction. Together these data are expected to impact our fundamental understanding of how the CNS, and CNS myelination specifically, contribute to the regulation urinary function and therein generate new knowledge on the basis of bladder dysfunction in disease.