Jose F. Abisambra - US grants

There is a fundamental gap in understanding why tau causes memory impairment in 20 known tauopathies. One pathological mechanism involves the association of aberrant tau with ribosomes. However, the consequences of this interaction are unknown. The long-term goal of this work is to better understand the link between tau abnormalities and memory impairment. The overall objective of this proposal is to determine the impact of pathological tau on ribosomal function. We will use in vitro and in vivo models that encompass studies of ribosomes in isolation, RNA translation and protein synthesis in cells, and protein production in mice brains. Our preliminary results substantiate that the tau-ribosome association reduces protein synthesis. Therefore, the central hypothesis is that pathological tau inhibits ribosomal function. The rationale for the proposed research is that understanding the tau-mediated mechanism of ribosomal dysfunction will aid in the design of therapeutic targets for tauopathies, which currently afflict a vast majority of the aging population. Our strong preliminary data serves as support for our hypothesis, which will be tested in the following specific aims: 1) Determine the extent to which endogenous pathological tau reduces ribosomal efficiency in vivo; and 2) Measure the effect of exogenous, tauopathic brain-derived tau seeds on ribosomal efficiency. Aim 1 is to measure the effects of endogenous pathological tau on ribosomes using rTg4510 tau transgenic mice. In this model, tau expression can be regulated with doxycycline. Tau expression will be manipulated in these mice, and protein synthesis will be measured in the brain using SUnSET in vivo, which has not been previously done. The anticipated results are that pathological tau levels will be inversely proportional with protein synthesis. Aim 2 is to investigate the impact of exogenous pathological tau on ribosomal function. We will isolate the most toxic tau variants from human tauopathic brains. In addition, we will isolate tau from brains at different stages of Alzheimer's disease and related tauopathies. The toxic tau species will be incubated with ribosomes in a cell-free environment (2.1), cell-based assays (immortalized and primary neurons) that measure protein synthesis: photoconversion-based method and non-radioactive pulse assay (SUnSET). These aims have the potential of extrinsic merit to be used as screening tools for modulators of ribosomal function. Our approach is innovative because it incorporates novel assays, which offer excellent sensitivity that is not achievable by more traditional approaches. This work is significant because it departs from the status quo by testing a new mechanism in which ribosomal function mediates tauopathic symptoms. This work is expected to advance the field by filling the gap in understanding of tau-mediated brain dysfunction. This knowledge will serve to better characterize the link between tau and memory impairment in order to develop novel therapeutic strategies.

AD and related diseases are the most crippling cognitive threat to our aging population. There is no cure for AD, and this is partly due to poor understanding of how aging and inflammation impacts tau pathogenesis. By 2050, it is expected that the United States will spend $1.2 trillion to maintain the constantly deteriorating quality of life of 16 million Americans with AD (Thies et al., 2013). As a result, there is an urgent need to identify effective therapeutic interventions with clear molecular mechanisms that ameliorate risk for AD in order to find therapeutic targets. Age is the primary risk factor for neurodegenerative diseases. One factor thought to contributes to a loss of resilience to pathological insults, such as tau, with age is a background of inflammation (Heneka et al., 2015). Unfortunately, the underlying molecular alterations that lead to inflammation with age and therapeutic approaches to improve resiliency are not fully understood (Bennet et al., 1996; Niccoli and Partridge, 2012; Michaud et al., 2013; Moll et al., 2014). One of the major contributors to this aging ?environment? is an activation of the innate immune system. We show that age is a critical factor contributing to tau pathology Further, we propose exosomes from human adipose derived stem cells (hASC?s) as a novel intervention to modulate inflammation that will be a useful therapeutic for AD and related tauopathies. The secretome of stem cells, including exosomes are powerful modulators of inflammation: Human adipose-derived stem cells (hASC?s) manifest a secretome containing exosomes that is capable of modulating immune function as part of their mechanism of action (Kim et al., 2016; Long et al., 2017). Of the cargo secreted in hASC exosomes, lncRNA MALAT1 controls key biological processes including modulating immune cell expression of TNFa and IL-6 following LPS stimulation (Zhao et al., 2016). The overall objective is to establish the therapeutic impact of hASC exosomes on tau pathogenesis in the background of aging. We will evaluate this in models of tauopathies, using age as a primary variable. We will express tau at different ages and evaluate functional outcomes, pathology, microglial phenotype. hASC exosome treatment will be induced with the hypothesis that this will reduce tau pathology. We will explore the molecular targets of hASC exosomes with age in microglia using proteomic profiling and ex vivo study of microglial function. We will further address the effect of age and hASC exosomes on tau pathology and tau spreading. Our data show that hASC exosomes reduce tau phosphorylation in Tg4510 mice. Thus, there is an intersection of direct and indirect actions on tau pathogenesis that is influenced by age.

This study will establish the molecular mechanism linking mild repetitive traumatic brain injury (mrTBI) and onset of tau pathology that is associated with Alzheimer?s disease (AD). Our preliminary data suggest that endoplasmic reticulum stress is a notable and long-lasting cascade that is activated by injury. ER stress activates a protein called PERK, which is responsible for initiating protective pathways that help restore ER function. However, long- term activation of PERK leads to cell death. Brain cells are particularly susceptible to PERK-mediated cell death. Indeed, a common sign between TBI and AD is PERK hyperactivity. We recently established that another com- mon pathological hallmark of TBI and AD, abnormal aggregation of the protein tau, is driven by chronic activation of PERK. PERK induces tau to adopt toxic conformations that are associated with disease. Therefore, the overall hypothesis of this project is that TBI induces long-lasting activation of PERK, which in turn catalyzes the formation of pathological tau species. This ultimately leads to increased risk for AD. We will test our hypotheses using mouse models in two aims. In Aim 1, we will determine the conditions under which mrTBI causes activation of PERK. To accomplish this objective, mice will be subjected to mrTBI at different intensities and for different time points, and the levels of active PERK will be measured. In addition, we will determine the extent of tissue that shows PERK activity. In Aim 2, we will manipulate PERK activity in mouse models of tauopathy that have suffered mrTBI. We expect that PERK activation will cause more tau pathology and induce damage to brain function. Conversely, PERK inhibition will restore brain function and prevent tau pathology. Aim 3 will determine the va- lidity of using PERK as a biomarker of TBI. Our preliminary data suggest that individuals who suffered one or more TBIs in their lifetime have two times more active/total PERK ratio in their blood. These data support our enthusiasm to expand our studies into a much larger cohort. If successful, this grant will not only identify a molecular mechanism that links injury and AD, but it will also highlight a key pathological pathway replete with therapeutic targets. Logical extensions of these studies involve testing inhibitors of the PERK pathway for po- tential therapeutic value. It will also offer relief to the 1.7 million people in the United States who suffer a TBI annually. Our expertise in ER stress, PERK, tau, AD, and TBI makes us uniquely suited to accomplish the pro- posed work. In addition, the unique resources available to my lab, such as small animal MR imaging, cohort biospecimens and clinical histories, and the UF Viral Production Core have strengthened the impact of our work and brought us closer to understanding the mechanisms of tau-mediated neurotoxic events stemming from the ER.