Terrence C. Town, Ph.D. - US grants

PROJECT SUMMARY While amyloid plaques and neurofibrillary tangles are Alzheimer's disease (AD) defining features, Alzheimer himself originally identified a third pathology? inflammation of the brain's glial support cells. Neuroinflammation in AD is characterized by reactive astrocytes and microglia that surround amyloid plaques and chronically secrete inflammatory innate immune cytokines. The dominant view for decades has been that all forms of inflammation damage the AD brain. Yet, non-steroidal anti-inflammatory drugs failed to produce a positive signal for AD primary prevention. This raises a fundamental question: should we be blocking or possibly even promoting inflammation as an AD therapeutic? While the focus has mainly been on pro-inflammatory cerebral innate immunity, little attention has been paid to factors that curtail peripheral innate immune responses. The unifying theme of our work is that `rebalancing' peripheral innate immunity to homeostasis by releasing immunosuppression will limit AD progression. Strikingly, our focus on innate immunity in AD has just recently been validated by genome-wide association studies. These results have taken the field by storm; identifying clusters of AD risk alleles in core peripheral macrophage pathways. As a key cytokine suppressor of innate immunity and inflammation, transforming growth factor-beta (TGF-?) mRNA abundance is increased in AD patient brains. We hypothesize that the AD brain over- compensates to pro-inflammatory signals by producing these abnormally high levels of TGF-?. Paradoxically, this sets up early, low-level and chronic neuroinflammation that fails to support amyloid-? (A?) clearance. I and my team have shown in published and preliminary data that genetic or pharmacologic blockade of TGF-?- Smad 2/3 signaling in peripheral macrophages leads to brain entry of these cells and A? phagocytosis; sparing neurons from injury and restoring learning and memory. To further explore this theme, we have now generated the TgF344-AD rat that recapitulates cognitive impairment and the full array of human AD pathological features: neuroinflammation, plaques, tangles, and frank neuronal loss. In AIM 1, we will use non-invasive longitudinal imaging approaches to determine whether early neuroinflammation preempts later cognitive impairment, A? deposition, structural connectivity changes and neuronal death in TgF344-AD rats. AIM 2 is designed to longitudinally evaluate if blocking peripheral innate immune TGF-? signaling licenses A? phagocytosis and mitigates AD-like changes by delivering cutting- edge nanoparticles containing small molecule TGF-?-Smad 2/3 signaling inhibitor payload to hematogenous macrophages. Finally, we will pharmacologically delete peripheral macrophages to definitively establish if they are responsible for the beneficial effects of TGF-? signaling inhibition. While AD animal model studies are typically limited by cross-sectional designs, this project will break this barrier by coupling the most advanced multimodal, longitudinal brain imaging with peripheral TGF-? signaling inhibition in the TgF344-AD rat.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common dementia, and is hallmarked by deposition of amyloid-¿ peptides as 'senile' ¿-amyloid plaques, neurofibrillary tangles comprised of abnormally phosphorylated tau protein, and neuronal dysfunction and loss. Currently available AD treatments have a quantitatively minor impact on the disease, doing little to improve the quality or duration of life of patients suffering from thi debilitating illness, which is clinically characterized by loss of pneumonic and higher cortical functions. A critical lynchpin for the development of an AD treatment that is both effective and safe is model systems that faithfully recapitulate the human syndrome. In this regard, transgenic mice harboring mutations in one or more genes that cause early-onset familial AD (fAD) have been enormously helpful, both in terms of interrogating potential therapeutic targets and also for understanding pathological mechanisms of disease. Yet, these models are necessarily limited due to their species, and it remains an open question as to whether the mouse will ever be able to faithfully model AD neuropathology as it occurs in the human. The central theme of our R21 grant application is to use an emerging technology with great promise for modeling human diseases: human induced pluripotent stem (hiPS) cells. The basic steps involve culturing skin fibroblasts from individuals bearing mutations in genes that cause fAD or from age-matched control relatives lacking disease, and reprogramming them into hiPS cells that are later differentiated into forebrain glutamatergic neurons. Once differentiated, these forebrain neurons will be functionally interrogated to specifically assess pathologic hallmarks of human AD. We propose to carry out this work in two parts. The focus of Specific Aim 1 is to establish iPS cell lines from four fAD mutant and four related control fibroblast cell lines. We will draw fibroblasts from ~175 lines derived from individuals bearing fAD mutations and age-matched control relatives, maintained through the NIH/NIA Aging Cell Culture Repository at the Coriell Institute for Medical Research. The main goal of Specific Aim 2 is to interrogate Alzheimer phenotypes in fAD mutant vs. control forebrain neurons differentiated from reprogrammed iPS cells. In Sub-Aim 2a, we hypothesize that Alzheimer disease phenotypes will occur and be exacerbated by experimental induction of excitotoxicity in fAD mutant vs. non-mutant hiPS-derived forebrain neurons. Sub-Aim 2b will test proof-of-concept for whether the current standard of care AD drug, memantine, will at least partially rescue Alzheimer phenotype(s) in differentiated fAD mutant forebrain neurons. Completion of this exploratory work is expected to lead to a 'disease-in-a-dish' model of human fAD. Such a model could pave the way toward understanding both basic pathologic mechanisms of the disease as well as potential therapeutic approaches. PUBLIC HEALTH RELEVANCE: There are now over 3 million Americans afflicted with Alzheimer's disease, a figure that is projected to increase to 9 million by 2050, underscoring a rapidly developing public health crisis. We propose to utilize cutting-edge human induced pluripotent stem (hiPS) cell technology to model this devastating disease in cultured neurons. If successful, this exciting disease-in-a-dish model could allow pre-clinical testing of therapeutic approaches.

? DESCRIPTION (provided by applicant): Pediatric brain tumors are the most common malignant solid tumors in children. Despite modern treatments having increased survival, current therapeutics often carry debilitating side effects with them, including lifelong intellectul and neurological disability. Because of this, there is a pressing need for novel therapeutic approaches. While transforming growth factor-beta (TGF-ß) is one of the most well-studied immune molecules in cancer biology, in regards to pediatric brain cancer, precious little is known. We recently showed that genetic blockade of T cell TGF-ß signaling promotes CD8 cytotoxic T lymphocyte (CTL)-mediated anti-tumor immunity in the smoothened (SmoA1) mouse model of medulloblastoma (MB). Furthermore, inhibiting T cell TGF-ß signaling led to generation of CD8+ short-lived effector T cells (SLECs) that were efficient tumor cell serial killers. These results uncover a cellular immune mechanism whereby TGF-ß signaling blockade licenses CD8 SLEC-CTLs to kill pediatric brain tumors. The central theme of this revised exploratory grant application is to develop an experimental pharmacological approach for the treatment of MB. Specifically, we seek to test whether nanoparticle-based blockade of TGF-ß signaling in the key cellular mediator of anti- tumor immunity, the CD8 T cell, is a viable MB therapeutic approach. To achieve this goal, we will: 1) test whether CD8 targeted nanoparticle inhibition of TGF-ß signaling in peripheral T cells will prevent or treat established MB in SmoA1 mice, and 2) elucidate the mechanism of CD8 SLEC-CTL activity in nanoparticle-treated CD8 T cells. Through a collaborative effort, we have already developed CD8- targeted nanoparticles consisting of the biodegradable polyester, poly(ethylene glycol) and poly(lactic-co-glycolic) acid (PEG-PLGA), encapsulating the small molecule TGF-ß-Smad 2/3 signaling inhibitor (SB-505124) and the non-toxic fluorescent tracer, Coumarin-6 (C6). In Specific Aim 1, we will utilize this pharmacologically-relevant method to target TGF-ß signaling in CD8 T cells in SmoA1 mice. Both disease prevention and active treatment study arms will be carried out. For Specific Aim 2, CD8 T cells will be isolated from SmoA1 animals treated with C6 or C6/SB nanoparticles and tumor cell killing will be read-out by an established CTL assay. Western blot analysis will be performed on isolated CD8 T cells from these animals to verify TGF-ß signaling inhibition. Immunophenotyping will be carried out to determine if CD8 T cell TGF-ß signaling blockade promotes conversion to SLEC-CTLs. We hypothesize that nanoparticle-mediated TGF-ß pathway blockade in CD8 T cells will prolong survival in a pre-clinical animal model of pediatric brain cancer by promoting the SLEC-CTL anti-tumor immune response.

PROJECT SUMMARY/ABSTRACT While amyloid plaques and neurofibrillary tangles are Alzheimer?s disease (AD) pathognomonic features, Alzheimer himself originally identified a third pathology?glial inflammation. AD neuroinflammation is characterized by reactive astrocytes and microglia that surround amyloid plaques and chronically secrete low levels of pro-inflammatory innate immune cytokines. The dominant view for decades has been that all forms of inflammation damage the AD brain. Yet, the simplistic notion that blocking inflammation is beneficial has not held up?non-steroidal anti-inflammatory drugs failed to produce a positive signal for AD primary prevention. This raises a fundamental question: could the innate immune system actually be harnessed as an AD therapeutic? While the focus has squarely been on pro-inflammatory molecules, little attention has been paid to factors that suppress the innate immune response. The unifying theme of our work is that ?rebalancing? cerebral innate immunity by releasing immunosuppression will limit AD progression. Our focus on innate immunity in AD has recently been validated by genome-wide association studies, which took the field by storm by identifying a cluster of AD risk alleles belonging to core innate immune and inflammation pathways. Toll-like receptors (TLRs), germline-encoded sensors of invading pathogens and endogenous danger- associated molecular patterns (DAMPs), are largely responsible for innate immune responses. Mounting evidence has shown that amyloid-? acts as a DAMP to provoke microglial TLR signaling. TLRs transduce their signals through interleukin-1 receptor-associated kinases (IRAKs), and IRAK-M is the only IRAK family member that suppresses TLR signaling. Importantly, IRAK-M is selectively expressed by mononuclear phagocytes (e.g., microglia and macrophages). Our preliminary data demonstrate that the IRAK-associated TRAF6/MEKK3 pathway is abnormally elevated in human AD, and IRAK-M deletion licenses A? phagocytosis. In this proposal, we will relieve TLR signaling inhibition by genetic deletion of IRAK-M in the APP/PS1 mouse model of cerebral amyloidosis. Our overarching hypothesis is that releasing IRAK-M inhibition of innate immunity will beneficially activate amyloid-? phagocytosis and restore cognitive function. In Specific Aim 1, cognitive impairment, AD-like pathology and A?/?-amyloid phagocytosis (using our novel q3DISM technology) will be evaluated in APP/PS1 x IRAK-M deficient mice. Further, we will isolate brain monocytes and perform innate immune phenotyping by transcriptomics (RNAseq). In Specific Aim 2, we will determine the relative contribution(s) of peripheral verses central innate immune compartments with bone marrow chimeras. Completion of this project will lead to a deeper, basic understanding of innate immunity in the context of AD.

PROJECT SUMMARY Microglia can be neuroprotective by phagocytizing amyloid-? (A?), but shift to a destructive phenotype that likely contributes to Alzheimer?s disease (AD) evolution. While greatest attention has been directed toward pro-inflammatory molecules in AD, we have demonstrated that blocking innate immune suppressors mitigates AD-like pathology. Genome-wide association studies have validated the cardinal anti-inflammatory cytokine, interleukin-10 (Il10), as a risk factor for late-onset AD. We show that Il10 deficiency activates mononuclear phagocytes that restrict A? deposits and preserve synaptic integrity and cognitive function. Yet, there is a gap in our knowledge regarding the impact of IL-10 receptor and its proximal downstream mediator, STAT3, on innate immunity in AD. Our overarching hypothesis is that blocking IL-10/STAT3 signaling will return the innate immune system to a physiological state to clear cerebral amyloid and restore cognitive function. We hypothesize that ?re-balancing? the brain inflammatory response to homeostasis by inhibiting innate immune IL-10/STAT3 signaling will restrict Alzheimer-like disease. We have already localized Il10 expression to plaque-associated glia with innate immune properties in the APP/PS1 mouse model of cerebral amyloidosis. AIM 1: To evaluate if mononuclear phagocytes are the major responders to IL-10 in the AD context, we will conditionally and inducibly delete (by cre/lox recombination) Il10 receptor (Il10r) or Stat3 in the APP/PS1 innate immune system. Mice will be assessed for behavioral impairment, synaptotoxicity, AD-like pathology and A? phagocytosis by quantitative 3D in silico modeling (q3DISM). RNAseq and ChIPseq on isolated populations of CNS or peripheral mononuclear phagocytes will inform expression and epigenetic modification of hub genes responsive to Il10r or Stat3 deletion. AIM 2: To separate systemic from CNS input to Il10r-dependent resolution of AD-like pathology, we will cre/lox inducibly delete Il10r in CNS resident vs. peripheral innate immune compartments of APP/PS1 mice. Brain Il10r deletion will be accomplished with a Cx3cr1-cre/lox approach. Head-sparing bone marrow chimeras will be combined with a Csfr1-cre/lox genetic approach to restrict Il10r deletion to peripheral mononuclear phagocytes in APP/PS1 mice. Cognitive impairment and AD- like pathology will be quantified. AIM 3: To assess if systemic or CNS STAT3 inhibition in a human AD rat model rebalances innate immunity to restrict Alzheimer-like disease, TgF344-AD rats developing the full spectrum of AD-like pathologies will be utilized. To distinguish central vs. peripheral innate immune involvement in this scenario, mononuclear phagocyte-targeting nanoparticles loaded with STAT3 inhibitor will be delivered directly into the brain or into the periphery in disease prevention or treatment paradigms. Cognition, AD-like pathology, and A? phagocytosis will be evaluated. This work will define the cellular and molecular mechanisms of innate immune IL-10/STAT3 signaling in AD.