Cell senescence is an irreversible proliferation arrest caused by a variety of stresses. It is characterized by the secretion of proinflammatory factors, the senescence associated secretory phenotype (SASP), which is believed to contribute to an important hallmark of aging, a chronic inflammatory state known as 'inflammaging' (1). The parent grant elucidated a novel mechanism which propagates cell senescence and its SASP and thus promotes inflammaging. This mechanism involves retrotransposable elements (RTEs), and in particular the long inter- spersed nuclear elements (LINE-1, or L1). These elements are activated in senescent cells and induce a type-I interferon (IFN-I) response, which in turn drives the SASP. The IFN-I response is triggered by innate immune system DNA sensors that detect cytoplasmic L1 cDNAs. The 4th and final aim of the parent grant was to explore possible strategies for therapeutic interventions. The class of drugs known as nucleoside reverse transcriptase inhibitors (NRTIs) were developed to treat HIV, and we found that several well-tolerated NRTI drugs potently inhibit the L1 reverse transcriptase (RT) enzyme. We have shown that treatment of senescent cells with NRTIs blocks the synthesis of L1 cDNA and induction of an IFN-I response (2, 3). More importantly, treatment of aged mice with NRTIs downregulated inflammaging in several tissues. Alzheimer's disease (AD) and AD-related dementias (ADRDs) are associated with neuroinflammation, which establishes an environment in the brain that is hostile for the function and survival of neurons. Many brain cell types, astrocytes and microglia in particular, can become senescent. The frequency of senescent cells increases in Alzheimer's and ADRDs (4-6), and removing senescent cells in mouse models of Alzheimer's reduces neuroinflammation, alleviates pathology and improves cognitive function (6, 7). While it is not yet clear if neuroinflammation is a cause of Alzheimer's, it is increasingly believed that alleviating neuroinflammatory processes might slow down Alzheimer's disease progression. On the basis of the research performed by the parent grant, as well as extensive evidence in the published literature, we believe that NRTI drugs should be investigated for repurposing to treat Alzheimer's and ADRDs. NRTIs have excellent bioavailability and blood brain barrier (BBB) permeability, and recent generations, such as Emtricitabine (FTC) show both high efficacy and safety. Based on the above premise, I applied, in collaboration with Stephen Salloway, MD, Director of the Memory and Aging Program at Butler Hospital (an affiliate of Brown Medical School), to the Alzheimer's Association of America to conduct a phase 1b clinical trial to establish the safety and tolerability of Emtricitabine in a geriatric population with mild cognitive impairment or early Alzheimer's. Our application was approved, see: www.brown.edu/news/2019-12-10/alzgrant; www.alz.org/news/2019/researchers-add-hiv-aids-diabetes-and-organ-trans; www.usnews.com/news/best-states/rhode-island/articles/2019-12-10/rhode-island-hospitals- brown-to-research-alzheimers-drug This supplement aims to accelerate the clinical translation of NRTIs for the treatment of AD and ADRDs. A key missing component are sensitive and accurate assays to directly detect and monitor L1 in accessible human specimens such as plasma or cerebrospinal fluid (CSF). These tools are needed not only to assess the presence and activity of L1 in disease, but also the effect of therapy on downregulating L1 (target engagement). This supplement seeks funds for the development of in vitro assays to detect the presence of L1 replication intermediates. It is within the scope of the parent grant because: 1) It will exploit our expertise of RTE biology which stems from the pursuit of the parent grant; 2) The experiments are based directly on the results obtained under the parent grant. In fact, many of the assays we propose to further develop and adapt here were already successfully used on human fibroblasts in the parent grant. The biological fluid specimens, CSF and plasma, will be de-identified and from deceased individuals. Their use thus does not constitute human subjects research. None of the funds from this supplement will be used for human subjects research, clinical tests, or patient care. Abstract of Proposed Supplement NRTI drugs are the mainstay of HIV therapy and are in general well tolerated. They are approved for clinical use, and hence can be explored for repurposing to treat conditions associated with inflammaging, in particular Alzheimer's disease and AD-related dementias. The drug we have chosen is Emtricitabine (marketed as Emtriva by Gilead Sciences), which is the most advanced and well tolerated NRTI. We have shown it also potently inhibits L1 RT. The crucial immediate need for further development are assays to directly assess L1 levels and activity in biospecimens (CSF, plasma). The goal of this supplement is to lay the groundwork for sensitive assays to quantify levels of L1 transcripts (mRNA) and cDNA (RNA/DNA heteroduplexes). These methodologies will form the basis for target engagement assays that will be needed to assess efficacy of treatment in future human trials.
We will develop in vitro assays to assess the expression of retrotransposable elements (RTES) in human biological fluids such as cerebrospinal fluid (CSF) and blood plasma. The assays will target replication intermediates of the elements. The health relevance of these assays will be to quantify the activation of RTEs in Alzheimer's disease (AD) and AD-related dementias (ADRDs), and their response to therapy.
