Different pharmacological and non-pharmacological interventions have been suggested for the management of blood glucose, among which are sodium-glucose cotransporter-2 (SGLT-2) inhibitors and exercise. SGLT-2 inhibitors (-gliflozins) are a class of drugs that reduceglucose reabsorption from the proximal renal tubule, thereby decreasing its levels in the blood, while increasing its excretion in the urine. Several studies have demonstrated cardio-, neuro-, and renoprotective benefits of these drugs, including reduced cardiovascular deaths, hospitalizations due to heart failure, rate of progression of kidney disease, cognitive deficits, reactive oxygen species, and progression of amyloid beta (Aβ) formation. Several additional mechanisms of action have been proposed for the observed benefits of SGLT-2 inhibitors. Irisin is a myokine released by skeletal muscles in response to exercise that results in browning of adipose tissue, that has been shown to have similar benefits to SGLT-2 inhibitors. Recent studies have shown that SGLT-2 inhibitors upregulate fibronectin type III domain-containing protein 5 (FNDC5) expression, from which irisin is derived, presumably by activating upstream regulators, such as adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), thereby mimicking exercise-induced pathways, contributing to their protective benefits across various organ systems. We hypothesize that activation of the PGC-1α - FNDC5/irisin axis represents a previously unrecognized downstream mediator of the pleiotropic cardiorenal and neuroprotective effects of SGLT-2 inhibitors. While current evidence is largely preclinical and associative, this framework generates a testable mechanistic model in which circulating irisin could serve both as a mediator and biomarker of SGLT-2 inhibitor-induced tissue protection.

Ketone bodies, particularly β-hydroxybutyrate (BHB), play an important role in the epigenetic regulation of gene expression in cardiac tissues, impacting both cardiac health and disease. This review explores the multifaceted influence of ketone bodies on epigenetic mechanisms, including histone acetylation, DNA methylation, ubiquitination, sirtuins activation, and RNA modulation. By acting as endogenous histone deacetylase inhibitors, ketone bodies enhance histone acetylation, thereby promoting the expression of genes involved in antioxidant defenses, anti-inflammatory responses, and metabolic regulation. Furthermore, BHB affects DNA methylation patterns by altering the availability of key metabolites such as S-adenosylmethionine. Ketogenic diet, which elevates BHB levels, has been shown to modulate gene expression, such as increasing FOXO3a and metallothionein 2, and improve cardiac function. This review highlights the therapeutic potential of ketone bodies in managing cardiac diseases through their epigenetic effects, underscoring the need for further research to elucidate the detailed molecular pathways and long-term impacts of these metabolic interventions.

Introduction:The renin-angiotensin-aldosterone system (RAAS) has been shown to be dysregulated in dementia, with elevated levels of angiotensin-converting enzyme (ACE), angiotensin (Ang) II, and Ang II type 1 receptors (AT1Rs). Cerebral amyloid angiopathy (CAA), a common cerebrovascular disease, currently has no treatment or cure available. We aimed to determine if a mouse model with CAA (Tg-SwDI) also exhibits elevated levels of AT1Rs and whether RAAS-targeting drugs (telmisartan and lisinopril) mitigate these effects. Materials and Methods:Tg-SwDI mice were treated with sub-depressor doses of either telmisartan or lisinopril from 3-8 months of age, with blood pressure being monitored 2 and 4 months after the start of treatment. Postmortem, receptor autoradiography was performed to determine levels of AT1R in 13 brain regions in untreated and treated Tg-SwDI mice compared to wild-type controls (C57Bl/6J). Results:No statistically significant differences among groups were observed in any of the 13 regions analyzed. However, trends with medium to large effect sizes were observed. Conclusions:CAA did not significantly dysregulate AT1R levels in the brains of Tg-SwDI mice compared to wild-type mice. Drug treatment caused no significant brain AT1R alterations. Further studies are required to determine if the trends observed are pathophysiological and pharmacologically significant.

Introduction The renin-angiotensin-aldosterone system (RAAS) has been shown to be dysregulated in dementia, with elevated levels of angiotensin-converting enzyme (ACE), angiotensin (Ang) II, and Ang II type 1 receptors (AT1Rs). Cerebral amyloid angiopathy (CAA), a dementia-causing cerebrovascular disease, currently has no treatment or cure available. We assessed whether a mouse model with CAA (Tg-SwDI) exhibits elevated levels of AT1Rs and whether RAAS-targeting drugs (telmisartan and lisinopril) affect these levels. Materials and methods Tg-SwDI mice were treated with sub-depressor doses of either telmisartan or lisinopril from 3 to 8 months of age, with blood pressure being monitored 2 and 4 months after the start of treatment. Post-mortem, receptor autoradiography was performed to determine levels of AT1R in 13 brain regions in untreated and treated Tg-SwDI mice compared to untreated wild-type controls (C57BL/6J mice). Results No statistically significant differences among groups were observed in AT1R levels in any of the 13 brain regions analyzed. Although sexual dimorphism in blood pressure was observed in both wild-type and Tg-SwDI mice (males > females), drug treatment did not significantly lower blood pressure in Tg-SwDI mice, confirming the delivery of sub-depressor doses. Conclusions Regional brain AT1R levels were not significantly dysregulated in Tg-SwDI mice compared to wild-type mice. Additionally, drug treatment with sub-depressor doses of the AT1R antagonist, telmisartan, or the ACE inhibitor, lisinopril, caused no significant brain AT1R alterations in Tg-SwDI mice. This suggests that pathophysiological changes in the brain renin-angiotensin system do not mediate the cognitive impairments and neuropathological changes associated with CAA in Tg-SwDI mice.

The melanocortin receptors are a class of centrally and peripherally expressed G protein-coupled receptors, of which the MC3R and MC4R subtypes are implicated in the regulation of appetite and energy homeostasis and can serve as potential therapeutic targets for disorders such as obesity and cachexia. An unbiased high-throughput mixture-based library screen was implemented to identify novel ligands with an emphasis on the identification of nanomolar-potent agonists of the mouse melanocortin-3 receptor. This screen yielded the discovery of an N-branched tricyclic guanidine scaffold (TPI2408) that contained three nanomolar potent mMC3R agonists and additional compounds that possessed antagonism for the mMC4R. The antagonist character of this scaffold library at the mMC4R was confirmed by a follow-up positional scanning antagonist screen. Additionally, molecular dynamics simulations herein provide mechanistic insight into the polypharmacological characteristics of melanocortin receptors. The disclosed materials have the potential to serve as important tools and SAR scaffolds in the study of melanocortin receptor function.

Cerebral amyloid angiopathy (CAA) is a cerebrovascular disease that results from beta-amyloid (Aβ) accumulation in the vessel walls that is associated with cognitive impairment and other neurological pathologies. There are currently no medications approved to treat CAA. This study investigated whether renin-angiotensin system (RAS)-targeting drugs, commonly prescribed to treat hypertension, can be repurposed to treat CAA, and whether their effects differ by sex. Male and female Tg-SwDI mice were treated for 5 months with sub-depressor doses of either telmisartan [angiotensin II receptor blocker (ARB)] or lisinopril [angiotensin-converting enzyme (ACE) inhibitor] starting at 3 months of age. Blood pressure monitoring was performed 2 and 4 months after the start of treatment, followed by behavior testing at 7 months of age. Histochemical analyses were conducted to determine vasculopathy, Aβ pathology, and neuroinflammation (microgliosis and astrogliosis). Outcomes in drug-treated and untreated Tg-SwDI mice were compared to each other and with wild-type (C57BL/6J) controls. Overall, both drugs were able to rescue some cognitive-behavioral functions; however, no reductions in Aβ levels were observed, and only limited improvements in vascular density and neuroinflammatory markers were detected. Notably, some treatment effects varied with sex, the specific behavioral task, and the brain region analyzed. These findings support the hypothesis that RAS-targeting drugs exert neuroprotective effects through mechanisms beyond blood pressure control offering a promising therapeutic avenue for CAA. ### Competing Interest Statement The authors have declared no competing interest. American Heart Association, https://ror.org/013kjyp64, 946666 National Institute on Aging, https://ror.org/049v75w11, R03 AG081865

Cerebral amyloid angiopathy (CAA), the accumulation of amyloid proteins in the cerebral vasculature, increases the risk of stroke and vascular cognitive impairment and dementia (VCID). Not only is there no treatment for CAA, but the condition is also highly comorbid with Alzheimer’s disease (AD), and its presence may serve as a contraindication to treating patients with anti-amyloid therapies due to an increased risk of hemorrhage and edema. Therefore, it is crucial to identify novel treatments for individuals with CAA. Epidemiological studies suggest that certain antihypertensive medications, including those that target the renin-angiotensin system (RAS), are associated with a decreased risk of dementia. This study assesses whether two FDA-approved RAS-targeting drugs: telmisartan [a moderately brain-penetrant angiotensin receptor blocker (ARB)], and lisinopril [a brain-penetrant angiotensin-converting enzyme (ACE) inhibitor]; can be repurposed for the treatment of CAA. At either ∼3 months (early intervention) or ∼8 months (later intervention) of age, male and female Tg-SwDI mice began treatment with either telmisartan (1 mg/kg/day) or lisinopril (15 mg/kg/day) dissolved in drinking water or received plain drinking water only. Age- and sex-matched C57BL/6J mice receiving plain drinking water served as wild-type controls. Following 4 months of treatment, mice underwent blood pressure measurement followed by behavioral testing prior to euthanasia. Voluntary oral consumption delivered doses similar to the target dose for both drugs. At the doses used, telmisartan and lisinopril treatment did not significantly reduce blood pressure in Tg-SwDI mice. Our findings thus far suggest that these drug treatments, particularly lisinopril, may mitigate cognitive-behavioral deficits observed in Tg-SwDI mice. Ongoing experiments are being completed to increase sample sizes and investigate the potential benefits of telmisartan and lisinopril to mitigate neuropathological and cognitive impairment in Tg-SwDI mice. If findings support our hypothesis, this will demonstrate that these drugs could be repurposed to prevent and/or treat CAA, reducing the worldwide burden of stroke and dementia.

Biological sex is a critical determinant in cardiovascular and renal disease outcomes. Although angiotensin II (Ang II) infusion is widely used to model hypertension in mice and rats, little is known about its effects in the Syrian hamster, a small rodent increasingly used for translational research. This study aimed to develop a model of chronic Ang II-induced hypertension in Syrian hamsters and investigate sex-specific differences in blood pressure, renal pathology, and components of the renin-angiotensin system (RAS). Male and female Syrian hamsters (8–9 weeks old) were infused subcutaneously with Ang II (200 ng/kg/min) or saline via osmotic minipumps for four weeks. Mean arterial pressure (MAP) and kidney wet weight were determined on the euthanasia day. The kidneys were analyzed for renal pathology; renal RAS enzymes (ACE and ACE2) were measured by colorimetric assay and qPCR; cytokines (IL-6 and IL-1β) were measured by qPCR; and the angiotensin receptor type 1 (AT1R) was measured by radioligand binding and qPCR. Ang II infusion increased MAP in both sexes but elicited a significantly greater response in females (+ 50 mmHg) than males (+ 27 mmHg, p < 0.005). Female hamsters exhibited pronounced kidney injury, including acute tubular necrosis, glomerular sclerosis, and vascular fibrinoid necrosis, along with a 2-fold increase in kidney weight normalized to body weight. Ang II significantly downregulated renal ACE, ACE2, and AT1R expression and activity in females but not in males. Renal IL-6 and IL-1β mRNA levels were elevated 20-fold and 3.9-fold, respectively, in females, compared to modest increases in males. Female Syrian hamsters exhibit heightened vulnerability to Ang II-induced hypertension and renal damage compared to males, marked by exaggerated blood pressure elevation, enhanced renal inflammation, and suppression of classical RAS components. This novel hamster model provides a unique platform for studying sex-specific mechanisms of hypertension and renal pathology, with translational relevance for subpopulations of women who are at increased risk of Ang II-dependent hypertension-associated renal disease. High blood pressure (also known as hypertension) is a major risk factor for heart and kidney disease. Men and women often experience these conditions differently, but scientists still don’t fully understand why. In this study, researchers focused on how male and female Syrian hamsters respond to a substance called angiotensin II, which is known to raise blood pressure and is commonly used in research to mimic high blood pressure in animals. Over four weeks, male and female hamsters were given either angiotensin II or a harmless salt solution. The researchers measured blood pressure and looked at changes in kidney health and in a system of hormones that helps control blood pressure, called the renin-angiotensin system. They found that angiotensin II caused a much bigger rise in blood pressure in female hamsters than in males. The female hamsters also showed more severe kidney damage, including scarring and inflammation. Important hormones and enzymes that usually help regulate blood pressure were found to be lower in females after treatment, while markers of inflammation were much higher. These results suggest that female hamsters are more sensitive to angiotensin II and may be more at risk of developing severe kidney problems from high blood pressure. This new hamster model offers a valuable way to study how men and women differ in their response to high blood pressure and could help researchers find better, more personalized treatments—especially for women who may be more vulnerable to certain types of hypertension-related kidney disease.

Background: Heart failure, characterized by a metabolic imbalance, leads to impaired mitochondrial ATP production. Ketone bodies not only serve as an alternate energy source but also mitigate heart failure by reducing cardiac remodeling and inflammation. The role of long noncoding RNAs (lncRNAs) in cardiac remodeling and mitochondrial metabolism is increasingly appreciated, and this study examines the effect of ketogenic diet on cardiac lncRNA regulation and mitochondrial protection. Methods and Results: In a six-month study, 10-week-old male and female C57BL/6J mice were assigned to either a control diet (10% fat, 20% protein, 70% carbohydrate) or a ketogenic diet (Keto; 80% fat, 15% protein, 5% carbohydrate). Data were analyzed using unpaired two-tailed t-tests (GraphPad Prism) and reported as mean ± SE. The keto diet significantly increased blood ketone bodies (n=36/group), indicating ketosis, and led to weight gain as well as a significant decrease in heart mass relative to body weight, suggesting cardiac metabolic adaptation (Fig 1A-B) . The cardiac expression analysis of lncRNAs Anril, Caren, Carmn, Gm15441, Malat1, Miat, and Oip5-as1 via qPCR (n=13/group) revealed significant upregulation of Anril and Malat1 in the Keto hearts ( Fig 1C-D ), indicating their potential regulatory roles under ketogenic conditions. Sex-specific differences were not observed in the lncRNA expressions. Key proteins involved in mitochondrial biogenesis, protection, and metabolic adaptation, including Nrf2 and PGC1α, were significantly increased at both mRNA and protein levels (n=6-10) in the hearts of mice on keto diet ( Fig 1E-H ). Conclusion: A six-month ketogenic diet in mice promotes cardiometabolic adaptations characterized by a reduction in relative heart mass and activation of key proteins involved in mitochondrial biogenesis and protection possibly through lncRNAs Anril and Malat1. These findings shed light on novel molecular targets and the potential of ketogenic diet/ketone bodies in treating heart failure.

Abstract Angiotensin II analogue and β‐arrestin biased agonist TRV027 (Sarcosine1, d‐Alanine8‐Angiotensin (Ang) II; SD Ang II), developed by Trevena, Inc. in the early 2010s, brought hopes of a novel treatment for cardiovascular diseases, due to its ability to simultaneously cause signaling through the β‐arrestin signaling pathway, while antagonizing the pathophysiological effects of Ang II mediated by the AT1 receptor G protein signaling cascades. However, a phase II clinical trial of this agent revealed no significant benefit compared to placebo treatment. Using 125I‐Sarcosine1, Isoleucine8‐Ang II (125I‐SI Ang II) radioligand receptor competition binding assays, we assessed the relative affinity of TRV027 compared to SI Ang II for liver AT1 receptors. We also compared radioiodinated TRV027 (125I‐SD Ang II) binding affinity for liver AT1 receptors with 125I‐SI Ang II. We found that despite its anticipated gain in metabolic stability, TRV027 and 125I‐SD Ang II had reduced affinity for the AT1 receptor compared with SI Ang II and 125I‐SI Ang II. Additionally, male–female comparisons showed that females have a higher AT1 receptor density, potentially attributed to tissue‐dependent estrogen and progesterone effects. Peptide drugs have become more popular over the years due to their increased bioavailability, fast onset of action, high specificity, and low toxicity. Even though Trevena®'s biased agonist peptide TRV027 offered greater stability and potency compared to earlier AT1R biased agonists, it failed its phase II clinical trial in 2016. Further refinements to AT1R biased agonist peptides to improve affinity, as seen with SI Ang II, with better stability and bioavailability, has the potential to achieve the anticipated biased agonism.

Introduction: In a previous retrospective study using postmortem human brain tissues, we demonstrated that loss of Cholinergic Receptor Muscarinic 1 (CHRM1) in the temporal cortex of a subset of Alzheimer's patients was associated with poor survival, whereas similar loss in the hippocampus showed no such association. Mitochondrial dysfunction underlies Alzheimer's pathogenesis. Therefore, to investigate the mechanistic basis of our findings, we evaluated cortical mitochondrial phenotypes in Chrm1 knockout (Chrm1-/-) mice. Cortical Chrm1 loss resulted in reduced respiration, reduced supramolecular assembly of respiratory protein complexes, and caused mitochondrial ultrastructural abnormalities. These mouse-based findings mechanistically linked cortical CHRM1 loss with poor survival of Alzheimer's patients. However, evaluation of the effect of Chrm1 loss on mouse hippocampal mitochondrial characteristics is necessary to fully understand our retrospective human tissue-based observations. This is the objective of this study. Methods: Enriched hippocampal and cortical mitochondrial fractions (EHMFs/ECMFs, respectively) derived from wild-type and Chrm1-/- mice were used to measure respiration by quantifying real-time oxygen consumption, supramolecular assembly of oxidative phosphorylation (OXPHOS)-associated proteins by blue native polyacrylamide gel electrophoresis, post-translational modifications (PTMs) by isoelectric focusing (IEF), and mitochondrial ultrastructure by electron microscopy. Results: In contrast to our previous observations in Chrm1-/- ECMFs, EHMFs of Chrm1-/- mice significantly increased respiration with a concomitant increase in the supramolecular assembly of OXPHOS-associated proteins, specifically Atp5a and Uqcrc2, with no mitochondrial ultrastructural alterations. IEF of ECMFs and EHMFs from Chrm1-/- mice showed a decrease and an increase, respectively in a negatively charged (pH∼3) fraction of Atp5a relative to the wild-type mice, with a corresponding decrease or increase in the supramolecular assembly of Atp5a and respiration indicating a tissue-specific signaling effect. Discussion: Our findings indicate that loss of Chrm1 in the cortex causes structural, and physiological alterations to mitochondria that compromise neuronal function, whereas Chrm1 loss in the hippocampus may benefit neuronal function by enhancing mitochondrial function. This brain region-specific differential effect of Chrm1 deletion on mitochondrial function supports our human brain region-based findings and Chrm1-/- mouse behavioral phenotypes. Furthermore, our study indicates that Chrm1-mediated brain region-specific differential PTMs of Atp5a may alter complex-V supramolecular assembly which in turn regulates mitochondrial structure-function.

Consumption of high fat diet (HFD) contributes to several neurological disorders and metabolic disease. Our previous work has shown that a HFD results in sex-specific effects in C57Bl/6J mice, including adult hippocampal neurogenesis, neuroinflammation, and performance on cognitive-behavioral tasks. In general, females are more negatively impacted compared to males, even when the metabolic effects of HFD are similar amongst the sexes. The ketogenic (Keto) diet is a popular fad diet that involves limiting the intake of carbohydrates to induce a state of “nutritional ketosis.” This state promotes ketogenesis while reducing gluconeogenesis. While the Keto diet is also very high in fat content, it appears to hold potential for neuroprotection against brain aging and neurodegenerative disorders, as well as boost mood and cognitive function in young healthy individuals. Further research on the effects of the Keto diet remains to be underexplored in a sex-specific manner. Also, most prior research using animal models has generally used a very strict Keto diet that is not ideal for translational relevance (~0% carbohydrates). Thus, the goal of this study is to compare the physiological and cognitive-behavioral effects of both a high fat and translationally relevant Keto diet to a low-fat control diet in adult male and female mice. Male and female C57Bl/6J mice were fed either a low fat (LF; 10% fat, 70% carbs), high fat (HF; 60% fat, 20% carbs), or Keto (Keto; 80% fat, 5% carbs) diet beginning at 2-3 months of age. Body weight, food intake, and fluid intake were measured weekly throughout the experiment. Five months into the diet intervention, mice underwent a battery of behavioral tests to assess potential changes in general activity levels, anxiety-like behavior, and several domains of cognitive function. Diabetic status and ketosis were assessed, and tissues and organs were collected and weighed at the end of the experiment to further determine physiological effects of the various diets. As expected, HFD resulted in increased weight gain and glucose intolerance compared to LF diet in both males and females. Sex-specific changes in fat accumulation and organ mass were also noted in response to HF diet. Keto diet induced a state of mild ketosis to a similar degree amongst the sexes, with fewer physiological alterations compared to HFD. While HF and Keto diets tended to reduce exploratory behavior in males, this effect was not seen in females. Of note, preliminary data suggests that while spatial working memory is not affected by any of these diet interventions, Keto diet may improve long-term spatial memory in male mice only. Taken together, these findings demonstrate the physiological and cognitive-behavioral effects of a HFD vs. Keto diet in males and females. Even a Keto diet that produces only a mild state of ketosis may improve some aspects of cognition in adulthood, albeit potentially in a sex-specific manner that favors males. Nova Southeastern University President's Faculty Research and Development Grant (LSR), Nova Southeastern University College of Psychology Faculty Research Fellowship (LSR), American Heart Association Award #946666 (LSR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The renin-angiotensin system (RAS) is a hormonal cascade that contributes to several disorders: systemic hypertension, heart failure, kidney disease, and neurodegenerative disease. Activation of the RAS can promote inflammation and fibrosis. Drugs that target the RAS can be classified into 3 categories, AT1 angiotensin receptor blockers (ARBs), angiotensin-converting enzyme (ACE) inhibitors, and renin inhibitors. The therapeutic efficacy of current RAS-inhibiting drugs is limited by poor penetration across the blood-brain barrier, low bioavailability, and to some extent, short half-lives. Nanoparticle-mediated drug delivery systems (DDSs) are possible emerging alternatives to overcome such limitations. Nanoparticles are ideally 1-100 nm in size and are considered efficient DDSs mainly due to their unique characteristics, including water dispersity, prolonged half-life in blood circulation, smaller size, and biocompatibility. Nano-scale DDSs can reduce the drug dosage frequency and acute toxicity of drugs while enhancing therapeutic success. Different types of nanoparticles, such as chitosan, polymeric, and nanofibers, have been examined in RAS-related studies, especially in hypertension, cardiovascular disease, and COVID-19. In this review article, we summarize the physical and chemical characteristics of each nanoparticle to elaborate on their potential use in RAS-related nano-drug delivery research and clinical application.

To the Editor: The article by Bhandari et al. (Dec. 1 issue)(1) does not distinguish between angiotensin-converting-enzyme (ACE) inhibitor therapy and angiotensin-receptor blocker (ARB) therapy in advanced chronic kidney disease (CKD). The mechanisms of action of ACE inhibitors and ARBs differ substantially. Blockade of conversion of angiotensin I to angiotensin II blocks activation of type 2 angiotensin (AT(2)) receptors, which mediate beneficial effects of angiotensin II,(2) as well as type 1 angiotensin (AT(1)) receptors. In addition, ACE inhibitors block degradation of bradykinin, a vasodilator and proinflammatory mediator, and other peptides with varying effects on renal function. We found that a . . .

INTRODUCTION:Non-angiotensin converting enzyme mechanisms of angiotensin II production remain underappreciated in part due to the success of current therapies to ameliorate the impact of primary hypertension and atherosclerotic diseases of the heart and the blood vessels. This review scrutinize the current literature to highlight chymase role as a critical participant in the pathogenesis of cardiovascular disease and heart failure. AREAS COVERED:We review the contemporaneous understanding of circulating and tissue biotransformation mechanisms of the angiotensins focusing on the role of chymase as an alternate tissue generating pathway for angiotensin II pathological mechanisms of action. EXPERT OPINION:While robust literature documents the singularity of chymase as an angiotensin II-forming enzyme, particularly when angiotensin converting enzyme is inhibited, this knowledge has not been fully recognized to clinical medicine. This review discusses the limitations of clinical trials' that explored the benefits of chymase inhibition in accounting for the failure to duplicate in humans what has been demonstrated in experimental animals.

OBJECTIVES/GOALS: The SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2), which underlies the current COVID-19 pandemic, among other tissues, also targets the central nervous system (CNS). The goal of this study is to investigate mechanisms of neuroinflammation in Lipopolysaccharides (LPS)-treated mouse model and SARS-CoV-2-infected hamsters. METHODS/STUDY POPULATION: In this research I will assay vascular reactivity of cerebral vessels to assess vascular dysfunction within the microcirculation. I will determine expression of proinflammatory cytokines, coagulation factors and AT1 receptors (AT1R) in isolated microvessels from the circle of Willis to assess inflammation, thrombosis and RAS activity in the microvasculature. LPS and SARS-CoV-2, are both associated with coagulopathies and because of that I will measure concentration of PAI-1, von Willebrand Factor, thrombin and D-dimer to assess the thrombotic pathway in the circulation. Histology and immunohistochemistry will assess immune cell type infiltration into the brain parenchyma, microglia activation and severity of neuroinflammation and neural injury. RESULTS/ANTICIPATED RESULTS: We hypothesize that under conditions of reduced ACE2 (e.g., SARS-CoV-2 infection), AT1R activity is upregulated in the microvasculature. In the presence of an inflammatory insult, these AT1Rs promote endothelialitis and immunothrombosis through pro-thrombotic pathways and pro-inflammatory cytokine production leading to endothelial dysfunction in the microvasculature, blood brain barrier (BBB) injury, deficits in cognition and increased anxiety. We will test this hypothesis through 2 aims: Aim 1: Determine the role of the pro-injury arm of the RAS in the pathophysiology of the brain in animal models of neuroinflammation and COVID-19. Aim 1: Determine the role of the protective arm of the RAS in the pathophysiology of the brain in animal models of neuroinflammation and COVID-19. DISCUSSION/SIGNIFICANCE: This study will provide insights that will complement on-going clinical trials on angiotensin type 1 receptor (AT1R) blockers (ARBs) in COVID-19. This research is a necessary first step in understanding mechanisms of brain pathogenesis that can set the groundwork for future studies of more complex models of disease.

BACKGROUND Dysfunction of cholinergic neurotransmission is a hallmark of Alzheimer's disease (AD); forming the basis for using acetylcholine (ACh) esterase (AChE) inhibitors to mitigate symptoms of ACh deficiency in AD. The Cholinergic Receptor Muscarinic 1 (CHRM1) is highly expressed in brain regions impaired by AD. Previous analyses of postmortem AD brains revealed unaltered CHRM1 mRNA expression compared to normal brains. However, the CHRM1 protein level in AD and other forms of dementia has not been extensively studied. Reduced expression of CHRM1 in AD patients may explain the limited clinical efficacy of AChE inhibitors. OBJECTIVE To quantify CHRM1 protein in the postmortem hippocampus and temporal cortex of AD, Parkinson's disease (PD), and frontotemporal dementia (FTD) patients. METHODS Western blotting was performed on postmortem hippocampus (N = 19/73/7/9: unaffected/AD/FTD/PD) and temporal cortex (N = 9/74/27: unaffected/AD/PD) using a validated anti-CHRM1 antibody. RESULTS Quantification based on immunoblotting using a validated anti-CHRM1 antibody revealed a significant loss of CHRM1 protein level (<50%) in the hippocampi (78% AD, 66% PD, and 85% FTD) and temporal cortices (56% AD and 42% PD) of dementia patients. Loss of CHRM1 in the temporal cortex was significantly associated with early death (<65-75 years) for both AD and PD patients. CONCLUSION Severe reduction of CHRM1 in a subset of AD and PD patients can explain the reported low efficacy of AChE inhibitors as a mitigating treatment for dementia patients. Based on this study, it can be suggested that future research should prioritize therapeutic restoration of CHRM1 protein levels in cholinergic neurons.