Novel brain-penetrant inhibitor of G9a methylase blocks Alzheimer's disease proteopathology for precision medication.

Current amyloid beta-targeting approaches for Alzheimer's disease (AD) therapeutics only slow cognitive decline for small numbers of patients. This limited efficacy exists because AD is a multifactorial disease whose pathological mechanism(s) and diagnostic biomarkers are largely unknown. Here we report a new mechanism of AD pathogenesis in which the histone methyltransferase G9a noncanonically regulates translation of a hippocampal proteome that defines the proteopathic nature of AD. Accordingly, we developed a novel brain-penetrant inhibitor of G9a, MS1262, across the blood-brain barrier to block this G9a-regulated, proteopathologic mechanism. Intermittent MS1262 treatment of multiple AD mouse models consistently restored both cognitive and noncognitive functions to healthy levels. Comparison of proteomic/phosphoproteomic analyses of MS1262-treated AD mice with human AD patient data identified multiple pathological brain pathways that elaborate amyloid beta and neurofibrillary tangles as well as blood coagulation, from which biomarkers of early stage of AD including SMOC1 were found to be affected by MS1262 treatment. Notably, these results indicated that MS1262 treatment may reduce or avoid the risk of blood clot burst for brain bleeding or a stroke. This mouse-to-human conservation of G9a-translated AD proteopathology suggests that the global, multifaceted effects of MS1262 in mice could extend to relieve all symptoms of AD patients with minimum side effect. In addition, our mechanistically derived biomarkers can be used for stage-specific AD diagnosis and companion diagnosis of individualized drug effects.

Novel brain-penetrant inhibitor of G9a methylase blocks Alzheimer's disease proteopathology for precision medication.

Current amyloid beta-targeting approaches for Alzheimer's disease (AD) therapeutics only slow cognitive decline for small numbers of patients. This limited efficacy exists because AD is a multifactorial disease whose pathological mechanism(s) and diagnostic biomarkers are largely unknown. Here we report a new mechanism of AD pathogenesis in which the histone methyltransferase G9a noncanonically regulates translation of a hippocampal proteome that defines the proteopathic nature of AD. Accordingly, we developed a novel brain-penetrant inhibitor of G9a, MS1262, across the blood-brain barrier to block this G9a-regulated, proteopathologic mechanism. Intermittent MS1262 treatment of multiple AD mouse models consistently restored both cognitive and noncognitive functions to healthy levels. Comparison of proteomic/phosphoproteomic analyses of MS1262-treated AD mice with human AD patient data identified multiple pathological brain pathways that elaborate amyloid beta and neurofibrillary tangles as well as blood coagulation, from which biomarkers of early stage of AD including SMOC1 were found to be affected by MS1262 treatment. Notably, these results indicated that MS1262 treatment may reduce or avoid the risk of blood clot burst for brain bleeding or a stroke. This mouse-to-human conservation of G9a-translated AD proteopathology suggests that the global, multifaceted effects of MS1262 in mice could extend to relieve all symptoms of AD patients with minimum side effect. In addition, our mechanistically derived biomarkers can be used for stage-specific AD diagnosis and companion diagnosis of individualized drug effects. One-Sentence Summary: A brain-penetrant inhibitor of G9a methylase blocks G9a translational mechanism to reverse Alzheimer's disease related proteome for effective therapy.

Activation of hypothalamic-enhanced adult-born neurons restores cognitive and affective function in Alzheimer's disease.

Patients with Alzheimer's disease (AD) exhibit progressive memory loss, depression, and anxiety, accompanied by impaired adult hippocampal neurogenesis (AHN). Whether AHN can be enhanced in impaired AD brain to restore cognitive and affective function remains elusive. Here, we report that patterned optogenetic stimulation of the hypothalamic supramammillary nucleus (SuM) enhances AHN in two distinct AD mouse models, 5×FAD and 3×Tg-AD. Strikingly, the chemogenetic activation of SuM-enhanced adult-born neurons (ABNs) rescues memory and emotion deficits in these AD mice. By contrast, SuM stimulation alone or activation of ABNs without SuM modification fails to restore behavioral deficits. Furthermore, quantitative phosphoproteomics analyses reveal activation of the canonical pathways related to synaptic plasticity and microglia phagocytosis of plaques following acute chemogenetic activation of SuM-enhanced (vs. control) ABNs. Our study establishes the activity-dependent contribution of SuM-enhanced ABNs in modulating AD-related deficits and informs signaling mechanisms mediated by the activation of SuM-enhanced ABNs.

Self-reported Learning and Study Strategies in First and Second Year Medical Students.

Medical school presents a unique challenge to the average learner as the instructional strategies used in medical curricula are often different than what the student has experienced prior. The large volume of information taught in medical school is delivered with a variety of techniques. After the educational material has been delivered, it is the student's responsibility to study and learn the information for future exams and for their future patients. The current study aims to explore what learning activities and teaching strategies first (M1) and second year (M2) medical students use and prefer. Additionally, the study aims to determine if there are cohort differences in classroom and study habits. A group of 95 M1 students and 109 M2 students were recruited to participate in this online survey study. The analyses indicated statistical differences between M1 and M2 student cohorts with M1 students preferring group work and small group discussions more than M2 students. Classic didactic lecturing was preferred by 71.6% of students surveyed. M1 students reported a greater tendency for self-testing and group study versus M2 students. GPA and study technique preference were not correlated. These findings indicate that medical students are not using research-based learning and study strategies at the possible detriment of long-term knowledge retention. Modeling of research-based learning and study strategies by medical educators is one possible solution to encourage medical students to change their study practice. Future work should focus on how medical student learning preferences change as they progress through medical school.

A retrospective and correlative analysis of academic and nonacademic predictors of COMLEX level 1 performance.

CONTEXT: National licensing exams (NLEs) including the Comprehensive Osteopathic Medical Licensing Examination (COMLEX) Level 1 evaluate student achievement. Scores have historically been utilized to stratify medical student applicants for residency. Grade point average (GPA), number of practice questions completed, and performance on practice exams have been shown to be predictive of NLE performance. Test anxiety and acute stress have been shown to negatively impact NLE performance. The role of study behaviors and other nonacademic factors in COMLEX Level 1 performance is unknown. OBJECTIVES: This study aims to evaluate academic and nonacademic factors and to correlate them with COMLEX Level 1 performance. Additional analysis is conducted to associate COMLEX Level 1 performance with academic and nonacademic factors when controlling for GPA. METHODS: An anonymous online survey was administered to third- (OMS III) and fourth-year (OMS IV) osteopathic medical students at Kansas City University that had completed the COMLEX Level 1 examination. In total, 72 students responded to the survey. Survey results were linked to student records of GPA and COMLEX Level 1 scores, resulting in 59 complete responses for analysis. Independent-sample t-tests and linear ordinary least squares regression were utilized to analyze the results. RESULTS: The majority of participants are male (62.7%) and OMS III (98.3%) with an average age of 27.14 ± 2.58 (mean ± standard deviation). Further demographic data reveal hours per week spent for personal time during dedicated study (n=46, 19.7 ± 18.53), hours of sleep per night during dedicated study (7.34 ± 0.92), and money spent on board preparation ($1,319.12 ± $689.17). High ($1,600-$3,000), average ($1,000-$1,500), and low ($100-$900) spenders do not statistically differ and COMLEX Level 1 performance is not related to the number of resources utilized (F statistics <1; p>0.05). Pearson correlations reveal a statistically significant relationship between COMLEX Level 1 scores with GPA (0.73, p<0.001), number of practice exams completed (0.39, p<0.001), number of questions completed (0.46, p<0.001), number of weeks of study (0.55, p<0.001), and preparation cost (0.28, p<0.05). The regression analysis revealed that money spent on board preparation, number of questions completed, and time spent studying accounted for 75.8% of the variance in COMLEX Level 1 scores after controlling for GPA. CONCLUSIONS: The data show the association of money spent on board preparation, numbers of questions competed, and time spent studying with a student's COMLEX Level 1 score. Additionally, these results highlight the amount of money students spend on extracurricular materials to prepare for COMLEX Level 1, yet the data show that the number of resources that students utilized is not related to a student's COMLEX Level 1 performance.

Epigenetic regulation in the neurogenic niche of the adult dentate gyrus.

The adult dentate gyrus (DG) of the hippocampal formation is a specialized region of the brain that creates new adult-born neurons from a pool of resident adult neural stem and progenitor cells (aNSPCs) throughout life. These aNSPCs undergo epigenetic and epitranscriptomic regulation, including 3D genome interactions, histone modifications, DNA modifications, noncoding RNA mechanisms, and RNA modifications, to precisely control the neurogenic process. Furthermore, the specialized neurogenic niche also uses epigenetic mechanisms in mature neurons and glial cells to communicate signals to direct the behavior of the aNSPCs. Here, we review recent advances of epigenetic regulation in aNSPCs and their surrounding niche cells within the adult DG.

CNS fibroblasts form a fibrotic scar in response to immune cell infiltration.

Fibrosis is a common pathological response to inflammation in many peripheral tissues and can prevent tissue regeneration and repair. Here, we identified persistent fibrotic scarring in the CNS following immune cell infiltration in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Using lineage tracing and single-cell sequencing in EAE, we determined that the majority of the fibrotic scar is derived from proliferative CNS fibroblasts, not pericytes or infiltrating bone marrow-derived cells. Ablating proliferating fibrotic cells using cell-specific expression of herpes thymidine kinase led to an increase in oligodendrocyte lineage cells within the inflammatory lesions and a reduction in motor disability. We further identified that interferon-gamma pathway genes are enriched in CNS fibrotic cells, and the fibrotic cell-specific deletion of Ifngr1 resulted in reduced fibrotic scarring in EAE. These data delineate a framework for understanding the CNS fibrotic response.

Pdgfrα-Cre mediated knockout of the aryl hydrocarbon receptor protects mice from high-fat diet induced obesity and hepatic steatosis.

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR's role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects.

This is Not Your Grandfather's Medical School: Novel Tools to Enhance Medical Education.

As new medical students start their journey to become the next generation of physicians, they are in awe of the wealth of knowledge at their fingertips as they begin medical school. Every student brings with them a unique story, and most bring with them a high tolerance for technology. The internet, smart phones, and the personal computer have shrunk the academic world and allowed students access to entire libraries that fit within their pockets. Medical school curricula continues to try to evolve to meet students in their increasingly technology filled world. How are medical schools evolving to integrate technology into their curricula? What follows is a review of the application of different technologies in medical education and a close look at the most efficient uses of technology within medical school curricula. This discussion is followed by perspectives from professors and a student on the use of a variety of different technologies for teaching and learning including podcasts, YouTube, Twitter, and varying online resources.

RABL6A inhibits tumor-suppressive PP2A/AKT signaling to drive pancreatic neuroendocrine tumor growth.

Hyperactivated AKT/mTOR signaling is a hallmark of pancreatic neuroendocrine tumors (PNETs). Drugs targeting this pathway are used clinically, but tumor resistance invariably develops. A better understanding of factors regulating AKT/mTOR signaling and PNET pathogenesis is needed to improve current therapies. We discovered that RABL6A, a new oncogenic driver of PNET proliferation, is required for AKT activity. Silencing RABL6A caused PNET cell-cycle arrest that coincided with selective loss of AKT-S473 (not T308) phosphorylation and AKT/mTOR inactivation. Restoration of AKT phosphorylation rescued the G1 phase block triggered by RABL6A silencing. Mechanistically, loss of AKT-S473 phosphorylation in RABL6A-depleted cells was the result of increased protein phosphatase 2A (PP2A) activity. Inhibition of PP2A restored phosphorylation of AKT-S473 in RABL6A-depleted cells, whereas PP2A reactivation using a specific small-molecule activator of PP2A (SMAP) abolished that phosphorylation. Moreover, SMAP treatment effectively killed PNET cells in a RABL6A-dependent manner and suppressed PNET growth in vivo. The present work identifies RABL6A as a new inhibitor of the PP2A tumor suppressor and an essential activator of AKT in PNET cells. Our findings offer what we believe is a novel strategy of PP2A reactivation for treatment of PNETs as well as other human cancers driven by RABL6A overexpression and PP2A inactivation.

Calcium and P-glycoprotein independent synergism between schweinfurthins and verapamil.

Schweinfurthins are intriguing natural products with anti-cancer activities and as yet incompletely understood mechanisms of action. We investigated whether inhibitors of P-glycoprotein (Pgp), in a manner analogous to other natural products, might enhance schweinfurthins' growth inhibitory actions by increasing intracellular schweinfurthin levels. Both the schweinfurthin-sensitive glioblastoma multiforme cell line SF-295 and relatively insensitive lung carcinoma cell line A549 were treated with 2 schweinfurthin analogs: 3-deoxyschweinfurthin B-p-nitro bis-stilbene (3dSB-PNBS) and 5'-methylschweinfurthin G (methyl-G). There was a synergistic enhancement of growth inhibition with the combination of the Pgp inhibitor verapamil and both analogs in SF-295 cells. Methyl-G, verapamil, and the combination did not result in alterations to intracellular calcium concentration. Verapamil increased the intracellular concentration of 3dSB-PNBS in both SF-295 and A549 cells in a Pgp-independent manner. Methyl-G, verapamil, and the combination do not result in increased ER stress. Methyl-G increased the intracellular concentration of a known Pgp substrate, Rhodamine 123 in SF-295 cells. Reduction of cellular cholesterol leads to the accumulation of Pgp substrates, as Pgp requires cholesterol for proper function. Since 3dSB enhances lovastatin-induced upregulation of the cholesterol efflux pump ABCA1, it is intriguing that co-treatment with cholesterol rescued the methyl-G-induced increase in Rhodamine 123 intracellular concentration. These studies support the hypothesis that verapamil potentiates the schweinfurthin growth inhibitory effect by increasing its intracellular concentration.

Functional evaluation of a fluorescent schweinfurthin: mechanism of cytotoxicity and intracellular quantification.

Schweinfurthins are potent inhibitors of cancer cell growth, especially against human central nervous system tumor lines such as SF-295 cells. However, the mechanisms through which these compounds impede cell growth are not fully understood. In an effort to understand the basis for the effects of schweinfurthins, we present a fluorescent schweinfurthin, 3-deoxyschweinfurthin B-like p-nitro-bis-stilbene (3dSB-PNBS), which displays biological activity similar to that of 3-deoxyschweinfurthin B (3dSB). These two schweinfurthins retain the unique differential activity of the natural schweinfurthins, as evidenced by the spindle-like morphological changes induced in SF-295 cells and the unaltered appearance of human lung carcinoma A549 cells. We demonstrate that incubation with 3dSB or 3dSB-PNBS results in cleavage of poly-ADP-ribose polymerase (PARP) and caspase-9, both markers of apoptosis. Coincubation of 3dSB or 3dSB-PNBS with the caspase-9 inhibitor (Z)-Leu-Glu(O-methyl)-His-Asp(O-methyl)-fluoromethylketone prevents PARP cleavage. Therapeutic agents that induce apoptosis often activate cellular stress pathways. A marker for multiple stress pathways is the phosphorylation of eukaryotic initiation factor 2α, which is phosphorylated in response to 3dSB and 3dSB-PNBS treatment. Glucose-regulated protein 78 and protein disulfide isomerase, both endoplasmic reticulum chaperones, are up-regulated with schweinfurthin exposure. Using the fluorescent properties of 3dSB-PNBS and dimethoxyphenyl-p-nitro-bis-stilbene (DMP-PNBS), a control compound, we show that the intracellular levels of 3dSB-PNBS are higher than those of Rhodamine 123 or DMP-PNBS in SF-295 and A549 cells.