Barriers to help-seeking in medical students with anxiety at the University of South Carolina School of Medicine Greenville.

Anxiety levels in medical students have been reported as higher than the aged-matched general population, yet medical students are less likely to seek care for mental health issues. Medical students carry high levels of self-stigma about their own mental health and fear the negative consequences of seeking care. The purpose of this study was to examine the student population at the University of South Carolina School of Medicine Greenville (UofSC SOMG) for anxiety levels and determine the self-stigma attitudes this population carries. UofSC SOMG students were surveyed using the GAD7, questions about mental health stigma, and open-ended questions on barriers to mental health care in medical students. Anxiety levels were compared to student responses. 31% of students reported moderate-severe anxiety levels. Stigma was the most frequently listed barrier to care, however, students with moderate-severe anxiety were more likely to report cost as a barrier to care than students with minimal anxiety levels. Despite free and accessible mental health care, medical students at UofSC SOMG still have anxiety at rates higher than the general population. Future work should help to provide interventions to the barriers of care, so medical students can better utilize mental health care resources.

Orchiectomy sensitizes cortical bone in male mice to the harmful effects of kynurenine.

Kynurenine (Kyn) is a tryptophan metabolite that increases with age and promotes musculoskeletal dysfunction. We previously found a sexually dimorphic pattern in how Kyn affects bone, with harmful effects more prevalent in females than males. This raises the possibility that male sex steroids might exert a protective effect that blunts the effects of Kyn in males. To test this, orchiectomy (ORX) or sham surgeries were performed on 6-month-old C57BL/6 mice, after which mice received Kyn (10 mg/kg) or vehicle via intraperitoneal injection, once daily, 5×/week, for four weeks. Bone histomorphometry, DXA, microCT, and serum marker analyses were performed after sacrifice. In vitro studies were performed to specifically test the effect of testosterone on activation of aryl hydrocarbon receptor (AhR)-mediated signaling by Kyn in mesenchymal-lineage cells. Kyn treatment reduced cortical bone mass in ORX- but not sham-operated mice. Trabecular bone was unaffected. Kyn's effects on cortical bone in ORX mice were attributed primarily to enhanced endosteal bone resorption activity. Bone marrow adipose tissue was increased in Kyn-treated ORX animals but was unchanged by Kyn in sham-operated mice. ORX surgery increased mRNA expression of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1a1 in the bone, suggesting a priming and/or amplification of AhR signaling pathways. Mechanistic in vitro studies revealed that testosterone blunted Kyn-stimulated AhR transcriptional activity and Cyp1a1 expression in mesenchymal-linage cells. These data suggest a protective role for male sex steroids in blunting the harmful effects of Kyn in cortical bone. Therefore, testosterone may play an important role in regulating Kyn/AhR signaling in musculoskeletal tissues, suggesting crosstalk between male sex steroids and Kyn signaling may influence age-associated musculoskeletal frailty.

The Glucocorticoid Receptor in Osterix-Expressing Cells Regulates Bone Mass, Bone Marrow Adipose Tissue, and Systemic Metabolism in Female Mice During Aging.

Hallmarks of aging-associated osteoporosis include bone loss, bone marrow adipose tissue (BMAT) expansion, and impaired osteoblast function. Endogenous glucocorticoid levels increase with age, and elevated glucocorticoid signaling, associated with chronic stress and dysregulated metabolism, can have a deleterious effect on bone mass. Canonical glucocorticoid signaling through the glucocorticoid receptor (GR) was recently investigated as a mediator of osteoporosis during the stress of chronic caloric restriction. To address the role of the GR in an aging-associated osteoporotic phenotype, the current study utilized female GR conditional knockout (GR-CKO; GRfl/fl :Osx-Cre+) mice and control littermates on the C57BL/6 background aged to 21 months and studied in comparison to young (3- and 6-month-old) mice. GR deficiency in Osx-expressing cells led to low bone mass and BMAT accumulation that persisted with aging. Surprisingly, however, GR-CKO mice also exhibited alterations in muscle mass (reduced % lean mass and soleus fiber size), accompanied by reduced voluntary physical activity, and also exhibited higher whole-body metabolic rate and elevated blood pressure. Moreover, increased lipid storage was observed in GR-CKO osteoblastic cultures in a glucocorticoid-dependent fashion despite genetic deletion of the GR, and could be reversed via pharmacological inhibition of the mineralocorticoid receptor (MR). These findings provide evidence of a role for the GR (and possibly the MR) in facilitating healthy bone maintenance with aging in females. The effects of GR-deficient bone on whole-body physiology also demonstrate the importance of bone as an endocrine organ and suggest evidence for compensatory mechanisms that facilitate glucocorticoid signaling in the absence of osteoblastic GR function; these represent new avenues of research that may improve understanding of glucocorticoid signaling in bone toward the development of novel osteogenic agents. © 2021 American Society for Bone and Mineral Research (ASBMR).

Absent Internal Carotid Artery With Intact Circle of Willis.

The internal carotid arteries are one of the primary suppliers of the Circle of Willis and cerebral blood flow, but the rare case of agenesis of the internal carotid artery can impair the functional redundancies of cerebral blood supply. In this study, routine, medical education-focused cadaveric dissection of an 80-year-old male cadaver (cause of death was ventricular tachycardia) was performed. A case of agenesis of the left internal carotid artery and the carotid canal was identified. Upon investigation, we found that the compensatory pattern of irrigation in the Circle of Willis did not conform to previously described cases in the scientific literature. Further literature review suggested that such agenesis can be associated with a wide range of conditions from stroke, migraine, tinnitus, and Horner's syndrome. Due to the altered blood flow pattern, we caution the reading physician regarding the potential for ischemia and iatrogenic damage, particularly of the pituitary gland and eye. We suggest the use of neuroangiographic imaging in cases of agenesis of an internal carotid artery.

Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells.

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging. We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p. Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3',4'-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis.

Picolinic acid, a tryptophan oxidation product, does not impact bone mineral density but increases marrow adiposity.

Tryptophan is an essential amino acid catabolized initially to kynurenine (kyn), an immunomodulatory metabolite that we have previously shown to promote bone loss. Kyn levels increase with aging and have also been associated with neurodegenerative disorders. Picolinic acid (PA) is another tryptophan metabolite downstream of kyn. However, in contrast to kyn, PA is reported to be neuroprotective and further, to promote osteogenesis in vitro. Thus, we hypothesized that PA might be osteoprotective in vivo. In an IACUC-approved protocol, we fed PA to aged (23-month-old) C57BL/6 mice for eight weeks. In an effort to determine potential interactions of PA with dietary protein we also fed PA in a low-protein diet (8%). The mice were divided into four groups: Control (18% dietary protein), +PA (700 ppm); Low-protein (8%), +PA (700 ppm). The PA feedings had no impact on mouse weight, body composition or bone density. At sacrifice bone and stem cells were collected for analysis, including µCT and RT-qPCR. Addition of PA to the diet had no impact on trabecular bone parameters. However, marrow adiposity was significantly increased in PA-fed mice, and in bone marrow stromal cells isolated from these mice increases in the expression of the lipid storage genes, Plin1 and Cidec, were observed. Thus, as a downstream metabolite of kyn, PA no longer showed kyn's detrimental effects on bone but instead appears to impact energy balance.

Kynurenine suppresses osteoblastic cell energetics in vitro and osteoblast numbers in vivo.

Aging is a progressive process associated with declining tissue function over time. Kynurenine, an oxidized metabolite of the essential amino acid tryptophan that increases in abundance with age, drives cellular processes of aging and dysfunction in many tissues, and recent work has focused on understanding the pathways involved in the harmful effects of kynurenine on bone. In this study, we sought to investigate the effects of controlled kynurenine administration on osteoblast bioenergetics, in vivo osteoblast abundance, and marrow fat accumulation. Additionally, as an extension of earlier studies with dietary administration of kynurenine, we investigated the effects of kynurenine on Hdac3 and NCoR1 expression and enzymatic deacetylase activity as potential mechanistic contributors to the effects of kynurenine on osteoblasts. Kynurenine administration suppressed cellular metabolism in osteoblasts at least in part through impaired mitochondrial respiration, and suppressed osteoblastic numbers in vivo with no concurrent effects on marrow adiposity. Deleterious effects of kynurenine treatment on osteoblasts were more pronounced in female models as compared to males. However, kynurenine treatment did not inhibit Hdac3's enzymatic deacetylase activity nor its repression of downstream glucocorticoid signaling. As such, future work will be necessary to determine the mechanisms by which increased kynurenine contributes to aging bone bioenergetics. The current study provides novel further support for the idea that kynurenine contributes to impaired osteoblastic function, and suggests that impaired matrix production by kynurenine-affected osteoblasts is attributed in part to impaired osteoblastic bioenergetics. As circulating kynurenine levels in increase with age, and human bone density inversely correlates with the serum kynurenine to tryptophan ratio, these mechanisms may have important relevance in the etiology and pathogenesis of osteoporosis in humans.

Accumulation of kynurenine elevates oxidative stress and alters microRNA profile in human bone marrow stromal cells.

Kynurenine, a metabolite of tryptophan breakdown, has been shown to increase with age, and plays a vital role in a number of age-related pathophysiological changes, including bone loss. Accumulation of kynurenine in bone marrow stromal cells (BMSCs) has been associated with a decrease in cell proliferation and differentiation, though the exact mechanism by which kynurenine mediates these changes is poorly understood. MiRNAs have been shown to regulate BMSC function, and accumulation of kynurenine may alter the miRNA expression profile of BMSCs. The aim of this study was to identify differentially expressed miRNAs in human BMSCs in response to treatment with kynurenine, and correlate miRNAs function in BMSCs biology through bioinformatics analysis. Human BMSCs were cultured and treated with and without kynurenine, and subsequent miRNA isolation was performed. MiRNA array was performed to identify differentially expressed miRNA. Microarray analysis identified 50 up-regulated, and 36 down-regulated miRNAs in kynurenine-treated BMSC cultures. Differentially expressed miRNA included miR-1281, miR-330-3p, let-7f-5p, and miR-493-5p, which are important for BMSC proliferation and differentiation. KEGG analysis found up-regulated miRNA targeting glutathione metabolism, a pathway critical for removing oxidative species. Our data support that the kynurenine dependent degenerative effect is partially due to changes in the miRNA profile of BMSCs.

Kynurenine signaling through the aryl hydrocarbon receptor: Implications for aging and healthspan.

The tryptophan metabolite kynurenine increases with aging and inflammation, and appears to contribute directly to the development and progression of several age-related conditions. Kynurenine is now known to signal through the aryl hydrocarbon receptor (Ahr) to modulate levels of reactive oxygen species (ROS). The Ahr promoter region contains several sites for NF-kB binding, indicating that inflammation is a key factor modulating Ahr expression. Furthermore, kynurenine activation of Ahr is observed to stimulate expression of the enzyme IDO1, which generates kynurenine by degrading tryptophan, representing a positive feedback loop that may link inflammation with ROS production. On the other hand, the antioxidant system-inducing transcription factor Nrf2 can be stimulated by Ahr, and Nrf2 can itself activate Ahr expression. The balance between pro- and antioxidant functions of Ahr mediated by kynurenine may therefore regulate healthy versus unhealthy aging in different tissues and organ systems. Potential therapeutic approaches to target this pathway include exercise to alter kynurenine production or molecules such as metformin or resveratrol that may suppress Ahr activity.

Kynurenine, a Tryptophan Metabolite That Increases with Age, Induces Muscle Atrophy and Lipid Peroxidation.

The cellular and molecular mechanisms underlying loss of muscle mass with age (sarcopenia) are not well-understood; however, heterochronic parabiosis experiments show that circulating factors are likely to play a role. Kynurenine (KYN) is a circulating tryptophan metabolite that is known to increase with age and is a ligand of the aryl hydrocarbon receptor (Ahr). Here, we tested the hypothesis that KYN activation of Ahr plays a role in muscle loss with aging. Results indicate that KYN treatment of mouse and human myoblasts increased levels of reactive oxygen species (ROS) 2-fold and KYN treatment in vivo reduced muscle size and strength and increased muscle lipid peroxidation in young mice. PCR array data indicate that muscle fiber size reduction with KYN treatment reduces protein synthesis markers whereas ubiquitin ligase gene expression is not significantly increased. KYN is generated by the enzyme indoleamine 2,3-dioxygenase (IDO), and aged mice treated with the IDO inhibitor 1-methyl-D-tryptophan showed an increase in muscle fiber size and muscle strength. Small-molecule inhibition of Ahr in vitro, and Ahr knockout in vivo, did not prevent KYN-induced increases in ROS, suggesting that KYN can directly increase ROS independent of Ahr activation. Protein analysis identified very long-chain acyl-CoA dehydrogenase as a factor activated by KYN that may increase ROS and lipid peroxidation. Our data suggest that IDO inhibition may represent a novel therapeutic approach for the prevention of sarcopenia and possibly other age-associated conditions associated with KYN accumulation such as bone loss and neurodegeneration.

Muscle-derived miR-34a increases with age in circulating extracellular vesicles and induces senescence of bone marrow stem cells.

Extracellular vesicles (EVs) are known to play important roles in cell-cell communication. Here we investigated the role of muscle-derived EVs and their microRNAs in the loss of bone stem cell populations with age. Aging in male and female C57BL6 mice was associated with a significant increase in expression of the senescence-associated microRNA miR-34a-5p (miR-34a) in skeletal muscle and in serum -derived EVs. Muscle-derived, alpha-sarcoglycan positive, EVs isolated from serum samples also showed a significant increase in miR-34a with age. EVs were isolated from conditioned medium of C2C12 mouse myoblasts and primary human myotubes after cells were treated with hydrogen peroxide to simulate oxidative stress. These EVs were shown to have elevated levels of miR-34a, and these EVs decreased viability of bone marrow mesenchymal (stromal) cells (BMSCs) and increased BMSC senescence. A lentiviral vector system was used to overexpress miR-34a in C2C12 cells, and EVs isolated from these transfected cells were observed to home to bone in vivo and to induce senescence and decrease Sirt1 expression of primary bone marrow cells ex vivo. These findings suggest that aged skeletal muscle is a potential source of circulating, senescence-associated EVs that may directly impact stem cell populations in tissues such as bone via their microRNA cargo.

Bone Marrow Derived Extracellular Vesicles Activate Osteoclast Differentiation in Traumatic Brain Injury Induced Bone Loss.

Traumatic brain injury (TBI) is a major source of worldwide morbidity and mortality. Patients suffering from TBI exhibit a higher susceptibility to bone loss and an increased rate of bone fractures; however, the underlying mechanisms remain poorly defined. Herein, we observed significantly lower bone quality and elevated levels of inflammation in bone and bone marrow niche after controlled cortical impact-induced TBI in in vivo CD-1 mice. Further, we identified dysregulated NF-κB signaling, an established mediator of osteoclast differentiation and bone loss, within the bone marrow niche of TBI mice. Ex vivo studies revealed increased osteoclast differentiation in bone marrow-derived cells from TBI mice, as compared to sham injured mice. We also found bone marrow derived extracellular vesicles (EVs) from TBI mice enhanced the colony forming ability and osteoclast differentiation efficacy and activated NF-κB signaling genes in bone marrow-derived cells. Additionally, we showed that miRNA-1224 up-regulated in bone marrow-derived EVs cargo of TBI. Taken together, we provide evidence that TBI-induced inflammatory stress on bone and the bone marrow niche may activate NF-κB leading to accelerated bone loss. Targeted inhibition of these signaling pathways may reverse TBI-induced bone loss and reduce fracture rates.

Activation of PERK Elicits Memory Impairment through Inactivation of CREB and Downregulation of PSD95 After Traumatic Brain Injury.

The PKR-like ER kinase (PERK), a transmembrane protein, resides in the endoplasmic reticulum (ER). Its activation serves as a key sensor of ER stress, which has been implicated in traumatic brain injury (TBI). The loss of memory is one of the most common symptoms after TBI, but the precise role of PERK activation in memory impairment after TBI has not been well elucidated. Here, we have shown that blocking the activation of PERK using GSK2656157 prevents the loss of dendritic spines and rescues memory deficits after TBI. To elucidate the molecular mechanism, we found that activated PERK phosphorylates CAMP response element binding protein (CREB) and PSD95 directly at the S129 and T19 residues, respectively. Phosphorylation of CREB protein prevents its interaction with a coactivator, CREB-binding protein, and subsequently reduces the BDNF level after TBI. Conversely, phosphorylation of PSD95 leads to its downregulation in pericontusional cortex after TBI in male mice. Treatment with either GSK2656157 or overexpression of a kinase-dead mutant of PERK (PERK-K618A) rescues BDNF and PSD95 levels in the pericontusional cortex by reducing phosphorylation of CREB and PSD95 proteins after TBI. Similarly, administration of either GSK2656157 or overexpression of PERK-K618A in primary neurons rescues the loss of dendritic outgrowth and number of synapses after treatment with a PERK activator, tunicamycin. Therefore, our study suggests that inhibition of PERK phosphorylation could be a potential therapeutic target to restore memory deficits after TBI.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) is the leading cause of death and disability around the world and affects 1.7 million Americans each year. Here, we have shown that TBI-activated PKR-like ER kinase (PERK) is responsible for memory deficiency, which is the most common problem in TBI patients. A majority of PERK's biological activities have been attributed to its function as an eIF2α kinase. However, our study suggests that activated PERK mediates its function via increasing phosphorylation of CAMP response element binding protein (CREB) and PSD95 after TBI. Blocking PERK phosphorylation rescues spine loss and memory deficits independently of phosphorylation of eIF2α. Therefore, our study suggests that CREB and PSD95 are novel substrates of PERK, so inhibition of PERK phosphorylation using GSK2656157 would be beneficial against memory impairment after TBI.