Antiretroviral treatment reveals a novel role for lysosomes in oligodendrocyte maturation.

White matter deficits are a common neuropathologic finding in neurologic disorders, including HIV-associated neurocognitive disorders (HAND). In HAND, the persistence of white matter alterations despite suppressive antiretroviral (ARV) therapy suggests that ARVs may be directly contributing to these impairments. Here, we report that a frontline ARV, bictegravir (BIC), significantly attenuates remyelination following cuprizone-mediated demyelination, a model that recapitulates acute demyelination, but has no impact on already formed mature myelin. Mechanistic studies utilizing primary rat oligodendrocyte precursor cells (OPCs) revealed that treatment with BIC leads to significant decrease in mature oligodendrocytes accompanied by lysosomal deacidification and impairment of lysosomal degradative capacity with no alterations in lysosomal membrane permeability or total lysosome number. Activation of the endolysosomal cation channel TRPML1 prevents both lysosomal deacidification and impairment of oligodendrocyte differentiation by BIC. Lastly, we show that deacidification of lysosomes by compounds that raise lysosomal pH is sufficient to prevent maturation of oligodendrocytes. Overall, this study has uncovered a critical role for lysosomal acidification in modulating oligodendrocyte function and has implications for neurologic diseases characterized by lysosomal dysfunction and white matter abnormalities.

Dietary vitamin D is a novel modulator of tumor engraftment through regulation of GC protein abundance.

The vitamin D binding protein, the GC protein, is a multifunctional protein that binds circulating vitamin D and also increases macrophage killing of tumor cells. Injecting exogenous GC protein concurrent with experimental tumor implant decreases tumor engraftment rate. Until now serum abundance of this protein was thought to be controlled by estrogen, glucocorticoids and inflammatory cytokines, but, not by vitamin D itself(1, 2). Nonetheless, increasing dietary vitamin D is thought to increase serum vitamin D, which is 98% bound by the GC protein. Based on the protection that excess GC protein offers we sought to determine if decreased GC protein abundance might decrease tumor immunity. Relatedly, we theorized, by contrast to the current model, that dietary vitamin D might affect serum abundance of GC protein. If exogenous vitamin D alters available GC levels, then this effect might indicate a novel pathway by which vitamin D enhances immunity. To examine these possibilities, we examined the effect of GC protein absence on tumor persistence or engraftment on two different and common tumor types (prostate cancer and breast cancer). We further examined the relationship between dietary vitamin D and serum GC abundance. We found that absence of GC protein allowed significantly more engraftment of breast tumor cells in female mice and of prostate tumor cells in male mice. Further, we found a U-shaped response of serum GC protein to dietary vitamin D dosage as well as to serum vitamin D, indicating the potential benefit of high exogenous doses to enhance immunity and reduce tumor burden.

High dose dietary vitamin D allocates surplus calories to muscle and growth instead of fat via modulation of myostatin and leptin signaling.

Obesity occurs because the body stores surplus calories as fat rather than as muscle. Fat secretes a hormone, leptin, that modulates energy balance at the brain. Changes in fat mass are mirrored by changes in serum leptin. Elevated leptin prompts the brain to decrease appetite and increase energy expenditure. In obesity, however, impaired leptin sensitivity mutes these leptin-mediated changes. We have limited understanding of what controls leptin production by fat or leptin sensitivity in the brain. Muscle produces a hormone, myostatin, that plays a role in muscle analogous to the one that leptin plays in fat. Absent myostatin leads to increased muscle mass and strength. As with leptin, we also do not know what controls myostatin production or sensitivity. Although fat mass and muscle mass are closely linked, the interplay between leptin and myostatin remains obscure. Here we describe an interplay linked thru vitamin D. Conventionally, it is thought that vitamin D improves strength via trophic effects at the muscle. However, we find here that high dose dietary vitamin D allocates excess calories to muscle and linear growth instead of storage as fat. Vitamin D mediates this allocation by decreasing myostatin production and increasing leptin production and sensitivity. That is, high dose vitamin D improves integration of organismal energy balance. Obesity, aging and other chronic inflammatory diseases are associated with increased fat mass and decreased muscle mass and function (e.g. sarcopenia). Our work provides a physiologic framework for how high-dose vitamin D would increase allocation of calories to muscle instead of fat in these pathologies. Additionally, our work reveals a novel link between the myostatin and leptin signaling whereby myostatin conveys energy needs to modulate leptin effects on calorie allocation. This result provides evidence to update the conventional model of energy stores sensing to a new model of energy balance sensing. In our proposed model, integration of leptin and myostatin signaling allows control of body composition independent of weight. Furthermore, our work reveals how physiologic seasonal variation in vitamin D may be important in controlling season-specific metabolism and calorie allocation to fat in winter and muscle and growth in summer.

Inhibition of lipid synthesis by the HIV integrase strand transfer inhibitor elvitegravir in primary rat oligodendrocyte cultures.

Combined antiretroviral therapy (cART) has greatly decreased mortality and morbidity among persons with HIV; however, neurologic impairments remain prevalent, in particular HIV-associated neurocognitive disorders (HANDs). White matter damage persists in cART-treated persons with HIV and may contribute to neurocognitive dysfunction as the lipid-rich myelin membrane of oligodendrocytes is essential for efficient nerve conduction. Because of the importance of lipids to proper myelination, we examined the regulation of lipid synthesis in oligodendrocyte cultures exposed to the integrase strand transfer inhibitor elvitegravir (EVG), which is administered to persons with HIV as part of their initial regimen. We show that protein levels of genes involved in the fatty acid pathway were reduced, which correlated with greatly diminished de novo levels of fatty acid synthesis. In addition, major regulators of cellular lipid metabolism, the sterol regulatory element-binding proteins (SREBP) 1 and 2, were strikingly altered following exposure to EVG. Impaired oligodendrocyte differentiation manifested as a marked reduction in mature oligodendrocytes. Interestingly, most of these deleterious effects could be prevented by adding serum albumin, a clinically approved neuroprotectant. These new findings, together with our previous study, strengthen the possibility that antiretroviral therapy, at least partially through lipid dysregulation, may contribute to the persistence of white matter changes observed in persons with HIV and that some antiretrovirals may be preferable as life-long therapy.

Differential Frequency of CYP2R1 Variants Across Populations Reveals Pathway Selection for Vitamin D Homeostasis.

CONTEXT: Normal vitamin D homeostasis is necessary to ensure optimal mineral metabolism. Dietary insufficiency of vitamin D and the lack of sunlight each have well understood roles in vitamin D deficiency; however, the extent to which common genetic variations in vitamin D metabolizing enzymes contribute to alterations in vitamin D homeostasis remains uncertain. OBJECTIVE: To examine the possibility that common coding variation in vitamin D metabolizing enzymes alters vitamin D homeostasis we determined the effect of 44 nonsynonymous polymorphisms in CYP2R1, the vitamin D 25-hydroxylase, on enzyme function. RESULTS: Twenty-one of these polymorphisms decreased activity, while 2 variants increased activity. The frequency of CYP2R1 alleles with decreased 25-hydroxylase activity is 3 in every 1000 Caucasians and 7 in every 1000 African Americans. In populations where exposure to sunlight is high, alleles with decreased function occur at a frequency as high as 8%. The pattern of selected variation as compared to nonselected variation is consistent with it being the result of positive selection for nonfunctional alleles closer to the equator. To examine this possibility, we examined the variation pattern in another protein in the vitamin D pathway, the vitamin D binding protein (GC protein). The pattern of selected variation in the GC protein as compared to nonselected variation is also consistent with it being the result of positive selection for nonfunctional alleles closer to the equator. CONCLUSIONS: CYP2R1 polymorphisms have important effects on vitamin D homeostasis, and the geographic variability of CYP2R1 alleles represents an adaptation to differential exposures to UVB irradiation from sunlight.

Obesity Decreases Hepatic 25-Hydroxylase Activity Causing Low Serum 25-Hydroxyvitamin D.

Normal vitamin D homeostasis is critical for optimal health; nevertheless, vitamin D deficiency is a worldwide public health problem. Vitamin D insufficiency is most commonly due to inadequate cutaneous synthesis of cholecalciferol and/or insufficient intake of vitamin D, but can also arise as a consequence of pathological states such as obesity. Serum concentrations of 25(OH)D (calcidiol) are low in obesity, and fail to increase appropriately after vitamin D supplementation. Although sequestration of vitamin D in adipose tissues or dilution of ingested or cutaneously synthesized vitamin D in the large fat mass of obese patients has been proposed to explain these findings, here we investigate the alternative mechanism that reduced capacity to convert parent vitamin D to 25(OH)D due to decreased expression of CYP2R1, the principal hepatic vitamin D 25-hydroxylase. To test this hypothesis, we isolated livers from female mice of 6 to 24 weeks of age, weaned onto either a normal chow diet or a high-fat diet, and determined the abundance of Cyp2r1 mRNA using digital droplet-quantitative PCR. We observed a significant (p < 0.001) decrease in Cyp2r1 mRNA in the liver of high-fat diet-fed mice relative to lean-chow-fed female mice. Moreover, there was a significant (p < 0.01) relationship between levels of Cyp2r1 mRNA and serum 25(OH)D concentrations as well as between Cyp2R1 mRNA and the ratio of circulating 25(OH)D3 to cholecalciferol (p < 0.0001). Using linear regression we determined a curve with 25(OH)D3/cholecalciferol versus normalized Cyp2R1 mRNA abundance with an R2 value of 0.85. Finally, we performed ex vivo activity assays of isolated livers and found that obese mice generated significantly less 25(OH)D3 than lean mice (p < 0.05). Our findings indicate that expression of CYP2R1 is reduced in obesity and accounts in part for the decreased circulating 25(OH)D. © 2019 American Society for Bone and Mineral Research.

Decreased Serum 25-Hydroxyvitamin D in Aging Male Mice Is Associated With Reduced Hepatic Cyp2r1 Abundance.

The prevalence of vitamin D deficiency, as determined by circulating levels of 25-hydroxycalciferol [25(OH)D], is greater in older individuals compared with the young. To examine the hypothesis that altered production or inactivation of 25(OH)D contributes to lower circulating levels of 25(OH)D, we measured the serum levels of parent vitamin D3 (cholecalciferol) and 25(OH)D. We also determined the relative abundance of transcripts encoding hepatic CYP2R1 and CYP27B1, the principal 25-hydroxylases, transcripts encoding enzymes that degrade 25(OH)D in the liver (Cyp3A11) and kidney (Cyp24A1) and transcripts encoding megalin and cubilin, proteins critical to vitamin D resorption in the kidney in mice at three different ages. We observed a significant decline in the relative abundance of Cyp2R1 in the liver with aging (one-way ANOVA, P = 0.0077). Concurrent with the decrease in mRNA, a significant decline in hepatic CYP2R1 protein (one-way ANOVA for trend, P = 0.007) and 25(OH)D (one-way ANOVA for trend, P = 0.002) and in the ratio of 25(OH)D3 to cholecalciferol (one-way ANOVA, P = 0.0003). By contrast, levels of the transcripts encoding Cyp3a11, Cyp24a1, and Cyp27b1 megalin and cubilin were unchanged with aging. A significant positive correlation was found between Cyp2r1 mRNA and 25(OH)D, and a stronger correlation was found between Cyp2r1 mRNA and the ratio of 25(OH)D3 to cholecalciferol. These results indicate that decreased expression of CYP2R1 contributes to the reduced serum levels of 25(OH)D in aging.

Ablation of Oxytocin Neurons Causes a Deficit in Cold Stress Response.

The paraventricular nucleus (PVN) is a critical locus of energy balance control. Three sets of neurons in the PVN are involved in regulating energy balance: oxytocin-expressing neurons (OXT-neurons), thyrotropin-releasing hormone-expressing neurons, and corticotrophin-releasing hormone-expressing neurons. To examine the role of OXT-neurons in energy balance, we ablated these neurons in mice by injecting diphtheria toxin into mice possessing both the oxytocin promoter driving cre expression and a cre-inducible diphtheria toxin receptor. Immunohistochemistry and real-time reverse transcriptase polymerase chain reaction confirmed that this injection caused a significant decrease in PVN OXT-neurons and OXT-mRNA abundance. OXT-neuron ablation did not alter food intake, weight, or energy expenditure at room temperature on either chow or a high-fat diet. To further characterize OXT-neuron-ablated mice, we examined their response to 1) intraperitoneal cholecystokinin (CCK) injection and 2) thermogenic stress. OXT-neuron-ablated mice had a blunted decrease in feeding response to CCK. When exposed to the extreme cold (4°C) for 3 hours, OXT-neuron-ablated mice had significant decreases in both rectal and brown adipose tissue temperature relative to controls, which was rescued by OXT treatment. Thermographic imaging revealed that OXT-neuron-ablated mice had increased body surface temperature. Thus, we report that OXT-neuron ablation shows no role for OXT-neurons in energy homeostasis at neutral temperature but reveals a heretofore unappreciated role for OXT-neurons and oxytocin specifically in regulating the thermogenic stress response.

Hormonal and molecular effects of restraint stress on formalin-induced pain-like behavior in male and female mice.

The evolutionary advantages to the suppression of pain during a stressful event (stress-induced analgesia (SIA)) are obvious, yet the reasoning behind sex-differences in the expression of this pain reduction are not. The different ways in which males and females integrate physiological stress responses and descending pain inhibition are unclear. A potential supraspinal modulator of stress-induced analgesia is the central nucleus of the amygdala (CeA). This limbic brain region is involved in both the processing of stress and pain; the CeA is anatomically and molecularly linked to regions of the hypothalamic pituitary adrenal (HPA) axis and descending pain network. The CeA exhibits sex-based differences in response to stress and pain that may differentially induce SIA in males and females. Here, sex-based differences in behavioral and molecular indices of SIA were examined following noxious stimulation. Acute restraint stress in male and female mice was performed prior to intraplantar injections of formalin, a noxious inflammatory agent. Spontaneous pain-like behaviors were measured for 60min following formalin injection and mechanical hypersensitivity was evaluated 120 and 180min post-injection. Restraint stress altered formalin-induced spontaneous behaviors in male and female mice and formalin-induced mechanical hypersensitivity in male mice. To assess molecular indices of SIA, tissue samples from the CeA and blood samples were collected at the 180min time point. Restraint stress prevented formalin-induced increases in extracellular signal regulated kinase 2 (ERK2) phosphorylation in the male CeA, but no changes associated with pERK2 were seen with formalin or restraint in females. Sex differences were also seen in plasma corticosterone concentrations 180min post injection. These results demonstrate sex-based differences in behavioral, molecular, and hormonal indices of acute stress in mice that extend for 180min after stress and noxious stimulation.

Postinflammatory hyperpigmentation after human cold pain testing.

Changes in cold temperature sensitivity are often associated with chronic pain conditions. Progress in understanding the neurobiological mechanism underlying these changes and resulting development of effective therapies has been slowed by the accessibility and affordability of devices used to measure thermal sensitivity in humans. To address this gap, we developed an inexpensive method to measure cold pain thresholds in healthy adult volunteers using dry ice and a thermode. However, early in preliminary testing, a subject presented with epidermal postinflammatory hyperpigmentation that lasted for >200 days. Although this response was unique among the small number of subjects in development of the assay, it raised questions as to the safety of the assay design.