Integrating sleep health into resilience research.

Sleep is a biological necessity that is a critical determinant of mental and physical well-being. Sleep may promote resilience by enhancing an individual's biological preparedness to resist, adapt and recover from a challenge or stressor. This report analyzes currently active National Institutes of Health (NIH) grants focussed on sleep and resilience, specifically examining the design of studies that explore sleep as a factor that promotes health maintenance, survivorship, or protective/preventive pathways. A search of NIH R01 and R21 research project grants that received funding in Fiscal Years (FY) 2016-2021 and focussed on sleep and resilience was conducted. A total of 16 active grants from six NIH institutes met the inclusion criteria. Most grants were funded in FY 2021 (68.8%), used the R01 mechanism (81.3%), were observational studies (75.0%), and measured resilience in the context of resisting a stressor/challenge (56.3%). Early adulthood and midlife were most commonly studied and over half of the grants focussed on underserved/underrepresented populations. NIH-funded studies focussed on sleep and resilience, or the ways in which sleep can influence an individual's ability to resist, adapt, or recover from a challenging event. This analysis highlights an important gap and the need to expand research focussed on sleep as a promotor of molecular, physiological, and psychological resilience.

Perspectives: on Precision Nutrition Research in Heart, Lung, and Blood Diseases and Sleep Disorders.

The release of the 2020-2030 Strategic Plan for NIH Nutrition Research (SPNR) and its emphasis on precision nutrition has provided an opportunity to identify future nutrition research that addresses individual variability in response to diet and nutrition across the life span-including those relevant to the Strategic Vision of the National Heart, Lung, and Blood Institute (NHLBI). The SPNR and the NHLBI's Strategic Vision were developed with extensive input from the extramural research community, and both have 4 overarching strategic goals within which are embedded several objectives for research. For the SPNR, these include 1) spur discovery science and normal biological functions (e.g., role of the microbiome in health and disease), 2) population science to understand individual differences (e.g., biomarkers including 'omics that predict disease status), 3) emerging scientific areas of investigation and their application (e.g., data science, artificial intelligence), and 4) cross-cutting themes (e.g., training the scientific workforce and minority health and health disparities). These strategic goals and objectives serve as blueprints for research and training. Nutrition remains important in the prevention and treatment of heart, lung, blood, and sleep (HLBS) disorders and diseases, and the NHLBI has played a pivotal role in supporting nutrition research. In this paper, we report important gaps in the scientific literature related to precision nutrition in HLBS diseases. Research opportunities that could stimulate precision nutrition and their alignment with the SPNR and the NHLBI Strategic Vision Objectives are provided. These opportunities include 1) exploring individual differences in response to varying dietary patterns and nutrients; 2) investigating genetic/epigenetic, biological (e.g., microbiome, biomarkers), social, psychosocial, and environmental underpinnings of individual variability in diet; 3) elucidating the role of circadian rhythm and chrononutrition; and 4) applying implementation science research methods in precision nutrition interventions relevant to HLBS diseases.

Altered glutamate clearance in ascorbate deficient mice increases seizure susceptibility and contributes to cognitive impairment in APP/PSEN1 mice.

Ascorbate (vitamin C) is critical as a first line of defense antioxidant within the brain, and specifically within the synapse. Ascorbate is released by astrocytes during glutamate clearance and disruption of this exchange mechanism may be critical in mediating glutamate toxicity within the synapse. This is likely even more critical in neurodegenerative disorders with associated excitotoxicity and seizures, in particular Alzheimer's disease, in which ascorbate levels are often low. Using Gulo-/- mice that are dependent on dietary ascorbate, we established that low brain ascorbate increased sensitivity to kainic acid as measured via behavioral observations, electroencephalography (EEG) measurements, and altered regulation of several glutamatergic system genes. Kainic acid-induced immobility was improved in wild-type mice following treatment with ceftriaxone, which upregulates glutamate transporter GLT-1. The same effect was not observed in ascorbate-deficient mice in which sufficient ascorbate is not available for release. A single, mild seizure event was sufficient to disrupt performance in the water maze in low-ascorbate mice and in APPSWE/PSEN1dE9 mice. Together, the data support the critical role of brain ascorbate in maintaining protection during glutamatergic hyperexcitation events, including seizures. The study further supports a role for mild, subclinical seizures in cognitive decline in Alzheimer's disease.

Oxidative Stress Levels in the Brain Are Determined by Post-Mortem Interval and Ante-Mortem Vitamin C State but Not Alzheimer's Disease Status.

The current study highlighted several changes in measures of oxidative stress and antioxidant status that take place in the mouse brain over the course of 24 h post-mortem. Ascorbic acid (vitamin C) and glutathione both decreased significantly in cortex in as little as 2 h and malondialdehyde levels increased. Further change from baseline was observed up to 24 h, including carbonyl and sulfhydryl formation. The greatest changes were observed in brains that began with low ascorbic acid levels (gulo−/− mice) compared to wild-type or 5XFAD mice. Cortical samples from nine Alzheimer’s Disease cases and five controls were also assayed under the same conditions. Post mortem intervals ranged from 6 to 47 h and all samples had low ascorbic acid levels at time of measurement. Malondialdehyde levels were lower in Alzheimer’s Disease cases. Despite a strong positive correlation between ascorbic acid and glutathione levels, no other correlations among oxidative stress measures or post mortem interval were observed. Together the data suggest that molecular changes occurring within the first hours of death may mask differences between patient groups. Care must be taken interpreting studies in human brain tissue where ante-mortem nutrient status is not known to avoid bias or confounding of results.

Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer's disease and a novel protective role for ascorbate.

Mitochondrial dysfunction is elevated in very early stages of Alzheimer's disease and exacerbates oxidative stress, which contributes to disease pathology. Mitochondria were isolated from 4-month-old wild-type mice, transgenic mice carrying the APPSWE and PSEN1dE9 mutations, mice with decreased brain and mitochondrial ascorbate (vitamin C) via heterozygous knockout of the sodium dependent vitamin C transporter (SVCT2+/-) and transgenic APP/PSEN1 mice with heterozygous SVCT2 expression. Mitochondrial isolates from SVCT2+/- mice were observed to consume less oxygen using high-resolution respirometry, and also exhibited decreased mitochondrial membrane potential compared to wild type isolates. Conversely, isolates from young (4 months) APP/PSEN1 mice consumed more oxygen, and exhibited an increase in mitochondrial membrane potential, but had a significantly lower ATP/ADP ratio compared to wild type isolates. Greater levels of reactive oxygen species were also produced in mitochondria isolated from both APP/PSEN1 and SVCT2+/- mice compared to wild type isolates. Acute administration of ascorbate to mitochondria isolated from wild-type mice increased oxygen consumption compared with untreated mitochondria suggesting ascorbate may support energy production. This study suggests that both presence of amyloid and ascorbate deficiency can contribute to mitochondrial dysfunction, even at an early, prodromal stage of Alzheimer's disease, although occurring via different pathways. Ascorbate may, therefore, provide a useful preventative strategy against neurodegenerative disease, particularly in populations most at risk for Alzheimer's disease in which stores are often depleted through mitochondrial dysfunction and elevated oxidative stress.

Reversal of high fat diet-induced obesity improves glucose tolerance, inflammatory response, ß-amyloid accumulation and cognitive decline in the APP/PSEN1 mouse model of Alzheimer's disease.

This study assessed the extent to which high fat diet (HFD)-induced ß-amyloid accumulation and cognitive decline in APP/PSEN1 mice are reversible through control of fat intake. Ten months of HFD (60% calories from fat) led to significant deficits in a 2-trial Y maze task, and nest building assay, and decreased voluntary locomotor activity. The HFD induced an inflammatory response, indicated by increased expression of several inflammatory markers. Substituting a low fat diet led to pronounced weight loss and correction of glucose intolerance, decreases in the inflammatory response, and improved performance on behavioral tasks in both wild-type and APP/PSEN1 transgenic mice. Insoluble ß-amyloid levels, and extent of tau phosphorylation were also lower following dietary reversal in APP/PSEN1 mice compared to high fat-fed animals, indicating that the inflammatory response may have contributed to key pathogenic pathways in the Alzheimer's disease model. The data suggest that weight loss can be a vital strategy for cognitive protection, but also highlight potential mechanisms for intervention when sustained weight loss is not possible.

Vitamin C deficiency in the brain impairs cognition, increases amyloid accumulation and deposition, and oxidative stress in APP/PSEN1 and normally aging mice.

Subclinical vitamin C deficiency is widespread in many populations, but its role in both Alzheimer's disease and normal aging is understudied. In the present study, we decreased brain vitamin C in the APPSWE/PSEN1deltaE9 mouse model of Alzheimer's disease by crossing APP/PSEN1(+) bigenic mice with SVCT2(+/-) heterozygous knockout mice, which have lower numbers of the sodium-dependent vitamin C transporter required for neuronal vitamin C transport. SVCT2(+/-) mice performed less well on the rotarod task at both 5 and 12 months of age compared to littermates. SVCT2(+/-) and APP/PSEN1(+) mice and the combination genotype SVCT2(+/-)APP/PSEN1(+) were also impaired on multiple tests of cognitive ability (olfactory memory task, Y-maze alternation, conditioned fear, Morris water maze). In younger mice, both low vitamin C (SVCT2(+/-)) and APP/PSEN1 mutations increased brain cortex oxidative stress (malondialdehyde, protein carbonyls, F2-isoprostanes) and decreased total glutathione compared to wild-type controls. SVCT2(+/-) mice also had increased amounts of both soluble and insoluble Aß1-42 and a higher Aß1-42/1-40 ratio. By 14 months of age, oxidative stress levels were similar among groups, but there were more amyloid-ß plaque deposits in both hippocampus and cortex of SVCT2(+/-)APP/PSEN1(+) mice compared to APP/PSEN1(+) mice with normal brain vitamin C. These data suggest that even moderate intracellular vitamin C deficiency plays an important role in accelerating amyloid pathogenesis, particularly during early stages of disease development, and that these effects are likely modulated by oxidative stress pathways.

Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1- and JMJD3-dependent epigenetic control manner.

Intracellular Vitamin C (VC) is maintained at high levels in the developing brain by the activity of sodium-dependent VC transporter 2 (Svct2), suggesting specific VC functions in brain development. A role of VC as a cofactor for Fe(II)-2-oxoglutarate-dependent dioxygenases has recently been suggested. We show that VC supplementation in neural stem cell cultures derived from embryonic midbrains greatly enhanced differentiation toward midbrain-type dopamine (mDA) neurons, the neuronal subtype associated with Parkinson's disease. VC induced gain of 5-hydroxymethylcytosine (5hmC) and loss of H3K27m3 in DA phenotype gene promoters, which are catalyzed by Tet1 and Jmjd3, respectively. Consequently, VC enhanced DA phenotype gene transcriptions in the progenitors by Nurr1, a transcription factor critical for mDA neuron development, to be more accessible to the gene promoters. Further mechanism studies including Tet1 and Jmjd3 knockdown/inhibition experiments revealed that both the 5hmC and H3K27m3 changes, specifically in the progenitor cells, are indispensible for the VC-mediated mDA neuron differentiation. We finally show that in Svct2 knockout mouse embryos, mDA neuron formation in the developing midbrain decreased along with the 5hmC/H3k27m3 changes. These findings together indicate an epigenetic role of VC in midbrain DA neuron development.

Islet α-, ß-, and δ-cell development is controlled by the Ldb1 coregulator, acting primarily with the islet-1 transcription factor.

Ldb1 and Ldb2 are coregulators that mediate Lin11-Isl1-Mec3 (LIM)-homeodomain (HD) and LIM-only transcription factor-driven gene regulation. Although both Ldb1 and Ldb2 mRNA were produced in the developing and adult pancreas, immunohistochemical analysis illustrated a broad Ldb1 protein expression pattern during early pancreatogenesis, which subsequently became enriched in islet and ductal cells perinatally. The islet-enriched pattern of Ldb1 was similar to pan-endocrine cell-expressed Islet-1 (Isl1), which was demonstrated in this study to be the primary LIM-HD transcription factor in developing and adult islet cells. Endocrine cell-specific removal of Ldb1 during mouse development resulted in a severe reduction of hormone⁺ cell numbers (i.e., α, ß, and δ) and overt postnatal hyperglycemia, reminiscent of the phenotype described for the Isl1 conditional mutant. In contrast, neither endocrine cell development nor function was affected in the pancreas of Ldb2(-/-) mice. Gene expression and chromatin immunoprecipitation (ChIP) analyses demonstrated that many important Isl1-activated genes were coregulated by Ldb1, including MafA, Arx, insulin, and Glp1r. However, some genes (i.e., Hb9 and Glut2) only appeared to be impacted by Ldb1 during development. These findings establish Ldb1 as a critical transcriptional coregulator during islet α-, ß-, and δ-cell development through Isl1-dependent and potentially Isl1-independent control.