Brain responses to intermittent fasting and the healthy living diet in older adults.

Diet may promote brain health in metabolically impaired older individuals. In an 8-week randomized clinical trial involving 40 cognitively intact older adults with insulin resistance, we examined the effects of 5:2 intermittent fasting and the healthy living diet on brain health. Although intermittent fasting induced greater weight loss, the two diets had comparable effects in improving insulin signaling biomarkers in neuron-derived extracellular vesicles, decreasing the brain-age-gap estimate (reflecting the pace of biological aging of the brain) on magnetic resonance imaging, reducing brain glucose on magnetic resonance spectroscopy, and improving blood biomarkers of carbohydrate and lipid metabolism, with minimal changes in cerebrospinal fluid biomarkers for Alzheimer's disease. Intermittent fasting and healthy living improved executive function and memory, with intermittent fasting benefiting more certain cognitive measures. In exploratory analyses, sex, body mass index, and apolipoprotein E and SLC16A7 genotypes modulated diet effects. The study provides a blueprint for assessing brain effects of dietary interventions and motivates further research on intermittent fasting and continuous diets for brain health optimization. For further information, please see ClinicalTrials.gov registration: NCT02460783.

Lipidomic Analysis of Plasma Extracellular Vesicles Derived from Alzheimer's Disease Patients.

Analysis of blood-based indicators of brain health could provide an understanding of early disease mechanisms and pinpoint possible intervention strategies. By examining lipid profiles in extracellular vesicles (EVs), secreted particles from all cells, including astrocytes and neurons, and circulating in clinical samples, important insights regarding the brain's composition can be gained. Herein, a targeted lipidomic analysis was carried out in EVs derived from plasma samples after removal of lipoproteins from individuals with Alzheimer's disease (AD) and healthy controls. Differences were observed for selected lipid species of glycerolipids (GLs), glycerophospholipids (GPLs), lysophospholipids (LPLs) and sphingolipids (SLs) across three distinct EV subpopulations (all-cell origin, derived by immunocapture of CD9, CD81 and CD63; neuronal origin, derived by immunocapture of L1CAM; and astrocytic origin, derived by immunocapture of GLAST). The findings provide new insights into the lipid composition of EVs isolated from plasma samples regarding specific lipid families (MG, DG, Cer, PA, PC, PE, PI, LPI, LPE, LPC), as well as differences between AD and control individuals. This study emphasizes the crucial role of plasma EV lipidomics analysis as a comprehensive approach for identifying biomarkers and biological targets in AD and related disorders, facilitating early diagnosis and potentially informing novel interventions.

Identification of Clinically Relevant Brain Endothelial Cell Biomarkers in Plasma.

BACKGROUND: Proteins expressed by brain endothelial cells (BECs), the primary cell type of the blood-brain barrier, may serve as sensitive plasma biomarkers for neurological and neurovascular conditions, including cerebral small vessel disease. METHODS: Using data from the BLSA (Baltimore Longitudinal Study of Aging; n=886; 2009-2020), BEC-enriched proteins were identified among 7268 plasma proteins (measured with SomaScanv4.1) using an automated annotation algorithm that filtered endothelial cell transcripts followed by cross-referencing with BEC-specific transcripts reported in single-cell RNA-sequencing studies. To identify BEC-enriched proteins in plasma most relevant to the maintenance of neurological and neurovascular health, we selected proteins significantly associated with 3T magnetic resonance imaging-defined white matter lesion volumes. We then examined how these candidate BEC biomarkers related to white matter lesion volumes, cerebral microhemorrhages, and lacunar infarcts in the ARIC study (Atherosclerosis Risk in Communities; US multisite; 1990-2017). Finally, we determined whether these candidate BEC biomarkers, when measured during midlife, were related to dementia risk over a 25-year follow-up period. RESULTS: Of the 28 proteins identified as BEC-enriched, 4 were significantly associated with white matter lesion volumes (CDH5 [cadherin 5], CD93 [cluster of differentiation 93], ICAM2 [intracellular adhesion molecule 2], GP1BB [glycoprotein 1b platelet subunit beta]), while another approached significance (RSPO3 [R-Spondin 3]). A composite score based on 3 of these BEC proteins accounted for 11% of variation in white matter lesion volumes in BLSA participants. We replicated the associations between the BEC composite score, CDH5, and RSPO3 with white matter lesion volumes in ARIC, and further demonstrated that the BEC composite score and RSPO3 were associated with the presence of ≥1 cerebral microhemorrhages. We also showed that the BEC composite score, CDH5, and RSPO3 were associated with 25-year dementia risk. CONCLUSIONS: In addition to identifying BEC proteins in plasma that relate to cerebral small vessel disease and dementia risk, we developed a composite score of plasma BEC proteins that may be used to estimate blood-brain barrier integrity and risk for adverse neurovascular outcomes.

Neuron-derived extracellular vesicles to examine brain mTOR target engagement with sirolimus in patients with multiple system atrophy.

INTRODUCTION: Impaired autophagy is a pathogenic mechanism in the synucleinopathies. Sirolimus, a potent mTOR inhibitor and autophagy activator, had no beneficial effects in a randomized placebo-controlled trial in patients with multiple system atrophy (MSA). Whether sirolimus effectively inhibited brain mTOR activity was unknown. We aimed to evaluate if patients with MSA treated with sirolimus had evidence of inhibited brain mTOR pathways by measuring neuron-derived serum extracellular vesicles (NEVs). METHODS: Serum samples were collected from participants of the sirolimus-MSA trial, which randomized patients to sirolimus (2-6 mg/day) or placebo for 48 weeks. NEVs were immunoprecipitated with three antibodies-against neurons. Brain mTOR engagement was quantified as the change in the NEV phosphorylated mTOR (p-mTOR) to total-mTOR (tot-mTOR) ratio after 48 weeks of sirolimus. RESULTS: Samples from 27 patients [mean (±SD) age, 59.2±7 years, 15 (55.5%) men] were analyzed (19 sirolimus, 8 placebo). Treated- and placebo-patients had similar p-mTOR:tot-mTOR ratio at 24 (placebo: 0.248 ± 0.03, sirolimus: 0.289 ± 0.02; P = 0.305) and 48 weeks (placebo: 0.299 ± 0.05, sirolimus: 0.261 ± 0.03; P = 0.544). The tot-mTOR, p-mTOR, or their ratio levels were not associated with Unified MSA Rating Scale (UMSARS) worsening. DISCUSSION: These results are consistent with no brain mTOR engagement by oral sirolimus up to 6 mg/day. NEV-based biomarkers are a rational approach to investigating target engagement in clinical trials of brain-targeted therapeutics.

Extracellular vesicle biomarkers for cognitive impairment in Parkinson's disease.

Besides motor symptoms, many individuals with Parkinson's disease develop cognitive impairment perhaps due to coexisting α-synuclein and Alzheimer's disease pathologies and impaired brain insulin signalling. Discovering biomarkers for cognitive impairment in Parkinson's disease could help clarify the underlying pathogenic processes and improve Parkinson's disease diagnosis and prognosis. This study used plasma samples from 273 participants: 103 Parkinson's disease individuals with normal cognition, 121 Parkinson's disease individuals with cognitive impairment (81 with mild cognitive impairment, 40 with dementia) and 49 age- and sex-matched controls. Plasma extracellular vesicles enriched for neuronal origin were immunocaptured by targeting the L1 cell adhesion molecule, then biomarkers were quantified using immunoassays. α-Synuclein was lower in Parkinson's disease compared to control individuals (P = 0.004) and in cognitively impaired Parkinson's disease individuals compared to Parkinson's disease with normal cognition (P < 0.001) and control (P < 0.001) individuals. Amyloid-ß42 did not differ between groups. Phosphorylated tau (T181) was higher in Parkinson's disease than control individuals (P = 0.003) and in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P < 0.001) and controls (P < 0.001). Total tau was not different between groups. Tyrosine-phosphorylated insulin receptor substrate-1 was lower in Parkinson's disease compared to control individuals (P = 0.03) and in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.02) and controls (P = 0.01), and also decreased with increasing motor symptom severity (P = 0.005); serine312-phosphorylated insulin receptor substrate-1 was not different between groups. Mechanistic target of rapamycin was not different between groups, whereas phosphorylated mechanistic target of rapamycin trended lower in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.05). The ratio of α-synuclein to phosphorylated tau181 was lower in Parkinson's disease compared to controls (P = 0.001), in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P < 0.001) and decreased with increasing motor symptom severity (P < 0.001). The ratio of insulin receptor substrate-1 phosphorylated serine312 to insulin receptor substrate-1 phosphorylated tyrosine was higher in Parkinson's disease compared to control individuals (P = 0.01), in cognitively impaired compared to cognitively normal Parkinson's disease individuals (P = 0.02) and increased with increasing motor symptom severity (P = 0.003). α-Synuclein, phosphorylated tau181 and insulin receptor substrate-1 phosphorylated tyrosine contributed in diagnostic classification between groups. These findings suggest that both α-synuclein and tau pathologies and impaired insulin signalling underlie Parkinson's disease with cognitive impairment. Plasma neuronal extracellular vesicles biomarkers may inform cognitive prognosis in Parkinson's disease.

Oral nicotinamide riboside raises NAD+ and lowers biomarkers of neurodegenerative pathology in plasma extracellular vesicles enriched for neuronal origin.

Declining nicotinamide adenine dinucleotide (NAD+ ) concentration in the brain during aging contributes to metabolic and cellular dysfunction and is implicated in the pathogenesis of aging-associated neurological disorders. Experimental therapies aimed at boosting brain NAD+ levels normalize several neurodegenerative phenotypes in animal models, motivating their clinical translation. Dietary intake of NAD+ precursors, such as nicotinamide riboside (NR), is a safe and effective avenue for augmenting NAD+ levels in peripheral tissues in humans, yet evidence supporting their ability to raise NAD+ levels in the brain or engage neurodegenerative disease pathways is lacking. Here, we studied biomarkers in plasma extracellular vesicles enriched for neuronal origin (NEVs) from 22 healthy older adults who participated in a randomized, placebo-controlled crossover trial (NCT02921659) of oral NR supplementation (500 mg, 2x /day, 6 weeks). We demonstrate that oral NR supplementation increases NAD+ levels in NEVs and decreases NEV levels of Aß42, pJNK, and pERK1/2 (kinases involved in insulin resistance and neuroinflammatory pathways). In addition, changes in NAD(H) correlated with changes in canonical insulin-Akt signaling proteins and changes in pERK1/2 and pJNK. These findings support the ability of orally administered NR to augment neuronal NAD+ levels and modify biomarkers related to neurodegenerative pathology in humans. Furthermore, NEVs offer a new blood-based window into monitoring the physiologic response of NR in the brain.

Sodium alginate microencapsulation of human mesenchymal stromal cells modulates paracrine signaling response and enhances efficacy for treatment of established osteoarthritis.

Mesenchymal stromal cells (MSCs) have shown promise as osteoarthritis (OA) treatments; however, effective translation has been limited by high variability and heterogeneity of MSCs, suboptimal delivery strategies, and poor understanding of critical quality and potency attributes. Furthermore, most pre-clinical studies of MSC therapeutics for OA have focused on delaying OA development and not on treating established OA, which brings added clinical relevance. Thus, the objective of the current study was to assess the effects of sodium alginate microencapsulation on human MSC (hMSC) secretion of immunomodulatory cytokines in an OA microenvironment and therapeutic efficacy in treating established OA. A Medial Meniscal Transection (MMT) pre-clinical model of OA was implemented. Three weeks post-surgery, after OA was established, intra-articular injections of encapsulated hMSCs or nonencapsulated hMSCs were administered. Six weeks post-surgery, microstructural changes in the knee joint were quantified using microCT. Encapsulated hMSCs reduced articular cartilage degeneration and subchondral bone remodeling. A multiplexed immunoassay panel was used to profile the in vitro secretome of hMSCs in response to IL-1ß. Nonencapsulated hMSCs showed an indiscriminate increase in all cytokines in response to IL-1ß while encapsulated hMSCs showed a targeted secretory response with increased expression of pro-inflammatory (IL-1ß, IL-6, IL-7, IL-8), anti-inflammatory (IL-1RA), and chemotactic (G-CSF, MDC, IP10) cytokines. These data show that sodium alginate microencapsulation can modulate hMSC paracrine signaling and enhance the therapeutic efficacy of the hMSCs in treating established OA. This cytokine profile provides a foundation for the identification of key factors affecting the overall potency of hMSC therapeutics for OA. STATEMENT OF SIGNIFICANCE: While there has been considerable interest in material based MSC encapsulation for treatment of OA, there are critical gaps in our translational understanding of these biomaterial-based technologies for OA. More specifically, previous studies have several important limitations: (1) they have been largely focused on preventing OA development, which limits their translational utility and (2) little prior work has been done to delineate potential routes/mechanisms by which material encapsulation alters MSC therapeutic action. In our manuscript, we aimed to fill these gaps in knowledge by testing the hypotheses that: (1) hMSC encapsulation can attenuate established disease progression, which is a more clinically relevant scenario and (2) hMSC encapsulation significantly changes the secreted paracrine factors from hMSCs.

Characterization of OA development between sexes in the rat medial meniscal transection model.

Objective: Osteoarthritis (OA) is a chronic degenerative disease of the joints characterized by articular cartilage degradation. While clear sex differences exist in human OA development, most pre-clinical research has been conducted solely in male animals, limiting generalizability of findings to both sexes. The objective of this study was to determine if sex impacts the progression and severity of OA in the rat medial meniscal tear (MMT) preclinical model used to surgically induce OA. It was hypothesized that differences would be observed between males and females following MMT surgery. Design: An MMT model was employed in male and female Lewis rats to induce OA. Animals were euthanized 3 weeks post-surgery and EPIC-µCT was used to quantitatively evaluate articular cartilage structure and composition, osteophyte volumes and subchondral bone structure. Results: Analysis of medial 1/3 articular cartilage, showed increased cartilage thickness and proteoglycan loss in the MMT of both sexes, when compared to sham. Both male and female MMT groups also saw increased subchondral bone mineral density and larger osteophyte volumes. Significant interactions between sex and OA development were seen in normalized cartilage volume (larger in females), and normalized total osteophyte volumes (larger in males). Conclusion: This study demonstrates the viability of both sexes in the rat MMT preclinical OA model. Though clear differences exist, this model can be used to model OA development and evaluate sex as a factor in the efficacy of OA therapeutics.