BACKGROUND:Approximately 60% of individuals with Down syndrome (DS) have sleep abnormalities independent of breathing obstruction. Previously, we demonstrated increased wakefulness and decreased NREM sleep in the Dp(16)1Yey+ (Dp16) DS model mouse. In this study, we determined if increased RCAN1 levels mediate sleep disruption in Dp16 mice. METHODS:We examined sleep architecture and electroencephalogram (EEG) patterns in young and aged Dp16 mice in which we genetically restored disomic levels of Rcan1 (Dp16 Rcan12N, shortened to Dp16-2N). Approximately equal numbers of male and female mice were used for each age group. We also examined gene expression and anxiety-like behaviors in aged mice. RESULTS:We found that young Dp16 and Dp16-2N mice differ slightly from WT mice in sleep architecture and EEG characteristics. However, with age, more severe sleep deficits manifest in Dp16 mice and are partially rescued by Rcan1 dosage correction. Aged Dp16 mice exhibit significantly less mean EEG total power across different sleep states and activity phases. In contrast, WT and Dp16-2N mice show no age-related differences across states and stages. Aged Dp16 mice diverge from aged WT mice across multiple wake and sleep frequency bands during dark and light phases. In contrast, the EEG characteristics of aged Dp16-2N differ only modestly from those of WT mice. CONCLUSIONS:Combined, our data demonstrate that restoring Rcan1 gene levels mitigates some sleep architecture disruptions and EEG differences observed in aged Dp16 mice.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ∼0.9 in as little as six-weeks with a mean firing rate of ∼13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of blood-brain barrier breakdown by using human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

Transactive response DNA binding protein 43 kilodaltons (TDP-43) is a DNA and RNA binding protein associated with severe neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), primarily affecting motor neurons in the brain and spinal cord. Partial knockdown of TDP-43 expression in a mouse model (the amiR-TDP-43 mice) leads to progressive, age-related motor dysfunction, as observed in ALS patients. Work in Caenorhabditis elegans suggests that TDP-43 dysfunction can lead to deficits in chromatin processing and double-stranded RNA (dsRNA) accumulation, potentially activating the innate immune system and promoting neuroinflammation. To test this hypothesis, we used immunostaining to investigate dsRNA accumulation and other signs of CNS pathology in the spinal cords of amiR-TDP-43 mice. Compared with wild-type controls, TDP-43 knockdown animals show increases in dsRNA deposition in the dorsal and ventral horns of the spinal cord. Additionally, animals with heavy dsRNA expression show markedly increased levels of astrogliosis and microgliosis. Interestingly, areas of high dsRNA expression and microgliosis overlap with regions of heavy neurodegeneration, indicating that activated microglia could contribute to the degeneration of spinal cord neurons. This study suggests that loss of TDP-43 function could contribute to neuropathology by increasing dsRNA deposition and subsequent innate immune system activation.

Aging is the primary risk factor for most neurodegenerative diseases, including Alzheimer's disease. Major hallmarks of brain aging include neuroinflammation/immune activation and reduced neuronal health/function. These processes contribute to cognitive dysfunction (a key risk factor for Alzheimer's disease), but their upstream causes are incompletely understood. Age-related increases in transposable element (TE) transcripts might contribute to reduced cognitive function with brain aging, as the reverse transcriptase inhibitor 3TC reduces inflammation in peripheral tissues and TE transcripts have been linked with tau pathology in Alzheimer's disease. However, the effects of 3TC on cognitive function with aging have not been investigated. Here, in support of a role for TE transcripts in brain aging/cognitive decline, we show that 3TC: (a) improves cognitive function and reduces neuroinflammation in old wild-type mice; (b) preserves neuronal health with aging in mice and Caenorhabditis elegans; and (c) enhances cognitive function in a mouse model of tauopathy. We also provide insight on potential underlying mechanisms, as well as evidence of translational relevance for these observations by showing that TE transcripts accumulate with brain aging in humans, and that these age-related increases intersect with those observed in Alzheimer's disease. Collectively, our results suggest that TE transcript accumulation during aging may contribute to cognitive decline and neurodegeneration, and that targeting these events with reverse transcriptase inhibitors like 3TC could be a viable therapeutic strategy.

Abstract Neurofilament light chain (NfL) is a biomarker indexing axonal integrity where small NfL variations may be associated with cognitive performance in early adulthood and high values associated with neurodegenerative disorders such as Alzheimer’s disease. In the Colorado Adoption/Twin Study of Lifespan behavioral development and cognitive aging (CATSLife1) individuals were tested at 28–49 years (M=33.1, SD=4.9). Quanterix Simoa assays of plasma NfL (pNfL) were measured in duplicate, and we included values for 1159 individuals where 1098 had available general cognitive ability scores and sociodemographic covariates. Unadjusted NfL values were consistent with other studies of early-mid adulthood (M = 5.9, SD = 3.1, range = 1.14 – 40.1 pg/mL) and 6% showed values outside expected normal reference limits (>10 pg/mL). After adjusting for technical covariates and skew, higher natural log-transformed pNfL was associated with age (r = 0.27) and female sex (r = 0.07). Moreover, adjusting for sociodemographic covariates, higher pNfL was associated with lower general cognitive ability (GCA) (r = -.06), where associations were more pronounced above the mean pNfL value (r = -.08). Multi-level regression analyses suggested that GCA-NfL associations were modified by age, whereby the worse performance was observed at higher ages and pNfL values (p <= 0.03), accounting for sibling relatedness and sociodemographic covariates. We observed small negative associations of higher plasma NfL and lower cognitive performance, where associations may become magnified with increasing age in early- to mid-adulthood.

The global obesity pandemic coupled with ever-growing life expectancies equates to hundreds of millions of individuals with potentially longer but not healthier lives. Aging is one of the risk factors for numerous maladies such as metabolic disorder and frailty, which are exacerbated under obesity. Thus, therapeutic approaches that address obesity to ultimately improve affected individuals’ quality of life and extend their lifespan are needed. We previously reported that the every other day (EOD) fasting initiated late-life improved metabolic, musculoskeletal, and cognitive endpoints in standard rodent diet-fed mice. In the present study, using the same dietary intervention methodology, we tested if 2.5 months of EOD fasting could improve metabolic, physiological, and cognitive endpoints in mice after an 18 month obesogenic high-fat diet (HFD). The positive effects of EOD fasting were generally consistent across the endpoints; EOD fasting decreased total body mass, maintained more %lean mass, improved glucose tolerance and utilization, and improved neuromuscular function. In contrast to our previous study, grip strength, hippocampal-dependent memory, and renal hydrogen sulfide (H 2 S) production were not improved by the HFD EOD fasting. Thus, efficacy for late-life initiated intermittent fasting to improve specific frailty markers may be partially dependent on nutritional compositions of the diet.

Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leading to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and it is causally linked to a range of neurodegenerative diseases such as Alzheimer's diseases (AD), Multiple Sclerosis (MS), Parkinson's disease (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier, making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our Nanoligomer Discovery Engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1β or IL-1β, tumor necrosis factor-alpha or TNF-α, TNF receptor 1 or TNFR1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-κβ) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification via a bioinformatics and artificial intelligence-based ranking method to design sequence-specific peptide molecules to up- or downregulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of Nanoligomers targeted to NF-κβ (SB.201.17D.8_NF-κβ1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low dose (5 mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy.

One hallmark of Alzheimer’s Disease (AD) pathology is hyperphosphorylation of tau protein, which leads to loss of its normal function and promotes aggregation into neurotoxic fibrillary tangles. While tau gain-of-toxic function is well-documented, the exact role of tau loss-of-function in AD pathogenesis remains uncharacterized. We hypothesize that tau loss-of-function contributes to AD pathogenesis by activating the cellular stress response in neurons and astrocytes, characterized by cytoplasmic double-stranded RNA (dsRNA) release, stress granule formation, and inflammation. We generated human induced pluripotent stem cell (hiPSC)-derived mixed cortical cultures and primary human fetal astrocyte cultures, allowing us to model the earliest stages of AD pathogenesis in a uniquely human system. In these cultures, we depleted tau through lentiviral transduction and CRISPR-Cas9 to model acute and chronic loss-of-function, respectively. We utilized quantitative real-time polymerase chain reactions, enzyme-linked immunosorbent assays, and immunocytochemistry to investigate the expression of genes and proteins implicated in stress and inflammation. Preliminary results indicate upregulation of genes and proteins associated with inflammation and stress granule formation in tau-depleted neurons and astrocytes. Tau-depleted cortical cultures exhibit increased secretion of chemoattractant cytokines, and tau-depleted astrocytes demonstrate increased glial fibrillary acidic protein expression suggestive of non-cell autonomous pro-inflammatory cytokine induction. Further, tau-depleted neurons show increased levels of stress chaperones and cytoplasmic dsRNA known to induce stress granules. Tau loss-of-function may contribute to AD pathogenesis through promoting the release of double-stranded RNA into the cytoplasm, stress granule formation, and inflammation.

ABSTRACT Background Dietary sulfur amino acid restriction (SAAR) improves body composition and metabolic health across several model organisms in part through induction of the integrated stress response (ISR). Objective We investigate the hypothesis that activating transcription factor 4 (ATF4) acts as a converging point in the ISR during SAAR. Methods Using liver-specific or global gene ablation strategies, in both female and male mice, we address the role of ATF4 during dietary SAAR. Results We show that ATF4 is dispensable in the chronic induction of the hepatokine fibroblast growth factor 21 while being essential for the sustained production of endogenous hydrogen sulfide. We also affirm that biological sex, independent of ATF4 status, is a determinant of the response to dietary SAAR. Conclusions Our results suggest that auxiliary components of the ISR, which are independent of ATF4, are critical for SAAR-mediated improvements in metabolic health in mice.

Global average life expectancy continues to rise. As aging increases the likelihood of frailty, which encompasses metabolic, musculoskeletal, and cognitive deficits, there is a need for effective anti-aging treatments. It is well established in model organisms that dietary restriction (DR), such as caloric restriction or protein restriction, enhances health and lifespan. However, DR is not widely implemented in the clinic due to patient compliance and its lack of mechanistic underpinnings. Thus, the present study tested the effects of a somewhat more clinically applicable and adoptable DR regimen, every-other-day (EOD) intermittent fasting, on frailty in 20-month-old male and female C57BL/6 mice. Frailty was determined by a series of metabolic, musculoskeletal, and cognitive tasks performed prior to and toward the end of the 2.5-month dietary intervention. Late-life EOD fasting attenuated overall energy intake, hypothalamic inflammatory gene expression, and frailty in males. However, it failed to reduce overall caloric intake and had a little positive effect in females. Given that the selected benefits of DR are dependent on augmented production of the gasotransmitter hydrogen sulfide (H 2 S) and that renal H 2 S production declines with age, we tested the effects of EOD fasting on renal H 2 S production capacity and its connection to frailty in males. EOD fasting boosted renal H 2 S production, which positively correlated with improvements in multiple components of frailty tasks. Therefore, late-life initiated EOD fasting is sufficient to reduce aging-related frailty, at least in males, and suggests that renal H 2 S production capacity may modulate the effects of late-life EOD fasting on frailty.

Aims: Peripubertal endocrine disruption has immediate and lifelong consequences on health, cognition, and lifespan. Disruption comes from dietary, environmental, and pharmaceutical sources. The plasticizer Bisphenol A (BPA) is one such endocrine disrupting chemical. However, it is unclear whether peripubertal BPA exposure incites long-lasting physiological, neuro-cognitive, and/or longevity-related metabolic impairments. Catabolism of cysteine via transsulfuration enzymes produces hydrogen sulfide (H2S), a redox-modulating gasotransmitter causative to endocrine and metabolic homeostasis and improved cognitive function with age. As thyroid hormone (TH) regulates hepatic H2S production and BPA is a TH receptor antagonist, we hypothesized that BPA exposure during peripubertal development impairs metabolic and neuro-cognitive/behavioral endpoints in aged mice, in part, due to altered peripheral and/or localized H2S production and redox status. Results: To test this, male C57BL/6J mice at 5 weeks of age were orally exposed daily for 5 weeks to 250 μg BPA/kg, defined as low dose group (LD BPA), or 250 mg BPA/kg, defined as high dose group (HD BPA). Both LD and HD BPA exposure decreased lean mass and increased fat mass accompanied by decreased serum total TH at advanced ages. In addition, LD BPA had an anxiogenic effect whereas HD BPA caused cognitive deficits. Notably, HD BPA disrupted tissue-specific H2S production capacities and/or protein persulfidation, with the former negatively correlated with memory deficits and oxidative stress. Innovation and Conclusion: These findings provide a potential mechanism of action for acute and long-term health impacts of BPA-induced peripubertal endocrine disruption and bolster the need for improved monitoring and limitation of adolescent BPA exposure.

Numerous reports have suggested that infectious agents could play a role in neurodegenerative diseases, but specific etiological agents have not been convincingly demonstrated. To search for candidate agents in an unbiased fashion, we have developed a bioinformatic pipeline that identifies microbial sequences in mammalian RNA-seq data, including sequences with no significant nucleotide similarity hits in GenBank. Effectiveness of the pipeline was tested using publicly available RNA-seq data and in a reconstruction experiment using synthetic data. We then applied this pipeline to a novel RNA-seq dataset generated from a cohort of 120 samples from amyotrophic lateral sclerosis patients and controls, and identified sequences corresponding to known bacteria and viruses, as well as novel virus-like sequences. The presence of these novel virus-like sequences, which were identified in subsets of both patients and controls, were confirmed by quantitative RT-PCR. We believe this pipeline will be a useful tool for the identification of potential etiological agents in the many RNA-seq datasets currently being generated.

Numerous studies have identified microbial sequences or epitopes in pathological and non-pathological human brain samples. It has not been resolved if these observations are artifactual, or truly represent population of the brain by microbes. Given the tempting speculation that resident microbes could play a role in the many neuropsychiatric and neurodegenerative diseases that currently lack clear etiologies, there is a strong motivation to determine the "ground truth" of microbial existence in living brains. Here I argue that the evidence for the presence of microbes in diseased brains is quite strong, but a compelling demonstration of resident microbes in the healthy human brain remains to be done. Dedicated animal models studies may be required to determine if there is indeed a "brain microbiome."

Hydrogen sulfide (H2S) is a cytoprotective redox-active metabolite that signals through protein persulfidation (R-SSnH). Despite the known importance of persulfidation, tissue-specific persulfidome profiles and their associated functions are not well characterized, specifically under conditions and interventions known to modulate H2S production. We hypothesize that dietary restriction (DR), which increases lifespan and can boost H2S production, expands tissue-specific persulfidomes. Here, we find protein persulfidation enriched in liver, kidney, muscle, and brain but decreased in heart of young and aged male mice under two forms of DR, with DR promoting persulfidation in numerous metabolic and aging-related pathways. Mice lacking cystathionine γ-lyase (CGL) have overall decreased tissue protein persulfidation numbers and fail to functionally augment persulfidomes in response to DR, predominantly in kidney, muscle, and brain. Here, we define tissue- and CGL-dependent persulfidomes and how diet transforms their makeup, underscoring the breadth for DR and H2S to impact biological processes and organismal health.

The amyloid beta (Aβ) peptide is believed to play a central role in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. However, the natural, evolutionarily selected functions of Aβ are incompletely understood. Here, we report that nanomolar concentrations of Aβ act synergistically with known cytokines to promote pro-inflammatory activation in primary human astrocytes (a cell type increasingly implicated in brain aging and AD). Using transcriptomics (RNA-seq), we show that Aβ can directly substitute for the complement component C1q in a cytokine cocktail previously shown to induce astrocyte immune activation. Furthermore, we show that astrocytes synergistically activated by Aβ have a transcriptional signature similar to neurotoxic "A1" astrocytes known to accumulate with age and in AD. Interestingly, we find that this biological action of Aβ at low concentrations is distinct from the transcriptome changes induced by the high/supraphysiological doses of Aβ often used in in vitro studies. Collectively, our results suggest an important, cytokine-like function for Aβ and a novel mechanism by which it may directly contribute to the neuroinflammation associated with brain aging and AD.

Aging is associated with declines in cognitive performance, which are mediated in part by neuroinflammation, characterized by astrocyte activation and higher levels of pro-inflammatory cytokines; however, the upstream drivers are unknown. We investigated the potential role of the gut microbiome–derived metabolite trimethylamine N -oxide (TMAO) in modulating neuroinflammation and cognitive function with aging. Study 1: In middle-aged and older humans (65 ± 7 years), plasma TMAO levels were inversely related to performance on NIH Toolbox Cognition Battery tests of memory and fluid cognition (both r 2 = 0.07, p < 0.05). Study 2: In mice, TMAO concentrations in plasma and the brain increased in parallel with aging ( r 2 = 0.60), suggesting TMAO crosses the blood-brain barrier. The greater TMAO concentrations in old mice (27 months) were associated with higher brain pro-inflammatory cytokines and markers of astrocyte activation vs. young adult mice (6 months). Study 3: To determine if TMAO independently induces an “aging-like” decline in cognitive function, young mice (6 months) were supplemented with TMAO in chow for 6 months. Compared with controls, TMAO-supplemented mice performed worse on the novel object recognition test, indicating impaired memory and learning, and had increased neuroinflammation and markers of astrocyte activation. Study 4: Human astrocytes cultured with TMAO vs. control media exhibited changes in cellular morphology and protein markers consistent with astrocyte activation, indicating TMAO directly acts on these cells. Our results provide translational insight into a novel pathway that modulates neuroinflammation and cognitive function with aging, and suggest that TMAO might be a promising target for prevention of neuroinflammation and cognitive decline with aging.

Abstract Markers of neurodegeneration such as neurofilament light chain (NfL) may be elevated with neurological diseases such as multiple sclerosis (MS) as well as Alzheimer’s disease. NfL is a marker of axonal integrity where higher values positively relate to the degree of damage. NfL shows variations in early adulthood among healthy individuals and may relate to executive function performance in otherwise healthy individuals aged 19-32 years. In the ongoing CATSLife (Colorado Adoption/Twin Study of Lifespan behavioral development and cognitive aging) Quanterix Simoa assays of NfL were measured in 34 individuals selected based on self-reported neuroinflammatory conditions (N = 5) or by APOE genotype (N_nonE4 = 18, N_E4 = 16). The distribution of NfL was consistent with other studies of early-mid adulthood (range = 1.3 - 22.3 pg/ml). Based on partial regression weights predicting log-transformed NfL, NfL was higher in cases (exp(b)=1.08 pg/ml), in males (exp(b)=1.25 pg/ml), by age (exp(b)=1.03 pg/ml per year) and in APOE E4 carriers (exp(b)=1.11 p/mg). Moreover, correlations partialed for age, sex, APOE e4 and case status suggest higher NfL may be associated with lower Full Scale IQ and general cognitive ability (r’s = -.18 and -. 28) overall and may be more evident among APOE E4 carriers (r’s = -.42 - .44, partialed for age, sex, case status). In this pilot study, the observed NfL associations with general cognitive ability, particularly among APOE E4 carriers, suggests NfL may be a salient biomarker of cognitive functioning by early- to mid-adulthood.

Abstract Global average life expectancy continues to rise. As aging increases likelihoods of exhibiting geriatric syndromes (a.k.a. frailty), there is a need for effective anti-aging treatments. Multiple studies have shown the positive effects of dietary restriction (DR) on lifespan in various model organisms. However, DR is not widely implemented in older adults due to issues with patient compliance and the overall lack of understanding on the effects of DR initiated later in life. Thus, the present study tested whether late-life DR, specifically Every-Other-Day (EOD) fasting, attenuates aging-related frailty using a modified and simplified frailty index in mice. Briefly, 20-month old male and female C57BL/6 mice (human equivalent of 65 years) that had been on a control chow diet ad libitum during adulthood were placed on EOD fasting or ad libitum feeding for 2.5 months. Their frailty index was identified using an indirect calorimeter, glucose tolerance test, novel object place recognition test, forelimb grip strength meter, and rotarod. We found that late-life EOD fasting decreased overall caloric intake in males but not in females. In addition, EOD fasting significantly improved metabolic, musculoskeletal, and cognitive endpoints in male mice, but enhanced only some of these in female mice. Furthermore, EOD fasting improved hydrogen sulfide (H2S) production capacity and its associated sulfhydration signaling in tissues, which positively correlated with improvements in frailty measures. We conclude that EOD fasting implemented late in life can have therapeutic potential in the clinic. We are currently investigating the necessity of H2S production for DR mediated benefits and longevity.