Novel small molecules inhibit proteotoxicity and inflammation: Mechanistic and therapeutic implications for Alzheimer's Disease, healthspan and lifespan- Aging as a consequence of glycolysis.

Inflammation drives many age-related, especially neurological, diseases, and likely mediates age-related proteotoxicity. For example, dementia due to Alzheimer's Disease (AD), cerebral vascular disease, many other neurodegenerative conditions is increasingly among the most devastating burdens on the American (and world) health system and threatens to bankrupt the American health system as the population ages unless effective treatments are developed. Dementia due to either AD or cerebral vascular disease, and plausibly many other neurodegenerative and even psychiatric conditions, is driven by increased age-related inflammation, which in turn appears to mediate Abeta and related proteotoxic processes. The functional significance of inflammation during aging is also supported by the fact that Humira, which is simply an antibody to the pro-inflammatory cytokine TNF-a, is the best-selling drug in the world by revenue. These observations led us to develop parallel high-throughput screens to discover small molecules which inhibit age-related Abeta proteotoxicity in a C. elegans model of AD AND LPS-induced microglial TNF-a. In the initial screen of 2560 compounds (Microsource Spectrum library) to delay Abeta proteotoxicity, the most protective compounds were, in order, phenylbutyrate, methicillin, and quetiapine, which belong to drug classes (HDAC inhibitors, beta lactam antibiotics, and tricyclic antipsychotics, respectably) already robustly implicated as promising to protect in neurodegenerative diseases, especially AD. RNAi and chemical screens indicated that the protective effects of HDAC inhibitors to reduce Abeta proteotoxicity are mediated by inhibition of HDAC2, also implicated in human AD, dependent on the HAT Creb binding protein (Cbp), which is also required for the protective effects of both dietary restriction and the daf-2 mutation (inactivation of IGF-1 signaling) during aging. In addition to methicillin, several other beta lactam antibiotics also delayed Abeta proteotoxicity and reduced microglial TNF-a. In addition to quetiapine, several other tricyclic antipsychotic drugs also delayed age-related Abeta proteotoxicity and increased microglial TNF-a, leading to the synthesis of a novel congener, GM310, which delays Abeta as well as Huntingtin proteotoxicity, inhibits LPS-induced mouse and human microglial and monocyte TNF-a, is highly concentrated in brain after oral delivery with no apparent toxicity, increases lifespan, and produces molecular responses highly similar to those produced by dietary restriction, including induction of Cbp inhibition of inhibitors of Cbp, and genes promoting a shift away from glycolysis and toward metabolism of alternate (e.g., lipid) substrates. GM310, as well as FDA-approved tricyclic congeners, prevented functional impairments and associated increase in TNF-a in a mouse model of stroke. Robust reduction of glycolysis by GM310 was functionally corroborated by flux analysis, and the glycolytic inhibitor 2-DG inhibited microglial TNF-a and other markers of inflammation, delayed Abeta proteotoxicity, and increased lifespan. These results support the value of phenotypic screens to discover drugs to treat age-related, especially neurological and even psychiatric diseases, including AD and stroke, and to clarify novel mechanisms driving neurodegeneration (e.g., increased microglial glycolysis drives neuroinflammation and subsequent neurotoxicity) suggesting novel treatments (selective inhibitors of microglial glycolysis).

Diets, genes, and drugs that increase lifespan and delay age-related diseases: Role of nutrient-sensing neurons and Creb-binding protein.

Discovery of interventions that delay or minimize age-related diseases is arguably the major goal of aging research. Conversely discovery of interventions based on phenotypic screens have often led to further elucidation of pathophysiological mechanisms. Although most hypotheses to explain lifespan focus on cell-autonomous processes, increasing evidence suggests that in multicellular organisms, neurons, particularly nutrient-sensing neurons, play a determinative role in lifespan and age-related diseases. For example, protective effects of dietary restriction and inactivation of insulin-like signaling increase lifespan and delay age-related diseases dependent on Creb-binding protein in GABA neurons, and Nrf2/Skn1 in just 2 nutrient-sensing neurons in C. elegans. Screens for drugs that increase lifespan also indicate that such drugs are predominantly active through neuronal signaling. Our own screens also indicate that neuroactive drugs also delay pathology in an animal model of Alzheimer's Disease, as well as inhibit cytokine production implicated in driving many age-related diseases. The most likely mechanism by which nutrient-sensing neurons influence lifespan and the onset of age-related diseases is by regulating metabolic architecture, particularly the relative rate of glycolysis vs. alternative metabolic pathways such as ketone and lipid metabolism. These results suggest that neuroactive compounds are a most promising class of drugs to delay or minimize age-related diseases.

RNA-sequencing Reveals a Gene Expression Signature in Skeletal Muscle of a Mouse Model of Age-associated Postoperative Functional Decline.

This study aimed to characterize the effects of laparotomy on postoperative physical function and skeletal muscle gene expression in male C57BL/6N mice at 3, 20, and 24 months of age to investigate late-life vulnerability and resiliency to acute surgical stress. Pre and postoperative physical functioning was assessed by forelimb grip strength on postoperative day (POD) 1 and 3 and motor coordination on POD 2 and 4. Laparotomy-induced an age-associated postoperative decline in forelimb grip strength that was the greatest in the oldest mice. While motor coordination declined with increasing age at baseline, it was unaffected by laparotomy. Baseline physical function as stratified by motor coordination performance (low functioning vs high functioning) in 24-month-old mice did not differentially affect postlaparotomy reduction in grip strength. RNA sequencing of soleus muscles showed that laparotomy-induced age-associated differential gene expression and canonical pathway activation with the greatest effects in the youngest mice. Examples of such age-associated, metabolically important pathways that were only activated in the youngest mice after laparotomy included oxidative phosphorylation and NRF2-mediated oxidative stress response. Analysis of lipid mediators in serum and gastrocnemius muscle showed alterations in profiles during aging and confirmed an association between such changes and functional status in gastrocnemius muscle. These findings demonstrate a mouse model of laparotomy which recapitulated some features of postoperative skeletal muscle decline in older adults, and identified age-associated, laparotomy-induced molecular signatures in skeletal muscles. Future research can build upon this model to study molecular mechanisms of late-life vulnerability and resiliency to acute surgical stress.

Biological age and environmental risk factors for dementia and stroke: Molecular mechanisms.

Since the development of antibiotics and vaccination, as well as major improvements in public hygiene, the main risk factors for morbidity and mortality are age and chronic exposure to environmental factors, both of which can interact with genetic predispositions. As the average age of the population increases, the prevalence and costs of chronic diseases, especially neurological conditions, are rapidly increasing. The deleterious effects of age and environmental risk factors, develop chronically over relatively long periods of time, in contrast to the relatively rapid deleterious effects of infectious diseases or accidents. Of particular interest is the hypothesis that the deleterious effects of environmental factors may be mediated by acceleration of biological age. This hypothesis is supported by evidence that dietary restriction, which universally delays age-related diseases, also ameliorates deleterious effects of environmental factors. Conversely, both age and environmental risk factors are associated with the accumulation of somatic mutations in mitotic cells and epigenetic modifications that are a measure of "biological age", a better predictor of age-related morbidity and mortality than chronological age. Here we review evidence that environmental risk factors such as smoking and air pollution may also drive neurological conditions, including Alzheimer's Disease, by the acceleration of biological age, mediated by cumulative and persistent epigenetic effects as well as somatic mutations. Elucidation of such mechanisms could plausibly allow the development of interventions which delay deleterious effects of both aging and environmental risk factors.

Serotonin and Dopamine Mimic Glucose-Induced Reinforcement in C. elegans: Potential Role of NSM Neurons and the Serotonin Subtype 4 Receptor.

Food produces powerful reinforcement that can lead to overconsumption and likely contributes to the obesity epidemic. The present studies examined molecular mechanisms mediating food-induced reinforcement in the model system C. elegans. After a 1-h training session during which food (bacteria) is paired with the odorant butanone, odor preference for butanone robustly increased. Glucose mimicked this effect of bacteria. Glucose-induced odor preference was enhanced similarly by prior food withdrawal or blocking glucose metabolism in the presence of food. Food- and glucose-induced odor preference was mimicked by serotonin signaling through the serotonin type-4 (5-HT4) receptor. Dopamine (thought to act primarily through a D1-like receptor) facilitated, whereas the D2 agonist bromocriptine blocked, food- and glucose-induced odor preference. Furthermore, prior food withdrawal similarly influenced reward produced by serotonin, dopamine, or food, implying post-synaptic enhancement of sensitivity to serotonin and dopamine. These results suggest that glucose metabolism plays a key role in mediating both food-induced reinforcement and enhancement of that reinforcement by prior food withdrawal and implicate serotonergic signaling through 5-HT4 receptor in the re-enforcing properties of food.

Biopsy During Minimally Invasive Intracerebral Hemorrhage Clot Evacuation.

BACKGROUND: The safety and efficacy of brain parenchyma biopsy during minimally invasive (MIS) intracerebral hemorrhage (ICH) clot evacuation has not been previously reported. The objective of this study was to establish the safety and diagnostic efficacy of brain biopsy during MIS ICH clot evacuation and to validate the modified Boston criteria as a predictor of cerebral amyloid angiopathy (CAA) in this cohort. METHODS: From October 2016 to March 2018, superficial and perihematomal biopsies were collected for 40 patients undergoing MIS ICH clot evacuation and analyzed by the pathology department to assess for various ICH etiologies. Additionally, the admission magnetic resonance imaging or computed tomography scan of each patient was analyzed and evaluated for the likelihood of a CAA etiology based on the modified Boston criteria. Student t test was used to analyze intergroup differences in continuous variables, and a 2-tailed Fisher exact test was used to determine intergroup differences of categorical variables, with significance set at P < 0.05. RESULTS: Two of the 40 patients (5%) experienced postoperative rebleed. Four of the 40 patients (10%) had evidence of CAA on biopsy. Patients with CAA on biopsy were older (P = 0.005) and had a higher prevalence of parietal lobe (P = 0.02) and occipital lobe (P = 0.001) hemorrhage. The modified Boston criteria had a sensitivity of 100% (95% confidence interval [CI], 39.6%-100%) and a specificity of 72.2% (95% CI, 54.6%-84.2%) for predicting CAA on biopsy. CONCLUSIONS: Brain biopsy in MIS ICH clot evacuation is safe and allows for the diagnosis of various ICH etiologies.

Glucose-Induced Transcriptional Hysteresis: Role in Obesity, Metabolic Memory, Diabetes, and Aging.

During differentiation transient, inducers produce permanent changes in gene expression. A similar phenomenon, transcriptional hysteresis, produced by transient or prolonged exposure to glucose, leads to cumulative, persistent, and largely irreversible effects on glucose-regulated gene expression, and may drive key aspects of metabolic memory, obesity, diabetes, and aging, and explain the protective effects of dietary restriction during aging. The most relevant effects of glucose-induced transcriptional hysteresis are the persistent effects of elevated glucose on genes that control glucose metabolism itself. A key observation is that, as with the lac operon, glucose induces genes that promote glycolysis and inhibits gene expression of alternative metabolic pathways including the pentose pathway, beta oxidation, and the TCA cycle. A similar pattern of metabolic gene expression is observed during aging, suggesting that cumulative exposure to glucose during aging produces this metabolic shift. Conversely, dietary restriction, which increases lifespan and delays age-related impairments, produces the opposite metabolic profile, leading to a shift away from glycolysis and toward the use of alternative substrates, including lipid and ketone metabolisms. The effect of glucose on gene expression leads to a positive feedback loop that leads to metastable persistent expression of genes that promote glycolysis and inhibit alternative pathways, a phenomenon first observed in the regulation of the lac operon. On the other hand, this pattern of gene expression can also be inhibited by activation of peroxisome proliferator activating receptor transcription factors that promote beta oxidation and inhibit metabolism of glucose-derived carbon bonds in the TCA cycle. Several pathological consequences may arise from glucose-induced transcriptional hysteresis. First, elevated glucose induces glycolytic genes in pancreatic beta cells, which induces a semi-stable persistent increase in insulin secretion, which could drive obesity and insulin resistance, and also due to glucose toxicity could eventually lead to beta-cell decompensation and diabetes. Diabetic complications persist even after complete normalization of glucose, a phenomenon known as metabolic memory. This too can be explained by persistent bistable expression of glucose-induced glycolytic genes.

Dietary Restriction and Glycolytic Inhibition Reduce Proteotoxicity and Extend Lifespan via NHR-49.

Mechanisms mediating protective effects of dietary restriction during aging are of great interest since activating such mechanisms protect against a wide range of age-related diseases. In mammals key metabolic responses to nutritional deprivation are mediated by the transcription factor PPAR-alpha, which is activated by free fatty acids and promotes lipid metabolism while inhibiting glucose metabolism. The C. elegans gene nhr-49 appears to function similarly in C. elegans. Here we report that protective effects of dietary restriction and inhibition of glucose metabolism to increase lifespan wild-type C. elegans and reduce toxicity in a polyQ model of Huntington's disease in C. elegans are dependent on NHR-49 and its co-activator CREB-Binding Protein (CBP). We have previously demonstrated that inhibition of cbp blocks protective effects of dietary restriction and blocks the molecular switch from glucose metabolism to alternative substrates. Conversely, increased glucose concentration and inhibition of cbp reduce lifespan and increase proteotoxicity. Lactate and inhibition of ETC complex II mimicked toxic effects of glucose on proteotoxicity whereas pyruvate and inhibition of ETC complex I protected against glucose-enhanced proteotoxicity. These results support that PPAR-alpha-like activity mediates protective effects of dietary restriction by reducing glucose metabolism via reducing production of NADH, and corroborate and extend recent studies demonstrating that PPPAR-alpha agonists increase lifespan in C. elegans dependent on NHR-49.

Dopamine D2 Receptor Signaling in the Nucleus Accumbens Comprises a Metabolic-Cognitive Brain Interface Regulating Metabolic Components of Glucose Reinforcement.

Appetitive drive is influenced by coordinated interactions between brain circuits that regulate reinforcement and homeostatic signals that control metabolism. Glucose modulates striatal dopamine (DA) and regulates appetitive drive and reinforcement learning. Striatal DA D2 receptors (D2Rs) also regulate reinforcement learning and are implicated in glucose-related metabolic disorders. Nevertheless, interactions between striatal D2R and peripheral glucose have not been previously described. Here we show that manipulations involving striatal D2R signaling coincide with perseverative and impulsive-like responding for sucrose, a disaccharide consisting of fructose and glucose. Fructose conveys orosensory (ie, taste) reinforcement but does not convey metabolic (ie, nutrient-derived) reinforcement. Glucose however conveys orosensory reinforcement but unlike fructose, it is a major metabolic energy source, underlies sustained reinforcement, and activates striatal circuitry. We found that mice with deletion of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) exclusively in D2R-expressing cells exhibited preferential D2R changes in the nucleus accumbens (NAc), a striatal region that critically regulates sucrose reinforcement. These changes coincided with perseverative and impulsive-like responding for sucrose pellets and sustained reinforcement learning of glucose-paired flavors. These mice were also characterized by significant glucose intolerance (ie, impaired glucose utilization). Systemic glucose administration significantly attenuated sucrose operant responding and D2R activation or blockade in the NAc bidirectionally modulated blood glucose levels and glucose tolerance. Collectively, these results implicate NAc D2R in regulating both peripheral glucose levels and glucose-dependent reinforcement learning behaviors and highlight the notion that glucose metabolic impairments arising from disrupted NAc D2R signaling are involved in compulsive and perseverative feeding behaviors.

Epigenetic mechanisms underlying lifespan and age-related effects of dietary restriction and the ketogenic diet.

Aging constitutes the central risk factor for major diseases including many forms of cancer, neurodegeneration, and cardiovascular diseases. The aging process is characterized by both global and tissue-specific changes in gene expression across taxonomically diverse species. While aging has historically been thought to entail cell-autonomous, even stochastic changes, recent evidence suggests that modulation of this process can be hierarchal, wherein manipulations of nutrient-sensing neurons (e.g., in the hypothalamus) produce peripheral effects that may modulate the aging process itself. The most robust intervention extending lifespan, plausibly impinging on the aging process, involves different modalities of dietary restriction (DR). Lifespan extension by DR is associated with broad protection against diseases (natural and engineered). Here we review potential epigenetic processes that may link lifespan to age-related diseases, particularly in the context of DR and (other) ketogenic diets, focusing on brain and hypothalamic mechanisms.

Role of Hypothalamic Creb-Binding Protein in Obesity and Molecular Reprogramming of Metabolic Substrates.

We have reported a correlation between hypothalamic expression of Creb-binding protein (Cbp) and lifespan, and that inhibition of Cbp prevents protective effects of dietary restriction during aging, suggesting that hypothalamic Cbp plays a role in responses to nutritional status and energy balance. Recent GWAS and network analyses have also implicated Cbp as the most connected gene in protein-protein interactions in human Type 2 diabetes. The present studies address mechanisms mediating the role of Cbp in diabetes by inhibiting hypothalamic Cbp using a Cre-lox strategy. Inhibition of hypothalamic Cbp results in profound obesity and impaired glucose homeostasis, increased food intake, and decreased body temperature. In addition, these changes are accompanied by molecular evidence in the hypothalamus for impaired leptin and insulin signaling, a shift from glucose to lipid metabolism, and decreased Pomc mRNA, with no effect on locomotion. Further assessment of the significance of the metabolic switch demonstrated that enhanced expression of hypothalamic Cpt1a, which promotes lipid metabolism, similarly resulted in increased body weight and reduced Pomc mRNA.

Discover the network underlying the connections between aging and age-related diseases.

Although our knowledge of aging has greatly expanded in the past decades, it remains elusive why and how aging contributes to the development of age-related diseases (ARDs). In particular, a global mechanistic understanding of the connections between aging and ARDs is yet to be established. We rely on a network modelling named "GeroNet" to study the connections between aging and more than a hundred diseases. By evaluating topological connections between aging genes and disease genes in over three thousand subnetworks corresponding to various biological processes, we show that aging has stronger connections with ARD genes compared to non-ARD genes in subnetworks corresponding to "response to decreased oxygen levels", "insulin signalling pathway", "cell cycle", etc. Based on subnetwork connectivity, we can correctly "predict" if a disease is age-related and prioritize the biological processes that are involved in connecting to multiple ARDs. Using Alzheimer's disease (AD) as an example, GeroNet identifies meaningful genes that may play key roles in connecting aging and ARDs. The top modules identified by GeroNet in AD significantly overlap with modules identified from a large scale AD brain gene expression experiment, supporting that GeroNet indeed reveals the underlying biological processes involved in the disease.

Mechanisms and significance of brain glucose signaling in energy balance, glucose homeostasis, and food-induced reward.

The concept that hypothalamic glucose signaling plays an important role in regulating energy balance, e.g., as instantiated in the so-called "glucostat" hypothesis, is one of the oldest in the field of metabolism. However the mechanisms by which neurons in the hypothalamus sense glucose, and the function of glucose signaling in the brain, has been difficult to establish. Nevertheless recent studies probing mechanisms of glucose signaling have also strongly supported a role for glucose signaling in regulating energy balance, glucose homeostasis, and food-induced reward.

Protection by dietary restriction in the YAC128 mouse model of Huntington's disease: Relation to genes regulating histone acetylation and HTT.

Huntington's disease (HD) is a fatal neurodegenerative disease characterized by metabolic, cognitive, and motor deficits. HD is caused by an expanded CAG repeat in the first exon of the HTT gene, resulting in an expanded polyglutamine section. Dietary restriction (DR) increases lifespan and ameliorates age-related pathologies, including in a model of HD, but the mechanisms mediating these protective effects are unknown. We report metabolic and behavioral effects of DR in the full-length YAC128 HD mouse model, and associated transcriptional changes in hypothalamus and striatum. DR corrected many effects of the transgene including increased body weight, decreased blood glucose, and impaired motor function. These changes were associated with reduced striatal human (but not mouse) HTT expression, as well as alteration in gene expression regulating histone acetylation modifications, particularly Hdac2. Other mRNAs related to Huntington's pathology in striatal tissue showed significant modulation by the transgene, dietary restriction or both. These results establish a protective role of DR in a transgenic model that contains the complete human HTT gene and for the first time suggest a role for DR in lowering HTT level, which correlates with severity of symptoms.

Synchronized age-related gene expression changes across multiple tissues in human and the link to complex diseases.

Aging is one of the most important biological processes and is a known risk factor for many age-related diseases in human. Studying age-related transcriptomic changes in tissues across the whole body can provide valuable information for a holistic understanding of this fundamental process. In this work, we catalogue age-related gene expression changes in nine tissues from nearly two hundred individuals collected by the Genotype-Tissue Expression (GTEx) project. In general, we find the aging gene expression signatures are very tissue specific. However, enrichment for some well-known aging components such as mitochondria biology is observed in many tissues. Different levels of cross-tissue synchronization of age-related gene expression changes are observed, and some essential tissues (e.g., heart and lung) show much stronger "co-aging" than other tissues based on a principal component analysis. The aging gene signatures and complex disease genes show a complex overlapping pattern and only in some cases, we see that they are significantly overlapped in the tissues affected by the corresponding diseases. In summary, our analyses provide novel insights to the co-regulation of age-related gene expression in multiple tissues; it also presents a tissue-specific view of the link between aging and age-related diseases.

Laparotomy in mice induces blood cell expression of inflammatory and stress genes.

Surgical trauma induces immune and stress responses although its effects on postsurgical inflammatory and stress gene expression remain poorly characterized. This study sought to improve current scientific knowledge by investigating the effects of laparotomy on mouse blood cell inflammatory and stress gene expression. Three-month-old male C57BL/6J mice were subjected to 2% isoflurane or 2% isoflurane with laparotomy and sacrificed 4 h postintervention. Blood was collected and blood cell expression of 158 genes central to inflammatory and stress responses was assayed using quantitative polymerase chain reaction arrays. Mice subjected to isoflurane with laparotomy, compared with mice receiving isoflurane alone, had >2-fold upregulation of genes in inflammation (Osm, IL1rn, IL1b, and Csf1), oxidative stress (Hmox1), heat shock (Hspa1b), growth arrest (Cdkn1a), and DNA repair (Ugt1a2). These genes demonstrated similar expression patterns by Pearson correlation and cluster analysis. Thus, laparotomy induces coordinated, postsurgical blood cell expression of unique inflammatory and stress genes whose roles in influencing surgical outcomes need further investigation.

Metabolic mystery: aging, obesity, diabetes, and the ventromedial hypothalamus.

We propose that energy balance, glucose homeostasis, and aging are all regulated largely by the same nutrient-sensing neurons in the ventromedial hypothalamus (VMH). Although the central role of these neurons in regulating energy balance is clear, their role in regulating glucose homeostasis has only recently become more clear. This latter function may be most relevant to aging and lifespan by controlling the rate of glucose metabolism. Specifically, glucose-sensing neurons in VMH promote peripheral glucose metabolism, and dietary restriction, by reducing glucose metabolism in these neurons, reduces glucose metabolism of the rest of the body, thereby increasing lifespan. Here we discuss recent studies demonstrating the key role of hypothalamic neurons in driving aging and age-related diseases.

Glucose-induced metabolic memory in Schwann cells: prevention by PPAR agonists.

A major barrier in reversing diabetic complications is that molecular and pathologic effects of elevated glucose persist despite normalization of glucose, a phenomenon referred to as metabolic memory. In the present studies we have investigated the effects of elevated glucose on Schwann cells, which are implicated in diabetic neuropathy. Using quantitative PCR arrays for glucose and fatty acid metabolism, we have found that chronic (>8 wk) 25 mM high glucose induces a persistent increase in genes that promote glycolysis, while inhibiting those that oppose glycolysis and alternate metabolic pathways such as fatty acid metabolism, the pentose phosphate pathway, and trichloroacetic acid cycle. These sustained effects were associated with decreased peroxisome proliferator-activated receptor (PPAR)γ binding and persistently increased reactive oxygen species, cellular NADH, and altered DNA methylation. Agonists of PPARγ and PPARα prevented select effects of glucose-induced gene expression. These observations suggest that Schwann cells exhibit features of metabolic memory that may be regulated at the transcriptional level. Furthermore, targeting PPAR may prevent metabolic memory and the development of diabetic complications.