The Hormesis Project
This project is aimed at understanding adaptive cellular stress response pathways in neural cells, how they are activated or inhibited by dietary and behavioral factors, and whether activation of such pathways can provide therapeutic benefit in animal models and human subjects.
Since its inception in 2000 the Cellular and Molecular Neurosciences Section (CMNS) has placed an increasing emphasis on elucidating adaptive stress response mechanisms that preserve or enhance brain function, and protect against disease. In this regard, we have made considerable progress in the “Hormesis Project” which tests the hypothesis that moderate levels of general stressors (e.g., exercise, dietary energy restriction) or specific stress-inducing chemicals can protect the nervous system against age- and disease-related dysfunction and degeneration. Our findings during the past 4 years are detailed below and have led to the conceptual framework for our research program shown in Figure 1.
We are focusing on understanding how a defined set of neural signaling pathways, known to promote cell survival and or plasticity (neurogenesis and synaptic plasticity), can be activated by behavioral and dietary modifications and specific low molecular weight chemicals. These pathways involve kinases (PI3 kinase, Akt, MAP kinases, CaMKII) and transcriptional regulators (NF-kB, Nrf-2, CREB, FOXOs and REST) and their target genes (Figure 2).
Figure 2. Adaptive cellular stress response pathways being investigated in models relevant to brain aging and disease.
1a. Discovery of plumbagin as a novel neuroprotective lifespan-extending phytochemical that activates the Nrf-2 pathway. We employed cultured human neuroblastoma cells expressing luciferase driven by either the antioxidant response element (ARE), NF-kB or FOXO promoters to screen a panel of more than 50 naturally occurring botanical pesticides to identify agents that activate one or more of these adaptive stress response signaling pathways. The chemicals were selected from a larger ‘library’ of botanical pesticides based upon their structure (medicinal chemistry-amenable representatives from a range of structural classes) and known potency as insect antifeedants. For each chemical we first performed cell survival analysis to establish a concentration-response cytotoxicity curve, and then determined whether subtoxic concentrations of the chemical activated ARE, NF-kB and/or FOXO. Among the chemicals evaluated, plumbagin was highly effective in activating ARE. Plumbagin increased nuclear localization and transcriptional activity of Nrf2 and induced the expression of the Nrf2/ARE-dependent genes such as heme oxygenase 1 and NQO1 in human neuroblastoma cells, and in primary mixed cultures from ARE-human placental alkaline phosphatase reporter mice. Exposure of neuroblastoma cells and primary cortical neurons to plumbagin provided protection against subsequent oxidative and metabolic insults in an Nrf2-dependent manner. Treatment of mice with plumbagin significantly reduced the amount of brain damage and ameliorated associated neurological deficits in a model of focal ischemic stroke (52). These initial findings establish precedence for the identification and characterization of neuroprotective phytochemicals based upon their ability to activate adaptive cellular stress response pathways.
1b. Dietary energy intake and age interact to modify cell stress pathways and stroke outcome. During the past 10 years we have demonstrated beneficial effects of dietary energy restriction, alternate day fasting (ADF) and limited daily feeding caloric restriction (CR) in reducing neuropathological processes and improving functional outcome in animal models of both acute and chronic neurodegenerative conditions. We showed that ADF and CR up-regulate the expression of genes in CNS cells that encode proteins that promote neuronal survival and plasticity (BDNF, HSP70, GRP78 and UCPs). We recently performed an experiment aimed at addressing two major unanswered questions of considerable importance: 1) Does advancing age alter that ability of dietary energy restriction to activate neuroprotective pathways? 2) How do age and energy intake affect the outcome in an animal model of stroke? We employed a novel microchip-based immunoaffinity capillary electrophoresis technology to measure a panel of neurotrophic factors, cytokines, and cellular stress resistance proteins in brain tissue samples from young, middle-aged, and old mice that had been maintained on control or ADF diets for 3 months prior to focal cerebral ischemia – reperfusion (3). Mortality from focal ischemic stroke was increased with advancing age and reduced by ADF. Brain damage and functional impairment were reduced by ADF in young and middle-aged mice, but not in old mice. The basal and poststroke levels of BDNF, bFGF, protein chaperones (heat shock protein 70 and glucose regulated protein 78), and the antioxidant enzyme heme oxygenase-1 were decreased, whereas levels of inflammatory cytokines were increased in the cerebral cortex and striatum of old mice compared with young and middle age mice. ADF coordinately increased levels of protective proteins and decreased inflammatory cytokines in young, but not in old mice. These findings suggest that the ability of ADF to activate adaptive neuronal stress response pathways and to suppress inflammation is impaired during the aging process, resulting in increased brain damage and poorer functional outcome.
1c. Evidence that dietary energy intake and exercise enhance brain cell stress response signaling. To elucidate if and how energy intake and expenditure affect the vulnerability of the brain to age-related dysfunction and neuronal degeneration we performed a series of studies using rodent models of overeating (db/db mice), energy restriction (ADF and CR) and exercise (voluntary wheel running). In one study we examined the effects of wheel running and CR on hippocampal neuron morphology and BDNF levels in db/db mice, a model of insulin resistant diabetes resulting from overeating (59). Running wheel activity, CR, or the combination of the two treatments increased levels of BDNF in the hippocampus of db/db mice. Enhancement of hippocampal BDNF was accompanied by increases in dendritic spine density on the secondary and tertiary dendrites of dentate granule neurons. We conclude that diabetes exerts detrimental effects on hippocampal structure, and that this state can be counteracted by energy restriction and exercise, possibly via a BDNF-mediated pathway.
We performed several studies aimed at elucidating the molecular underpinnings of the neuroprotective effects of energy restriction and exercise. As part of the AGEMAP study (76) we measured transcripts of 16,896 genes in 5 CNS regions from cohorts of young, middle-aged and old male and female mice that had been maintained on either a control diet or a CR diet (73). Each CNS region (cerebral cortex, hippocampus, striatum, cerebellum and spinal cord) exhibited a distinct transcriptome fingerprint that was independent of age, gender and energy intake. Less than 10% of genes were significantly affected by age, diet or gender, with most of these changes occurring between middle and old age. The transcriptome of the spinal cord was the most responsive to age, diet and gender, while the striatal transcriptome was the least responsive. Gender and energy restriction had particularly robust influences on the hippocampal transcriptome of middle-aged mice suggesting that energy intake during midlife may have a particularly strong influence on cognitive function in old age. Prominent functional groups of age- and energy-sensitive genes were those encoding proteins involved in DNA damage responses (Werner and telomere-associated proteins), mitochondrial and proteasome functions, cell fate determination (Wnt and Notch signaling) and synaptic vesicle trafficking. Based upon the prominent decrease in Werner expression in brain cells during aging, and amelioration of this decline by energy restriction, we have generated transgenic mice that overexpress Werner only in neurons to establish if and how Werner might affect neuronal vulnerability in aging and neurodegenerative disorder models.
In a related study we performed a gene array analysis of the hippocampus in male and female rats that had been maintained for 6 months on either ad libitum (control), 20% CR, 40% CR, ADF or high fat/high glucose (HFG) diets (39). Interestingly, the energy restricted diets significantly increased the size of the hippocampus of females, but not males. Functional physiological pathway analyses showed that the 20% CR diet down-regulated genes involved in glycolysis and mitochondrial ATP production in males, whereas these metabolic pathways were up-regulated in females. The 40% CR diet up-regulated genes involved in glycolysis, protein deacetylation, PGC-1a and mTOR pathways in both sexes. ADF down-regulated many genes in males including those involved in protein degradation and apoptosis, but up-regulated many genes in females including those involved in cellular energy metabolism, cell cycle regulation and protein deacetylation. Genes involved in energy metabolism, oxidative stress responses and cell death were affected by the HFG diet in both males and females. The gender-specific molecular genetic responses of hippocampal cells to variations in dietary energy intake identified in this study may mediate differential behavioral responses of males and females to differences in energy availability, and possibly differential vulnerability of males and females to neurodegenerative disease.
Physical activity may preserve cognition during aging, but the mechanisms remain obscure. To identify candidate genes and pathways responsible for the preservation of cognitive function by exercise, we trained mice that had been exposed to running or sedentary lifestyle for 16 months in the hippocampus-dependent water maze (55). After water maze training, we analyzed the expression of 24,000 genes in the hippocampus using Illumina bead microarray. Runners showed greater activation of genes associated with synaptic plasticity and mitochondrial function, and also exhibited significant down-regulation of genes associated with oxidative stress and lipid metabolism. Running also modified the effects of learning on the expression of genes involved in cell excitability, energy metabolism, and insulin, MAP kinase and Wnt signaling. These results suggest that the enhancement of cognitive function by lifelong exercise is associated with an altered transcriptional profile following learning.
Another of our recent advances resulted from a collaboration with Jean Cadet, a neighbor at NIDA. Because the olfactory system plays a major role in food consumption, and because 'food addiction' and associated morbidities have reached epidemic proportions, we tested the hypothesis that dietary energy restriction can modify adverse effects of cocaine on behavior and olfactory cellular and molecular plasticity (74). Mice maintained on an ADF diet exhibited heightened baseline locomotion and increased cocaine-sensitized locomotion during cocaine conditioning, despite no change in cocaine conditioned place preference, compared with mice fed ad libitum. Levels of dopamine and its metabolites in the olfactory bulb (OB) were suppressed in mice on the ADF diet compared with mice on the control diet, independent of acute or chronic cocaine treatment. The expression of several enzymes involved in dopamine metabolism including tyrosine hydroxylase, monoamine oxidases A and B, and catechol-O-methyltransferase were significantly reduced in OBs of mice on the ADF diet. Both acute and chronic administration of cocaine suppressed the production of new OB cells, and this effect of cocaine was attenuated in mice on the ADF diet. Cocaine administration to mice on the control diet resulted in up-regulation of OB genes involved in mitochondrial energy metabolism, synaptic plasticity, cellular stress responses, and calcium- and cAMP-mediated signaling, whereas multiple olfactory receptor genes were down-regulated by cocaine treatment. ADF abolished most of the effects of cocaine on OB gene expression. Our findings reveal that dietary energy intake modifies the neural substrates underlying some of the behavioral and physiological responses to repeated cocaine treatment, and also suggest novel roles for the olfactory system in addiction. The data further suggest that modification of dietary energy intake could provide a novel potential approach to treatments for addiction and neurodegenerative disorders that involve olfactory deficits including AD and PD.
1d. Neurons efficiently repair glutamate-induced oxidative DNA damage by a process involving CREB-mediated up-regulation of APE1. We found that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using sub-toxic levels of glutamate (75). This DNA damage was prevented by intracellular Ca2+ chelation, by the mitochondrial superoxide dismutase mimetic MnTMPyP, and by blockade of the mitochondrial permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knock-down induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A CREB binding sequence is present in the APE1 gene promoter and treatment of neurons with a Ca2+/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference technology prevented glutamate-induced up-regulation of APE1. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.