Jacob Raber, Ph.D. - US grants

DESCRIPTION (provided by the applicant): The three major human apolipoprotein (apo) E isoforms are encoded by distinct alleles (E2, e3, and c4). Compared with c2 and F3, F4 increases the risk of cognitive impairments and of developing Alzheimer's disease (AD). ApoE4 interacts with female gender, resulting in an even greater risk of developing AD. Understanding apoE-gender interactions is important for developing AD treatments. To assess how interactions between gender and apoE isoforms affect cognition, we study transgenic mice expressing human apoE isoforms in the brain and lacking mouse apoE (apoe-/-). As they age, female, but not male, apoE4 mice develop progressive impairments in spatial learning and memory in the water maze, compared with age- and sex-matched wild-type, apoe-/- or apoE3 mice. This could be relevant to cognitive impairments in human 4 carriers. Spatial memory is impaired in AD. Because the cognitive impairments are observed in female apoE4 mice that express apoE4 in neurons or astrocytes, they are independent of the cellular source of apoE. Adult female mice that express both apoE3 and apoB4 do not show cognitive deficits in the water maze, indicating that apoE3 antagonizes the effects of apoE4 on cognition. We hypothesize that sex steroids contribute to the gender-dependent behavioral alterations in apoE4 mice and that androgens antagonize the apoE4-induced behavioral alterations. Our preliminary data show that testosterone and dihydrotestosterone antagonize the memory retention deficits of adult apoE4 female mice in the water maze. We further hypothesize that androgen receptors (ARs) mediate protection against apoE4-induced cognitive impairments. ApoE4 male mice, which do not show cognitive deficits in the water maze, developed cognitive impairments after blockade of androgen receptors, whereas apoE3 male mice did not. The Specific Aims are: (1) To determine whether apoE4 has gender-specific effects on performance in hippocampus dependent tests and whether sex steroids contribute to these effects; (2) To determine whether ARs protect against apoE4-induced cognitive impairments; (3) To determine whether gender- and isoform-specific effects of apoE on AR function in the hippocampus contribute to cognitive impairments; and (4) To determine whether apoE3 antagonizes the effects of apoE4 on cognitive function. The proposed study will help to elucidate the roles of apoE isoforms in the brain and their interactions with sex steroids in cognition. This is important for our understanding of cognitive function in health as well as in diseases characterized by cognitive impairments and will likely advance the development of therapeutic intervention to prevent or even reverse cognitive impairments.

apolipoprotein E; cognition disorders; learning; aging; memory; emotions; stress; gender difference; testosterone; clinical research; human subject; human old age (65+);

DESCRIPTION (provided by applicant): The fast actions of the excitatory neurotransmitter glutamate are mediated by glutamate-gated ion channels (ionotropic Glu receptors). Metabotropic glutamate receptors (mGluRs) are coupled to second messenger pathways via G proteins and modulate glutamatergic and GABAergic neurotransmission. Of the eight different types of mGluRs (mGluR1-mGluR8), mGluR8 is a member of group III, which are generally located presynaptically and regulate neurotransmitter release. Because of their role in modulating excitatory neurotransmission, mGluRs are attractive targets for therapies aimed at treating anxiety disorders. Our preliminary data show increased measures of anxiety in the open field and elevated plus maze in 6-month- old C57BI6/J mice lacking mGluR8 (mGluR8-/-). Further, the specific mGluRS agonist (S)-3,4,- dicarboxyphenylglycine (DCPG) induced neuronal activity marker c-Fos in GABA-ergic inhibitory neurons in the central nucleus of the amygdala (CeA) and suppressed excitatory synaptic transmission in the bed nucleus of the stria terminalis (BNST), brain areas involved in the regulation of anxiety. The effects of mGluRS on measures of anxiety might be mediated by the neuropeptides corticotropin-releasing factor (CRF) and arginine vasopressin (AVP). The amygdala, BNST, ventral hippocampus, and lateral septum, express these neuropeptides implicated in the regulation of anxiety. Our preliminary data show mGluRS immunoreactivity in the amygdala and BNST of C57BI6/J wild-type mice. We hypothesize that mGluRS modulates measures of anxiety in the amygdaloid complex and that effects of mGiuRS signaling on the expression of CRF and AVP contribute to increased measures of anxiety in mGluR8-/- mice. The Specific Aims are: (1) To determine if mGluR8-/- mice show increased measures of anxiety;(2) To determine if acute stimulation of mGluR8 reduces measures of anxiety and alters expression of CRF and AVP in wild-type mice;(3) To localize mGluR8 in neural structures implicated in the regulation of anxiety;and (4) To determine the role of mGluR8 in synaptic function in the basolateral nucleus of the amygdala (BLA) and extended amygdala (BNST and CeA). Elucidating the role of mGluR8 in anxiety and assessing the potential of pharmacological mGluR8 stimulation to reduce anxiety might be especially valuable for patients with anxiety disorders resistant to treatment with benzodiazepine.

Project Summary ApoE (apolipoprotein E) is an essential component of lipoprotein particles. Three human APOE isoforms exist: E2, E3, and E4. Compared to E3, E2 is protective with regard to susceptibility to develop Alzheimer's disease (AD), is more prevalent among centenarians, and is associated with improved episodic memory performance, larger hippocampal volume, and reduced hippocampal atrophy rate. Little is known, however, about the mechanisms involved in the neuroprotection of E2 in aging, including slowing AD progression. In this proposal, we will use targeted replacement mice and an unbiased epigenomics approach to identify pathway changes that underlie the protective role of E2. The mouse strains to be used include human ApoE and AD genotypes. We have mouse strains expressing human E2 E3, and E4 under the mouse apoE promoter (apoE TR mice). These strains will be crossed with human APP knock in mice developed by Dr. Takaomi Saido (Riken Research Brain Institute) that contain the Swedish and Iberian mutations (APP NL-F) or contain these mutations in combination with the Artic mutation (APP NL-G-F). APP NL-G-F mice accumulate neuropathology and show cognitive impairments one year earlier than the APP NL-F mice (6 versus 18 months). This difference in disease onset will allow us to examine the interaction between neuropathological and aging processes and the E2 isoform. We hypothesize that E2 slows the development of cognitive injury and AD pathology because it enhances neuroprotective and aging protective pathways. To test this hypothesis, three Specific Aims are proposed. The first Aim will examine the protective effect of the E2 isoform, relative to E3 and E4, with regard to early onset histopathological and behavioral and cognitive changes in the NL-G-F mouse strain. We expect to observe less change in the E2 background. Aim 2 will resemble Aim 1, with the exception of crossing the ApoE mice with the slower acting APP NL-F gene. Again, we expect protection from the AD phenotype in the E2 background, which may be relatively greater than in Aim 1 if aging processes are also slowed in the E2 background. Because the association of E2 with reduced AD risk is seen in men but not women, we will examine male and female mice in Aims 1 and 2. For Aim 3, we will use omics approaches to examine DNA methylation (both methylcytosine and hydroxymethylcytosine) and RNA expression profiles in the different strain backgrounds to identify neuroprotective and aging protective pathways that are enhanced in the E2 mice and help explain the protective effect that is conferred by this isoform. Overall, we expect that the work and analyses proposed in this application to identify disease-related pathways that change more slowly and neuroprotective pathways that will be more pronounced in the E2 background, facilitating the identification of diagnostic markers and the development of novel therapeutic targets for AD and other age-related cognitive disorders.

Project Summary/Abstract Cognitive impairments including those associated with metabolic syndrome, mild cognitive impairment, and Alzheimer's disease (AD), are increasingly associated with deficient insulin action in the central nervous system (CNS). Evidence exists for both decreased brain insulin levels and impaired brain insulin signaling (i.e., insulin resistance) as causes for this decreased insulin action. Intranasal insulin therapy can improve cognition in both healthy subjects as well as those suffering from cognitive impairments. However, being an apolipoprotein E4 (apoE4) carrier, female, or obese is associated with decreased CNS insulin action, including a decreased therapeutic response to CNS insulin. In this application, using mice expressing human E3 or E4 under control of the mouse apoE promoter, we will investigate the mechanisms by which the apoE genotype, sex, and diet can affect insulin action in the brain. We will examine the three main mechanisms that can underlie differences in CNS insulin action: 1) insulin transport, 2) insulin retention and degradation, and 3) insulin receptor activation. In SA1, we will assess brain pharmacokinetics, including measuring the transport and brain distribution of insulin after intravenous and intranasal administration. In SA2, insulin availability will be investigated by measuring the association of apoE with the insulin receptor and by measuring levels of insulin degradation. In SA3, insulin action at the receptor level will be investigated amongst the groups and we will investigate which factor (apoE4, sex, and diet) most predominantly impacts cognition in response to chronic intranasal insulin treatment. Overall, these studies will for the first time investigate the combinatorial roles and mechanisms involved in the effects of apoE isoform, sex, and diet on decreased CNS insulin activity and cognition.

Project Summary There is growing evidence that non-smokers who were exposed to second-hand smoke (SHS) are at an increased risk of mild cognitive impairment (MCI), often a preclinical form of dementia. Patients with MCI have a significantly higher rate of exposure to SHS than dementia patients and most MCI patients eventually develop dementia. Environmental factors ranging from previous head trauma to educational level to one's early life experiences (e.g., altered brain growth and development), including exposure to toxins through the diet (e.g., nitrosamines) or by inhalation (e.g., cigarette smoke), are major risk factors for the sporadic forms of dementia. It is becoming increasingly clear that environmental factors play an important role in the etiology of dementia, but which of them is poorly understood. SHS might be an important environmental trigger of MCI and dementia. Studies that focus on exploring the influence of SHS on cognitive function and the underlying metabolic processes could advance understanding of the etiopathogenesis of MCI and sporadic dementia and possibly reveal novel approaches for their effective treatment. MCI is characterized by a dysfunction in brain glucose metabolism as well as the accumulation of pathological proteins (e.g., tau, amyloid), biomarkers that correlate with disease severity. Tau misregulation is an important underlying mechanism of the progressive cognitive and neuropathological changes that develop in patients with MCI or dementia. Smoke inhaled through a cigarette (active or mainstream smoking) is an important risk factor for MCI and dementia. SHS doubles the risk for dementia among individuals who never smoked and it is 2-6 times more toxic and tumorigenic to humans than mainstream smoke. Thus, SHS might have a greater effect on brain metabolism and cognitive function than mainstream smoking. Studies strongly suggest that SHS increases the risk of MCI or dementia by perturbing brain metabolism (i.e., insulin signaling, oxidative stress) and the accumulation of pathological proteins (i.e, tau, amyloid). We hypothesize that chronic exposure to SHS induces changes in brain metabolism that lead to tau misregulation, amyloid accumulation and cognitive injury. We also hypothesize that these SHS-induced cognitive and pathological changes will be accelerated in susceptible individuals (i.e., those with misregulated tau such as htau mice). These hypotheses will be tested by comparing the effect of chronic (12-month) SHS exposure on cognitive function of wild-type and htau mice. Following behavioral and cognitive testing, brain areas pertinent to the behavioral and cognitive changes will be assessed for metabolic changes, tau and amyloid pathology, and neuronal loss. These studies will provide important information about the role of SHS in the cognitive injury and early metabolic changes in MCI. Importantly, this animal model may be especially useful for developing therapeutic agents to counteract the metabolic, pathological and cognitive changes following human exposure to SHS.

Abstract of proposed studies under the Administrative Supplement in response to NOT- AG-20-022 Age is a significant risk factor in the recent SARS-CoV-2 pandemic and this risk might be more profound in middle-aged people with age-related cognitive decline, neurodegeneration and Alzheimer?s disease. While working directly with the SARS-CoV-2 virus is valuable, it requires working in an ABSL-3 level facility and substantially restricts potential behavioral and cognitive studies, brain imaging experiments, and postmortem studies with non-perfused tissues. SARS- CoV-2 recombinant virus-like particles (VLPs) offer an attractive way to study the pathogenesis of SARS-CoV-2 in animals, without the involvement of replicating viruses. In this study, we expose wild-type and human tau mice to SARS-CoV-2 VLPs to study the effects on cognitive performance, brain activity, markers of inflammation, and the effect of sex on the expected degeneration. This study will provide the first data on potential central nervous system complications of Alzheimer?s disease patients, as well as an age-matched population, which are exposed to SARS-CoV-2 VLPs, and will provide an experimental platform to test potential treatments.