Jeffrey S. Johnson - US grants

DESCRIPTION: (ADAPTED FROM APPLICANT'S ABSTRACT) Induction of NAD(P)H:quinone oxidoreductase (QR) by tert-butylhydroquinone (tBHQ) prior to glutamate treatment significantly decreased glutamate-mediated cytotoxicity. Stable overexpression of QR, however, did not protect cells from glutamate-, dopamine or H2O2-induced apoptosis. Thus, the protection afforded by tBHQ is not due simply to an increase in QR, but the coordinate regulation of multiple genes by a common mechanism. Our laboratory has shown that tBHQ increases QR through activation of its antioxidant response element (ARE) in human neuroblastoma cells and primary glial cell cultures. We hypothesize that increased expression of ARE-driven genes block oxidative stress-induced cell death. The specific aims of this proposal are to: 1) Determine the molecular mechanism(s) by which tBHQ activates the ARE and increases QR in human neuroblstoma cells; 2) Characterize the expression pattern and regulation of the ARE in vivo; 3) Determine the effect of overexpresion of amyloid precursor protein on QR and ARE in transgenic mouse models of alzheimer's disease; 4) Characterize the expression pattern of QR in human brains from control and AD patients. Increased oxidative stress is associated with neuronal cell death following acute insults such as epilepsy, ischemia, and hypoglycemia. Oxidative stress is also believed to be a principle factor in the development of many chronic neurodegenerative diseases such as Alzheimer's, Parkinson's Huntington's and Amyotrophic Lateral Sclerosis. Oxidative stress is an imbalance in which free radicals and their products exceed the capacity of antioxidant defense mechanisms. A gain in product formation and /or loss in protective mechanisms can disturb this equilibrium. Presently, we have little knowledge of how or by neurodegenerative diseases such as Alzheimer's disease (Specific Aims 3 and 4). Elucidating the molecular mechanisms (s) regulating ARE genes in brain, therefore, may be crucial for developing therapeutic approaches to mitigate, or prevent, neurotoxicity.

neurotoxicology; neuroprotectants; hyperbilirubinemia; glutathione transferase; brain metabolism; age difference; glia; enzyme induction /repression; cerebellar Purkinje cell; bilirubin; cerebellum; brain mapping; cell type; isozymes; developmental neurobiology; tissue /cell culture; immunocytochemistry; laboratory rat; transfection;

[unreadable] DESCRIPTION (provided by applicant): Oxidative stress is an imbalance in which free radicals and their products exceed the capacity of antioxidant defense mechanisms. The harmful reactive compounds generated by oxidative stress are associated with neuronal cell death following acute insults and are also believed to be a principle factor in the development of many chronic neurodegenerative diseases such as Alzheimer's, Parkinson's (PD), Huntington's and Amyotrophic Lateral Sclerosis. The expression of many neuroprotective phase II detoxification enzymes and/or antioxidant genes is governed by the antioxidant responsive element (ARE). ARE-dependent gene expression is induced by the transcriptional factor, Nrf2, and is considered to be a novel and important pathway that confers protection to a variety of oxidative stress-related neurodegenerative insults. The long- range objective of the laboratory is to evaluate the regulation and cell-specific expression of ARE-driven genes and the potential role of these genes in prevention of neurodegeneration. In order to develop potential therapeutic strategies targeting Parkinson's disease through activation of the ARE, small molecules that penetrate the blood-brain barrier and robustly activate ARE will be required. We will use primary cortical cultures from Nrf2 knockout and wild-type mice as an in vitro system to examine potential chemical activators of the ARE. We will test whether compounds identified as potent ARE activators can attenuate nigrostriatal lesions in both 6-hydroxydopamine (6-OHDA) and MPTP induced Parkinson's models in Nrf2-/- and wild-type mice. Finally, we will examine Nrf2-mediated neuroprotection by infecting astrocyte cultures with adenovirus Nrf2 constructs and transplanting those cells into the striata of Nrf2-/- and wildtype mice after 6-OHDA or MPTP lesions. The specific aims of this proposal are: Specific Aim 1. Characterize Nrf2-dependent ARE activation by chemical activators and evaluate their neuroprotective potential in vitro. Specific Aim 2. Determine that chemical activators of the Nrf2-ARE pathway confer protection from the 6-OHDA and MPTP-induced nigrostriatal lesions and loss of dopaminergic (DA) neurons. Specific Aim 3. Determine that transplantation of Nrf2 overexpressing neural stem cells and/or astrocytes confers protection from 6-OHDA or MPTP-induced nigrostriatal lesions and loss of DA neurons. [unreadable] [unreadable] [unreadable]

Transthyretin's Regulatory Role in Beta-Amyloid Aggregation and Toxicity Alzheimer's disease (AD) has been linked to deposition of beta-amyloid (A[unreadable]) as amyloid plaques in the brain. Transgenic mice expressing the human A[unreadable] precursor protein (APP) produce high levels of A[unreadable] and develop amyloid plaques, but they do not suffer the extensive neuronal cell death that is characteristic of AD. Recent studies have uncovered a possible explanation: these transgenic mice greatly increase the synthesis of the transport protein transthyretin (TTR), and TTR appears to protect the mice from the neurotoxic effects of A[unreadable]. The long-term goals of this project are to answer three questions that arise from these intriguing results: (1) how does TTR exert its protective activity? (2) why does this natural protective activity fail in AD? (3) can it be restored? Our hypothesis is that subtle changes in TTR tertiary and/or quaternary structure strongly modulate TTR- A[unreadable] interactions. In specific aim 1, several TTR mutants that differ in their tertiary and/or quaternary structure and stability will be produced and characterized. Each mutant will be screened for its ability to interfere with A[unreadable] aggregation, and to inhibit A[unreadable] toxicity in an in vitro cell culture model. The data will be analyzed to identify correlations between TTR structure and stability with its ability to alter A[unreadable] aggregation and toxicity. In specific aim 2, a detailed examination of the interaction between TTR (both wildtype and selected mutants) and A[unreadable] will be undertaken. Biophysical and biochemical tools such as circular dichroism, fluorescence, static and dynamic light scattering, crosslinking, and kinetic modeling will be employed. From these data will emerge molecular-level mechanistic insights into the nature of TTR-A[unreadable] association and the means by which TTR affects A[unreadable] aggregation kinetics. The goal of specific aim 3 is to identify small ligands that stabilize TTR and determine their influence on TTR's ability to modulate A[unreadable] aggregation and toxicity. In aim 4, TTR (wildtype and mutants) along with TTR-binding ligands will be tested for protection against A[unreadable] toxicity in ex vivo and in vivo mouse models. This will be achieved by using a newly developed assay in which stereotactic injection of A[unreadable] into mouse brain leads to loss of CA1 and dentate gyrus neurons, and by infection of astrocytes with adenovirus-TTR constructs. The project spans from characterization of the structure and stability of TTR (and mutants), through in vitro assessment of TTR's effect on A[unreadable] aggregation and toxicity, to in vivo evaluation of TTR efficacy at preventing neuronal cell death. These studies will provide a sound and rational basis for developing novel pharmacological approaches to preventing AD by enhancement of the natural defenses provided by TTR.

DESCRIPTION (provided by applicant): Nrf2 (NF-E2-related factor 2) has been demonstrated to play a central role in the gene expression of detoxification enzymes and antioxidant genes through binding the antioxidant responsive element (ARE). Importantly, in in vitro and in vivo models, this system has been shown to be effective at blocking neurotoxicity resulting from glutathione depletion, lipid peroxidation, intracellular calcium overload, excitotoxins, and disruption of the mitochondrial electron transport chain. In particular, increased levels of glutathione (GSH) seem to be a major component of the protection observed by Nrf2 activation. Glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits make up the rate-limiting enzyme complex for GSH synthesis and Nrf2 transcriptionally regulated both enzymes. Interestingly, the Nrf2-ARE pathway activation is isolated to astrocytes that confer resistance to neurons. Several studies have demonstrated that secreted GSH can protect neurons acting as an antioxidant in the extracellular compartment and boosting GSH levels in neurons by increasing the availability of precursors for GSH synthesis. We are particularly intrigued by astrocyte-motor neuron interaction and how the Nrf2-ARE pathway could be involved in this intimate relationship, as well as the level of importance of GSH to motor neuron survival, in amyotrophic lateral sclerosis (ALS). Some of the inherited forms of ALS are associated with mutations in human Cu/Zn superoxide dismutase 1 (hSOD1). Mouse models have been generated using mutant forms of hSOD1 that recapitulate much of the cellular and phenotypic changes observed in human ALS patients. We hypothesize that Nrf2-ARE activation and GSH production in the astrocyte will protect motor neurons in models of ALS in vitro and in vivo. We have generated glial fibrillary acidic protein (GFAP)-Nrf2 mice that selectively overexpress Nrf2 in astrocytes. Preliminary data in co-cultures of astrocytes and motor neurons indicate that hSOD1G93A astrocytes are "sick" and that motor neurons die when plated on these "sick" astrocytes. Strikingly, if Nrf2 is overexpressed in the same "sick" astrocytes, the motor neurons do not die. Initial in vivo studies show an increase in lifespan of the hSOD1G93A mice when crossed with GFAP-Nrf2 mice. Finally, ongoing experiments using the GCLM-/- mice are underway. In this case, hSOD1G93A mice on GCLM-/- background show dramatic muscle weakness (0 sec on wire hang) and initial signs of paralysis by 45 days of age compared to 120 days of age on a GCLM+/+ background. The specific aims of this proposal are: Aim 1. To determine the significance of Nrf2 and GSH in astrocyte-mediated motor neuron survival in vitro;Aim 2. To evaluate the role of GSH in the Nrf2- mediated delay in onset and progression of disease in hSOD1G93A mice;and Aim 3. Test the efficacy of clinically relevant approaches for the treatment o ALS using viral-mediated delivery of Nrf2 or non- viral delivery of Keap1 siRNA. PUBLIC HEALTH RELEVANCE Pound 5,600 people in the United States are diagnosed with ALS every year and there are about 30,000 Americans with the disease at any given time. Approximately 80% of ALS suffers will die within 5 years of diagnosis. Thus, the lack of any significant therapeutic intervention for ALS is an important medical issue. The majority of ALS cases are sporadic with unknown cause. Environmental exposure is thought to play a major role in sporadic disease and is believed to be a principal component behind the increased incidence in ALS among Gulf War veterans and people serving in the military. Irrespective of the underlying cause of ALS, the approaches outlined in proposal are focused on a pathologic processes seen in both sporadic and familial disease, and could lead to new ways to treat ALS. In addition to looking at the onset of the disease in these animal models, specific experiments will examine disease progression that more accurately represent the human clinical situation. The hope is to gain a greater understanding of the disease process and discover ways to treat ALS that will stop or at least slow disease progression.

DESCRIPTION (provided by applicant): EEG recording studies in humans have revealed the presence of oscillatory rhythms in brain activity, and D oscillations in different frequency bands have been associated with cognitive processes such as working D memory. Additionally, alterations of oscillatory activity are found in psychiatric illnesses such as D schizophrenia. Despite their apparent relevance, however, the role of neural oscillations in cognitive D processes are as yet poorly understood. The proposed project explores these issues using simultaneous D transcranial magnetic stimulation (TMS) and EEG recording. Specifically, the present proposal assesses D two proposals regarding the mechanisms by which TMS influences ongoing neural activity and hence D behavior. The first proposal holds that TMS achieves it effects by injecting electrical noise into task-related D neural areas, producing characteristic declines in performance. However, this proposal is inconsistent with D findings of TMS-related improvements in performance, which have been reported in several studies. Thus, C an alternative account holds that TMS interacts in subtle ways with ongoing neural activity related to the D performance of specific tasks, producing disruptions or improvements in performance depending on the D nature of the interaction. This issue is explored in an experiment comparing the effects of 10-Hz repetitive D (r)TMS on neural activity, to the effects of 10-Hz visual flicker, which is known to produce widespread D entrainment (i.e. disruption) of neural activity to the flicker frequency. Two additional experiments explore 7 task-related changes in neural oscillations, and how TMS can be used to alter such oscillations, producing u improvements or disruptions in performance at the individual subject level. D D The proposed project will help to clarify how neurostimulation methods such as transcranial magnetic D stimulation (TMS) influence ongoing neural activity, and will contribute decisively to our understanding of D the role of neural oscillations in cognitive processes such as working memory. Moreover, the methods D developed here will point the way towards the use of TMS in the exploration and treatment of abnormal D oscillations found in psychiatric illnesses such as schizophrenia.

DESCRIPTION (provided by applicant): Beta-amyloid (Aß) is the primary protein component of Alzheimer's disease (AD) amyloid plaques, and there is substantial evidence to support the hypothesis that soluble Aß aggregates are neurotoxic. Transgenic mice expressing the Swedish mutation of the human Aß precursor protein (APPSw) produce high levels of Aß and develop amyloid plaques, but surprisingly they do not suffer the extensive neuronal cell death characteristic of AD. Recent studies have uncovered a possible explanation: APPSw mice upregulate synthesis of transthyretin (TTR), a transport protein found in plasma and cerebrospinal fluid, and TTR appears to protect the mice from the neurotoxic effects of Aß. The long-term goals of this project are to answer three questions that arise from this intriguing discovery: (1) how does TTR exert its protective activity? (2) why does this natural protective activity fail in AD? (3) can it be restored or replaced? In aim 1, the specific residues on TTR involved with binding to Aß will be identified. From mass spectrometry analysis coupled with peptide array binding studies, residues on the G strand and near the EF helix of TTR were implicated. Further definition of the binding site will be obtained by screening for Aß binding to peptide library derived from overlapping sequences of TTR, and by targeted alanine mutagenesis. Compounds that mimic the TTR binding sites will be synthesized and tested for Aß binding as well as inhibition of in vitro toxicity. In aim 2, the effect of TTR and variants on Aß aggregation will be characterized. Preliminary data show that TTR quaternary structure and stability, oxidation, and binding of natural ligands all influence the extent of Aß binding to TTR. The greatest Aß binding is observed at intermediate aggregation states. Put another way, TTR may be a natural scavenger for the most toxic Aß aggregates. Aggregation of Aß in the presence of TTR will be characterized by a combination of dynamic and static light scattering, and nanoparticle tracking. These complementary methods, which are particularly suited for examining soluble aggregates, will yield data on aggregate size, size distribution, number, morphology, and aggregation rate. Synthetic mimics, developed in aim 1, will also be characterized for their effect on Aß aggregation. In aim 3, further validation of TTR's neuroprotective action will be sought. Astrocytes will be transfected to secrete TTR (wt and monomeric), and inhibition of Aß toxicity will be tested in vitro with mixed cortical cultures, or y adding medium from secreting astrocytes to highly enriched cortical neurons. Transgenic mice that overexpress TTR in astrocytes on an APP/PS1 background will be generated, and pathological endpoints will be evaluated to ascertain the level of in vivo protection afforded by TTR. Finally, initial screening of promising TTR mimics from Aim 1 will be tested in a stereotactic injection assay. These studies, which integrate chemical, biophysical, and biological approaches, will provide a rational basis for developing novel pharmacological approaches to preventing AD by enhancement of TTR's natural defenses.

PROJECT 1 - PROJECT SUMMARY / ABSTRACT Alzheimer's disease (AD), an age associated neurodegenerative disorder, is pathologically characterized by plaques consisting of aggregated beta-amyloid (A?) and intracellular neurofibrillary tangles (NFT) formed by hyperphosphorylated tau. A? is formed by the sequential action of ? and ? secretase on the C-terminal side of amyloid precursor protein (APP). Oxidative stress has been implicated in human AD patients and several studies have shown A?-mediated oxidative stress to be central to the AD pathogenesis and progression. In addition to, or associated with oxidative stress, a reduction/dysfunction of autophagy is observed in AD. Autophagic vacuoles (AVs) that are rarely observed in neurons of healthy individuals are found to accumulate in the dystrophic and less affected neuritis in AD. Indeed, autophagy is hypothesized to be a major mechanism mediating APP turnover normally and generation of intracellular A? when dysfunctional. NF-E2- related factor 2 (Nrf2) has been implicated to be involved, not only in mitigating oxidative stress, but also an important factor in regulating and responding directly and indirectly to the changes in autophagy in vivo and in vitro. There is little work examining the role of Nrf2 in APP processing/turnover and A? production/degradation in AD with no information existing in the animal models of AD or human AD tissue. Recently, data from our laboratory found that overexpression of Nrf2 in astrocytes (GFAP-Nrf2) dramatically delays autophagic dysfunction in neurons of alpha synuclein A53T mice. This correlated with a highly significant delay in disease onset and extension of lifespan. Initial crosses of our GFAP-Nrf2 mice with APP/PS1 mice demonstrate a reduced plaque density and a dramatic reduction in intracellular APP or cleavage products. Thus, the specific aims of this proposal are: Aim 1. To determine the effect of astrocytic Nrf2 overexpression on APP processing/turnover and A? production/degradation in APP/PS1 mice. Aim 2. To elucidate the mechanism involved in astrocytic Nrf2-mediated changes in APP processing/turnover and A? production/degradation in astrocyte-neuron cultures derived from APP/PS1 mice. Aim 3. To determine changes in astrocytic Nrf2 activation in possible/early and definitive/late stage human AD brain. Completion of the proposed studies will solidify the significance of astrocytic Nrf2 activation as a modulator of AD pathology.

DESCRIPTION (provided by applicant): Working memory (WM) is a core psychological construct that plays a central role in everyday activities, from relatively simple tasks such as th temporary retention of task-relevant information, to the manipulation and use of this information in complex cognitive tasks and in the control and guidance of adaptive behaviors. Additionally, an individual's ability to retain and manipulate information in WM has been shown to be an important factor underlying individual differences across a broad spectrum of experimental and real world measures, and WM dysfunction has been strongly implicated in the cognitive deficits observed in psychiatric illnesses, most notably schizophrenia. A prominent theme in current cognitive neuroscience research is the idea that WM is supported by causal interactions between specialized brain areas linked together into functional networks. However, much of the evidence supporting this proposal has come from functional connectivity analysis of neural data (fMRI, PET, EEG), which gives a correlational rather than causal indication of the functional properties of particular brain areas, and of how network interactions might support cognition. Furthermore, a causal and mechanistic description of how causal network interactions give rise to WM functions is lacking. The proposed research addresses these issues using non-invasive brain stimulation (TMS), and neural recording (EEG) to directly probe causal interregional interactions during retention in WM, and neurally plausible modeling to provide a mechanistic description of how such interactions vary as a function of cognitive state changes. Specific Aim 1 uses combined TMS-EEG to clarify the role of causal interactions between frontal and posterior brain areas in the short-term retention of visual information. Prior work has suggested a general role for the prefrontal cortex (PFC) in the top-down control of activity in posterior sensory areas. Although the PFC has been shown to regulate WM maintenance by filtering out distracting information, PFC activation and increased frontal-posterior connectivity in the absence of distraction suggests that such interactions might also be involved in pure storage functions. This possibility will be examined by stimulating the frontal cortex during the retention of information in WM and recording the resulting response, both within the stimulated area and at distal cortical sites, using EEG. If frontal-posterior functional interactions play a direct rol in maintenance, we expect the strength of connectivity to increase with the amount of information being held in WM. Similarly, we expect to observe systematic changes in the spatial spread of TMS-evoked activations to posterior brain areas when the PFC is stimulated during the delay period of tasks requiring the storage of information that is coded in distinct posterior brain area. Specific Aim 2 will use an existing neural model of WM and change detection to implement a systems-level model of connectivity that can capture EEG signatures of WM maintenance and account for the observed state-dependence of TMS/EEG expected in Aim 1. Establishing this link between neural modeling and TMS/EEG will lay the groundwork for future work aimed at formally testing different predictions regarding how causal network interactions support WM and other cognitive functions, and will provide a basis for examining possible aberrant network interactions underlying the symptoms of debilitating mental illnesses, such as schizophrenia.