Ari E. Berman

Nuclear Factor Erythroid 2-Related Factor 2 Facilitates Neuronal Glutathione Synthesis by Upregulating Neuronal Excitatory Amino Acid Transporter 3 Expression

Astrocytes support neuronal antioxidant capacity by releasing glutathione, which is cleaved to cysteine in brain extracellular space. Free cysteine is then taken up by neurons through excitatory amino acid transporter 3 [EAAT3; also termed Slc1a1 (solute carrier family 1 member 1)] to support de novo glutathione synthesis. Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway by oxidative stress promotes astrocyte release of glutathione, but it remains unknown how this release is coupled to neuronal glutathione synthesis. Here we evaluated transcriptional regulation of the neuronal cysteine transporter EAAT3 by the Nrf2-ARE pathway. Nrf2 activators and Nrf2 overexpression both produced EAAT3 transcriptional activation in C6 cells. A conserved ARE-related sequence was found in the EAAT3 promoter of several mammalian species. This ARE-related sequence was bound by Nrf2 in mouse neurons in vivo as observed by chromatin immunoprecipitation. Chemical activation of the Nrf2-ARE pathway in mouse brain increased both neuronal EAAT3 levels and neuronal glutathione content, and these effects were abrogated in mice genetically deficient in either Nrf2 or EAAT3. Selective overexpression of Nrf2 in brain neurons by lentiviral gene transfer was sufficient to upregulate both neuronal EAAT3 protein and glutathione content. These findings identify a mechanism whereby Nrf2 activation can coordinate astrocyte glutathione release with neuronal glutathione synthesis through transcriptional upregulation of neuronal EAAT3 expression.

Inhibition of poly(ADP-ribose) polymerase suppresses inflammation and promotes recovery after ischemic injury

The brain inflammatory response induced by stroke contributes to cell death and impairs neurogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1) is a coactivator of the transcription factor NF-κB and required for NF-κB-mediated inflammatory responses. Here we evaluated PARP inhibition as a means of suppressing post-stroke inflammation and improving outcome after stroke. Rats were subjected to bilateral carotid occlusion-reperfusion, and treatment with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) was begun 48 h later. PJ34 was found to rapidly suppress the ischemia-induced microglial activation and astrogliosis. Behavioral tests performed 6 to 8 weeks after ischemia showed deficits in spatial memory and learning that were lessened by the PJ34 treatment. Immunohistochemical evaluation of hippocampus at 8 weeks after ischemia showed increased neuronal density in CA1 layer of PJ34-treated animals relative to vehicle-treated animals. Bromodeoxyuridine labeling showed formation of new neurons in hippocampal CA1 area in PJ34-treated animals, but not in vehicle-treated animals. Together, these results suggest that treatment with a PARP inhibitor for several days after ischemia enhances long-term neuronal survival and neurogenesis by reducing inflammation.

Transcriptional Signatures of Cellular Plasticity in Mice Lacking the α1 Subunit of GABAA Receptors

GABAA receptors mediate the majority of inhibitory neurotransmission in the CNS. Genetic deletion of the α1 subunit of GABAA receptors results in a loss of α1-mediated fast inhibitory currents and a marked reduction in density of GABAA receptors. A grossly normal phenotype of α1-deficient mice suggests the presence of neuronal adaptation to these drastic changes at the GABA synapse. We used cDNA microarrays to identify transcriptional fingerprints of cellular plasticity in response to altered GABAergic inhibition in the cerebral cortex and cerebellum of α1 mutants. In silico analysis of 982 mutation-regulated transcripts highlighted genes and functional groups involved in regulation of neuronal excitability and synaptic transmission, suggesting an adaptive response of the brain to an altered inhibitory tone. Public gene expression databases permitted identification of subsets of transcripts enriched in excitatory and inhibitory neurons as well as some glial cells, providing evidence for cellular plasticity in individual cell types. Additional analysis linked some transcriptional changes to cellular phenotypes observed in the knock-out mice and suggested several genes, such as the early growth response 1 (Egr1), small GTP binding protein Rac1 (Rac1), neurogranin (Nrgn), sodium channel β4 subunit (Scn4b), and potassium voltage-gated Kv4.2 channel (Kcnd2) as cell type-specific markers of neuronal plasticity. Furthermore, transcriptional activation of genes enriched in Bergman glia suggests an active role of these astrocytes in synaptic plasticity. Overall, our results suggest that the loss of α1-mediated fast inhibition produces diverse transcriptional responses that act to regulate neuronal excitability of individual neurons and stabilize neuronal networks, which may account for the lack of severe abnormalities in α1 null mutants.

Poly(ADP-ribose)polymerase-1 modulates microglial responses to amyloid β

BackgroundAmyloid β (Aβ) accumulates in Alzheimers disease (AD) brain. Microglial activation also occurs in AD, and this inflammatory response may contribute to disease progression. Microglial activation can be induced by Aβ, but the mechanisms by which this occurs have not been defined. The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) regulates microglial activation in response to several stimuli through its interactions with the transcription factor, NF-κB. The purpose of this study was to evaluate whether PARP-1 activation is involved in Aβ-induced microglial activation, and whether PARP-1 inhibition can modify microglial responses to Aβ.MethodshAPPJ20 mice, which accumulate Aβ with ageing, were crossed with PARP-1-/- mice to assess the effects of PARP-1 depletion on microglial activation, hippocampal synaptic integrity, and cognitive function. Aβ peptide was also injected into brain of wt and PARP-1-/- mice to directly determine the effects of PARP-1 on Aβ-induced microglial activation. The effect of PARP-1 on Aβ-induced microglial cytokine production and neurotoxicity was evaluated in primary microglia cultures and in microglia-neuron co-cultures, utilizing PARP-1-/- cells and a PARP-1 inhibitor. NF-κB activation was evaluated in microglia infected with a lentivirus reporter gene.ResultsThe hAPPJ20 mice developed microglial activation, reduced hippocampal CA1 calbindin expression, and impaired novel object recognition by age 6 months. All of these features were attenuated in hAPPJ20/PARP-1-/- mice. Similarly, Aβ1-42 injected into mouse brain produced a robust microglial response in wild-type mice, and this was blocked in mice lacking PARP-1 expression or activity. Studies using microglial cultures showed that PARP-1 activity was required for Aβ-induced NF-κB activation, morphological transformation, NO release, TNFα release, and neurotoxicity. Conversely, PARP-1 inhibition increased release of the neurotrophic factors TGFβ and VEGF, and did not impair microglial phagocytosis of Aβ peptide.ConclusionsThese results identify PARP-1 as a requisite and previously unrecognized factor in Aβ-induced microglial activation, and suggest that the effects of PARP-1 are mediated, at least in part, by its interactions with NF-κB. The suppression of Aβ-induced microglial activation and neurotoxicity by PARP-1 inhibition suggests this approach could be useful in AD and other disorders in which microglial neurotoxicity may contribute.

DIGITALLY QUANTIFYING CEREBRAL HEMORRHAGE USING PHOTOSHOP® AND IMAGE J

A spectrophotometric hemoglobin assay is widely used to estimate the extent of brain hemorrhage by measuring the amount of hemoglobin in the brain. However, this method requires using the entire brain sample, leaving none for histology or other assays. Other widely used measures of gross brain hemorrhage are generally semi-quantitative and can miss subtle differences. Semi-quantitative brain hemorrhage scales may also be subject to bias. Here, we present a method to digitally quantify brain hemorrhage using Photoshop and Image J, and compared this method to the spectrophotometric hemoglobin assay. Male Sprague-Dawley rats received varying amounts of autologous blood injected into the cerebral hemispheres in order to generate different sized hematomas. 24h later, the brains were harvested, sectioned, photographed then prepared for the hemoglobin assay. From the brain section photographs, pixels containing hemorrhage were identified by Photoshop and the optical intensity was measured by Image J. Identification of hemorrhage size using optical intensities strongly correlated to the hemoglobin assay (R=0.94). We conclude that our method can accurately quantify the extent of hemorrhage. An advantage of this technique is that brain tissue can be used for additional studies.

Alcohol trait and transcriptional genomic analysis of C57BL/6 substrains

C57BL/6 inbred mice have been widely used as research models; however, widespread demand has led to the creation of several B6 substrains with markedly different phenotypes. In this study, we report that two substrains of C57BL/6 mice, C57BL/6J (B6J) and C57BL/6NCrl (B6C), separated over 50 years ago at two different breeding facilities differ significantly in alcohol consumption and alcohol preference. The genomes of these two substrains are estimated to differ by only 1–2% of all gene loci, providing a unique opportunity to extract particular expression signatures between these substrains that are associated with quantifiable behavioral differences. Expression profiling of the cortex and striatum, hippocampus, cerebellum and the ventral brain region from alcohol‐naïve B6C and B6J mice showed intervals on three chromosomes that are enriched in clusters of coregulated transcripts significantly divergent between the substrains. Additional analysis identified two genomic regions containing putative copy number differences between the substrains. One such region on chromosome 14 contained an estimated 3n copy number in the B6J genome compared with B6C. Within this interval, a gene of unknown function, D14Ertd449e, was found to be both associated with alcohol preference and vary in copy number across several inbred strain lineages. H2afz, Psen1, Wdfy1 and Clu were also identified as candidate genes that may be involved in influencing alcohol consumption.

EAAC1 Gene Deletion Alters Zinc Homeostasis and Exacerbates Neuronal Injury after Transient Cerebral Ischemia

EAAC1 is a neuronal glutamate and cysteine transporter. EAAC1 uptake of cysteine provides substrate for neuronal glutathione synthesis, which plays a key role in both antioxidant defenses and intracellular zinc binding. Here we evaluated the role of EAAC1 in neuronal resistance to ischemia. EAAC1−/− mice subjected to transient cerebral ischemia exhibited twice as much hippocampal neuronal death as wild-type mice and a corresponding increase in microglial activation. EAAC1−/− mice also had elevated vesicular and cytosolic zinc concentrations in hippocampal CA1 neurons and an increased zinc translocation to postsynaptic neurons after ischemia. Treatment of the EAAC1−/− mice with N-acetyl cysteine restored neuronal glutathione concentrations and normalized basal zinc levels in the EAAC1−/− mice. Treatment of the EAAC1−/− mice with either N-acetyl cysteine or with zinc chelators reduced ischemia-induced zinc translocation, superoxide production, and neuron death. These findings suggest that cysteine uptake by EAAC1 is important for zinc homeostasis and neuronal antioxidant function under ischemic conditions.

Expression Profiling in Alcoholism Research.

This article represents the proceedings of a symposium at the 2004 International Society for Biomedical Research on Alcoholism in Mannheim, Germany, organized and co-chaired by Susan E. Bergeson and Wolfgang Sommer. The presentations and presenter were (1) Gene Expression in Brains of Alcohol-Preferring and Non-Preferring Rats, by Howard J. Edenberg (2) Candidate Treatment Targets for Alcoholism: Leads from Functional Genomics Approaches, by Wolfgang Sommer (3) Microarray Analysis of Acute and Chronic Alcohol Response in Brain, by Susan E. Bergeson (4) On the Integration of QTL and Gene Expression Analysis, by Robert J. Hitzemann (5) Microarray and Proteomic Analysis of the Human Alcoholic Brain, by Peter R. Dodd.