Saumyendra N. Sarkar

Estrogen Actions on Mitochondria-Physiological and Pathological Implications

Estrogens are potent neuroprotective hormones and mitochondria are the site of cellular life-death decisions. As such, it is not surprising that we and others have shown that estrogens have remarkable effects on mitochondrial function. Herein we provide evidence for a primary effect of estrogens on mitochondrial function, achieved in part by the import of estrogen receptor beta (ERbeta) into the mitochondria where it mediates a number of estrogen actions on this vital organelle. ERbeta is imported into the mitochondria, through tethering to cytosolic chaperone protein and/or through direct interaction with mitochondrial import proteins. In the mitochondria, ERbeta can affect transcription of critical mitochondrial genes through the interaction with estrogen response elements (ERE) or through protein-protein interactions with mitochondrially imported transcription factors. The potent effects of estrogens on mitochondrial function, particularly during mitochondrial stress, argues for a role of estrogens in the treatment of mitochondrial defects in chronic neurodegenerative diseases like Alzheimers disease (AD) and Parkinsons disease (PD) and more acute conditions of mitochondrial compromise, like cerebral ischemia and traumatic brain injury.

Estrogens directly potentiate neuronal L-type Ca2+ channels

L-type voltage-gated Ca2+channels (VGCC) play an important role in dendritic development, neuronal survival, and synaptic plasticity. Recent studies have demonstrated that the gonadal steroid estrogen rapidly induces Ca2+ influx in hippocampal neurons, which is required for neuroprotection and potentiation of LTP. The mechanism by which estrogen rapidly induces this Ca2+ influx is not clearly understood. We show by electrophysiological studies that extremely low concentrations of estrogens acutely potentiate VGCC in hippocampal neurons, hippocampal slices, and HEK-293 cells transfected with neuronal L-type VGCC, in a manner that was estrogen receptor (ER)-independent. Equilibrium, competitive, and whole-cell binding assays indicate that estrogen directly interacts with the VGCC. Furthermore, a L-type VGCC antagonist to the dihydropyridine site displaced estrogen binding to neuronal membranes, and the effects of estrogen were markedly attenuated in a mutant, dihydropyridine-insensitive L-type VGCC, demonstrating a direct interaction of estrogens with L-type VGCC. Thus, estrogen-induced potentiation of calcium influx via L-type VGCC may link electrical events with rapid intracellular signaling seen with estrogen exposure leading to modulation of synaptic plasticity, neuroprotection, and memory formation.

Estrogen Receptor β as a Mitochondrial Vulnerability Factor

We recently demonstrated mitochondrial localization of estrogen receptor β (ERβ). We herein confirm the mitochondrial localization of ERβ by the loss of mitochondrial ERβ immunoreactivity in ERβ knockdown cells. A phenotype change characterized as an increase in resistance to oxidative stressors is associated with ERβ knockdown. ERβ knockdown results in a lower resting mitochondrial membrane potential (Δψm) and increase in resistance to hydrogen peroxide-induced Δψm depolarization in both immortal hippocampal cells and primary hippocampal neurons. ERβ knockdown cells maintained ATP concentrations despite insults that compromise ATP production and produce less mitochondrial superoxide under oxidative stress. Furthermore, similar mitochondrial phenotype changes were identified in primary hippocampal neurons derived from ERβ knock-out mice. These data demonstrate that ERβ is expressed in mitochondria and function as a mitochondrial vulnerability factor involved in Δψm maintenance, potentially through a mitochondrial transcription dependent mechanism.

Expression of microRNA-34a in Alzheimer's disease brain targets genes linked to synaptic plasticity, energy metabolism, and resting state network activity.

Polygenetic risk factors and reduced expression of many genes in late-onset Alzheimers disease (AD) impedes identification of a target(s) for disease-modifying therapies. We identified a single microRNA, miR-34a that is over expressed in specific brain regions of AD patients as well as in the 3xTg-AD mouse model. Specifically, increased miR-34a expression in the temporal cortex region compared to age matched healthy control correlates with severity of AD pathology. miR-34a over expression in patients tissue and forced expression in primary neuronal culture correlates with concurrent repression of its target genes involved in synaptic plasticity, oxidative phosphorylation and glycolysis. The repression of oxidative phosphorylation and glycolysis related proteins correlates with reduced ATP production and glycolytic capacity, respectively. We also found that miR-34a overexpressed neurons secrete miR-34a containing exosomes that are taken up by neighboring neurons. Furthermore, miR-34a targets dozens of genes whose expressions are known to be correlated with synchronous activity in resting state functional networks. Our analysis of human genomic sequences from the tentative promoter of miR-34a gene shows the presence of NFκB, STAT1, c-Fos, CREB and p53 response elements. Together, our results raise the possibilities that pathophysiology-induced activation of specific transcription factor may lead to increased expression of miR-34a gene and miR-34a mediated concurrent repression of its target genes in neural networks may result in dysfunction of synaptic plasticity, energy metabolism, and resting state network activity. Thus, our results provide insights into polygenetic AD mechanisms and disclose miR-34a as a potential therapeutic target for AD.

Estrogen-induced signaling attenuates soluble Aβ peptide-mediated dysfunction of pathways in synaptic plasticity.

Neuromodulation of synaptic plasticity by 17β-estradiol (E2) is thought to influence information processing and storage in the cortex and hippocampus. Because E2 rapidly affects cortical memory and synaptic plasticity, we examined its effects on phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase (ERK), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) [AMPA-type glutamate receptor subunit 1 (GluR1 subunit)], all of which are important for the induction and maintenance of synaptic plasticity and memory. Acute E2 treatment resulted in an increased temporal and spatial phosphorylation pattern of CaMKII, ERK, and AMPAR (GluR1 subunit). By using inhibitors, we were able to attribute GluR1 phosphorylation to CaMKII at serine 831, and we also found that E2 treatment increased GluR1 insertion into the surface membrane. Because soluble amyloid-beta (Aβ) oligomers inhibit CaMKII and ERK activation, which is necessary for synaptic plasticity, we also tested E2s ability to ameliorate Aβ-induced dysfunction of synaptic plasticity. We found that estrogen treatment in neuronal culture, slice culture, and in vivo, ameliorated Aβ oligomer-induced inhibition of CaMKII, ERK, and AMPAR phosphorylation, and also ameliorated the Aβ oligomer-induced reduction of dendritic spine density in a CaMKII-dependent manner. These phosphorylation events are correlated with the early stage of inhibitory avoidance learning, and our data show that E2 improved inhibitory avoidance memory deficits in animals treated with soluble Aβ oligomers. This study identifies E2-induced signaling that attenuates soluble Aβ peptide-mediated dysfunction of pathways in synaptic plasticity.

MiR-34a regulates blood–brain barrier permeability and mitochondrial function by targeting cytochrome c

The blood–brain barrier is composed of cerebrovascular endothelial cells and tight junctions, and maintaining its integrity is crucial for the homeostasis of the neuronal environment. Recently, we discovered that mitochondria play a critical role in maintaining blood–brain barrier integrity. We report for the first time a novel mechanism underlying blood–brain barrier integrity: miR-34a mediated regulation of blood–brain barrier through a mitochondrial mechanism. Bioinformatics analysis suggests miR-34a targets several mitochondria-associated gene candidates. We demonstrated that miR-34a triggers the breakdown of blood–brain barrier in cerebrovascular endothelial cell monolayer in vitro, paralleled by reduction of mitochondrial oxidative phosphorylation and adenosine triphosphate production, and decreased cytochrome c levels.

Estrogen amelioration of Aβ-induced defects in mitochondria is mediated by mitochondrial signaling pathway involving ERβ, AKAP and Drp1

Perturbations in dynamic properties of mitochondria including fission, fusion, and movement lead to disruption of energy supply to synapses contributing to neuropathology and cognitive dysfunction in Alzheimer׳s disease (AD). The molecular mechanisms underlying these defects are still unclear. Previously, we have shown that ERβ is localized in the mitochondria and ERβ knock down disrupts mitochondrial functions. Because a selective ERβ modulator (DPN) can activate PKA, and localized PKA signaling in the mitochondrial membrane regulates mitochondrial structure and functions, we reasoned that ERβ signaling in the mitochondrial membrane rescues many of the mitochondrial defects caused by soluble Aβ oligomer. We now report that DPN treatment in primary hippocampal neurons attenuates soluble Aβ-oligomer induced dendritic mitochondrial fission and reduced mobility. Additionally, Aβ treatment reduced the respiratory reserve capacity of hippocampal neuron and inhibited phosphorylation of Drp1 at its PKA site, which induces excessive mitochondrial fission, and DPN treatment ameliorates these inhibitions. Finally, we discovered a direct interaction of ERβ with a mitochondrial resident protein AKAP1, which induces the PKA-mediated local signaling pathway involved in increased oxidative phosphorylation and inhibition of mitochondrial fission. Taken together, our findings highlight the possibility that ERβ signaling pathway may be a useful mitochondria-directed therapeutic target for AD.

MiR-34a and stroke: Assessment of non-modifiable biological risk factors in cerebral ischemia

Aging of the nervous system, and the occurrence of age-related brain diseases such as stroke, are associated with changes to a variety of cellular processes controlled by many distinct genes. MicroRNAs (miRNAs), short non-coding functional RNAs that can induce translational repression or site-specific cleavage of numerous target mRNAs, have recently emerged as important regulators of cellular senescence, aging, and the response to neurological insult. Here, we focused on the assessment of the role of miR-34a in stroke. We noted increases in miR-34a expression in the blood of stroke patients as well as in blood and brain of mice subjected to experimental stroke. Our methodical genetic manipulation of miR-34a expression substantially impacted stroke-associated preclinical outcomes and we have in vitro evidence that these changes may be driven at least in part by disruptions to blood brain barrier integrity and mitochondrial oxidative phosphorylation in endothelial cells. Finally, aging, independent of brain injury, appears to be associated with shifts in circulating miRNA profiles. Taken together, these data support a role for miRNAs, and specifically miR-34a, in brain aging and the physiological response to age-related neurological insult, and lay the groundwork for future investigation of this novel therapeutic target.

MicroRNAs and the Genetic Nexus of Brain Aging, Neuroinflammation, Neurodegeneration, and Brain Trauma

Aging is a complex and integrated gradual deterioration of cellular activities in specific organs of the body, which is associated with increased mortality. This deterioration is the primary risk factor for major human pathologies, including cancer, diabetes, cardiovascular disorders, neurovascular disorders, and neurodegenerative diseases. There are nine tentative hallmarks of aging. In addition, several of these hallmarks are increasingly being associated with acute brain injury conditions. In this review, we consider the genes and their functional pathways involved in brain aging as a means of developing new strategies for therapies targeted to the neuropathological processes themselves, but also as targets for many age-related brain diseases. A single microRNA (miR), which is a short, non-coding RNA species, has the potential for targeting many genes simultaneously and, like practically all other cellular processes, genes associated with many features of brain aging and injury are regulated by miRs. We highlight how certain miRs can mediate deregulation of genes involved in neuroinflammation, acute neuronal injury and chronic neurodegenerative diseases. Finally, we review the recent progress in the development of effective strategies to block specific miR functions and discuss future approaches with the prediction that anti-miR drugs may soon be used in the clinic.

TNF-ñ and Beyond: Rapid Mitochondrial Dysfunction Mediates TNF-ñ-InducedNeurotoxicity

This short communication describes our research which demonstrates that TNF-α causes a rapid decline in mitochondrial function, leading to neuronal cell death. As such, this neurotoxic proinflammatory cytokine may play a role in brain damage from stroke and neurodegeneration in chronic conditions such as Alzheimer’s disease (AD) and Parkinson’s disease. We have extended this initial observation by demonstrating that TNF-α stimulates a microRNA (miR-34a) which we have shown reduces five key proteins in the mitochondrial electron transport chain through base-pair complementarity. miR-34a is increased in affected brain regions of Alzheimer’s patients and transgenic AD mouse models. We have further shown that oligomeric amyloid beta 42 (oAβ42) stimulates miR-34a. Collectively, these data suggest that TNF-α, oAβ42, and miR-34a participate in a vicious cycle, resulting in mitochondrial dysfunction, which is critical to the neuropathology of AD.