Jing-Qiong Kang

Erythropoietin Is a Novel Vascular Protectant Through Activation of Akt1 and Mitochondrial Modulation of Cysteine Proteases

Background—Erythropoietin (EPO) is a critical regulator for the proliferation of immature erythroid precursors, but its role as a potential cytoprotectant in the cerebrovasculature system has not been defined. Methods and Results—We examined the ability of EPO to regulate a cascade of apoptotic death-related cellular pathways during anoxia-induced vascular injury in endothelial cells (ECs). EC injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine (PS) exposure, protein kinase B activity, mitochondrial membrane potential, and cysteine protease induction. Exposure to anoxia alone rapidly increased genomic DNA fragmentation from 2±1% to 40±5% and membrane PS exposure from 3±2% to 56±5% over 24 hours. Administration of a cytoprotective concentration of EPO (10 ng/mL) prevented DNA destruction and PS exposure. Cytoprotection by EPO was completely abolished by cotreatment with anti-EPO neutralizing antibody, which suggests that EPO was necessary and sufficient for the prevention of apoptosis. Protection by EPO was intimately dependent on the activation of protein kinase B (Akt1) and the maintenance of mitochondrial membrane potential. Subsequently, EPO inhibited caspase 8-, caspase 1-, and caspase 3-like activities that were linked to mitochondrial cytochrome c release. Conclusions—The present work serves to illustrate that EPO can offer novel cytoprotection during ischemic vascular injury through direct modulation of Akt1 phosphorylation, mitochondrial membrane potential, and cysteine protease activity.

Hematopoietic Factor Erythropoietin Fosters Neuroprotection through Novel Signal Transduction Cascades

In addition to promoting the survival, proliferation, and differentiation of immature erythroid cells, erythropoietin and the erythropoietin receptor have recently been shown to modulate cellular signal transduction pathways that extend beyond the erythropoietic function of erythropoietin. In particular, erythropoietin has been linked to the prevention of programmed cell death in neuronal systems. Although this work is intriguing, the underlying molecular mechanisms that serve to mediate neuroprotection by erythropoietin are not well understood. Further analysis illustrates that erythropoietin modulates two distinct components of programmed cell death that involve the degradation of DNA and the externalization of cellular membrane phosphatidylserine residues. Initiation of the cascades that modulate protection by erythropoietin and its receptor may begin with the activation of the Janus tyrosine kinase 2 protein. Subsequent downstream mechanisms appear to lead to the activation of multiple signal transduction pathways that include transcription factor STAT5 (signal transducers and activators of transcription), Bcl-2, protein kinase B, cysteine proteases, mitogen-activated protein kinases, proteintyrosine phosphatases, and nuclear factor-κB. New knowledge of the cellular pathways regulated by erythropoietin in neuronal environments will potentially solidify the development and initiation of therapeutic strategies against nervous system disorders.

Erythropoietin fosters both intrinsic and extrinsic neuronal protection through modulation of microglia, Akt1, Bad, and caspase-mediated pathways

Erythropoietin (EPO) plays a significant role in the hematopoietic system, but the function of EPO as a neuroprotectant and anti‐inflammatory mediator requires further definition. We therefore examined the cellular mechanisms that mediate protection by EPO during free radical injury in primary neurons and cerebral microglia. Neuronal injury was evaluated by trypan blue, DNA fragmentation, phosphatidylserine (PS) exposure, Akt1 phosphorylation, Bad phosphorylation, mitochondrial membrane potential, and cysteine protease activity. Microglial activation was assessed through proliferating cell nuclear antigen and PS receptor expression. EPO provides intrinsic neuronal protection that is both necessary and sufficient to prevent acute genomic DNA destruction and subsequent membrane PS exposure, since protection by EPO is completely abolished by cotreatment with an anti‐EPO neutralizing antibody. Extrinsic protection by EPO is offered through the inhibition of cerebral microglial activation and the suppression of microglial PS receptor expression for the prevention of neuronal phagocytosis. In regards to microglial chemotaxis, EPO modulates neuronal poptotic membrane PS exposure necessary for microglial activation primarily through the regulation of caspase 1. EPO increases Akt1 activity, phosphorylates Bad, and maintains neuronal nuclear DNA integrity through the downstream modulation of mitochrondrial membrane potential, cytochrome c release, and caspase 1, 3, and 8‐like activities. Elucidating the intrinsic and extrinsic protective pathways of EPO that mediate both neuronal integrity and inflammatory microglial activation may enhance the development of future therapies directed against acute neuronal injury.

Mutations in GABAA receptor subunits associated with genetic epilepsies

Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking.

Apaf-1, Bcl-xL, Cytochrome c, and Caspase-9 Form the Critical Elements for Cerebral Vascular Protection by Erythropoietin

Erythropoietin (EPO) plays a prominent role in the regulation of the hematopoietic system, but the potential function of this trophic factor as a cytoprotectant in the cerebral vascular system is not known. The authors examined the ability of EPO to modulate a series of death-related cellular pathways during free radical–induced injury in cerebral microvascular endothelial cells (ECs). Endothelial cell injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine exposure, apoptotic protease–activating factor-1 (Apaf-1), and Bcl-xL expression, mitochondrial membrane potential, cytochrome c release, and cysteine protease activity. They show that constitutive EPO is present in ECs but is insufficient to prevent cellular injury. Signaling through the EPO receptor, however, remains biologically responsive to exogenous EPO administration to offer significant protection against nitric oxide–induced injury. Exogenous EPO maintains both genomic DNA integrity and cellular membrane asymmetry through parallel pathways that prevent the induction of Apaf-1 and preserve mitochondrial membrane potential in conjunction with enhanced Bcl-xL expression. Consistent with the modulation of Apaf-1 and the release of cytochrome c, EPO also inhibits the activation of caspase-9 and caspase-3–like activities. Identification of novel cytoprotective pathways used by EPO may serve as therapeutic targets for cerebral vascular disease.

Erythropoietin prevents early and late neuronal demise through modulation of Akt1 and induction of caspase 1, 3, and 8

Erythropoietin (EPO) modulates primarily the proliferation of immature erythroid precursors, but little is known of the potential protective mechanisms of EPO in the central nervous system. We therefore examined the ability of EPO to modulate a series of death‐related cellular pathways during anoxia and free radical induced neuronal degeneration. Neuronal injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine exposure, protein kinase B phosphorylation, cysteine protease activity, mitochondrial membrane potential, and mitogen‐activated protein (MAP) kinase phosphorylation. We demonstrate that constitutive neuronal EPO is insufficient to prevent cellular injury, but that signaling through the EPO receptor remains biologically responsive to exogenous EPO administration. Exogenous EPO is both necessary and sufficient to prevent acute genomic DNA destruction and subsequent phagocytosis through membrane PS exposure, because neuronal protection by EPO is completely abolished by co‐treatment with an anti‐EPO neutralizing antibody. Through pathways that involve the initial activation of protein kinase B, EPO maintains mitochondrial membrane potential. Subsequently, EPO inhibits caspase 8‐, caspase 1‐, and caspase 3‐like activities linked to cytochrome c release through mechanisms that are independent from the MAP kinase systems of p38 and JNK. Elucidating some of the novel neuroprotective pathways employed by EPO may further the development of new therapeutic strategies for neurodegenerative disorders.

Why Does Fever Trigger Febrile Seizures? GABAA Receptor γ2 Subunit Mutations Associated with Idiopathic Generalized Epilepsies Have Temperature-Dependent Trafficking Deficiencies

With a worldwide incidence as high as 6.7% of children, febrile seizures are one of the most common reasons for seeking pediatric care, but the mechanisms underlying generation of febrile seizures are poorly understood. Febrile seizures have been suspected to have a genetic basis, and recently, mutations in GABAA receptor and sodium channel genes have been identified that are associated with febrile seizures and generalized seizures with febrile seizures plus pedigrees. Pentameric GABAA receptors mediate the majority of fast synaptic inhibition in the brain and are composed of combinations of α(1–6), β(1–3), and γ(1–3) subunits. In αβγ2 GABAA receptors, the γ2 subunit is critical for receptor trafficking, clustering, and synaptic maintenance, and mutations in the γ2 subunit have been monogenically associated with autosomal dominant transmission of febrile seizures. Here, we report that whereas trafficking of wild-type α1β2γ2 receptors was slightly temperature dependent, trafficking of mutant α1β2γ2 receptors containing γ2 subunit mutations [γ2(R43Q), γ2(K289M), and γ2(Q351X)] associated with febrile seizures was highly temperature dependent. In contrast, trafficking of mutant α1β2γ2 receptors containing an α1 subunit mutation [α1(A322D)] not associated with febrile seizures was not highly temperature dependent. Brief increases in temperature from 37 to 40°C rapidly (<10 min) impaired trafficking and/or accelerated endocytosis of heterozygous mutant α1β2γ2 receptors containing γ2 subunit mutations associated with febrile seizures but not of wild-type α1β2γ2 receptors or heterozygous mutant α1(A322D)β2γ2 receptors, suggesting that febrile seizures may be produced by a temperature-induced dynamic reduction of susceptible mutant surface GABAA receptors in response to fever.

The GABAA Receptor γ2 Subunit R43Q Mutation Linked to Childhood Absence Epilepsy and Febrile Seizures Causes Retention of α1β2γ2S Receptors in the Endoplasmic Reticulum

The GABAA receptor γ2 subunit mutation R43Q is an autosomal dominant mutation associated with childhood absence epilepsy and febrile seizures. Previously, we demonstrated that homozygous α1β3γ2L(R43Q) receptor whole-cell currents had reduced amplitude with unaltered time course, suggesting reduced cell surface expression of functional receptors. In human embryonic kidney 293-T cells, we demonstrate that both heterozygous and homozygous α1β2γ2S(R43Q) GABAA receptor current amplitudes were reduced when receptors were assembled from coexpressed α1, β2, and γ2S subunits and from β2-α1 tandem subunits coexpressed with the γ2L subunit. Using fluorescence confocal microscopy, we demonstrated that mutant receptors containing enhanced yellow fluorescent protein-tagged γ2S subunits had reduced surface expression and were retained in the endoplasmic reticulum. In addition, using biotinylation of surface receptors and immunoblotting, we confirmed that α1β2γ2S(R43Q) receptors had reduced surface expression. These results provide evidence that the γ2S(R43Q) mutation impaired GABAA receptor function by compromising receptor trafficking and reducing surface expression.

Akt1 protects against inflammatory microglial activation through maintenance of membrane asymmetry and modulation of cysteine protease activity

In several cell systems, protein kinase B (Akt1) can promote cell growth and development, but the “antiapoptotic” pathways of this kinase that may offer protection against cellular inflammatory demise have not been defined. Given that early cellular membrane phosphatidylserine exposure is a critical component of apoptosis, we investigated the role of Akt1 during neuronal apoptotic injury. By employing differentiated SH‐SY5Y neuronal cells that overexpress a constitutively active form of Akt1 (myristoylated Akt1), free radical‐induced cell injury was assessed through trypan blue dye exclusion, DNA fragmentation, membrane phosphatidylserine exposure, protein kinase B phosphorylation, cysteine protease activity, and mitochondrial membrane potential. Membrane phosphatidylserine exposure was both necessary and sufficient for microglial activation, insofar as cotreatment with an antiphosphatidylserine receptor‐neutralizing antibody could prevent microglial activity following neuronal loss of membrane asymmetry. Furthermore, expression of myristoylated Akt1 not only prevented cell injury through the prevention of membrane phosphatidylserine exposure and genomic DNA fragmentation but also inhibited microglial activation and proliferation that required the inhibition of caspase 9‐, caspase 3‐, and caspase 1‐like activities linked to cytochrome c release. Interestingly, Akt1 modulation of membrane phosphatidylserine exposure was primarily through caspase 1 activity. Removal of Akt1 activity abolished neuronal protection, suggesting that Akt1 functions as a critical pathway for the maintenance of cellular integrity and the prevention of phagocytic cellular removal during neurodegenerative insults.

δ Subunit Susceptibility Variants E177A and R220H Associated with Complex Epilepsy Alter Channel Gating and Surface Expression of α4β2δ GABAA Receptors

Most human idiopathic generalized epilepsies (IGEs) are polygenic, but virtually nothing is known of the molecular basis for any of the complex epilepsies. Recently, two GABAA receptor δ subunit variants (E177A, R220H) were proposed as susceptibility alleles for generalized epilepsy with febrile seizures plus and juvenile myoclonic epilepsy. In human embryonic kidney 293T cells, recombinant hα1β2δ(E177A) and hα1β2δ(R220H) receptor currents were reduced, but the basis for the current reduction was not determined. We examined the mechanistic basis for the current reduction produced by these variants using the hα4β2δ receptor, an isoform more physiologically relevant and linked to epileptogenesis, by characterizing the effects of these variants on receptor cell surface expression and single-channel gating properties. Expression of variant α4β2δ(R220H) receptors resulted in a decrease in surface receptor proteins, and a smaller, but significant, reduction was observed for variant α4β2δ(E177A) receptors. For both variants, no significant alterations of surface expression were observed for mixed population of wild-type and variant receptors. The mean open durations of α4β2δ(E177A) and α4β2δ(R220H) receptor single-channel currents were both significantly decreased compared to wild-type receptors. These data suggest that both δ(E177A) and δ(R220H) variants may result in disinhibition in IGEs by similar cellular and molecular mechanisms, and in heterozygously affected individuals, a reduction in channel open duration of δ subunit-containing GABAA receptors may be the major contributor to the epilepsy phenotypes.