Mir Ahamed Hossain

Neuroprotection by scatter factor/hepatocyte growth factor and FGF-1 in cerebellar granule neurons is phosphatidylinositol 3-kinase/Akt-dependent and MAPK/CREB-independent

Neuroprotective actions of scatter factor/hepatocyte growth factor (SF/HGF) have not been described. We examined the effects of SF/HGF in comparison to acidic fibroblast growth factor‐1 (FGF‐1) on N‐methyl‐d‐aspartate (NMDA) and quinolinic acid (QUIN)‐induced excitotoxicity in primary cerebellar granule neurons. Exposure to NMDA or QUIN for 24 h resulted in concentration‐dependent cell death (p < 0.001) that was completely attenuated (p < 0.001) by pre‐treatment of cells with SF/HGF (50 ng/mL) or FGF‐1 (40 ng/mL). SF/HGF and FGF‐1 activated both Akt and MAP‐kinase > threefold (p < 0.001). Neither SF/HGF nor FGF‐1 activated cyclic AMP‐response element binding protein (CREB), a downstream target of MAP‐kinase, whereas brain‐derived neurotrophic factor (BDNF) activated both MAP‐kinase and CREB in granule neurons. Neuroprotection against NMDA or QUIN by SF/HGF and FGF‐1 was negated by the addition of LY294002 (10 µm) or wortmannin (100 nm), two distinct inhibitors of phosphatidylinositol 3‐kinase (PI3‐K), but not by the MAP‐kinase kinase (MEK) inhibitor PD98059 (33 µm). Likewise, expression of a dominant‐negative mutant of Akt (Akt‐kd) completely prevented the neuroprotective actions of SF/HGF and FGF‐1. Overexpression of a constitutively activated Akt (Akt‐myr) or wild‐type Akt (wtAkt) attenuated excitotoxic cell death. These data show that both SF/HGF and FGF‐1 protect cerebellar granule neurons against excitotoxicity with similar potency in a PI3‐K/Akt‐dependent and MAP‐kinase/CREB‐independent manner.

Hypoxic-Ischemic Injury in Neonatal Brain: Involvement of a Novel Neuronal Molecule in Neuronal Cell Death and Potential Target for Neuroprotection.

Perinatal hypoxia‐ischemia (HI) is the most common cause of various neurological disabilities in children with high societal cost. Hypoxic‐ischemic brain damage is an evolving process and ample evidence suggests distinct difference between the immature and mature brain in the pathology and consequences of brain injury. Therefore, it is of utmost importance to better understand the mechanisms underlying the hypoxic‐ischemic injury in neonatal brain to devise effective therapeutic strategies. Nonetheless, the mechanism(s) involved in this pathology in the developing brain remain inadequately understood. Effective neuroprotective strategies will include either inhibition of the death effector pathways or induction of their regulatory and survival promoting cellular proteins. Neuronal pentraxins (NPs) define a family of novel proteins “long pentraxins” that are exclusively expressed in the central neurons, and are homologous to the C‐reactive and acute‐phase proteins in the immune system. NPs have been shown to be involved in the excitatory synaptic remodeling. We found that the neuronal protein ‘neuronal pentraxin 1′ (NP1) is induced in neonatal rat brain following HI, and NP1 induction preceded the time of actual tissue loss in brain. In demonstrating this we also found that NP1 gene silencing is neuroprotective against hypoxia‐induced neuronal death. This is the first evidence for a pathophysiological function of NP1 in central neurons. Our results suggest that NP1 is part of a death program triggered by HI. Most importantly, our findings of specific interactions of NP1 with the excitatory glutamate receptors AMPA GluR1 subunit and their co‐localization suggest a role for this novel neuronal protein NP1 in the excitotoxic cascade. Blockade of AMPA‐induced neuronal death following inhibition of NP1 expression further implicates a regulatory interaction between NP1 and AMPA glutamate receptors. Subsequent experiments using NP1 loss‐of‐function strategies, we have demonstrated specific requirements of NP1 induction in HI‐induced neuronal death. Together our findings clearly identify a novel role for NP1 in the coupling between HI and cerebral cell death. Thus, NP1 could be a new molecular target in the central neurons for preventing hypoxic‐ischemic neuronal death in developing brain. These very novel results could lead to more effective neuroprotective strategies against hypoxic‐ischemic brain injury in neonates.

Nuclear translocation of X‐linked inhibitor of apoptosis (XIAP) determines cell fate after hypoxia ischemia in neonatal brain

The inhibitors of apoptosis (IAPs) are emerging as key proteins in the control of cell death. In this study, we evaluated the expression and subcellular distribution of the antiapoptotic protein X‐linked IAP (XIAP), and its interactions with the XIAP‐associated factor 1 (XAF1) in neonatal rat brain following hypoxia‐ischemia (HI). HI triggered the mitochondrial release of cytochrome c, Smac/DIABLO, and caspase 3 activation. Confocal microscopy detected XIAP‐specific immunofluorescence in the cytoplasm under normal condition, which exhibited a diffuse distribution at 6 h post‐HI and by 12 h the majority of XIAP was redistributed into the nucleus. XIAP nuclear translocation was confirmed by subcellular fractionations and by expressing FLAG‐tagged XIAP in primary cortical neurons. Over‐expression of XIAP significantly reduced, whereas XIAP gene silencing further enhanced cell death, demonstrating a specific requirement of cytoplasmic XIAP for cell survival. An elevated level of cytosolic XIAP was also evident under the conditions of neuroprotection by fibroblast growth factor‐1. XAF1 expression was increased temporally and there was increased nuclear co‐localization with XIAP in hypoxic‐ischemic cells. XIAP co‐immunoprecipitated > 9‐fold XAF1 protein concurrent with decreased association with caspases 9 and 3. This is evidenced by the enhanced caspase 3 activity and neuronal death. Our findings implicate XIAP nuclear translocation in neuronal death and point to a novel mechanism in the regulation of hypoxic‐ischemic brain injury.

Enhanced cell death in MeCP2 null cerebellar granule neurons exposed to excitotoxicity and hypoxia.

Rett syndrome (RTT) is associated with mutations in the transcriptional repressor gene MeCP2. Although the clinical and neuropathological signs of RTT suggest disrupted synaptic function, the specific role of methyl-CpG binding protein 2 (MeCP2) in postmitotic neurons remains relatively unknown. We examined whether MeCP2 deficiency in central neurons contributes to the neuropathogenesis in RTT. Primary cerebellar granule neuronal cultures from wild-type (WT) and MeCP2-/- mice were exposed to N-methyl-d-aspartate (NMDA) and AMPA-induced excitotoxicity and hypoxic-ischemic insult. The magnitude of cell death in MeCP2-/- cells after excitotoxicity and hypoxia was greater than in the WT littermate control cultures and occurred after shorter exposures that usually, in the WT, would not cause cell death. Pretreatment with the growth factor fibroblast growth factor 1 (FGF-1) under conditions at which WT cells showed complete neuroprotection, only partially protected MeCP2-/- cells. To elucidate specifically the effects of MeCP2 knockout (KO) on cell death, we examined two death cascade pathways. MeCP2-/- neurons exposed to 6 h of hypoxia exhibited enhanced activation of the proapoptotic caspase-3 and increased mitochondrial release of apoptosis inducing factor (AIF) compared with WT neurons, which did not show significant changes. However, pretreatment with the caspase inhibitor ZVAD-FMK had little or no effect on AIF release and its subcellular translocation to the nucleus, suggesting caspase-independent AIF release and their independent contribution to hypoxia-induced cell death. Reintroduction of intact MeCP2 gene in MeCP2-/- cells or MeCP2 gene silencing by MeCP2siRNA in WT cells further confirmed the differential sensitivity of the WT and MeCP2-/- cells and suggest a direct role of MeCP2 in cell death. These results clearly demonstrate increased cell death occurred in neurons lacking MeCP2 expression via both caspase- and AIF-dependent apoptotic mechanisms. Our findings suggest a novel, yet unknown, role for MeCP2 in central neurons in the control of neuronal response to cell death.

Vascular endothelial growth factor mediates vasogenic edema in acute lead encephalopathy.

Brain injury from inorganic Pb2+ is considered the most important environmental childhood health hazard worldwide. The microvasculature of the developing brain is uniquely susceptible to high level Pb2+ toxicity (ie, Pb2+ encephalopathy) characterized by cerebellar hemorrhage, increased blood–brain barrier permeability, and vasogenic edema. However, the specific molecular mediators of Pb2+ encephalopathy have been elusive. We found that Pb2+ induces vascular endothelial growth factor/vascular permeability factor (VEGF) in cultured astrocytes (J Biol Chem, 2000;275:27874–27882). The study presented here asks if VEGF dysregulation contributes mechanistically to Pb2+ encephalopathy. Neonatal rats exposed to 4% Pb‐carbonate develop the histopathological features of Pb2+ encephalopathy seen in children. Cerebellar VEGF expression increased approximately twofold (p < 0.01) concurrent with the development of cerebellar microvascular hemorrhage, enhanced vascular permeability to serum albumin, and vasogenic cerebellar edema (p < 0.01). No change in VEGF expression occurred in cerebral cortex that does not develop these histopathological complications of acute Pb2+ intoxication. Pb2+ exposure increased phosphorylation of cerebellar Flk‐1 VEGF receptors and the Flk‐1 inhibitor CEP‐3967 completely blocked cerebellar edema formation without affecting microhemorrhage formation or blood–brain barrier permeability. This establishes that Pb2+‐induced vasogenic edema formation develops via a Flk‐1–dependent mechanism and suggests that the vascular permeability caused by Pb2+ is Flk‐1 independent.

Sex-specific activation of cell death signalling pathways in cerebellar granule neurons exposed to oxygen glucose deprivation followed by reoxygenation

Neuronal death pathways following hypoxia–ischaemia are sexually dimorphic, but the underlying mechanisms are unclear. We examined cell death mechanisms during OGD (oxygen-glucose deprivation) followed by Reox (reoxygenation) in segregated male (XY) and female (XX) mouse primary CGNs (cerebellar granule neurons) that are WT (wild-type) or Parp-1 [poly(ADP-ribose) polymerase 1] KO (knockout). Exposure of CGNs to OGD (1.5 h)/Reox (7 h) caused cell death in XY and XX neurons, but cell death during Reox was greater in XX neurons. ATP levels were significantly lower after OGD/Reox in WT-XX neurons than in XY neurons; this difference was eliminated in Parp-1 KO-XX neurons. AIF (apoptosis-inducing factor) was released from mitochondria and translocated to the nucleus by 1 h exclusively in WT-XY neurons. In contrast, there was a release of Cyt C (cytochrome C) from mitochondria in WT-XX and Parp-1 KO neurons of both sexes; delayed activation of caspase 3 was observed in the same three groups. Thus deletion of Parp-1 shunted cell death towards caspase 3-dependent apoptosis. Delayed activation of caspase 8 was also observed in all groups after OGD/Reox, but was much greater in XX neurons, and caspase 8 translocated to the nucleus in XX neurons only. Caspase 8 activation may contribute to increased XX neuronal death during Reox, via caspase 3 activation. Thus, OGD/Reox induces death of XY neurons via a PARP-1-AIF-dependent mechanism, but blockade of PARP-1-AIF pathway shifts neuronal death towards a caspase-dependent mechanism. In XX neurons, OGD/Reox caused prolonged depletion of ATP and delayed activation of caspase 8 and caspase 3, culminating in greater cell death during Reox.

Critical role of neuronal pentraxin 1 in mitochondria-mediated hypoxic-ischemic neuronal injury

Developing brain is highly susceptible to hypoxic-ischemic (HI) injury leading to severe neurological disabilities in surviving infants and children. Previously, we have reported induction of neuronal pentraxin 1 (NP1), a novel neuronal protein of long-pentraxin family, following HI neuronal injury. Here, we investigated how this specific signal is propagated to cause the HI neuronal death. We used wild-type (WT) and NP1 knockout (NP1-KO) mouse hippocampal cultures, modeled in vitro following exposure to oxygen glucose deprivation (OGD), and in vivo neonatal (P9-10) mouse model of HI brain injury. Our results show induction of NP1 in primary hippocampal neurons following OGD exposure (4-8 h) and in the ipsilateral hippocampal CA1 and CA3 regions at 24-48 h post-HI compared to the contralateral side. We also found increased PTEN activity concurrent with OGD time-dependent (4-8 h) dephosphorylation of Akt (Ser473) and GSK-3β (Ser9). OGD also caused a time-dependent decrease in the phosphorylation of Bad (Ser136), and Bax protein levels. Immunofluorescence staining and subcellular fractionation analyses revealed increased mitochondrial translocation of Bad and Bax proteins from cytoplasm following OGD (4 h) and simultaneously increased release of Cyt C from mitochondria followed by activation of caspase-3. NP1 protein was immunoprecipitated with Bad and Bax proteins; OGD caused increased interactions of NP1 with Bad and Bax, thereby, facilitating their mitochondrial translocation and dissipation of mitochondrial membrane potential (ΔΨ(m)). This NP1 induction preceded the increased mitochondrial release of cytochrome C (Cyt C) into the cytosol, activation of caspase-3 and OGD time-dependent cell death in WT primary hippocampal neurons. In contrast, in NP1-KO neurons there was no translocation of Bad and Bax from cytosol to the mitochondria, and no evidence of ΔΨ(m) loss, increased Cyt C release and caspase-3 activation following OGD; which resulted in significantly reduced neuronal death. Our results indicate a regulatory role of NP1 in Bad/Bax-dependent mitochondrial release of Cyt C and caspase-3 activation. Together our findings demonstrate a novel mechanism by which NP1 regulates mitochondria-driven hippocampal cell death; suggesting NP1 as a potential therapeutic target against HI brain injury in neonates.

Neuronal Pentraxin 1 Induction in Hypoxic Ischemic Neuronal Death is Regulated via a Glycogen Synthase Kinase-3α/β Dependent Mechanism

Intracellular signaling pathways that regulate the production of lethal proteins in central neurons are not fully characterized. Previously, we reported induction of a novel neuronal protein neuronal pentraxin 1 (NP1) in neonatal brain injury following hypoxia-ischemia (HI); however, how NP1 is induced in hypoxic-ischemic neuronal death remains elusive. Here, we have elucidated the intracellular signaling regulation of NP1 induction in neuronal death. Primary cortical neurons showed a hypoxic-ischemia time-dependent increase in cell death and that NP1 induction preceded the actual neuronal death. NP1 gene silencing by NP1-specific siRNA significantly reduced neuronal death. The specificity of NP1 induction in neuronal death was further confirmed by using NP1 (-/-) null primary cortical neurons. Declines in phospho-Akt (i.e. deactivation) were observed concurrent with decreased phosphorylation of its downstream substrate GSK-3α/β (at Ser21/Ser9) (i.e. activation) and increased GSK-3α and GSK-3β kinase activities, which occurred prior to NP1 induction. Expression of a dominant-negative inhibitor of Akt (Akt-kd) blocked phosphorylation of GSK-3α/β and subsequently enhanced NP1 induction. Whereas, overexpression of constitutively activated Akt (Akt-myr) or wild-type Akt (wtAkt) increased GSK-α/β phosphorylation and attenuated NP1 induction. Transfection of neurons with GSK-3α siRNA completely blocked NP1 induction and cell death. Similarly, overexpression of the GSK-3β inhibitor Frat1 or the kinase mutant GSK-3βKM, but not the wild-type GSK-3βWT, blocked NP1 induction and rescued neurons from death. Our findings clearly implicate both GSK-3α- and GSK-3β-dependent mechanism of NP1 induction and point to a novel mechanism in the regulation of hypoxic-ischemic neuronal death.

Genetic deletion of neuronal pentraxin 1 expression prevents brain injury in a neonatal mouse model of cerebral hypoxia-ischemia

Neonatal hypoxic-ischemic (HI) brain injury is a leading cause of mortality and morbidity in infants and children for which there is no promising therapy at present. Previously, we reported induction of neuronal pentraxin 1 (NP1), a novel neuronal protein of the long-pentraxin family, following HI injury in neonatal brain. Here, we report that genetic deletion of NP1 expression prevents HI injury in neonatal brain. Elevated expression of NP1 was observed in neurons, not in astrocytes, of the ipsilateral cortical layers (I-IV) and in the hippocampal CA1 and CA3 areas of WT brains following hypoxia-ischemia; brain areas that developed infarcts (at 24-48 h), showed significantly increased numbers of TUNEL-(+) cells and tissue loss (at 7 days). In contrast, NP1-KO mice showed no evidence of brain infarction and tissue loss after HI. The immunofluorescence staining of brain sections with mitochondrial protein COX IV and subcellular fractionation analysis showed increased accumulation of NP1 in mitochondria, pro-death protein Bax activation and NP1 co-localization with activated caspase-3 in WT, but not in the NP1-KO brains; corroborating NP1 interactions with the mitochondria-derived pro-death pathways. Disruption of NP1 translocation to mitochondria by NP1-siRNA in primary cortical cultures significantly reduced ischemic neuronal death. NP1 was immunoprecipitated with activated Bax [6A7] proteins; HI caused increased interactions of NP1 with Bax, thereby, facilitating Bax translocation to mitochondrial and neuronal death. To further delineate the specificity of NPs, we found that NP1 but not the NP2 induction is specifically involved in brain injury mechanisms and that knockdown of NP1 only results in neuroprotection. Furthermore, live in vivo T2-weighted magnetic resonance imaging (MRI) including fractional anisotropy (FA) mapping showed no sign of delayed brain injury or tissue loss in the NP1-KO mice as compared to the WT at different post-HI periods (4-24 weeks) examined; indicating a long-term neuroprotective efficacy of NP1 gene deletion. Collectively, our results demonstrate a novel mechanism of neuronal death and predict that inhibition of NP1 expression is a promising strategy to prevent hypoxic-ischemic injury in immature brain.

Osteoblast function and bone histomorphometry in a murine model of Rett syndrome

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder due to mutations affecting the neural transcription factor MeCP2. Approximately 50% of affected females have decreased bone mass. We studied osteoblast function using a murine model of RTT. Female heterozygote (HET) and male Mecp2-null mice were compared to wild type (WT) mice. Micro-CT of tibia from 5 week-old Mecp2-null mice showed significant alterations in trabecular bone including reductions in bone volume fraction (-29%), number (-19%), thickness (-9%) and connectivity density (-32%), and increases in trabecular separation (+28%) compared to WT. We also found significant reductions in cortical bone thickness (-18%) and in polar moment of inertia (-45%). In contrast, cortical and trabecular bone from 8 week-old WT and HET female mice were not significantly different. However, mineral apposition rate, mineralizing surface and bone formation rate/bone surface were each decreased in HET and Mecp2-null mice compared to WT mice. Histomorphometric analysis of femurs showed decreased numbers of osteoblasts but similar numbers of osteoclasts compared to WT, altered osteoblast morphology and decreased tissue synthesis of alkaline phosphatase in Mecp2-null and HET mice. Osteoblasts cultured from Mecp2-null mice, which unlike WT osteoblasts did not express MeCP2, had increased growth rates, but reductions in mRNA expression of type I collagen, Runx2 and Osterix compared to WT osteoblasts. These results indicate that MeCP2 deficiency leads to altered bone growth. Osteoblast dysfunction was more marked in Mecp2-null male than in HET female mice, suggesting that expression of MeCP2 plays a critical role in bone development.