The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons

To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.     

Activity‐dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live‐cell imaging

Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning‐related synaptic plasticity and potentiating N‐methyl‐D ‐aspartate (NMDA) receptor‐dependent signaling. These findings suggest that tPA may localize to, and undergo activity‐dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization‐induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense‐core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca⁺² concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse‐pinch‐linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA‐type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

The serine protease inhibitor neuroserpin regulates the growth and maturation of hippocampal neurons through a non-inhibitory mechanism

Neuroserpin is a brain-specific serine protease inhibitor that is expressed in the developing and adult nervous system. Its expression profile led to suggestions that it played roles in neuronal growth and connectivity. In this study, we provide direct evidence to support a role for neuroserpin in axon and dendritic growth. We report that axon growth is enhanced while axon and dendrite diameter are reduced following neuroserpin treatment of hippocampal neurons. More complex effects are seen on dendritic growth and branching with neuroserpin-stimulating dendritic growth and branching in young neurons but switching to an inhibitory response in older neurons. The protease inhibitory activity of neuroserpin is not required to activate changes in neuronal morphology and a proportion of responses are modulated by an antagonist to the LRP1 receptor. Collectively, these findings support a key role for neuroserpin as a regulator of neuronal development through a non-inhibitory mechanism and suggest a basis for neuroserpin's effects on complex emotional behaviours and recent link to schizophrenia.     

Sex differences in the nicotinic excitation of principal neurons within the developing hippocampal formation

The hippocampal formation (HF) plays an important role to facilitate higher order cognitive functions. Cholinergic activation of heteromeric nicotinic acetylcholine receptors (nAChRs) within the HF is critical for the normal development of principal neurons within this brain region. However, previous research investigating the expression and function of heteromeric nAChRs in principal neurons of the HF is limited to males or does not differentiate between the sexes. We used whole‐cell electrophysiology to show that principal neurons in the CA1 region of the female mouse HF are excited by heteromeric nAChRs throughout postnatal development, with the greatest response occurring during the first two weeks of postnatal life. Excitability responses to heteromeric nAChR stimulation were also found in principal neurons in the CA3, dentate gyrus, subiculum, and entorhinal cortex layer VI (ECVI) of young postnatal female HF. A direct comparison between male and female mice found that principal neurons in ECVI display greater heteromeric nicotinic passive and active excitability responses in females. This sex difference is likely influenced by the generally more excitable nature of ECVI neurons from female mice, which display a higher resting membrane potential, greater input resistance, and smaller afterhyperpolarization potential of medium duration (mAHP). These findings demonstrate that heteromeric nicotinic excitation of ECVI neurons differs between male and female mice during a period of major circuitry development within the HF, which may have mechanistic implications for known sex differences in the development and function of this cognitive brain region.     

Participation of protein kinase C alpha isoform and extracellular signal-regulated kinase in neurite outgrowth of GT1 hypothalamic neurons

In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform-selective inhibitors and transfection-based expression of enhanced green fluorescence protein (EGFP)-fused PKC isoforms. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingolTM(a PKCalpha-selective inhibitor), but not by rottlerinTM (a PKCdelta-selective inhibitor), indicating that PKCalpha may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and beta-tubulin. To define the differential localization of PKC isoforms, EGFP-tagged PKCalpha, PKCgamma, and PKCdelta were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKCalpha-EGFP to growth cones and cell-cell adhesion sites, PKCgamma-EGFP to the nucleus, and PKCdelta-EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKCalpha (PKCalpha-Cat-EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKCalpha enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKCalpha-Cat-EGFP alone increased the neurite-outgrowth and doxycycline treatment further augmented the number of neurite-containing cells. We also examined the involvement of the extracellular signal-regulated kinase (ERK) MAP kinase in TPA-induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKCalpha with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase-1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKCalpha isoform-specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway.     

Role of moving growth cone–like “wave” structures in the outgrowth of cultured hippocampal axons and dendrites

Hippocampal neurons exhibit periodically recurring growth cone–like structures, referred to as “waves,” that emerge at the base of neurites and travel distally to the tip. As a wave nears the tip, the neurite undergoes retraction, and when it reaches the tip, the neurite undergoes a burst of growth. At 1 day in culture, during early axon outgrowth, axons undergo an average 7.5‐μm retraction immediately preceding wave arrival at the tip followed by 12‐μm growth immediately after arrival (an average net growth of 4.5 μm). In branched axons, waves often selectively travel down one branch or the other. Growth selectively occurs in the branch chosen by the wave. In dendrites, which grow much slower on average, wave‐associated retractions are much greater, resulting in less net growth. In the presence of Brefeldin A, which disrupts membrane traffic through the Golgi apparatus and leads to retraction of the axon, axonal waves continue to be associated with both growth spurts and retractions. The magnitude of the growth spurts is not significantly different from untreated axons, but wave‐associated retractions are significantly increased. The close association between waves and cyclical elongation suggests that waves may act to bring about this pattern of growth. Our results also show that modulation of regularly occurring retraction phases plays a prominent role in determining average outgrowth rates. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 97–106, 1999     

An intracellular domain with a novel sequence regulates cell surface expression and synaptic clustering of leucine‐rich repeat transmembrane proteins in hippocampal neurons

Leucine‐rich repeat transmembrane proteins (LRRTMs) are single‐spanning transmembrane proteins that belong to the family of synaptically localized adhesion molecules that play various roles in the formation, maturation, and function of synapses. LRRTMs are highly localized in the post‐synaptic density; however, the mechanisms and significance of LRRTM synaptic clustering remain unclear. Here, we focus on the intracellular domain of LRRTMs and investigate its role in cell surface expression and synaptic clustering. The deletion of 55–56 residues in the cytoplasmic tail caused significantly reduced synaptic clustering of LRRTM1–4 in rat hippocampal neurons, whereas it simultaneously resulted in augmented LRRTM1–2 cell surface expression. A series of deletions and further single amino acid substitutions in the intracellular domain of LRRTM2 demonstrated that a previously uncharacterized sequence at the region of ‐16 to ‐13 from the C‐terminus was responsible for efficient synaptic clustering and proper cell surface trafficking of LRRTMs. Furthermore, the clustering‐deficient LRRTM2 mutant lost the ability to promote the accumulation of post‐synaptic density protein‐95 (PSD‐95). These results suggest that trafficking to the cell surface and synaptic clustering of LRRTMs are regulated by a specific mechanism through this novel sequence in the intracellular domain that underlies post‐synaptic molecular assembly and maturation.

     

Anti-Thy-1 antibody-induced neurite outgrowth in cultured dorsal root ganglionic neurons is mediated by the c-Src-MEK signaling pathway

Our previous study has shown that anti-Thy-1 antibody promotes neurite outgrowth of cultured dorsal root ganglion (DRG) neurons in a protein kinase A (PKA)-dependent manner. The present study provided another intracellular signaling pathway for the neurotrophic effect of anti-Thy-1 antibody. In DMSO-treated control cells, Thy-1 was enriched in microdomain-like structures on cell membranes by immunofluorescence observation. Treatment of DRG neurons with anti-Thy-1 antibody not only stimulated neurite outgrowth, but also increased the branching complexity of the neurites in both small and large neurons. We have previously shown that anti-Thy-1 antibody causes a time-dependent activation of mitogen-activated protein kinase (MEK) and of cyclic AMP response-element binding protein (CREB). Here, anti-Thy-1 antibody elicited a transient activation of c-Src kinase, and the activation of c-Src kinase appeared occurring upstream of the activation of MEK and CREB, since pretreatment with the Src kinase inhibitor, PP2, effectively abolished the anti-Thy-1 antibody-induced neurite outgrowth and the phosphorylation of MEK and CREB. CREB phosphorylation might result in upregulation of certain neurite outgrowth-related proteins. We therefore conclude that anti-Thy-1 antibody activates the c-Src kinase-MEK-CREB cascade and overcomes the inhibitory effect of Thy-1 on neurite outgrowth in DRG neurons.     

Ribosomal protein L10 interacts with the SH3 domain and regulates GDNF-induced neurite growth in SH-SY-5y cells

The 24.5 kDa ribosomal protein L10 (RP-L10), which was encoded by QM gene, was known to interact with the SH3 domain of Yes kinase. Herein, we demonstrate that RP-L10 interacts with the SH3 domain of Src and activates the binding of the Nck1 adaptor protein with skeletal proteins such as the Wiskott-Aldrich Syndrome Protein (WASP) and WASP interacting protein (WIP) in neuroblastoma cell line, SH-SY-5y. The RP-L10 was associated with the SH3 domains of Src and Yes. It is shown that two different regions of RP-L10 are associated with the Src-SH3. The effect of ectopic RP-L10 expression on neuronal cell scaffolding was explored in cells transiently transfected with QM. SH-SY-5y human neuroblastoma cells transfected with QM were considerably more susceptible to neurite outgrowth induced by glial cell line-derived neurotrophic factor (GDNF). However, RP-L10 did not directly interact with actin assembly. Taken together, these results suggest that the RP-L10 may positively regulate the GDNF/Ret-mediated signaling of neurite outgrowth in the neuroblastoma cell line, SH-SY-5y.     

Nicotine activates the extracellular signal-regulated kinase 1/2 via the alpha7 nicotinic acetylcholine receptor and protein kinase A, in SH-SY5Y cells and hippocampal neurones

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate many cellular mechanisms, such as cell survival and memory processing, which are also influenced by the serine/threonine protein kinases ERK1/2. In SH-SY5Y cells and hippocampal neurones, nicotine (100 microM) increased the activity of ERK1/2. This effect was Ca2+ dependent, and prevented by the alpha7 nAChR antagonist alpha-bungarotoxin (alpha-Bgt) and an inhibitor (PD98059) of the upstream kinase MEK. To determine the intervening steps linking Ca2+ entry to MEK-ERK1/2 activation, inhibitors of Ca2+-dependent kinases were deployed. In SH-SY5Y cells, selective blockers for PKC (Ro 31-8220), CaM kinase II (KN-62) or PI3 kinase (LY 294002) failed to inhibit the nicotine-evoked increase in ERK1/2 activity. In contrast, two structurally different inhibitors of PKA (KT 5720 and H-89) completely prevented the nicotine-dependent increase in ERK1/2 activity. Inhibition of the nicotine-evoked increase in ERK1/2 activity by H-89 was also observed in hippocampal cultures. Down stream of PKA, the activity of B-Raf was significantly decreased by nicotine in SH-SY5Y cells, as determined by direct measurement of MEK1 phosphorylation or in vitro kinase assays, whereas the modulation of MEK1 phosphorylation by Raf-1 tended to increase. Thus, this study provides evidence for a novel signalling route coupling the stimulation of alpha7 nAChR to the activation of ERK1/2, in a Ca2+ and PKA dependent manner.     

Role of PKA in the anti-Thy-1 antibody-induced neurite outgrowth of dorsal root ganglionic neurons

Thy-1 is highly expressed in the mammalian nervous system. Our previous study showed that addition of anti-Thy-1 antibody to cultured dorsal root ganglionic (DRG) neurons promotes neurite outgrowth. In this study, we identified a novel signaling pathway mediating this event. Treatment with function-blocking anti-Thy-1 antibodies enhanced neurite outgrowth of DRG neurons in terms of total neurite length, longest neurite length, and total neurite branching points. To elucidate the possible signal transduction pathway involved, activation of kinases was evaluated by Western blotting. Transient phosphorylation of protein kinase A (PKA) and mitogen-activated kinase kinase (MEK) was induced after 15 min of anti-Thy-1 antibody treatment. Pretreatment with a PKA inhibitor (PKI) or an MEK inhibitor, PD98059, significantly decreased the neurite outgrowth response triggered by anti-Thy-1 antibody, indicating the involvement of both kinases. In addition, anti-Thy-1 antibody treatment also induced transient phosphorylation of cyclic AMP-response element-binding protein (CREB) and this effect was also blocked by a PKI or PD98059. Furthermore, the fact that PKI abolished anti-Thy-1 antibody-induced MEK phosphorylation showed that PKA acts upstream of the MEK-CREB cascade. In summary, the PKA-MEK-CREB pathway is a new pathway involved in the neurite outgrowth-promoting effect of anti-Thy-1 antibody.     

The steroid-induced microRNA let-7 regulates developmental growth by targeting cdc7 in the Drosophila fat body

In insects, 20-hydroxyecdysone (20E) limits systemic growth by triggering developmental transitions. Previous studies have shown that 20E-induced let-7 exhibits crosstalk with the cell cycle. Here, we examined the underlying molecular mechanisms and physiological functions of 20E-induced let-7 in the fat body, an organ for energy storage and nutrient mobilization which plays a critical role in the larval growth. First, the overexpression of let-7 decreased the body size and led to the reduction of both nucleolus and cell sizes in the larval fat body. In contrast, the overexpression of let-7-Sponge increased the nucleolus and cell sizes. Moreover, we found that cdc7, encoding a conserved protein kinase that controls the endocycle, is a target of let-7. Notably, the mutation of cdc7 in the fat body resulted in growth defects. Overall, our findings revealed a novel role of let-7 in the control of endoreduplication-related growth during larval-prepupal transition in Drosophila.     

Reduction in glutamate uptake is associated with extrasynaptic NMDA and metabotropic glutamate receptor activation at the hippocampal CA1 synapse of aged rats

This study aims to determine whether the regulation of extracellular glutamate is altered during aging and its possible consequences on synaptic transmission and plasticity. A decrease in the expression of the glial glutamate transporters GLAST and GLT‐1 and reduced glutamate uptake occur in the aged (24–27 months) Sprague–Dawley rat hippocampus. Glutamatergic excitatory postsynaptic potentials recorded extracellularly in ex vivo hippocampal slices from adult (3–5 months) and aged rats are depressed by DL‐TBOA, an inhibitor of glutamate transporter activity, in an N‐Methyl‐d‐ Aspartate (NMDA)‐receptor‐dependent manner. In aged but not in young rats, part of the depressing effect of DL‐TBOA also involves metabotropic glutamate receptor (mGluRs) activation as it is significantly reduced by the specific mGluR antagonist d‐methyl‐4‐carboxy‐phenylglycine (MCPG). The paired‐pulse facilitation ratio, a functional index of glutamate release, is reduced by MCPG in aged slices to a level comparable to that in young rats both under control conditions and after being enhanced by DL‐TBOA. These results suggest that the age‐associated glutamate uptake deficiency favors presynaptic mGluR activation that lowers glutamate release. In parallel, 2 Hz‐induced long‐term depression is significantly decreased in aged animals and is fully restored by MCPG. All these data indicate a facilitated activation of extrasynaptic NMDAR and mGluRs in aged rats, possibly because of an altered distribution of glutamate in the extrasynaptic space. This in turn affects synaptic transmission and plasticity within the aged hippocampal CA1 network.     

DCLK1 phosphorylates the microtubule‐associated protein MAP7D1 to promote axon elongation in cortical neurons

Doublecortin‐like kinase 1 (DCLK1) is a member of the neuronal microtubule‐associated doublecortin (DCX) family and functions in multiple stages of neural development including radial migration and axon growth of cortical neurons. DCLK1 is suggested to play the roles in part through its protein kinase activity, yet the kinase substrates of DCLK1 remain largely unknown. Here we have identified MAP7D1 (microtubule‐associated protein 7 domain containing 1) as a novel substrate of DCLK1 by using proteomic analysis. MAP7D1 is expressed in developing cortical neurons, and knockdown of MAP7D1 in layer 2/3 cortical neurons results in a significant impairment of callosal axon elongation, but not of radial migration, in corticogenesis. We have further defined the serine 315 (Ser 315) of MAP7D1 as a DCLK1‐induced phosphorylation site and shown that overexpression of a phosphomimetic MAP7D1 mutant in which Ser 315 is substituted with glutamic acid (MAP7D1 S315E), but not wild‐type MAP7D1, fully rescues the axon elongation defects in Dclk1 knockdown neurons. These data demonstrate that DCLK1 phosphorylates MAP7D1 on Ser 315 to facilitate axon elongation of cortical neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

MicroRNA-424 regulates epithelial-mesenchymal transition of endometrial carcinoma by directly targeting insulin-like growth factor 1 receptor

Although numerous miRNAs are reported to contribute to the carcinogenesis of malignant tumor, the specific role of miR-424 in endometrial carcinoma is seldom reported. To explore the effect of miR-424 on epithelial-mesenchymal transition and its underlying mechanism, we detected miR-424 expression in endometrial carcinoma tissue and cells. We found that miR-424 was significantly downregulated in endometrial carcinoma tissues and cells, especially in HEC-1B cells. To perform the functional analysis, we transfected HEC-1B with miR-424-mi, miR-424-inh, mi-control, and inh-control, respectively. We found that overexpression of miR-424 significantly decreases cell proliferation and migration, accompanied with the increased E-cadherin/Vimentin expression and the transition of mesenchymal to epithelial cell phenotype. We identified that insulin-like growth factor-1 receptor (IGF-1R) was a potential target of miR-424 by computational analysis followed by luciferase reporter assays. Of note, we found that the downregulation of miR-424 in HEC-1B cells enhanced endogenous IGF-1R expression. Further mechanistic analysis revealed that forced expression of IGF-1R in miR-424-mim transfected cells remedied the weakened migration resulting from overexpression of IGF-1R. Taken together, the results of the current study demonstrated that miR-424 was a tumor suppressor for endometrial carcinoma and a favorable factor against tumor progression through targeting IGF-1R, thus providing a target for the treatment of endometrial carcinoma.     

Involvement of α2‐antiplasmin in dendritic growth of hippocampal neurons

The α2‐Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule‐associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP^?/? mice than in the neurons from α2AP^+/+ mice. Exogenous treatment with α2AP enhanced the microtubule‐associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP‐induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen^?/? mice. The activation of p38 MAPK was involved in the α2AP‐induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.     

Overexpression of alpha7 nicotinic acetylcholine receptor prevents G1-arrest and DNA fragmentation in PC12 cells after hypoxia

We investigated the neuroprotective function of alpha7 nicotinic acetylcholine receptor (alpha7nAChR) after transient hypoxia (12 h) and reoxygenation (0-72 h), comparing rat pheochromocytoma (PC12) cells overexpressing FLAG-tagged alpha7nAChR (alpha7pCMV cells) and control PC12 cells (non-transfected or transfected with vector only) in medium with and without nicotine. Plasma membrane degradation in the early phase after hypoxia was inhibited in PC12 cells with nicotine, and more profoundly in alpha7pCMV cells with nicotine. Inhibition of DNA fragmentation in the late phase after hypoxia was most remarkable in alpha7pCMV cells with nicotine, but, surprisingly, it was more remarkable in alpha7pCMV cells without nicotine than in PC12 cells with nicotine. G1-arrest of the cell cycle, observed in control PC12 cells at 12 h after hypoxia, preceding DNA fragmentation, was not evident in alpha7pCMV cells, with or without nicotine. Furthermore, in alpha7pCMV cells with and without nicotine, the basal expression levels of total Akt were approximately 1.5-fold higher, and the up-regulation of Akt phosphorylated at Ser473 after hypoxia was strikingly enhanced, compared with control PC12 cells. These findings suggest that alpha7nAChR functions constitutively in PC12 cells, that its overexpression raises tolerance against G1-arrest and DNA fragmentation after hypoxia, and that it can be considered a candidate target for treatment against hypoxia-induced acute membrane degradation and delayed DNA fragmentation in neurons.