Biochemical and immunocytochemical characterization of calsenilin in mouse brain

Mutations in the presenilin 1 and 2 genes cause the majority of early onset familial forms of Alzheimer's disease. Here we describe the biochemical and immunohistochemical characterization of calsenilin, a novel calcium binding protein that we have previously shown to interact with presenilins 1 and 2, in mouse brain. The co-immunoprecipitation of endogenous calsenilin and presenilin 1 demonstrates that these proteins are physiologic binding partners. Although calsenilin has been predicted to be a soluble protein, we have found that the majority of it is tightly associated with the cytoplasmic face of intracellular membranes and that it can only be dissociated using harsh treatments such as urea. In addition, we have demonstrated that calsenilin is a developmentally regulated protein that is mainly present in the brain, where it localizes to both the hippocampus and cerebellum. Calsenilin staining co-localized with the somatodendritic marker microtubule-associated protein-2 primarily in the granular cell layer of the cerebellum, indicating that calsenilin expression is primarily neuronal. In primary cultured neurons, calsenilin immunoreactivity was observed in cell bodies as well as in some neuronal processes. Co-localization experiments using specific axonal and dendritic markers indicate that these processes were mainly axonal in nature, although a smaller subset of dendrites also appears to contain calsenilin. In summary, we have established that calsenilin and presenilin 1 can interact at physiologic levels, and that calsenilin is a developmentally regulated protein that is expressed primarily in the cerebellum and hippocampus. Although calsenilin is a soluble protein, it is tightly associated with the membrane. Finally, the expression pattern of calsenilin, which is similar to that of the presenilin(s), suggests that the common locations of these two proteins provide an opportunity for physical interaction in vivo.     

The Notch pathway mediates expansion of a progenitor pool and neuronal differentiation in adult neural progenitor cells after stroke

Molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Using neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat subjected to focal cerebral ischemia, we investigated the Notch pathway in regulating proliferation and differentiation of adult neural progenitor cells after stroke. During proliferation of neural progenitor cells, ischemic neural progenitor cells exhibited substantially increased levels of Notch, Notch intracellular domain (NICD), and hairy enhancer of split (Hes) 1, which was associated with a significant increase of proliferating cells. Blockage of the Notch pathway by short interfering ribonucleic acid (siRNA) against Notch or a γ secretase inhibitor significantly reduced Notch, NICD and Hes1 expression and cell proliferation induced by stroke. During differentiation of neural progenitor cells, Notch and Hes1 expression was downregulated in ischemic neural progenitor cells, which was coincident with a significant increase of neuronal population. Inhibition of the Notch pathway with a γ secretase inhibitor further substantially increased neurons, but did not alter astrocyte population in ischemic neural progenitor cells. These data suggest that the Notch signaling pathway mediates adult SVZ neural progenitor cell proliferation and differentiation after stroke.     

Role for notch signaling in salivary acinar cell growth and differentiation

The Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. The Notch signaling is essential for Drosophila salivary gland development but its role in mammalian salivary gland remains unclear. The human salivary epithelial cell line, HSG, was studied to determine the role of Notch signaling in salivary epithelial cell differentiation. HSG expressed Notch 1 to 4, and the Notch ligands Jagged 1 and 2 and Delta 1. Treatment of HSG cells with inhibitors of γ‐secretase, which is required for Notch cleavage and activation, blocked vimentin and cystatin S expression, an indicator of HSG differentiation. HSG differentiation was also associated with Notch downstream signal Hes‐1 expression, and Hes‐1 expression was inhibited by γ‐secretase inhibitors. siRNA corresponding to Notch 1 to 4 was used to show that silencing of all four Notch receptors was required to inhibit HSG differentiation. Normal human submandibular gland expressed Notch 1 to 4, Jagged 1 and 2, and Delta 1, with nuclear localization indicating Notch signaling in vivo. Hes‐1 was also expressed in the human tissue, with staining predominantly in the ductal cells. In salivary tissue from rats undergoing and recovering from ductal obstruction, we found that Notch receptors and ligands were expressed in the nucleus of the regenerating epithelial cells. Taken together, these data suggest that Notch signaling is critical for normal salivary gland cell growth and differentiation. Developmental Dynamics 238:724–731, 2009. © 2009 Wiley‐Liss, Inc.     

Neuronal vulnerability of CLN3 deletion to calcium-induced cytotoxicity is mediated by calsenilin

Calsenilin/DREAM/KChIP3, a neuronal Ca(2+)-binding protein, has multifunctions in nucleus and cytosol. Here, we identified CLN3 as a calsenilin-binding partner whose mutation or deletion is observed in Batten disease. In vitro binding and immunoprecipitation assays show that calsenilin interacts with the C-terminal region of CLN3 and the increase of Ca(2+) concentration in vitro and in cells causes significant dissociation of calsenilin from CLN3. Ectopic expression of CLN3 or its deletion mutant containing only the C-terminus (153-438) and capable of binding to calsenilin suppresses thapsigargin or A23187-induced death of neuronal cells. In contrast, CLN3 deletion mutant containing the N-terminus (1-153) or (1-263), which is frequently found in Batten disease, induces the perturbation of Ca(2+) transient and fails to inhibit the cell death. In addition, the expression of calsenilin is increased in the brain tissues of CLN3 knock-out mice and SH-SY5Y/CLN3 knock-down cells. Down-regulation of CLN3 expression sensitizes SH-SY5Y cells to thapsigargin or A23187. However, additional decrease of calsenilin expression rescues the sensitivity of SH-SY5Y/CLN3 knock-down cells to Ca(2+)-mediated cell death. These results suggest that the vulnerability of CLN3 knock-out or CLN3 deletion (1-153)-expressing neuronal cells to Ca(2+)-induced cell death may be mediated by calsenilin.     

Upregulated miR-29b promotes neuronal cell death by inhibiting Bcl2L2 after ischemic brain injury

It is increasingly clear that microRNAs (miRNAs) play an important role in controlling cell survival. However, the functional significance of miRNAs in ischemic brain injury remains poorly understood. In the present study, we assayed the expression levels of miR-29b after ischemic brain injury, and defined the target genes and biological functions of miR-29b. We found that the miR-29b levels were significantly increased in rat brain after transient middle cerebral artery occlusion and neurons after oxygen–glucose deprivation. Moreover, ectopic expression of miR-29b promoted neuronal cell death, whereas its repression decreased cell death. Furthermore, we verified that miR-29b directly targeted and inhibited Bcl2L2 gene expression, and then increased neuronal cell death. Importantly, Bcl2L2 overexpression rescued neuronal cell death induced by miR-29b. These results suggest an important role of miR-29b in regulating neuronal cell death, thus offering a new target for the development of therapeutic agents against ischemic brain injury.     

Long‐term survival and regeneration of neuronal and vasculature cells inside the core region after ischemic stroke in adult mice

Focal cerebral ischemia results in an ischemic core surrounded by the peri‐infarct region (penumbra). Most research attention has been focused on penumbra while the pattern of cell fates inside the ischemic core is poorly defined. In the present investigation, we tested the hypothesis that, inside the ischemic core, some neuronal and vascular cells could survive the initial ischemic insult while regenerative niches might exist many days after stroke in the adult brain. Adult mice were subjected to focal cerebral ischemia induced by permanent occlusion of distal branches of the middle cerebral artery (MCA) plus transient ligations of bilateral common carotid artery (CCA). The ischemic insult uniformly reduced the local cerebral blood flow (LCBF) by 90%. Massive cell death occurred due to multiple mechanisms and a significant infarction was cultivated in the ischemic cortex 24 h later. Nevertheless, normal or even higher levels of brain‐derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) persistently remained in the core tissue, some NeuN‐positive and Glut‐1/College IV‐positive cells with intact ultrastructural features resided in the core 7‐14 days post stroke. BrdU‐positive but TUNEL‐negative neuronal and endothelial cells were detected in the core where extensive extracellular matrix infrastructure developed. Meanwhile, GFAP‐positive astrocytes accumulated in the penumbra and Iba‐1‐positive microglial/macrophages invaded the core several days after stroke. The long term survival of neuronal and vascular cells inside the ischemic core was also seen after a severe ischemic stroke induced by permanent embolic occlusion of the MCA. We demonstrate that a therapeutic intervention of pharmacological hypothermia could save neurons/endothelial cells inside the core. These data suggest that the ischemic core is an actively regulated brain region with residual and newly formed viable neuronal and vascular cells acutely and chronically after at least some types of ischemic strokes.     

Role of Cytotoxic Protease Granzyme‐b in Neuronal Degeneration During Human Stroke

Infiltration of leukocytes into post‐ischemic cerebrum is a well‐described phenomenon in stroke injury. Because CD‐8⁺ T‐lymphocytes secrete cytotoxic proteases, including granzyme‐b (Gra‐b) that exacerbates post‐ischemic brain damage, we investigated roles of Gra‐b in human stroke. To study the role of Gra‐b in stroke, ischemic and non‐ischemic tissues (from post‐mortem stroke patients) were analyzed using immunoblotting, co‐immunoprecipitation, terminal deoxy uridine nick end labeling (TUNEL) and Annexin–V immunostaining, and in vitro neuron survival assays. Activated CG‐SH cells and supernatants were used to model leukocyte‐dependent injury. Non‐ischemic brain tissues were used as non‐pathological controls. Non‐activated CG‐SH cells and supernatants were used as controls for in vitro experiments. Human stroke (ischemic) samples contained significantly higher levels of Gra‐b and interferon‐gamma inducible protein‐10 (IP‐10/CXCL10) than non‐ischemic controls. In stroke, poly (ADP‐ribose) polymerase‐1 and heat shock protein‐70 were cleaved to canonical proteolytic “signature” fragments by Gra‐b. Gra‐b was also found to bind to Bid and caspase‐3. Gra‐b also co‐localized with Annexin‐V⁺/TUNEL⁺ in degenerating neurons. Importantly, Gra‐b inhibition protected both normal and ischemia‐reperfused neurons against in vitro neurotoxicity mediated by activated CG‐SH cells and supernatants. These results suggest that increased leukocyte infiltration and elevated Gra‐b levels in the post‐stroke brain can induce contact‐dependent and independent post‐ischemic neuronal death to aggravate stroke injury.     

Targeting Notch Signaling and Autophagy Increases Cytotoxicity in Glioblastoma Neurospheres

Glioblastomas are highly aggressive tumors that contain treatment resistant stem‐like cells. Therapies targeting developmental pathways such as Notch eliminate many neoplastic glioma cells, including those with stem cell features, but their efficacy can be limited by various mechanisms. One potential avenue for chemotherapeutic resistance is the induction of autophagy, but little is known how it might modulate the response to Notch inhibitors. We used the γ‐secretase inhibitor MRK003 to block Notch pathway activity in glioblastoma neurospheres and assessed its effects on autophagy. A dramatic, several fold increase of LC3B‐II/LC3B‐I autophagy marker was noted on western blots, along with the emergence of punctate LC3B immunostaining in cultured cells. By combining the late stage autophagy inhibitor chloroquine (CQ) with MRK003, a significant induction in apoptosis and reduction in growth was noted as compared to Notch inhibition alone. A similar beneficial effect on inhibition of cloogenicity in soft agar was seen using the combination treatment. These results demonstrated that pharmacological Notch blockade can induce protective autophagy in glioma neurospheres, resulting in chemoresistance, which can be abrogated by combination treatment with autophagy inhibitors.     

The Recent Updates of Therapeutic Approaches Against Aβ for the Treatment of Alzheimer's Disease

One of the main neuropathological lesions observed in brain autopsy of Alzheimer's disease (AD) patients is the extracellular senile plaques mainly composed of amyloid‐beta (Aβ) peptide. Recently, treatment strategies have focused on modifying the formation, clearance, and accumulation of this potentially neurotoxic peptide. β‐ and γ‐secretase are responsible for the cleavage of amyloid precursor protein (APP) and the generation of Aβ peptide. Treatments targeting these two critical secretases may therefore reduce Aβ peptide levels and positive impact on AD. Vaccination is also an advanced approach against Aβ. This review focuses on recent advances of our understanding of this key peptide, with emphasis on Aβ peptide synthesis, accumulation and neurotoxicity, and current therapies including vaccination and two critical secretase inhibitors. MicroRNAs (miRNAs) are a class of conserved endogenous small noncoding RNAs, known to regulate the expression of complementary messenger RNAs, involved in AD development. We therefore address the relationship of miRNAs in the brain and Aβ generation, as a novel therapeutic approach to the treatment of AD while also providing new insights on the etiology of this neurological disorder. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Neuronal Rho GTPase Rac1 elimination confers neuroprotection in a mouse model of permanent ischemic stroke

The Rho GTPase Rac1 is a multifunctional protein involved in distinct pathways ranging from development to pathology. The aim of the present study was to unravel the contribution of neuronal Rac1 in regulating the response to brain injury induced by permanent focal cerebral ischemia (pMCAO). Our results show that pMCAO significantly increased total Rac1 levels in wild type mice, mainly through rising nuclear Rac1, while a reduction in Rac1 activation was observed. Such changes preceded cell death induced by excitotoxic stress. Pharmacological inhibition of Rac1 in primary neuronal cortical cells prevented the increase in oxidative stress induced after overactivation of glutamate receptors. However, this was not sufficient to prevent the associated neuronal cell death. In contrast, RNAi‐mediated knock down of Rac1 in primary cortical neurons prevented cell death elicited by glutamate excitotoxicity and decreased the activity of NADPH oxidase. To test whether in vivo down regulation of neuronal Rac1 was neuroprotective after pMCAO, we used tamoxifen‐inducible neuron‐specific conditional Rac1‐knockout mice. We observed a significant 50% decrease in brain infarct volume of knockout mice and a concomitant increase in HIF‐1α expression compared to littermate control mice, demonstrating that ablation of Rac1 in neurons is neuroprotective. Transmission electron microscopy performed in the ischemic brain showed that lysosomes in the infarct of Rac1‐ knockout mice were preserved at similar levels to those of non‐infarcted tissue, while littermate mice displayed a decrease in the number of lysosomes, further corroborating the notion that Rac1 ablation in neurons is neuroprotective. Our results demonstrate that Rac1 plays important roles in the ischemic pathological cascade and that modulation of its levels is of therapeutic interest.     

Neuronal susceptibility to beta‐amyloid toxicity and ischemic injury involves histone deacetylase‐2 regulation of endophilin‐B1

Histone deacetylases (HDACs) catalyze acetyl group removal from histone proteins, leading to altered chromatin structure and gene expression. HDAC2 is highly expressed in adult brain, and HDAC2 levels are elevated in Alzheimer's disease (AD) brain. We previously reported that neuron‐specific splice isoforms of Endophilin‐B1 (Endo‐B1) promote neuronal survival, but are reduced in human AD brain and mouse models of AD and stroke. Here, we demonstrate that HDAC2 suppresses Endo‐B1 expression. HDAC2 knockdown or knockout enhances expression of Endo‐B1. Conversely, HDAC2 overexpression decreases Endo‐B1 expression. We also demonstrate that neurons exposed to beta‐amyloid increase HDAC2 and reduce histone H3 acetylation while HDAC2 knockdown prevents Aβ induced loss of histone H3 acetylation, mitochondrial dysfunction, caspase‐3 activation, and neuronal death. The protective effect of HDAC2 knockdown was abrogated by Endo‐B1 shRNA and in Endo‐B1‐null neurons, suggesting that HDAC2‐induced neurotoxicity is mediated through suppression of Endo‐B1. HDAC2 overexpression also modulates neuronal expression of mitofusin2 (Mfn2) and mitochondrial fission factor (MFF), recapitulating the pattern of change observed in AD. HDAC2 knockout mice demonstrate reduced injury in the middle cerebral artery occlusion with reperfusion (MCAO/R) model of cerebral ischemia demonstrating enhanced neuronal survival, minimized loss of Endo‐B1, and normalized expression of Mfn2. These findings support the hypothesis that HDAC2 represses Endo‐B1, sensitizing neurons to mitochondrial dysfunction and cell death in stroke and AD.     

The voltage-gated proton channel Hv1 enhances brain damage from ischemic stroke

Phagocytic cell NADPH oxidase (NOX) generates reactive oxygen species (ROS) as part of innate immunity. Unfortunately, ischemia can also induce this pathway and inflict damage on native cells. The voltage-gated proton channel Hv1 enables NOX function by compensating cellular loss of electrons with protons. Accordingly, we investigated whether NOX-mediated brain damage in stroke can be inhibited by suppression of Hv1. We found that mouse and human brain microglia, but not neurons or astrocytes, expressed large Hv1-mediated currents. Hv1 was required for NOX-dependent ROS generation in brain microglia in situ and in vivo. Mice lacking Hv1 were protected from NOX-mediated neuronal death and brain damage 24 h after stroke. These results indicate that Hv1-dependent ROS production is responsible for a substantial fraction of brain damage at early time points after ischemic stroke and provide a rationale for Hv1 as a therapeutic target for the treatment of ischemic stroke.     

N-terminally cleaved Bcl-xL mediates ischemia-induced neuronal death

Transient global ischemia in rats induces delayed death of hippocampal CA1 neurons. Early events include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. However, whether these events have a causal role in ischemia-induced neuronal death is unclear. We found that the Bcl-2 and Bcl-x(L) inhibitor ABT-737, which enhances death of tumor cells, protected rats against neuronal death in a clinically relevant model of brain ischemia. Bcl-x(L) is prominently expressed in adult neurons and can be cleaved by caspases to generate a pro-death fragment, ΔN-Bcl-x(L). We found that ABT-737 administered before or after ischemia inhibited ΔN-Bcl-x(L)-induced mitochondrial channel activity and neuronal death. To establish a causal role for ΔN-Bcl-x(L), we generated knock-in mice expressing a caspase-resistant form of Bcl-x(L). The knock-in mice exhibited markedly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death. These findings suggest that truncated Bcl-x(L) could be a potentially important therapeutic target in ischemic brain injury.     

Tumor necrosis factor-like weak inducer of apoptosis and fibroblast growth factor-inducible 14 mediate cerebral ischemia-induced poly(ADP-ribose) polymerase-1 activation and neuronal death

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Fn14) are expressed in neurons. Here we demonstrate that TWEAK induces a dose-dependent increase in neuronal death and that this effect is independent of tumor necrosis factor alpha (TNF-α) and mediated by nuclear factor-kappa B (NF-κB) pathway activation. Incubation with TWEAK induces apoptotic cell death in wild-type (Wt) but not in Fn14 deficient (Fn14(-/-)) neurons. Intracerebral injection of TWEAK induces accumulation of poly(ADP-ribose) polymers (PAR) in Wt but not in Fn14(-/-) mice. Exposure to oxygen-glucose deprivation (OGD) conditions increases TWEAK and Fn14 mRNA expression in Wt neurons, and decreases cell survival in Wt but not in Fn14(-/-) or TWEAK deficient (TWEAK(-/-)) neurons. Experimental middle cerebral artery occlusion (MCAO) increases the expression of TWEAK and Fn14 mRNA and active caspase-3, and the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) with accumulation of PAR in the ischemic area in Wt but not Fn14(-/-) mice. Together, these results suggest a model where in response to hypoxia/ischemia the interaction between TWEAK and Fn14 in neurons induces PARP-1 activation with accumulation of PAR polymers and cell death via NF-κB pathway activation. This is a novel pathway for hypoxia/ischemia-induced TWEAK-mediated cell death and a potential therapeutic target for ischemic stroke.