Pharmacological Inhibition of the Asparaginyl Endopeptidase (AEP) in an Alzheimer's Disease Model Improves the Survival and Efficacy of Transplanted Neural Stem Cells.

Stem-cell-based therapy is very promising for Alzheimer's disease (AD), yet has not become a reality. A critical challenge is the transplantation microenvironment, which impacts the therapeutic effect of stem cells. In AD brains, amyloid-beta (Aß) peptides and inflammatory cytokines continuously poison the tissue microenvironment, leading to low survival of grafted cells and restricted efficacy. It is necessary to create a growth-supporting microenvironment for transplanted cells. Recent advances in AD studies suggest that the asparaginyl endopeptidase (AEP) is a potential intervention target for modifying pathological changes. We here chose APP/PS1 mice as an AD model and employed pharmacological inhibition of the AEP for one month to improve the brain microenvironment. Thereafter, we transplanted neural stem cells (NSCs) into the hippocampus and maintained therapy for one more month. We found that inhibition of AEPs resulted in a significant decrease of Aß, TNF-α, IL-6 and IL-1ß in their brains. In AD mice receiving NSC transplantation alone, the survival of NSCs was at a low level, while in combination with AEP inhibition pre-treatment the survival rate of engrafted cells was doubled. Within the 2-month treatment period, implantation of NSCs plus pre-inhibition of the AEP significantly enhanced neural plasticity of the hippocampus and rescued cognitive impairment. Neither NSC transplantation alone nor AEP inhibition alone achieved significant efficacy. In conclusion, pharmacological inhibition of the AEP ameliorated brain microenvironment of AD mice, and thus improved the survival and therapeutic efficacy of transplanted stem cells.

δ-Secretase-cleaved Tau stimulates Aß production via upregulating STAT1-BACE1 signaling in Alzheimer's disease.

δ-Secretase, an age-dependent asparagine protease, cleaves both amyloid precursor protein (APP) and Tau and is required for amyloid plaque and neurofibrillary tangle pathologies in Alzheimer's disease (AD). However, whether δ-secretase activation is sufficient to trigger AD pathogenesis remains unknown. Here we show that the fragments of δ-secretase-cleavage, APP (586-695) and Tau(1-368), additively drive AD pathogenesis and cognitive dysfunctions. Tau(1-368) strongly augments BACE1 expression and Aß generation in the presence of APP. The Tau(1-368) fragment is more robust than full-length Tau in binding active STAT1, a BACE1 transcription factor, and promotes its nuclear translocation, upregulating BACE1 and Aß production. Notably, Aß-activated SGK1 or JAK2 kinase phosphorylates STAT1 and induces its association with Tau(1-368). Inhibition of these kinases diminishes stimulatory effect of Tau(1-368). Knockout of STAT1 abolishes AD pathologies induced by δ-secretase-generated APP and Tau fragments. Thus, we show that Tau may not only be a downstream effector of Aß in the amyloid hypothesis, but also act as a driving force for Aß, when cleaved by δ-secretase.

The Asparaginyl Endopeptidase Legumain: An Emerging Therapeutic Target and Potential Biomarker for Alzheimer's Disease.

Alzheimer's disease (AD) is incurable dementia closely associated with aging. Most cases of AD are sporadic, and very few are inherited; the pathogenesis of sporadic AD is complex and remains to be elucidated. The asparaginyl endopeptidase (AEP) or legumain is the only recognized cysteine protease that specifically hydrolyzes peptide bonds after asparagine residues in mammals. The expression level of AEPs in healthy brains is far lower than that of peripheral organs. Recently, growing evidence has indicated that aging may upregulate and overactivate brain AEPs. The overactivation of AEPs drives the onset of AD through cleaving tau and amyloid precursor proteins (APP), and SET, an inhibitor of protein phosphatase 2A (PP2A). The AEP-mediated cleavage of these peptides enhances amyloidosis, promotes tau hyperphosphorylation, and ultimately induces neurodegeneration and cognitive impairment. Upregulated AEPs and related deleterious reactions constitute upstream events of amyloid/tau toxicity in the brain, and represent early pathological changes in AD. Thus, upregulated AEPs are an emerging drug target for disease modification and a potential biomarker for predicting preclinical AD. However, the presence of the blood-brain barrier greatly hinders establishing body-fluid-based methods to measure brain AEPs. Research on AEP-activity-based imaging probes and our recent work suggest that the live brain imaging of AEPs could be used to evaluate its predictive efficacy as an AD biomarker. To advance translational research in this area, AEP imaging probes applicable to human brain and AEP inhibitors with good druggability are urgently needed.

Suppressing Effect of Na+/Ca2+ Exchanger (NCX) Inhibitors on the Growth of Melanoma Cells.

The role of calcium ion (Ca2+) signaling in tumorigenicity has received increasing attention in melanoma research. Previous Ca2+ signaling studies focused on Ca2+ entry routes, but rarely explored the role of Ca2+ extrusion. Functioning of the Na+/Ca2+ exchanger (NCX) on the plasma membrane is the major way of Ca2+ extrusion, but very few associations between NCX and melanoma have been reported. Here, we explored whether pharmacological modulation of the NCX could suppress melanoma and promise new therapeutic strategies. Methods included cell viability assay, Ca2+ imaging, immunoblotting, and cell death analysis. The NCX inhibitors SN-6 and YM-244769 were used to selectively block reverse operation of the NCX. Bepridil, KB-R7943, and CB-DMB blocked either reverse or forward NCX operation. We found that blocking the reverse NCX with SN-6 or YM-244769 (5-100 µM) did not affect melanoma cells or increase cytosolic Ca2+. Bepridil, KB-R7943, and CB-DMB all significantly suppressed melanoma cells with IC50 values of 3-20 µM. Bepridil and KB-R7943 elevated intracellular Ca2+ level of melanoma. Bepridil-induced melanoma cell death came from cell cycle arrest and enhanced apoptosis, which were all attenuated by the Ca2+ chelator BAPTA-AM. As compared with melanoma, normal melanocytes had lower NCX1 expression and were less sensitive to the cytotoxicity of bepridil. In conclusion, blockade of the forward but not the reverse NCX leads to Ca2+-related cell death in melanoma and the NCX is a potential drug target for cancer therapy.

Imaging asparaginyl endopeptidase (AEP) in the live brain as a biomarker for Alzheimer's disease.

BACKGROUND: Discovery of early-stage biomarkers is a long-sought goal of Alzheimer's disease (AD) diagnosis. Age is the greatest risk factor for most AD and accumulating evidence suggests that age-dependent elevation of asparaginyl endopeptidase (AEP) in the brain may represent a new biological marker for predicting AD. However, this speculation remains to be explored with an appropriate assay method because mammalian AEP exists in many organs and the level of AEP in body fluid isn't proportional to its concentration in brain parenchyma. To this end, we here modified gold nanoparticle (AuNPs) into an AEP-responsive imaging probe and choose transgenic APPswe/PS1dE9 (APP/PS1) mice as an animal model of AD. Our aim is to determine whether imaging of brain AEP can be used to predict AD pathology. RESULTS: This AEP-responsive imaging probe AuNPs-Cy5.5-A&C consisted of two particles, AuNPs-Cy5.5-AK and AuNPs-Cy5.5-CABT, which were respectively modified with Ala-Ala-Asn-Cys-Lys (AK) and 2-cyano-6-aminobenzothiazole (CABT). We showed that AuNPs-Cy5.5-A&C could be selectively activated by AEP to aggregate and emit strong fluorescence. Moreover, AuNPs-Cy5.5-A&C displayed a general applicability in various cell lines and its florescence intensity correlated well with AEP activity in these cells. In the brain of APP/PS1 transgenic mice , AEP activity was increased at an early disease stage of AD that precedes formation of senile plaques and cognitive impairment. Pharmacological inhibition of AEP with δ-secretase inhibitor 11 (10 mg kg-1, p.o.) reduced production of ß-amyloid (Aß) and ameliorated memory loss. Therefore, elevation of AEP is an early sign of AD onset. Finally, we showed that live animal imaging with this AEP-responsive probe could monitor the up-regulated AEP in the brain of APP/PS1 mice. CONCLUSIONS: The current work provided a proof of concept that assessment of brain AEP activity by in vivo imaging assay is a potential biomarker for early diagnosis of AD.

Optochemogenetic Stimulation of Transplanted iPS-NPCs Enhances Neuronal Repair and Functional Recovery after Ischemic Stroke.

Cell transplantation therapy provides a regenerative strategy for neural repair. We tested the hypothesis that selective excitation of transplanted induced pluripotent stem cell-derived neural progenitor cells (iPS-NPCs) could recapitulate an activity-enriched microenvironment that confers regenerative benefits for the treatment of stroke. Mouse iPS-NPCs were transduced with a novel optochemogenetics fusion protein, luminopsin 3 (LMO3), which consisted of a bioluminescent luciferase, Gaussia luciferase, and an opsin, Volvox Channelrhodopsin 1. These LMO3-iPS-NPCs can be activated by either photostimulation using light or by the luciferase substrate coelenterazine (CTZ). In vitro stimulations of LMO3-iPS-NPCs increased expression of synapsin-1, postsynaptic density 95, brain derived neurotrophic factor (BDNF), and stromal cell-derived factor 1 and promoted neurite outgrowth. After transplantation into the ischemic cortex of mice, LMO3-iPS-NPCs differentiated into mature neurons. Synapse formation between implanted and host neurons was identified using immunogold electron microscopy and patch-clamp recordings. Stimulation of transplanted cells with daily intranasal administration of CTZ enhanced axonal myelination, synaptic transmission, improved thalamocortical connectivity, and functional recovery. Patch-clamp and multielectrode array recordings in brain slices showed that CTZ or light stimulation facilitated synaptic transmission and induced neuroplasticity mimicking the LTP of EPSPs. Stroke mice received the combined LMO3-iPS-NPC/CTZ treatment, but not cell or CTZ alone, showed enhanced neural network connections in the peri-infarct region, promoted optimal functional recoveries after stroke in male and female, young and aged mice. Thus, excitation of transplanted cells via the noninvasive optochemogenetics treatment provides a novel integrative cell therapy with comprehensive regenerative benefits after stroke.SIGNIFICANCE STATEMENT Neural network reconnection is critical for repairing damaged brain. Strategies that promote this repair are expected to improve functional outcomes. This study pioneers the generation and application of an optochemogenetics approach in stem cell transplantation therapy after stroke for optimal neural repair and functional recovery. Using induced pluripotent stem cell-derived neural progenitor cells (iPS-NPCs) expressing the novel optochemogenetic probe luminopsin (LMO3), and intranasally delivered luciferase substrate coelenterazine, we show enhanced regenerative properties of LMO3-iPS-NPCs in vitro and after transplantation into the ischemic brain of different genders and ages. The noninvasive repeated coelenterazine stimulation of transplanted cells is feasible for clinical applications. The synergetic effects of the combinatorial cell therapy may have significant impacts on regenerative approach for treatments of CNS injuries.

Psychological stress responses to COVID-19 and adaptive strategies in China.

The novel coronavirus disease 2019 (COVID-19) developed into a pandemic on March 11. COVID-19 not only brought life crisis, but also incurred psychological stress: tension, anxiety, fear and despair among affected populations. How to help people overcome traumatic stress reactions and get out of psychological crisis has become a public concern that needs to be resolved in time. This article reported the psychological responses caused by the COVID-19 epidemic in China based on relevant experience and studies. The anti-epidemic measures of self-quarantine and social-distancing were deployed to contain the spread of COVID-19, but inevitably caused a certain extent of side effect: frustration and anxiety in the general public. Especially, the front-line medical rescue staff and COVID-19 patients were more susceptible to developing psychological disorders. Correspondingly, adaptive strategies and public health policies were rapidly implemented in China to deal with outbreak-caused mental stress. The psychological impact of COVID-19 and coping strategies adopted in China provided warning and reference for countries that are and going to be affected by this natural disaster.

Intrinsic strain-induced segregation in multiply twinned Cu-Pt icosahedra.

We present an atomistic simulation study on the compositional arrangements throughout Cu-Pt icosahedra, with a specific focus on the effects of inherent strain on general segregation trends. The coexistence of radial and site-selective segregation patterns is found in bimetallic nanoparticles for a broad range of sizes and compositions, consistent with prior analytical and atomistic models. Through a thorough comparison between the composition patterns and strain-related patterns, it is suggested that the presence of gradient and site-selective segregation is natural to largely relieve the inherent strain by preferential segregation of big atoms at tensile sites and vice versa, as previously hypothesized in the literature. Analogous to the case of single crystal particles, Cu-rich surface and damped oscillations can also be found in the outer shells of icosahedra, which are dominated by the lowering of both the surface energy and the chemical energy. The thermodynamic stability of segregated icosahedra is similar to segregated cuboctahedra but higher than disordered bulk alloys, validating prior thinking that element segregation driven by strain relief can extend the stability range of multiply-twinned nanoparticles. Our work sheds new light on understanding strain-induced segregation in multiply-twinned nanosystems that have elements with large lattice mismatch and strong alloying ability.

Optogenetic stimulation of glutamatergic neuronal activity in the striatum enhances neurogenesis in the subventricular zone of normal and stroke mice.

Neurogenesis in the subventricular zone (SVZ) of the adult brain may contribute to tissue repair after brain injuries. Whether SVZ neurogenesis can be upregulated by specific neuronal activity in vivo and promote functional recovery after stroke is largely unknown. Using the spatial and cell type specific optogenetic technique combined with multiple approaches of in vitro, ex vivo and in vivo examinations, we tested the hypothesis that glutamatergic activation in the striatum could upregulate SVZ neurogenesis in the normal and ischemic brain. In transgenic mice expressing the light-gated channelrhodopsin-2 (ChR2) channel in glutamatergic neurons, optogenetic stimulation of the glutamatergic activity in the striatum triggered glutamate release into SVZ region, evoked membrane currents, Ca2+ influx and increased proliferation of SVZ neuroblasts, mediated by AMPA receptor activation. In ChR2 transgenic mice subjected to focal ischemic stroke, optogenetic stimuli to the striatum started 5days after stroke for 8days not only promoted cell proliferation but also the migration of SVZ neuroblasts into the peri-infarct cortex with increased neuronal differentiation and improved long-term functional recovery. These data provide the first morphological and functional evidence showing a unique striatum-SVZ neuronal regulation via a semi-phasic synaptic mechanism that can boost neurogenic cascades and stroke recovery. The benefits from stimulating endogenous glutamatergic activity suggest a novel regenerative strategy after ischemic stroke and other brain injuries.

Disrupted Ionic Homeostasis in Ischemic Stroke and New Therapeutic Targets.

BACKGROUND: Stroke is a leading cause of long-term disability. All neuroprotectants targeting excitotoxicity have failed to become stroke medications. In order to explore and identify new therapeutic targets for stroke, we here reviewed present studies of ionic transporters and channels that are involved in ischemic brain damage. METHOD: We surveyed recent literature from animal experiments and clinical reports in the databases of PubMed and Elsevier ScienceDirect to analyze ionic mechanisms underlying ischemic cell damage and suggest promising ideas for stroke therapy. RESULTS: Dysfunction of ionic transporters and disrupted ionic homeostasis are most early changes that underlie ischemic brain injury, thus receiving sustained attention in translational stroke research. The Na+/K+-ATPase, Na+/Ca2+ Exchanger, ionotropic glutamate receptor, acid-sensing ion channels (ASICs), sulfonylurea receptor isoform 1 (SUR1)-regulated NCCa-ATP channels, and transient receptor potential (TRP) channels are critically involved in ischemia-induced cellular degenerating processes such as cytotoxic edema, excitotoxicity, necrosis, apoptosis, and autophagic cell death. Some ionic transporters/channels also act as signalosomes to regulate cell death signaling. For acute stroke treatment, glutamate-mediated excitotoxicity must be interfered within 2 hours after stroke. The SUR1-regulated NCCa-ATP channels, Na+/K+-ATPase, ASICs, and TRP channels have a much longer therapeutic window, providing new therapeutic targets for developing feasible pharmacological treatments toward acute ischemic stroke. CONCLUSION: The next generation of stroke therapy can apply a polypharmacology strategy for which drugs are designed to target multiple ion transporters/channels or their interaction with neurotoxic signaling pathways. But a successful translation of neuroprotectants relies on in-depth analyses of cell death mechanisms and suitable animal models resembling human stroke.

iPSC Transplantation increases regeneration and functional recovery after ischemic stroke in neonatal rats.

Limited treatments are available for perinatal/neonatal stroke. Induced pluripotent stem cells (iPSCs) hold therapeutic promise for stroke treatment, but the benefits of iPSC transplantation in neonates are relatively unknown. We hypothesized that transplanted iPSC-derived neural progenitor cells (iPSC-NPCs) would increase regeneration after stroke. Mouse pluripotent iPSCs were differentiated into neural progenitors using a retinoic acid protocol. Differentiated neural cells were characterized by using multiple criteria and assessments. Ischemic stroke was induced in postnatal day 7 (P7) rats by occluding the right middle cerebral artery and right common carotid artery. iPSC-NPCs (400,000 in 4 µl) were transplanted into the penumbra via intracranial injection 7 days after stroke. Trophic factor expression in the peri-infarct tissue was measured using Western blot analysis. Animals received daily bromodeoxyuridine (BrdU) injections and were sacrificed 21 days after stroke for immunohistochemistry. The vibrissae-elicited forelimb placement test was used to evaluate functional recovery. Differentiated iPSCs expressed mature neuronal markers, functional sodium and potassium channels, and fired action potentials. Several angiogenic and neurogenic trophic factors were identified in iPSC-NPCs. Animals that received iPSC-NPC transplantation had greater expression of stromal cell-derived factor 1-α (SDF-1α) and vascular endothelial growth factor (VEGF) in the peri-infarct region. iPSC-NPCs stained positive for neuronal nuclei (NeuN) or glial fibrillary acidic protein (GFAP) 14 days after transplantation. iPSC-NPC-transplanted animals showed greater numbers of BrdU/NeuN and BrdU/Collagen IV colabeled cells in the peri-infarct area compared with stroke controls and performed better in a sensorimotor functional test after stroke. iPSC-NPC therapy may play multiple therapeutic roles after stroke by providing trophic factors, increasing angiogenesis and neurogenesis, and providing new cells for tissue repair.

Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA.

BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. METHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. CONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.

Inhibition of Na+/K+-ATPase induces hybrid cell death and enhanced sensitivity to chemotherapy in human glioblastoma cells.

BACKGROUND: Glioblastoma multiforme (GBM) is very difficult to treat with conventional anti-cancer/anti-apoptotic drugs. We tested the hypothesis that inhibition of Na+/K+-ATPase causes a mixed or hybrid form of concurrent apoptosis and necrosis and therefore should enhance anti-cancer effects of chemotherapy on glioblastoma cells. METHODS: In human LN229 and drug-resistant T98G glioblastoma cell cultures, cell death and signal pathways were measured using immunocytochemistry and Western blotting. Fluorescent dyes were applied to measure intracellular Ca2+, Na+ and K+ changes. RESULTS: The specific Na+/K+-ATPase blocker ouabain (0.1 - 10 µM) induced cell death and disruption of K+ homeostasis in a time- and concentration-dependent manner. Annexin-V translocation and caspase-3 activation indicated an apoptotic component in ouabain cytoxicity, which was accompanied with reduced Bcl-2 expression and mitochondrial membrane potential. Ouabain-induced cell death was partially attenuated by the caspase inhibitor Z-VAD (100 µM). Consistently, the K+ ionophore valinomycin initiated apoptosis in LN229 cells in a K+ efflux-dependent manner. Ouabain caused an initial cell swell, which was followed by a sustained cell volume decrease. Electron microscopy revealed ultrastructural features of both apoptotic and necrotic alterations in the same cells. Finally, human T98G glioblastoma cells that are resistant to the chemotherapy drug temozolomide (TMZ) showed a unique high expression of the Na+/K+-ATPase α2 and α3 subunits compared to the TMZ-sensitive cell line LN229 and normal human astrocytes. At low concentrations, ouabain selectively killed T98G cells. Knocking down the α3 subunit sensitized T98G cells to TMZ and caused more cell death. CONCLUSION: This study suggests that inhibition of Na+/K+-ATPase triggers hybrid cell death and serves as an underlying mechanism for an enhanced chemotherapy effect on glioblastoma cells.

Efficient neuronal differentiation of mouse ES and iPS cells using a rotary cell culture protocol.

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells hold great promise in regenerative medicine for the treatment of neurodegenerative diseases. Current neuronal differentiation protocols however, are not optimized yet for the high scale production of neural precursors and terminally differentiated neurons. The present investigation reports a novel technique for the scalable production of highly uniformed neurospheres, neural precursors and terminal neurons from mouse ES and iPS cells using retinoic acid and a mechanical rotation procedure. We compared embryoid bodies (EB) and neurosphere morphology, yield of neural precursors and quality of neurons between rotary and static suspension cultures of mouse ES and iPS cells undergoing neural differentiation. Analysis of neurospheres formed under continuous rotation showed increased neurosphere uniformity and a high yield of neural precursors after neurosphere dissociation. Neurospheres formed under rotation conditions were relatively smaller, more uniform and had less dead cells and higher proliferation compared to those formed under static conditions. Neural precursors under rotation conditions matured faster, survived better, differentiated to functional neurons that stained positively for mature neuronal markers, and fired action potentials similar to the statically cultured neurons. This report thus provides a technique for the scalable production of neurons from ES and iPS cells and we suggest that rotation culture procedure can be a routine technique for stem cell neural and neuronal differentiation.

SRPK2 phosphorylates tau and mediates the cognitive defects in Alzheimer's disease.

Serine-arginine protein kinases 2 (SRPK2) is a cell cycle-regulated kinase that phosphorylates serine/arginine domain-containing proteins and mediates pre-mRNA splicing with unclear function in neurons. Here, we show that SRPK2 phosphorylates tau on S214, suppresses tau-dependent microtubule polymerization, and inhibits axonal elongation in neurons. Depletion of SRPK2 in dentate gyrus inhibits tau phosphorylation in APP/PS1 mouse and alleviates the impaired cognitive behaviors. The defective LTP in APP/PS1 mice is also improved after SRPK2 depletion. Moreover, active SRPK2 is increased in the cortex of APP/PS1 mice and the pathological structures of human Alzheimer's disease (AD) brain. Therefore, our study suggests SRPK2 may contribute to the formation of hyperphosphorylated tau and the pathogenesis of AD.

Regulatory roles of the NMDA receptor GluN3A subunit in locomotion, pain perception and cognitive functions in adult mice.

Since the discovery of the glutamate NMDA receptor subunit 3A (GluN3A), the functional role of this unique inhibitory subunit has been largely obscure. GluN3A expression is high in the neonatal brain but declines to a low level in the adult brain; it is thus commonly believed that GluN3A does not have a major functional impact in adulthood. Using wild-type (WT) and GluN3A knockout (KO) mice, we show here that deletion of GluN3A affected multiple behavioural functions in adult animals. GluN3A KO mice showed impaired locomotor activity on a variety of motor function tests, and increased sensitivity to acute and sub-acute inflammatory pain. GluN3A KO mice also showed enhanced recognition and spatial learning and memory functions. Hippocampal slices from juvenile and adult GluN3A KO mice showed greater long-term potentiation (LTP) compared with WT slices. GluN3A deletion resulted in increased expression of Ca(2+)/calmodulin-dependent kinase II (CaMKII) in the forebrain, and the phosphorylated CaMKII level upon LTP induction was significantly higher in the GluN3A KO hippocampus compared with WT controls. CaMKII inhibition abrogated the enhanced LTP in GluN3A KO slices. These data reveal for the first time that the presence of GluN3A may have profound impacts on several functional/behavioural activities in adult animals, and could be a therapeutic target for neurological disorders associated with NMDA receptor functions.

Pharmacological inhibition of asparaginyl endopeptidase by δ-secretase inhibitor 11 mitigates Alzheimer's disease-related pathologies in a senescence-accelerated mouse model.

BACKGROUND: Currently, there is no cure for Alzheimer's disease (AD). Therapeutics that can modify the early stage of AD are urgently needed. Recent studies have shown that the pathogenesis of AD is closely regulated by an endo/lysosomal asparaginyl endopeptidase (AEP). Inhibition of AEP has been reported to prevent neural degeneration in transgenic mouse models of AD. However, more than 90% of AD cases are age-related sporadic AD rather than hereditary AD. The therapeutic efficacy of AEP inhibition in ageing-associated sporadic AD remains unknown. METHODS: The senescence-accelerated mouse prone 8 (SAMP8) was chosen as an approximate model of sporadic AD and treated with a selective AEP inhibitor,: δ-secretase inhibitor 11. Activation of AEP was determined by enzymatic activity assay. Concentration of soluble amyloid ß (Aß) in the brain was determined by ELISA. Morris water maze test was performed to assess the learning and memory-related cognitive ability. Pathological changes in the brain were explored by morphological and western blot analyses. RESULTS: The enzymatic activity of AEP in the SAMP8 mouse brain was significantly higher than that in the age-matched SAMR1 mice. The half maximal inhibitory concentration (IC50) for δ-secretase inhibitor 11 to inhibit AEP in vitro is was around 150 nM. Chronic treatment with δ-secretase inhibitor 11 markedly decreased the brain AEP activity, reduced the generation of Aß1-40/42 and ameliorated memory loss. The inhibition of AEP with this reagent not only reduced the AEP-cleaved tau fragments and tau hyperphosphorylation, but also attenuated neuroinflammation in the form of microglial activation. Moreover, treatment with δ-secretase inhibitor 11 prevented the synaptic loss and alleviated dendritic disruption in SAMP8 mouse brain. CONCLUSIONS: Pharmacological inhibition of AEP can intervene and prevent AD-like pathological progress in the model of sporadic AD. The up-regulated AEP in the brain could be a promising target for early treatment of AD. The δ-secretase inhibitor 11 can be used as a lead compound for translational development of AD treatment.

Peripheral cholinoceptor antagonist anisodamine counteracts cholinergic adverse effects and facilitates cognitive amelioration of rivastigmine.

Rivastigmine is a potent acetyl- and butyrylcholinesterase inhibitor widely used for cognitive improvement in Alzheimer's disease (AD) therapy. However, dose-limiting adverse effects restrict its tolerability and clinical outcomes. This study explored new combined therapy, in which peripheral cholinergic adverse effects and central cognitive amelioration of rivastigmine were differentiated by a peripheral cholinoceptor antagonist anisodamine. The results demonstrated that rivastigmine (0.75 and 2.0 mg/kg) could significantly reverse the scopolamine-induced cognitive deficit in mice through passive avoidance test. Nevertheless, a high dose of rivastigmine (3.25 mg/kg) would compromise cognitive amelioration and produce obvious adverse effects, including hypersalivation, intestinal hyperperistalsis and muscle cramp. Interestingly, concomitant administration of anisodamine (10 mg/kg) effectively counteracted both the muscarinergic and nicotinergic adverse effects, while facilitating cognitive amelioration of rivastigmine (3.25 mg/kg). These findings provide an insight into the feasibility of combined therapy with cholinesterase inhibitors and peripheral cholinoceptor antagonists for the treatment of AD.

Blockade of the forward Na+ /Ca2+ exchanger suppresses the growth of glioblastoma cells through Ca2+ -mediated cell death.

BACKGROUND AND PURPOSE: The Na+ /Ca2+ exchanger (NCX) working in either forward or reverse mode participates in maintaining intracellular Ca2+ ([Ca2+ ]i ) homeostasis, which is essential for determining cell fate. Previously, numerous blockers targeting reverse or forward NCX have been developed and studied in ischaemic tissue injury but barely examined in glioblastoma for the purpose of anti-tumour therapy. We assessed the effect of NCX blockers on glioblastoma growth and whether NCX can become a therapeutic target. EXPERIMENTAL APPROACH: Patch-clamp recording, Ca2+ imaging, flow cytometry, and Western blot were used to study the effects of specific and non-specific NCX blockers on cultured glioblastoma cells. In vivo bioluminescent imaging was used to measure effects on grafted glioblastoma. KEY RESULTS: Selectively blocking the reverse NCX with SEA0400, SN-6, and YM-244769 did not affect tumour cell viability. Blocking the forward NCX with bepridil, CB-DMB, or KB-R7943 elevated [Ca2+ ]i and killed glioblastoma cells. Bepridil and CB-DMB caused Ca2+ -dependent cell cycle arrest together with apoptosis, which were all attenuated by a Ca2+ chelator BAPTA-AM. Systemic administration of bepridil inhibited growth of brain-grafted glioblastoma. Bepridil did not appear to have a cytotoxic effect on human astrocytes, which have higher functional expression of NCX than glioblastoma cells. CONCLUSIONS AND IMPLICATIONS: Low expression of the NCX makes glioblastoma cells sensitive to disturbance of [Ca2+ ]i . Interventions designed to block the forward NCX can cause Ca2+ -mediated injury to glioblastoma thus having therapeutic potential. Bepridil could be a lead compound for developing new anti-tumour drugs.

Differential neuropsychopharmacological influences of naturally occurring tropane alkaloids anisodamine versus scopolamine.

Two naturally occurring tropane alkaloids, anisodamine and scopolamine, structurally dissimilar in one OH group, are well established as muscarinic acetylcholine receptor (mAChR) antagonists in clinic and basic research. However, experimental evidence for central effects of anisodamine is limited and conflicting compared with that of scopolamine. In the present study, Morris water maze test, long-term potentiation (LTP) recording and receptor radioligand binding assays were used to explore the disparity in neuropsychopharmacological influences of anisodamine versus scopolamine and possible mechanisms. Anisodamine, at 10-40-fold higher doses than those of scopolamine, did not produce any spatial cognitive deficits as scopolamine, but tended to improve cognition at the repeated high doses. LTP in vivo was then adopted to predict BBB permeability of the muscarinic antagonists following systemic drug administration. Contrary to scopolamine, anisodamine did not influence the formation of LTP in the CA(1) region of rat hippocampus at 40-fold higher dose than that of scopolamine. Additionally, receptor radioligand binding assays (RRLBA) revealed that the binding affinity of anisodamine to mice brain mAChR was much lower than that of scopolamine. The findings suggested that anisodamine did not impair cognition nor depress LTP primarily due to its poor BBB permeability. This work enlarged knowledge of structure-activity relationship among tropane alkaloids, meanwhile providing evidence for more reasonable drug prescription in clinic.