Mingke Song

Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer's disease

Neurofibrillary tangles (NFTs), composed of truncated and hyperphosphorylated tau, are a common feature of numerous aging-related neurodegenerative diseases, including Alzheimers disease (AD). However, the molecular mechanisms mediating tau truncation and aggregation during aging remain elusive. Here we show that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is activated during aging and proteolytically degrades tau, abolishes its microtubule assembly function, induces tau aggregation and triggers neurodegeneration. AEP is upregulated and active during aging and is activated in human AD brain and tau P301S–transgenic mice with synaptic pathology and behavioral impairments, leading to tau truncation in NFTs. Tau P301S–transgenic mice with deletion of the gene encoding AEP show substantially reduced tau hyperphosphorylation, less synapse loss and rescue of impaired hippocampal synaptic function and cognitive deficits. Mice infected with adeno-associated virus encoding an uncleavable tau mutant showed attenuated pathological and behavioral defects compared to mice injected with adeno-associated virus encoding tau P301S. Together, these observations indicate that AEP acts as a crucial mediator of tau-related clinical and neuropathological changes. Inhibition of AEP may be therapeutically useful for treating tau-mediated neurodegenerative diseases.

7,8-Dihydroxyflavone Prevents Synaptic Loss and Memory Deficits in a Mouse Model of Alzheimer's Disease

Synaptic loss in the brain correlates well with disease severity in Alzheimer disease (AD). Deficits in brain-derived neurotrophic factor/tropomyosin-receptor-kinase B (TrkB) signaling contribute to the synaptic dysfunction of AD. We have recently identified 7,8-dihydroxyflavone (7,8-DHF) as a potent TrkB agonist that displays therapeutic efficacy toward various neurological diseases. Here we tested the effect of 7,8-DHF on synaptic function in an AD model both in vitro and in vivo. 7,8-DHF protected primary neurons from Aβ-induced toxicity and promoted dendrite branching and synaptogenesis. Chronic oral administration of 7,8-DHF activated TrkB signaling and prevented Aβ deposition in transgenic mice that coexpress five familial Alzheimer’s disease mutations (5XFAD mice). Moreover, 7,8-DHF inhibited the loss of hippocampal synapses, restored synapse number and synaptic plasticity, and prevented memory deficits. These results suggest that 7,8-DHF represents a novel oral bioactive therapeutic agent for treating AD.

Delta-secretase cleaves amyloid precursor protein and regulates the pathogenesis in Alzheimer's disease.

The age-dependent deposition of amyloid-β peptides, derived from amyloid precursor protein (APP), is a neuropathological hallmark of Alzheimers disease (AD). Despite age being the greatest risk factor for AD, the molecular mechanisms linking ageing to APP processing are unknown. Here we show that asparagine endopeptidase (AEP), a pH-controlled cysteine proteinase, is activated during ageing and mediates APP proteolytic processing. AEP cleaves APP at N373 and N585 residues, selectively influencing the amyloidogenic fragmentation of APP. AEP is activated in normal mice in an age-dependent manner, and is strongly activated in 5XFAD transgenic mouse model and human AD brains. Deletion of AEP from 5XFAD or APP/PS1 mice decreases senile plaque formation, ameliorates synapse loss, elevates long-term potentiation and protects memory. Blockade of APP cleavage by AEP in mice alleviates pathological and behavioural deficits. Thus, AEP acts as a δ-secretase, contributing to the age-dependent pathogenic mechanisms in AD.

Vector-Free and Transgene-Free Human iPS Cells Differentiate into Functional Neurons and Enhance Functional Recovery after Ischemic Stroke in Mice

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice.

Restoration of Intracortical and Thalamocortical Circuits After Transplantation of Bone Marrow Mesenchymal Stem Cells Into the Ischemic Brain of Mice

Transplantation of bone marrow mesenchymal stem cells (BMSCs) provides a promising regenerative medicine for stroke. Whether BMSC therapy could repair ischemia-damaged neuronal circuits and recover electrophysiological activity has largely been unknown. To address this issue, BMSCs were implanted into the ischemic barrel cortex of adult mice 1 and 7 days after focal barrel cortex stroke. Two days after the first transplantation (3 days after stroke), the infarct volume determined by TTC staining was significantly smaller in BMSC-treated compared to vehicle-treated stroke mice. The behavioral corner test showed better long-term recovery of sensorimotor function in BMSC-treated mice. Six weeks poststroke, thalamocortical slices were prepared and neuronal circuit activity in the peri-infarct region of the barrel cortex was determined by extracellular recordings of evoked field potentials. In BMSC-transplanted brain slices, the ischemia-disrupted intracortical activity from layer 4 to layer 2/3 was noticeably recovered, and the thalamocortical circuit connection was also partially restored. In contrast, much less and slower recovery was seen in control animals of barrel cortex stroke. Immunohistochemical staining disclosed that the density of neurons, axons, and blood vessels in the peri-infarct region was significantly higher in BMSC-treated mice, accompanied with enhanced local blood flow recovery. Western blotting showed that BMSC treatment increased the expression of stromal cell-derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) in the peri-infarct region. Moreover, the expression of the axonal growth associated protein-43 (GAP-43) was markedly increased, whereas the axonal growth inhibiting proteins ROCK II and NG2 were suppressed in the BMSC-treated brains. BMSC transplantation also promoted directional migration and survival of doublecortin (DCX)-positive neuroblasts in the peri-infarct region. The present investigation thus provides novel evidence that BMSC transplantation has the potential to repair the ischemia-damaged neural networks and restore lost neuronal connections. The recovered circuit activity likely contributes to the improved sensorimotor function after focal ischemic stroke and BMSC transplantation.

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. Stem Cells 2014;32:3075–3087

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.

Highly efficient differentiation of neural precursors from human embryonic stem cells and benefits of transplantation after ischemic stroke in mice.

IntroductionIschemic stroke is a leading cause of death and disability, but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study, we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model.MethodsNeural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention.ResultsAfter 11 days of neural induction by using the small-molecule protocol, over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude, repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals.ConclusionsHuman embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo, enhance regenerative activities, and improve sensory recovery after ischemic stroke.

The TRPC channel blocker SKF 96365 inhibits glioblastoma cell growth by enhancing reverse mode of the Na+/Ca2+ exchanger and increasing intracellular Ca2+

SKF 96365 is well known for its suppressing effect on human glioblastoma growth by inhibiting pre‐activated transient receptor potential canonical (TRPC) channels and Ca2+ influx. The effect of SKF 96363 on glioblastoma cells, however, may be multifaceted and this possibility has been largely ignored.

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

•  Adult glutamate NMDA receptor subunit 3A (GluN3A) knockout (KO) mice showed slow locomotor activity, motor deficits and increased pain sensation. •  Hippocampal slices from juvenile and adult GluN3A KO mice showed greater long‐term potentiation compared with wild‐type slices. •  Adult GluN3A KO mice showed enhanced abilities in learning and memory tasks. •  GluN3A deletion resulted in increased expression and/or phosphorylation of Ca2+/calmodulin‐ dependent kinase II in the brain. •  This is the first investigation showing that deletion of GluN3A in the embryonic stage has imperative impacts on multiple behaviours in adults.