The Ca2+ hypothesis for Alzheimer's disease (AD) conceives Ca2+ dyshomeostasis as a common mechanism of AD; the cause of Ca2+ dysregulation, however, is obscure. Meanwhile, hyperactivities of N-Methyl-D-aspartate receptors (NMDARs), the primary mediator of Ca2+ influx, are reported in AD. GluN3A (NR3A) is an NMDAR inhibitory subunit. We hypothesize that GluN3A is critical for sustained Ca2+ homeostasis and its deficiency is pathogenic for AD. Cellular, molecular, and functional changes were examined in adult/aging GluN3A knockout (KO) mice. The GluN3A KO mouse brain displayed age-dependent moderate but persistent neuronal hyperactivity, elevated intracellular Ca2+, neuroinflammation, impaired synaptic integrity/plasticity, and neuronal loss. GluN3A KO mice developed olfactory dysfunction followed by psychological/cognitive deficits prior to amyloid-β/tau pathology. Memantine at preclinical stage prevented/attenuated AD syndromes. AD patients’ brains show reduced GluN3A expression. We propose that chronic “degenerative excitotoxicity” leads to sporadic AD, while GluN3A represents a primary pathogenic factor, an early biomarker, and an amyloid-independent therapeutic target.

Spinal cord injury (SCI) is a debilitating condition with neurological deficits and loss of motor function that, depending on the severity, may lead to paralysis. The only treatment currently available is methylprednisolone, which is widely used and renders limited efficacy in SCI. Therefore, other therapeutic agents must be developed. The neuroprotective efficacy of estrogen in SCI was studied with a pre-clinical and pro-translational perspective. Acute SCI was induced in rats that were treated with low doses of estrogen (1, 5, 10, or 100 μg/kg) and compared with vehicle-treated injured rats or laminectomy control (sham) rats at 48 h post-SCI. Changes in gliosis and other pro-inflammatory responses, expression and activity of proteolytic enzymes (e.g., calpain, caspase-3), apoptosis of neurons in SCI, and cell death were monitored via Western blotting and immunohistochemistry. Negligible pro-inflammatory responses or proteolytic events and very low levels of neuronal death were found in sham rats. In contrast, vehicle-treated SCI rats showed profound pro-inflammatory responses with reactive gliosis, elevated expression and activity of calpain and caspase-3, elevated Bax:Bcl-2 ratio, and high levels of neuronal death in lesion and caudal regions of the injured spinal cord. Estrogen treatment at each dose reduced pro-inflammatory and proteolytic activities and protected neurons in the caudal penumbra in acute SCI. Estrogen treatment at 10 μg was found to be as effective as 100 μg in ameliorating the above parameters in injured animals. Results from this investigation indicated that estrogen at a low dose could be a promising therapeutic agent for treating acute SCI. Experimental studies with low dose estrogen therapy in acute spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes. Estrogen has been found to ameliorate several degenerative pathways following SCI. Thus, such early protective effects may even lead to functional recovery in long term injury. Studies are underway in chronic SCI in a follow up manuscript. Experimental studies with low dose estrogen therapy in acute spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes. Estrogen has been found to ameliorate several degenerative pathways following SCI. Thus, such early protective effects may even lead to functional recovery in long term injury. Studies are underway in chronic SCI in a follow up manuscript.

The generation of neural stem and progenitor cells following injury is critical for the function of the central nervous system, but the molecular mechanisms modulating this response remain largely unknown. We have previously identified the G protein-coupled receptor 37 (GPR37) as a modulator of ischemic damage in a mouse model of stroke. Here we demonstrate that GPR37 functions as a critical negative regulator of progenitor cell dynamics and gliosis following ischemic injury. In the central nervous system, GPR37 is enriched in mature oligodendrocytes, but following injury we have found that its expression is dramatically increased within a population of Sox2-positive progenitor cells. Moreover, the genetic deletion of GPR37 did not alter the number of mature oligodendrocytes following injury but did markedly increase the number of both progenitor cells and injury-induced Olig2-expressing glia. Alterations in the glial environment were further evidenced by the decreased activation of oligodendrocyte precursor cells. These data reveal that GPR37 regulates the response of progenitor cells to ischemic injury and provides new perspectives into the potential for manipulating endogenous progenitor cells following stroke.

Stroke and late-onset Alzheimer's disease (AD) are risk factors for each other; the comorbidity of these brain disorders in aging individuals represents a significant challenge in basic research and clinical practice. The similarities and differences between stroke and AD in terms of pathogenesis and pathophysiology, however, have rarely been comparably reviewed. Here, we discuss the research background and recent progresses that are important and informative for the comorbidity of stroke and late-onset AD and related dementia (ADRD). Glutamatergic NMDA receptor (NMDAR) activity and NMDAR-mediated Ca2+ influx are essential for neuronal function and cell survival. An ischemic insult, however, can cause rapid increases in glutamate concentration and excessive activation of NMDARs, leading to swift Ca2+ overload in neuronal cells and acute excitotoxicity within hours and days. On the other hand, mild upregulation of NMDAR activity, commonly seen in AD animal models and patients, is not immediately cytotoxic. Sustained NMDAR hyperactivity and Ca2+ dysregulation lasting from months to years, nevertheless, can be pathogenic for slowly evolving events, i.e. degenerative excitotoxicity, in the development of AD/ADRD. Specifically, Ca2+ influx mediated by extrasynaptic NMDARs (eNMDARs) and a downstream pathway mediated by transient receptor potential cation channel subfamily M member (TRPM) are primarily responsible for excitotoxicity. On the other hand, the NMDAR subunit GluN3A plays a "gatekeeper" role in NMDAR activity and a neuroprotective role against both acute and chronic excitotoxicity. Thus, ischemic stroke and AD share an NMDAR- and Ca2+-mediated pathogenic mechanism that provides a common receptor target for preventive and possibly disease-modifying therapies. Memantine (MEM) preferentially blocks eNMDARs and was approved by the Federal Drug Administration (FDA) for symptomatic treatment of moderate-to-severe AD with variable efficacy. According to the pathogenic role of eNMDARs, it is conceivable that MEM and other eNMDAR antagonists should be administered much earlier, preferably during the presymptomatic phases of AD/ADRD. This anti-AD treatment could simultaneously serve as a preconditioning strategy against stroke that attacks ≥ 50% of AD patients. Future research on the regulation of NMDARs, enduring control of eNMDARs, Ca2+ homeostasis, and downstream events will provide a promising opportunity to understand and treat the comorbidity of AD/ADRD and stroke.

Asparagine endopeptidase (AEP), a newly identified delta-secretase, simultaneously cleaves both APP and Tau, promoting Alzheimer's disease (AD) pathologies. However, its pathological role in AD remains incompletely understood. Here we show that delta-secretase cleaves BACE1, a rate-limiting protease in amyloid-β (Aβ) generation, escalating its enzymatic activity and enhancing senile plaques deposit in AD. Delta-secretase binds BACE1 and cuts it at N294 residue in an age-dependent manner and elevates its protease activity. The cleaved N-terminal motif is active even under neutral pH and associates with senile plaques in human AD brains. Subcellular fractionation reveals that delta-secretase and BACE1 reside in the endo-lysosomes. Interestingly, truncated BACE1 enzymatic domain (1-294) augments delta-secretase enzymatic activity and accelerates Aβ production, facilitating AD pathologies and cognitive impairments in APP/PS1 AD mouse model. Uncleavable BACE1 (N294A) inhibits delta-secretase activity and Aβ production and decreases AD pathologies in 5XFAD mice, ameliorating cognitive dysfunctions. Hence, delta- and beta- secretases’ crosstalk aggravates each other's roles in AD pathogenesis.

Ischemic stroke is a leading cause of morbidity and mortality, with limited treatments that can facilitate brain regeneration. Neural progenitor cells (NPCs) hold promise for replacing tissue lost to stroke, and biomaterial approaches may improve their efficacy to overcome hurdles in clinical translation. The immune response and its role in stroke pathogenesis and regeneration may interplay with critical mechanisms of stem cell and biomaterial therapies. Cellular therapy can modulate the immune response to reduce toxic neuroinflammation early after ischemia. However, few studies have attempted to harness the regenerative effects of neuroinflammation to augment recovery. Our previous studies demonstrated that intracerebrally transplanted NPCs encapsulated in a chondroitin sulfate-A hydrogel (CS-A + NPCs) can improve vascular regeneration after stroke. In this paper, we found that CS-A + NPCs affect the microglia/macrophage response to promote a regenerative phenotype following stroke in mice. Following transplantation, PPARγ-expressing microglia/macrophages, and MCP-1 and IL-10 protein levels are enhanced. Secreted immunomodulatory factor expression of other factors was altered compared to NPC transplantation alone. Post-stroke depression-like behavior was reduced following cellular and material transplantation. Furthermore, we showed in cultures that microglia/macrophages encapsulated in CS-A had increased expression of angiogenic and arteriogenic mediators. Neutralization with anti-IL-10 antibody negated these effects in vitro. Cumulatively, this work provides a framework for understanding the mechanisms by which immunomodulatory biomaterials can enhance the regenerative effects of cellular therapy for ischemic stroke and other brain injuries.

OBJECTIVE: Stroke is a leading cause of human death and disability. Effective early treatments with reasonable therapeutic windows remain critically important to improve the outcomes of stroke. Transcranial magnetic stimulation (TMS) is an established noninvasive technique that has been applied clinically and in animal research for multiple brain disorders, but few studies have examined acute neuroprotection against ischemic stroke. The present investigation tested the novel approach of low-frequency repetitive TMS (rTMS) as an acute treatment after ischemic stroke. METHODS: Adult male rats received focal ischemic surgery through occlusion of the right middle cerebral artery for 60 minutes. The rats received either rTMS or sham treatment with 1.5-, 3-, 4-, or 7-hour delay after the onset of stroke. Low-frequency and low-intensity rTMS was applied to the rat brain for two 30-minute episodes separated by a 1-hour interval. RESULTS: Three days after stroke, compared to stroke controls, rats receiving rTMS treatment with a 1.5-hour delay showed a 35% reduction of infarct volume. Protective effects were also seen with 3- or 4-hour-delayed treatments by rTMS, shown as reduced infarct volume and cell death. rTMS treatment upregulated the antiapoptotic factor Bcl-2 and downregulated the proapoptotic caspase-3 cleavage, expressions of Bax and matrix metallopeptidase-9. In sensorimotor functional assessments 3 to 21 days after stroke, rats receiving rTMS treatment with a 1.5- or 3-hour delay showed significantly better performance compared to stroke controls. INTERPRETATION: These results support the inference that low-frequency rTMS may be feasible as a neuroprotective acute treatment after ischemic stroke. ANN NEUROL 2023;93:336-347.

Alzheimer’s disease has a higher incidence in older women, with a spike in cognitive decline that tracks with visceral adiposity, dysregulated energy homeostasis and bone loss during the menopausal transition1,2. Inhibiting the action of follicle-stimulating hormone (FSH) reduces body fat, enhances thermogenesis, increases bone mass and lowers serum cholesterol in mice3–7. Here we show that FSH acts directly on hippocampal and cortical neurons to accelerate amyloid-β and Tau deposition and impair cognition in mice displaying features of Alzheimer’s disease. Blocking FSH action in these mice abrogates the Alzheimer’s disease-like phenotype by inhibiting the neuronal C/EBPβ–δ-secretase pathway. These data not only suggest a causal role for rising serum FSH levels in the exaggerated Alzheimer’s disease pathophysiology during menopause, but also reveal an opportunity for treating Alzheimer’s disease, obesity, osteoporosis and dyslipidaemia with a single FSH-blocking agent.

Atherosclerosis (ATH) and Alzheimer’s disease (AD) are both age-dependent inflammatory diseases, associated with infiltrated macrophages and vascular pathology and overlapping molecules. C/EBPβ, an Aβ or inflammatory cytokine-activated transcription factor, and AEP (asparagine endopeptidase) are intimately implicated in both ATH and AD; however, whether C/EBPβ/AEP signaling couples ATH to AD pathogenesis remains incompletely understood. Here we show that C/EBPβ/AEP pathway mediates ATH pathology and couples ATH to AD. Deletion of C/EBPβ or AEP from primary macrophages diminishes cholesterol load, and inactivation of this pathway reduces foam cell formation and lesions in aorta in ApoE−/− mice, fed with HFD (high-fat-diet). Knockout of ApoE from 3xTg AD mouse model augments serum LDL and increases lesion areas in the aorta. Depletion of C/EBPβ or AEP from 3xTg/ApoE−/− mice substantially attenuates these effects and elevates cerebral blood flow and vessel length, improving cognitive functions. Strikingly, knockdown of ApoE from the hippocampus of 3xTg mice decreases the cerebral blood flow and vessel length and aggravates AD pathologies, leading to cognitive deficits. Inactivation of C/EBPβ/AEP pathway alleviates these events and restores cognitive functions. Hence, our findings demonstrate that C/EBPβ/AEP signaling couples ATH to AD via mediating vascular pathology.

Previous investigations suggest that DL-3-n-butylphthalide (NBP) is a promising multifaceted drug for the treatment of stroke. It is not clear whether NBP can treat traumatic brain injury (TBI) and what could be the mechanisms of therapeutic benefits. To address these issues, TBI was induced by a controlled cortical impact in adult male mice. NBP (100 mg/kg) or saline was intraperitoneally administered within 5 min after TBI. One day after TBI, apoptotic events including caspase-3/9 activation, cytochrome c release from the mitochondria, and apoptosis-inducing factor (AIF) translocation into the nucleus in the pericontusion region were attenuated in NBP-treated mice compared to TBI-saline controls. In the assessment of the nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) pathway, NBP ameliorated the p65 expression and the p-IκB-α/IκB-α ratio, indicating reduced NF-κB activation. Consistently, NBP reduced the upregulation of proinflammatory cytokines such as tumor necrotizing factor-alpha (TNF-α) and interleukin-1beta (IL-1β) after TBI. In sub-acute treatment experiments, NBP was intranasally delivered once daily for 3 days. At 3 days after TBI, this repeated NBP treatment significantly reduced the contusion volume and cell death in the pericontusion region. In chronic experiments up to 21 days after TBI, continues daily intranasal NBP treatment increased neurogenesis, angiogenesis, and arteriogenesis in the post-TBI brain, accompanied with upregulations of regenerative genes including brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), endothelial-derived nitric oxide synthase (eNOS), and matrix metallopeptidase 9 (MMP-9). The NBP treatment significantly improved sensorimotor functional recovery and reduced post-TBP depressive behavior. These new findings demonstrate that NBP shows multiple therapeutic benefits after TBI.