hUC-MSCs-derived MFGE8 ameliorates locomotor dysfunction via inhibition of ITGB3/ NF-κB signaling in an NMO mouse model.

Neuromyelitis optica (NMO) is a severe autoimmune inflammatory disease of the central nervous system that affects motor function and causes relapsing disability. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have been used extensively in the treatment of various inflammatory diseases, due to their potent regulatory roles that can mitigate inflammation and repair damaged tissues. However, their use in NMO is currently limited, and the mechanism underlying the beneficial effects of hUC-MSCs on motor function in NMO remains unclear. In this study, we investigate the effects of hUC-MSCs on the recovery of motor function in an NMO systemic model. Our findings demonstrate that milk fat globule epidermal growth 8 (MFGE8), a key functional factor secreted by hUC-MSCs, plays a critical role in ameliorating motor impairments. We also elucidate that the MFGE8/Integrin αvß3/NF-κB signaling pathway is partially responsible for structural and functional recovery, in addition to motor functional enhancements induced by hUC-MSC exposure. Taken together, these findings strongly support the involvement of MFGE8 in mediating hUC-MSCs-induced improvements in motor functional recovery in an NMO mouse model. In addition, this provides new insight on the therapeutic potential of hUC-MSCs and the mechanisms underlying their beneficial effects in NMO.

CHI3L1 signaling impairs hippocampal neurogenesis and cognitive function in autoimmune-mediated neuroinflammation.

Chitinase-3-like protein 1 (CHI3L1) is primarily secreted by activated astrocytes in the brain and is known as a reliable biomarker for inflammatory central nervous system (CNS) conditions such as neurodegeneration and autoimmune disorders like neuromyelitis optica (NMO). NMO is an astrocyte disease caused by autoantibodies targeting the astroglial protein aquaporin 4 (AQP4) and leads to vision loss, motor deficits, and cognitive decline. In this study examining CHI3L1's biological function in neuroinflammation, we found that CHI3L1 expression correlates with cognitive impairment in our NMO patient cohort. Activated astrocytes secrete CHI3L1 in response to AQP4 autoantibodies, and this inhibits the proliferation and neuronal differentiation of neural stem cells. Mouse models showed decreased hippocampal neurogenesis and impaired learning behaviors, which could be rescued by depleting CHI3L1 in astrocytes. The molecular mechanism involves CHI3L1 engaging the CRTH2 receptor and dampening ß-catenin signaling for neurogenesis. Blocking this CHI3L1/CRTH2/ß-catenin cascade restores neurogenesis and improves cognitive deficits, suggesting the potential for therapeutic development in neuroinflammatory disorders.

Pleiotrophin ameliorates age-induced adult hippocampal neurogenesis decline and cognitive dysfunction.

Cognitive impairment has been associated with an age-related decline in adult hippocampal neurogenesis (AHN). The molecular basis of declining neurogenesis in the aging hippocampus remains to be elucidated. Here, we show that pleiotrophin (PTN) expression is decreased with aging in neural stem and progenitor cells (NSPCs). Mice lacking PTN exhibit impaired AHN accompanied by poor learning and memory. Mechanistically, we find that PTN engages with protein tyrosine phosphatase receptor type Z1 (PTPRZ1) to promote NSPC proliferation and differentiation by activating AKT signaling. PTN overexpression or pharmacological activation of AKT signaling in aging mice restores AHN and alleviates relevant memory deficits. Importantly, we also find that PTN overexpression improves impaired neurogenesis in senescence-accelerated mouse prone 8 (SAMP8) mice. We further confirm that PTN is required for enriched environment-induced increases in AHN. These results corroborate the significance of AHN in aging and reveal a possible therapeutic intervention by targeting PTN.

Inhibition of complement C3 signaling ameliorates locomotor and visual dysfunction in autoimmune inflammatory diseases.

Neuromyelitis optica (NMO) is an autoimmune inflammatory disease of the central nervous system (CNS) characterized by transverse myelitis and optic neuritis. The pathogenic serum IgG antibody against the aquaporin-4 (AQP4) on astrocytes triggers the activation of the complement cascade, causing astrocyte injury, followed by oligodendrocyte injury, demyelination, and neuronal loss. Complement C3 is positioned as a central player that relays upstream initiation signals to activate downstream effectors, potentially stimulating and amplifying host immune and inflammatory responses. However, whether targeting the inhibition of C3 signaling could ameliorate tissue injury, locomotor defects, and visual impairments in NMO remains to be investigated. In this study, using the targeted C3 inhibitor CR2-Crry led to a significant decrease in complement deposition and demyelination in both slice cultures and focal intracerebral injection models. Moreover, the treatment downregulated the expression of inflammatory cytokines and improved motor dysfunction in a systemic NMO mouse model. Similarly, employing serotype 2/9 adeno-associated virus (AAV2/9) to induce permanent expression of CR2-Crry resulted in a reduction in visual dysfunction by attenuating NMO-like lesions. Our findings reveal the therapeutic value of inhibiting the complement C3 signaling pathway in NMO.

Remyelination in neuromyelitis optica spectrum disorder is promoted by edaravone through mTORC1 signaling activation.

Neuromyelitis optica spectrum disorder (NMOSD) is a severe inflammatory autoimmune disease of the central nervous system that is manifested as secondary myelin loss. Oligodendrocyte progenitor cells (OPCs) are the principal source of myelinating oligodendrocytes (OLs) and are abundant in demyelinated regions of NMOSD patients, thus possibly representing a cellular target for pharmacological intervention. To explore the therapeutic compounds that enhance myelination due to endogenous OPCs, we screened the candidate drugs in mouse neural progenitor cell (NPC)-derived OPCs. We identified drug edaravone, which is approved by the Food and Drug Administration (FDA), as a promoter of OPC differentiation into mature OLs. Edaravone enhanced remyelination in organotypic slice cultures and in mice, even when edaravone was administered following NMO-IgG-induced demyelination, and ameliorated motor impairment in a systemic mouse model of NMOSD. The results of mechanistic studies in NMO-IgG-treated mice and the biopsy samples of the brain tissues of NMOSD patients indicated that the mTORC1 signaling pathway was significantly inhibited, and edaravone promoted OPC maturation and remyelination by activating mTORC1 signaling. Furthermore, pharmacological activation of mTORC1 signaling significantly enhanced myelin regeneration in NMOSD. Thus, edaravone is a potential therapeutic agent that promotes lesion repair in NMOSD patients by enhancing OPC maturation.

Astrocytic Piezo1-mediated mechanotransduction determines adult neurogenesis and cognitive functions.

Adult brain activities are generally believed to be dominated by chemical and electrical transduction mechanisms. However, the importance of mechanotransduction mediated by mechano-gated ion channels in brain functions is less appreciated. Here, we show that the mechano-gated Piezo1 channel is expressed in the exploratory processes of astrocytes and utilizes its mechanosensitivity to mediate mechanically evoked Ca2+ responses and ATP release, establishing Piezo1-mediated mechano-chemo transduction in astrocytes. Piezo1 deletion in astrocytes causes a striking reduction of hippocampal volume and brain weight and severely impaired (but ATP-rescuable) adult neurogenesis in vivo, and it abolishes ATP-dependent potentiation of neural stem cell (NSC) proliferation in vitro. Piezo1-deficient mice show impaired hippocampal long-term potentiation (LTP) and learning and memory behaviors. By contrast, overexpression of Piezo1 in astrocytes sufficiently enhances mechanotransduction, LTP, and learning and memory performance. Thus, astrocytes utilize Piezo1-mediated mechanotransduction mechanisms to robustly regulate adult neurogenesis and cognitive functions, conceptually highlighting the importance of mechanotransduction in brain structure and function.

Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells.

Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2Cas9). We selected endothelial ß-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2Cas9 transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.

Brain endothelial PTEN/AKT/NEDD4-2/MFSD2A axis regulates blood-brain barrier permeability.

The low level of transcytosis is a unique feature of cerebrovascular endothelial cells (ECs), ensuring restrictive blood-brain barrier (BBB) permeability. Major facilitator superfamily domain-containing 2a (MFSD2A) is a key regulator of the BBB function by suppressing caveolae-mediated transcytosis. However, the mechanisms regulating MFSD2A at the BBB have been barely explored. Here, we show that cerebrovascular EC-specific deletion of Pten (phosphatase and tensin homolog) results in a dramatic increase in vesicular transcytosis by the reduction of MFSD2A, leading to increased transcellular permeability of the BBB. Mechanistically, AKT signaling inhibits E3 ubiquitin ligase NEDD4-2-mediated MFSD2A degradation. Consistently, cerebrovascular Nedd4-2 overexpression decreases MFSD2A levels, increases transcytosis, and impairs BBB permeability, recapitulating the phenotypes of Pten-deficient mice. Furthermore, Akt deletion decreases phosphorylated NEDD4-2 levels, restores MFSD2A levels, and normalizes BBB permeability in Pten-mutant mice. Altogether, our work reveals the essential physiological function of the PTEN/AKT/NEDD4-2/MFSD2A axis in the regulation of BBB permeability.

Hepatocyte growth factor-regulated tyrosine kinase substrate is essential for endothelial cell polarity and cerebrovascular stability.

AIMS: Hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs), a key component of the endosomal sorting complex required for transport (ESCRT), has been implicated in many essential biological processes. However, the physiological role of endogenous Hgs in the vascular system has not previously been explored. Here, we have generated brain endothelial cell (EC) specific Hgs knockout mice to uncover the function of Hgs in EC polarity and cerebrovascular stability. METHODS AND RESULTS: Knockout of Hgs in brain ECs led to impaired endothelial apicobasal polarity and brain vessel collapse in mice. We determined that Hgs is essential for recycling of vascular endothelial (VE)-cadherin to the plasma membrane, since loss of Hgs blocked trafficking of endocytosed VE-cadherin from early endosomes to recycling endosomes, and impaired the motility of recycling endosomes. Supportively, overexpression of the motor kinesin family member 13A (KIF13A) restored endosomal recycling and rescued abrogated polarized trafficking and distribution of VE-cadherin in Hgs knockdown ECs. CONCLUSION: These data uncover a novel physiological function of Hgs and support an essential role for the ESCRT machinery in the maintenance of EC polarity and cerebrovascular stability.

Brain Endothelial Cells Maintain Lactate Homeostasis and Control Adult Hippocampal Neurogenesis.

Increased understanding of the functions of lactate has suggested a close relationship between lactate homeostasis and normal brain activity because of its importance as an energy source and signaling molecule. Here we show that lactate levels affect adult hippocampal neurogenesis. Cerebrovascular-specific deletion of PTEN causes learning and memory deficits and disrupts adult neurogenesis with accompanying lactate accumulation. Consistently, administering lactate to wild-type animals impairs adult hippocampal neurogenesis. The endothelial PTEN/Akt pathway increases monocarboxylic acid transporter 1 (MCT1) expression to enhance lactate transport across the brain endothelium. Moreover, cerebrovascular overexpression of MCT1 or deletion of Akt1 restores MCT1 expression, decreases lactate levels, and normalizes hippocampal neurogenesis and cognitive function in PTEN mutant mice. Together, these findings delineate how the brain endothelium maintains lactate homeostasis and contributes to adult hippocampal neurogenesis and cognitive functions.

SUMO-specific protease 1 modulates cadmium-augmented transcriptional activity of androgen receptor (AR) by reversing AR SUMOylation.

Cadmium is a potential prostate carcinogen and can mimic the effects of androgen by a mechanism that involves the hormone-binding domain of the androgen receptor (AR), which is a key transcriptional factor in prostate carcinogenesis. We focused on transcriptional activity of AR to investigate the toxicity of cadmium exposure on human prostate cell lines. Cadmium increased the proliferative index of LNCaP and the proliferative effect was obstructed significantly by AR blocking agent. In luciferase assay, cadmium activated the transcriptional activity of AR in 293T cells co-transfected with wild-type AR and an ARE (AR response elements)-luciferase reporter gene. Cadmium also increased expression of PSA, a downstream gene of AR, whereas the metal had no significant effect on AR amount. AR is regulated by multiple posttranslational modifications including SUMOylation. SUMOylated AR shows a lower transcriptional activity. SUMO-specific protease 1 (SENP1) decreases AR SUMOylation by deconjugating AR-SUMO covalent bond. We detected that cadmium increased the amount of SENP1 in a dose and time dependent manner. Knocking down of SENP1 by RNAi led to decrease of PSA expression and transcriptional activity of AR in luciferase assay. Furthermore, co-immunoprecipitation (Co-IP) results showed that SUMOylation level of AR was decreased after cadmium treatment. In conclusion, our results indicated that cadmium-induced SENP1 enhanced AR transcriptional activity by decreasing AR SUMOylation.

Comparison of freely-moving telemetry Chinese Miniature Experiment Pigs (CMEPs) to beagle dogs in cardiovascular safety pharmacology studies.

INTRODUCTION: Telemetry beagle dogs are the most frequently used species in cardiovascular telemetry assessments. However, beagle dogs may not be always suitable for all of the tests. Recently minipigs have received increased attention for these studies. Differences between the two species regarding the response of their cardiovascular systems to environmental stimuli are unclear. This study investigates how the telemetry minipig compares to beagle dog as a test subject and also refines the experimental protocols necessary to obtain accurate data. METHODS: Beagle dogs and Chinese Miniature Experiment Pigs (CMEPs) were implanted with telemetry transmitters and the influences of gavage, feeding and the circadian cycle on various cardiovascular parameters were investigated. RESULTS: ECG signal quality from CMEPs was superior to that of the beagle dogs. Poor ECG signal quality, elevated HR, BP and locomotor activity associated with gavage and feeding were observed in both species. ECG signal quality, BP and locomotor activity recovered more quickly in the CMEPs than in the beagle dogs. Residual elevation of HR found in CMEPs lasted approximately 4h post-feeding, which has a profound influence on the circadian cycle. A diurnal rhythm in CMEP with a significant increase of body temperature during the dark period and a clear circadian rhythm of locomotor activity in both species were observed. DISCUSSION: The present data demonstrated that gavage, feeding and circadian cycle were having an enormous influence on BP, HR and locomotor activity in both species. If drug-induced effects are expected rapidly after oral administration and feeding, CMEP seems to be a favorable choice. Also, due to the effects of feeding on HR, CMEPs should fast at least 5h before the start of recording or should not be fed during the study where the Tmax of a given compound might occur very late. It also should be taken into consideration when the test article has a potential effect on body temperature by using CMEPs. In summary, the telemetry CMEP is a valuable alternative to the beagle dog for cardiovascular telemetry studies.

Citrinin reduces testosterone secretion by inducing apoptosis in rat Leydig cells.

A previous study has shown that CTN (Citrinin) inhibits mouse testosterone production. In this study, the mechanism by which testosterone production is inhibited by CTN in rat Leydig cells was investigated, and the morphological evidence of apoptosis, including nuclei fragmentation and phosphatidylserine (PS) exposure on cell surfaces, was clearly observed 36h after CTN exposure. The results showed that citrinin at 50 and 100µM significantly suppressed testosterone secretion by human chorionic gonadotropin (hCG) at 10IU/ml. Western blotting results showed that CTN induced formation of processed p53, caspase-9, and caspase-3 proteins in a dose-dependent manner; CTN also induced a dose-dependent increase in caspase-3 catalytic activity. Western blot assays also showed that CTN decreased expression of three key enzymes (P450scc, 3ß-HSD-1, and StAR) of testosterone production. Taken together, these results suggested that CTN reduced testosterone secretion by inducing apoptosis in rat Leydig cells, a mechanism that might account for CTN stimulation of p53 expression followed by activation of multiple caspases.