Mutations of RNF213 are responsible for sporadic cerebral cavernous malformation and lead to a mulberry-like cluster in zebrafish.

Although familial forms of cerebral cavernous malformation are mainly attributed to three CCM genes (KRIT1, CCM2 and PDCD10), no mutation is identified in sporadic cerebral cavernous malformation cases with a unique lesion, indicating additional genes for sporadic cerebral cavernous malformation. To screen the candidate genes, we conducted whole exome sequencing in 31 sporadic cerebral cavernous malformation patients and 32 healthy controls, and identified 5 affected individuals carrying 6 heterozygous deleterious mutations in RNF213 but no RNF213 mutation in healthy individuals. To further confirm RNF213 was associated with cerebral cavernous malformation, we generated rnf213a homozygous knockout zebrafish and found mutation of rnf213a in zebrafish led to a mulberry-like cluster of disordered-flow vascular channels which was reminiscent of human cerebral cavernous malformation. In addition, we revealed kbtbd7 and anxa6 were significantly downregulated due to rnf213a mutation through transcriptomic sequencing and RT-qPCR analysis. Based on the mulberry-like phenotype partly rescued by mRNA of kbtbd7 as well as anxa6, we suggested that rnf213a promoted mulberry-like cluster via downregulation of kbtbd7 and anxa6. Altogether, we firstly demonstrate RNF213is a novel candidate gene for sporadic cerebral cavernous malformation and the mutation of rnf213a is responsible for the mulberry-like cluster in zebrafish.

Oral P. gingivalis impairs gut permeability and mediates immune responses associated with neurodegeneration in LRRK2 R1441G mice.

BACKGROUND: The R1441G mutation in the leucine-rich repeat kinase 2 (LRRK2) gene results in late-onset Parkinson's disease (PD). Peripheral inflammation and gut microbiota are closely associated with the pathogenesis of PD. Chronic periodontitis is a common type of peripheral inflammation, which is associated with PD. Porphyromonas gingivalis (Pg), the most common bacterium causing chronic periodontitis, can cause alteration of gut microbiota. It is not known whether Pg-induced dysbiosis plays a role in the pathophysiology of PD. METHODS: In this study, live Pg were orally administrated to animals, three times a week for 1 month. Pg-derived lipopolysaccharide (LPS) was used to stimulate mononuclear cells in vitro. The effects of oral Pg administration on the gut and brain were evaluated through behaviors, morphology, and cytokine expression. RESULTS: Dopaminergic neurons in the substantia nigra were reduced, and activated microglial cells were increased in R1441G mice given oral Pg. In addition, an increase in mRNA expression of tumor necrosis factor (TNF-α) and interleukin-1ß (IL-1ß) as well as protein level of α-synuclein together with a decrease in zonula occludens-1 (Zo-1) was detected in the colon in Pg-treated R1441G mice. Furthermore, serum interleukin-17A (IL-17A) and brain IL-17 receptor A (IL-17RA) were increased in Pg-treated R1441G mice. CONCLUSIONS: These findings suggest that oral Pg-induced inflammation may play an important role in the pathophysiology of LRRK2-associated PD.

hUCMSCs Mitigate LPS-Induced Trained Immunity in Ischemic Stroke.

Innate immune memory is a part of the innate immune system that facilitates the elimination of pathogens. However, it may exacerbate neuropathology. In this study, we found that innate immune memory is detrimental in stroke, because it promotes the acute immune response and exacerbates ischemic infarcts. Mesenchymal stem cell therapy has been widely studied for its therapeutic potential in various diseases including stroke, but whether it diminishes innate immune memory has not been studied. Here, our study demonstrates that, after the activation of innate immune memory by lipopolysaccharide, mesenchymal stem cell therapy can diminish innate immune memory though down-regulation of H3 methylation and subsequently protect against stroke. Our results demonstrate that innate immune memory is detrimental in stroke, and we describe a novel potential therapeutic target involving the use of mesenchymal stem cells to treat stroke patients.

Overexpression of Slit2 decreases neuronal excitotoxicity, accelerates glymphatic clearance, and improves cognition in a multiple microinfarcts model.

BACKGROUND: Cerebral microinfarcts (MIs) lead to progressive cognitive impairments in the elderly, and there is currently no effective preventative strategy due to uncertainty about the underlying pathogenic mechanisms. One possibility is the dysfunction of GABAergic transmission and ensuing excitotoxicity. Dysfunction of GABAergic transmission induces excitotoxicity, which contributes to stroke pathology, but the mechanism has kept unknown. The secreted leucine-rich repeat (LRR) family protein slit homologue 2 (Slit2) upregulates GABAergic activity and protects against global cerebral ischemia, but the neuroprotective efficacy of Slit2 against MIs has not been examined. METHODS: Middle-aged Wild type (WT) and Slit2-Tg mice were divided into sham and MI treatment groups. MIs were induced in parietal cortex by laser-evoked arteriole occlusion. Spatial memory was then compared between sham and MI groups using the Morris water maze (MWM) task. In addition, neuronal activity, blood brain barrier (BBB) permeability, and glymphatic clearance in peri-infarct areas were compared using two-photon imaging, while GABAergic transmission, microglial activation, neuronal loss, and altered cortical connectivity were compared by immunofluorescent staining or western blotting. RESULTS: Microinfarcts increased the amplitude and frequency of spontaneous intracellular Ca2+ signals, reduced neuronal survival and connectivity within parietal cortex, decreased the number of GABAergic interneurons and expression of vesicular GABA transporter (VGAT), induced neuroinflammation, and impaired both glymphatic clearance and spatial memory. Alternatively, Slit2 overexpression attenuated dysfunctional neuronal Ca2+ signaling, protected against neuronal death in the peri-infarct area as well as loss of parietal cortex connectivity, increased GABAergic interneuron number and VGAT expression, attenuated neuroinflammation, and improved both glymphatic clearance and spatial memory. CONCLUSION: Our results strongly suggest that overexpression of Slit2 protected against the dysfunction in MIs, which is a potential therapeutic target for cognition impairment in the elderly.

Mesenchymal stem cells alleviate AQP-4-dependent glymphatic dysfunction and improve brain distribution of antisense oligonucleotides in BACHD mice.

Huntington's disease (HD) is a neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene that results in the production of neurotoxic mutant HTT (mHTT) protein. Suppressing HTT production with antisense oligonucleotides (ASOs) is a promising treatment strategy for HD; however, the difficulty of delivering ASOs to deep brain structures is a major barrier for its clinical application. The glymphatic system of astrocytes involving aquaporin 4 (AQP-4) controls the entry of macromolecules from the cerebrospinal fluid into the brain. Mesenchymal stem cells (MSCs) target astrocytes to inhibit neuroinflammation. Here we examined the glymphatic distribution of ASO in the brain and the therapeutic potential of combining intravenously injection of mesenchymal stem cells (IV-MSC) and ASOs for the treatment of HD. Our results show that Cy3-labeled ASOs entered the brain parenchyma via the perivascular space following cisternal injection, but the brain distribution was significantly lower in AQP-4-/- as compared with wild-type mice. Downregulation of the AQP-4 M23 isoform was accompanied by decreased brain levels of ASOs in BACHD mice as well as an increase in astrogliosis and phosphorylation of nuclear factor κB (NF-κB) p65. IV-MSC treatment restored AQP-4 M23 expression, attenuated astrogliosis, and decreased NF-κB p65 phosphorylation; it also increased the brain distribution of ASOs and enhanced the suppression of mHTT in BACHD mice. These results suggest that modulating glymphatic activity using IV-MSC is a novel strategy for improving the potency of ASO in the treatment of HD.

Extracellular ASC exacerbated the recurrent ischemic stroke in an NLRP3-dependent manner.

Using a photothrombotic mouse model of single stroke, we show that a single stroke onset increases the nuclear factor-κB (NF-κB), NLR family CARD domain containing protein 4 (NLRC4), and absent in melanoma 2 (AIM2) inflammasomes, as well as the mRNA levels of NLRP3. Next, using a photothrombotic mouse model of recurrent stroke, we found that recurrent strokes increased the activation of NLRP3, exacerbated the brain damage and the pro-inflammatory response in wild type (WT) mice, but not in NLRP3 knockout (NLRP3 KO) mice. Additionally, we found that apoptosis-associated speck-like protein containing a CARD (ASC) protein level surrounding the infarct area was comparatively increased, but that ASC specks outside of microglia in both the ipsilateral and contralateral of stroke site were decreased in NLRP3 KO mice relative to wild-type (WT) controls, and the number of ASC specks surrounding the second infarct area was positively correlated to the damage scores. Mechanistically, we found that recombinant ASC (RecASC) activated NLRP3 and induced pro-inflammatory responses, exacerbating the outcome of ischemic stroke, in WT mice, but not in NLRP3 KO mice. We therefore conclude that the NLRP3 inflammasome is activated by two attacks of stroke, which act together with ASC to exacerbate recurrent strokes.

Xyloketal derivative C53N protects against mild traumatic brain injury in mice.

PURPOSE: Mild traumatic brain injury (mTBI), the most common type of TBI, can result in prolonged cognitive impairment, mood disorders, and behavioral problems. Reducing oxidative stress and inflammation can rescue the neurons from mTBI-induced cell death. Xyloketal B, a natural product from mangrove fungus, has shown good antioxidative and neuroprotective effects in several disease models. Here, we investigated the potential protection afforded by a xyloketal derivative, C53N, in a closed-skull mTBI model. MATERIALS AND METHODS: Skulls of mice were thinned to 20-30 µm thickness, following which they were subjected to a slight compression injury to induce mTBI. One hour after TBI, mice were intraperitoneally injected with C53N, which was solubilized in 0.5% dimethyl sulfoxide in saline. In vivo two-photon laser scanning microscopy was used to image cell death in injured parenchyma in each mouse over a 12-hour period (at 1, 3, 6, and 12 hours). Water content and oxidation index, together with pathological analysis of glial reactivity, were assessed at 24 hours to determine the effect of C53N on mTBI. RESULTS: Cell death, oxidative stress, and glial reactivity increased in mTBI mice compared with sham-injured mice. Treatment with 40 or 100 mg/kg C53N 1 hour after mTBI significantly attenuated oxidative stress and glial reactivity and reduced parenchymal cell death at the acute phase after mTBI. CONCLUSION: The present study highlights the therapeutic potential of the xyloketal derivative C53N for pharmacological intervention in mTBI.

Chronic colitis induces meninges traffic of gut-derived T cells, unbalances M1 and M2 microglia/macrophage and increases ischemic brain injury in mice.

Ischemic stroke is one of the most common diseases leading to death and is the primary cause of physical handicap. Recent studies have reported that chronic colitis increases the risk of ischemic stroke, but it is unknown whether chronic colitis participates in ischemic brain injury directly. A combined mouse model of chronic colitis induced by dextran sodium sulfate (DSS) and ischemic stroke induced by photochemical infarction was used in this study. We demonstrated that chronic colitis significantly increased the infarction volume, activated microglia/macrophage numbers, proliferation of M1 microglia/macrophage, non-gut-derived CD4+ T lymphocyte penetration and decreased neuron numbers in the peri-infarction at 7 d after stroke. Furthermore, gut-derived CD4+ T cell accumulation on the meninges was observed at 7 d after stroke. In addition, selective depletion of meningeal macrophages resulted in a reduction of infarction volume and the non-gut-derived CD4+ T lymphocyte penetration. We concluded that chronic colitis exacerbated ischemic stroke by promoting CD4+ T cell migration from the gut to the meninges and disequilibrium of M1 and M2 microglia/macrophages. We speculated that the gut-derived CD4+ T cells may interact with meningeal macrophages and result in non-gut-derived CD4+ T lymphocyte infiltration that aggravated brain injury in ischemic stroke.

Transplanted human neural precursor cells integrate into the host neural circuit and ameliorate neurological deficits in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) is to date one of the major critical conditions causing death and disability worldwide. Exogenous neural stem/precursor cells (NSCs/NPCs) hold great promise for improving neurological dysfunction, but their functional properties in vivo remain unknown. Human neural precursor cells (hNPCs) carrying one fluorescent reporter gene (DsRed) can be observed directly in vivo using two-photon laser-scanning microscope. Therefore, we evaluated the neural integration and potential therapeutic effect of hNPCs on mice with TBI. Behavioral tests were performed by rotarod task and Morris Water Maze task. Neural integration was detected by fluorometric Ca2+ imaging and nerve tracing. We found that motor and cognition functions were significantly improved in mice with hNPCs injection compared to mice with vehicle treatment, and hNPCs integrated into the host circuit and differentiated toward neuronal lineage. Our study provided reliable evidence for further hNPCs transplantation in clinical practice.

Intravenous PEP-1-GDNF is protective after focal cerebral ischemia in rats.

Glial cell line-derived neurotrophic factor (GDNF) is a potential therapeutic protein on a variety of central nervous system diseases including ischemic stroke. However, GDNF is a large molecule that cannot cross the blood-brain barrier (BBB), which is still intact in the early hours after stroke when neural rescue is possible. PEP-1 protein transduction domain can deliver protein cargo across the cell membrane and the BBB. In the present study, we generated a novel fusion protein PEP-1-GDNF and examined whether PEP-1-GDNF is protective in focal cerebral ischemia. PEP-1-GDNF (200 µg/kg) or PBS was intravenously applied over 5 min immediately after reperfusion of 90 min transient middle cerebral artery occlusion (MCAO). After 28 days, rats were deeply anesthetized and decapitated. Behavioral tests were performed during this period. The results showed that PEP-1-GDNF significantly reduced the infarct volume and improved behavioral function. Further, PEP-1-GDNF promoted the cell proliferation and differentiation in the dentate gyrus of the hippocampus and attenuated ischemia-induced learning and memory damage.

Allopurinol protects against ischemic insults in a mouse model of cortical microinfarction.

Microinfarcts are common in patients with cognitive decline and dementia. Allopurinol (ALLO), a xanthine oxidase (XO) enzyme inhibitor, has been found to reduce proinflammatory molecules and oxidative stress in the vasculature. We here examined the effect of pre-treatment with allopurinol on the cortical microinfarction. C57BL/6J mice were subjected to a permanent single penetrating arteriole occlusion induced by two-photon laser irradiation. Infarction volume, the activation of glial cells and nitrosative stress in the ischemic brain was assessed using immunohistochemistry. Pre-treatment with ALLO achieved 42% reduction of infarct volume and significantly reduced microglia infiltration, astrocyte proliferation and nitrosative stress in the ischemic brain. These data indicate that ALLO protects against microinfarcts possibly through inhibition of nitrosative stress and attenuation of microglia infiltration as well as astrocytes reactivation.

Identification of marine neuroactive molecules in behaviour-based screens in the larval zebrafish.

High-throughput behavior-based screen in zebrafish is a powerful approach for the discovery of novel neuroactive small molecules for treatment of nervous system diseases such as epilepsy. To identify neuroactive small molecules, we first screened 36 compounds (1-36) derived from marine natural products xyloketals and marine isoprenyl phenyl ether obtained from the mangrove fungus. Compound 1 demonstrated the most potent inhibition on the locomotor activity in larval zebrafish. Compounds 37-42 were further synthesized and their potential anti-epilepsy action was then examined in a PTZ-induced epilepsy model in zebrafish. Compound 1 and compounds 39, 40 and 41 could significantly attenuate PTZ-induced locomotor hyperactivity and elevation of c-fos mRNA in larval zebrafish. Compound 40 showed the most potent inhibitory action against PTZ-induced hyperactivity. The structure-activity analysis showed that the OH group at 12-position played a critical role and the substituents at the 13-position were well tolerated in the inhibitory activity of xyloketal derivatives. Thus, these derivatives may provide some novel drug candidates for the treatment of epilepsy.

Protective effects of traditional Chinese medicine formula NaoShuanTong capsule on haemorheology and cerebral energy metabolism disorders in rats with blood stasis.

NaoShuanTong capsule (NSTC), an oral traditional Chinese medicine formula, is composed of Pollen Typhae, Radix Paeoniae Rubra, Rhizoma Gastrodiae, Radix Rhapontici and Radix Curcumae. It has been widely used to treat ischemic stroke in clinic for many years in China. In addition to neuronal apoptosis, haemorheology and cerebral energy metabolism disorders also play an important role in the pathogenesis and development of ischemic stroke. The present study was designed to evaluate the in vivo protective effects of NSTC on haemorheology and cerebral energy metabolism disorders in rats with blood stasis. Sixty specific pathogen-free sprague-dawley rats, male only, were randomly divided into six groups (control group, model group, aspirin (100 mg/kg/d) group, NSTC low-dose (400 mg/kg/d) group, NSTC intermediate-dose (800 mg/kg/d) group, NSTC high-dose (1600 mg/kg/d) group) with 10 animals in each. The rats except those in the control group were placed in ice-cold water (0-4 °C) for 5 min during the time interval (4 h) of two adrenaline hydrochloride injections (0.8 mg/kg) to induce blood stasis. After treatment, whole blood viscosity at three shear rates, plasma viscosity and erythrocyte sedimentation rate significantly decreased in NSTC intermediate- and high-dose groups; erythrocyte aggregation index and red corpuscle electrophoresis index significantly decreased in all the three dose NSTC groups. Moreover, treatment with high-dose NSTC could significantly improve Na+-K+ adenosine triphosphatase (ATPase) and Ca2+ ATPase activity, as well as lower lactic acid level in brain tissues. These results demonstrated the protective effects of NSTC on haemorheology and cerebral energy metabolism disorders, which may provide scientific information for the further understanding of mechanism(s) of NSTC as a clinical treatment for ischemic stroke. Furthermore, the protective effects of activating blood circulation as observed in this study might create valuable insight for the utilisation of NSTC to be a feasible alternative therapeutic agent for patients with blood stasis.