An increase in ER stress and unfolded protein response in iPSCs-derived neuronal cells from neuronopathic Gaucher disease patients.

Gaucher disease (GD) is a lysosomal storage disorder caused by a mutation in the GBA1 gene, responsible for encoding the enzyme Glucocerebrosidase (GCase). Although neuronal death and neuroinflammation have been observed in the brains of individuals with neuronopathic Gaucher disease (nGD), the exact mechanism underlying neurodegeneration in nGD remains unclear. In this study, we used two induced pluripotent stem cells (iPSCs)-derived neuronal cell lines acquired from two type-3 GD patients (GD3-1 and GD3-2) to investigate the mechanisms underlying nGD by biochemical analyses. These iPSCs-derived neuronal cells from GD3-1 and GD3-2 exhibit an impairment in endoplasmic reticulum (ER) calcium homeostasis and an increase in unfolded protein response markers (BiP and CHOP), indicating the presence of ER stress in nGD. A significant increase in the BAX/BCL-2 ratio and an increase in Annexin V-positive cells demonstrate a notable increase in apoptotic cell death in GD iPSCs-derived neurons, suggesting downstream signaling after an increase in the unfolded protein response. Our study involves the establishment of iPSCs-derived neuronal models for GD and proposes a possible mechanism underlying nGD. This mechanism involves the activation of ER stress and the unfolded protein response, ultimately leading to apoptotic cell death in neurons.

Establishment of MUi030-A: A human induced pluripotent stem cell line carrying homozygous L444P mutation in the GBA1 gene to study type-3 Gaucher disease.

Gaucher disease (GD) is a common lysosomal storage disease resulting from mutations in the glucocerebrosidase (GBA1) gene. This genetic disorder manifests with symptoms affecting multiple organs, yet the underlying mechanisms leading to pathology remain elusive. In this study, we successfully generated the MUi030-A human induced pluripotent stem cell (hiPSC) line using a non-integration method from a male type-3 GD patient with a homozygous c.1448T>C (L444P) mutation. These hiPSCs displayed a normal karyotype and pluripotency markers and the remarkable ability to differentiate into cells representing all three germ layers. This resourceful model holds significant promise for illuminating GD's underlying pathogenesis.

The Effects of PP2A Disruption on ER-Mitochondria Contact and Mitochondrial Functions in Neuronal-like Cells.

Mitochondria-associated membranes (MAMs) regulate several cellular processes, including calcium homeostasis and mitochondrial function, and dynamics. While MAMs are upregulated in Alzheimer's disease (AD), the mechanisms underlying this increase remain unknown. A possible mechanism may include dysregulation of protein phosphatase 2A (PP2A), which is reduced in the AD brain. Furthermore, PP2A has been previously reported to modulate MAM formation in hepatocytes. However, it is unknown whether PP2A and MAMs are linked in neuronal cells. Here, to test the correlation between PP2A and MAMs, we inhibited the activity of PP2A to mimic its low levels in AD brains and observed MAM formation, function, and dynamics. MAMs were significantly increased after PP2A inhibition, which correlated with elevated mitochondrial Ca2+ influx and disrupted mitochondrial membrane potential and mitochondrial fission. This study highlights the essential role PP2A plays in regulating MAM formation and mitochondrial function and dynamics for the first time in neuronal-like cells.

Neuroprotective effect of short-chain fatty acids against oxidative stress-induced SH-SY5Y injury via GPR43-dependent pathway.

A neurodegenerative disorder is a condition that causes a degeneration of neurons in the central nervous system, leading to cognitive impairment and movement disorders. An accumulation of oxidative stress in neurons contributes to the pathogenesis of neurodegenerative disorders. Over the past few years, several studies have suggested that short-chain fatty acids, metabolites of the gut microbiota, might have a beneficial effect in neurodegenerative disorders. A G protein-coupled receptor 43 (GPR43) plays an important role in modulating oxidative stress and inflammatory processes in several tissues. Interestingly, the downstream signaling pathways activated by GPR43 to modulate oxidative stress differ among tissues. Moreover, the cellular mechanisms underlying GPR43 activation in neuronal cells to handle oxidative stress remain unclear. In this present study, we tested the role of GPR43, which is activated by short-chain fatty acids or a specific GPR43 agonist, in an oxidative stress-induced neuronal cell line (SH-SY5Y) injury. Our findings suggest that a combination of short-chain fatty acids with a physiological function could protect neurons from H2 O2 -induced cell damage. The effect of short-chain fatty acids mixture was abolished by pretreatment with a GPR43 antagonist, indicating this protective effect is a GPR43-dependent mechanism. In addition, a specific GPR43 agonist shows a similar result to that found in short-chain fatty acids mixture. Furthermore, our findings indicate that the downstream activation of GPR43 to protect against oxidative stress-induced neuronal injury is a biased Gq activation signaling of GPR43, which results in the prevention of H2 O2 -induced neuronal apoptosis. In conclusion, our results show new insight into the cellular mechanism of GPR43 and its neuroprotective effect. Taken together, this newly discovered finding suggests that activation of the biased Gq signaling pathway of GPR43 might be a potential therapeutic target for aging-related neurodegeneration.

Pharmacological Activities of Fingerroot Extract and Its Phytoconstituents Against SARS-CoV-2 Infection in Golden Syrian Hamsters.

Background: The outbreak of COVID-19 has led to the suffering of people around the world, with an inaccessibility of specific and effective medication. Fingerroot extract, which showed in vitro anti-SARS-CoV-2 activity, could alleviate the deficiency of antivirals and reduce the burden of health systems. Aim of Study: In this study, we conducted an experiment in SARS-CoV-2-infected hamsters to determine the efficacy of fingerroot extract in vivo. Materials and Methods: The infected hamsters were orally administered with vehicle control, fingerroot extract 300 or 1000 mg/kg, or favipiravir 1000 mg/kg at 48 h post-infection for 7 consecutive days. The hamsters (n = 12 each group) were sacrificed at day 2, 4 and 8 post-infection to collect the plasma and lung tissues for analyses of viral output, lung histology and lung concentration of panduratin A. Results: All animals in treatment groups reported no death, while one hamster in the control group died on day 3 post-infection. All treatments significantly reduced lung pathophysiology and inflammatory mediators, PGE2 and IL-6, compared to the control group. High levels of panduratin A were found in both the plasma and lung of infected animals. Conclusion: Fingerroot extract was shown to be a potential of reducing lung inflammation and cytokines in hamsters. Further studies of the full pharmacokinetics and toxicity are required before entering into clinical development.

A generation of human induced pluripotent stem cell line (MUi031-A) from a type-3 Gaucher disease patient carrying homozygous mutation on GBA1 gene.

Gaucher disease (GD) is one of the most prevalent lysosomal storage diseases caused by mutation of glucocerebrosidase (GBA1) gene. GD patients develop symptoms in various organs of the body; however, the underlying mechanisms causing pathology are still elusive. Thus, a suitable disease model is important in order to facilitate subsequent investigations. Here, we established MUi031-A human induced pluripotent stem cell (hiPSC) line from CD34+ hematopoietic stem cells of a female type-3 GD patient with homozygous c.1448 T > C (L444P) mutation. The cells exhibited embryonic stem cell-like characteristics and expressed pluripotency markers with capability to differentiate into three germ layers.

In vivo mechanisms of cortical network dysfunction induced by systemic inflammation.

Peripheral inflammation is known to impact brain function, resulting in lethargy, loss of appetite and impaired cognitive abilities. However, the channels for information transfer from the periphery to the brain, the corresponding signaling molecules and the inflammation-induced interaction between microglia and neurons remain obscure. Here, we used longitudinal in vivo two-photon Ca2+ imaging to monitor neuronal activity in the mouse cortex throughout the early (initiation) and late (resolution) phases of peripheral inflammation. Single peripheral lipopolysaccharide injection induced a substantial but transient increase in ongoing neuronal activity, restricted to the initiation phase, whereas the impairment of visual processing was selectively observed during the resolution phase of systemic inflammation. In the frontal/motor cortex, the initiation phase-specific cortical hyperactivity was seen in the deep (layer 5) and superficial (layer 2/3) pyramidal neurons but not in the axons coming from the somatosensory cortex, and was accompanied by reduced activity of layer 2/3 cortical interneurons. Moreover, the hyperactivity was preserved after depletion of microglia and in NLRP3-/- mice but absent in TNF-α-/- mice. Together, these data identify microglia-independent and TNF-α-mediated reduction of cortical inhibition as a likely cause of the initiation phase-specific cortical hyperactivity and reveal the resolution phase-specific impairment of sensory processing, presumably caused by activated microglia.

Targeting the complement system in neuromyelitis optica spectrum disorder.

INTRODUCTION: Neuromyelitis optica spectrum disorder (NMOSD) is characterized by central nervous system inflammation and demyelination. In AQP4-IgG seropositive NMOSD, circulating immunoglobulin G (IgG) autoantibodies against astrocyte water channel aquaporin-4 (AQP4) cause tissue injury. Compelling evidence supports a pathogenic role for complement activation following AQP4-IgG binding to AQP4. Clinical studies supported the approval of eculizumab, an inhibitor of C5 cleavage, in AQP4-IgG seropositive NMOSD. AREAS COVERED: This review covers in vitro, animal models, and human evidence for complement-dependent and complement-independent tissue injury in AQP4-IgG seropositive NMOSD. Complement targets are discussed, including complement proteins, regulators and anaphylatoxin receptors, and corresponding drug candidates. EXPERT OPINION: Though preclinical data support a central pathogenic role of complement activation in AQP4-IgG seropositive NMOSD, they do not resolve the relative contributions of complement-dependent vs. complement-independent disease mechanisms such as antibody-dependent cellular cytotoxicity, T cell effector mechanisms, and direct AQP4-IgG-induced cellular injury. The best evidence that complement-dependent mechanisms predominate in AQP4-IgG seropositive NMOSD comes from eculizumab clinical data. Various drug candidates targeting distinct complement effector mechanisms may offer improved safety and efficacy. However, notwithstanding the demonstrated efficacy of complement inhibition in AQP4-IgG seropositive NMOSD, the ultimate niche for complement inhibition is not clear given multiple drug options with alternative mechanisms of action.Abbreviations: AAV2, Adeno-associated virus 2; ADCC, antibody-dependent cellular cytotoxicity; ANCA, antineutrophilic cytoplasmic autoantibody; AQP4, aquaporin-4; AQP4-IgG, AQP4-immunoglobulin G; C1-INH, C1-esterase inhibitor; C3aR, C3a receptor; C4BP, C4 binding protein; C5aR, C5a receptor; CDC, complement-dependent cytotoxicity; CFHR1, complement factor H related 1; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; EndoS, endoglycosidase S; FHL-1, factor-H-like protein 1; GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor protein-1; IgG, immunoglobulin G; IVIG, intravenous human immunoglobulin G; MAC, membrane attack complex; MBL, maltose-binding lectin; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; NK cell, natural killer cell; NMOSD, neuromyelitis optica spectrum disorder; OAP, orthogonal arrays of particles; PNH, paroxysmal nocturnal hemoglobinuria.

Cell motility and migration as determinants of stem cell efficacy.

BACKGROUND: Stem cells` (SC) functional heterogeneity and its poorly understood aetiology impedes clinical development of cell-based therapies in regenerative medicine and oncology. Recent studies suggest a strong correlation between the SC migration potential and their therapeutic efficacy in humans. Designating SC migration as a denominator of functional SC heterogeneity, we sought to identify highly migrating subpopulations within different SC classes and evaluate their therapeutic properties in comparison to the parental non-selected cells. METHODS: We selected highly migrating subpopulations from mesenchymal and neural SC (sMSC and sNSC), characterized their features including but not limited to migratory potential, trophic factor release and transcriptomic signature. To assess lesion-targeted migration and therapeutic properties of isolated subpopulations in vivo, surgical transplantation and intranasal administration of MSCs in mouse models of glioblastoma and Alzheimer's disease respectively were performed. FINDINGS: Comparison of parental non-selected cells with isolated subpopulations revealed superior motility and migratory potential of sMSC and sNSC in vitro. We identified podoplanin as a major regulator of migratory features of sMSC/sNSC. Podoplanin engineering improved oncovirolytic activity of virus-loaded NSC on distantly located glioblastoma cells. Finally, sMSC displayed more targeted migration to the tumour site in a mouse glioblastoma model and remarkably higher potency to reduce pathological hallmarks and memory deficits in transgenic Alzheimer's disease mice. INTERPRETATION: Functional heterogeneity of SC is associated with their motility and migration potential which can serve as predictors of SC therapeutic efficacy. FUNDING: This work was supported in part by the Robert Bosch Stiftung (Stuttgart, Germany) and by the IZEPHA grant.

Emerging therapeutic targets for neuromyelitis optica spectrum disorder.

Introduction: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease of the central nervous system affecting primarily the spinal cord and optic nerves. Most NMOSD patients are seropositive for immunoglobulin G autoantibodies against astrocyte water channel aquaporin-4, called AQP4-IgG, which cause astrocyte injury leading to demyelination and neurological impairment. Current therapy for AQP4-IgG seropositive NMOSD includes immunosuppression, B cell depletion, and plasma exchange. Newer therapies target complement, CD19 and IL-6 receptors.Areas covered: This review covers early-stage pre-clinical therapeutic approaches for seropositive NMOSD. Targets include pathogenic AQP4-IgG autoantibodies and their binding to AQP4, complement-dependent and cell-mediated cytotoxicity, blood-brain barrier, remyelination and immune effector and regulatory cells, with treatment modalities including small molecules, biologics, and cells.Expert opinion: Though newer NMOSD therapies appear to have increased efficacy in reducing relapse rate and neurological deficit, increasingly targeted therapies could benefit NMOSD patients with ongoing relapses and could potentially be superior in efficacy and safety. Of the various early-stage therapeutic approaches, IgG inactivating enzymes, aquaporumab blocking antibodies, drugs targeting early components of the classical complement system, complement regulator-targeted drugs, and Fc-based multimers are of interest. Curative strategies, perhaps involving AQP4 tolerization, remain intriguing future possibilities.

Healthy Brain Aging Modifies Microglial Calcium Signaling In Vivo.

Brain aging is characterized by a chronic, low-grade inflammatory state, promoting deficits in cognition and the development of age-related neurodegenerative diseases. Malfunction of microglia, the brain-resident immune cells, was suggested to play a critical role in neuroinflammation, but the mechanisms underlying this malfunctional phenotype remain unclear. Specifically, the age-related changes in microglial Ca2+ signaling, known to be linked to its executive functions, are not well understood. Here, using in vivo two-photon imaging, we characterize intracellular Ca2+ signaling and process extension of cortical microglia in young adult (2⁻4-month-old), middle-aged (9⁻11-month-old), and old (18⁻21-month-old) mice. Our data revealed a complex and nonlinear dependency of the properties of intracellular Ca2+ signals on an animal's age. While the fraction of cells displaying spontaneous Ca2+ transients progressively increased with age, the frequencies and durations of the spontaneous Ca2+ transients followed a bell-shaped relationship, with the most frequent and largest Ca2+ transients seen in middle-aged mice. Moreover, in old mice microglial processes extending toward an ATP source moved faster but in a more disorganized manner, compared to young adult mice. Altogether, these findings identify two distinct phenotypes of aging microglia: a reactive phenotype, abundantly present in middle-aged animals, and a dysfunctional/senescent phenotype ubiquitous in old mice.

Role of intracellular Ca2+ stores for an impairment of visual processing in a mouse model of Alzheimer's disease.

Besides deficits in memory and cognition, impaired visual processing is common for Alzheimer's disease (AD) patients and mouse models of AD but underlying mechanisms still remain unclear. Using in vivo Ca2+ imaging of the mouse primary visual cortex (V1) we tested whether such impairment is caused by neuronal hyperactivity, an emerging functional hallmark of AD. Profound neuronal hyperactivity was indeed found in V1 of APPSWE/PS1G384A and even of PS1G384A mice, presenting neither with plaque accumulation nor with neuroinflammation. This hyperactivity was accompanied by over-responsiveness to visual stimuli and impaired visual tuning properties of individual neurons, largely caused by insufficient suppression of responses to non-preferred orientation/direction stimuli. Moreover, visual stimulation robustly suppressed the ongoing spontaneous activity in WT but not in APPSWE/PS1G384A mice. Emptying intracellular Ca2+ stores significantly reduced neuronal hyperactivity and the pathological over-responsiveness to visual stimuli, but could not rescue stimulus-induced suppression of spontaneous activity and impaired tuning properties of individual cells. Thus, our data identify the AD-mediated dysfunction of intracellular Ca2+ stores as a main cause of pathologically increased visual responsiveness in APPSWE/PS1G384A mice. At the same time, the impairment of visual tuning and the stimulus-induced suppression of spontaneous activity, identified in this study, are likely caused by different mechanisms as, for example, dysfunction of local interneurons.

Intracellular Ca2+ stores control in vivo neuronal hyperactivity in a mouse model of Alzheimer's disease.

Neuronal hyperactivity is the emerging functional hallmark of Alzheimer's disease (AD) in both humans and different mouse models, mediating an impairment of memory and cognition. The mechanisms underlying neuronal hyperactivity remain, however, elusive. In vivo Ca2+ imaging of somatic, dendritic, and axonal activity patterns of cortical neurons revealed that both healthy aging and AD-related mutations augment neuronal hyperactivity. The AD-related enhancement occurred even without amyloid deposition and neuroinflammation, mainly due to presenilin-mediated dysfunction of intracellular Ca2+ stores in presynaptic boutons, likely causing more frequent activation of synaptic NMDA receptors. In mutant but not wild-type mice, store emptying reduced both the frequency and amplitude of presynaptic Ca2+ transients and, most importantly, normalized neuronal network activity. Postsynaptically, the store dysfunction was minor and largely restricted to hyperactive cells. These findings identify presynaptic Ca2+ stores as a key element controlling AD-related neuronal hyperactivity and as a target for disease-modifying treatments.

Activation of liver X receptor inhibits OCT2-mediated organic cation transport in renal proximal tubular cells.

Liver X receptor (LXR) is transcriptional factor that plays an important role in the regulation of energy metabolism such as cholesterol, lipid, and glucose metabolism as well as membrane transporters and channels. Using both in vitro and in vivo models, LXR regulation of the expression and function of renal organic cation transporter 2 (OCT2) was observed. Synthetic LXR agonist (GW3965) and endogenous LXR agonist (22R-hydroxycholesterol) significantly reduced the uptake of 3H-MPP+, a prototypic substrate of OCT2, in both OCT2- Chinese hamster ovary K1 and human renal proximal tubular cells (RPTEC/TERT1). GW3965 decreased transport activity of OCT2 via a reduction of the maximal transport rate of MPP+ without affecting transporter affinity. The inhibitory effect of GW3965 was attenuated by co-treatment with LXR antagonist (fenofibrate) indicating the inhibition was LXR-dependent mechanism. In addition, co-treatment with a retinoic X receptor (RXR) ligand, 9-cis retinoic acid enhanced the inhibitory effect of GW3965, indicating negative regulation of OCT2 transport activity by the LXR/RXR complex. Treatment RPTEC/TERT1 cells with GW3965 significantly reduced OCT2 protein expression without changing mRNA expression. In parallel, the effect of LXR activation on OCT2 function was investigated in intact mouse kidney. Treating mice with 50 mg/kg BW T0901317 for 14 days significantly decreased 3H-MPP+ uptake into renal cortical slices, correlating with decreased OCT2 protein expression in renal cortex without changes in mRNA expression levels. Taken together, LXR/RXR activation downregulates the protein expression and function of OCT2 in renal proximal tubule, suggesting LXR might affect the total profile of renal excretion of cationic compounds.

Bridging the gap between clinicians and systems biologists: from network biology to translational biomedical research.

With the wealth of data accumulated from completely sequenced genomes and other high-throughput experiments, global studies of biological systems, by simultaneously investigating multiple biological entities (e.g. genes, transcripts, proteins), has become a routine. Network representation is frequently used to capture the presence of these molecules as well as their relationship. Network biology has been widely used in molecular biology and genetics, where several network properties have been shown to be functionally important. Here, we discuss how such methodology can be useful to translational biomedical research, where scientists traditionally focus on one or a small set of genes, diseases, and drug candidates at any one time. We first give an overview of network representation frequently used in biology: what nodes and edges represent, and review its application in preclinical research to date. Using cancer as an example, we review how network biology can facilitate system-wide approaches to identify targeted small molecule inhibitors. These types of inhibitors have the potential to be more specific, resulting in high efficacy treatments with less side effects, compared to the conventional treatments such as chemotherapy. Global analysis may provide better insight into the overall picture of human diseases, as well as identify previously overlooked problems, leading to rapid advances in medicine. From the clinicians' point of view, it is necessary to bridge the gap between theoretical network biology and practical biomedical research, in order to improve the diagnosis, prevention, and treatment of the world's major diseases.

Potential therapeutic benefit of C1-esterase inhibitor in neuromyelitis optica evaluated in vitro and in an experimental rat model.

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system in which binding of anti-aquaporin-4 (AQP4) autoantibodies (NMO-IgG) to astrocytes causes complement-dependent cytotoxicity (CDC) and inflammation resulting in oligodendrocyte and neuronal injury. There is compelling evidence for a central role of complement in NMO pathogenesis. Here, we evaluated the potential of C1-esterase inhibitor (C1-inh) for complement-targeted therapy of NMO. C1-inh is an anti-inflammatory plasma protein with serine protease inhibition activity that has a broad range of biological activities on the contact (kallikrein), coagulation, fibrinolytic and complement systems. C1-inh is approved for therapy of hereditary angioedema (HAE) and has been studied in a small safety trial in acute NMO relapses (NCT 01759602). In vitro assays of NMO-IgG-dependent CDC showed C1-inh inhibition of human and rat complement, but with predicted minimal complement inhibition activity at a dose of 2000 units in humans. Inhibition of complement by C1-inh was potentiated by ∼10-fold by polysulfated macromolecules including heparin and dextran sulfate. In rats, intravenous C1-inh at a dose 30-fold greater than that approved to treat HAE inhibited serum complement activity by <5%, even when supplemented with heparin. Also, high-dose C1-inh did not reduce pathology in a rat model of NMO produced by intracerebral injection of NMO-IgG. Therefore, although C1r and C1s are targets of C1-inh, our in vitro data with human serum and in vivo data in rats suggest that the complement inhibition activity of C1-inh in serum is too low to confer clinical benefit in NMO.

Neuromyelitis optica pathology in rats following intraperitoneal injection of NMO-IgG and intracerebral needle injury.

INTRODUCTION: Animal models of neuromyelitis optica (NMO) are needed for drug testing and evaluation of NMO disease pathogenesis mechanisms. RESULTS: We describe a novel passive-transfer model of NMO in which rats made seropositive for human anti-aquaporin-4 (AQP4) immunoglobulin G antibody (NMO-IgG) by intraperitoneal (IP) injections were subject to intracerebral needle injury. Following a single IP injection, NMO-IgG distributed rapidly to peripheral AQP4-expressing cells (kidney collecting duct, gastric glands, airways, skeletal muscle) and area postrema in brain, but not elsewhere in the central nervous system; however, no pathology was seen in brain, spinal cord, optic nerve or peripheral tissues. After testing various maneuvers to produce NMO-IgG-dependent pathology in brain, we found that transient puncture of brain parenchyma with a 28-gauge needle in NMO-IgG seropositive rats produced robust NMO pathology around the needle track, with loss of AQP4 and glial fibrillary acidic protein, granulocyte and macrophage infiltration, centrovascular deposition of activated complement, and blood-brain barrier disruption, with demyelination by 5 days. Pathology was not seen in rats receiving control (non-NMO) human IgG or in NMO-IgG-seropositive rats made complement-deficient by cobra venom factor. Interestingly, at 1 day a reversible, multifocal astrocytopathy was seen with loss of AQP4 and GFAP (but not myelin) in areas away from the needle track. CONCLUSIONS: NMO-IgG-seropositivity alone is not sufficient to cause NMO pathology in rats, but a single intracerebral needle insertion, without pre-existing inflammation or infusion of pro-inflammatory factors, was sufficient to produce robust NMO pathology in seropositive rats.

Neuroprotective effect of aquaporin-4 deficiency in a mouse model of severe global cerebral ischemia produced by transient 4-vessel occlusion.

Astrocyte water channel aquaporin-4 (AQP4) facilitates water movement across the blood-brain barrier and into injured astrocytes. We previously showed reduced cytotoxic brain edema with improved neurological outcome in AQP4 knockout mice in water intoxication, infection and cerebral ischemia. Here, we established a 4-vessel transient occlusion model to test the hypothesis that AQP4 deficiency in mice could improve neurological outcome following severe global cerebral ischemia as occurs in cardiac arrest/resuscitation. Mice were subjected to 10-min transient bilateral carotid artery occlusion at 24h after bilateral vertebral artery cauterization. Cerebral blood flow was reduced during occlusion by >94% in both AQP4(+/+) and AQP4(-/-) mice. The primary outcome, neurological score, was remarkably better at 3 and 5 days after occlusion in AQP4(-/-) than in AQP4(+/+) mice, and survival was significantly improved as well. Brain water content was increased by 2.8±0.4% in occluded AQP4(+/+) mice, significantly greater than that of 0.3±0.6% in AQP4(-/-) mice. Histological examination and immunofluorescence of hippocampal sections at 5 days showed significantly greater neuronal loss in the CA1 region of hippocampus in AQP4(+/+) than AQP4(-/-) mice. The neuroprotection in mice conferred by AQP4 deletion following severe global cerebral ischemia provides proof-of-concept for therapeutic AQP4 inhibition to improve neurological outcome in cardiac arrest.

Experimental mouse model of optic neuritis with inflammatory demyelination produced by passive transfer of neuromyelitis optica-immunoglobulin G.

BACKGROUND: Although optic neuritis (ON) is a defining feature of neuromyelitis optica (NMO), appropriate animal models of NMO ON are lacking. Most NMO patients are seropositive for immunoglobulin G autoantibodies (NMO-IgG) against the astrocyte water channel aquaporin-4 (AQP4). METHODS: Several approaches were tested to develop a robust, passive-transfer mouse model of NMO ON, including NMO-IgG and complement delivery by: (i) retrobulbar infusion; (ii) intravitreal injection; (iii) a single intracranial injection near the optic chiasm; and (iv) 3-days continuous intracranial infusion near the optic chiasm. RESULTS: Little ON or retinal pathology was seen using approaches (i) to (iii). Using approach (iv), however, optic nerves showed characteristic NMO pathology, with loss of AQP4 and glial fibrillary acidic protein immunoreactivity, granulocyte and macrophage infiltration, deposition of activated complement, demyelination and axonal injury. Even more extensive pathology was created in mice lacking complement inhibitor protein CD59, or using a genetically modified NMO-IgG with enhanced complement effector function, including significant loss of retinal ganglion cells. In control studies, optic nerve pathology was absent in treated AQP4-deficient mice, or in wild-type mice receiving control (non-NMO) IgG and complement. CONCLUSION: Passive transfer of NMO-IgG and complement by continuous infusion near the optic chiasm in mice is sufficient to produce ON with characteristic NMO pathology. The mouse model of NMO ON should be useful in further studies of NMO pathogenesis mechanisms and therapeutics.