Neurotoxicity of ischemic astrocytes involves STAT3-mediated metabolic switching and depends on glycogen usage.

Astrocytic responses are critical for the maintenance of neuronal networks in health and disease. In stroke, reactive astrocytes undergo functional changes potentially contributing to secondary neurodegeneration, but the mechanisms of astrocyte-mediated neurotoxicity remain elusive. Here, we investigated metabolic reprogramming in astrocytes following ischemia-reperfusion in vitro, explored their role in synaptic degeneration, and verified the key findings in a mouse model of stroke. Using indirect cocultures of primary mouse astrocytes and neurons, we demonstrate that transcription factor STAT3 controls metabolic switching in ischemic astrocytes promoting lactate-directed glycolysis and hindering mitochondrial function. Upregulation of astrocytic STAT3 signaling associated with nuclear translocation of pyruvate kinase isoform M2 and hypoxia response element activation. Reprogrammed thereby, the ischemic astrocytes induced mitochondrial respiration failure in neurons and triggered glutamatergic synapse loss, which was prevented by inhibiting astrocytic STAT3 signaling with Stattic. The rescuing effect of Stattic relied on the ability of astrocytes to utilize glycogen bodies as an alternative metabolic source supporting mitochondrial function. After focal cerebral ischemia in mice, astrocytic STAT3 activation was associated with secondary synaptic degeneration in the perilesional cortex. Inflammatory preconditioning with LPS increased astrocytic glycogen content, reduced synaptic degeneration, and promoted neuroprotection post stroke. Our data indicate the central role of STAT3 signaling and glycogen usage in reactive astrogliosis and suggest novel targets for restorative stroke therapy.

Neural precursor cell delivery induces acute post-ischemic cerebroprotection, but fails to promote long-term stroke recovery in hyperlipidemic mice due to mechanisms that include pro-inflammatory responses associated with brain hemorrhages.

BACKGROUND: The intravenous delivery of adult neural precursor cells (NPC) has shown promising results in enabling cerebroprotection, brain tissue remodeling, and neurological recovery in young, healthy stroke mice. However, the translation of cell-based therapies to clinical settings has encountered challenges. It remained unclear if adult NPCs could induce brain tissue remodeling and recovery in mice with hyperlipidemia, a prevalent vascular risk factor in stroke patients. METHODS: Male mice on a normal (regular) diet or on cholesterol-rich Western diet were exposed to 30 min intraluminal middle cerebral artery occlusion (MCAO). Vehicle or 106 NPCs were intravenously administered immediately after reperfusion, at 3 day and 7 day post-MCAO. Neurological recovery was evaluated using the Clark score, Rotarod and tight rope tests over up to 56 days. Histochemistry and light sheet microscopy were used to examine ischemic injury and brain tissue remodeling. Immunological responses in peripheral blood and brain were analyzed through flow cytometry. RESULTS: NPC administration reduced infarct volume, blood-brain barrier permeability and the brain infiltration of neutrophils, monocytes, T cells and NK cells in the acute stroke phase in both normolipidemic and hyperlipidemic mice, but increased brain hemorrhage formation and neutrophil, monocyte and CD4+ and CD8+ T cell counts and activation in the blood of hyperlipidemic mice. While neurological deficits in hyperlipidemic mice were reduced by NPCs at 3 day post-MCAO, NPCs did not improve neurological deficits at later timepoints. Besides, NPCs did not influence microglia/macrophage abundance and activation (assessed by morphology analysis), astroglial scar formation, microvascular length or branching point density (evaluated using light sheet microscopy), long-term neuronal survival or brain atrophy in hyperlipidemic mice. CONCLUSIONS: Intravenously administered NPCs did not have persistent effects on post-ischemic neurological recovery and brain remodeling in hyperlipidemic mice. These findings highlight the necessity of rigorous investigations in vascular risk factor models to fully assess the long-term restorative effects of cell-based therapies. Without comprehensive studies in such models, the clinical potential of cell-based therapies cannot be definitely determined.

Postacute Delivery of GABAA α5 Antagonist Promotes Postischemic Neurological Recovery and Peri-infarct Brain Remodeling.

Background and Purpose- Poststroke, neuronal excitability is tonically reduced in peri-infarct tissue via inhibitory influences of extrasynaptic GABAA receptors. We hypothesized that GABAA α5 blockade by the competitive antagonist S44819 enhances postischemic neurological recovery, brain remodeling, and neuroplasticity. Methods- In an explorative study followed by a confirmation study, male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. Starting 72 hours poststroke, vehicle or S44819 (3 or 10 mg/kg, BID) was delivered orally for 28 days. Neurological recovery, perilesional tissue remodeling, and contralesional pyramidal tract plasticity were evaluated for 42 days, that is, 14 days after completion of S44819 delivery. Results- S44819, delivered at 10 but not 3 mg/kg, persistently improved motor coordination and spatial memory in both studies. Striatal atrophy was reduced by 10 mg/kg S44819 at 42 days post-treatment onset, and neuronal long-term survival in the peri-infarct striatum was increased. Delayed neuroprotection was associated with reduced peri-infarct astrogliosis, increased peri-infarct brain capillary density, and increased neural precursor cell proliferation and differentiation in proximity to the ipsilesional subventricular zone. Contralesional pyramidal tract plasticity, evaluated by anterograde tract tracing at the level of the red nucleus, was not influenced by S44819. Concentrations of neurotrophic (brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor) and angiogenic (vascular endothelial growth factor and basic fibroblast growth factor) growth factors were elevated by 10 mg/kg S44819 in peri-infarct but not contralesional brain tissue. Conclusions- Our data demonstrate that S44819 enhances neurological recovery and peri-infarct brain remodeling in the postacute stroke phase.

Sustained neurological recovery induced by resveratrol is associated with angioneurogenesis rather than neuroprotection after focal cerebral ischemia.

According to the French paradox, red wine consumption reduces the incidence of vascular diseases even in the presence of highly saturated fatty acid intake. This phenomenon is widely attributed to the phytoalexin resveratrol, a red wine ingredient. Experimental studies suggesting that resveratrol has neuroprotective properties mostly used prophylactic delivery strategies associated with short observation periods. These studies did not allow conclusions to be made about resveratrol's therapeutic efficacy post-stroke. Herein, we systematically analyzed effects of prophylactic, acute and post-acute delivery of resveratrol (50mg/kg) on neurological recovery, tissue survival, and angioneurogenesis after focal cerebral ischemia induced by intraluminal middle cerebral artery occlusion in mice. Over an observation period of four weeks, only prolonged post-acute resveratrol delivery induced sustained neurological recovery as assessed by rota rod, tight rope and corner turn tests. Although prophylactic and acute resveratrol delivery reduced infarct volume and enhanced blood-brain-barrier integrity at 2 days post-ischemia by elevating resveratrol's downstream signal sirtuin-1, increasing cell survival signals (phosphorylated Akt, heme oxygenase-1, Bcl-2) and decreasing cell death signals (Bax, activated caspase-3), a sustained reduction of infarct size on day 28 was not observed in any of the three experimental conditions. Instead, enhanced angiogenesis and neurogenesis were noted in animals receiving post-acute resveratrol delivery, which were associated with elevated concentrations of GDNF and VEGF in the brain. Thus, sustained neurological recovery induced by resveratrol depends on successful brain remodeling rather than structural neuroprotection. The recovery promoting effect of delayed resveratrol delivery opens promising perspectives for stroke therapy.

Acid sphingomyelinase inhibition induces cerebral angiogenesis post-ischemia/reperfusion in an oxidative stress-dependent way and promotes endothelial survival by regulating mitochondrial metabolism.

Acid sphingomyelinase (ASM) inhibitors are widely used for the treatment of post-stroke depression. They promote neurological recovery in animal stroke models via neurorestorative effects. In a previous study, we found that antidepressants including amitriptyline, fluoxetine, and desipramine increase cerebral angiogenesis post-ischemia/reperfusion (I/R) in an ASM-dependent way. To elucidate the underlying mechanisms, we investigated the effects of the functional ASM inhibitor amitriptyline in two models of I/R injury, that is, in human cerebral microvascular endothelial hCMEC/D3 cells exposed to oxygen-glucose deprivation and in mice exposed to middle cerebral artery occlusion (MCAO). In addition to our earlier studies, we now show that amitriptyline increased mitochondrial reactive oxygen species (ROS) formation in hCMEC/D3 cells and increased ROS formation in the vascular compartment of MCAO mice. ROS formation was instrumental for amitriptyline's angiogenic effects. ROS formation did not result in excessive endothelial injury. Instead, amitriptyline induced a profound metabolic reprogramming of endothelial cells that comprised reduced endothelial proliferation, reduced mitochondrial energy metabolism, reduced endoplasmic reticulum stress, increased autophagy/mitophagy, stimulation of antioxidant responses and inhibition of apoptotic cell death. Specifically, the antioxidant heme oxygenase-1, which was upregulated by amitriptyline, mediated amitriptyline's angiogenic effects. Thus, heme oxygenase-1 knockdown severely compromised angiogenesis and abolished amitriptyline's angiogenic responses. Our data demonstrate that ASM inhibition reregulates a complex network of metabolic and mitochondrial responses post-I/R that contribute to cerebral angiogenesis without compromising endothelial survival.

Stroke and myocardial infarction induce neutrophil extracellular trap release disrupting lymphoid organ structure and immunoglobulin secretion.

Post-injury dysfunction of humoral immunity accounts for infections and poor outcomes in cardiovascular diseases. Among immunoglobulins (Ig), IgA, the most abundant mucosal antibody, is produced by plasma B cells in intestinal Peyer's patches (PP) and lamina propria. Here we show that patients with stroke and myocardial ischemia (MI) had strongly reduced IgA blood levels. This was phenocopied in experimental mouse models where decreased plasma and fecal IgA were accompanied by rapid loss of IgA-producing plasma cells in PP and lamina propria. Reduced plasma IgG was detectable in patients and experimental mice 3-10 d after injury. Stroke/MI triggered the release of neutrophil extracellular traps (NETs). Depletion of neutrophils, NET degradation or blockade of NET release inhibited the loss of IgA+ cells and circulating IgA in experimental stroke and MI and in patients with stroke. Our results unveil how tissue-injury-triggered systemic NET release disrupts physiological Ig secretion and how this can be inhibited in patients.

MicroRNA-124 protects against focal cerebral ischemia via mechanisms involving Usp14-dependent REST degradation.

MicroRNAs (miRNAs) are highly conserved non-coding RNAs modulating gene expression via mRNA binding. Recent work suggests an involvement of miRNAs in cardiovascular diseases including stroke. As such, the brain-abundant miR-124 and its transcriptional repressor RE1-silencing transcription factor (REST) do not only have elementary roles in the developing and the adult brain, but also alter expression upon cerebral ischemia. However, the therapeutic potential of miR-124 against stroke and the mechanisms involved remain elusive. Here, we analyzed the therapeutic potential of ectopic miR-124 against stroke and its underlying mechanisms with regard to the interaction between miR-124 and REST. Our results show that viral vector-mediated miR-124 delivery increased the resistance of cultured oxygen-glucose-deprived cortical neurons in vitro and reduced brain injury as well as functional impairment in mice submitted to middle cerebral artery occlusion. Likewise, miR-124 induced enhanced neurovascular remodeling leading to increased angioneurogenesis 8 weeks post-stroke. While REST abundance increased upon stroke, the increase was prevented by miR-124 despite a so far unknown negative feedback loop between miR-124 and REST. Rather, miR-124 decreased the expression of the deubiquitinating enzyme Usp14, which has two conserved miR-124-binding sites in the 3'UTR of its mRNA, and thereby mediated reduced REST levels. The down-regulation of REST by miR-124 was also mimicked by the Usp14 inhibitor IU-1, suggesting that miR-124 promotes neuronal survival under ischemic conditions via Usp14-dependent REST degradation. Ectopic miR-124 expression, therefore, appears as an attractive and novel tool in stroke treatment, mediating neuroprotection via a hitherto unknown mechanism that involves Usp14-dependent REST degradation.

The novel proteasome inhibitor BSc2118 protects against cerebral ischaemia through HIF1A accumulation and enhanced angioneurogenesis.

Only a minority of stroke patients receive thrombolytic therapy. Therefore, new therapeutic strategies focusing on neuroprotection are under review, among which, inhibition of the proteasome is attractive, as it affects multiple cellular pathways. As proteasome inhibitors like bortezomib have severe side effects, we applied the novel proteasome inhibitor BSc2118, which is putatively better tolerated, and analysed its therapeutic potential in a mouse model of cerebral ischaemia. Stroke was induced in male C57BL/6 mice using the intraluminal middle cerebral artery occlusion model. BSc2118 was intrastriatally injected 12 h post-stroke in mice that had received normal saline or recombinant tissue-plasminogen activator injections during early reperfusion. Brain injury, behavioural tests, western blotting, MMP9 zymography and analysis of angioneurogenesis were performed for up to 3 months post-stroke. Single injections of BSc2118 induced long-term neuroprotection, reduced functional impairment, stabilized blood-brain barrier through decreased MMP9 activity and enhanced angioneurogenesis when given no later than 12 h post-stroke. On the contrary, recombinant tissue-plasminogen activator enhanced brain injury, which was reversed by BSc2118. Protein expression of the transcription factor HIF1A was significantly increased in saline-treated and recombinant tissue-plasminogen activator-treated mice after BSc2118 application. In contrast, knock-down of HIF1A using small interfering RNA constructs or application of the HIF1A inhibitor YC1 (now known as RNA-binding motif, single-stranded-interacting protein 1 (RBMS1)) reversed BSc2118-induced neuroprotection. Noteworthy, loss of neuroprotection after combined treatment with BSc2118 and YC1 in recombinant tissue-plasminogen activator-treated animals was in the same order as in saline-treated mice, i.e. reduction of recombinant tissue-plasminogen activator toxicity through BSc2118 did not solely depend on HIF1A. Thus, the proteasome inhibitor BSc2118 is a promising new candidate for stroke therapy, which may in addition alleviate recombinant tissue-plasminogen activator-induced brain toxicity.

Comparative methylation profiles and telomerase biology of mouse multipotent adult germline stem cells and embryonic stem cells.

Recently, several groups described the isolation of mouse spermatogonial stem cells (SSCs) and their potential to develop to embryonic stem cell (ESC)-like cells, so-called multipotent germline stem cells (mGSCs). We were the first to derive such mGSCs from SSCs isolated from adult mouse testis and, therefore, called these mGSCs multipotent adult germline stem cells (maGSCs). Here, we comparatively analyzed gene-specific and global DNA methylation profiles as well as the telomerase biology of several maGSC and male ESC lines. We show that undifferentiated maGSCs are very similar to undifferentiated male ESCs with regard to global DNA methylation, methylation of pluripotency marker gene loci, telomerase activity and telomere length. Imprinted gene methylation levels were generally lower in undifferentiated maGSCs than in undifferentiated male ESCs, but, compared with undifferentiated mGSCs derived by other groups, more similar to those of male ESCs. Differentiation of maGSCs increased the methylation of three of the four analyzed imprinted genes to almost somatic methylation patterns, but dramatically decreased global DNA methylation. Our findings further substantiate the pluripotency of maGSCs and their potential for regenerative medicine.

Very Delayed Remote Ischemic Post-conditioning Induces Sustained Neurological Recovery by Mechanisms Involving Enhanced Angioneurogenesis and Peripheral Immunosuppression Reversal.

Ischemic conditioning is defined as a transient and subcritical period of ischemia integrated in an experimental paradigm that involves a stimulus of injurious ischemia, activating endogenous tissue repair mechanisms that lead to cellular protection under pathological conditions like stroke. Whereas ischemic pre-conditioning is irrelevant for stroke treatment, ischemic post-conditioning, and especially non-invasive remote ischemic post-conditioning (rPostC) is an innovative and potential strategy for stroke treatment. Although rPostC has been shown to induce neuroprotection in stroke models before, resulting in some clinical trials on the way, fundamental questions with regard to its therapeutic time frame and its underlying mechanisms remain elusive. Hence, we herein used a model of non-invasive rPostC of hind limbs after cerebral ischemia in male C57BL6 mice, studying the optimal timing for the application of rPostC and its underlying mechanisms for up to 3 months. Mice undergoing rPostC underwent three different paradigms, starting with the first cycle of rPostC 12 h, 24 h, or 5 days after stroke induction, which is a very delayed time point of rPostC that has not been studied elsewhere. rPostC as applied within 24 h post-stroke induces reduction of infarct volume on day three. On the contrary, very delayed rPostC does not yield reduction of infarct volume on day seven when first applied on day five, albeit long-term brain injury is significantly reduced. Likewise, very delayed rPostC yields sustained neurological recovery, whereas early rPostC (i.e., <24 h) results in transient neuroprotection only. The latter is mediated via heat shock protein 70 that is a well-known signaling protein involved in the pathophysiological cellular cascade of cerebral ischemia, leading to decreased proteasomal activity and decreased post-stroke inflammation. Very delayed rPostC on day five, however, induces a pleiotropic effect, among which a stimulation of angioneurogenesis, a modulation of the ischemic extracellular milieu, and a reversal of the stroke-induced immunosuppression occur. As such, very delayed rPostC appears to be an attractive tool for future adjuvant stroke treatment that deserves further preclinical attention before large clinical trials are in order, which so far have predominantly focused on early rPostC only.

Lithium-induced neuroprotection in stroke involves increased miR-124 expression, reduced RE1-silencing transcription factor abundance and decreased protein deubiquitination by GSK3ß inhibition-independent pathways.

Lithium promotes acute poststroke neuronal survival, which includes mechanisms that are not limited to GSK3ß inhibition. However, whether lithium induces long-term neuroprotection and enhanced brain remodeling is unclear. Therefore, mice were exposed to transient middle cerebral artery occlusion and lithium (1 mg/kg bolus followed by 2 mg/kg/day over up to 7 days) was intraperitoneally administered starting 0-9 h after reperfusion onset. Delivery of lithium no later than 6 h reduced infarct volume on day 2 and decreased brain edema, leukocyte infiltration, and microglial activation, as shown by histochemistry and flow cytometry. Lithium-induced neuroprotection persisted throughout the observation period of 56 days and was associated with enhanced neurological recovery. Poststroke angioneurogenesis and axonal plasticity were also enhanced by lithium. On the molecular level, lithium increased miR-124 expression, reduced RE1-silencing transcription factor abundance, and decreased protein deubiquitination in cultivated cortical neurons exposed to oxygen-glucose deprivation and in brains of mice exposed to cerebral ischemia. Notably, this effect was not mimicked by pharmacological GSK3ß inhibition. This study for the first time provides efficacy data for lithium in the postacute ischemic phase, reporting a novel mechanism of action, i.e. increased miR-124 expression facilitating REST degradation by which lithium promotes postischemic neuroplasticity and angiogenesis.

Conditioned Medium Derived from Neural Progenitor Cells Induces Long-term Post-ischemic Neuroprotection, Sustained Neurological Recovery, Neurogenesis, and Angiogenesis.

Adult neural progenitor cells (NPCs) induce post-ischemic long-term neuroprotection and brain remodeling by releasing of survival- and plasticity-promoting mediators. To evaluate whether secreted factors may mimic neuroprotective and restorative effects of NPCs, we exposed male C57BL6 mice to focal cerebral ischemia and intravenously applied conditioned medium (CM) derived from subventricular zone NPCs. CM dose-dependently reduced infarct volume and brain leukocyte infiltration after 48 h when delivered up to 12 h after focal cerebral ischemia. Neuroprotection persisted in the post-acute stroke phase yielding enhanced neurological recovery that lasted throughout the 28-day observation period. Increased Bcl-2, phosphorylated Akt and phosphorylated STAT-3 abundance, and reduced caspase-3 activity and Bax abundance were noted in ischemic brains of CM-treated mice at 48 h post-stroke, indicative of enhanced cell survival signaling. Long-term neuroprotection was associated with increased brain glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF) concentrations at 28 days resulting in increased neurogenesis and angiogenesis. The observation that NPC-derived CM induces sustained neuroprotection and neurological recovery suggests that cell transplantation may be dispensable when secreted factors are instead administered.

Ischemic Post-Conditioning Induces Post-Stroke Neuroprotection via Hsp70-Mediated Proteasome Inhibition and Facilitates Neural Progenitor Cell Transplantation.

In view of the failure of pharmacological therapies, alternative strategies promoting post-stroke brain repair are needed. Post-conditioning is a potentially promising therapeutic strategy, which induces acute neuroprotection against ischemic injury. To elucidate longer lasting actions of ischemic post-conditioning, mice were exposed to a 60-min stroke and post-conditioning by an additional 10-min stroke that was induced 10 min after reperfusion onset. Animals were sacrificed 24 h or 28 days post-stroke. Post-conditioning reduced infarct volume and neurological deficits 24 h post-stroke, enhancing blood-brain barrier integrity, reducing brain leukocyte infiltration, and reducing oxidative stress. On the molecular level, post-conditioning yielded increased Hsp70 expression, whereas nuclear factor (NF)-κB and proteasome activities were decreased. Reduced infarct volume and proteasome inhibition were reversed by Hsp70 knockdown, suggesting a critical role of the Hsp70 proteasome pathway in ischemic post-conditioning. The survival-promoting effects of ischemic post-conditioning, however, were not sustainable as neuroprotection and neurological recovery were lost 28 days post-stroke. Although angioneurogenesis was not increased by post-conditioning, the favorable extracellular milieu facilitated intracerebral transplantation of neural progenitor cells 6 h post-stroke, resulting in persisted neuroprotection and neurological recovery. Thus, post-conditioning might support brain repair processes, but in view of its transient, neuroprotection is unlikely useful as stroke therapy in its current form.

Association of Plasma ß-Amyloid with Cognitive Performance and Decline in Chronic Kidney Disease.

Decreased ß-amyloid (Aß) clearance from the brain has been suggested to contribute to cerebral Aß accumulation in Alzheimer's disease. Based on the idea of a dynamic Aß equilibrium in different body compartments, plasma Aß levels have been investigated as biomarker candidates for preclinical Alzheimer's pathology, yet with inconsistent results. Since the kidneys are involved in Aß elimination from the blood, we evaluated how chronic kidney disease (CKD) affects the association between plasma Aß and cognitive deficits and cognitive decline. In 28 CKD patients, stages 3-5D, and 26 control subjects with comparable vascular risk profile from the New Tools for the Prevention of Cardiovascular Disease in Chronic Kidney Disease (NTCVD) cohort, plasma total Aß was determined with a highly sensitive electrochemiluminescence immunoassay. Cognition was evaluated using a comprehensive battery of ten neuropsychological tests at baseline and 2-year follow-up. Subjects with high plasma Aß level (above median) demonstrated a significantly worse baseline cognitive performance than subjects exhibiting low Aß level (summary score of global cognitive performance at baseline z = -0.46 ± 0.76 vs z = -0.08 ± 0.57, p = 0.045). Cognitive performance moderately decreased over the 2-year follow-up in subjects with high plasma Aß level (Δz = -0.13 ± 0.51), but increased in subjects with low plasma Aß level (Δz = 0.16 ± 0.41, p = 0.023). In linear regression analyses, baseline plasma Aß was significantly associated with cognitive decline both in unadjusted analyses (ß = -0.28, 95% CI = -0.55 to -0.01) and analyses adjusted for age (ß = -0.27, 95% CI = -0.54 to -0.01). Our results suggest the utility of plasma Aß level in predicting cognitive decline in patients suffering from CKD.

Systemic Proteasome Inhibition Induces Sustained Post-stroke Neurological Recovery and Neuroprotection via Mechanisms Involving Reversal of Peripheral Immunosuppression and Preservation of Blood-Brain-Barrier Integrity.

In view of its profound effect on cell survival and function, the modulation of the ubiquitin-proteasome-system has recently been shown to promote neurological recovery and brain remodeling after focal cerebral ischemia. Hitherto, local intracerebral delivery strategies were used, which can hardly be translated to human patients. We herein analyzed effects of systemic intraperitoneal delivery of the proteasome inhibitor BSc2118 on neurological recovery, brain injury, peripheral and cerebral immune responses, neurovascular integrity, as well as cerebral neurogenesis and angiogenesis in a mouse model of transient intraluminal middle cerebral artery occlusion. Systemic delivery of BSc2118 induced acute neuroprotection reflected by reduced infarct volume when delivered up to 9 h post-stroke. The latter was associated with reduced brain edema and stabilization of blood-brain-barrier integrity, albeit cerebral proteasome activity was only mildly reduced. Neuronal survival persisted in the post-acute stroke phase up to 28 days post-stroke and was associated with improved neurological recovery when the proteasome inhibitor was continuously delivered over 7 days. Systemic proteasome inhibition prevented stroke-induced acute leukocytosis in peripheral blood and reversed the subsequent immunosuppression, namely, the reduction of blood lymphocyte and granulocyte counts. On the contrary, post-ischemic brain inflammation, cerebral HIF-1α abundance, cell proliferation, neurogenesis, and angiogenesis were not influenced by the proteasome inhibitor. The modulation of peripheral immune responses might thus represent an attractive target for the clinical translation of proteasome inhibitors.

Factors Responsible for Plasma ß-Amyloid Accumulation in Chronic Kidney Disease.

Disturbed brain-to-blood elimination of ß-amyloid (Aß) promotes cerebral Aß accumulation in Alzheimer's disease. Considering that the kidneys are involved in Aß elimination from the blood, we evaluated how chronic kidney disease (CKD) affects plasma Aß. In 106 CKD patients stages 3-5 (including 19 patients on hemodialysis and 15 kidney recipients), 53 control subjects with comparable vascular risk profile and 10 kidney donors, plasma Aß was determined using electrochemiluminescence immunoassay and gel electrophoresis followed by Western blotting. Plasma Aß increased with CKD stage (control = 182.98 ± 76.73 pg/ml; CKD3A = 248.34 ± 103.77 pg/ml; CKD3B = 259.25 ± 97.74 pg/ml; CKD4 = 489.16 ± 154.16 pg/ml; CKD5 = 721.19 ± 291.69 pg/ml) and was not influenced by hemodialysis (CKD5D = 697.97 ± 265.91 pg/ml). Renal transplantation reduced plasma Aß (332.57 ± 162.82 pg/ml), whereas kidney donation increased it (251.51 ± 34.34 pg/ml). Gel electrophoresis confirmed stage-dependent elevation namely of Aß1-40, the most abundant Aß peptide. In a multivariable regression including age, sex, estimated glomerular filtration rate (eGFR), potassium, hemoglobin, urine urea, and urine total protein, the factors eGFR (ß = -0.42, p < 0.001), hemoglobin (ß = -0.17, p = 0.020), and urine protein (ß = 0.26, p = 0.008) were associated with plasma Aß. In a regression including age, sex, eGFR, potassium, hemoglobin and the vascular risk factors systolic blood pressure, smoking, LDL, HDL, HbA1c, body mass index, brain-derived natriuretic peptide and fibrinogen, the factors eGFR (ß = -0.53, p < 0.001), body mass index (ß = -0.17, p = 0.022), and fibrinogen (ß = 0.18, p = 0.024) were associated with plasma Aß. Our results demonstrate a stage-dependent plasma Aß increase that is augmented by loss of glomerulotubular integrity, low body weight, and inflammation, demonstrating a multifaceted role of renal dysfunction in Aß retention.

Rapid Regulation of Depression-Associated Genes in a New Mouse Model Mimicking Interferon-α-Related Depression in Hepatitis C Virus Infection.

Major depression is a serious side effect of interferon-α (IFN-α), which is used in the therapy of hepatitis C virus (HCV) infection. Due to the lack of reproducible animal models, the mechanisms underlying IFN-α-related depression are largely unknown. We herein established a mouse model, in which murine IFN-α (250 IU/day) and polyinosinic/polycytidylic acid (poly(I:C); 1 µg/day), a toll-like receptor-3 (TLR3) agonist that mimics the effect of HCV double-strand RNA, were continuously infused into the lateral ventricle via miniosmotic pumps over up to 14 days. The delivery of IFN-α and poly(I:C), but not of IFN-α or poly(I:C) alone, resulted in a reproducible depression-like state that was characterized by reduced exploration behavior in open-field tests, increased immobility in tail suspension and forced swimming tests, and a moderate loss of body weight. In the hippocampus and prefrontal cortex, the pro-inflammatory genes TNF-α, IL-6, tissue inhibitor of metalloproteinases-1 (Timp-1), CXC motif ligand-1 (Cxcl1), Cxcl10, and CC motif ligand-5 (Ccl5) were synergistically induced by IFN-α and poly(I:C), most pronounced after 14-day exposure. In comparison, the interferon-inducible genes of signal transducer and activator of transcription-1 (Stat1), guanylate binding protein-1 (Gbp1), proteasome subunit-ß type-9 (Psmb9), ubiquitin-conjugating enzyme E2L-6 (Ube2l6), receptor transporter protein-4 (Rtp4), and GTP cyclohydrolase-1 (Gch1), which had previously been elevated in the blood of IFN-α-treated patients developing depression, in the brains of suicidal individuals, and in primary neurons exposed to IFN-α and poly(I:C), were induced even earlier, reaching maximum levels mostly after 24 hours. We propose that interferon-inducible genes might be useful markers of imminent depression.

The indirect NMDAR inhibitor flupirtine induces sustained post-ischemic recovery, neuroprotection and angioneurogenesis.

N-methyl-D-aspartate receptor (NMDAR) activation induces excitotoxicity, contributing to post-stroke brain injury. Hitherto, NMDAR deactivation failed in clinical trials due to insufficient pre-clinical study designs and drug toxicity. Flupirtine is an indirect NMDAR antagonist being used as analgesic in patients. Taking into account its tolerability profile, we evaluated effects of flupirtine on post-stroke tissue survival, neurological recovery and brain remodeling.Mice were exposed to stroke and intraperitoneally treated with saline (control) or flupirtine at various doses (1-10 mg/kg) and time-points (0-12 hours). Tissue survival and cell signaling were studied on day 2, whereas neurological recovery and tissue remodeling were analyzed until day 84.Flupirtine induced sustained neuroprotection, when delivered up to 9 hours. The latter yielded enhanced neurological recovery that persisted over three months and which was accompanied by enhanced angioneurogenesis. On the molecular level, inhibition of calpain activation was noted, which was associated with increased signal-transducer-and-activator-of-transcription-6 (STAT6) abundance, reduced N-terminal-Jun-kinase and NF-κB activation, as well as reduced proteasomal activity. Consequently, blood-brain-barrier integrity was stabilized, oxidative stress was reduced and brain leukocyte infiltration was diminished.In view of its excellent tolerability, considering its sustained effects on neurological recovery, brain tissue survival and remodeling, flupirtine is an attractive candidate for stroke therapy.

Post-stroke transplantation of adult subventricular zone derived neural progenitor cells--A comprehensive analysis of cell delivery routes and their underlying mechanisms.

With neuroprotective approaches having failed until recently, current focus on experimental stroke research has switched towards manipulation of post-ischemic neuroregeneration. Transplantation of subventricular zone (SVZ) derived neural progenitor cells (NPCs) is a promising strategy for promotion of neurological recovery. Yet, fundamental questions including the optimal cell delivery route still have to be addressed. Consequently, male C57BL6 mice were exposed to transient focal cerebral ischemia and allowed to survive for as long as 84 days post-stroke. At 6h post-stroke, NPCs were grafted using six different cell delivery routes, i.e., intravenous, intraarterial, ipsilateral intrastriatal, contralateral intrastriatal, ipsilateral intraventricular and ipsilateral intracortical injection. Control mice received PBS only using the aforementioned delivery routes. Intralesional numbers of GFP(+) NPCs were high only after ipsilateral intrastriatal transplantation, whereas other injection paradigms only yielded comparatively small numbers of grafted cells. However, acute neuroprotection and improved functional outcome were observed after both systemic (i.e., intraarterial and intravenous) and ipsilateral intrastriatal transplantation only. Whereas systemic cell delivery induced acute and long-term neuroprotection, reduction of brain injury after ipsilateral intrastriatal cell grafting was only temporary, in line with the loss of transplanted NPCs in the brain. Both systemic and ipsilateral intrastriatal NPC delivery reduced microglial activation and leukocyte invasion, thus reducing free radical formation within the ischemic brain. On the contrary, only systemic NPC administration stabilized the blood-brain-barrier and reduced leukocytosis in the blood. Although intraarterial NPC transplantation was as effective as intravenous cell grafting, mortality of stroke mice was high using the intraarterial delivery route. Consequently, intravenous delivery of native NPCs in our experimental model is an attractive and effective strategy for stroke therapy that deserves further proof-of-concept studies.

The indirect NMDAR antagonist acamprosate induces postischemic neurologic recovery associated with sustained neuroprotection and neuroregeneration.

Cerebral ischemia stimulates N-methyl-d-aspartate receptors (NMDARs) resulting in increased calcium concentration and excitotoxicity. Yet, deactivation of NMDAR failed in clinical studies due to poor preclinical study designs or toxicity of NMDAR antagonists. Acamprosate is an indirect NMDAR antagonist used for patients with chronic alcohol dependence. We herein analyzed the therapeutic potential of acamprosate on brain injury, neurologic recovery and their underlying mechanisms. Mice were exposed to cerebral ischemia, treated with intraperitoneal injections of acamprosate or saline (controls), and allowed to survive until 3 months. Acamprosate yielded sustained neuroprotection and increased neurologic recovery when given no later than 12 hours after stroke. The latter was associated with increased postischemic angioneurogenesis, albeit acamprosate did not stimulate angioneurogenesis itself. Rather, increased angioneurogenesis was due to inhibition of calpain-mediated pro-injurious signaling cascades. As such, acamprosate-mediated reduction of calpain activity resulted in decreased degradation of p35, increased abundance of the pro-survival factor STAT6, and reduced N-terminal-Jun-kinase activation. Inhibition of calpain was associated with enhanced stability of the blood-brain barrier, reduction of oxidative stress and cerebral leukocyte infiltration. Taken into account its excellent tolerability, its sustained effects on neurologic recovery, brain tissue survival, and neural remodeling, acamprosate is an intriguing candidate for adjuvant future stroke treatment.