Yuriy Pomeshchik

Nrf2 Regulates Neurogenesis and Protects Neural Progenitor Cells Against Aβ Toxicity

Neural stem/progenitor cells (NPCs) proliferate and produce new neurons in neurogenic areas throughout the lifetime. While these cells represent potential therapeutic treatment of neurodegenerative diseases, regulation of neurogenesis is not completely understood. We show that deficiency of nuclear factor erythroid 2‐related factor (Nrf2), a transcription factor induced in response to oxidative stress, prevents the ischemia‐induced increase in newborn neurons in the subgranular zone of the dentate gyrus. Consistent with this finding, the growth of NPC neurospheres was increased by lentivirus‐mediated overexpression of Nrf2 gene or by treatment with pyrrolidine dithiocarbamate (PDTC), an Nrf2 activating compound. Also, neuronal differentiation of NPCs was increased by Nrf2 overexpression or PDTC treatment but reduced by Nrf2 deficiency. To investigate the impact of Nrf2 on NPCs in Alzheimers disease (AD), we treated NPCs with amyloid beta (Aβ), a toxic peptide associated with neurodegeneration and cognitive abnormalities in AD. We found that Aβ1–42‐induced toxicity and reduction in neurosphere proliferation were prevented by Nrf2 overexpression, while Nrf2 deficiency enhanced the Aβ1–42‐induced reduction of neuronal differentiation. On the other hand, Aβ1–40 had no effect on neurosphere proliferation in wt NPCs but increased the proliferation of Nrf2 overexpressing neurospheres and reduced it in Nrf2‐deficient neurospheres. These results suggest that Nrf2 is essential for neuronal differentiation of NPCs, regulates injury‐induced neurogenesis and provides protection against Aβ‐induced NPC toxicity. Stem Cells 2014;32:1904–1916

Interleukin-33 treatment reduces secondary injury and improves functional recovery after contusion spinal cord injury

Interleukin-33 (IL-33) is a member of the interleukin-1 cytokine family and highly expressed in the naïve mouse brain and spinal cord. Despite the fact that IL-33 is known to be inducible by various inflammatory stimuli, its cellular localization in the central nervous system and role in pathological conditions is controversial. Administration of recombinant IL-33 has been shown to attenuate experimental autoimmune encephalomyelitis progression in one study, yet contradictory reports also exist. Here we investigated for the first time the pattern of IL-33 expression in the contused mouse spinal cord and demonstrated that after spinal cord injury (SCI) IL-33 was up-regulated and exhibited a nuclear localization predominantly in astrocytes. Importantly, we found that treatment with recombinant IL-33 alleviated secondary damage by significantly decreasing tissue loss, demyelination and astrogliosis in the contused mouse spinal cord, resulting in dramatically improved functional recovery. We identified both central and peripheral mechanisms of IL-33 action. In spinal cord, IL-33 treatment reduced the expression of pro-inflammatory tumor necrosis factor-alpha and promoted the activation of anti-inflammatory arginase-1 positive M2 microglia/macrophages, which chronically persisted in the injured spinal cord for up to at least 42 days after the treatment. In addition, IL-33 treatment showed a tendency towards reduced T-cell infiltration into the spinal cord. In the periphery, IL-33 treatment induced a shift towards the Th2 type cytokine profile and reduced the percentage and absolute number of cytotoxic, tumor necrosis factor-alpha expressing CD4+ cells in the spleen. Additionally, IL-33 treatment increased expression of T-regulatory cell marker FoxP3 and reduced expression of M1 marker iNOS in the spleen. Taken together, these results provide the first evidence that IL-33 administration is beneficial after CNS trauma. Treatment with IL33 may offer a novel therapeutic strategy for patients with acute contusion SCI.

Effects of human intravenous immunoglobulin on amyloid pathology and neuroinflammation in a mouse model of Alzheimer's disease

BackgroundHuman intravenous immunoglobulin (hIVIG) preparation is indicated for treating primary immunodeficiency disorders associated with impaired humoral immunity. hIVIG is known for its anti-inflammatory properties and a decent safety profile. Therefore, by virtue of its constituent natural anti-amyloid beta antibodies and anti-inflammatory effects, hIVIG is deemed to mediate beneficial effects to patients of Alzheimer’s disease (AD). Here, we set out to explore the effects of hIVIG in a mouse model of AD.MethodsWe treated APP/PS1dE9 transgenic and wild-type mice with weekly injections of a high hIVIG dose (1 g/kg) or saline for 3 or 8 months. Treatment effect on brain amyloid pathology and microglial reactivity was assessed by ELISA, immunohistochemistry, RT-PCR, and confocal microscopy.ResultsWe found no evidence for reduction in Aβ pathology; instead 8 months of hIVIG treatment significantly increased soluble levels of Aβ40 and Aβ42. In addition, we noticed a significant reduction in CD45 and elevation of Iba-1 markers in specific sub-populations of microglial cells. Long-term hIVIG treatment also resulted in significant suppression of TNF-α and increase in doublecortin positive adult-born neurons in the dentate gyrus.ConclusionsOur data indicate limited ability of hIVIG to impact amyloid burden but shows changes in microglia, pro-inflammatory gene expression, and neurogenic effects. Immunomodulation by hIVIG may account for its beneficial effect in AD patients.

Applications of the Keap1-Nrf2 system for gene and cell therapy.

Oxidative stress has been implicated to play a role in a number of acute and chronic diseases including acute injuries of the central nervous system, neurodegenerative and cardiovascular diseases, and cancer. The redox-activated transcription factor Nrf2 has been shown to protect many different cell types and organs from a variety of toxic insults, whereas in many cancers, unchecked Nrf2 activity increases the expression of cytoprotective genes and, consequently, provides growth advantage to cancerous cells. Herein, we discuss current preclinical gene therapy approaches to either increase or decrease Nrf2 activity with a special reference to neurological diseases and cancer. In addition, we discuss the role of Nrf2 in stem cell therapy for neurological disorders.

Transplanted Human Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Do Not Promote Functional Recovery of Pharmacologically Immunosuppressed Mice With Contusion Spinal Cord Injury.

Improved functional recovery after spinal cord injury by transplantation of induced pluripotent stem cell-derived neural stem/progenitor cells (iPSC-NPCs) has been reported. However, beneficial effects of iPSC-based therapy have so far been produced mostly using genetically immunodeficient rodents. Because of the long time required for generation and characterization of iPSCs, the use of autologous iPSCs for treating patients with acute spinal cord injury (SCI) is not feasible. Therefore, it is of utmost importance to investigate the effect of iPSC-based therapy on functional recovery after SCI using pharmacologically immunosuppressed, immunocompetent animal models. Here we studied the functional outcome following subacute transplantation of human iPSC-derived NPCs into contused mouse spinal cord when tacrolimus was used as an immunosuppressive agent. We show that human iPSC-derived NPCs transplanted into pharmacologically immunosuppressed C57BL/6J mice exhibited poor long-term survival and failed to improve functional recovery after SCI as measured by Basso Mouse Scale (BMS) for locomotion and CatWalk gait analysis when compared to vehicle-treated animals. The scarce effect of iPSC-based therapy observed in the current study may be attributable to insufficient immunosuppressive effect, provided by monotherapy with tacrolimus in combination with immunogenicity of transplanted cells and complex microenvironment of the injured spinal cord. Our results highlight the importance of extensive preclinical studies of transplanted cells before the clinical application of iPSC-based cell therapy is achieved.

Anti‐inflammatory effects of ADAMTS‐4 in a mouse model of ischemic stroke

ADAMTS‐4 (a disintegrin and metalloproteinase with thrombospondin motifs type 4) is a metalloprotease capable to degrade chondroitin sulfate proteoglycans leading to cartilage destruction during arthritis or to neuroplasticity during spinal cord injury (SCI). Although ADAMTS‐4 is an inflammatory‐regulated enzyme, its role during inflammation has never been investigated. The aim of this study was to investigate the role of ADAMTS‐4 in neuroinflammation. First, we evidenced an increase of ADAMTS‐4 expression in the ischemic brain hemisphere of mouse and human patients suffering from ischemic stroke. Then, we described that ADAMTS‐4 has predominantly an anti‐inflammatory effect in the CNS. Treatment of primary microglia or astrocyte cultures with low doses of a human recombinant ADAMTS‐4 prior to LPS exposure decreased NO production and the synthesis/release of pro‐inflammatory cytokines including NOS2, CCL2, TNF‐α, IL‐1β and MMP‐9. Accordingly, when cell cultures were transfected with silencing siRNA targeting ADAMTS‐4 prior to LPS exposure, the production of NO and the synthesis/release of pro‐inflammatory cytokines were increased. Finally, the feasibility of ADAMTS‐4 to modulate neuroinflammation was investigated in vivo after permanent middle cerebral artery occlusion in mice. Although ADAMTS‐4 treatment did not influence the lesion volume, it decreased astrogliosis and macrophage infiltration, and increased the number of microglia expressing arginase‐1, a marker of alternatively activated cells with inflammation inhibiting functions. Additionally, ADAMTS‐4 increased the production of IL‐10 and IL‐6 in the peri‐ischemic area. By having anti‐inflammatory and neuroregenerative roles, ADAMTS‐4 may represent an interesting target to treat acute CNS injuries, such as ischemic stroke, SCI or traumatic brain injury. GLIA 2016;64:1492–1507

The Copper bis(thiosemicarbazone) Complex CuII(atsm) Is Protective Against Cerebral Ischemia Through Modulation of the Inflammatory Milieu

Developing new therapies for stroke is urgently needed, as this disease is the leading cause of death and disability worldwide, and the existing treatment is only available for a small subset of patients. The interruption of blood flow to the brain during ischemic stroke launches multiple immune responses, characterized by infiltration of peripheral immune cells, the activation of brain microglial cells, and the accumulation of immune mediators. Copper is an essential trace element that is required for many critical processes in the brain. Copper homeostasis is disturbed in chronic neurodegenerative diseases and altered in stroke patients, and targeted copper delivery has been shown to be protective against chronic neurodegeneration. This study was undertaken to assess whether the copper bis(thiosemicarbazone) complex, CuII(atsm), is beneficial in acute brain injury, in preclinical mouse models of ischemic stroke. We demonstrate that the copper complex CuII(atsm) protects neurons from excitotoxicity and N2a cells from OGD in vitro, and is protective in permanent and transient ischemia models in mice as measured by functional outcome and lesion size. Copper delivery in the ischemic brains modulates the inflammatory response, specifically affecting the myeloid cells. It reduces CD45 and Iba1 immunoreactivity, and alters the morphology of Iba1 positive cells in the ischemic brain. CuII(atsm) also protects endogenous microglia against ischemic insult and reduces the proportion of invading monocytes. These results demonstrate that the copper complex CuII(atsm) is an inflammation-modulating compound with high therapeutic potential in stroke and is a strong candidate for the development of therapies for acute brain injury.

ADAMTS-4 promotes neurodegeneration in a mouse model of amyotrophic lateral sclerosis

BackgroundA disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteoglycanases are specialized in the degradation of chondroitin sulfate proteoglycans and participate in mechanisms mediating neuroplasticity. Despite the beneficial effect of ADAMTS-4 on neurorepair after spinal cord injury, the functions of ADAMTS proteoglycanases in other CNS disease states have not been studied. Therefore, we investigated the expression, effects and associated mechanisms of ADAMTS-4 during amyotrophic lateral sclerosis (ALS) in the SOD1G93A mouse model.ResultsADAMTS-4 expression and activity were reduced in the spinal cord of SOD1G93A mice at disease end-stage when compared to WT littermates. To counteract the loss of ADAMTS-4, SOD1G93A and WT mice were treated with saline or a recombinant ADAMTS-4 before symptom onset. Administration of ADAMTS-4 worsened the prognosis of SOD1G93A mice by accelerating clinical signs of neuromuscular dysfunctions. The worsened prognosis of ADAMTS-4-treated SOD1G93A mice was accompanied by increased degradation of perineuronal nets enwrapping motoneurons and increased motoneuron degeneration in the lumbar spinal cord. Motoneurons of ADAMTS-4-treated SOD1G93A mice were more vulnerable to degeneration most likely due to the loss of their extracellular matrix envelopes. The decrease of neurotrophic factor production induced by ADAMTS-4 in vitro and in vivo may also contribute to a hostile environment for motoneuron especially when devoid of a net.ConclusionsThis study suggests that the reduction of ADAMTS-4 activity during the progression of ALS pathology may be an adaptive change to mitigate its neurodegenerative impact in CNS tissues. Therapies compensating the compromized ADAMTS-4 activity are likely not promising approaches for treating ALS.

HX600, a synthetic agonist for RXR-Nurr1 heterodimer complex, prevents ischemia-induced neuronal damage

Ischemic stroke is amongst the leading causes of death and disabilities. The available treatments are suitable for only a fraction of patients and thus novel therapies are urgently needed. Blockage of one of the cerebral arteries leads to massive and persisting inflammatory reaction contributing to the nearby neuronal damage. Targeting the detrimental pathways of neuroinflammation has been suggested to be beneficial in conditions of ischemic stroke. Nuclear receptor 4A-family (NR4A) member Nurr1 has been shown to be a potent modulator of harmful inflammatory reactions, yet the role of Nurr1 in cerebral stroke remains unknown. Here we show for the first time that an agonist for the dimeric transcription factor Nurr1/retinoid X receptor (RXR), HX600, reduces microglia expressed proinflammatory mediators and prevents inflammation induced neuronal death in in vitro co-culture model of neurons and microglia. Importantly, HX600 was protective in a mouse model of permanent middle cerebral artery occlusion and alleviated the stroke induced motor deficits. Along with the anti-inflammatory capacity of HX600 in vitro, treatment of ischemic mice with HX600 reduced ischemia induced Iba-1, p38 and TREM2 immunoreactivities, protected endogenous microglia from ischemia induced death and prevented leukocyte infiltration. These anti-inflammatory functions were associated with reduced levels of brain lysophosphatidylcholines (lysoPCs) and acylcarnitines, metabolites related to proinflammatory events. These data demonstrate that HX600 driven Nurr1 activation is beneficial in ischemic stroke and propose that targeting Nurr1 is a novel candidate for conditions involving neuroinflammatory component.

Effects of human intravenous immunoglobulins on amyloid pathology and inflammation in a mouse model of Alzheimer's disease

of Alzheimer’s disease (AD). IVIG contains conformation-selective antiamyloid antibodies and has immunomodulatory properties of potential relevance to treating AD. To better understand IVIG’s mechanisms of action in AD, we measured plasma cytokine levels of AD subjects enrolled in a Phase 2 placebo-controlled IVIG clinical trial. We hypothesized that IVIG treatment alters cytokines involved in the inflammatory neuropathology of AD. Methods: 24 subjects with mild to moderate AD (MMSE 14-26) were randomly assigned to receive either placebo or Gammagard IVIG at one of four doses. Plasma samples were obtained prior the first infusion and one week after the month 6 infusion. A panel of 31 plasma cytokines was assayed using the Luminex platform. Evaluable data was obtained from 22 of 24 enrolled subjects. Every plasma sample was assayed twice and replicates were averaged. The significance of changes between baseline and six months was determined by t-test. Correlation analysis was used to explore relationship between cytokine alterations and clinical outcomes. Results: The expression of nine cytokines increased significantly following IVIG treatment compared to placebo including IL-1A, IL-4, IL-5, IL-6, IL-8, IL-13, GCSF, EGF and VEGF. Increases in 3 other cytokines (IL-17, MIP-1A and IL-12P70) trended towards significance. The magnitude of the cytokines changes varied with the dose of IVIG administered. Increases in IL-5 and IL-8 best correlated with 6 month cognitive, behavioral and functional outcomes while increases in IL-13 and GCSF correlated with the global outcome (CGIC). Conclusions: IVIG administration to AD patients for six months resulted in significant increases in 9 of 31 plasma cytokines tested. Since IVIG does not contain cytokines, these changes likely represent the immunologic consequences of IVIG treatment. Changes in three pro-inflammatory (Il5, IL-8, GCSF) and one anti-inflammatory (IL-13) cytokine correlated with clinical outcomes at 6 months. These molecules are associated with TH-2 type immune responses. Although sample size was limited, this study provides evidence that plasma cytokines are altered in AD by chronic immunoglobulin administration. Immunomodulation by cytokines may be part of the therapeutic mechanism of action of IVIG in AD.