Moses Zhang

Anti-lysophosphatidic acid antibodies improve traumatic brain injury outcomes

BackgroundLysophosphatidic acid (LPA) is a bioactive phospholipid with a potentially causative role in neurotrauma. Blocking LPA signaling with the LPA-directed monoclonal antibody B3/Lpathomab is neuroprotective in the mouse spinal cord following injury.FindingsHere we investigated the use of this agent in treatment of secondary brain damage consequent to traumatic brain injury (TBI). LPA was elevated in cerebrospinal fluid (CSF) of patients with TBI compared to controls. LPA levels were also elevated in a mouse controlled cortical impact (CCI) model of TBI and B3 significantly reduced lesion volume by both histological and MRI assessments. Diminished tissue damage coincided with lower brain IL-6 levels and improvement in functional outcomes.ConclusionsThis study presents a novel therapeutic approach for the treatment of TBI by blocking extracellular LPA signaling to minimize secondary brain damage and neurological dysfunction.

Ceruloplasmin and β-amyloid precursor protein confer neuroprotection in traumatic brain injury and lower neuronal iron

Traumatic brain injury (TBI) is in part complicated by pro-oxidant iron elevation independent of brain hemorrhage. Ceruloplasmin (CP) and β-amyloid protein precursor (APP) are known neuroprotective proteins that reduce oxidative damage through iron regulation. We surveyed iron, CP, and APP in brain tissue from control and TBI-affected patients who were stratified according to time of death following injury. We observed CP and APP induction after TBI accompanying iron accumulation. Elevated APP and CP expression was also observed in a mouse model of focal cortical contusion injury concomitant with iron elevation. To determine if changes in APP or CP were neuroprotective we employed the same TBI model on APP(-/-) and CP(-/-) mice and found that both exhibited exaggerated infarct volume and iron accumulation postinjury. Evidence supports a regulatory role of both proteins in defence against iron-induced oxidative damage after TBI, which presents as a tractable therapeutic target.

The σ1 receptor agonist 4-PPBP elicits ERK1/2 phosphorylation in primary neurons: A possible mechanism of neuroprotective action

Although sigma 1 (σ(1)) receptors and mitogen-activated protein kinases (MAPKs) are known modulators of neuroprotection, a role for MAPK signaling pathways in σ receptor-mediated neuroprotection has not been investigated in detail.The present study aims to investigate the possible link between σ(1) receptors and MAPKs in neuroprotection. Primary mixed cortical and hippocampal neurons were treated with σ(1) receptor agonists PRE-084 or 4-PPBP in a time- and concentration-dependent manner; and in another set of experiments, cells were pre-incubated with σ(1) receptor antagonist BD1047 or MEK inhibitor PD98059 in a concentration-dependent manner prior to PRE-084 or 4-PPBP treatment. Levels of phosphorylated and total ERK1/2, JNK and p38-MAPK were determined with western blotting and ERK1/2 phosphorylation was confirmed with immunofluorescence. To investigate neuroprotection by σ(1) receptors, cells were pre-treated with PRE-084 or 4-PPBP and glucose-starved for various times: in the presence or absence of pre-incubated BD1047 or PD98059. Cell viability was then measured with MTT assay. Both PRE-084 and 4-PPBP caused phosphorylation of ERK1/2, but not p38-MAPK and JNK. ERK1/2 phosphorylation was inhibited by BD1047 and PD98059 in a concentration-dependent manner. Immunofluorescence confirmed the phosphorylation of ERK1/2 by PRE-084 and 4-PPBP and its inhibition by BD1047 and PD98059. Pre-treating glucose-deprived neurons with 4-PPBP, but not PRE-084; caused neuroprotection which was inhibited by BD1047 and PD98059. 4-PPBP, but not PRE-084; causes ERK1/2 phosphorylation-mediated neuroprotection. This presents a novel mechanism by σ(1) receptors in modulating neuroprotection.

Type-1 interferons contribute to the neuroinflammatory response and disease progression of the MPTP mouse model of Parkinson's disease

Type‐1 interferons (IFNs) are pleiotropic cytokines with a critical role in the initiation and regulation of the pro‐inflammatory response. However, the contribution of the type‐1 IFNs to CNS disorders, specifically chronic neuropathologies such as Parkinsons disease is still unknown. Here, we report increased type‐1 IFN signaling in both post mortem human Parkinsons disease samples and in the 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) mouse model. In response to MPTP, mice lacking the type‐1 IFN receptor (IFNAR1−/−) displayed decreased type‐1 IFN signaling, an attenuated pro‐inflammatory response and reduced loss of dopaminergic neurons. The neuroprotective potential of targeting the type‐1 IFN pathway was confirmed by reduced neuroinflammation and DA cell death in mice treated with a blocking monoclonal IFNAR1 (MAR‐1) antibody. The MPTP/MAR‐1 treated mice also displayed increased striatal dopamine levels and improved behavioural outcomes compared to their MPTP/IgG controls. These data, implicate for the first time, a deleterious role for the type‐1 IFNs as key modulators of the early neuroinflammatory response and therefore the neuronal cell death in Parkinsons disease. GLIA 2016;64:1590–1604

MyD88 is a critical regulator of hematopoietic cell-mediated neuroprotection seen after stroke.

Neuroinflammation is critical in the neural cell death seen in stroke. It has been shown that CNS and peripheral responses drive this neuroinflammatory response in the brain. The Toll-like receptors (TLRs) are important regulators of inflammation in response to both exogenous and endogenous stressors. Taking advantage of a downstream adapter molecule that controls the majority of TLR signalling, this study investigated the role of the TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the control of CNS and peripheral inflammation. Reversible middle-cerebral artery occlusion was used as the model of stroke in vivo; in vitro primary cultured neurons and glia were subject to four hours of oxygen and glucose deprivation (OGD). Both in vitro and in vivo Myd88−/− animals or cells were compared with wild type (WT). We found that after stroke Myd88−/− animals have a larger infarct volume compared to WT animals. Interestingly, in vitro there was no difference between the survival of Myd88−/− and WT cells following OGD, suggesting that peripheral responses were influencing stroke outcome. We therefore generated bone marrow chimeras and found that Myd88−/− animals have a smaller stroke infarct than their radiation naive counterparts if their hematopoietic cells are WT. Furthermore, WT animals have a larger stroke than their radiation naive counterparts if the hematopoietic cells are Myd88−/−. We have demonstrated that MyD88-dependent signalling in the hematopoietic cell lineage reduces infarct size following stroke and that infiltrating cells to the site of neuroinflammation are neuroprotective following stroke.

Soluble amyloid triggers a myeloid differentiation factor 88 and interferon regulatory factor 7 dependent neuronal type-1 interferon response in vitro

BackgroundNeuro-inflammation has long been implicated as a contributor to the progression of Alzheimer’s disease in both humans and animal models. Type-1 interferons (IFNs) are pleiotropic cytokines critical in mediating the innate immune pro-inflammatory response. The production of type-1 IFNs following pathogen detection is, in part, through the activation of the toll-like receptors (TLRs) and subsequent signalling through myeloid differentiation factor-88 (Myd88) and interferon regulatory factors (IRFs). We have previously identified that neuronal type-1 IFN signalling, through the type-1 interferon alpha receptor-1 (IFNAR1), is detrimental in models of AD. Using an in vitro approach, this study investigated the TLR network as a potential production pathway for neuronal type-1 IFNs in response to Aβ.MethodsWildtype and Myd88−/− primary cultured cortical and hippocampal neurons were treated with 2.5 μM Aβ1-42 for 24 to 72 h or 1 to 10 μM Aβ1-42 for 72 h. Human BE(2)M17 neuroblastoma cells stably expressing an IRF7 small hairpin RNA (shRNA) or negative control shRNA construct were subjected to 7.5 μM Aβ1-42/Aβ42-1 for 24 to 96 h, 2.5 to 15 μM Aβ1-42 for 96 h or 100 ng/ml LPS for 0.5 to 24 h. Q-PCR was used to analyse IFNα, IFNβ, IL-1β, IL-6 and TNFα mRNA transcript levels. Phosphorylation of STAT-3 was detected by Western blot analysis, and cell viability was assessed by MTS assay.ResultsReduced IFNα, IFNβ, IL-1β, IL-6 and TNFα expression was detected in Aβ1-42-treated Myd88−/− neurons compared to wildtype cells. This correlated with reduced phosphorylation of STAT-3, a downstream type-1 IFN signalling mediator. Significantly, Myd88−/− neuronal cultures were protected against Aβ1-42-induced neurotoxicity compared to wildtype as determined by MTS assay. Knockdown of IRF7 in M17 cells was sufficient in blocking IFNα, IFNβ and p-STAT-3 induction to both Aβ1-42 and the TLR4 agonist LPS. M17 IRF7 KD cells were also protected against Aβ1-42-induced cytotoxicity.ConclusionsThis study confirms that the neuronal type-1 IFN response to soluble amyloid is mediated primarily through TLRs. This production is dependent upon Myd88 and IRF7 signalling. This study suggests that targeting this pathway to modulate neuronal type-1 IFN levels may be beneficial in controlling Aβ-induced neurotoxicity.

Ablation of Type-1 IFN Signaling in Hematopoietic Cells Confers Protection Following Traumatic Brain Injury

Abstract Type-1 interferons (IFNs) are pleiotropic cytokines that signal through the type-1 IFN receptor (IFNAR1). Recent literature has implicated the type-1 IFNs in disorders of the CNS. In this study, we have investigated the role of type-1 IFNs in neuroinflammation following traumatic brain injury (TBI). Using a controlled cortical impact model, TBI was induced in 8- to 10-week-old male C57BL/6J WT and IFNAR1−/− mice and brains were excised to study infarct volume, inflammatory mediator release via quantitative PCR analysis and immune cell profile via immunohistochemistry. IFNAR1−/− mice displayed smaller infarcts compared with WT mice after TBI. IFNAR1−/− mice exhibited an altered anti-inflammatory environment compared with WT mice, with significantly reduced levels of the proinflammatory mediators TNFα, IL-1β and IL-6, an up-regulation of the anti-inflammatory mediator IL-10 and an increased activation of resident and peripheral immune cells after TBI. WT mice injected intravenously with an anti-IFNAR1 blocking monoclonal antibody (MAR1) 1 h before, 30 min after or 30 min and 2 d after TBI displayed significantly improved histological and behavioral outcome. Bone marrow chimeras demonstrated that the hematopoietic cells are a peripheral source of type-1 IFNs that drives neuroinflammation and a worsened TBI outcome. Type-1 IFN mRNA levels were confirmed to be significantly altered in human postmortem TBI brains. Together, these data demonstrate that type-1 IFN signaling is a critical pathway in the progression of neuroinflammation and presents a viable therapeutic target for the treatment of TBI.

Type-1 interferons contribute to oxygen glucose deprivation induced neuro-inflammation in BE(2) M17 human neuroblastoma cells

BackgroundHypoxic-ischaemic injuries such as stroke and traumatic brain injury exhibit features of a distinct neuro-inflammatory response in the hours and days post-injury. Microglial activation, elevated pro-inflammatory cytokines and macrophage infiltration contribute to core tissue damage and contribute to secondary injury within a region termed the penumbra. Type-1 interferons (IFNs) are a super-family of pleiotropic cytokines that regulate pro-inflammatory gene transcription via the classical Jak/Stat pathway; however their role in hypoxia-ischaemia and central nervous system neuro-inflammation remains unknown. Using an in vitro approach, this study investigated the role of type-1 IFN signalling in an inflammatory setting induced by oxygen glucose deprivation (OGD).MethodsHuman BE(2)M17 neuroblastoma cells or cells expressing a type-1 interferon-α receptor 1 (IFNAR1) shRNA or negative control shRNA knockdown construct were subjected to 4.5 h OGD and a time-course reperfusion period (0 to 24 h). Q-PCR was used to evaluate IFNα, IFNβ, IL-1β, IL-6 and TNF-α cytokine expression levels. Phosphorylation of signal transducers and activators of transcription (STAT)-1, STAT-3 and cleavage of caspase-3 was detected by western blot analysis. Post-OGD cellular viability was measured using a MTT assay.ResultsElevated IFNα and IFNβ expression was detected during reperfusion post-OGD in parental M17 cells. This correlated with enhanced phosphorylation of STAT-1, a downstream type-1 IFN signalling mediator. Significantly, ablation of type-1 IFN signalling, through IFNAR1 knockdown, reduced IFNα, IFNβ, IL-6 and TNF-α expression in response to OGD. In addition, MTT assay confirmed the IFNAR1 knockdown cells were protected against OGD compared to negative control cells with reduced pro-apoptotic cleaved caspase-3 levels.ConclusionsThis study confirms a role for type-1 IFN signalling in the neuro-inflammatory response following OGD in vitro and suggests its modulation through therapeutic blockade of IFNAR1 may be beneficial in reducing hypoxia-induced neuro-inflammation.

Type-I interferons mediate the neuroinflammatory response and neurotoxicity induced by rotenone.

Evidence from post‐mortem human brains, animal studies and cell culture models has implicated neuroinflammation in the aetiology of chronic neuropathologies including Alzheimers and Parkinsons diseases. Although the neuroinflammatory response is considered detrimental in contributing to these pathologies, the underlying mechanisms are still not well understood. The type‐I interferons (IFNs) have been well characterised in the periphery and are known to initiate/modulate the immune response. Recently, they have been implicated in ageing and we have also demonstrated increased type‐I IFN expression in post‐mortem human Alzheimers and Parkinsons disease brains. We hypothesise that the type‐I IFNs are key drivers of the damaging, self‐perpetuating pro‐inflammatory response that contributes to these chronic neuropathologies. In support of this, we have recently confirmed in models of Alzheimers and Parkinsons disease that mice lacking the type‐I IFN receptor (IFNAR1), display an attenuated neuroinflammatory response with subsequent neuroprotection. To further investigate type‐I IFN‐mediated neuroinflammation and the specific CNS cell types involved, this study treated primary cultured wild‐type and IFNAR1−/− neurons or mixed glia with the mitochondrial complex I inhibitor, rotenone. Wild‐type neurons and glia treated with 3 nM and 25 nM rotenone, respectively, exhibited a pro‐inflammatory response, including increased type‐I IFN expression that was attenuated in cells lacking IFNAR1. Reduced type‐I IFN signalling in IFNAR1−/− neurons also conferred protection against caspase‐3‐mediated rotenone‐induced cell death. Further, this reduced pro‐inflammatory response in the IFNAR1−/− glia subsequently diminished their neurotoxic effects to wild‐type neurons. In support of this, we confirmed that therapeutically targeting the type‐I IFN glial response to rotenone through a specific IFNAR1 blocking monoclonal antibody was neuroprotective. Our data has confirmed that both neurons and glia contribute to the pro‐inflammatory response induced by rotenone with attenuation of this response beneficial in reducing neuronal cell death.

Weight-Bearing Locomotion in the Developing Opossum, Monodelphis domestica following Spinal Transection: Remodeling of Neuronal Circuits Caudal to Lesion

Complete spinal transection in the mature nervous system is typically followed by minimal axonal repair, extensive motor paralysis and loss of sensory functions caudal to the injury. In contrast, the immature nervous system has greater capacity for repair, a phenomenon sometimes called the infant lesion effect. This study investigates spinal injuries early in development using the marsupial opossum Monodelphis domestica whose young are born very immature, allowing access to developmental stages only accessible in utero in eutherian mammals. Spinal cords of Monodelphis pups were completely transected in the lower thoracic region, T10, on postnatal-day (P)7 or P28 and the animals grew to adulthood. In P7-injured animals regrown supraspinal and propriospinal axons through the injury site were demonstrated using retrograde axonal labelling. These animals recovered near-normal coordinated overground locomotion, but with altered gait characteristics including foot placement phase lags. In P28-injured animals no axonal regrowth through the injury site could be demonstrated yet they were able to perform weight-supporting hindlimb stepping overground and on the treadmill. When placed in an environment of reduced sensory feedback (swimming) P7-injured animals swam using their hindlimbs, suggesting that the axons that grew across the lesion made functional connections; P28-injured animals swam using their forelimbs only, suggesting that their overground hindlimb movements were reflex-dependent and thus likely to be generated locally in the lumbar spinal cord. Modifications to propriospinal circuitry in P7- and P28-injured opossums were demonstrated by changes in the number of fluorescently labelled neurons detected in the lumbar cord following tracer studies and changes in the balance of excitatory, inhibitory and neuromodulatory neurotransmitter receptors’ gene expression shown by qRT-PCR. These results are discussed in the context of studies indicating that although following injury the isolated segment of the spinal cord retains some capability of rhythmic movement the mechanisms involved in weight-bearing locomotion are distinct.