Toxoplasma infection induces an aged neutrophil population in the CNS that is associated with neuronal protection.

BACKGROUND: Infection with the protozoan parasite Toxoplasma gondii leads to the formation of lifelong cysts in neurons that can have devastating consequences in the immunocompromised. In the immunocompetent individual, anti-parasitic effector mechanisms and a balanced immune response characterized by pro- and anti-inflammatory cytokine production establishes an asymptomatic infection that rarely leads to neurological symptoms. Several mechanisms are known to play a role in this successful immune response in the brain including T cell production of IFNγ and IL-10 and the involvement of CNS resident cells. This limitation of clinical neuropathology during chronic infection suggests a balance between immune response and neuroprotective mechanisms that collectively prevent clinical manifestations of disease. However, how these two vital mechanisms of protection interact during chronic Toxoplasma infection remains poorly understood. MAIN TEXT: This study demonstrates a previously undescribed connection between innate neutrophils found chronically in the brain, termed "chronic brain neutrophils" (CBNeuts), and neuroprotective mechanisms during Toxoplasma infection. Lack of CBNeuts during chronic infection, accomplished via systemic neutrophil depletion, led to enhanced infection and deleterious effects on neuronal regeneration and repair mechanisms in the brain. Phenotypic and transcriptomic analysis of CBNeuts identified them as distinct from peripheral neutrophils and revealed two main subsets of CBNeuts that display heterogeneity towards both classical effector and neuroprotective functions in an age-dependent manner. Further phenotypic profiling defined expression of the neuroprotective molecules NRG-1 andErbB4 by these cells, and the importance of this signaling pathway during chronic infection was demonstrated via NRG-1 treatment studies. CONCLUSIONS: In conclusion, this work identifies CBNeuts as a heterogenous population geared towards both classical immune responses and neuroprotection during chronic Toxoplasma infection and provides the foundation for future mechanistic studies of these cells.

Regulation of NRG-1-ErbB4 signaling and neuroprotection by exogenous neuregulin-1 in a mouse model of epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Dysregulation of glutamate transporters has been a common finding across animal models of epilepsy and in patients with TLE. In this study, we investigate NRG-1/ErbB4 signaling in epileptogenesis and the neuroprotective effects of NRG-1 treatment in a mouse model of temporal lobe epilepsy. Using immunohistochemistry, we report the first evidence for NRG-1/ErbB4-dependent selective upregulation of glutamate transporter EAAC1 and bihemispheric neuroprotection by exogeneous NRG-1 in the intrahippocampal kainic acid (IHKA) model of TLE. Our findings provide evidence that dysregulation of glutamate transporter EAAC1 contributes to the development of epilepsy and can be therapeutically targeted to reduce neuronal death following IHKA-induced status epilepticus (SE).

Bilateral gene interaction hierarchy analysis of the cell death gene response emphasizes the significance of cell cycle genes following unilateral traumatic brain injury.

BACKGROUND: Delayed or secondary cell death that is caused by a cascade of cellular and molecular processes initiated by traumatic brain injury (TBI) may be reduced or prevented if an effective neuroprotective strategy is employed. Microarray and subsequent bioinformatic analyses were used to determine which genes, pathways and networks were significantly altered 24 h after unilateral TBI in the rat. Ipsilateral hemi-brain, the corresponding contralateral hemi-brain, and naïve (control) brain tissue were used for microarray analysis. RESULTS: Ingenuity Pathway Analysis showed cell death and survival (CD) to be a top molecular and cellular function associated with TBI on both sides of the brain. One major finding was that the overall gene expression pattern suggested an increase in CD genes in ipsilateral brain tissue and suppression of CD genes contralateral to the injury which may indicate an endogenous protective mechanism. We created networks of genes of interest (GOI) and ranked the genes by the number of direct connections each had in the GOI networks, creating gene interaction hierarchies (GIHs). Cell cycle was determined from the resultant GIHs to be a significant molecular and cellular function in post-TBI CD gene response. CONCLUSIONS: Cell cycle and apoptosis signalling genes that were highly ranked in the GIHs and exhibited either the inverse ipsilateral/contralateral expression pattern or contralateral suppression were identified and included STAT3, CCND1, CCND2, and BAX. Additional exploration into the remote suppression of CD genes may provide insight into neuroprotective mechanisms that could be used to develop therapies to prevent cell death following TBI.

Regulation of inflammatory responses by neuregulin-1 in brain ischemia and microglial cells in vitro involves the NF-kappa B pathway.

BACKGROUND: We previously demonstrated that neuregulin-1 (NRG-1) was neuroprotective in rats following ischemic stroke. Neuroprotection by NRG-1 was associated with the suppression of pro-inflammatory gene expression in brain tissues. Over-activation of brain microglia can induce pro-inflammatory gene expression by activation of transcriptional regulators following stroke. Here, we examined how NRG-1 transcriptionally regulates inflammatory gene expression by computational bioinformatics and in vitro using microglial cells. METHODS: To identify transcriptional regulators involved in ischemia-induced inflammatory gene expression, rats were sacrificed 24 h after middle cerebral artery occlusion (MCAO) and NRG-1 treatment. Gene expression profiles of brain tissues following ischemia and NRG-1 treatment were examined by microarray technology. The Conserved Transcription Factor-Binding Site Finder (CONFAC) bioinformatics software package was used to predict transcription factors associated with inflammatory genes induced following stroke and suppressed by NRG-1 treatment. NF-kappa B (NF-kB) was identified as a potential transcriptional regulator of NRG-1-suppressed genes following ischemia. The involvement of specific NF-kB subunits in NRG-1-mediated inflammatory responses was examined using N9 microglial cells pre-treated with NRG-1 (100 ng/ml) followed by lipopolysaccharide (LPS; 10 µg/ml) stimulation. The effects of NRG-1 on cytokine production were investigated using Luminex technology. The levels of the p65, p52, and RelB subunits of NF-kB and IkB-α were determined by western blot analysis and ELISA. Phosphorylation of IkB-α was investigated by ELISA. RESULTS: CONFAC identified 12 statistically over-represented transcription factor-binding sites (TFBS) in our dataset, including NF-kBP65. Using N9 microglial cells, we observed that NRG-1 significantly inhibited LPS-induced TNFα and IL-6 release. LPS increased the phosphorylation and degradation of IkB-α which was blocked by NRG-1. NRG-1 also prevented the nuclear translocation of the NF-kB p65 subunit following LPS administration. However, NRG-1 increased production of the neuroprotective cytokine granulocyte colony-stimulating factor (G-CSF) and the nuclear translocation of the NF-kB p52 subunit, which is associated with the induction of anti-apoptotic and suppression of pro-inflammatory gene expression. CONCLUSIONS: Neuroprotective and anti-inflammatory effects of NRG-1 are associated with the differential regulation of NF-kB signaling pathways in microglia. Taken together, these findings suggest that NRG-1 may be a potential therapeutic treatment for treating stroke and other neuroinflammatory disorders.

Persistent neuroinflammation and cognitive impairment in a rat model of acute diisopropylfluorophosphate intoxication.

BACKGROUND: Acute intoxication with organophosphorus (OP) cholinesterase inhibitors can trigger convulsions that progress to life-threatening status epilepticus. Survivors face long-term morbidity including mild-to-severe decline in memory. It is posited that neuroinflammation plays a key role in the pathogenesis of OP-induced neuropsychiatric deficits. Rigorous testing of this hypothesis requires preclinical models that recapitulate relevant phenotypic outcomes. Here, we describe a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) that exhibits persistent neuroinflammation and cognitive impairment. METHODS: Neuroinflammation, neurodegeneration, and cognitive function were compared in adult male Sprague Dawley rats injected with an acutely toxic dose of DFP vs. vehicle controls at multiple time points up to 36 days post-exposure. Neuroinflammation was quantified using immunohistochemical biomarkers of microglia (ionized calcium-binding adapter molecule 1, IBA1) and activated astrocytes (glial fibrillary acidic protein, GFAP) and positron emission tomography (PET) imaging of [11C]-(R)-PK11195, a ligand for the 18-kDa mitochondrial membrane translocator protein (TSPO). FluoroJade-B staining was used to assess neurodegeneration; Pavlovian conditioning, to assess cognitive function. RESULTS: Animals exhibited moderate-to-severe seizures within minutes of DFP injection that continued for up to 6 h post-injection. As indicated by IBA1 and GFAP immunoreactivity and by PET imaging of TSPO, acute DFP intoxication triggered neuroinflammation in the hippocampus and cortex during the first 3 days that peaked at 7 days and persisted to 21 days post-exposure in most animals. Neurodegeneration was detected in multiple brain regions from 1 to 14 days post-exposure. All DFP-intoxicated animals exhibited significant deficits in contextual fear conditioning at 9 and 20 days post-exposure compared to vehicle controls. Whole-brain TSPO labeling positively correlated with seizure severity score, but did not correlate with performance in the contextual fear-conditioning task. CONCLUSIONS: We describe a preclinical model in which acute DFP intoxication causes seizures, persistent neuroinflammation, neurodegeneration, and memory impairment. The extent of the neuroinflammatory response is influenced by seizure severity. However, the observation that a subset of animals with moderate seizures and minimal TSPO labeling exhibited cognitive deficits comparable to those of animals with severe seizures and significant TSPO labeling suggests that DFP may impair learning and memory circuitry via mechanisms independent of seizures or neuroinflammation.

Neuregulin-1 inhibits neuroinflammatory responses in a rat model of organophosphate-nerve agent-induced delayed neuronal injury.

BACKGROUND: Neuregulin-1 (NRG-1) has been shown to act as a neuroprotectant in animal models of nerve agent intoxication and other acute brain injuries. We recently demonstrated that NRG-1 blocked delayed neuronal death in rats intoxicated with the organophosphate (OP) neurotoxin diisopropylflurophosphate (DFP). It has been proposed that inflammatory mediators are involved in the pathogenesis of OP neurotoxin-mediated brain damage. METHODS: We examined the influence of NRG-1 on inflammatory responses in the rat brain following DFP intoxication. Microglial activation was determined by immunohistchemistry using anti-CD11b and anti-ED1 antibodies. Gene expression profiling was performed with brain tissues using Affymetrix gene arrays and analyzed using the Ingenuity Pathway Analysis software. Cytokine mRNA levels following DFP and NRG-1 treatment was validated by real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: DFP administration resulted in microglial activation in multiple brain regions, and this response was suppressed by treatment with NRG-1. Using microarray gene expression profiling, we observed that DFP increased mRNA levels of approximately 1,300 genes in the hippocampus 24 h after administration. NRG-1 treatment suppressed by 50% or more a small fraction of DFP-induced genes, which were primarily associated with inflammatory responses. Real-time RT-PCR confirmed that the mRNAs for pro-inflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were significantly increased following DFP exposure and that NRG-1 significantly attenuated this transcriptional response. In contrast, tumor necrosis factor α (TNFα) transcript levels were unchanged in both DFP and DFP + NRG-1 treated brains relative to controls. CONCLUSION: Neuroprotection by NRG-1 against OP neurotoxicity is associated with the suppression of pro-inflammatory responses in brain microglia. These findings provide new insight regarding the molecular mechanisms involved in the neuroprotective role of NRG-1 in acute brain injuries.

Neuregulin-1 attenuates mortality associated with experimental cerebral malaria.

BACKGROUND: Cerebral Malaria (CM) is a diffuse encephalopathy caused by Plasmodium falciparum infection. Despite availability of antimalarial drugs, CM-associated mortality remains high at approximately 30% and a subset of survivors develop neurological and cognitive disabilities. While antimalarials are effective at clearing Plasmodium parasites they do little to protect against CM pathophysiology and parasite-induced brain inflammation that leads to seizures, coma and long-term neurological sequelae in CM patients. Thus, there is urgent need to explore therapeutics that can reduce or prevent CM pathogenesis and associated brain inflammation to improve survival. Neuregulin-1 (NRG-1) is a neurotrophic growth factor shown to protect against brain injury associated with acute ischemic stroke (AIS) and neurotoxin exposure. However, this drug has not been tested against CM-associated brain injury. Since CM-associated brain injuries and AIS share similar pathophysiological features, we hypothesized that NRG-1 will reduce or prevent neuroinflammation and brain damage as well as improve survival in mice with late-stage experimental cerebral malaria (ECM). METHODS: We tested the effects of NRG-1 on ECM-associated brain inflammation and mortality in P. berghei ANKA (PbA)-infected mice and compared to artemether (ARM) treatment; an antimalarial currently used in various combination therapies against malaria. RESULTS: Treatment with ARM (25 mg/kg/day) effectively cleared parasites and reduced mortality in PbA-infected mice by 82%. Remarkably, NRG-1 therapy (1.25 ng/kg/day) significantly improved survival against ECM by 73% despite increase in parasite burden within NRG-1-treated mice. Additionally, NRG-1 therapy reduced systemic and brain pro-inflammatory factors TNFalpha, IL-6, IL-1alpha and CXCL10 and enhanced anti-inflammatory factors, IL-5 and IL-13 while decreasing leukocyte accumulation in brain microvessels. CONCLUSIONS: This study suggests that NRG-1 attenuates ECM-associated brain inflammation and injuries and may represent a novel supportive therapy for the management of CM.

The case for neuregulin-1 as a clinical treatment for stroke.

Ischemic stroke is the leading cause of serious long-term disability and the 5th leading cause of death in the United States. Revascularization of the occluded cerebral artery, either by thrombolysis or endovascular thrombectomy, is the only effective, clinically-approved stroke therapy. Several potentially neuroprotective agents, including glutamate antagonists, anti-inflammatory compounds and free radical scavenging agents were shown to be effective neuroprotectants in preclinical animal models of brain ischemia. However, these compounds did not demonstrate efficacy in clinical trials with human patients following stroke. Proposed reasons for the translational failure include an insufficient understanding on the cellular and molecular pathophysiology of ischemic stroke, lack of alignment between preclinical and clinical studies and inappropriate design of clinical trials based on the preclinical findings. Therefore, novel neuroprotective treatments must be developed based on a clearer understanding of the complex spatiotemporal mechanisms of ischemic stroke and with proper clinical trial design based on the preclinical findings from specific animal models of stroke. We and others have demonstrated the clinical potential for neuregulin-1 (NRG-1) in preclinical stroke studies. NRG-1 significantly reduced ischemia-induced neuronal death, neuroinflammation and oxidative stress in rodent stroke models with a therapeutic window of >13 h. Clinically, NRG-1 was shown to be safe in human patients and improved cardiac function in multisite phase II studies for heart failure. This review summarizes previous stroke clinical candidates and provides evidence that NRG-1 represents a novel, safe, neuroprotective strategy that has potential therapeutic value in treating individuals after acute ischemic stroke.

Gene expression patterns following unilateral traumatic brain injury reveals a local pro-inflammatory and remote anti-inflammatory response.

BACKGROUND: Traumatic brain injury (TBI) results in irreversible damage at the site of impact and initiates cellular and molecular processes that lead to secondary neural injury in the surrounding tissue. We used microarray analysis to determine which genes, pathways and networks were significantly altered using a rat model of TBI. Adult rats received a unilateral controlled cortical impact (CCI) and were sacrificed 24 h post-injury. The ipsilateral hemi-brain tissue at the site of the injury, the corresponding contralateral hemi-brain tissue, and naïve (control) brain tissue were used for microarray analysis. Ingenuity Pathway Analysis (IPA) software was used to identify molecular pathways and networks that were associated with the altered gene expression in brain tissues following TBI. RESULTS: Inspection of the top fifteen biological functions in IPA associated with TBI in the ipsilateral tissues revealed that all had an inflammatory component. IPA analysis also indicated that inflammatory genes were altered on the contralateral side, but many of the genes were inversely expressed compared to the ipsilateral side. The contralateral gene expression pattern suggests a remote anti-inflammatory molecular response. We created a network of the inversely expressed common (i.e., same gene changed on both sides of the brain) inflammatory response (IR) genes and those IR genes included in pathways and networks identified by IPA that changed on only one side. We ranked the genes by the number of direct connections each had in the network, creating a gene interaction hierarchy (GIH). Two well characterized signaling pathways, toll-like receptor/NF-kappaB signaling and JAK/STAT signaling, were prominent in our GIH. CONCLUSIONS: Bioinformatic analysis of microarray data following TBI identified key molecular pathways and networks associated with neural injury following TBI. The GIH created here provides a starting point for investigating therapeutic targets in a ranked order that is somewhat different than what has been presented previously. In addition to being a vehicle for identifying potential targets for post-TBI therapeutic strategies, our findings can also provide a context for evaluating the potential of therapeutic agents currently in development.

Neuregulin-1 is neuroprotective in a rat model of organophosphate-induced delayed neuronal injury.

Current medical countermeasures against organophosphate (OP) nerve agents are effective in reducing mortality, but do not sufficiently protect the CNS from delayed brain damage and persistent neurological symptoms. In this study, we examined the efficacy of neuregulin-1 (NRG-1) in protecting against delayed neuronal cell death following acute intoxication with the OP diisopropylflurophosphate (DFP). Adult male Sprague-Dawley rats were pretreated with pyridostigmine (0.1 mg/kg BW, i.m.) and atropine methylnitrate (20 mg/kg BW, i.m.) prior to DFP (9 mg/kg BW, i.p.) intoxication to increase survival and reduce peripheral signs of cholinergic toxicity but not prevent DFP-induced seizures or delayed neuronal injury. Pretreatment with NRG-1 did not protect against seizures in rats exposed to DFP. However, neuronal injury was significantly reduced in most brain regions by pretreatment with NRG-1 isoforms NRG-EGF (3.2 µg/kg BW, i.a) or NRG-GGF2 (48 µg/kg BW, i.a.) as determined by FluroJade-B labeling in multiple brain regions at 24 h post-DFP injection. NRG-1 also blocked apoptosis and oxidative stress-mediated protein damage in the brains of DFP-intoxicated rats. Administration of NRG-1 at 1h after DFP injection similarly provided significant neuroprotection against delayed neuronal injury. These findings identify NRG-1 as a promising adjuvant therapy to current medical countermeasures for enhancing neuroprotection against acute OP intoxication.

Spatial Analysis of Neural Cell Proteomic Profiles Following Ischemic Stroke in Mice Using High-Plex Digital Spatial Profiling.

Stroke is ranked as the fifth leading cause of death and the leading cause of adult disability in the USA. The progression of neuronal damage after stroke is recognized to be a complex integration of glia, neurons, and the surrounding extracellular matrix, therefore potential treatments must target the detrimental effects created by these interactions. In this study, we examined the spatial cellular and neuroinflammatory mechanisms occurring early after ischemic stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. Male C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO) and sacrificed at 3 days post-ischemia. Spatial distinction of the ipsilateral hemisphere was studied according to the regions of interest: the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in comparison to the contralateral hemisphere. We demonstrated that the ipsilateral hemisphere initiates distinct spatial regulatory proteomic profiles with DSP technology that can be identified consistently with the immunohistochemical markers, FJB, GFAP, and Iba-1. The core border profile demonstrated an induction of neuronal death, apoptosis, autophagy, immunoreactivity, and early degenerative proteins. Most notably, the core border resulted in a decrease of the neuronal proteins Map2 and NeuN; an increase in the autophagy proteins BAG3 and CTSD; an increase in the microglial and peripheral immune invasion proteins Iba1, CD45, CD11b, and CD39; and an increase in the neurodegenerative proteins BACE1, APP, amyloid ß 1-42, ApoE, and hyperphosphorylated tau protein S-199. The peri-infarct region demonstrated increased astrocytic, immunoreactivity, apoptotic, and neurodegenerative proteomic profiles, with an increase in BAG3, GFAP, and hyperphosphorylated tau protein S-199. The PiNT region displayed minimal changes compared to the contralateral cortex with only an increase in GFAP. In this study, we showed that mechanisms known to be associated with stroke, such as apoptosis and inflammation, occur in distinct spatial domains of the injured brain following ischemia. We also demonstrated the dysregulation of specific autophagic pathways that may lead to neurodegeneration in peri-infarct brain tissues. Taken together, these data suggest that identifying post-ischemic mechanisms occurring in a spatiotemporal manner may lead to more precise targets for successful therapeutic interventions to treat stroke.

Short-term exposure to an obesogenic diet during adolescence elicits anxiety-related behavior and neuroinflammation: modulatory effects of exogenous neuregulin-1.

Childhood obesity leads to hippocampal atrophy and altered cognition. However, the molecular mechanisms underlying these impairments are poorly understood. The neurotrophic factor neuregulin-1 (NRG1) and its cognate ErbB4 receptor play critical roles in hippocampal maturation and function. This study aimed to determine whether exogenous NRG1 administration reduces hippocampal abnormalities and neuroinflammation in rats exposed to an obesogenic Western-like diet (WD). Lewis rats were randomly divided into four groups (12 rats/group): (1) control diet+vehicle (CDV); (2) CD + NRG1 (CDN) (daily intraperitoneal injections: 5 µg/kg/day; between postnatal day, PND 21-PND 41); (3) WD + VEH (WDV); (4) WD + NRG1 (WDN). Neurobehavioral assessments were performed at PND 43-49. Brains were harvested for MRI and molecular analyses at PND 49. We found that NRG1 administration reduced hippocampal volume (7%) and attenuated hippocampal-dependent cued fear conditioning in CD rats (56%). NRG1 administration reduced PSD-95 protein expression (30%) and selectively reduced hippocampal cytokine levels (IL-33, GM-CSF, CCL-2, IFN-γ) while significantly impacting microglia morphology (increased span ratio and reduced circularity). WD rats exhibited reduced right hippocampal volume (7%), altered microglia morphology (reduced density and increased lacunarity), and increased levels of cytokines implicated in neuroinflammation (IL-1α, TNF-α, IL-6). Notably, NRG1 synergized with the WD to increase hippocampal ErbB4 phosphorylation and the tumor necrosis alpha converting enzyme (TACE/ADAM17) protein levels. Although the results did not provide sufficient evidence to conclude that exogenous NRG1 administration is beneficial to alleviate obesity-related outcomes in adolescent rats, we identified a potential novel interaction between obesogenic diet exposure and TACE/ADAM17-NRG1-ErbB4 signaling during hippocampal maturation. Our results indicate that supraoptimal ErbB4 activities may contribute to the abnormal hippocampal structure and cognitive vulnerabilities observed in obese individuals.

Spatiotemporal pattern of neuronal injury induced by DFP in rats: a model for delayed neuronal cell death following acute OP intoxication.

Organophosphate (OP) neurotoxins cause acute cholinergic toxicity and seizures resulting in delayed brain damage and persistent neurological symptoms. Testing novel strategies for protecting against delayed effects of acute OP intoxication has been hampered by the lack of appropriate animal models. In this study, we characterize the spatiotemporal pattern of cellular injury after acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague-Dawley rats received pyridostigmine (0.1 mg/kg, im) and atropine methylnitrate (20mg/kg, im) prior to DFP (9 mg/kg, ip) administration. All DFP-treated animals exhibited moderate to severe seizures within minutes after DFP injection but survived up to 72 h. AChE activity was significantly depressed in the cortex, hippocampus, subcortical brain tissue and cerebellum at 1h post-DFP injection and this inhibition persisted for up to 72 h. Analysis of neuronal injury by Fluoro-Jade B (FJB) labeling revealed delayed neuronal cell death in the hippocampus, cortex, amygdala and thalamus, but not the cerebellum, starting at 4h and persisting until 72 h after DFP treatment, although temporal profiles varied between brain regions. At 24h post-DFP injection, the pattern of FJB labeling corresponded to TUNEL staining in most brain regions, and FJB-positive cells displayed reduced NeuN immunoreactivity but were not immunopositive for astrocytic (GFAP), oligodendroglial (O4) or macrophage/microglial (ED1) markers, demonstrating that DFP causes a region-specific delayed neuronal injury mediated in part by apoptosis. These findings indicate the feasibility of this model for testing neuroprotective strategies, and provide insight regarding therapeutic windows for effective pharmacological intervention following acute OP intoxication.

Neuroprotection by Exogenous and Endogenous Neuregulin-1 in Mouse Models of Focal Ischemic Stroke.

Identifying novel neuroprotectants that can halt or reverse the neurological effects of stroke is of interest to both clinicians and scientists. We and others previously showed the pre-clinical neuroprotective efficacy of neuregulin-1 (NRG-1) in rats following focal brain ischemia. In this study, we examined neuroprotection by exogenous and endogenous NRG-1 using a mouse model of ischemic stroke. C57BL6 mice were subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion. NRG-1 or vehicle was infused intra-arterially (i.a.) or intravenously (i.v.) after MCAO and before the onset of reperfusion. NRG-1 treatment (16 µg/kg; i.a.) reduced cerebral cortical infarct volume by 72% in mice when delivered post-ischemia. NRG-1 also inhibited neuronal injury as measured by Fluoro Jade B labeling and rescued NeuN immunoreactivity in neurons. Neuroprotection by NRG-1 was also observed in mice when administered i.v. (100 µg/kg) in both male and female mice. We investigated whether endogenous NRG-1 was neuroprotective using male and female heterozygous NRG-1 knockout mice (NRG-1+/-) compared with wild-type mice (WT) littermates. NRG-1+/- and WT mice were subjected to MCAO for 45 min, and infarct size was measured 24 h following MCAO. NRG-1+/- mice displayed a sixfold increase in cortical infarct size compared with WT mice. These results demonstrate that NRG-1 treatment mitigates neuronal damage following cerebral ischemia. We further showed that reduced endogenous NRG-1 results in exacerbated neuronal injury in vivo. These findings suggest that NRG-1 represents a promising therapy to treat stroke in human patients.

Spontaneous Isomerization of Long-Lived Proteins Provides a Molecular Mechanism for the Lysosomal Failure Observed in Alzheimer's Disease.

Proteinaceous aggregation is a well-known observable in Alzheimer's disease (AD), but failure and storage of lysosomal bodies within neurons is equally ubiquitous and actually precedes bulk accumulation of extracellular amyloid plaque. In fact, AD shares many similarities with certain lysosomal storage disorders though establishing a biochemical connection has proven difficult. Herein, we demonstrate that isomerization and epimerization, which are spontaneous chemical modifications that occur in long-lived proteins, prevent digestion by the proteases in the lysosome (namely, the cathepsins). For example, isomerization of aspartic acid into l-isoAsp prevents digestion of the N-terminal portion of Aß by cathepsin L, one of the most aggressive lysosomal proteases. Similar results were obtained after examination of various target peptides with a full series of cathepsins, including endo-, amino-, and carboxy-peptidases. In all cases peptide fragments too long for transporter recognition or release from the lysosome persisted after treatment, providing a mechanism for eventual lysosomal storage and bridging the gap between AD and lysosomal storage disorders. Additional experiments with microglial cells confirmed that isomerization disrupts proteolysis in active lysosomes. These results are easily rationalized in terms of protease active sites, which are engineered to precisely orient the peptide backbone and cannot accommodate the backbone shift caused by isoaspartic acid or side chain dislocation resulting from epimerization. Although Aß is known to be isomerized and epimerized in plaques present in AD brains, we further establish that the rates of modification for aspartic acid in positions 1 and 7 are fast and could accrue prior to plaque formation. Spontaneous chemistry can therefore provide modified substrates capable of inducing gradual lysosomal failure, which may play an important role in the cascade of events leading to the disrupted proteostasis, amyloid formation, and tauopathies associated with AD.

Analysis of transcriptional profiles and functional clustering of global cerebellar gene expression in PCD3J mice.

The Purkinje cell degeneration (PCD) mutant mouse is characterized by a degeneration of cerebellar Purkinje cells and progressive ataxia. To identify the molecular mechanisms that lead to the death of Purkinje neurons in PCD mice, we used Affymetrix microarray technology to compare cerebellar gene expression profiles in pcd3J mutant mice 14 days of age (prior to Purkinje cell loss) to unaffected littermates. Microarray analysis, Ingenuity Pathway Analysis (IPA) and expression analysis systematic explorer (EASE) software were used to identify biological and molecular pathways implicated in the progression of Purkinje cell degeneration. IPA analysis indicated that mutant pcd3J mice showed dysregulation of specific processes that may lead to Purkinje cell death, including several molecules known to control neuronal apoptosis such as Bad, CDK5 and PTEN. These findings demonstrate the usefulness of these powerful microarray analysis tools and have important implications for understanding the mechanisms of selective neuronal death and for developing therapeutic strategies to treat neurodegenerative disorders.

Neuroprotective effects of neuregulin-1 on B35 neuronal cells following ischemia.

We previously showed that neuregulin-1 (NRG-1) protected neurons from death in vivo following focal ischemia. The goal of this study was to develop an in vitro rat ischemia model to examine the cellular and molecular mechanisms involved in the neuroprotective effects of NRG-1 on ischemia-induced neuronal death. Rat B-35 neuroblastoma cells differentiated by serum withdrawal, developed enhanced neuronal characteristics including, neurite extension and upregulation of neuronal markers of differentiation. When B35 neurons were subjected to oxygen glucose deprivation (OGD)/reoxygenation or glutamate, widespread neuronal death was seen after both treatments. Treatment with NRG-1 immediately after OGD significantly increased neuronal survival. NRG-1 administration also resulted in a significant decrease in annexin V, an early marker of apoptosis. However, the neurotoxic actions of glutamate were unaffected by NRG-1. The neuroprotective effects of NRG-1 were prevented by an inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. These results provide a new model to gain insight into the mechanisms employed by NRG-1 to protect neurons from ischemic brain injury.

Evaluation of neuregulin-1's neuroprotection against ischemic injury in rats using diffusion tensor imaging.

Stroke is a devastating neurovascular disorder that results in damage to neurons and white matter tracts. It has been previously demonstrated that neuregulin-1 (NRG-1) protects neurons from ischemic injury following stroke. Here, diffusion tensor imaging (DTI) was utilized to characterize the effects of NRG-1 treatment on cererbral infarction and integrity of white matter after ischemic insult using a permanent middle celebral artery occlusion (pMCAo) rat model. In the present study, sixteen Sprague-Dawley rats underwent pMCAo surgery and received either a single intra-arterial bolus (20 µg/kg) dose of NRG-1 or saline immediately prior to pMCAo. MRI including T2-weighted imaging and DTI was performed in the first 3 h post stroke, and repeated 48 h later. It is found that the stroke infarction was significantly reduced in the NRG-1 treated group. Also, NRG-1 prevented the reduction of fractional anisotropy (FA) in white matter tracts of fornix and corpus callosum (CC), indicating its protection of CC and fornix white matter bundles from ischemia insult. As a conclusion, the present DTI results demonstrate that NRG-1 has significantly neuroprotective effects in both cerebral cortex and white matter including corpus callosum and fornix during acute stroke. In particular, NRG-1 is more effective on stroke lesion with mild ischemia. As CC and fornix white matter bundles play critical roles in transcallosal connectivity and hippocampal projections respectively in the central nervous system, the findings could provide complementary information for better understanding the biological mechanism of NRG-1's neuroprotection in ischemic tissues and neurobehavioral effects.

Transcriptomic analysis of neuregulin-1 regulated genes following ischemic stroke by computational identification of promoter binding sites: A role for the ETS-1 transcription factor.

Ischemic stroke is a major cause of mortality in the United States. We previously showed that neuregulin-1 (NRG1) was neuroprotective in rat models of ischemic stroke. We used gene expression profiling to understand the early cellular and molecular mechanisms of NRG1's effects after the induction of ischemia. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO). Rats were allocated to 3 groups: (1) control, (2) MCAO and (3) MCAO + NRG1. Cortical brain tissues were collected three hours following MCAO and NRG1 treatment and subjected to microarray analysis. Data and statistical analyses were performed using R/Bioconductor platform alongside Genesis, Ingenuity Pathway Analysis and Enrichr software packages. There were 2693 genes differentially regulated following ischemia and NRG1 treatment. These genes were organized by expression patterns into clusters using a K-means clustering algorithm. We further analyzed genes in clusters where ischemia altered gene expression, which was reversed by NRG1 (clusters 4 and 10). NRG1, IRS1, OPA3, and POU6F1 were central linking (node) genes in cluster 4. Conserved Transcription Factor Binding Site Finder (CONFAC) identified ETS-1 as a potential transcriptional regulator of NRG1 suppressed genes following ischemia. A transcription factor activity array showed that ETS-1 activity was increased 2-fold, 3 hours following ischemia and this activity was attenuated by NRG1. These findings reveal key early transcriptional mechanisms associated with neuroprotection by NRG1 in the ischemic penumbra.