Targeting SRSF3 restores immune mRNA translation in microglia/macrophages following cerebral ischemia.

We recently described a novel ribosome-based regulatory mechanism/checkpoint that controls innate immune gene translation and microglial activation in non-sterile inflammation orchestrated by RNA binding protein SRSF3. Here we describe a role of SRSF3 in the regulation of microglia/macrophage activation phenotypes after experimental stroke. Using a model-system for analysis of the dynamic translational state of microglial ribosomes we show that 24 h after stroke highly upregulated immune mRNAs are not translated resulting in a marked dissociation of mRNA and protein networks in activated microglia/macrophages. Next, microglial activation after stroke was characterized by a robust increase in pSRSF3/SRSF3 expression levels. Targeted knockdown of SRSF3 using intranasal delivery of siRNA 24 h after stroke caused a marked knockdown of endogenous protein. Further analyses revealed that treatment with SRSF3-siRNA alleviated translational arrest of selected genes and induced a transient but significant increase in innate immune signaling and IBA1+ immunoreactivity peaking 5 days after initial injury. Importantly, delayed SRSF3-mediated increase in immune signaling markedly reduced the size of ischemic lesion measured 7 days after stroke. Together, our findings suggest that targeting SRSF3 and immune mRNA translation may open new avenues for molecular/therapeutic reprogramming of innate immune response after ischemic injury.

Chronically activated microglia in ALS gradually lose their immune functions and develop unconventional proteome.

Neuroinflammation and chronic activation of microglial cells are the prominent features of amyotrophic lateral sclerosis (ALS) pathology. While alterations in the mRNA profile of diseased microglia have been well documented, the actual microglia proteome remains poorly characterized. Here we performed a functional characterization together with proteome analyses of microglial cells at different stages of disease in the SOD1-G93A model of ALS. Functional analyses of microglia derived from the lumbar spinal cord of symptomatic mice revealed: (i) remarkably high mitotic index (close to 100% cells are Ki67+) (ii) significant decrease in phagocytic capacity when compared to age-matched control microglia, and (iii) diminished response to innate immune challenges in vitro and in vivo. Proteome analysis revealed a development of two distinct molecular signatures at early and advanced stages of disease. While at early stages of disease, we identified several proteins implicated in microglia immune functions such as GPNMB, HMBOX1, at advanced stages of disease microglia signature at protein level was characterized with a robust upregulation of several unconventional proteins including rootletin, major vaults proteins and STK38. Upregulation of GPNMB and rootletin has been also found in the spinal cord samples of sporadic ALS. Remarkably, the top biological functions of microglia, in particular in the advanced disease, were not related to immunity/immune response, but were highly enriched in terms linked to RNA metabolism. Together, our results suggest that, over the course of disease, chronically activated microglia develop unconventional protein signatures and gradually lose their immune identity ultimately turning into functionally inefficient immune cells.

Therapeutic Trial of anle138b in Mouse Models of Genetic Prion Disease.

Phenotypic screening has yielded small-molecule inhibitors of prion replication that are effective in vivo against certain prion strains but not others. Here, we sought to test the small molecule anle138b in multiple mouse models of prion disease. In mice inoculated with the RML strain of prions, anle138b doubled survival and durably suppressed astrogliosis measured by live-animal bioluminescence imaging. In knock-in mouse models of the D178N and E200K mutations that cause genetic prion disease, however, we were unable to identify a clear, quantifiable disease endpoint against which to measure therapeutic efficacy. Among untreated animals, the mutations did not impact overall survival, and bioluminescence remained low out to >20 months of age. Vacuolization and PrP deposition were observed in some brain regions in a subset of mutant animals but appeared to be unable to carry the weight of a primary endpoint in a therapeutic study. We conclude that not all animal models of prion disease are suited to well-powered therapeutic efficacy studies, and care should be taken in choosing the models that will support drug development programs. IMPORTANCE There is an urgent need to develop drugs for prion disease, a currently untreatable neurodegenerative disease. In this effort, there is a debate over which animal models can best support a drug development program. While the study of prion disease benefits from excellent animal models because prions naturally afflict many different mammals, different models have different capabilities and limitations. Here, we conducted a therapeutic efficacy study of the drug candidate anle138b in mouse models with two of the most common mutations that cause genetic prion disease. In a more typical model where prions are injected directly into the brain, we found anle138b to be effective. In the genetic models, however, the animals never reached a clear, measurable point of disease onset. We conclude that not all prion disease animal models are ideally suited to drug efficacy studies, and well-defined, quantitative disease metrics should be a priority.

Distinct Plasma Immune Profile in ALS Implicates sTNFR-II in pAMPK/Leptin Homeostasis.

Amyotrophic lateral sclerosis (ALS) is a clinically highly heterogeneous disease with a survival rate ranging from months to decades. Evidence suggests that a systemic deregulation of immune response may play a role and affect disease progression. Here, we measured 62 different immune/metabolic mediators in plasma of sporadic ALS (sALS) patients. We show that, at the protein level, the majority of immune mediators including a metabolic sensor, leptin, were significantly decreased in the plasma of sALS patients and in two animal models of the disease. Next, we found that a subset of patients with rapidly progressing ALS develop a distinct plasma assess immune-metabolic molecular signature characterized by a differential increase in soluble tumor necrosis factor receptor II (sTNF-RII) and chemokine (C-C motif) ligand 16 (CCL16) and further decrease in the levels of leptin, mostly dysregulated in male patients. Consistent with in vivo findings, exposure of human adipocytes to sALS plasma and/or sTNF-RII alone, induced a significant deregulation in leptin production/homeostasis and was associated with a robust increase in AMP-activated protein kinase (AMPK) phosphorylation. Conversely, treatment with an AMPK inhibitor restored leptin production in human adipocytes. Together, this study provides evidence of a distinct plasma immune profile in sALS which affects adipocyte function and leptin signaling. Furthermore, our results suggest that targeting the sTNF-RII/AMPK/leptin pathway in adipocytes may help restore assess immune-metabolic homeostasis in ALS.

Prion protein lowering is a disease-modifying therapy across prion disease stages, strains and endpoints.

Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that <25% PrP suppression is sufficient to extend survival and delay symptoms in a prophylactic paradigm. Rise in both neuroinflammation and neuronal injury markers can be reversed by a single dose of PrP-lowering ASO administered after the detection of pathological change. Chronic ASO-mediated suppression of PrP beginning at any time up to early signs of neuropathology confers benefit similar to constitutive heterozygous PrP knockout. Remarkably, even after emergence of frank symptoms including weight loss, a single treatment prolongs survival by months in a subset of animals. These results support ASO-mediated PrP lowering, and PrP-lowering therapeutics in general, as a promising path forward against prion disease.

Microglia-derived galectin-3 in neuroinflammation; a bittersweet ligand?

Galectins are soluble ß-galactoside-binding proteins found in all multicellular organisms. Galectins may act as danger-associated molecular patterns in innate immunity and/or as pattern-recognition receptors that bind to pathogen-associated molecular patterns. Among different galectin family members, galectin-3 has been the focus of studies in neurodegenerative diseases in recent years. This lectin modulates brain innate immune responses, microglia activation patterns in physiological and pathophysiological settings in a context-dependent manner. Galectin-3 is considered as a pivotal tuner of macrophage and microglial activity. Indeed galectin-3 acts as a double edged sword in neuroinflammatory context and this multimodal lectin has diverse roles in physiological and pathophysiological conditions. Better understanding of galectin-3 physiology (its extracellular and intracellular actions) and structure (its C terminus vs. N terminus) is instrumental to design molecules that selectively modulate galectin-3 function toward neuroprotective phenotypes. Several experimental studies using different approaches and methods have demonstrated both protective and deleterious effects of galectin-3 in neuroinflammatory diseases. According to the crucial role of galectin-3 in modulation of innate immune response in brain, it is an attractive target in drug discovery of neurodegenerative diseases. The current insight attempts to provide an updated and balanced discussion on the role of galectin-3 as a complex endogenous immune modulator. This helps to have a better insight into the development of galectin-3 modulators with translational value in different neurological disorders including stroke and neurodegenerative diseases, such as Alzheimer's disease, Huntington's disease and Parkinson's disease.

Differential Expression Patterns of TDP-43 in Single Moderate versus Repetitive Mild Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) is a disabling disorder and a major cause of death and disability in the world. Both single and repetitive traumas affect the brain acutely but can also lead to chronic neurodegenerative changes. Clinical studies have shown some dissimilarities in transactive response DNA binding protein 43 (TDP-43) expression patterns following single versus repetitive TBI. We explored the acute cortical post-traumatic changes of TDP-43 using the lateral fluid percussion injury (LFPI) model of single moderate TBI in adult male mice and investigated the association of TDP-43 with post-traumatic neuroinflammation and synaptic plasticity. In the ipsilateral cortices of animals following LFPI, we found changes in the cytoplasmic and nuclear levels of TDP-43 and the decreased expression of postsynaptic protein 95 within the first 3 d post-injury. Subacute pathological changes of TDP-43 in the hippocampi of animals following LFPI and in mice exposed to repetitive mild TBI (rmTBI) were studied. Changes in the hippocampal TDP-43 expression patterns at 14 d following different brain trauma procedures showed pathological alterations only after single moderate, but not following rmTBI. Hippocampal LFPI-induced TDP-43 pathology was not accompanied by the microglial reaction, contrary to the findings after rmTBI, suggesting that different types of brain trauma may cause diverse pathophysiological changes in the brain, specifically related to the TDP-43 protein as well as to the microglial reaction. Taken together, our findings may contribute to a better understanding of the pathophysiological events following brain trauma.

Long-Term Effects of Repetitive Mild Traumatic Injury on the Visual System in Wild-Type and TDP-43 Transgenic Mice.

Little is known about the impairments and pathological changes in the visual system in mild brain trauma, especially repetitive mild traumatic brain injury (mTBI). The goal of this study was to examine and compare the effects of repeated head impacts on the neurodegeneration, axonal integrity, and glial activity in the optic tract (OT), as well as on neuronal preservation, glial responses, and synaptic organization in the lateral geniculate nucleus (LGN) and superior colliculus (SC), in wild-type mice and transgenic animals with overexpression of human TDP-43 mutant protein (TDP-43G348C) at 6 months after repeated closed head traumas. Animals were also assessed in the Barnes maze (BM) task. Neurodegeneration, axonal injury, and gliosis were detected in the OT of the injured animals of both genotypes. In the traumatized mice, myelination of surviving axons was mostly preserved, and the expression of neurofilament light chain was unaffected. Repetitive mTBI did not induce changes in the LGN and the SC, nor did it affect the performance of the BM task in the traumatized wild-type and TDP-43 transgenic mice. Differences in neuropathological and behavioral assessments between the injured wild-type and TDP-43G348C mice were not revealed. Results of the current study suggest that repetitive mTBI was associated with chronic damage and inflammation in the OT in wild-type and TDP-43G348C mice, which were not accompanied with behavioral problems and were not affected by the TDP-43 genotype, while the LGN and the SC remained preserved in the used experimental conditions.

Age-related deregulation of TDP-43 after stroke enhances NF-κB-mediated inflammation and neuronal damage.

BACKGROUND: TDP-43 has been identified as a disease-associated protein in several chronic neurodegenerative disorders and increasing evidence suggests its potentially pathogenic role following brain injuries. Normally expressed in nucleus, under pathological conditions TDP-43 forms cytoplasmic ubiquitinated inclusions in which it is abnormally phosphorylated and cleaved to generate a 35 and a 25 kDa C-terminal fragments. In the present study, we investigated age-related expression patterns of TDP-43 in neurons and glia and its role as modulator of inflammation following ischemic injury. METHODS: Wild-type and TDP-43 transgenic mice of different age groups were subjected to transient middle cerebral artery occlusion. The role of TDP-43 in modulation of inflammation was assessed using immunofluorescence, Western blot analysis, and in vivo bioluminescence imaging. Finally, post-mortem stroke human brain sections were analyzed for TDP-43 protein by immunohistochemistry. RESULTS: We report here an age-related increase and formation of ubiquitinated TDP-43 cytoplasmic inclusions after stroke. The observed deregulation in TDP-43 expression patterns was associated with an increase in microglial activation and innate immune signaling as revealed by in vivo bioluminescence imaging and immunofluorescence analysis. The presence of ubiquitinated TDP-43 aggregates and its cleaved TDP-35 and TDP-25 fragments was markedly increased in older, 12-month-old mice leading to larger infarctions and a significant increase in in neuronal death. Importantly, unlike the hallmark neuropathological features associated with chronic neurodegenerative disorders, the TDP-43-positive cytoplasmic inclusions detected after stroke were not phosphorylated. Next, we showed that an increase and/or overexpression of the cytoplasmic TDP-43 drives the pathogenic NF-κB response and further increases levels of pro-inflammatory markers and ischemic injury after stroke in age-dependent manner. Finally, analyses of the post-mortem stroke brain tissues revealed the presence of the cytoplasmic TDP-43 immunoreactive structures after human stroke. CONCLUSION: Together, our findings suggest that the level of cytoplasmic TDP-43 increases with aging and may act as an age-related mediator of inflammation and neuronal injury after stroke. Thus, targeting cytoplasmic TDP-43 may have a therapeutic potential after stroke.

IL-10 Controls Early Microglial Phenotypes and Disease Onset in ALS Caused by Misfolded Superoxide Dismutase 1.

While reactive microgliosis is a hallmark of advanced stages of amyotrophic lateral sclerosis (ALS), the role of microglial cells in events initiating and/or precipitating disease onset is largely unknown. Here we provide novel in vivo evidence of a distinct adaptive shift in functional microglial phenotypes in preclinical stages of superoxide dismutase 1 (SOD1)-mutant-mediated disease. Using a mouse model for live imaging of microglial activation crossed with SOD1(G93A) and SOD1(G37R) mouse models, we discovered that the preonset phase of SOD1-mediated disease is characterized by development of distinct anti-inflammatory profile and attenuated innate immune/TLR2 responses to lipopolysaccharide (LPS) challenge. This microglial phenotype was associated with a 16-fold overexpression of anti-inflammatory cytokine IL-10 in baseline conditions followed by a 4.5-fold increase following LPS challenge. While infusion of IL-10R blocking antibody, initiated at day 60, caused a significant increase in markers of microglial activation and precipitated clinical onset of disease, a targeted overexpression of IL-10 in microglial cells, delivered via viral vectors expressed under CD11b promoter, significantly delayed disease onset and increased survival of SOD1(G93A) mice. We propose that the high IL-10 levels in resident microglia in early ALS represent a homeostatic and compensatory "adaptive immune escape" mechanism acting as a nonneuronal determinant of clinical onset of disease. Significance statement: We report here for the first time that changing the immune profile of brain microglia may significantly affect clinical onset and duration of disease in ALS models. We discovered that in presymptomatic disease microglial cells overexpress anti-inflammatory cytokine IL-10. Given that IL-10 is major homeostatic cytokine and its production becomes deregulated with aging, this may suggest that the capacity of microglia to adequately produce IL-10 may be compromised in ALS. We show that blocking IL-10 increased inflammation and precipitated clinical disease onset, whereas overexpression of IL-10 in microglia using a gene therapy approach significantly delayed disease onset and increased survival of ALS mice. Based on our results, we propose that targeted overexpression of IL-10 in microglia may have therapeutic potential in ALS.

Molecular imaging of nestin in neuroinflammatory conditions reveals marked signal induction in activated microglia.

BACKGROUND: Nestin is a known marker of neuronal progenitor cells in the adult brain. Following neuro- and gliogenesis, nestin is replaced by cell type-specific intermediate filaments, e.g., neurofilaments for panneuronal expression and glial fibrillary acidic protein as a specific marker of mature astrocytes. While previous work have been mostly focused on the neuronal fate of nestin-positive progenitors, in the present study, we sought to investigate in real time how nestin signals and cellular expression patterns are controlled in the context of neuroinflammatory challenge and ischemic brain injury. METHODS: To visualize effects of neuroinflammation on neurogenesis/gliogenesis, we created a transgenic model bearing the dual reporter system luciferase and GFP under transcriptional control of the murine nestin promoter. In this model, transcriptional activation of nestin was visualized from the brains of living animals using biophotonic/bioluminescence molecular imaging and a high resolution charged coupled device camera. Nestin induction profiles in vivo and in tissue sections were analyzed in two different experimental paradigms: middle cerebral artery occlusion and lipopolysaccharide-induced innate immune stimuli. RESULTS: We report here a context- and injury-dependent induction and cellular expression profile of nestin. While in the baseline conditions the nestin signal and/or GFP expression was restricted to neuronal progenitors, the cellular expression patterns of nestin following innate immune challenge and after stroke markedly differed shifting the cellular expression patterns towards activated microglia/macrophages and astrocytes. CONCLUSIONS: Our results suggest that nestin may serve as a context-dependent biomarker of inflammatory response in glial cells including activated microglia/macrophages.

Live imaging of the innate immune response in neonates reveals differential TLR2 dependent activation patterns in sterile inflammation and infection.

Activation of microglial cells in response to brain injury and/or immune stimuli is associated with a marked induction of Toll-like receptors (TLRs). While in adult brain, the contribution of individual TLRs, including TLR2, in pathophysiological cascades has been well established, their role and spatial and temporal induction patterns in immature brain are far less understood. To examine whether infectious stimuli and sterile inflammatory stimuli trigger distinct TLR2-mediated innate immune responses, we used three models in postnatal day 9 (P9) mice, a model of infection induced by systemic endotoxin injection and two models of sterile inflammation, intra-cortical IL-1ß injection and transient middle cerebral artery occlusion (tMCAO). We took advantage of a transgenic mouse model bearing the dual reporter system luciferase/GFP under transcriptional control of a murine TLR2 promoter (TLR2-luc-GFP) to visualize the TLR2 response in the living neonatal brain and then determined neuroinflammation, microglial activation and leukocyte infiltration. We show that in physiological postnatal brain development the in vivo TLR2-luc signal undergoes a marked ∼30-fold decline and temporal-spatial changes during the second and third postnatal weeks. We then show that while endotoxin robustly induces the in vivo TLR2-luc signal in the living brain and increases levels of several inflammatory cytokines and chemokines, the in vivo TLR2-luc signal is reduced after both IL-1ß and tMCAO and the inflammatory response is muted. Immunofluorescence revealed that microglial cells are the predominant source of TLR2 production during postnatal brain development and in all three neonatal models studied. Flow cytometry revealed developmental changes in CD11b+/CD45+ and CD11b+/Ly6C+ cell populations, involvement of cells of the monocyte lineage, but lack of Ly6G+ neutrophils or CD3+ cells in acutely injured neonatal brains. Cumulatively, our results suggest distinct TLR2 induction patterns following PAMP and DAMP - mediated inflammation in immature brain.

A novel combinational approach of microstimulation and bioluminescence imaging to study the mechanisms of action of cerebral electrical stimulation in mice.

Deep brain stimulation (DBS) is used to treat a number of neurological conditions and is currently being tested to intervene in neuropsychiatric conditions. However, a better understanding of how it works would ensure that side effects could be minimized and benefits optimized. We have thus developed a unique device to perform brain stimulation (BS) in mice and to address fundamental issues related to this methodology in the pre-clinical setting. This new microstimulator prototype was specifically designed to allow simultaneous live bioluminescence imaging of the mouse brain, allowing real time assessment of the impact of stimulation on cerebral tissue. We validated the authenticity of this tool in vivo by analysing the expression of toll-like receptor 2 (TLR2), corresponding to the microglial response, in the stimulated brain regions of TLR2-fluc-GFP transgenic mice, which we further corroborated with post-mortem analyses in these animals as well as in human brains of patients who underwent DBS to treat their Parkinson's disease. In the present study, we report on the development of the first BS device that allows for simultaneous live in vivo imaging in mice. This tool opens up a whole new range of possibilities that allow a better understanding of BS and how to optimize its effects through its use in murine models of disease.

Adeno-associated virus-mediated delivery of a recombinant single-chain antibody against misfolded superoxide dismutase for treatment of amyotrophic lateral sclerosis.

There is emerging evidence that the misfolding of superoxide dismutase 1 (SOD1) may represent a common pathogenic event in both familial and sporadic amyotrophic lateral sclerosis (ALS). To reduce the burden of misfolded SOD1 species in the nervous system, we have tested a novel therapeutic approach based on adeno-associated virus (AAV)-mediated tonic expression of a DNA construct encoding a secretable single-chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the AAV encoding the single-chain antibody in SOD1(G93A) mice at 45 days of age resulted in sustained expression of single-chain antibodies in the spinal cord, and it delayed disease onset and extension of life span by up to 28%, in direct correlation with scFv titers in the spinal cord. The treatment caused attenuation of neuronal stress signals and reduction in levels of misfolded SOD1 in the spinal cord of SOD1(G93A) mice. From these results, we propose that an immunotherapy based on intrathecal inoculation of AAV encoding a secretable scFv against misfolded SOD1 should be considered as potential treatment for ALS, especially for individuals carrying SOD1 mutations.

Enriched housing down-regulates the Toll-like receptor 2 response in the mouse brain after experimental stroke.

Post-ischemic inflammation plays an important role in the evolution of brain injury, recovery and repair after stroke. Housing rodents in an enriched environment provides multisensory stimulation to the brain and enhances functional recovery after experimental stroke, also depressing the release of cytokines and chemokines in the peri-infarct. In order to identify targets for late stroke treatment, we studied the dynamics of inflammation and the contribution of resident Toll-like receptor 2 (TLR2) expressing microglia cells. We took advantage of the biophotonic/bioluminescent imaging technique using the reporter mouse-expressing luciferase and GFP reporter genes under transcriptional control of the murine TLR2 promoter (TLR2-luc/GFP mice) for non-invasive in vivo analysis of TLR2 activation/response in photothrombotic stroke after differential housing. Real-time imaging at 1day after stroke, revealed up-regulation of TLR2 in response to photothrombotic stroke that subsequently declined over time of recovery (14days). The inflammatory response was persistently down-regulated within days of enriched housing, enhancing recovery of lost sensori-motor function in TLR2-luc mice without affecting infarct size. The number of YM1-expressing microglia in the peri-infarct and areas remote from the infarct was also markedly attenuated. Using a live imaging approach, we demonstrate that multisensory stimulation rapidly, persistently and generally attenuates brain inflammation after experimental stroke, reducing the TLR2 response and leading to improved neurological outcome. TLR2-expressing microglia cells may provide targets for new stroke therapeutics.

Toll-like receptor expression in the blood and brain of patients and a mouse model of Parkinson's disease.

BACKGROUND: Accumulating evidence supports a role for the immune system in the pathogenesis of Parkinson's disease. Importantly, recent preclinical studies are now suggesting a specific contribution of inflammation to the α-synuclein-induced pathology seen in this condition. METHODS: We used flow cytometry and western blots to detect toll-like receptor 2 and 4 expression in blood and brain samples of Parkinson's disease patients and mice overexpressing human α-synuclein. To further assess the effects of α-synuclein overexpression on the innate immune system, we performed a longitudinal study using Thy1.2-α-synuclein mice that expressed a bicistronic DNA construct (reporter genes luciferase and green fluorescent protein) under the transcriptional control of the murine toll-like receptor 2 promoter. RESULTS: Here, we report increases in toll-like receptors 2 and 4 expression in circulating monocytes and of toll-like receptor 4 in B cells and in the caudate/putamen of Parkinson's disease patients. Monthly bioluminescence imaging of Thy1.2-α-synuclein mice showed increasing toll-like receptor 2 expression from 10 months of age, although no change in toll-like receptor 2 and 4 expression was observed in the blood and brain of these mice at 12 months of age. Dexamethasone treatment starting at 5 months of age for 1 month significantly decreased the microglial response in the brain of these mice and promoted functional recovery as observed using a wheel-running activity test. CONCLUSION: Our results show that toll-like receptors 2 and 4 are modulated in the blood and brain of Parkinson's disease patients and that overexpression of α-synuclein leads to a progressive microglial response, the inhibition of which has a beneficial impact on some motor phenotypes of an animal model of α-synucleinopathy.

Inhibition of NF-κB with an Analog of Withaferin-A Restores TDP-43 Homeostasis and Proteome Profiles in a Model of Sporadic ALS.

The current knowledge on pathogenic mechanisms in amyotrophic lateral sclerosis (ALS) has widely been derived from studies with cell and animal models bearing ALS-linked genetic mutations. However, it remains unclear to what extent these disease models are of relevance to sporadic ALS. Few years ago, we reported that the cerebrospinal fluid (CSF) from sporadic ALS patients contains toxic factors for disease transmission in mice via chronic intracerebroventricular (i.c.v.) infusion. Thus a 14-day i.c.v. infusion of pooled CSF samples from ALS cases in mice provoked motor impairment as well as ALS-like pathological features. This offers a unique paradigm to test therapeutics in the context of sporadic ALS disease. Here, we tested a new Withaferin-A analog (IMS-088) inhibitor of NF-κB that was found recently to mitigate disease phenotypes in mouse models of familial disease expressing TDP-43 mutant. Our results show that oral intake of IMS-088 ameliorated motor performance of mice infused with ALS-CSF and it alleviated pathological changes including TDP-43 proteinopathy, neurofilament disorganization, and neuroinflammation. Moreover, CSF infusion experiments were carried out with transgenic mice having neuronal expression of tagged ribosomal protein (hNfL-RFP mice), which allowed immunoprecipitation of neuronal ribosomes for analysis by mass spectrometry of the translational peptide signatures. The results indicate that treatment with IMS-088 prevented many proteomic alterations associated with exposure to ALS-CSF involving pathways related to cytoskeletal changes, inflammation, metabolic dysfunction, mitochondria, UPS, and autophagy dysfunction. The effective disease-modifying effects of this drug in a mouse model based on i.c.v. infusion of ALS-CSF suggest that the NF-κB signaling pathway represents a compelling therapeutic target for sporadic ALS.

Abnormal regenerative responses and impaired axonal outgrowth after nerve crush in TDP-43 transgenic mouse models of amyotrophic lateral sclerosis.

Tar DNA binding protein 43 (TDP-43) mislocalization and aggregation is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. Moreover, TDP-43 mRNA was found to be upregulated by ∼2.5-fold in the spinal cord of sporadic ALS subjects. Here we have examined the effects of nerve injury in new transgenic mouse models overexpressing by approximately threefold wild-type or mutant (G348C) TDP-43 species. Four weeks after axonal crush of sciatic nerve, TDP-43 transgenic mice remained paralyzed at the injured limb unlike control mice, which had regained most of their normal mobility. In contrast to normal mice, TDP-43 transgenic mice exhibited sustained elevation of TDP-43 cytoplasmic levels in motor neurons after nerve crush, and the relocalization of TDP-43 to the nucleus was delayed by several weeks. After crush, peripherin and ubiquitin levels remained also significantly elevated in TDP-43 transgenic mice compared with control mice. Analysis of the sciatic nerve at 11 d after nerve crush showed that the number of regenerating axons in the distal portion of the lesion was considerably reduced in TDP-43 transgenic mice, especially in TDP-43(G348C) mice, which exhibited a reduction of ∼40%. In addition, markers of neuroinflammation were detected at much higher levels in TDP-43 transgenic mice. These results suggest that a deregulation of TDP-43 expression in ALS is a phenomenon that can affect the regenerative responses to neuronal injury and regrowth potential of axons.

Galectin-3 is required for resident microglia activation and proliferation in response to ischemic injury.

Growing evidence suggests that galectin-3 is involved in fine tuning of the inflammatory responses at the periphery, however, its role in injured brain is far less clear. Our previous work demonstrated upregulation and coexpression of galectin-3 and IGF-1 in a subset of activated/proliferating microglial cells after stroke. Here, we tested the hypothesis that galectin-3 plays a pivotal role in mediating injury-induced microglial activation and proliferation. By using a galectin-3 knock-out mouse (Gal-3KO), we demonstrated that targeted disruption of the galectin-3 gene significantly alters microglia activation and induces ∼4-fold decrease in microglia proliferation. Defective microglia activation/proliferation was further associated with significant increase in the size of ischemic lesion, ∼2-fold increase in the number of apoptotic neurons, and a marked deregulation of the IGF-1 levels. Next, our results revealed that contrary to WT cells, the Gal3-KO microglia failed to proliferate in response to IGF-1. Moreover, the IGF-1-mediated mitogenic microglia response was reduced by N-glycosylation inhibitor tunicamycine while coimmunoprecipitation experiments revealed galectin-3 binding to IGF-receptor 1 (R1), thus suggesting that interaction of galectin-3 with the N-linked glycans of receptors for growth factors is involved in IGF-R1 signaling. While the canonical IGF-1 signaling pathways were not affected, we observed an overexpression of IL-6 and SOCS3, suggesting an overactivation of JAK/STAT3, a shared signaling pathway for IGF-1/IL-6. Together, our findings suggest that galectin-3 is required for resident microglia activation and proliferation in response to ischemic injury.

CD36 neutralisation blunts TLR2-IRF7 but not IRF3 pathway in neonatal mouse brain and immature human microglia following innate immune challenge.

Innate immune response in neonatal brain is associated with a robust microglial activation and induction of Toll-like Receptors (TLRs). To date, the role of the scavenger receptor CD36 in TLRs modulation, particularly TLR2 signaling, has been well established in adult brain. However, the crosstalk between TLR4, TLR2 and CD36 and its immunogenic influence in the neonatal brain remains unclear. In this study, using a CD36 blocking antibody (anti-CD36) at post-natal day 8, we evaluated the response of neonates to systemic endotoxin (lipopolysaccharide; LPS) challenge. We visualized the TLR2 response by bioluminescence imaging using the transgenic mouse model bearing the dual reporter system luciferase/green fluorescent protein under transcriptional control of a murine TLR2 promoter. The anti-CD36 treatment modified the LPS induced inflammatory profile in neonatal brains, causing a significant decrease in inflammatory cytokine levels and the TLR2 and TLR3 mediated signalling.The interferon regulatory factor 3 (IRF3) pathway remained unaffected. Treatment of the LPS-challenged human immature microglia with anti-CD36 induced a marked decrease in TLR2/TLR3 expression levels while TLR4 and IRF3 expression was not affected, suggesting the shared CD36 regulatory mechanisms in human and mouse microglia. Collectively, our results indicate that blocking CD36 alters LPS-induced inflammatory profile of mouse and human microglia, suggesting its role in fine-tuning of neuroinflammation.