Inflammatory bowel disease induces pathological α-synuclein aggregation in the human gut and brain.

AIMS: According to Braak's hypothesis, it is plausible that Parkinson's disease (PD) originates in the enteric nervous system (ENS) and spreads to the brain through the vagus nerve. In this work, we studied whether inflammatory bowel diseases (IBDs) in humans can progress with the emergence of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. METHODS: We have analysed the gut and the ventral midbrain from subjects previously diagnosed with IBD and form a DSS-based rat model of gut inflammation in terms of α-syn pathology. RESULTS: Our data support the existence of pathogenic α-syn in both the gut and the brain, thus reinforcing the potential role of the ENS as a contributing factor in PD aetiology. Additionally, we have analysed the effect of a DSS-based rat model of gut inflammation to demonstrate (i) the appearance of P-α-syn inclusions in both Auerbach's and Meissner's plexuses (gut), (ii) an increase in α-syn expression in the ventral mesencephalon (brain) and (iii) the degeneration of nigral dopaminergic neurons, which all are considered classical hallmarks in PD. CONCLUSION: These results strongly support the plausibility of Braak's hypothesis and emphasise the significance of peripheral inflammation and the gut-brain axis in initiating α-syn aggregation and transport to the substantia nigra, resulting in neurodegeneration.

Galectin-3 is upregulated in frontotemporal dementia patients with subtype specificity.

INTRODUCTION: Neuroinflammation is a major contributor to the progression of frontotemporal dementia (FTD). Galectin-3 (Gal-3), a microglial activation regulator, holds promise as a therapeutic target and potential biomarker. Our study aimed to investigate Gal-3 levels in patients with FTD and assess its diagnostic potential. METHODS: We examined Gal-3 levels in brain, serum, and cerebrospinal fluid (CSF) samples of patients with FTD and controls. Multiple linear regressions between Gal-3 levels and other FTD markers were explored. RESULTS: Gal-3 levels were increased significantly in patients with FTD, mainly across brain tissue and CSF, compared to controls. Remarkably, Gal-3 levels were higher in cases with tau pathology than TAR-DNA Binding Protein 43 (TDP-43) pathology. Only MAPT mutation carriers displayed increased Gal-3 levels in CSF samples, which correlated with total tau and 14-3-3. DISCUSSION: Our findings underscore the potential of Gal-3 as a diagnostic marker for FTD, particularly in MAPT cases, and highlights the relation of Gal-3 with neuronal injury markers.

Galectin-3 shapes toxic alpha-synuclein strains in Parkinson's disease.

Parkinson's Disease (PD) is a neurodegenerative and progressive disorder characterised by intracytoplasmic inclusions called Lewy bodies (LB) and degeneration of dopaminergic neurons in the substantia nigra (SN). Aggregated α-synuclein (αSYN) is known to be the main component of the LB. It has also been reported to interact with several proteins and organelles. Galectin-3 (GAL3) is known to have a detrimental function in neurodegenerative diseases. It is a galactose-binding protein without known catalytic activity and is expressed mainly by activated microglial cells in the central nervous system (CNS). GAL3 has been previously found in the outer layer of the LB in post-mortem brains. However, the role of GAL3 in PD is yet to be elucidated. In post-mortem samples, we identified an association between GAL3 and LB in all the PD subjects studied. GAL3 was linked to less αSYN in the LB outer layer and other αSYN deposits, including pale bodies. GAL3 was also associated with disrupted lysosomes. In vitro studies demonstrate that exogenous recombinant Gal3 is internalised by neuronal cell lines and primary neurons where it interacts with endogenous αSyn fibrils. In addition, aggregation experiments show that Gal3 affects spatial propagation and the stability of pre-formed αSyn fibrils resulting in short, amorphous toxic strains. To further investigate these observations in vivo, we take advantage of WT and Gal3KO mice subjected to intranigral injection of adenovirus overexpressing human αSyn as a PD model. In line with our in vitro studies, under these conditions, genetic deletion of GAL3 leads to increased intracellular αSyn accumulation within dopaminergic neurons and remarkably preserved dopaminergic integrity and motor function. Overall, our data suggest a prominent role for GAL3 in the aggregation process of αSYN and LB formation, leading to the production of short species to the detriment of larger strains which triggers neuronal degeneration in a mouse model of PD.

LPS priming before plaque deposition impedes microglial activation and restrains Aß pathology in the 5xFAD mouse model of Alzheimer's disease.

Microglia have an innate immunity memory (IIM) with divergent functions in different animal models of neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized by chronic neuroinflammation, neurodegeneration, tau tangles and ß-amyloid (Aß) deposition. Systemic inflammation has been implicated in contributing to the progression of AD. Multiple reports have demonstrated unique microglial signatures in AD mouse models and patients. However, the proteomic profiles of microglia modified by IIM have not been well-documented in an AD model. Therefore, in the present study, we investigate whether lipopolysaccharide (LPS)-induced IIM in the pre-clinical stage of AD alters the microglial responses and shapes the neuropathology. We accomplished this by priming 5xFAD and wild-type (WT) mice with an LPS injection at 6 weeks (before the robust development of plaques). 140 days later, we evaluated microglial morphology, activation, the microglial barrier around Aß, and Aß deposition in both 5xFAD primed and unprimed mice. Priming induced decreased soma size of microglia and reduced colocalization of PSD95 and Synaptophysin in the retrosplenial cortex. Priming appeared to increase phagocytosis of Aß, resulting in fewer Thioflavin S+ Aß fibrils in the dentate gyrus. RIPA-soluble Aß 40 and 42 were significantly reduced in Primed-5xFAD mice leading to a smaller size of MOAB2+ Aß plaques in the prefrontal cortex. We also found that Aß-associated microglia in the Primed-5xFAD mice were less activated and fewer in number. After priming, we also observed improved memory performance in 5xFAD. To further elucidate the molecular mechanism underlying these changes, we performed quantitative proteomic analysis of microglia and bone marrow monocytes. A specific pattern in the microglial proteome was revealed in primed 5xFAD mice. These results suggest that the imprint signatures of primed microglia display a distinctive phenotype and highlight the potential for a beneficial adaption of microglia when intervention occurs in the pre-clinical stage of AD.

Galectin-3 is elevated in CSF and is associated with Aß deposits and tau aggregates in brain tissue in Alzheimer's disease.

Galectin-3 (Gal-3) is a beta-galactosidase binding protein involved in microglial activation in the central nervous system (CNS). We previously demonstrated the crucial deleterious role of Gal-3 in microglial activation in Alzheimer's disease (AD). Under AD conditions, Gal-3 is primarily expressed by microglial cells clustered around Aß plaques in both human and mouse brain, and knocking out Gal-3 reduces AD pathology in AD-model mice. To further unravel the importance of Gal-3-associated inflammation in AD, we aimed to investigate the Gal-3 inflammatory response in the AD continuum. First, we measured Gal-3 levels in neocortical and hippocampal tissue from early-onset AD patients, including genetic and sporadic cases. We found that Gal-3 levels were significantly higher in both cortex and hippocampus in AD subjects. Immunohistochemistry revealed that Gal-3+ microglial cells were associated with amyloid plaques of a larger size and more irregular shape and with neurons containing tau-inclusions. We then analyzed the levels of Gal-3 in cerebrospinal fluid (CSF) from AD patients (n = 119) compared to control individuals (n = 36). CSF Gal-3 levels were elevated in AD patients compared to controls and more strongly correlated with tau (p-Tau181 and t-tau) and synaptic markers (GAP-43 and neurogranin) than with amyloid-ß. Lastly, principal component analysis (PCA) of AD biomarkers revealed that CSF Gal-3 clustered and associated with other CSF neuroinflammatory markers, including sTREM-2, GFAP, and YKL-40. This neuroinflammatory component was more highly expressed in the CSF from amyloid-ß positive (A+), CSF p-Tau181 positive (T+), and biomarker neurodegeneration positive/negative (N+/-) (A + T + N+/-) groups compared to the A + T-N- group. Overall, Gal-3 stands out as a key pathological biomarker of AD pathology that is measurable in CSF and, therefore, a potential target for disease-modifying therapies involving the neuroinflammatory response.

Correlative imaging to resolve molecular structures in individual cells: Substrate validation study for super-resolution infrared microspectroscopy.

Light microscopy has been a favorite tool of biological studies for almost a century, recently producing detailed images with exquisite molecular specificity achieving spatial resolution at nanoscale. However, light microscopy is insufficient to provide chemical information as a standalone technique. An increasing amount of evidence demonstrates that optical photothermal infrared microspectroscopy (O-PTIR) is a valuable imaging tool that can extract chemical information to locate molecular structures at submicron resolution. To further investigate the applicability of sub-micron infrared microspectroscopy for biomedical applications, we analyzed the contribution of substrate chemistry to the infrared spectra acquired from individual neurons grown on various imaging substrates. To provide an example of correlative immunofluorescence/O-PTIR imaging, we used immunofluorescence to locate specific organelles for O-PTIR measurement, thus capturing molecular structures at the sub-cellular level directly in cells, which is not possible using traditional infrared microspectroscopy or immunofluorescence microscopy alone.

Acute systemic LPS-exposure impairs perivascular CSF distribution in mice.

BACKGROUND: The exchange of cerebrospinal (CSF) and interstitial fluid is believed to be vital for waste clearance in the brain. The sleep-dependent glymphatic system, which is comprised of perivascular flow of CSF and is largely dependent on arterial pulsatility and astrocytic aquaporin-4 (AQP4) expression, facilitates much of this brain clearance. During the last decade, several observations have indicated that impaired glymphatic function goes hand in hand with neurodegenerative diseases. Since pathologies of the brain carry inflammatory components, we wanted to know how acute inflammation, e.g., with lipopolysaccharide (LPS) injections, would affect the glymphatic system. In this study, we aim to measure the effect of LPS on perivascular CSF distribution as a measure of glymphatic function. METHODS: Three hours after injection of LPS (1 mg/kg i.p.), C57bl/6 mice were (1) imaged for two CSF tracers, injected into cisterna magna, (2) transcardially perfused with buffer, or (3) used for physiological readouts. Tracer flow was imaged using a low magnification microscope on fixed brains, as well as using vibratome-cut slices for measuring tracer penetration in the brain. Cytokines, glial, and BBB-permeability markers were measured with ELISAs, Western blots, and immunohistochemistry. Cerebral blood flow was approximated using laser Doppler flowmetry, respiration and heart rate with a surgical monitor, and AQP4-polarization was quantified using confocal microscopy of immunolabeled brain sections. RESULTS: LPS-injections significantly lowered perivascular CSF tracer flow and penetration into the parenchyma. No differences in AQP4 polarization, cytokines, astroglial and BBB markers, cerebral blood flow, or respiration were detected in LPS-injected mice, although LPS did elevate cortical Iba1+ area and heart rate. CONCLUSIONS: This study reports another physiological response after acute exposure to the bacterial endotoxin LPS, namely the statistically significant decrease in perivascular distribution of CSF. These observations may benefit our understanding of the role of systemic inflammation in brain clearance.

Neuronal spreading and plaque induction of intracellular Aß and its disruption of Aß homeostasis.

The amyloid-beta peptide (Aß) is thought to have prion-like properties promoting its spread throughout the brain in Alzheimer's disease (AD). However, the cellular mechanism(s) of this spread remains unclear. Here, we show an important role of intracellular Aß in its prion-like spread. We demonstrate that an intracellular source of Aß can induce amyloid plaques in vivo via hippocampal injection. We show that hippocampal injection of mouse AD brain homogenate not only induces plaques, but also damages interneurons and affects intracellular Aß levels in synaptically connected brain areas, paralleling cellular changes seen in AD. Furthermore, in a primary neuron AD model, exposure of picomolar amounts of brain-derived Aß leads to an apparent redistribution of Aß from soma to processes and dystrophic neurites. We also observe that such neuritic dystrophies associate with plaque formation in AD-transgenic mice. Finally, using cellular models, we propose a mechanism for how intracellular accumulation of Aß disturbs homeostatic control of Aß levels and can contribute to the up to 10,000-fold increase of Aß in the AD brain. Our data indicate an essential role for intracellular prion-like Aß and its synaptic spread in the pathogenesis of AD.

The MHC class II transactivator modulates seeded alpha-synuclein pathology and dopaminergic neurodegeneration in an in vivo rat model of Parkinson's disease.

BACKGROUND: Abnormal folding, aggregation and spreading of alpha-synuclein (αsyn) is a mechanistic hypothesis for the progressive neuropathology in Parkinson's disease (PD). Spread of αsyn between cells is supported by clinical, neuropathological and experimental evidence. It has been proposed that a pro-inflammatory micro-environment in response to αsyn can promote its aggregation. We have previously shown that allelic differences in the major histocompatibility complex class two transactivator (Mhc2ta) gene, located in the VRA4 locus, alter MHCII expression levels, microglial activation and antigen presentation capacity in rats upon human αsyn over-expression. In addition, Mhc2ta regulated dopaminergic neurodegeneration and the extent of motor impairment. The purpose of this study was to determine whether Mhc2ta regulates αsyn aggregation, propagation and dopaminergic pathology in an αsyn pre-formed fibril (PFF)-seeded in vivo model of PD. METHODS: The DA and DA.VRA4 congenic rat strains share background genome but display differential microglial antigen presenting capacity due to different Mhc2ta alleles in the VRA4 locus. PFFs of human αsyn or BSA solution were injected unilaterally to the striatum of DA and DA.VRA4 rats two weeks after ipsilateral administration of recombinant adeno-associated virus (rAAV) vectors carrying human αsyn or GFP to the substantia nigra pars compacta. Behavioural assessment was performed at 2, 5 and 8 weeks while histological evaluation of αsyn pathology, inflammation and neurodegeneration as well as determination of serum cytokine profiles were performed at 8 weeks. RESULTS: rAAV-mediated expression of human αsyn in nigral dopaminergic neurons combined with striatal PFF administration induced enhanced αsyn pathology in DA.VRA4 compared to DA rats. Mhc2ta thus significantly regulated the seeding, propagation and toxicity of αsyn in vivo. This was reflected in terms of wider extent and anatomical distribution of αsyn inclusions, ranging from striatum to the forebrain, midbrain, hindbrain and cerebellum in DA.VRA4. Compared to DA rats, DA.VRA4 also displayed enhanced motor impairment and dopaminergic neurodegeneration as well as higher levels of the proinflammatory cytokines IL-2 and TNFα in serum. CONCLUSIONS: We conclude that the key regulator of MHCII expression, Mhc2ta, modulates neuroinflammation, αsyn-seeded Lewy-like pathology, dopaminergic neurodegeneration and motor impairment. This makes Mhc2ta and microglial antigen presentation promising therapeutic targets for reducing the progressive neuropathology and clinical manifestations in PD.

Amyloid Structural Changes Studied by Infrared Microspectroscopy in Bigenic Cellular Models of Alzheimer's Disease.

Alzheimer's disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of ß-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to neurotoxicity remains unknown. This lack of knowledge is due to the challenging nature of characterizing the secondary structure of amyloids in cells. To overcome this and investigate the molecular changes in proteins directly in cells, we used synchrotron-based infrared microspectroscopy, a label-free and non-destructive technique available for in situ molecular imaging, to detect structural changes in proteins and lipids. Specifically, we evaluated the formation of ß-sheet structures in different monogenic and bigenic cellular models of Alzheimer's disease that we generated for this study. We report on the possibility to discern different amyloid signatures directly in cells using infrared microspectroscopy and demonstrate that bigenic (amyloid-ß, α-synuclein) and (amyloid-ß, Tau) neuron-like cells display changes in ß-sheet load. Altogether, our findings support the notion that different molecular mechanisms of amyloid aggregation, as opposed to a common mechanism, are triggered by the specific cellular environment and, therefore, that various mechanisms lead to the development of Alzheimer's disease.

The Ubiquitin Proteasome System in Neuromuscular Disorders: Moving Beyond Movement.

Neuromuscular disorders (NMDs) affect 1 in 3000 people worldwide. There are more than 150 different types of NMDs, where the common feature is the loss of muscle strength. These disorders are classified according to their neuroanatomical location, as motor neuron diseases, peripheral nerve diseases, neuromuscular junction diseases, and muscle diseases. Over the years, numerous studies have pointed to protein homeostasis as a crucial factor in the development of these fatal diseases. The ubiquitin-proteasome system (UPS) plays a fundamental role in maintaining protein homeostasis, being involved in protein degradation, among other cellular functions. Through a cascade of enzymatic reactions, proteins are ubiquitinated, tagged, and translocated to the proteasome to be degraded. Within the ubiquitin system, we can find three main groups of enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin-protein ligases). Only the ubiquitinated proteins with specific chain linkages (such as K48) will be degraded by the UPS. In this review, we describe the relevance of this system in NMDs, summarizing the UPS proteins that have been involved in pathological conditions and neuromuscular disorders, such as Spinal Muscular Atrophy (SMA), Charcot-Marie-Tooth disease (CMT), or Duchenne Muscular Dystrophy (DMD), among others. A better knowledge of the processes involved in the maintenance of proteostasis may pave the way for future progress in neuromuscular disorder studies and treatments.

Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease in which the formation of extracellular aggregates of amyloid beta (Aß) peptide, fibrillary tangles of intraneuronal tau and microglial activation are major pathological hallmarks. One of the key molecules involved in microglial activation is galectin-3 (gal3), and we demonstrate here for the first time a key role of gal3 in AD pathology. Gal3 was highly upregulated in the brains of AD patients and 5xFAD (familial Alzheimer's disease) mice and found specifically expressed in microglia associated with Aß plaques. Single-nucleotide polymorphisms in the LGALS3 gene, which encodes gal3, were associated with an increased risk of AD. Gal3 deletion in 5xFAD mice attenuated microglia-associated immune responses, particularly those associated with TLR and TREM2/DAP12 signaling. In vitro data revealed that gal3 was required to fully activate microglia in response to fibrillar Aß. Gal3 deletion decreased the Aß burden in 5xFAD mice and improved cognitive behavior. Interestingly, a single intrahippocampal injection of gal3 along with Aß monomers in WT mice was sufficient to induce the formation of long-lasting (2 months) insoluble Aß aggregates, which were absent when gal3 was lacking. High-resolution microscopy (stochastic optical reconstruction microscopy) demonstrated close colocalization of gal3 and TREM2 in microglial processes, and a direct interaction was shown by a fluorescence anisotropy assay involving the gal3 carbohydrate recognition domain. Furthermore, gal3 was shown to stimulate TREM2-DAP12 signaling in a reporter cell line. Overall, our data support the view that gal3 inhibition may be a potential pharmacological approach to counteract AD.

Loss of Regulator of G-Protein Signaling 5 Leads to Neurovascular Protection in Stroke.

Background and Purpose- In ischemic stroke, breakdown of the blood-brain barrier (BBB) aggravates brain damage. Pericyte detachment contributes to BBB disruption and neurovascular dysfunction, but little is known about its regulation in stroke. Here, we investigated how loss of RGS5 (regulator of G protein signaling 5) in pericytes affects BBB breakdown in stroke and its consequences. Method- We used RGS5 knockout and control mice and applied a permanent middle cerebral occlusion model. We analyzed pericyte numbers, phenotype, and vessel morphology using immunohistochemistry and confocal microscopy. We investigated BBB breakdown by measuring endothelial coverage, tight junctions, and AQP4 (aquaporin 4) in addition to BBB permeability (fluorescent-conjugated dextran extravasation). Tissue hypoxia was assessed with pimonidazole hydrochloride and neuronal death quantified with the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results- We demonstrate that loss of RGS5 increases pericyte numbers and their endothelial coverage, which is associated with higher capillary density and length, and significantly less BBB damage after stroke. Loss of RGS5 in pericytes results in reduced vascular leakage and preserved tight junctions and AQP4, decreased cerebral hypoxia, and partial neuronal protection in the infarct area. Conclusions- Our findings show that loss of RGS5 affects pericyte-related BBB preservation in stroke and identifies RGS5 as an important target for neurovascular protection.

Inflammation leads to distinct populations of extracellular vesicles from microglia.

BACKGROUND: Activated microglia play an essential role in inflammatory responses elicited in the central nervous system (CNS). Microglia-derived extracellular vesicles (EVs) are suggested to be involved in propagation of inflammatory signals and in the modulation of cell-to-cell communication. However, there is a lack of knowledge on the regulation of EVs and how this in turn facilitates the communication between cells in the brain. Here, we characterized microglial EVs under inflammatory conditions and investigated the effects of inflammation on the EV size, quantity, and protein content. METHODS: We have utilized western blot, nanoparticle tracking analysis (NTA), and mass spectrometry to characterize EVs and examine the alterations of secreted EVs from a microglial cell line (BV2) following lipopolysaccharide (LPS) and tumor necrosis factor (TNF) inhibitor (etanercept) treatments, or either alone. The inflammatory responses were measured with multiplex cytokine ELISA and western blot. We also subjected TNF knockout mice to experimental stroke (permanent middle cerebral artery occlusion) and validated the effect of TNF inhibition on EV release. RESULTS: Our analysis of EVs originating from activated BV2 microglia revealed a significant increase in the intravesicular levels of TNF and interleukin (IL)-6. We also observed that the number of EVs released was reduced both in vitro and in vivo when inflammation was inhibited via the TNF pathway. Finally, via mass spectrometry, we identified 49 unique proteins in EVs released from LPS-activated microglia compared to control EVs (58 proteins in EVs released from LPS-activated microglia and 37 from control EVs). According to Gene Ontology (GO) analysis, we found a large increase of proteins related to translation and transcription in EVs from LPS. Importantly, we showed a distinct profile of proteins found in EVs released from LPS treated cells compared to control. CONCLUSIONS: We demonstrate altered EV production in BV2 microglial cells and altered cytokine levels and protein composition carried by EVs in response to LPS challenge. Our findings provide new insights into the potential roles of EVs that could be related to the pathogenesis in neuroinflammatory diseases.

Focal, but not global, cerebral ischaemia causes loss of myenteric neurons and upregulation of vasoactive intestinal peptide in mouse ileum.

Reduced blood flow to the brain induces cerebral ischaemia, potentially causing central injury and peripheral complications including gastrointestinal (GI) dysfunction. The pathophysiology behind GI symptoms is suspected to be neuropathy in the enteric nervous system (ENS), which is essential in regulating GI function. This study investigates if enteric neuropathy occurs after cerebral ischaemia, by analysing neuronal survival and relative numbers of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) expressing neurons in mouse ileum after three types of cerebral ischaemia. Focal cerebral ischaemia, modelled by permanent middle cerebral artery occlusion (pMCAO) and global cerebral ischaemia, modelled with either transient occlusion of both common carotid arteries followed by reperfusion (GCIR) or chronic cerebral hypoperfusion (CCH) was performed on C56BL/6 mice. Sham-operated mice for each ischaemia model served as control. Ileum was collected after 1-17 weeks, depending on model, and analysed using morphometry and immunocytochemistry. For each group, intestinal mucosa and muscle layer thicknesses, neuronal numbers and relative proportions of neurons immunoreactive (IR) for nNOS or VIP were estimated. No alterations in mucosa or muscle layer thicknesses were noted in any of the groups. Loss of myenteric neurons and an increased number of VIP-IR submucous neurons were found in mouse ileum 7 days after pMCAO. None of the global ischaemia models showed any alterations in neuronal survival or relative numbers of VIP- and nNOS-IR neurons. We conclude that focal cerebral ischaemia and global cerebral ischaemia influence enteric neuronal survival differently. This is suggested to reflect differences in peripheral neuro-immune responses.

Caspase signalling controls microglia activation and neurotoxicity.

Activation of microglia and inflammation-mediated neurotoxicity are suggested to play a decisive role in the pathogenesis of several neurodegenerative disorders. Activated microglia release pro-inflammatory factors that may be neurotoxic. Here we show that the orderly activation of caspase-8 and caspase-3/7, known executioners of apoptotic cell death, regulate microglia activation through a protein kinase C (PKC)-δ-dependent pathway. We find that stimulation of microglia with various inflammogens activates caspase-8 and caspase-3/7 in microglia without triggering cell death in vitro and in vivo. Knockdown or chemical inhibition of each of these caspases hindered microglia activation and consequently reduced neurotoxicity. We observe that these caspases are activated in microglia in the ventral mesencephalon of Parkinson's disease (PD) and the frontal cortex of individuals with Alzheimer's disease (AD). Taken together, we show that caspase-8 and caspase-3/7 are involved in regulating microglia activation. We conclude that inhibition of these caspases could be neuroprotective by targeting the microglia rather than the neurons themselves.

Cell therapy centered on IL-1Ra is neuroprotective in experimental stroke.

Cell-based therapies are emerging as new promising treatments in stroke. However, their functional mechanism and therapeutic potential during early infarct maturation has so far received little attention. Here, we asked if cell-based delivery of the interleukin-1 receptor antagonist (IL-1Ra), a known neuroprotectant in stroke, can promote neuroprotection, by modulating the detrimental inflammatory response in the tissue at risk. We show by the use of IL-1Ra-overexpressing and IL-1Ra-deficient mice that IL-1Ra is neuroprotective in stroke. Characterization of the cellular and spatiotemporal production of IL-1Ra and IL-1α/ß identifies microglia, not infiltrating leukocytes, as the major sources of IL-1Ra after experimental stroke, and shows IL-1Ra and IL-1ß to be produced by segregated subsets of microglia with a small proportion of these cells co-expressing IL-1α. Reconstitution of whole body irradiated mice with IL-1Ra-producing bone marrow cells is associated with neuroprotection and recruitment of IL-1Ra-producing leukocytes after stroke. Neuroprotection is also achieved by therapeutic injection of IL-1Ra-producing bone marrow cells 30 min after stroke onset, additionally improving the functional outcome in two different stroke models. The IL-1Ra-producing bone marrow cells increase the number of IL-1Ra-producing microglia, reduce the availability of IL-1ß, and modulate mitogen-activated protein kinase (MAPK) signaling in the ischemic cortex. The importance of these results is underlined by demonstration of IL-1Ra-producing cells in the human cortex early after ischemic stroke. Taken together, our results attribute distinct neuroprotective or neurotoxic functions to segregated subsets of microglia and suggest that treatment strategies increasing the production of IL-1Ra by infiltrating leukocytes or microglia may also be neuroprotective if applied early after stroke onset in patients.

Change in autoantibody and cytokine responses during the evolution of neuromyelitis optica in patients with systemic lupus erythematosus: A preliminary study.

BACKGROUND: Neuromyelitis optica (NMO)-systemic lupus erythematosus (SLE) association is a rare condition characterized by multiple autoantibodies. OBJECTIVE: To examine if, during the evolution of NMO, anti-AQP4 responses are part of polyclonal B cell activation, and if T cell responses contribute. METHODS: In 19 samples of six patients who developed NMO during SLE, we examined the correlation of AQP4-IgG1 and IgM with (i) anti-MOG IgG and IgM, (ii) anti-nuclear, anti-nucleosome and anti-dsDNA IgG antibodies, (iii) cytokines and chemokines in the serum and (iv) longitudinal relation to NMO relapses/remission. RESULTS: AQP4-IgG1 was present 1-2-5 years before the first NMO relapse. During relapse, AQP4-IgG1, ANA, anti-dsDNA and anti-nucleosome antibodies were elevated. Anti-MOG IgG/IgM and AQP4-IgM antibodies were not detected. AQP4-IgG1 antibodies correlated with concentration of anti-nucleosome, IFN-γ,interferon-gamma-induced CCL10/IP-10 and CCL17/TARC (p<0.05, respectively). CCL17/TARC correlated with levels of anti-nucleosome and anti-dsDNA (p<0.05, respectively). Compared to healthy subjects, concentration of IFN-γ and CCL17/TARC was higher in NMO/SLE (p<0.05). CONCLUSIONS: AQP4-IgG1 antibodies are present in the sera years before the first NMO attack in patients with SLE; elevation of anti-AQP4 is part of a polyclonal B cell response during NMO relapses; in spite of multiple autoantibodies in the serum, MOG antibodies were not present; Th1 responses accompany autoantibody responses in NMO/SLE.

Who let the dogs out?: detrimental role of Galectin-3 in hypoperfusion-induced retinal degeneration.

BACKGROUND: Retinal ischemia results in a progressive degeneration of neurons and a pathological activation of glial cells, resulting in vision loss. In the brain, progressive damage after ischemic insult has been correlated to neuroinflammatory processes involving microglia. Galectin-3 has been shown to mediate microglial responses to ischemic injury in the brain. Therefore, we wanted to explore the contribution of Galectin-3 (Gal-3) to hypoperfusion-induced retinal degeneration in mice. METHODS: Gal-3 knockout (Gal-3 KO) and wildtype (WT) C57BL/6 mice were subjected to chronic cerebral hypoperfusion by bilateral narrowing of the common carotid arteries using metal coils resulting in a 30% reduction of blood flow. Sham operated mice served as controls. After 17 weeks, the mice were sacrificed and the eyes were analyzed for retinal architecture, neuronal cell survival, and glial reactivity using morphological staining and immunohistochemistry. RESULTS: Hypoperfusion caused a strong increase in Gal-3 expression and microglial activation in WT mice, coupled with severe degenerative damage to all retinal neuronal subtypes, remodeling of the retinal lamination and Müller cell gliosis. In contrast, hypoperfused Gal-3 KO mice displayed a retained laminar architecture, a significant preservation of photoreceptors and ganglion cell neurons, and an attenuation of microglial and Müller cell activation. CONCLUSION: Moderate cerebral blood flow reduction in the mouse results in severe retinal degenerative damage. In mice lacking Gal-3 expression, pathological changes are significantly attenuated. Gal-3 is thereby a potential target for treatment and prevention of hypoperfusion-induced retinal degeneration and a strong candidate for further research as a factor behind retinal degenerative disease.