The aging mouse CNS is protected by an autophagy-dependent microglia population promoted by IL-34.

Microglia harness an unutilized health-promoting potential in age-related neurodegenerative and neuroinflammatory diseases, conditions like progressive multiple sclerosis (MS). Our research unveils an microglia population emerging in the cortical brain regions of aging mice, marked by ERK1/2, Akt, and AMPK phosphorylation patterns and a transcriptome indicative of activated autophagy - a process critical for cellular adaptability. By deleting the core autophagy gene Ulk1 in microglia, we reduce this population in the central nervous system of aged mice. Notably, this population is found dependent on IL-34, rather than CSF1, although both are ligands for CSF1R. When aging mice are exposed to autoimmune neuroinflammation, the loss of autophagy-dependent microglia leads to neural and glial cell death and increased mortality. Conversely, microglial expansion mediated by IL-34 exhibits a protective effect. These findings shed light on an autophagy-dependent neuroprotective microglia population as a potential target for treating age-related neuroinflammatory conditions, including progressive MS.

Thrombolytic tPA-induced hemorrhagic transformation of ischemic stroke is mediated by PKCß phosphorylation of occludin.

The current standard of care for moderate to severe ischemic stroke is thrombolytic therapy with tissue plasminogen activator (tPA). Treatment with tPA can significantly improve neurologic outcomes; however, thrombolytic therapy is associated with an increased risk of intracerebral hemorrhage (ICH). The risk of hemorrhage significantly limits the use of thrombolytic therapy, and identifying pathways induced by tPA that increase this risk could provide new therapeutic options to extend thrombolytic therapy to a wider patient population. Here, we investigate the role of protein kinase Cß (PKCß) phosphorylation of the tight junction protein occludin during ischemic stroke and its role in cerebrovascular permeability. We show that activation of this pathway by tPA is associated with an increased risk of ICH. Middle cerebral artery occlusion (MCAO) increased phosphorylation of occludin serine 490 (S490) in the ischemic penumbra in a tPA-dependent manner, as tPA-/- mice were significantly protected from MCAO-induced occludin phosphorylation. Intraventricular injection of tPA in the absence of ischemia was sufficient to induce occludin phosphorylation and vascular permeability in a PKCß-dependent manner. Blocking occludin phosphorylation, either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCß, reduced MCAO-induced permeability and improved functional outcome. Furthermore, inhibiting PKCß after MCAO prevented ICH associated with delayed thrombolysis. These results show that PKCß phosphorylation of occludin is a downstream mediator of tPA-induced cerebrovascular permeability and suggest that PKCß inhibitors could improve stroke outcome and prevent ICH associated with delayed thrombolysis, potentially extending the window for thrombolytic therapy in stroke.

Identification of four novel T cell autoantigens and personal autoreactive profiles in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which pathological T cells, likely autoimmune, play a key role. Despite its central importance, the autoantigen repertoire remains largely uncharacterized. Using a novel in vitro antigen delivery method combined with the Human Protein Atlas library, we screened for T cell autoreactivity against 63 CNS-expressed proteins. We identified four previously unreported autoantigens in MS: fatty acid-binding protein 7, prokineticin-2, reticulon-3, and synaptosomal-associated protein 91, which were verified to induce interferon-γ responses in MS in two cohorts. Autoreactive profiles were heterogeneous, and reactivity to several autoantigens was MS-selective. Autoreactive T cells were predominantly CD4+ and human leukocyte antigen-DR restricted. Mouse immunization induced antigen-specific responses and CNS leukocyte infiltration. This represents one of the largest systematic efforts to date in the search for MS autoantigens, demonstrates the heterogeneity of autoreactive profiles, and highlights promising targets for future diagnostic tools and immunomodulatory therapies in MS.

A Call to Use the Multicomponent Exercise Tai Chi to Improve Recovery From COVID-19 and Long COVID.

Approximately 10% of all COVID patients develop long COVID symptoms, which may persist from 1 month up to longer than 1 year. Long COVID may affect any organ/system and manifest in a broad range of symptoms such as shortness of breath, post-exercise malaise, cognitive decline, chronic fatigue, gastrointestinal disorders, musculoskeletal pain and deterioration of mental health. In this context, health institutions struggle with resources to keep up with the prolonged rehabilitation for the increasing number of individuals affected by long COVID. Tai Chi is a multicomponent rehabilitation approach comprising correct breathing technique, balance and neuromuscular training as well as stress- and emotional management. In addition, practicing Tai Chi elicits the relaxation response and balances the autonomic nervous system thus regulating respiration, heart rate, blood pressure and vitality in general. Moreover, Tai Chi has been shown to increase lung capacity, improve cognitive status and mental health, and thereby even the quality of life in diseases such as chronic obstructive pulmonary disease (COPD). Hence, we advocate Tai Chi as potent and suitable rehabilitation tool for post-COVID-19-affected individuals.

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund's adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

Effect of Vitamin D on Experimental Autoimmune Neuroinflammation Is Dependent on Haplotypes Comprising Naturally Occurring Allelic Variants of CIITA (Mhc2ta).

An increasing body of evidence associates low vitamin D levels with increased risk of multiple sclerosis (MS), suggesting the possibility of a gene-environment interaction for this environmental factor in MS pathogenesis. Moreover, it has been shown that vitamin D downregulates major histocompatibility complex (MHC) class II expression in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We here report about the impact of a dietary vitamin D supplementation on EAE in the rat strains having functionally relevant allelic variations in the CIITA (Mhc2ta) gene, a master regulator of MHC class II expression. Full length myelin oligodendrocyte glycoprotein (MOG)-EAE was induced in DA.PVGav1-Vra4 congenic rats harboring the Vra4 locus from PVG strain in the EAE- susceptible DA background, and compared to the parental strains. The congenic rats fed with either vitamin D supplemented, deprived or regular diet developed an intermediate clinical EAE phenotype, in contrast to DA and PVG strains. Immunopathological studies revealed vitamin D dose-dependent effect on demyelination and inflammatory infiltration of the central nervous system (CNS), expression of MHC class II and CIITA, as well as downregulation of a range of pro-inflammatory genes. Taken together, our findings demonstrate an impact of vitamin D on the target tissue pathology and peripheral immune response during EAE in DA.PVGav1-Vra4 congenic strain. Thereby, our data provide evidence of a modulatory effect of vitamin D in context of genetic variances in the Vra4 locus/Mhc2ta gene in MS-like neuroinflammation, with potential relevance for the human demyelinating disease.

Blocking PDGF-CC signaling ameliorates multiple sclerosis-like neuroinflammation by inhibiting disruption of the blood-brain barrier.

Disruption of blood-brain barrier (BBB) integrity is a feature of various neurological disorders. Here we found that the BBB is differently affected during the preclinical, progression and remission phase of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We have identified an upregulation of pro-inflammatory and pro-angiogenic factors in the BBB transcriptome and down-regulation of endothelial tight junction members coinciding with elevated BBB leakage specifically during the progression phase. These changes were antagonized by blocking PDGFRα signaling with the small tyrosine kinase inhibitor imatinib. Moreover, targeting the PDGFRα ligand PDGF-CC using a neutralizing antibody, facilitated recovery of BBB integrity and improvement of EAE symptoms. Intracerebroventricular injection of PDGF-CC induced upregulation, whereas blocking PDGF-CC during EAE led to downregulation of Tnfa and Il1a at the BBB. Our findings suggest that blocking PDGF-CC counteracts fundamental aspects of endothelial cell activation and disruption of the BBB by decreasing Tnfa and Il1a expression. We also demonstrate that both PDGF-CC and its receptor PDGFRα were upregulated in MS lesions indicating that blocking PDGF-CC may be considered a novel treatment for MS.

Microglial autophagy-associated phagocytosis is essential for recovery from neuroinflammation.

Multiple sclerosis (MS) is a leading cause of incurable progressive disability in young adults caused by inflammation and neurodegeneration in the central nervous system (CNS). The capacity of microglia to clear tissue debris is essential for maintaining and restoring CNS homeostasis. This capacity diminishes with age, and age strongly associates with MS disease progression, although the underlying mechanisms are still largely elusive. Here, we demonstrate that the recovery from CNS inflammation in a murine model of MS is dependent on the ability of microglia to clear tissue debris. Microglia-specific deletion of the autophagy regulator Atg7, but not the canonical macroautophagy protein Ulk1, led to increased intracellular accumulation of phagocytosed myelin and progressive MS-like disease. This impairment correlated with a microglial phenotype previously associated with neurodegenerative pathologies. Moreover, Atg7-deficient microglia showed notable transcriptional and functional similarities to microglia from aged wild-type mice that were also unable to clear myelin and recover from disease. In contrast, induction of autophagy in aged mice using the disaccharide trehalose found in plants and fungi led to functional myelin clearance and disease remission. Our results demonstrate that a noncanonical form of autophagy in microglia is responsible for myelin degradation and clearance leading to recovery from MS-like disease and that boosting this process has a therapeutic potential for age-related neuroinflammatory conditions.

VEGF-B signaling impairs endothelial glucose transcytosis by decreasing membrane cholesterol content.

Regulation of endothelial nutrient transport is poorly understood. Vascular endothelial growth factor B (VEGF-B) signaling in endothelial cells promotes uptake and transcytosis of fatty acids from the bloodstream to the underlying tissue, advancing pathological lipid accumulation and lipotoxicity in diabetic complications. Here, we demonstrate that VEGF-B limits endothelial glucose transport independent of fatty acid uptake. Specifically, VEGF-B signaling impairs recycling of low-density lipoprotein receptor (LDLR) to the plasma membrane, leading to reduced cholesterol uptake and membrane cholesterol loading. Reduced cholesterol levels in the membrane leads to a decrease in glucose transporter 1 (GLUT1)-dependent endothelial glucose uptake. Inhibiting VEGF-B in vivo reconstitutes membrane cholesterol levels and restores glucose uptake, which is of particular relevance for conditions involving insulin resistance and diabetic complications. In summary, our study reveals a mechanism whereby VEGF-B regulates endothelial nutrient uptake and highlights the impact of membrane cholesterol for regulation of endothelial glucose transport.

Preclinical toxicological assessment of a novel monoclonal antibody targeting human platelet-derived growth factor CC (PDGF-CC) in PDGF-CChum mice.

Platelet-derived growth factor CC (PDGF-CC) is important during foetal development but also in pathogenesis of neurologic diseases, cancer and fibrosis. We have previously demonstrated that blocking the PDGF-CC/PDGF receptor alpha (PDGFRα) axis resulted in reduction of stroke volume and cerebrovascular permeability after experimentally induced stroke. Recently, we could translate these findings into the clinic showing that imatinib, a small tyrosine kinase inhibitor targeting PDGF receptors, can significantly improve neurological outcome after ischemic stroke in human. Herein we report preclinical toxicological analyses of our newly generated monoclonal anti-human PDGF-CC antibody 6B3 (mAb 6B3) in PDGF-CC humanized mice. Beside histological organ assessment, we also analysed serum, urine, haematological parameters and the general health status of the treated mice. We could not find any indications that mAb 6B3 is toxic or has other significant side effects neither in short, nor in long treatment regimens. Our results indicate that mAb 6B3 can be further developed for clinical use. This opens up the possibility to assess the therapeutic potential of blocking PDGF-CC in diverse pathological conditions such as neurologic diseases, cancer and fibrosis.

Development of monoclonal anti-PDGF-CC antibodies as tools for investigating human tissue expression and for blocking PDGF-CC induced PDGFRα signalling in vivo.

PDGF-CC is a member of the platelet-derived growth factor (PDGF) family that stimulates PDGFRα phosphorylation and thereby activates intracellular signalling events essential for development but also in cancer, fibrosis and neuropathologies involving blood-brain barrier (BBB) disruption. In order to elucidate the biological and pathological role(s) of PDGF-CC signalling, we have generated high affinity neutralizing monoclonal antibodies (mAbs) recognizing human PDGF-CC. We determined the complementarity determining regions (CDRs) of the selected clones, and mapped the binding epitope for clone 6B3. Using the monoclonal 6B3, we determined the expression pattern for PDGF-CC in different human primary tumours and control tissues, and explored its ability to neutralize PDGF-CC-induced phosphorylation of PDGFRα. In addition, we showed that PDGF-CC induced disruption of the blood-retinal barrier (BRB) was significantly reduced upon intraperitoneal administration of a chimeric anti-PDGF-CC antibody. In summary, we report on high affinity monoclonal antibodies against PDGF-CC that have therapeutic efficacy in vivo.

Widespread cortical demyelination of both hemispheres can be induced by injection of pro-inflammatory cytokines via an implanted catheter in the cortex of MOG-immunized rats.

Cortical demyelination is a common finding in patients with chronic multiple sclerosis (MS) and contributes to disease progression and overall disability. The exact pathomechanism that leads to cortical lesions is not clear. Research is limited by the fact that standard animal models of multiple sclerosis do not commonly affect the cortex, or if they do in some variants, the cortical demyelination is rather sparse and already remyelinated within a few days. In an attempt to overcome these limitations we implanted a tissue-compatible catheter into the cortex of Dark Agouti rats. After 14days the rats were immunized with 5µg myelin oligodendrocyte glycoprotein (MOG) in incomplete Freund's Adjuvant, which did not cause any clinical signs but animals developed a stable anti-MOG antibody titer. Then the animals received an injection of proinflammatory cytokines through the catheter. This led to a demyelination of cortical and subcortical areas starting from day 1 in a cone-like pattern spreading from the catheter area towards the subarachnoid space. On day 3 cortical demyelination already expanded to the contralateral hemisphere and reached its peak between days 9-15 after cytokine injection with a widespread demyelination of cortical and subcortical areas of both hemispheres. Clinically the animals showed only discrete signs of fatigue and recovered completely after day 15. Even on day 30 we still were able to detect demyelination in subpial and intracortical areas along with areas of partial and complete remyelination. Loss of cortical myelin was accompanied with marked microglia activation. A second injection of cytokines through the catheter on day 30 led to a second demyelination phase with the same symptoms, but again no detectable motor dysfunction. Suffering of the animals appeared minor compared to standard Experimental Autoimmune Encephalomyelitis and therefore, even long-term observation and repeated demyelination phases seem ethically acceptable.

Functional genomics analysis of vitamin D effects on CD4+ T cells in vivo in experimental autoimmune encephalomyelitis ‬.

Vitamin D exerts multiple immunomodulatory functions and has been implicated in the etiology and treatment of several autoimmune diseases, including multiple sclerosis (MS). We have previously reported that in juvenile/adolescent rats, vitamin D supplementation protects from experimental autoimmune encephalomyelitis (EAE), a model of MS. Here we demonstrate that this protective effect associates with decreased proliferation of CD4+ T cells and lower frequency of pathogenic T helper (Th) 17 cells. Using transcriptome, methylome, and pathway analyses in CD4+ T cells, we show that vitamin D affects multiple signaling and metabolic pathways critical for T-cell activation and differentiation into Th1 and Th17 subsets in vivo. Namely, Jak/Stat, Erk/Mapk, and Pi3K/Akt/mTor signaling pathway genes were down-regulated upon vitamin D supplementation. The protective effect associated with epigenetic mechanisms, such as (i) changed levels of enzymes involved in establishment and maintenance of epigenetic marks, i.e., DNA methylation and histone modifications; (ii) genome-wide reduction of DNA methylation, and (iii) up-regulation of noncoding RNAs, including microRNAs, with concomitant down-regulation of their protein-coding target RNAs involved in T-cell activation and differentiation. We further demonstrate that treatment of myelin-specific T cells with vitamin D reduces frequency of Th1 and Th17 cells, down-regulates genes in key signaling pathways and epigenetic machinery, and impairs their ability to transfer EAE. Finally, orthologs of nearly 50% of candidate MS risk genes and 40% of signature genes of myelin-reactive T cells in MS changed their expression in vivo in EAE upon supplementation, supporting the hypothesis that vitamin D may modulate risk for developing MS.

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS). Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

Lipocalin-2 as an Infection-Related Biomarker to Predict Clinical Outcome in Ischemic Stroke.

OBJECTIVES: From previous data in animal models of cerebral ischemia, lipocalin-2 (LCN2), a protein related to neutrophil function and cellular iron homeostasis, is supposed to have a value as a biomarker in ischemic stroke patients. Therefore, we examined LCN2 expression in the ischemic brain in an animal model and measured plasma levels of LCN2 in ischemic stroke patients. METHODS: In the mouse model of transient middle cerebral artery occlusion (tMCAO), LCN2 expression in the brain was analyzed by immunohistochemistry and correlated to cellular nonheme iron deposition up to 42 days after tMCAO. In human stroke patients, plasma levels of LCN2 were determined one week after ischemic stroke. In addition to established predictive parameters such as age, National Institutes of Health Stroke Scale and thrombolytic therapy, LCN2 was included into linear logistic regression modeling to predict clinical outcome at 90 days after stroke. RESULTS: Immunohistochemistry revealed expression of LCN2 in the mouse brain already at one day following tMCAO, and the amount of LCN2 subsequently increased with a maximum at 2 weeks after tMCAO. Accumulation of cellular nonheme iron was detectable one week post tMCAO and continued to increase. In ischemic stroke patients, higher plasma levels of LCN2 were associated with a worse clinical outcome at 90 days and with the occurrence of post-stroke infections. CONCLUSIONS: LCN2 is expressed in the ischemic brain after temporary experimental ischemia and paralleled by the accumulation of cellular nonheme iron. Plasma levels of LCN2 measured in patients one week after ischemic stroke contribute to the prediction of clinical outcome at 90 days and reflect the systemic response to post-stroke infections.

Expression of vascular endothelial growth factor (VEGF)-B and its receptor (VEGFR1) in murine heart, lung and kidney.

Metabolic diseases, such as obesity and diabetes, are a serious burden for the health system. Vascular endothelial growth factor (VEGF)-B has been shown to regulate tissue uptake and accumulation of fatty acids and is thus involved in these metabolic diseases. However, the cell-type-specific expression pattern of Vegfb and its receptor (VEGFR1, gene Flt1) remains unclear. We explore the expression of Vegfb and Flt1 in the murine heart, lung and kidney by utilizing ß-galactosidase knock-in mouse models and combining the analysis of reporter gene expression and immunofluorescence microscopy. Furthermore, Flt1 heterozygous mice were analyzed with regard to muscular fatty acid accumulation and peripheral insulin sensitivity. Throughout the heart, Vegfb expression was found in cardiomyocytes with a postnatal ventricular shift corresponding to known changes in energy requirements. Vegfb expression was also found in the pulmonary myocardium of the lung and in renal epithelial cells of the thick ascending limb of Henle's loop, the connecting tubule and the collecting duct. In all analyzed organs, VEGFR1 expression was restricted to endothelial cells. We also show that reduced expression of VEGFR1 resulted in decreased cardiac fatty acid accumulation and increased peripheral insulin sensitivity, possibly as a result of attenuated VEGF-B/VEGFR1 signaling. Our data therefore support a tightly controlled, paracrine signaling mechanism of VEGF-B to VEGFR1. The identified cell-specific expression pattern of Vegfb and Flt1 might form the basis for the development of cell-type-targeted research models and contributes to the understanding of the physiological and pathological role of VEGF-B/VEGFR1 signaling.

Presymptomatic activation of the PDGF-CC pathway accelerates onset of ALS neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with unknown origins. Neurodegeneration in ALS mouse models occurs together with signs of disrupted blood-spinal cord barrier (BSCB) and regressed capillary network, but the molecular pathways contributing to these vascular pathologies remain unknown. We show that motor neurons of human sporadic ALS patients (n = 12) have increased gene expression of PDGFC and its activator PLAT and presymptomatic activation of the PDGF-CC pathway in SOD1 (G93A) mice leads to BSCB dysfunction. Decrease of Pdgfc expression in SOD1 (G93A) mice restored vascular barrier properties, reduced motor neuron loss and delayed symptom onset by up to 3 weeks. Similarly, lower expression levels of PDGFC or PLAT in motor neurons of sporadic ALS patients were correlated with older age at disease onset. PDGF-CC inhibition and restoration of BSCB integrity did not prevent capillary regression at disease end stage. Lower vessel density was found in spinal cords of sporadic ALS patients and the degree of regression in SOD1 (G93A) mice correlated with more aggressive progression after onset regardless of BSCB status. We conclude that PDGF-CC-induced BSCB dysfunction can contribute to timing of ALS onset, allow insight into disease origins and development of targeted novel therapies.

Role of Tumor Pericytes in the Recruitment of Myeloid-Derived Suppressor Cells.

BACKGROUND: Pericytes are members of the tumor stroma; however, little is known about their origin, function, or interaction with other tumor components. Emerging evidence suggest that pericytes may regulate leukocyte transmigration. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with powerful inhibitory effects on T-cell-mediated antitumor reactivity. METHODS: We generated subcutaneous tumors in a genetic mouse model of pericyte deficiency (the pdgfb (ret/ret) mouse) and littermate control mice (n = 6-25). Gene expression profiles from 253 breast cancer patients (stage I-III) were evaluated for clinic-pathological parameters and survival using Cox proportional hazard ratios (HRs) and 95% confidence intervals (CIs) based on a two-sided Wald test. RESULTS: We report that pericyte deficiency leads to increased transmigration of Gr1(+)/CD11b(+) cells in experimentally induced tumors. Pericyte deficiency produced defective tumor vasculature, resulting in a more hypoxic microenvironment promoting IL-6 upregulation in the malignant cells. Silencing IL-6 expression in tumor cells attenuated the observed differences in MDSC transmigration. Restoring the pericyte coverage in tumors abrogated the increased MDSC trafficking to pericyte-deficient tumors. MDSC accumulation in tumors led to increases in tumor growth and in circulating malignant cells. Finally, gene expression analysis from human breast cancer patients revealed increased expression of the human MDSC markers CD33 and S100A9 with concomitant decreased expression of pericyte genes and was associated with poor prognosis (HR = 1.88, 95% CI = 1.08 to 3.25, P = .03). CONCLUSIONS: Our data uncovers a novel paracrine interaction between tumor pericytes and inflammatory cells and delineates the cellular events resulting in the recruitment of MDSC to tumors. Furthermore, we propose for the first time a role for tumor pericytes in modulating the expression of immune mediators in malignant cells by promoting a hypoxic microenvironment.