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Apolipoprotein E4 (APOE4), the main susceptibility gene for Alzheimer’s disease (AD), leads to vascular dysfunction, amyloid-β pathology, neurodegeneration and dementia. How these different pathologies contribute to advanced-stage AD remains unclear. Using aged APOE knock-in mice crossed with 5xFAD mice, we show that, compared to APOE3, APOE4 accelerates blood–brain barrier (BBB) breakdown, loss of cerebral blood flow, neuronal loss and behavioral deficits independently of amyloid-β. BBB breakdown was associated with activation of the cyclophilin A-matrix metalloproteinase-9 BBB-degrading pathway in pericytes. Suppression of this pathway improved BBB integrity and prevented further neuronal loss and behavioral deficits in APOE4;5FAD mice while having no effect on amyloid-β pathology. Thus, APOE4 accelerates advanced-stage BBB breakdown and neurodegeneration in Alzheimer’s mice via the cyclophilin A pathway in pericytes independently of amyloid-β, which has implication for the pathogenesis and treatment of vascular and neurodegenerative disorder in AD. This study shows that APOE4, one of the largest genetic risk factors for Alzheimer’s disease, promotes advanced-stage vascular dysfunction and neurodegeneration in old mice via activation of the cyclophilin A pathway in pericytes and independently of the presence of amyloid-β.

Under physiological conditions in vivo astrocytes internalize and degrade neuronal mitochondria in a process called transmitophagy. Mitophagy is widely reported to be impaired in neurodegeneration but it is unknown whether and how transmitophagy is altered in Alzheimer’s disease (AD). Here we report that the internalization and degradation of neuronal mitochondria are significantly increased in astrocytes isolated from aged AD mouse brains. We also demonstrate for the first time a similar phenomenon between human neurons and AD astrocytes, and in murine hippocampi in vivo. The results suggest the involvement of S100a4 in impaired mitochondrial transfer between neurons and aged AD astrocytes. Significant increases in the mitophagy regulator Ambra1 were observed in the aged AD astrocytes. These findings demonstrate altered neuron-supporting functions of aged AD astrocytes and provide a starting point for studying the molecular mechanisms of transmitophagy in AD.

Exposure to ambient air pollution particulate matter (PM) is associated with increased risk of dementia and accelerated cognitive loss. Vascular contributions to cognitive impairment are well recognized. Chronic cerebral hypoperfusion (CCH) promotes neuroinflammation and blood-brain barrier weakening, which may augment neurotoxic effects of PM.This study examined interactions of nanoscale particulate matter (nPM; fine particulate matter with aerodynamic diameternPM was collected using a particle sampler near a Los Angeles, California, freeway. Mice were exposed to 10 wk of reaerosolized nPM or filtered air (FA) for 150 h. CCH was induced by BCAS surgery. Mice (C57BL/6J males) were randomized to four exposure paradigms:The jointOur data suggest that nPM and CCH contribute to white matter injury in a synergistic manner in a mouse model. Adverse neurological effects may be aggravated in a susceptible population exposed to air pollution. https://doi.org/10.1289/EHP8792.

Subcortical white matter (WM) stroke accounts for 25% of all strokes and is the second leading cause of dementia. Despite such clinical importance, we still do not have an effective treatment for ischemic WM stroke, and the mechanisms of WM postischemic neuroprotection remain elusive. 3K3A-activated protein C (APC) is a signaling-selective analogue of endogenous blood protease APC that is currently in development as a neuroprotectant for ischemic stroke patients. Here, we show that 3K3A-APC protects WM tracts and oligodendrocytes from ischemic injury in the corpus callosum in middle-aged mice by activating protease-activated receptor 1 (PAR1) and PAR3. We show that PAR1 and PAR3 were also required for 3K3A-APC’s suppression of post–WM stroke microglia and astrocyte responses and overall improvement in neuropathologic and functional outcomes. Our data provide new insights into the neuroprotective APC pathway in the WM and illustrate 3K3A-APC’s potential for treating WM stroke in humans, possibly including multiple WM strokes that result in vascular dementia.

Pericytes are positioned between brain capillary endothelial cells, astrocytes and neurons. They degenerate in multiple neurological disorders. However, their role in the pathogenesis of these disorders remains debatable. Here we generate an inducible pericyte-specific Cre line and cross pericyte-specific Cre mice with iDTR mice carrying Cre-dependent human diphtheria toxin receptor. After pericyte ablation with diphtheria toxin, mice showed acute blood-brain barrier breakdown, severe loss of blood flow, and a rapid neuron loss that was associated with loss of pericyte-derived pleiotrophin (PTN), a neurotrophic growth factor. Intracerebroventricular PTN infusions prevented neuron loss in pericyte-ablated mice despite persistent circulatory changes. Silencing of pericyte-derived Ptn rendered neurons vulnerable to ischemic and excitotoxic injury. Our data demonstrate a rapid neurodegeneration cascade that links pericyte loss to acute circulatory collapse and loss of PTN neurotrophic support. These findings may have implications for the pathogenesis and treatment of neurological disorders that are associated with pericyte loss and/or neurovascular dysfunction.

We studied the effects of systemic infection on brain cytokine level and cerebral vascular function in Alzheimer's disease and vascular dementia, in superior temporal cortex (Brodmann area 22) from Alzheimer's disease patients (n = 75), vascular dementia patients (n = 22) and age-matched control subjects (n = 46), stratified according to the presence or absence of terminal systemic infection. Brain cytokine levels were measured using Mesoscale Discovery Multiplex Assays and markers of cerebrovascular function were assessed by ELISA. Multiple brain cytokines were elevated in Alzheimer's disease and vascular dementia: IL-15 and IL-17A were maximally elevated in end-stage Alzheimer's disease (Braak tangle stage V-VI) whereas IL-2, IL-5, IL12p40 and IL-16 were highest in intermediate Braak tangle stage III-IV disease. Several cytokines (IL-1β, IL-6, TNF-α, IL-8 and IL-15) were further raised in Alzheimer's disease with systemic infection. Cerebral hypoperfusion-indicated by decreased MAG:PLP1 and increased vascular endothelial growth factor-A (VEGF)-and blood-brain barrier leakiness, indicated by raised levels of fibrinogen, were exacerbated in Alzheimer's disease and vascular dementia patients, and also in non-dementia controls, with systemic infection. Amyloid-β42 level did not vary with infection or in association with brain cytokine levels. In controls, cortical perfusion declined with increasing IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-13 and tumour necrosis factor-α (TNF-α) but these relationships were lost with progression of Alzheimer's disease, and with infection (even in Braak stage 0-II brains). Cortical platelet-derived growth factor receptor-β (PDGFRβ), a pericyte marker, was reduced, and endothelin-1 (EDN1) level was increased in Alzheimer's disease; these were related to amyloid-β level and disease progression and only modestly affected by systemic infection. Our findings indicate that systemic infection alters brain cytokine levels and exacerbates cerebral hypoperfusion and blood-brain barrier leakiness associated with Alzheimer's disease and vascular dementia, independently of the level of insoluble amyloid-β, and highlight systemic infection as an important contributor to dementia, requiring early identification and treatment in the elderly population.

A recombinant variant of activated protein C, 3K3A-APC, was recently tested in a successful phase II clinical trial for ischemic stroke (RHAPSODY), which demonstrated that the treatment may reduce hemorrhages and is safe in patients that were treated with tPA and/or thrombectomy. Animal models of large ischemic stroke (middle cerebral artery occlusion) and neurodegeneration have shown that the effects of 3K3A-APC are mediated by preserved APC cell signaling activities (anti-apoptotic neuronal protection, protection of blood-brain barrier (BBB) integrity and anti-inflammatory activities). Because recent studies have suggested a role of protease activated receptor 1 (PAR1) in oligodendrocyte recovery after white matter damage, we wanted to test whether biased PAR1 signaling mediated by 3K3A-APC would protect oligodendrocytes in in vivo and ex vivo models of ischemic white matter stroke. First, we used a mouse model of white matter stroke induced by injecting a vasoconstrictor N 5 -(1-Iminoethyl)-L-ornithine (L-NIO) into the corpus callosum. Our results show that treatment with recombinant murine 3K3A-APC (0.2 mg/kg, starting 4h after stroke) protected oligodendrocytes (OLS, Olig2+CNPase+) and oligodendrocyte precursor cells (OPCs, Olig2+PDGFRα+) from cell death at 1 and 7 days after stroke. In vitro , 3K3A-APC protected primary mouse oligodendrocytes from apoptosis when exposed to OGD conditions at nanomolar concentrations (EC50 concentration was 5.2 nM). Improved oligodendrocyte viability in vivo was associated by about ~67% reduction in overall cell death (cresyl violet staining, T2w and diffusion tensor MRI) and reduced BBB leakage (fibrin staining and DCE-MRI) as well as improved functional recovery (adhesive removal and grid walking tests). Protective effects of 3K3A-APC were abolished by silencing of PAR1 with small interfering RNA (siRNA) in vivo and in vitro . In conclusion, PAR1 mediated signaling initiated by 3K3A-APC protects oligodendrocyte pools in models of ischemic white matter damage with preservation of neurovascular unit integrity.

Lipid peroxidation-initiated ferroptosis is an iron-dependent mechanism of programmed cell death taking place in neurological diseases. Here we show that a condensed benzo[b]thiazine derivative small molecule with an arylthiazine backbone (ADA-409-052) inhibits tert-Butyl hydroperoxide (TBHP)-induced lipid peroxidation (LP) and protects against ferroptotic cell death triggered by glutathione (GSH) depletion or glutathione peroxidase 4 (GPx4) inhibition in neuronal cell lines. In addition, ADA-409-052 suppresses pro-inflammatory activation of BV2 microglia and protects N2a neuronal cells from cell death induced by pro-inflammatory RAW 264.7 macrophages. Moreover, ADA-409-052 efficiently reduces infarct volume, edema and expression of pro-inflammatory genes in a mouse model of thromboembolic stroke. Targeting ferroptosis may be a promising therapeutic strategy in neurological diseases involving severe neuronal death and neuroinflammation.

Endothelial low-density lipoprotein receptor–related protein 1 (LRP1) protects against neurodegeneration by inhibiting the proinflammatory cyclophilin A–matrix metalloproteinase-9 pathway at the blood–brain barrier. These findings have implications for the pathophysiology and treatment of neurodegenerative disorders linked to vascular dysfunction., The low-density lipoprotein receptor–related protein 1 (LRP1) is an endocytic and cell signaling transmembrane protein. Endothelial LRP1 clears proteinaceous toxins at the blood–brain barrier (BBB), regulates angiogenesis, and is increasingly reduced in Alzheimer’s disease associated with BBB breakdown and neurodegeneration. Whether loss of endothelial LRP1 plays a direct causative role in BBB breakdown and neurodegenerative changes remains elusive. Here, we show that LRP1 inactivation from the mouse endothelium results in progressive BBB breakdown, followed by neuron loss and cognitive deficits, which is reversible by endothelial-specific LRP1 gene therapy. LRP1 endothelial knockout led to a self-autonomous activation of the cyclophilin A–matrix metalloproteinase-9 pathway in the endothelium, causing loss of tight junctions underlying structural BBB impairment. Cyclophilin A inhibition in mice with endothelial-specific LRP1 knockout restored BBB integrity and reversed and prevented neuronal loss and behavioral deficits. Thus, endothelial LRP1 protects against neurodegeneration by inhibiting cyclophilin A, which has implications for the pathophysiology and treatment of neurodegeneration linked to vascular dysfunction.

Cerebral small vessel disease (SVD) is a major health burden, yet the pathophysiology remains poorly understood with no effective treatment. Since much of SVD develops silently and insidiously, non-invasive neuroimaging such as MRI is fundamental to detecting and understanding SVD in humans. Several relevant SVD rodent models are established for which MRI can monitor in vivo changes over time prior to histological examination. Here, we critically review the MRI methods pertaining to salient rodent models and evaluate synergies with human SVD MRI methods. We found few relevant publications, but argue there is considerable scope for greater use of MRI in rodent models, and opportunities for harmonisation of the rodent-human methods to increase the translational potential of models to understand SVD in humans. We summarise current MR techniques used in SVD research, provide recommendations and examples and highlight practicalities for use of MRI SVD imaging protocols in pre-selected, relevant rodent models. Electronic supplementary material The online version of this article (10.1007/s12975-020-00843-8) contains supplementary material, which is available to authorised users.

3K3A-APC is a recombinant variant of activated protein C (APC) with > 90% reduced anticoagulant activity but preserved cell signaling activity. Benefical effects are mediated by its antiapoptotic direct neuronal protective and vasculoprotective effects including protection of blood-brain barrier (BBB) integrity as well as anti-inflammatiory activity as shown in multiple animal models of large ischemic stroke (middle cerebral artery occlusion) and neurodegeneration. These preclinical studies led to a successfully completed phase II clinical trial of 3K3A-APC for ischemic stroke (RHAPSODY). RHAPSODY trial showed that 3K3A-APC is safe and may reduce hemorrhage in patients that were treated with tPA and/or thrombectomy standard of care therapy for stroke. Even though the protective effects of 3K3A-APC are well known to be mediated by biased signaling via protease activated receptor 1 (PAR1) activation, the role of PAR1 has not been studied in ischemic white matter stroke. In the present study, we used a mouse model of white matter stroke induced by injecting a vasoconstrictor N 5 -(1-Iminoethyl)-L-ornithine (L-NIO) into the corpus callosum. Our results suggest that 4 hrs post-treatment with 3K3A-APC (0.2 mg/kg) reduced lesion volumes by about ~67% (cresyl violet staining and T2w MRI), which was associated with reduced BBB leakage (fibrinogen staining and DCE-MRI), and improved function of oligodendrocytes (OLS, Olig2+CNPase+) and oligodendrocyte precursor cell (OPCs, Olig2+PDGFRα+) at 1 and 7 days after stroke. Beneficial effects of 3K3A-APC were associated with reduced axonal damage on diffusion tensor MRI, and improved behavioral performance on adhesive removal and grid walking tests. We next showed that PAR1 is expressed in all major cell types in the white matter (e.g., OLS, OPCs, microglia and astrocytes) and that silencing PAR1 with small interfering RNA (siRNA) abolishes the protective effect of 3K3A-APC in this model. Taken together, our data suggest that 3K3A-APC is protective in ischemic white matter disease via PAR1 mediated signaling, which improves oligovascular integrity and neuronal survival.

Pericytes play a key role in maintaining the blood-brain barrier (BBB) integrity. BBB disruption occurs during early stages after ischemic stroke. However, the role of pericytes in the pathogenesis of ischemic stroke remains still understudied. 3K3A-APC, a recombinant variant of activated protein C, has shown benefits in preclinical models of ischemic stroke and has favorable safety profile and reduces hemorrhage in Phase 2 study in ischemic stroke patients (RHAPSODY). In the present study, we used PDGFRβ heterozygous knockout (PDGFRβ+/-) mice to investigate the effects of pericyte deficiency on ischemic brain injury using transient proximal middle cerebral artery occlusion (tMCAO). Additionally, we investigated the effects of 3K3A-APC therapy (0.2mg/kg i.v. 4h after stroke) in this model. Compared to controls, pericyte deficiency in PDGFRβ+/- mice resulted in ~35% increase in the infarct and edema volumes, reduction in pericyte coverage from 58% to 25%, and increased IgG and fibrin deposition suggesting accelerated BBB breakdown 24h after stroke. Additionally, PDGFRβ+/- mice showed by 36% more degenerating Fluoro-Jade+ neurons and exhibited accelerated neurobehavioral abnormalities. 3K3A-APC improved neuropathological changes and functional deficits. Our results suggest that pericyte deficiency worsens brain damage and functional outcome after ischemic stroke in mice suggesting that pericytes may play an important role in protecting brain from post-ischemic. We also suggests that 3K3A-APC protects pericyte function in stroked mice which could contribute to its overall neuroprotective effects.

CLN5 disease is a rare form of late-infantile neuronal ceroid lipofuscinosis (NCL) caused by mutations in the CLN5 gene that encodes a protein whose primary function and physiological roles remains unresolved. Emerging lines of evidence point to mitochondrial dysfunction in the onset and progression of several forms of NCL, offering new insights into putative biomarkers and shared biological processes. In this work, we employed cellular and murine models of the disease, in an effort to clarify disease pathways associated with CLN5 depletion. A mitochondria-focused quantitative proteomics approach followed by functional validations using cell biology and immunofluorescence assays revealed an impairment of mitochondrial functions in different CLN5 KO cell models and in Cln5−/− cerebral cortex, which well correlated with disease progression. A visible impairment of autophagy machinery coupled with alterations of key parameters of mitophagy activation process functionally linked CLN5 protein to the process of neuronal injury. The functional link between impaired cellular respiration and activation of mitophagy pathways in the human CLN5 disease condition was corroborated by translating organelle-specific proteome findings to CLN5 patients’ fibroblasts. Our study highlights the involvement of CLN5 in activation of mitophagy and mitochondrial homeostasis offering new insights into alternative strategies towards the CLN5 disease treatment.

Introduction Blood-brain barrier (BBB) breakdown is an early independent biomarker of human cognitive dysfunction, as found using gadolinium (Gd) as a contrast agent. Whether Gd accumulates in brains of individuals with an age-dependent BBB breakdown and/or mild cognitive impairment remains unclear. Methods We analyzed T1-weighted magnetic resonance imaging (MRI) scans from 52 older participants with BBB breakdown in the hippocampus 19-28 months after either cyclic or linear Gd agent. Results There was no change in T1-weighted signal intensity between the baseline contrast MRI and unenhanced MRI on re-examination in any of the studied 10 brain regions with either Gd agent suggesting undetectable Gd brain retention. Discussion Gd does not accumulate in brains of older individuals with a BBB breakdown in the hippocampus. Thus, Gd agents can be used without risk of brain retention within a ∼2-year follow-up to study BBB in the aging human brain in relation to cognition and/or other pathologies.

Background DHCR24, involved in the de novo synthesis of cholesterol and protection of neuronal cells against different stress conditions, has been shown to be selectively downregulated in neurons of the affected brain areas in Alzheimer’s disease. Methods Here, we investigated whether the overexpression of DHCR24 protects neurons against inflammation-induced neuronal death using co-cultures of mouse embryonic primary cortical neurons and BV2 microglial cells upon acute neuroinflammation. Moreover, the effects of DHCR24 overexpression on dendritic spine density and morphology in cultured mature mouse hippocampal neurons and on the outcome measures of ischemia-induced brain damage in vivo in mice were assessed. Results Overexpression of DHCR24 reduced the loss of neurons under inflammation elicited by LPS and IFN-γ treatment in co-cultures of mouse neurons and BV2 microglial cells but did not affect the production of neuroinflammatory mediators, total cellular cholesterol levels, or the activity of proteins linked with neuroprotective signaling. Conversely, the levels of post-synaptic cell adhesion protein neuroligin-1 were significantly increased upon the overexpression of DHCR24 in basal growth conditions. Augmentation of DHCR24 also increased the total number of dendritic spines and the proportion of mushroom spines in mature mouse hippocampal neurons. In vivo, overexpression of DHCR24 in striatum reduced the lesion size measured by MRI in a mouse model of transient focal ischemia. Conclusions These results suggest that the augmentation of DHCR24 levels provides neuroprotection in acute stress conditions, which lead to neuronal loss in vitro and in vivo., published version, peerReviewed

Vasculature has an essential role both in central nervous system health and diseases. A new optimized imaging and analysis pipeline helps to characterize blood vessel network topology and plasticity across large regions in detail.

The Finnish-variant late infantile neuronal ceroid lipofuscinosis, also known as CLN5 disease, is caused by mutations in the CLN5 gene. Cln5 is strongly expressed in the developing brain and expression continues into adulthood. CLN5, a protein of unknown function, is implicated in neurodevelopment but detailed investigation is lacking. Using Cln5−/− embryos of various ages and cells harvested from Cln5−/− brains we investigated the hitherto unknown role of Cln5 in the developing brain. Loss of Cln5 results in neuronal differentiation deficits and delays in interneuron development during in utero period. Specifically, the radial thickness of dorsal telencephalon was significantly decreased in Cln5−/− mouse embryos at embryonic day 14.5 (E14.5), and expression of Tuj1, an important neuronal marker during development, was down-regulated. An interneuron marker calbindin and a mitosis marker p-H3 showed down-regulation in ganglionic eminences. Neurite outgrowth was compromised in primary cortical neuronal cultures derived from E16 Cln5−/− embryos compared with WT embryos. We show that the developmental deficits of interneurons may be linked to increased levels of the repressor element 1-silencing transcription factor, which we report to bind to glutamate decarboxylase (Gad1), which encodes GAD67, a rate-limiting enzyme in the production of gamma-aminobutyric acid (GABA). Indeed, adult Cln5−/− mice presented deficits in hippocampal parvalbumin-positive interneurons. Furthermore, adult Cln5−/− mice presented deficits in hippocampal parvalbumin-positive interneurons and showed age-independent cortical hyper excitability as measured by electroencephalogram and auditory-evoked potentials. This study highlights the importance of Cln5 in neurodevelopment and suggests that in contrast to earlier reports, CLN5 disease is likely to develop during embryonic stages.

Introduction: Subcortical white matter stroke (WMS) accounts for 25% of all incidences of stroke and results in severe motor and cognitive disability. WMS stands as the second leading cause of dementia and is notably prevalent in older adults. Activated protein C (APC), a plasma serine protease with potent antithrombotic and cell-signaling activities, was engineered to reduce bleeding risk while retaining normal cell-signaling activities (anti-inflammatory, anti-apoptotic, and neuroregenerative, etc. activities). The engineered variant, 3K3A-APC, is neuroprotective in preclinical models of ischemic stroke with large infarcts involving common large arteries and gray matter. A multicenter Phase 2 randomized clinical trial enrolled 110 patients to assess the safety, pharmacokinetics, and efficacy of human recombinant 3K3A-APC in acute ischemic stroke patients following endovascular treatments (NCT02222714). Methods: In this new study, a murine WMS was induced by the vasoconstrictor N5-(1-iminoethyl)-L-ornithine, dihydrochloride (L-Nio) by injection into the subcortical white matter ventral to the mouse forelimb motor cortex using the Neurostar motorized ultra-precise small animal stereotaxic instrument. Mice received intravenously 0.2 mg/kg recombinant murine 3K3A-APC or saline at 4, 24, 48 and 72 h after L-Nio. Then MRI scans were performed using a 7T PET/MRI scanner (MR Solutions) and a standard quadrature coil at 1 and 7 days after the stroke. Results: Both MRI and histological results showed that 3K3A-APC significantly reduced the lesion volume, increased the viability of oligodendrocytes and neurons, and protected against blood brain barrier damage at 1 and 7 days after stroke. Behavioral tests showed that 3K3A-APC improved functional recovery. Conclusions: These results demonstrated that 3K3A-APC might be an effective treatment for white matter stroke.

Astrocytes are the gatekeepers of neuronal energy supply. In neurodegenerative diseases, bio-energetics demand increases and becomes reliant upon fatty acid oxidation as a source of energy. Defective fatty acid oxidation and mitochondrial dysfunctions correlate with hippocampal neurodegeneration and memory deficits in Alzheimer’s disease (AD), but it is unclear whether energy metabolism can be targeted to prevent or treat the disease. Here we show for the first time an impairment in fatty acid oxidation in human astrocytes derived from induced pluripotent stem cells of AD patients. The impairment was corrected by treatment with a synthetic peroxisome proliferator activated receptor delta (PPARβ/δ) agonist GW0742 which acts to regulate an array of genes governing cellular metabolism. GW0742 enhanced the expression of CPT1a, the gene encoding for a rate-limiting enzyme of fatty acid oxidation. Similarly, treatment of a mouse model of AD, the APP/PS1-mice, with GW0742 increased the expression of Cpt1a and concomitantly reversed memory deficits in a fear conditioning test. Although the GW0742-treated mice did not show altered astrocytic glial fibrillary acidic protein-immunoreactivity or reduction in amyloid beta (Aβ) load, GW0742 treatment increased hippo-campal neurogenesis and enhanced neuronal differentiation of neuronal progenitor cells. Furthermore, GW0742 prevented Aβ-induced impairment of long-term potentiation in hippocampal slices. Collectively, these data suggest that PPARβ/δ-agonism alleviates AD related deficits through increasing fatty acid oxidation in astrocytes and improves cognition in a transgenic mouse model of AD.

Ischemic stroke is amongst the leading causes of death and disabilities. The available treatments are suitable for only a fraction of patients and thus novel therapies are urgently needed. Blockage of one of the cerebral arteries leads to massive and persisting inflammatory reaction contributing to the nearby neuronal damage. Targeting the detrimental pathways of neuroinflammation has been suggested to be beneficial in conditions of ischemic stroke. Nuclear receptor 4A-family (NR4A) member Nurr1 has been shown to be a potent modulator of harmful inflammatory reactions, yet the role of Nurr1 in cerebral stroke remains unknown. Here we show for the first time that an agonist for the dimeric transcription factor Nurr1/retinoid X receptor (RXR), HX600, reduces microglia expressed proinflammatory mediators and prevents inflammation induced neuronal death in in vitro co-culture model of neurons and microglia. Importantly, HX600 was protective in a mouse model of permanent middle cerebral artery occlusion and alleviated the stroke induced motor deficits. Along with the anti-inflammatory capacity of HX600 in vitro, treatment of ischemic mice with HX600 reduced ischemia induced Iba-1, p38 and TREM2 immunoreactivities, protected endogenous microglia from ischemia induced death and prevented leukocyte infiltration. These anti-inflammatory functions were associated with reduced levels of brain lysophosphatidylcholines (lysoPCs) and acylcarnitines, metabolites related to proinflammatory events. These data demonstrate that HX600 driven Nurr1 activation is beneficial in ischemic stroke and propose that targeting Nurr1 is a novel candidate for conditions involving neuroinflammatory component.

Background Ischemic stroke is one of the main causes of death and disability worldwide. It is caused by the cessation of cerebral blood flow resulting in the insufficient delivery of glucose and oxygen to the neural tissue. The inflammatory response initiated by ischemic stroke in order to restore tissue homeostasis in the acute phase of stroke contributes to delayed brain damage. Methods By using in vitro models of neuroinflammation and in vivo model of permanent middle cerebral artery occlusion, we demonstrate the neuroprotective and anti-inflammatory effects of sulfosuccinimidyl oleate sodium (SSO). Results SSO significantly reduced the lipopolysaccharide/interferon-γ-induced production of nitric oxide, interleukin-6 and tumor necrosis factor-α, and the protein levels of inflammatory enzymes including nitric oxide synthase 2, cyclooxygenase-2 (COX-2), and p38 mitogen-activated protein kinase (MAPK) in microglia, without causing cell toxicity. Although SSO failed to directly alleviate glutamate-induced excitotoxicity in murine cortical neurons, it prevented inflammation-induced neuronal death in microglia-neuron co-cultures. Importantly, oral administration of SSO in Balb/c mice subjected to permanent occlusion of the middle cerebral artery reduced microglial activation in the peri-ischemic area and attenuated brain damage. This in vivo neuroprotective effect of SSO was associated with a reduction in the COX-2 and heme oxygenase-1 immunoreactivities. Conclusions Our results suggest that SSO is an anti-inflammatory and a possible therapeutic candidate in diseases such as stroke where inflammation is a central hallmark., published version, peerReviewed

Developing new therapies for stroke is urgently needed, as this disease is the leading cause of death and disability worldwide, and the existing treatment is only available for a small subset of patients. The interruption of blood flow to the brain during ischemic stroke launches multiple immune responses, characterized by infiltration of peripheral immune cells, the activation of brain microglial cells, and the accumulation of immune mediators. Copper is an essential trace element that is required for many critical processes in the brain. Copper homeostasis is disturbed in chronic neurodegenerative diseases and altered in stroke patients, and targeted copper delivery has been shown to be protective against chronic neurodegeneration. This study was undertaken to assess whether the copper bis(thiosemicarbazone) complex, CuII(atsm), is beneficial in acute brain injury, in preclinical mouse models of ischemic stroke. We demonstrate that the copper complex CuII(atsm) protects neurons from excitotoxicity and N2a cells from OGD in vitro, and is protective in permanent and transient ischemia models in mice as measured by functional outcome and lesion size. Copper delivery in the ischemic brains modulates the inflammatory response, specifically affecting the myeloid cells. It reduces CD45 and Iba1 immunoreactivity, and alters the morphology of Iba1 positive cells in the ischemic brain. CuII(atsm) also protects endogenous microglia against ischemic insult and reduces the proportion of invading monocytes. These results demonstrate that the copper complex CuII(atsm) is an inflammation-modulating compound with high therapeutic potential in stroke and is a strong candidate for the development of therapies for acute brain injury.

Stroke is a highly debilitating, often fatal disorder for which current therapies are suitable for only a minor fraction of patients. Discovery of novel, effective therapies is hampered by the fact that advanced age, primary age-related tauopathy or comorbidities typical to several types of dementing diseases are usually not taken into account in preclinical studies, which predominantly use young, healthy rodents. Here we investigated for the first time the neuroprotective potential of bexarotene, an FDA-approved agent, in a co-morbidity model of stroke that combines high age and tauopathy with thromboembolic cerebral ischemia. Following thromboembolic stroke bexarotene enhanced autophagy in the ischemic brain concomitantly with a reduction in lesion volume and amelioration of behavioral deficits in aged transgenic mice expressing the human P301L-Tau mutation. In in vitro studies bexarotene increased the expression of autophagy markers and reduced autophagic flux in neuronal cells expressing P301L-Tau. Bexarotene also restored mitochondrial respiration deficits in P301L-Tau neurons. These newly described actions of bexarotene add to the growing amount of compelling data showing that bexarotene is a potent neuroprotective agent, and identify a novel autophagy-modulating effect of bexarotene.

Collagens are key structural components of basement membranes, providing a scaffold for other components or adhering cells. Collagens and collagen-derived active fragments contribute to biological activities such as cell growth, differentiation and migration. Here, we report that collagen XV knock-out (ColXV KO) mice are resistant to experimental ischemic stroke. Interestingly, the infarcts of ColXV KO mice were as small as those of wild-type (WT) mice thrombolysed with recombinant tissue plasminogen activator (rtPA), the actual treatment for ischemic stroke. Importantly, there were no differences in the architecture of cerebrovascular anatomy between WT and ColXV KO mice. We found a twofold increase of the most potent pro-angiogenic factor, type A vascular growth endothelial factor (VEGF-A) in the ipsilateral cortex of rtPA-treated ischemic WT mice compared with untreated ischemic and sham-operated counterparts. A similar increase of VEGF-A was also found in both rtPA and untreated ischemic ColXV KO mice compared with sham ColXV KO mice. Finally, we evidenced that the levels of ColXV were increased in the plasma of WT mice treated with rtPA compared with untreated ischemic counterparts. Altogether, this study indicates that the lack ColXV is protective after stroke and that the degradation of endothelial ColXV may contribute to the beneficial effect of rtPA after ischemic stroke. The neuroprotection observed in ColXV KO mice may be attributed to the increased VEGF-A production following stroke in the ischemic territory. Stroke is a leading cause of death and long-term disability worldwide.1 Ischemic strokes represent 80% of cerebral strokes and result from the obstruction of a major cerebral artery by a thrombus or an embolus which reduces the blood flow in downstream targeted brain regions, leading to brain damage. The establishment of the primary ischemic lesion is followed by a series of secondary events that worsens tissue damage, including vascular, cellular and molecular events.2 Nowadays, the only treatment for ischemic stroke is reperfusion with intravenous administration of recombinant tissue plasminogen activator (rtPA; Alteplase) within a strict time frame up to 4.5 h, leading to an improved functional recovery and reduced neurological deficits.3 Additionally to its narrowed therapeutic window, rtPA also increases the risk of intracerebral hemorrhagic transformations and is not efficient at degrading platelet-rich thrombi. Taking together all the limitations for the use of rtPA, only 5% of ischemic stroke patients are eligible for rtPA-induced thrombolysis.4 Therefore, it is essential to better understand stroke pathophysiology in order to find safer and more efficient approaches for therapy. Collagens are well-known to be critical extracellular components in vascular stability and functions. Some collagens such as ColXV, ColXVIII and ColXIX have also been shown to be essential for motor axon guidance and neuromuscular development.5, 6, 7 The structurally homologous ColXV and ColXVIII constitute the so called multiplexin family of non-fibrillar collagens, characterized by multiple triple helix interruptions and similar non-collagenous sequences.8 Nevertheless, they differ in their functional properties and expression patterns and for example, contrary to ColXVIII, ColXV predominantly carries chondroitin sulfate chains and was therefore classified as a chondroitin sulfate proteoglycan (CSPG).9 It is mainly produced by skeletal and cardiac muscle, and endothelial cells, occurring at the basement membranes adjacent to these cells. ColXV knock-out (KO) mice suffer from mild skeletal and cardiac myopathy10, 11 and defective myelination of peripheral nerves.12 In this study, we report for the first time the neuroprotective effect of ColXV deficiency in mice suffering from ischemic stroke. Accordingly, we found an increase of type A vascular endothelial growth factor (VEGF-A) in the ischemic cortex of ColXV KO mice. Additionally, we showed that rtPA increased the presence of unbound ColXV in the plasma of wild-type (WT) mice after stroke., published version, peerReviewed