Glutamate Transporter Glt1 Expression In Alzheimer Disease And Dementia With Lewy Bodies

Glutamate transporter solute carrier family 1, member 2 (GLT1/EAAT2), a major modulator of glutamate homeostasis in astrocytes, is assessed in post-mortem human brain samples of frontal cortex area 8 in advanced stages of Alzheimer disease (AD) and terminal stages of dementia with Lewy bodies (DLB) in order to gain understanding of astrogliopathy in diseases manifested by dementia. Glial fibrillary acidic protein (GFAP) mRNA expression is significantly increased in AD but not in DLB, whereas GLT1, vesicular glutamate transporter 1 (vGLUT1) and aldehyde dehydrogenase 1 family member 1 (ALDH1L1) are not modified in AD and DLB when compared with controls. GLT1 protein levels are not altered in AD and DLB but GFAP and ALDH1L1 are significantly increased in AD, and GFAP in DLB. As a result, a non-significant decrease in the ratio between GLT1 and GFAP, and between GLT1 and ALDH1L1, is found in both AD and DLB. Double-labeling immunofluorescence and confocal microscopy revealed no visible reduction of GLT1 immunoreactivity in relation to beta-amyloid plaques in AD. These data suggest a subtle imbalance between GLT1, and GFAP and ALDH1L1 expression, with limited consequences in glutamate transport

Mechanisms involved in the remyelinating effect of sildenafil /

Multiple Sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system characterized by a coordinated inflammatory attack on the myelin sheaths with ensuing damage to the underlying axons. Using myelin oligodendrocyte glycoprotein (MOG)-induced chronic experimental autoimmune encephalomyelitis (EAE) as a MS model, it has been previously demonstrated that daily administration of the PDE-5 inhibitor sildenafil starting at peak disease rapidly ameliorates clinical symptoms whereas administration at the onset of symptoms prevents disease progression. These beneficial effects involved down-regulation of adaptive and innate immune responses and protection of axons and oligodendrocytes and promotion of remyelination. The aim of this work was confirm the remyelinating potential of sildenafil treatment and investigate mechanisms involved in this effect in CNS cells. Results show that sildenafil induces remyelination in EAE mice even when the administration of the drug starts during the chronic stage of the disease. Sildenafil also stimulates remyelination in cerebellar organotypic cultures demyelinated with lysophosphatidylcholine and this effect is prevented by inhibitors of nitric oxide-dependent guanylyl cyclase (NO-GC), NO synthase type 2 (NOS-2) and cGMP-dependent protein kinase (PKG), indicating the involvement of the endogenous NO-cGMP-PKG pathway. Maturation of oligodendrocytes as a potential mechanism implicated in the remyelinating effect of sildenafil was investigated by immunostaining for transcription factors involved in different stages of oligodendrocyte development. Results in the EAE model show that sildenafil treatment increases oligodendrocyte precursor cells (OPCs; Nkx2.2+ cells) and promotes the final stage of oligodendrocyte maturation (olig2-/MBP+ cells). These later result was confirmed in LPC-demyelinated cerebellar slices treated with cGMP increasing compounds. This work also shows that expression of the neurotrophic factor CNTF, that has been implicated in oligodendrocyte maturation, is increased in astroytes of sildenafil-treated EAE mice spinal cord, as well as in cerebellar slice cultures. Results also show that cGMP-increasing treatments alter expression of inflammatory phenotype markers (COX-2 and Arg-1) in microglia in demyelinated slice cultures. The potential of cGMP-increasing treatments for regulating the inflammatory phenotype of monocytes was confirmed in bone marrow derived macrophages (BMDM). In these cells sildenafil treatment induces arginase activity and potentiates the effect of IL-4 suggesting the promotion of an M2 phenotype. Analysis by flow cytometry of BMDM confirmed that cGMP augments the number of cells expressing an M2 phenotype marker (CD206). This work further demonstrates that sildenafil significantly increases the myelin phagocytic capacity of microglia/macrophages in EAE mice and in BMDM. Taken together these data suggest that promotion of oligodendrocyte maturation, growth factor expression, modulation of the inflammatory process and clearance of myelin debris may be relevant mechanisms involved in sildenafil enhancement of remyelination in demyelinated tissue

Sporadic Creutzfeldt-Jakob disease with glial PrPRes nuclear and perinuclear immunoreactivity

Proteinase K-resistant prion protein (PrPRes ) nuclear and perinuclear immunoreactivity in oligodendrocytes of the frontal cortex is found in one case of otherwise typical sporadic Creutzfeldt-Jakob disease (sCJD) type VV2a. The PrP nature of the inclusions is validated with several anti-PrP antibodies directed to amino acids 130-160 (12F10), 109-112 (3F4), 97-102 (8G8) and the octarepeat region (amino acids 59-89: SAF32). Cellular identification and subcellular localization were evaluated with double- and triple-labeling immunofluorescence and confocal microscopy using antibodies against PrP, glial markers, and histone H3. Based on review of the literature and our own experience, this is a very odd situation that deserves further validation in other cases. Keywords: Creutzfeldt-Jakob disease; astrocytes; oligodendrocytes; prion

Cerebrospinal fluid lipocalin 2 as a novel biomarker for the differential diagnosis of vascular dementia

The clinical diagnosis of vascular dementia (VaD) is based on imaging criteria, and specific biochemical markers are not available. Here, we investigated the potential of cerebrospinal fluid (CSF) lipocalin 2 (LCN2), a secreted glycoprotein that has been suggested as mediating neuronal damage in vascular brain injuries. The study included four independent cohorts with a total n = 472 samples. LCN2 was significantly elevated in VaD compared to controls, Alzheimer's disease (AD), other neurodegenerative dementias, and cognitively unimpaired patients with cerebrovascular disease. LCN2 discriminated VaD from AD without coexisting VaD with high accuracy. The main findings were consistent over all cohorts. Neuropathology disclosed a high percentage of macrophages linked to subacute infarcts, reactive astrocytes, and damaged blood vessels in multi-infarct dementia when compared to AD. We conclude that CSF LCN2 is a promising candidate biochemical marker in the differential diagnosis of VaD and neurodegenerative dementias

Aging-related tau astrogliopathy (ARTAG): not only tau phosphorylation in astrocytes

Aging-related tau astrogliopathy (ARTAG) is defined by the presence of two types of tau-bearing astrocytes: thorn-shaped astrocytes (TSAs) and granular/fuzzy astrocytes in the brain of old-aged individuals. The present study is focused on TSAs in rare forms of ARTAG with no neuronal tau pathology or restricted to entorhinal and transentorhinal cortices, to avoid bias from associated tauopathies. TSAs show 4Rtau phosphorylation at several specific sites and abnormal tau conformation, but they lack ubiquitin and they are not immunostained with tau-C3 antibodies which recognize truncated tau at Asp421. Astrocytes in ARTAG have atrophic processes, reduced glial fibrillary acidic protein (GFAP) and increased superoxide dismutase 2 (SOD2) immunoreactivity. Gel electrophoresis and western blotting of sarkosyl-insoluble fractions reveal a pattern of phospho-tau in ARTAG characterized by two bands of 68 and 64 kDa, and several middle bands between 35 and 50 kDa which differ from what is seen in AD. Phosphoproteomics of dissected vulnerable regions identifies an increase of phosphorylation marks in a large number of proteins in ARTAG compared with controls. GFAP, aquaporin 4, several serine-threonine kinases, microtubule associated proteins and other neuronal proteins are among the differentially phosphorylated proteins in ARTAG thus suggesting a hyper-phosphorylation background that affects several molecules, including many kinases and proteins from several cell compartments and various cell types. Finally, present results show for the first time that tau seeding is produced in neurons of the hippocampal complex, astrocytes, oligodendroglia and along fibers of the corpus callosum, fimbria and fornix following inoculation into the hippocampus of wild type mice of sarkosyl-insoluble fractions enriched in hyper-phosphorylated tau from selected ARTAG cases. These findings show astrocytes as crucial players of tau seeding in tauopathies

Ykl-40 in the brain and cerebrospinal fluid of neurodegenerative dementias

Background: YKL-40 (also known as Chitinase 3-like 1) is a glycoprotein produced by inflammatory, cancer and stem cells. Its physiological role is not completely understood but YKL-40 is elevated in the brain and cerebrospinal fluid (CSF) in several neurological and neurodegenerative diseases associated with inflammatory processes. Yet the precise characterization of YKL-40 in dementia cases is missing. Methods: In the present study, we comparatively analysed YKL-40 levels in the brain and CSF samples from neurodegenerative dementias of different aetiologies characterized by the presence of cortical pathology and disease-specific neuroinflammatory signatures. Results: YKL-40 was normally expressed in fibrillar astrocytes in the white matter. Additionally YKL-40 was highly and widely expressed in reactive protoplasmic cortical and perivascular astrocytes, and fibrillar astrocytes in sporadic Creutzfeldt-Jakob disease (sCJD). Elevated YKL-40 levels were also detected in Alzheimer's disease (AD) but not in dementia with Lewy bodies (DLB). In AD, YKL-40-positive astrocytes were commonly found in clusters, often around beta-amyloid plaques, and surrounding vessels with beta-amyloid angiopathy; they were also distributed randomly in the cerebral cortex and white matter. YKL-40 overexpression appeared as a pre-clinical event as demonstrated in experimental models of prion diseases and AD pathology. CSF YKL-40 levels were measured in a cohort of 288 individuals, including neurological controls (NC) and patients diagnosed with different types of dementia. Compared to NC, increased YKL-40 levels were detected in sCJD (p 0.05, AUC = 0.71) or in DLB/Parkinson's disease dementia (PDD) (p > 0.05, AUC = 0.70). Further, two independent patient cohorts were used to validate the increased CSF YKL-40 levels in sCJD. Additionally, increased YKL-40 levels were found in genetic prion diseases associated with the PRNP-D178N (Fatal Familial Insomnia) and PRNP-E200K mutations. Conclusions: Our results unequivocally demonstrate that in neurodegenerative dementias, YKL-40 is a disease-specific marker of neuroinflammation showing its highest levels in prion diseases. Therefore, YKL-40 quantification might have a potential for application in the evaluation of therapeutic intervention in dementias with a neuroinflammatory component

TREM2 expression in the brain and biological fluids in prion diseases

Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune cell surface receptor that regulates microglial function and is involved in the pathophysiology of several neurodegenerative diseases. Its soluble form (sTREM2) results from shedding of the TREM2 ectodomain. The role of TREM2 in prion diseases, a group of rapidly progressive dementias remains to be elucidated. In the present study, we analysed the expression of TREM2 and its main sheddase ADAM10 in the brain of sporadic Creutzfeldt-Jakob disease (sCJD) patients and evaluated the role of CSF and plasma sTREM2 as a potential diagnostic marker of prion disease. Our data indicate that, compared to controls, TREM2 is increased in sCJD patient brains at the mRNA and protein levels in a regional and subtype dependent fashion, and expressed in a subpopulation of microglia. In contrast, ADAM10 is increased at the protein, but not the mRNA level, with a restricted neuronal expression. Elevated CSF sTREM2 is found in sCJD, genetic CJD with mutations E200K and V210I in the prion protein gene (PRNP), and iatrogenic CJD, as compared to healthy controls (HC) (AUC = 0.78-0.90) and neurological controls (AUC = 0.73-0.85), while CSF sTREM2 is unchanged in fatal familial insomnia. sTREM2 in the CSF of cases with Alzheimer's disease, and multiple sclerosis was not significantly altered in our series. CSF sTREM2 concentrations in sCJD are PRNP codon 129 and subtype-related, correlate with CSF 14-3-3 positivity, total-tau and YKL-40, and increase with disease progression. In plasma, sTREM2 is increased in sCJD compared with HC (AUC = 0.80), displaying positive correlations with plasma total-tau, neurofilament light, and YKL-40. We conclude that comparative study of TREM2 in brain and biological fluids of prion diseases reveals TREM2 to be altered in human prion diseases with a potential value in target engagement, patient stratification, and disease monitoring

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Mechanisms involved in the remyelinating effect of sildenafil

Multiple Sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system characterized by a coordinated inflammatory attack on the myelin sheaths with ensuing damage to the underlying axons. Using myelin oligodendrocyte glycoprotein (MOG)-induced chronic experimental autoimmune encephalomyelitis (EAE) as a MS model, it has been previously demonstrated that daily administration of the PDE-5 inhibitor sildenafil starting at peak disease rapidly ameliorates clinical symptoms whereas administration at the onset of symptoms prevents disease progression. These beneficial effects involved down-regulation of adaptive and innate immune responses and protection of axons and oligodendrocytes and promotion of remyelination. The aim of this work was confirm the remyelinating potential of sildenafil treatment and investigate mechanisms involved in this effect in CNS cells. Results show that sildenafil induces remyelination in EAE mice even when the administration of the drug starts during the chronic stage of the disease. Sildenafil also stimulates remyelination in cerebellar organotypic cultures demyelinated with lysophosphatidylcholine and this effect is prevented by inhibitors of nitric oxide-dependent guanylyl cyclase (NO-GC), NO synthase type 2 (NOS-2) and cGMP-dependent protein kinase (PKG), indicating the involvement of the endogenous NO-cGMP-PKG pathway. Maturation of oligodendrocytes as a potential mechanism implicated in the remyelinating effect of sildenafil was investigated by immunostaining for transcription factors involved in different stages of oligodendrocyte development. Results in the EAE model show that sildenafil treatment increases oligodendrocyte precursor cells (OPCs; Nkx2.2+ cells) and promotes the final stage of oligodendrocyte maturation (olig2-/MBP+ cells). These later result was confirmed in LPC-demyelinated cerebellar slices treated with cGMP increasing compounds. This work also shows that expression of the neurotrophic factor CNTF, that has been implicated in oligodendrocyte maturation, is increased in astroytes of sildenafil-treated EAE mice spinal cord, as well as in cerebellar slice cultures. Results also show that cGMP-increasing treatments alter expression of inflammatory phenotype markers (COX-2 and Arg-1) in microglia in demyelinated slice cultures. The potential of cGMP-increasing treatments for regulating the inflammatory phenotype of monocytes was confirmed in bone marrow derived macrophages (BMDM). In these cells sildenafil treatment induces arginase activity and potentiates the effect of IL-4 suggesting the promotion of an M2 phenotype. Analysis by flow cytometry of BMDM confirmed that cGMP augments the number of cells expressing an M2 phenotype marker (CD206). This work further demonstrates that sildenafil significantly increases the myelin phagocytic capacity of microglia/macrophages in EAE mice and in BMDM. Taken together these data suggest that promotion of oligodendrocyte maturation, growth factor expression, modulation of the inflammatory process and clearance of myelin debris may be relevant mechanisms involved in sildenafil enhancement of remyelination in demyelinated tissue

Glutamate Transporter Glt1 Expression In Alzheimer Disease And Dementia With Lewy Bodies

Glutamate transporter solute carrier family 1, member 2 (GLT1/EAAT2), a major modulator of glutamate homeostasis in astrocytes, is assessed in post-mortem human brain samples of frontal cortex area 8 in advanced stages of Alzheimer disease (AD) and terminal stages of dementia with Lewy bodies (DLB) in order to gain understanding of astrogliopathy in diseases manifested by dementia. Glial fibrillary acidic protein (GFAP) mRNA expression is significantly increased in AD but not in DLB, whereas GLT1, vesicular glutamate transporter 1 (vGLUT1) and aldehyde dehydrogenase 1 family member 1 (ALDH1L1) are not modified in AD and DLB when compared with controls. GLT1 protein levels are not altered in AD and DLB but GFAP and ALDH1L1 are significantly increased in AD, and GFAP in DLB. As a result, a non-significant decrease in the ratio between GLT1 and GFAP, and between GLT1 and ALDH1L1, is found in both AD and DLB. Double-labeling immunofluorescence and confocal microscopy revealed no visible reduction of GLT1 immunoreactivity in relation to beta-amyloid plaques in AD. These data suggest a subtle imbalance between GLT1, and GFAP and ALDH1L1 expression, with limited consequences in glutamate transport