Mutual Induction of TGFβ1 and NGF after Treatment with NGF or TGFβ1 in Grafted Chromaffin Cells of the Adrenal Medulla

Chromaffin cells have been recognized for their ability to transform into sympathetic ganglion-like cells in response to nerve growth factor (NGF) or to stimulation of other neurotrophic factors. Transforming growth factor β (TGFβ) family members have been shown to potentiate the effect of different trophic factors. The aim of this study was to investigate if TGFβ may influence NGF-induced neuronal transformation and regulation of NGF, TGFβ1, and their receptors in the adult rat chromaffin tissue after grafting. Intraocular transplantation of adult chromaffin tissue was employed and grafts were treated with TGFβ1 and/or NGF. Graft survival time was 18 days after which the grafts were processed for TGFβ luciferase detection assay, NGF enzyme immunoassay, or in situ hybridization. In grafts stimulated with NGF, increased levels of TGFβ1 and TGFβ1 mRNA were detected. When grafts instead were treated with TGFβ1, enhanced levels of NGF protein were found. Furthermore, a positive mRNA signal corresponding to the transforming growth factor II receptor (TβRII) was found in the chromaffin cells of the normal adrenal medulla as well as after grafting. No increase of TβRII mRNA levels was detected after transplantation or after TGFβ1 treatment. Instead a reduction of TβRII mRNA expression was noted after NGF treatment. NGF stimulation of grafts increased the message for NGF receptors p75 and trkA in the chromaffin transplants. Grafts processed for evaluations of neurite outgrowth were allowed to survive for 28 days and were injected weekly with NGF and/or TGFβ1. NGF treatment resulted in a robust innervation of the host irides. TGFβ1 had no additive effect on nerve fiber formation when combined with NGF. Combined treatment of NGF and anti-TGFβ1 resulted in a significantly larger area of reinnervation. In conclusion, it was found that NGF and TGFβ1 may regulate the expression of each other's protein in adult chromaffin grafts. Furthermore, TβRII mRNA was present in the adult rat chromaffin cells and became downregulated as a result of NGF stimulation. Although no synergistic effects of TGFβ1 were found on NGF-induced neurite outgrowth, it was found that TGFβ1 and NGF signaling are closely linked in the chromaffin cells of the adrenal medulla.     

TGFβ1 increases microglia‐mediated engulfment of apoptotic cells via upregulation of the milk fat globule‐EGF factor 8

Milk fat globule‐epidermal growth factor‐factor 8 (Mfge8) has been described as an essential molecule during microglia‐mediated clearance of apoptotic cells via binding to phosphatidylserine residues and subsequent phagocytosis. Impaired uptake of apoptotic cells by microglia results in prolonged inflammatory responses and damage of healthy cells. Although the mechanisms of Mfge8‐mediated engulfment of apoptotic cells are well understood, endogenous or exogenous factors that regulate Mfge8 expression remain elusive. Here, we describe that TGFβ1 increases the expression of Mfge8 and enhances the engulfment of apoptotic cells by primary mouse microglia in a Mfge8‐dependent manner. Further, apoptotic cells are capable of increasing microglial TGFβ expression and release and shift the microglia phenotype toward alternative activation. Moreover, we provide evidence that Mfge8 expression is differentially regulated in microglia after classical and alternative activation and that Mfge8 is not able to exert direct antiinflammatory effects on LPS‐treated primary microglia. Together, these results underline the importance of TGFβ1 as a regulatory factor for microglia and suggest that increased TGFβ1 expression in models of neurodegeneration might be involved in clearance of apoptotic cells via regulation of Mfge8 expression. GLIA 2015;63:142–153     

Smad3‐dependent signaling underlies the TGF‐β1‐mediated enhancement in astrocytic iNOS expression

We previously demonstrated that transforming growth factor‐β1 (TGF‐β1), while having no effect alone, enhances nitric oxide (NO) production in primary, purified mouse astrocytes induced by lipopolysaccharide (LPS) plus interferon‐γ (IFN‐γ), by recruiting a latent population of astrocytes to respond, thereby enhancing the total number of cells that express Nos2. In this investigation, we evaluated the molecular signaling pathway by which this occurs. We found that purified murine primary astrocytes express mRNA for TGFβRII as well as the TGFβRI subunit activin‐like kinase 5 (ALK5), but not ALK1. Immunofluorescence microscopy confirmed the expression of TGFβRII and ALK5 protein in astrocytes. Consistent with ALK5 signaling, Smad3 accumulated in the nucleus of astrocytes as early as 30 min after TGF‐β1 (3 ng/mL) treatment and persisted upto 32 hr after TGF‐β1 administration. Addition of ALK5 inhibitors prevented TGF‐β1‐mediated Smad3 nuclear accumulation and NO production when given prior to the Nos2 induction stimuli, but not after. Finally, astrocyte cultures derived from Smad3 null mutant mice did not exhibit a TGF‐β1‐mediated increase in iNOS expression. Overall, this data suggests that ALK5 signaling and Smad3 nuclear accumulation is required for optimal enhancement of LPS plus IFNγ‐induced NO production in astrocytes by TGF‐β1. © 2010 Wiley‐Liss, Inc.     

BMP‐ and TGFβ‐signaling regulate the formation of Müller glia‐derived progenitor cells in the avian retina

Müller glia‐derived progenitor cells (MGPCs) have the capability to regenerate neurons in the retinas of different vertebrate orders. The formation of MGPCs is regulated by a network of cell‐signaling pathways. The purpose of this study was to investigate how BMP/Smad1/5/8‐ and TGFβ/Smad2/3‐signaling are coordinated to influence the formation of MGPCs in the chick model system. We find that pSmad1/5/8 is selectively up‐regulated in the nuclei of Müller glia following treatment with BMP4, FGF2, or NMDA‐induced damage, and this up‐regulation is blocked by a dorsomorphin analogue DMH1. By comparison, Smad2/3 is found in the nuclei of Müller glia in untreated retinas, and becomes localized to the cytoplasm following NMDA‐ or FGF2‐treatment. These findings suggest a decrease in TGFβ‐ and increase in BMP‐signaling when MGPCs are known to form. In both NMDA‐damaged and FGF2‐treated retinas, inhibition of BMP‐signaling suppressed the proliferation of MGPCs, whereas inhibition of TGFβ‐signaling stimulated the proliferation of MGPCs. Consistent with these findings, TGFβ2 suppressed the formation of MGPCs in NMDA‐damaged retinas. Our findings indicate that BMP/TGFβ/Smad‐signaling is recruited into the network of signaling pathways that controls the formation of proliferating MGPCs. We conclude that signaling through BMP4/Smad1/5/8 promotes the formation of MGPCs, whereas signaling through TGFβ/Smad2/3 suppresses the formation of MGPCs.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

Genetic ablation of steroid receptor coactivator‐3 promotes PPAR‐β‐mediated alternative activation of microglia in experimental autoimmune encephalomyelitis

Steroid receptor coactivator‐3 (SRC‐3) has been demonstrated to regulate lipid metabolism by inhibiting adipocyte differentiation. In this study, the potential role of SRC‐3 in experimental autoimmune encephalomyelitis (EAE), which characterized by inflammatory demyelination in central nervous system (CNS), was examined by analyzing disease progression in SRC‐3‐deficient (SRC‐3^−/−) mice. We found that SRC‐3 deficiency significantly attenuated the disease severity of EAE along with decreased inflammatory infiltration and demyelination. However, these effects are not caused by inhibition of peripheral T cell response, but by upregulated expression of peroxisome proliferator‐activated receptor (PPAR)‐β in CNS, which induced an alternative activation state of microglia in SRC‐3^−/− mice. These alternatively activated microglia inhibited CNS inflammation through inhibition of proinflammatory cytokines and chemokines, such as TNF‐α, IFN‐γ, CCL2, CCL3, CCL5, and CXCL10, as well as upregulation of anti‐inflammatory cytokine IL‐10 and opsonins, such as C1qa and C1qb. Moreover, microglia alternative activation promoted myelin regeneration through increased accumulation of oligodendrocyte precursors in white matter and elevated expression of myelin genes in the spinal cords of SRC‐3^−/− mice. Our results build up a link between lipid metabolic regulation and immune functions, and the modulation of the expression of SRC‐3 or PPAR‐β may hopefully has therapeutic modality in MS and possibly other neurodegenerative diseases. © 2010 Wiley‐Liss, Inc.     

GSK3β regulates oligodendrogenesis in the dorsal microdomain of the subventricular zone via Wnt‐β‐catenin signaling

Oligodendrocytes, the myelinating cells of the CNS, are derived postnatally from oligodendrocyte precursors (OPs) of the subventricular zone (SVZ). However, the mechanisms that regulate their generation from SVZ neural stem cells (NSC) are poorly understood. Here, we have examined the role of glycogen synthase kinase 3β (GSK3β), an effector of multiple converging signaling pathways in postnatal mice. The expression of GSK3β by rt‐qPCR was most prominent in the SVZ and in the developing white matter, around the first 1–2 weeks of postnatal life, coinciding with the peak periods of OP differentiation. Intraventricular infusion of the GSK3β inhibitor ARA‐014418 in mice aged postnatal day (P) 8–11 significantly increased generation of OPs in the dorsal microdomain of the SVZ, as shown by expression of cell specific markers using rt‐qPCR and immunolabelling. Analysis of stage specific markers revealed that the augmentation of OPs occurred via increased specification from earlier SVZ cell types. These effects of GSK3β inhibition on the dorsal SVZ were largely attributable to stimulation of the canonical Wnt/β‐catenin signaling pathway over other pathways. The results indicate GSK3β is a key endogenous factor for specifically regulating oligodendrogenesis from the dorsal SVZ microdomain under the control of Wnt‐signaling. GLIA 2014;62:778–789     

Glycogen synthase kinase‐3β phosphorylates synphilin‐1 in vitro

α‐Synuclein is known to be a major component of Lewy bodies and glial cytoplasmic inclusions in the brains of patients with α‐synucleinopathies. Synphilin‐1, an α‐synuclein‐associated protein, is also present in these inclusions. However, little is known about the post‐translational modifications of synphilin‐1. In the present study, it is reported that synphilin‐1 is phosphorylated by glycogen synthase kinase‐3βin vitro. It is well known that protein phosphorylation is involved in various physiological phenomena, including signal transduction and protein degradation. Therefore, phosphorylation of synphilin‐1 may play an important role in the function of this protein in the brain.     

Toll‐Interleukin 1 Receptor domain‐containing adaptor protein positively regulates BV2 cell M1 polarization

Microglial activation, including classical (M1) and alternative (M2) activation, plays important roles in the development of several central nervous system disorders and promotes tissue reconstruction. Toll‐like receptor (TLR)4 is important for microglial polarization. TIR domain‐containing adaptor protein (TIRAP) is an intracellular adaptor protein, which is responsible for the early phase of TLR4 activation. The role of TIRAP in BV2 cell M1 polarization is still unknown. In this study, we showed that TIRAP expression is greatly elevated in lipopolysaccharide (LPS)/interferon (IFN)‐γ‐treated microglia. TIRAP overexpression promoted BV2 microglial M1 polarization by increasing M1‐related marker production (inducible nitric oxide synthase, CD86, interleukin‐6, interleukin‐1β and tumour necrosis factor‐α). In contrast, TIRAP knockdown prevented M1‐related marker production. Mechanistically, TIRAP could interact with TNF Receptor‐Associated Factor 6 (TRAF6) to increase M1‐related marker production in TIRAP overexpressed and LPS/IFN‐γ‐treated BV2 cells. In addition, silencing of TIRAP effectively inhibited the activation of the Transforming Growth Factor‐Beta‐Activated Kinase 1/I‐Kappa‐B Kinase /Nuclear Factor of Kappa Light Polypeptide Gene Enhancer in B‐Cells (TAK1/IKK/NF‐κB) signalling pathway and the phosphorylation of Akt and mitogen‐activated protein kinases, which were activated by LPS/IFN‐γ stimulation. Thus, our results suggest that TIRAP positively regulated BV2 microglial M1 polarization through TLR4‐mediated TAK1/IKK/NF‐κB, mitogen‐activated protein kinases and Akt signalling pathways.     

Implication of Akt‐dependent Prp19α/14‐3‐3β/Cdc5L complex formation in neuronal differentiation

PRP19α and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19α/14‐3‐3β/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)‐induced neuronal differentiation of PC12 cells. Analysis of PRP19α mutants revealed that the phosphorylation of PRP19α at Thr 193 by Akt was critical for its binding with 14‐3‐3β to translocate into the nuclei and for PRP19α/14‐3‐3β/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19α or sense 14‐3‐3β RNAs promoted NGF‐induced neuronal differentiation, whereas down‐regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19αT193A lost its binding ability with 14‐3‐3β and acted as a dominant‐negative mutant in neuronal differentiation. These results imply that Akt‐dependent phosphorylation of PRP19α at Thr193 triggers PRP19α/14‐3‐3β/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre‐mRNAs. © 2010 Wiley‐Liss, Inc.     

TGFα and the Blood–Brain Barrier: Accumulation in Cerebral Vasculature

Transforming growth factor α (TGFα) is a cytokine that belongs to the epidermal growth factor (EGF) family of growth factors. EGF has a fast and saturable entry from blood to brain that is inhibitable by TGFα (18). In this report, we studied the passage of TGFα from blood to brain after an i.v. bolus injection. Using radioactively labeled peptide, we found that TGFα had an apparent rate of entry of 0.7 μl/g/min. However, most of the TGFα was trapped in the capillary endothelial cells of the cerebral vasculature rather than entering the brain parenchyma. No saturation was detected. TGFα was relatively stable in blood for 20 min after i.v. injection, but dissociation of the isotope ¹²⁵I was more evident in brain. The accumulation of TGFα in the cerebral vasculature was similar to that of amyloid-β protein_1–40. Therefore, we conclude that TGFα from the periphery interacts with the blood–brain barrier without substantial uptake into brain parenchyma. This raises the possibility that TGFα might be involved in intracranial vascular disorders such as angiopathy.     

TGF‐β1 Pathway as a New Target for Neuroprotection in Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disorder that affects more than 37 million people worldwide. Current drugs for AD are only symptomatic, but do not interfere with the underlying pathogenic mechanisms of the disease. AD is characterized by the presence of ß‐amyloid (Aβ) plaques, neurofibrillary tangles, and neuronal loss. The identification of the molecular determinants underlying AD pathogenesis is a fundamental step to design new disease‐modifying drugs. Recently, a specific impairment of transforming‐growth‐factor‐β1 (TGF‐β1) signaling pathway has been demonstrated in AD brain. The deficiency of TGF‐β1 signaling has been shown to increase both Aβ accumulation and Aβ‐induced neurodegeneration in AD models. The loss of function of TGF‐ß1 pathway seems also to contribute to tau pathology and neurofibrillary tangle formation. Growing evidence suggests a neuroprotective role for TGF‐β1 against Aβ toxicity both in vitro and in vivo models of AD. Different drugs, such as lithium or group II mGlu receptor agonists are able to increase TGF‐β1 levels in the central nervous system (CNS), and might be considered as new neuroprotective tools against Aβ‐induced neurodegeneration. In the present review, we examine the evidence for a neuroprotective role of TGF‐β1 in AD, and discuss the TGF‐β1 signaling pathway as a new pharmacological target for the treatment of AD.