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.     

Enhanced IL‐1β production in response to the activation of hippocampal glial cells impairs neurogenesis in aged mice

A variety of mechanisms that contribute to the accumulation of age‐related damage and the resulting brain dysfunction have been identified. Recently, decreased neurogenesis in the hippocampus has been recognized as one of the mechanisms of age‐related brain dysfunction. However, the molecular mechanism of decreased neurogenesis with aging is still unclear. In the present study, we investigated whether aging decreases neurogenesis accompanied by the activation of microglia and astrocytes, which increases the expression of IL‐1β in the hippocampus, and whether in vitro treatment with IL‐1β in neural stem cells directly impairs neurogenesis. Ionized calcium‐binding adaptor molecule 1 (Iba1)‐positive microglia and glial fibrillary acidic protein (GFAP)‐positive astrocytes were increased in the dentate gyrus of the hippocampus of 28‐month‐old mice. Furthermore, the mRNA level of IL‐1β was significantly increased without related histone modifications. Moreover, a significant increase in lysine 9 on histone H3 (H3K9) trimethylation at the promoter of NeuroD (a neural progenitor cell marker) was observed in the hippocampus of aged mice. In vitro treatment with IL‐1β in neural stem cells prepared from whole brain of E14.5 mice significantly increased H3K9 trimethylation at the NeuroD promoter. These findings suggest that aging may decrease hippocampal neurogenesis via epigenetic modifications accompanied by the activation of microglia and astrocytes with the increased expression of IL‐1β in the hippocampus. Synapse 64:721–728, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Involvement of cathepsin B in the processing and secretion of interleukin‐1β in chromogranin A‐stimulated microglia

Cathepsin B (CB) is a cysteine lysosomal protease implicated in a number of inflammatory diseases. Although it is now evident that caspase‐1, an essential enzyme for maturation of interleukin‐1β (IL‐1β), can be activated through the inflammasome, there is still evidence suggesting the existence of lysosomal‐proinflammatory caspase pathways. In the present study, a marked induction of pro‐IL‐1β, its processing to the mature form and secretion were observed in the primary cultured microglia prepared from wild‐type mice after stimulation with chromogranin A (CGA). Although pro‐IL‐1β also markedly increased in microglia prepared from CB‐deficient mice, CB‐deficiency abrogated the pro‐IL‐1β processing. CA‐074Me, a specific inhibitor for CB, inhibited the pro‐IL‐1β maturation and its release from microglia. Furthermore, the caspase‐1 activation was also inhibited by CA‐074Me and E‐64d, a broad cysteine protease inhibitor. After treatment with CGA, CB was markedly induced at both protein and mRNA levels. The induced pro‐CB was rapidly processed to its mature form. The immunoreactivity for CB co‐localized with both that for caspase‐1 and the cleaved IL‐1β, in the acidic enlarged lysosomes. Inconsistent with these in vitro observations, the immunoreactivity for the cleaved IL‐1β was markedly observed in microglia of the hippocampus from aged wild‐type but not CB‐deficient mice. These observations strongly suggest that CB plays a key role in the pro‐IL‐1β maturationthrough the caspase‐1 activation in enlarged lysosomes ofCGA‐treated microglia. Therefore, either pharmacological or genetic inhibition of CB may provide therapeutic intervention in inflammation‐associated neurological diseases. © 2009 Wiley‐Liss, Inc.     

δ‐Catenin/NPRAP: A new member of the glycogen synthase kinase‐3β signaling complex that promotes β‐catenin turnover in neurons

Through a multiprotein complex, glycogen synthase kinase‐3β (GSK‐3β) phosphorylates and destabilizes β‐catenin, an important signaling event for neuronal growth and proper synaptic function. δ‐Catenin, or NPRAP (CTNND2), is a neural enriched member of the β‐catenin superfamily and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ‐catenin expression is also affected by GSK‐3β signaling and participates in the molecular complex regulating β‐catenin turnover in neurons. Immunofluorescent light microscopy revealed colocalization of δ‐catenin with members of the molecular destruction complex: GSK‐3β, β‐catenin, and adenomatous polyposis coli proteins in rat primary neurons. GSK‐3β formed a complex with δ‐catenin, and its inhibition resulted in increased δ‐catenin and β‐catenin expression levels. LY294002 and amyloid peptide, known activators of GSK‐3β signaling, reduced δ‐catenin expression levels. Furthermore, δ‐catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ‐catenin, like that of β‐catenin, is regulated by proteasome‐mediated degradation. Coimmunoprecipitation experiments showed that δ‐catenin overexpression promoted GSK‐3β and β‐catenin interactions. Primary cortical neurons and PC12 cells expressing δ‐catenin treated with proteasome inhibitors showed increased ubiquitinated β‐catenin forms. Consistent with the hypothesis that δ‐catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ‐catenin showed enhanced β‐catenin turnover. These studies identify δ‐catenin as a new member of the GSK‐3β signaling pathway and further suggest that δ‐catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β‐catenin in neuronal cells. © 2010 Wiley‐Liss, Inc.     

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.     

IL‐1β induces hypomyelination in the periventricular white matter through inhibition of oligodendrocyte progenitor cell maturation via FYN/MEK/ERK signaling pathway in septic neonatal rats

Neuroinflammation elicited by microglia plays a key role in periventricular white matter (PWM) damage (PWMD) induced by infectious exposure. This study aimed to determine if microglia‐derived interleukin‐1β (IL‐1β) would induce hypomyelination through suppression of maturation of oligodendrocyte progenitor cells (OPCs) in the developing PWM. Sprague‐Dawley rats (1‐day old) were injected with lipopolysaccharide (LPS) (1 mg/kg) intraperitoneally, following which upregulated expression of IL‐1β and IL‐1 receptor 1 (IL‐1R₁) was observed. This was coupled with enhanced apoptosis and suppressed proliferation of OPCs in the PWM. The number of PDGFR‐α and NG2‐positive OPCs was significantly decreased in the PWM at 24 h and 3 days after injection of LPS, whereas it was increased at 14 days and 28 days. The protein expression of Olig1, Olig2, and Nkx2.2 was significantly reduced, and mRNA expression of Tcf4 and Axin2 was upregulated in the developing PWM after LPS injection. The expression of myelin basic protein (MBP) and 2',3'‐cyclic‐nucleotide 3"‐phosphodiesterase (CNPase) was downregulated in the PWM at 14 days and 28 days after LPS injection; this was linked to reduction of the proportion of myelinated axons and thinner myelin sheath as revealed by electron microscopy. Primary cultured OPCs treated with IL‐1β showed the failure of maturation and proliferation. Furthermore, FYN/MEK/ERK signaling pathway was involved in suppression of maturation of primary OPCs induced by IL‐1β administration. Our results suggest that following LPS injection, microglia are activated and produce IL‐1β in the PWM in the neonatal rats. Excess IL‐1β inhibits the maturation of OPCs via suppression of FYN/MEK/ERK phosphorylation thereby leading to axonal hypomyelination. GLIA 2016;64:583–602     

A critical role of TRPM2 channel in Aβ42‐induced microglial activation and generation of tumor necrosis factor‐α

Amyloid β (Aβ)‐induced neuroinflammation plays an important part in Alzheimer's disease (AD). Emerging evidence supports a role for the transient receptor potential melastatin‐related 2 (TRPM2) channel in Aβ‐induced neuroinflammation, but how Aβ induces TRPM2 channel activation and this relates to neuroinflammation remained poorly understood. We investigated the mechanisms by which Aβ₄₂ activates the TRPM2 channel in microglial cells and the relationships to microglial activation and generation of tumor necrosis factor‐α (TNF‐α), a key cytokine implicated in AD. Exposure to 10–300 nM Aβ₄₂ induced concentration‐dependent microglial activation and generation of TNF‐α that were ablated by genetically deleting (TRPM2 knockout ;TRPM2‐KO) or pharmacologically inhibiting the TRPM2 channel, revealing a critical role of this channel in Aβ₄₂‐induced microglial activation and generation of TNF‐α. Mechanistically, Aβ₄₂ activated the TRPM2 channel via stimulating generation of reactive oxygen species (ROS) and activation of poly(ADPR) polymerase‐1 (PARP‐1). Aβ₄₂‐induced generation of ROS and activation of PARP‐1 and TRPM2 channel were suppressed by inhibiting protein kinase C (PKC) and NADPH oxidases (NOX). Aβ₄₂‐induced activation of PARP‐1 and TRPM2 channel was also reduced by inhibiting PYK2 and MEK/ERK. Aβ₄₂‐induced activation of PARP‐1 was attenuated by TRPM2‐KO and moreover, the remaining PARP‐1 activity was eliminated by inhibiting PKC and NOX, but not PYK2 and MEK/ERK. Collectively, our results suggest that PKC/NOX‐mediated generation of ROS and subsequent activation of PARP‐1 play a role in Aβ₄₂‐induced TRPM2 channel activation and TRPM2‐dependent activation of the PYK2/MEK/ERK signalling pathway acts as a positive feedback to further facilitate activation of PARP‐1 and TRPM2 channel. These findings provide novel insights into the mechanisms underlying Aβ‐induced AD‐related neuroinflammation.     

Increased levels of proinflammatory cytokines in the aged rat brain attenuate injury‐induced cytokine response after excitotoxic damage

In order to evaluate proinflammatory cytokine levels and their producing cell types in the control aged rat brain and after acute excitotoxic damage, both adult and aged male Wistar rats were injected with N‐methyl‐D ‐aspartate in the striatum. At different survival times between 6 hr and 7 days after lesioning, interleukin‐1 beta (IL‐1β), interleukin‐6 (IL‐6), and tumor necrosis factor alpha (TNF‐α) were analyzed by enzyme‐linked immunosorbent assay and by double immunofluorescence of cryostat sections by using cell‐specific markers. Basal cytokine expression was attributed to astrocytes and was increased in the normal aged brain showing region specificity: TNF‐α and IL‐6 displayed age‐dependent higher levels in the aged cortex, and IL‐1β and IL‐6 in the aged striatum. After excitotoxic striatal damage, notable age‐dependent differences in cytokine induction in the aged vs. the adult were seen. The adult injured striatum exhibited a rapid induction of all cytokines analyzed, but the aged injured striatum showed a weak induction of cytokine expression: IL‐1β showed no injury‐induced changes at any time, TNF‐α presented a late induction at 5 days after lesioning, and IL‐6 was only induced at 6 hr after lesioning. At both ages, in the lesion core, all cytokines were early expressed by neurons and astrocytes, and by microglia/macrophages later on. However, in the adjacent lesion border, cytokines were found in reactive astrocytes. This study highlights the particular inflammatory response of the aged brain and suggests an important role of increased basal levels of proinflammatory cytokines in the reduced ability to induce their expression after damage. © 2009 Wiley‐Liss, Inc.     

Neuronal TIMP‐1 release accompanies astrocytic MMP‐9 secretion and enhances astrocyte proliferation induced by β‐amyloid 25–35 fragment

The neuropathology of Alzheimer's disease (AD) is accompanied by an inflammatory response that includes neurodegeneration and glial reactivity. Tissue remodeling proteins, such as matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), are inflammatory mediators that might play a dual role in the AD brain. We aimed to investigate the effects of β‐amyloid (Abeta) on the MMP‐9/TIMP‐1 balance and its involvement in Abeta toxicity in neurons and glial cells. Our results demonstrate that the neurotoxic 25–35 Abeta fragment induces the activation of MMP‐9 and the increase of proMMP‐2/9 secretion and promotes the release of TIMP‐1 in a mixed cortical neuroglial culture. The same treatments performed in pure neuronal or astrocytic cultures confirm that astroglial cells are the major source of MMP‐9, whereas increased TIMP‐1 levels have a neuronal origin. Moreover, 25–35 Abeta fragment not only induced a release of these molecules but also caused expressional changes in MMP‐9 and TIMP‐1, correlated with the neurotoxicity process. We also show that TIMP‐1 promoted cell proliferation in a mixed neuroglial culture, and we confirm this effect in primary cultured astrocytes induced by rTIMP‐1 and 25–35 Abeta. Because the proliferative effect caused by Abeta 25–35 was enhanced by the presence of TIMP‐1, we suggest that the astroglial reactivity induced by chronic exposure of the peptide might be mediated in part by TIMP‐1, which is secreted mainly by injured neurons. In conclusion, our data suggest that the Abeta 25–35 fragment stimulates the MMP‐9–TIMP‐1 pathway, promoting gliosis, in a self‐defensive attempt to eliminate amyloid deposition from AD brains. © 2009 Wiley‐Liss, Inc.     

Effects of antiparkinsonian agents on β‐amyloid and α‐synuclein oligomer formation in vitro

The aggregation of β‐amyloid protein (Aβ) and α‐synuclein (αS) are hypothesized to be the key pathogenic event in Alzheimer's disease (AD) and Lewy body diseases (LBD), with oligomeric assemblies thought to be the most neurotoxic. Inhibitors of oligomer formation, therefore, could be valuable therapeutics for patients with AD and LBD. Here, we examined the effects of antiparkinsonian agents (dopamine, levodopa, trihexyphenidyl, selegiline, zonisamide, bromocriptine, peroxide, ropinirole, pramipexole, and entacapone) on the in vitro oligomer formation of Aβ40, Aβ42, and αS using a method of photo‐induced cross‐linking of unmodified proteins (PICUP), electron microscopy, and atomic force microscopy. The antiparkinsonian agents except for trihexyphenidyl inhibited both Aβ and αS oligomer formations, and, among them, dopamine, levodopa, pramipexole, and entacapone had the stronger in vitro activity. Circular dichroism and thioflavin T(S) assays showed that secondary structures of Aβ and αS assemblies inhibited by antiparkinsonian agents were statistical coil state and that their seeding activities had disappeared. The antiparkinsonian agents could be potential therapeutic agents to prevent or delay AD and LBD progression. © 2013 Wiley Periodicals, Inc.     

Α‐synuclein induces microglial cell migration through stimulating HIF‐1α accumulation

Microglial cell migration and infiltration plays a critical role in spinal cord injury after thoracoabdominal aortic surgery. In our previous study, α‐synuclein, a presynaptic protein was shown to be released from injured neurons and cause microglial cell activation. Here, we aimed to explore the effect of α‐synuclein on microglial cell migration. Primary microglial cells were isolated from Sprague–Dawley rats and then exposed different doses (0.2, 0.4, and 0.6 μM) of α‐synuclein oligomers. The mRNA and protein levels of HIF‐1α were then analyzed by qRT‐PCR and Western blot. Cell migration was examined by a 96‐well Boyden chamber. Moreover, toll‐like receptor (TLR) 2‐expression as well as TLR7/8‐expression was inhibited by specific siRNA transfection. HIF‐1α was overexpressed by Ad‐HIF‐1α transfection. In the results, α‐synuclein was found to stimulate HIF‐1α accumulation in microglial cells in a dose‐dependent manner. Silencing HIF‐1α expression dampened α‐synuclein induced microglial cell migration. Furthermore, blockade of TLR7/8 expression but not TLR2 expression reduced HIF‐1α accumulation in microglial cells. In addition, overexpressed HIF‐1α, along with Src, prompted caveolin‐1 expression and phosphorylation, as well as migration in microglial cells. Α‐synuclein acts via TLR7/8 and enhances HIF‐1α expression, which might play a regulatory role in microglial cell migration. © 2017 Wiley Periodicals, Inc.     

Role of ERK map kinase and CRM1 in IL‐1β‐stimulated release of HMGB1 from cortical astrocytes

Reactive astrocytes are traditionally thought to impede brain plasticity after stroke. However, we previously showed that reactive astrocytes may also contribute to stroke recovery, partly via the release of a nuclear protein called high‐mobility group box 1 (HMGB1). Here, we investigate the mechanisms that allow stimulated astrocytes to release HMGB1. Exposure of rat primary astrocytes to IL‐1β for 24 h elicited a dose‐dependent HMGB1 response. Immunostaining and western blots of cell lysates showed increased intracellular levels of HMGB1. Western blots confirmed that IL‐1β induced a release of HMGB1 into astrocyte conditioned media. MAP kinase signaling was involved. Levels of phospho‐ERK were increased by IL‐1β, and the MEK/ERK inhibitor U0126 decreased HMGB1 upregulation in the stimulated astrocytes. Since HMGB1 is a nuclear protein, the role of the nuclear protein exporter, chromosome region maintenance 1 (CRM1), was assessed as a candidate mechanism for linking MAP kinase signaling to HMGB1 release. IL‐1β increased CRM1 expression in concert with a translocation of HMGB1 from nucleus into cytoplasm. Blockade of IL‐1β‐stimulated HMGB1 release with the ERK inhibitor U0126 was accompanied by a downregulation of CRM1. Our findings reveal that IL‐1β stimulates the release of HMGB1 from activated astrocytes via ERK MAP kinase and CRM1 signaling. These data suggest a novel pathway by which inflammatory cytokines may enhance the ability of reactive astrocytes to release prorecovery mediators after stroke. © 2010 Wiley‐Liss, Inc.     

[18F]Nifene test–retest reproducibility in first‐in‐human imaging of α4β2* nicotinic acetylcholine receptors

The aim of this study was to examine the suitability of [¹⁸ F]nifene, a novel α4β2* nicotinic acetylcholine receptor (nAChR) radiotracer, for in vivo brain imaging in a first‐in‐human study. Methods : Eight healthy subjects (4 M,4 F;21–69,44 ± 21 yrs) underwent a [¹⁸F]nifene positron emission tomography scan (200 ± 3.7 MBq), and seven underwent a second scan within 58 ± 31 days. Regional estimates of DVR were measured using the multilinear reference tissue model (MRTM2) with the corpus callosum as reference region. DVR reproducibility was evaluated with test–retest variability (TRV) and intraclass correlation coefficient (ICC). Results : The DVR ranged from 1.3 to 2.5 across brain regions with a TRV of 0–7%, and did not demonstrate a systematic difference between test and retest. The ICCs ranged from 0.2 to 0.9. DVR estimates were stable after 40 min. Conclusion : The binding profile and tracer kinetics of [¹⁸F]nifene make it a promising α4β2* nAChR radiotracer for scientific research in humans, with reliable DVR test–retest reproducibility.