MAB1501 Sigma-AldrichAnti-Actin Antibody, clone C4

Alzheimer disease (AD) is a disease of lost memories. Mushroom postsynaptic spines play a key role in memory storage, and loss of mushroom spines has been proposed to be linked to memory loss in AD. Generation of amyloidogenic peptides and accumulation of amyloid plaques is one of the pathological hallmarks of AD. It is important to evaluate effects of amyloid on stability of mushroom spines.In this study we used in vitro and in vivo models of amyloid synaptotoxicity to investigate effects of amyloid peptides on hippocampal mushroom spines. We discovered that application of Aβ42 oligomers to hippocampal cultures or injection of Aβ42 oligomers directly into hippocampal region resulted in reduction of mushroom spines and activity of synaptic calcium-calmodulin-dependent kinase II (CaMKII). We further discovered that expression of STIM2 protein rescued CaMKII activity and protected mushroom spines from amyloid toxicity in vitro and in vivo.Obtained results suggest that downregulation of STIM2-dependent stability of mushroom spines and reduction in activity of synaptic CaMKII is a mechanism of hippocampal synaptic loss in AD model of amyloid synaptotoxicity and that modulators/activators of this pathway may have a potential therapeutic value for treatment of AD.

Atherosclerotic plaque formation is associated with irregular distribution of wall shear stress (WSS) that modulates endothelial function and integrity. Polycystins (PC)-1/-2 constitute a flow-sensing protein complex in endothelial cells, able to respond to WSS and induce cell-proliferation changes leading to atherosclerosis. An endothelial cell-culture system of measurable WSS was established to detect alterations in PCs expression under conditions of low- and high-oscillatory shear stress in vitro. PCs expression and p53 activation as a regulator of cell proliferation were further evaluated in vivo and in 69 advanced human carotid atherosclerotic plaques (AAPs). Increased PC-1/PC-2 expression was observed at 30-60 min of low shear stress (LSS) in endothelial cells. Elevated PC-1 expression at LSS was followed by p53 potentiation. PCs immunoreactivity localizes in areas with macrophage infiltration and neovascularization. PC-1 mRNA and protein levels were significantly higher than PC-2 in stable fibroatherotic (V) and unstable/complicated (VI) AAPs. Elevated PC-1 immunostaining was detected in AAPs from patients with diabetes mellitus, dyslipidemia, hypertension and carotid stenosis, at both arteries (50%) or in one artery (90%). PCs seem to participate in plaque formation and progression. Since PC-1 upregulation coincides with p38 and p53 activation, a potential interplay of these molecules in atherosclerosis induction is posed.

Axonal injury of the optic nerve (ON) is involved in various ocular diseases, such as glaucoma and traumatic optic neuropathy, which leads to apoptotic death of retinal ganglion cells (RGCs) and loss of vision. Caspases have been implicated in RGC pathogenesis. However, the role of caspase-7, a functionally unique caspase, in ON injury and RGC apoptosis has not been reported previously. The purpose of this study is to evaluate the role of caspase-7 in ON injury-induced RGC apoptosis.C57BL/6 (wildtype, WT) and caspase-7 knockout (Casp7(-/-)) mice were used. We show that ON crush activated caspase-7 and calpain-1, an upstream activator of caspase-7, in mouse RGCs, as well as hydrolysis of kinectin and co-chaperone P23, specific substrates of caspase-7. ON crush caused a progressive loss of RGCs to 28 days after injury. Knockout of caspase-7 partially and significantly protected against the ON injury-induced RGC loss; RGC density at 28 days post ON crush in Casp7(-/-) mice was approximately twice of that in WT ON injured retinas. Consistent with changes in RGC counts, spectral-domain optical coherence tomography analysis revealed that ON crush significantly reduced the in vivo thickness of the ganglion cell complex layer (including ganglion cell layer, nerve fiber layer, and inner plexiform layer) in the retina. The ON crush-induced thinning of retinal layer was significantly ameliorated in Casp7(-/-) mice when compared to WT mice. Moreover, electroretinography analysis demonstrated a decline in the positive component of scotopic threshold response amplitude in ON crushed eyes of the WT mice, whereas this RGC functional response was significantly higher in Casp7(-/-) mice at 28 days post injury.Altogether, our findings indicate that caspase-7 plays a critical role in ON injury-induced RGC death, and inhibition of caspase-7 activity may be a novel therapeutic strategy for glaucoma and other neurodegenerative diseases of the retina.

Stem cells reside in specialized niches that are critical for their function. Quiescent hair follicle stem cells (HFSCs) are confined within the bulge niche, but how the molecular composition of the niche regulates stem cell behaviour is poorly understood. Here we show that integrin-linked kinase (ILK) is a key regulator of the bulge extracellular matrix microenvironment, thereby governing the activation and maintenance of HFSCs. ILK mediates deposition of inverse laminin (LN)-332 and LN-511 gradients within the basement membrane (BM) wrapping the hair follicles. The precise BM composition tunes activities of Wnt and transforming growth factor-β pathways and subsequently regulates HFSC activation. Notably, reconstituting an optimal LN microenvironment restores the altered signalling in ILK-deficient cells. Aberrant stem cell activation in ILK-deficient epidermis leads to increased replicative stress, predisposing the tissue to carcinogenesis. Overall, our findings uncover a critical role for the BM niche in regulating stem cell activation and thereby skin homeostasis.

Certain missense mutations in optineurin/OPTN and amplification of TBK1 are associated with normal tension glaucoma. A glaucoma-associated variant of OPTN, M98K, induces autophagic degradation of transferrin receptor (TFRC) and death in retinal cells. Here, we have explored the role of Tbk1 in M98K-OPTN-induced autophagy and cell death, and the effect of Tbk1 overexpression in retinal cells. Cell death induced by M98K-OPTN was dependent on Tbk1 as seen by the effect of Tbk1 knockdown and blocking of Tbk1 activity by a chemical inhibitor. Inhibition of Tbk1 also restores M98K-OPTN-induced transferrin receptor degradation. M98K-OPTN-induced autophagosome formation, autophagy and cell death were dependent on its phosphorylation at S177 by Tbk1. Knockdown of OPTN reduced starvation-induced autophagosome formation. M98K-OPTN expressing cells showed higher levels of Tbk1 activation and enhanced phosphorylation at Ser177 compared to WT-OPTN expressing cells. M98K-OPTN-induced activation of Tbk1 and its ability to be phosphorylated better by Tbk1 was dependent on ubiquitin binding. Phosphorylated M98K-OPTN localized specifically to autophagosomes and endogenous Tbk1 showed increased localization to autophagosomes in M98K-OPTN expressing cells. Overexpression of Tbk1 induced cell death and caspase-3 activation that were dependent on its catalytic activity. Tbk1-induced cell death possibly involves autophagy, as shown by the effect of Atg5 knockdown, and requirement of autophagic function of OPTN. Our results show that phosphorylation of Ser177 plays a crucial role in M98K-OPTN-induced autophagosome formation, autophagy flux and retinal cell death. In addition, we provide evidence for cross talk between two glaucoma associated proteins and their inter-dependence to mediate autophagy-dependent cell death.

Antiretroviral therapy has increased the life span of HIV+ individuals; however, HIV-associated neurocognitive disorder (HAND) occurrence is increasing in aging HIV patients. Previous studies suggest HIV infection alters autophagy function in the aging CNS and HIV-1 proteins affect autophagy in monocyte-derived cells. Despite these findings, the mechanisms leading to dysregulated autophagy in the CNS remain unclear. Here we sought to determine how HIV Tat dysregulates autophagy in neurons. Tat caused a dose-dependent decrease in autophagosome markers, microtubule-associated protein-1 light chain β II (LC3II), and sequestosome 1(SQSTM1), in a membrane-enriched fraction, suggesting Tat increases autophagic degradation. Bafilomycin A1 increased autophagosome number, LC3II, and SQSTM1 accumulation; Tat cotreatment diminished this effect. Tat had no effect when 3-methyladenine or knockdown of beclin 1 blocked early stages of autophagy. Tat increased numbers of LC3 puncta and resulted in the formation of abnormal autophagosomes in vitro. Likewise, in vivo studies in GFAP-Tat tg mice showed increased autophagosome accumulation in neurons, altered LC3II levels, and neurodegeneration. These effects were reversed by rapamycin treatment. Tat colocalized with autophagosome and lysosomal markers and enhanced the colocalization of autophagosome with lysosome markers. Furthermore, co-IP studies showed that Tat interacts with lysosomal-associated membrane protein 2A (LAMP2A) in vitro and in vivo, and LAMP2A overexpression reduces Tat-induced neurotoxicity. Hence, Tat protein may induce autophagosome and lysosome fusion through interaction with LAMP2A leading to abnormal neuronal autophagy function and dysregulated degradation of critical intracellular components. Therapies targeting Tat-mediated autophagy alterations may decrease neurodegeneration in aging patients with HAND.

This study aimed to determine the effects of electroacupuncture stimulation at the Baihui (GV20) and Fengfu (GV16) acupoints, at frequencies of 5Hz (EA-5Hz) and 25Hz (EA-25Hz), 7 days after cerebral ischemia-reperfusion (I/R) injury, and to evaluate the possible signaling mechanisms involved in mitogen-activated protein kinase (MAPK) pathways.Rats were subjected to 30 min of middle cerebral artery occlusion (MCAo) followed by 7 days of reperfusion. EA-5Hz or EA-25Hz was applied immediately after MCAo and then once daily for 7 consecutive days.Results indicated that EA-5Hz and EA-25Hz both markedly attenuated cerebral infarction and neurological deficits. EA-5Hz and EA-25Hz both markedly downregulated cytosolic glial fibrillary acidic protein (GFAP), mitochondrial Bax, mitochondrial and cytosolic second mitochondrial-derived activator of caspase/direct inhibitor of apoptosis protein-binding protein with low isoelectric point (Smac/DIABLO), and cytosolic cleaved caspase-3 expression, and effectively restored cytosolic phospho-p38 MAPK (p-p38 MAPK), cytosolic cAMP response element-binding protein (CREB), mitochondrial Bcl-xL, and cytosolic X-linked inhibitor of apoptosis protein (XIAP) expression, in the ischemic cortical penumbra 7 days after reperfusion. Both EA-5Hz and EA-25Hz also significantly increased the ratios of mitochondrial Bcl-xL/Bax and Bcl-2/Bax, respectively.Both EA-5Hz and EA-25Hz effectively downregulate reactive astrocytosis to provide neuroprotection against cerebral infarction, most likely by activating the p38 MAPK/CREB signaling pathway. The modulating effects of EA-5Hz and EA-25Hz on Bax-mediated apoptosis are possibly due to the activation of p38 MAPK/CREB/Bcl-xL and p38 MAPK/CREB/Bcl-2 signaling pathways, respectively, and eventually contribute to the prevention of Smac/DIABLO translocation and subsequent restoration of XIAP-mediated suppression of caspase-3 in the cortical periinfarct area 7 days after reperfusion.

RNA interference (RNAi) is a powerful gene-silencing platform for cancer treatment. Previously, we demonstrated that ribophorin II (RPN2), which is part of the N-oligosaccharyl transferase complex, regulates docetaxel sensitivity and tumor lethal phenotypes in breast cancer. However, the molecular functions and clinical relevance of RPN2 in non-small-cell lung cancer (NSCLC) remain unknown. Here, we examined RPN2 expression in tumor specimens from recurrent NSCLC patients after resection (n = 32 and = 177) and assessed the correlation between RPN2 expression and various clinical features. We also investigated whether RPN2 affects cancer malignancy in vitro and tumor growth and drug resistance in vivo. Our data show that RPN2 expression confers early and distant recurrence as well as poor survival in NSCLC patients. Furthermore, RPN2 silencing suppressed cell proliferation and invasiveness, and increased the sensitivity to chemotherapeutic drugs in vitro. Remarkably, we found that intrinsic apoptosis signaling is the mechanism of cell death involved with RPN2 knockdown. Strikingly, RPN2 silencing repressed tumorigenicity and sensitized the tumors to cisplatin treatment, which led to the longer survival of NSCLC-bearing mice. In conclusion, these data suggest that RPN2 is involved in the regulation of lethal cancer phenotypes and represents a promising new target for RNAi-based medicine against NSCLC.

Dendritic protein synthesis and actin cytoskeleton reorganization are important events required for the consolidation of hippocampal LTP and memory. However, the temporal and spatial relationships between these two processes remain unclear. Here, we report that treatment of adult rat hippocampal slices with BDNF or with tetraethylammonium (TEA), which induces a chemical form of LTP, produces a rapid and transient increase in RhoA protein levels. Changes in RhoA were restricted to dendritic spines of CA3 and CA1 and require de novo protein synthesis regulated by mammalian target of rapamycin (mTOR). BDNF-mediated stimulation of RhoA activity, cofilin phosphorylation, and actin polymerization were completely suppressed by protein synthesis inhibitors. Furthermore, intrahippocampal injections of RhoA antisense oligodeoxynucleotides inhibited theta burst stimulation (TBS)-induced RhoA upregulation in dendritic spines and prevented LTP consolidation. Addition of calpain inhibitors after BDNF or TEA treatment maintained RhoA levels elevated and prolonged the effects of BDNF and TEA on actin polymerization. Finally, the use of isoform-selective calpain inhibitors revealed that calpain-2 was involved in RhoA synthesis, whereas calpain-1 mediated RhoA degradation. Overall, this mechanism provides a novel link between dendritic protein synthesis and reorganization of the actin cytoskeleton in hippocampal dendritic spines during LTP consolidation.

Pituitary somatotroph adenomas result in dysregulated growth hormone (GH) hypersecretion and acromegaly; however, regulatory mechanisms that promote GH hypersecretion remain elusive. Here, we provide evidence that STAT3 directly induces somatotroph tumor cell GH. Evaluation of pituitary tumors revealed that STAT3 expression was enhanced in human GH-secreting adenomas compared with that in nonsecreting pituitary tumors. Moreover, STAT3 and GH expression were concordant in a somatotroph adenoma tissue array. Promoter and expression analysis in a GH-secreting rat cell line (GH3) revealed that STAT3 specifically binds the Gh promoter and induces transcription. Stable expression of STAT3 in GH3 cells induced expression of endogenous GH, and expression of a constitutively active STAT3 further enhanced GH production. Conversely, expression of dominant-negative STAT3 abrogated GH expression. In primary human somatotroph adenoma-derived cell cultures, STAT3 suppression with the specific inhibitor S3I-201 attenuated GH transcription and reduced GH secretion in the majority of derivative cultures. In addition, S3I-201 attenuated somatotroph tumor growth and GH secretion in a rat xenograft model. GH induced STAT3 phosphorylation and nuclear translocation, indicating a positive feedback loop between STAT3 and GH in somatotroph tumor cells. Together, these results indicate that adenoma GH hypersecretion is the result of STAT3-dependent GH induction, which in turn promotes STAT3 expression, and suggest STAT3 as a potential therapeutic target for pituitary somatotroph adenomas.