Delivery of mRNA Encoding Interleukin-12 and a Stimulator of Interferon Genes Agonist Potentiates Antitumor Efficacy through Reversing T Cell Exhaustion.

T cell exhaustion has emerged as a major hurdle that impedes the clinical translation of stimulator of interferon genes (STING) agonists. It is crucial to explore innovative strategies to rejuvenate exhausted T cells and potentiate the antitumor efficacy. Here, we propose an approach utilizing MSA-2 as a STING agonist, along with nanoparticle-mediated delivery of mRNA encoding interleukin-12 (IL-12) to restore the function of T cells. We developed a lipid nanoparticle (DMT7-IL12 LNP) that encapsulated IL12 mRNA. Our findings convincingly demonstrated that the combination of MSA-2 and DMT7-IL12 LNP can effectively reverse the exhausted T cell phenotype, as evidenced by the enhanced secretion of cytokines, such as tumor necrosis factor alpha, interferon gamma, and Granzyme B, coupled with reduced levels of inhibitory molecules such as T cell immunoglobulin and mucin domain-3 and programmed cell death protein-1 on CD8+ T cells. Furthermore, this approach led to improved survival and tumor regression without causing any systemic toxicity in melanoma and lung metastasis models. These findings suggest that mRNA encoding IL-12 in conjunction with STING agonists has the potential to confer superior clinical outcomes, representing a promising advancement in cancer immunotherapy.

Targeted mRNA Nanoparticles Ameliorate Blood-Brain Barrier Disruption Postischemic Stroke by Modulating Microglia Polarization.

The ischemic stroke is a major global health concern, with high mortality and disability rates. Unfortunately, there is a dearth of effective clinical interventions for managing poststroke neuroinflammation and blood-brain barrier (BBB) disruption that are crucial for the brain injury evolving and neurological deficits. By leveraging the pathological progression of an ischemic stroke, we developed an M2 microglia-targeting lipid nanoparticle (termed MLNP) approach that can selectively deliver mRNA encoding phenotype-switching interleukin-10 (mIL-10) to the ischemic brain, creating a beneficial feedback loop that drives microglial polarization toward the protective M2 phenotypes and augments the homing of mIL-10-loaded MLNPs (mIL-10@MLNPs) to ischemic regions. In a transient middle cerebral artery occlusion (MCAO) mouse model of an ischemic stroke, our findings demonstrate that intravenously injected mIL-10@MLNPs induce IL-10 production and enhance the M2 polarization of microglia. The resulting positive loop reinforces the resolution of neuroinflammation, restores the impaired BBB, and prevents neuronal apoptosis after stroke. Using a permanent distal MCAO mouse model of an ischemic stroke, the neuroprotective effects of mIL-10@MLNPs have been further validated by the attenuation of the sensorimotor and cognitive neurological deficits. Furthermore, the developed mRNA-based targeted therapy has great potential to extend the therapeutic time window at least up to 72 h poststroke. This study depicts a simple and versatile LNP platform for selective delivery of mRNA therapeutics to cerebral lesions, showcasing a promising approach for addressing an ischemic stroke and associated brain conditions.

Caspase-3 cleaved tau impairs mitochondrial function through the opening of the mitochondrial permeability transition pore.

Mitochondrial dysfunction is a significant factor in the development of Alzheimer's disease (AD). Previous studies have demonstrated that the expression of tau cleaved at Asp421 by caspase-3 leads to mitochondrial abnormalities and bioenergetic impairment. However, the underlying mechanism behind these alterations and their impact on neuronal function remains unknown. To investigate the mechanism behind mitochondrial dysfunction caused by this tau form, we used transient transfection and pharmacological approaches in immortalized cortical neurons and mouse primary hippocampal neurons. We assessed mitochondrial morphology and bioenergetics function after expression of full-length tau and caspase-3-cleaved tau. We also evaluated the mitochondrial permeability transition pore (mPTP) opening and its conformation as a possible mechanism to explain mitochondrial impairment induced by caspase-3 cleaved tau. Our studies showed that pharmacological inhibition of mPTP by cyclosporine A (CsA) prevented all mitochondrial length and bioenergetics abnormalities in neuronal cells expressing caspase-3 cleaved tau. Neuronal cells expressing caspase-3-cleaved tau showed sustained mPTP opening which is mostly dependent on cyclophilin D (CypD) protein expression. Moreover, the impairment of mitochondrial length and bioenergetics induced by caspase-3-cleaved tau were prevented in hippocampal neurons obtained from CypD knock-out mice. Interestingly, previous studies using these mice showed a prevention of mPTP opening and a reduction of mitochondrial failure and neurodegeneration induced by AD. Therefore, our findings showed that caspase-3-cleaved tau negatively impacts mitochondrial bioenergetics through mPTP activation, highlighting the importance of this channel and its regulatory protein, CypD, in the neuronal damage induced by tau pathology in AD.

Modulating Plaque Inflammation via Targeted mRNA Nanoparticles for the Treatment of Atherosclerosis.

Atherosclerosis is a common pathology present in many cardiovascular diseases. Although the current therapies (including statins and inhibitors of the serine protease PCSK9) can effectively reduce low-density lipoprotein (LDL) cholesterol levels to guideline-recommended levels, major adverse cardiovascular events still occur frequently. Indeed, the subendothelial retention of lipoproteins in the artery wall triggers multiple events of inflammation in macrophages and is a major contributor to the pathological progression of atherosclerosis. It has been gradually recognized that modulating inflammation is, therefore, an attractive avenue to forestall and treat atherosclerosis and its complications. Unfortunately, challenges with specificity and efficacy in managing plaque inflammation have hindered progress in atherosclerosis treatment. Herein, we report an NP-mediated mRNA therapeutic approach to target atherosclerotic lesional macrophages, modulating inflammation in advanced atherosclerotic lesions for the treatment of atherosclerosis. We demonstrated that the targeted NPs containing IL-10 mRNA colocalized with M2-like macrophages and induced IL-10 production in atherosclerotic plaques following intravenous administration to Western diet (WD)-fed Ldlr-/- mice. Additionally, the lesions showed a significantly alleviated inflammatory response, as evidenced by reduced oxidative stress and macrophage apoptosis, resulting in decreased lipid deposition, diminished necrotic areas, and increased fiber cap thickness. These results demonstrate the successful delivery of mRNA therapeutics to macrophage-enriched plaques in a preclinical model of advanced atherosclerosis, showing that this targeted NP inflammation management approach has great potential for translation into a wide range of clinical applications.

mRNA lipid nanoparticle-mediated pyroptosis sensitizes immunologically cold tumors to checkpoint immunotherapy.

Synergistically improving T-cell responsiveness is promising for favorable therapeutic outcomes in immunologically cold tumors, yet current treatments often fail to induce a cascade of cancer-immunity cycle for effective antitumor immunity. Gasdermin-mediated pyroptosis is a newly discovered mechanism in cancer immunotherapy; however, cleavage in the N terminus is required to activate pyroptosis. Here, we report a single-agent mRNA nanomedicine-based strategy that utilizes mRNA lipid nanoparticles (LNPs) encoding only the N-terminus of gasdermin to trigger pyroptosis, eliciting robust antitumor immunity. In multiple female mouse models, we show that pyroptosis-triggering mRNA/LNPs turn cold tumors into hot ones and create a positive feedback loop to promote antitumor immunity. Additionally, mRNA/LNP-induced pyroptosis sensitizes tumors to anti-PD-1 immunotherapy, facilitating tumor growth inhibition. Antitumor activity extends beyond the treated lesions and suppresses the growth of distant tumors. We implement a strategy for inducing potent antitumor immunity, enhancing immunotherapy responses in immunologically cold tumors.

Targeted delivery of a STING agonist to brain tumors using bioengineered protein nanoparticles for enhanced immunotherapy.

Immunotherapy is emerging as a powerful tool for combating many human diseases. However, the application of this life-saving treatment in serious brain diseases, including glioma, is greatly restricted. The major obstacle is the lack of effective technologies for transporting therapeutic agents across the blood-brain barrier (BBB) and achieving targeted delivery to specific cells once across the BBB. Ferritin, an iron storage protein, traverses the BBB via receptor-mediated transcytosis by binding to transferrin receptor 1 (TfR1) overexpressed on BBB endothelial cells. Here, we developed bioengineered ferritin nanoparticles as drug delivery carriers that enable the targeted delivery of a small-molecule immunomodulator to achieve enhanced immunotherapeutic efficacy in an orthotopic glioma-bearing mouse model. We fused different glioma-targeting moieties on self-assembled ferritin nanoparticles via genetic engineering, and RGE fusion protein nanoparticles (RGE-HFn NPs) were identified as the best candidate. Furthermore, RGE-HFn NPs encapsulating a stimulator of interferon genes (STING) agonist (SR717@RGE-HFn NPs) maintained stable self-assembled structure and targeting properties even after traversing the BBB. In the glioma-bearing mouse model, SR717@RGE-HFn NPs elicited a potent local innate immune response in the tumor microenvironment, resulting in significant tumor growth inhibition and prolonged survival. Overall, this biomimetic brain delivery platform offers new opportunities to overcome the BBB and provides a promising approach for brain drug delivery and immunotherapy in patients with glioma.

BAG3 Regulation of RAB35 Mediates the Endosomal Sorting Complexes Required for Transport/Endolysosome Pathway and Tau Clearance.

BACKGROUND: Declining proteostasis with aging contributes to increased susceptibility to neurodegenerative diseases, including Alzheimer's disease (AD). Emerging studies implicate impairment of the endosome-lysosome pathway as a significant factor in the pathogenesis of these diseases. Previously, we demonstrated that BAG3 regulates phosphorylated tau clearance. However, we did not fully define how BAG3 regulates endogenous tau proteostasis, especially in the early stages of disease progression. METHODS: Mass spectrometric analyses were performed to identify neuronal BAG3 interactors. Multiple biochemical assays were used to investigate the BAG3-HSP70-TBC1D10B (EPI64B)-RAB35-HRS regulatory networks. Live-cell imaging was used to study the dynamics of the endosomal pathway. Immunohistochemistry and immunoblotting were performed in human AD brains and in P301S tau transgenic mice with BAG3 overexpressed. RESULTS: The primary group of neuronal BAG3 interactors identified are involved in the endocytic pathway. Among them were key regulators of small GTPases, such as the RAB35 GTPase-activating protein TBC1D10B. We demonstrated that a BAG3-HSP70-TBC1D10B complex attenuates the ability of TBC1D10B to inactivate RAB35. Thus, BAG3 interacts with TBC1D10B to support the activation of RAB35 and recruitment of HRS, initiating endosomal sorting complex required for transport-mediated endosomal tau clearance. Furthermore, TBC1D10B shows significantly less colocalization with BAG3 in AD brains than in age-matched controls. Overexpression of BAG3 in P301S tau transgenic mice increased the colocalization of phosphorylated tau with the endosomal sorting complex required for transport III protein CHMP2B and reduced the levels of the mutant human tau. CONCLUSIONS: We identified a novel BAG3-TBC1D10B-RAB35 regulatory axis that modulates endosomal sorting complex required for transport-dependent protein degradation machinery and tau clearance. Dysregulation of BAG3 could contribute to the pathogenesis of AD.

Lipoprotein Lipase Regulates Microglial Lipid Droplet Accumulation.

Microglia become increasingly dysfunctional with aging and contribute to the onset of neurodegenerative disease (NDs) through defective phagocytosis, attenuated cholesterol efflux, and excessive secretion of pro-inflammatory cytokines. Dysfunctional microglia also accumulate lipid droplets (LDs); however, the mechanism underlying increased LD load is unknown. We have previously shown that microglia lacking lipoprotein lipase (LPL KD) are polarized to a pro-inflammatory state and have impaired lipid uptake and reduced fatty acid oxidation (FAO). Here, we also show that LPL KD microglia show excessive accumulation of LD-like structures. Moreover, LPL KD microglia display a pro-inflammatory lipidomic profile, increased cholesterol ester (CE) content, and reduced cholesterol efflux at baseline. We also show reduced expression of genes within the canonical cholesterol efflux pathway. Importantly, PPAR agonists (rosiglitazone and bezafibrate) rescued the LD-associated phenotype in LPL KD microglia. These data suggest that microglial-LPL is associated with lipid uptake, which may drive PPAR signaling and cholesterol efflux to prevent inflammatory lipid distribution and LD accumulation. Moreover, PPAR agonists can reverse LD accumulation, and therefore may be beneficial in aging and in the treatment of NDs.

Genetic Variants of Lipoprotein Lipase and Regulatory Factors Associated with Alzheimer's Disease Risk.

Lipoprotein lipase (LPL) is a key enzyme in lipid and lipoprotein metabolism. The canonical role of LPL involves the hydrolysis of triglyceride-rich lipoproteins for the provision of FFAs to metabolic tissues. However, LPL may also contribute to lipoprotein uptake by acting as a molecular bridge between lipoproteins and cell surface receptors. Recent studies have shown that LPL is abundantly expressed in the brain and predominantly expressed in the macrophages and microglia of the human and murine brain. Moreover, recent findings suggest that LPL plays a direct role in microglial function, metabolism, and phagocytosis of extracellular factors such as amyloid- beta (Aß). Although the precise function of LPL in the brain remains to be determined, several studies have implicated LPL variants in Alzheimer's disease (AD) risk. For example, while mutations shown to have a deleterious effect on LPL function and expression (e.g., N291S, HindIII, and PvuII) have been associated with increased AD risk, a mutation associated with increased bridging function (S447X) may be protective against AD. Recent studies have also shown that genetic variants in endogenous LPL activators (ApoC-II) and inhibitors (ApoC-III) can increase and decrease AD risk, respectively, consistent with the notion that LPL may play a protective role in AD pathogenesis. Here, we review recent advances in our understanding of LPL structure and function, which largely point to a protective role of functional LPL in AD neuropathogenesis.

BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes.

A major cellular catabolic pathway in neurons is macroautophagy/autophagy, through which misfolded or aggregation-prone proteins are sequestered into autophagosomes that fuse with lysosomes, and are degraded. MAPT (microtubule-associated protein tau) is one of the protein clients of autophagy. Given that accumulation of hyperphosphorylated MAPT contributes to the pathogenesis of Alzheimer disease and other tauopathies, decreasing endogenous MAPT levels has been shown to be beneficial to neuronal health in models of these diseases. A previous study demonstrated that the HSPA/HSP70 co-chaperone BAG3 (BCL2-associated athanogene 3) facilitates endogenous MAPT clearance through autophagy. These findings prompted us to further investigate the mechanisms underlying BAG3-mediated autophagy in the degradation of endogenous MAPT. Here we demonstrate for the first time that BAG3 plays an important role in autophagic flux in the neurites of mature neurons (20-24 days in vitro [DIV]) through interaction with the post-synaptic cytoskeleton protein SYNPO (synaptopodin). Loss of either BAG3 or SYNPO impeded the fusion of autophagosomes and lysosomes predominantly in the post-synaptic compartment. A block of autophagy leads to accumulation of the autophagic receptor protein SQSTM1/p62 (sequestosome 1) as well as MAPT phosphorylated at Ser262 (p-Ser262). Furthermore, p-Ser262 appears to accumulate in autophagosomes at post-synaptic densities. Overall these data provide evidence of a novel role for the co-chaperone BAG3 in synapses. In cooperation with SYNPO, it functions as part of a surveillance complex that facilitates the autophagic clearance of MAPT p-Ser262, and possibly other MAPT species at the post-synapse. This appears to be crucial for the maintenance of a healthy, functional synapse.Abbreviations: aa: amino acids; ACTB: actin beta; BafA1: bafilomycin A1; BAG3: BCL2 associated athanogene 3; CQ chloroquine; CTSL: cathepsin L; DIV: days in vitro; DLG4/PSD95: discs large MAGUK scaffold protein 4; HSPA/HSP70: heat shock protein family A (Hsp70); MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2; MAPT: microtubule associated protein tau; p-Ser262: MAPT phosphorylated at serine 262; p-Ser396/404: MAPT phosphorylated at serines 396 and 404; p-Thr231: MAPT phosphorylated at threonine 231; PBS: phosphate buffered saline; PK: proteinase K; scr: scrambled; shRNA: short hairpin RNA; SQSTM1/p62 sequestosome 1; SYN1: synapsin I; SYNPO synaptopodin; SYNPO2/myopodin: synaptopodin 2; VPS: vacuolar protein sorting.

Tau Clearance Mechanisms.

Efficient quality control mechanisms are essential for a healthy, functional neuron. Recognition and degradation of misfolded, damaged, or potentially toxic proteins, is a crucial aspect of protein quality control. Tau is a protein that is highly expressed in neurons, and plays an important role in modulating a number of physiological processes. Maintaining appropriate levels of tau is key for neuronal health; hence perturbations in tau clearance mechanisms are likely significant contributors to neurodegenerative diseases such as Alzheimer's disease and frontotemporal lobar degeneration. In this chapter we will first briefly review the two primary degradative mechanisms that mediate tau clearance: the proteasome system and the autophagy-lysosome pathway. This will be followed by a discussion about what is known about the contribution of each of these pathways to tau clearance. We will also present recent findings on tau degradation through the endolysosomal system. Further, how deficits in these degradative systems may contribute to the accumulation of dysfunctional or toxic forms of tau in neurodegenerative conditions is considered.

A tau homeostasis signature is linked with the cellular and regional vulnerability of excitatory neurons to tau pathology.

Excitatory neurons are preferentially impaired in early Alzheimer's disease but the pathways contributing to their relative vulnerability remain largely unknown. Here we report that pathological tau accumulation takes place predominantly in excitatory neurons compared to inhibitory neurons, not only in the entorhinal cortex, a brain region affected in early Alzheimer's disease, but also in areas affected later by the disease. By analyzing RNA transcripts from single-nucleus RNA datasets, we identified a specific tau homeostasis signature of genes differentially expressed in excitatory compared to inhibitory neurons. One of the genes, BCL2-associated athanogene 3 (BAG3), a facilitator of autophagy, was identified as a hub, or master regulator, gene. We verified that reducing BAG3 levels in primary neurons exacerbated pathological tau accumulation, whereas BAG3 overexpression attenuated it. These results define a tau homeostasis signature that underlies the cellular and regional vulnerability of excitatory neurons to tau pathology.

Nuclear transglutaminase 2 directly regulates expression of cathepsin S in rat cortical neurons.

Transglutaminase 2 (TG2) is a protein that modulates neuronal survival processes. Although TG2 is primarily cytosolic, data have suggested the nuclear localization of TG2 is strongly associated with neuronal viability. Depletion of TG2 in neurons results in neurite retraction and loss of viability, which is likely due to a dysregulation in gene expression. To begin to understand how TG2 regulates neuronal gene expression, chromatin immunoprecipitation was performed in neurons with TG2 overexpression. The resulting genomic DNA was recovered and sequenced. Bioinformatics analyses revealed that a signature DNA motif was enriched in the TG2 immunoprecipitated genomic DNA. In particular, this motif strongly mapped to a region proximate to the gene Ctss (cathepsin S). Knockdown of TG2 resulted in a significant increase in cathepsin S expression, which preceded the loss of neuronal viability. This is the first demonstration that TG2 directly associates with genomic DNA and regulates gene expression in neurons. Given that expression of cathepsin S is increased in neurological disease states, our data suggest that TG2 may play a role in promoting neuron health in part by repressing the expression of cathepsin S. Overall these data provide new insights into the function of nuclear TG2 in neurons.

Nrf2 mediates the expression of BAG3 and autophagy cargo adaptor proteins and tau clearance in an age-dependent manner.

During aging, decreased efficiency of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation and autophagic processes in the brain may be a contributing factor in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. Therefore, we analyzed the expression of Bcl-2-associated athanogene 3, a cochaperone that mediates autophagy, and the autophagy adaptors NBR1, NDP52, and sequestosome 1/p62 in the brains of 4-, 8-, and 12-month-old wild-type and Nrf2 knockout (-/-) mice. We also analyzed the levels of total tau and phospho-tau species. There were minimal differences in the expression of autophagy-related genes or tau species in 4-month-old animals; however, by 12 months, all of these autophagy-associated genes were expressed at significantly lower levels in the Nrf2 (-/-) mice. The decreases in the autophagy-associated genes were accompanied by significantly elevated levels of phospho-tau species in the 12-month-old Nrf2 (-/-) brains. These findings indicate that Nrf2 regulation of autophagy-related genes likely plays a greater role in mediating the clearance of tau as an organism ages.

Assessing the degradation of tau in primary neurons: The role of autophagy.

Tau is a neuronal cytosolic, highly regulated protein. Although first identified as a protein that binds and stabilizes microtubules, it is now clear that tau plays numerous other roles in neurons. In addition to its key physiological roles in neuronal structure and function, tau is also involved in the pathogenesis of Alzheimer's disease and numerous other neurodegenerative disorders. In all tauopathies, there are pathogenic accumulations of tau. Given that tau homeostasis requires a balance of synthesis and degradation, understanding the pathways that mediate tau clearance and regulate this process in the disease state is of fundamental importance. In neurons, macroautophagy (referred to as autophagy in this chapter) plays a pivotal role in clearing damaged or misfolded proteins under normal conditions. However, in the disease state autophagy is impaired and tau may not be efficiently targeted for degradation which contributes to the increases in pathological tau species. Therefore, establishing model systems that allow for the analysis of tau clearance by autophagy and quantitative assessment of interventions that increase autophagy and tau clearance are needed. Of particular importance is the use of primary neurons as a model system, as they are more reflective of the relevant in vivo autophagy pathway than clonal or immortalized cell models. In this chapter we present detailed methods for the preparation of neurons, immunoblotting and imaging analyses, genetic and pharmacological manipulation of autophagy with analyses, and methods to quantitatively measure changes in tau and phospho-tau levels.

GSK-3/CREB pathway involved in the gx-50's effect on Alzheimer's disease.

Aggregation of amyloid-beta (Aß) fragments is one of the major pathological hallmarks of Alzheimer's disease (AD). Our previous study has demonstrated that a novel compound named N-[2-(3, 4-dimethoxyphenyl) ethyl]-3-phenyl-acrylamide (gx-50) can decrease the accumulation of Aß oligomers in the cerebral cortex and improve the cognitive abilities in transgenic demented mice. To further study the mechanism of the neuroprotective effect of gx-50 against AD, we employed microarray to investigate the gene expression profile of the primary cultured neurons treated with gx-50 or/and Aß. Microarray disclosed 351 genes associated with AD in the gx-50 plus Aß treated group, out of the 22,523 probes. 217 of the 351 genes were significantly up-regulated, 134 of them were down-regulated. The 351 genes were mainly involved in neurotransmission, signal transduction, nervous system development, protein phosphorylation, transcription and apoptosis. By the Onto-pathway analysis, a network involved two molecules - GSK-3, CREB and another two closely linked proteins - AKT, BDNF was discovered. The GSK/CREB pathway was further studied at the gene and protein level both in vivo and in vitro. Western blot and immunohistochemistry analysis showed that the gx-50 elevated the AKT phosphorylation and inhibited its downstream protein - GSK-3's activity, then restored the CREB's transcriptional activity, and finally enhanced the expression of the CREB target gene - BDNF. In addition, the real-time PCR results displayed the same tendency. In conclusion, studies in this research indicated that the gx-50 may improve the cognitive ability of AD via the GSK-3/CREB pathway.

The suppressive effects of gx-50 on Aß-induced chemotactic migration of microglia.

Microglia, the main immune cells of the central nervous system (CNS), play a vital role in the development of AD. Once microglia are activated, they migrate to neuritic plaques and persistently release pro-inflammatory mediators that lead to neuroinflammation and neuronal degeneration, accelerating the progression of AD. In this study, we analyzed whether an AD candidate drug, N-[2-(3,4-dimethoxyphenyl)ethyl]-3-phenyl-acrylamide (gx-50), a compound extracted from Sichuan pepper (Zanthoxylum bungeanum), exhibited suppressive effects on the chemotactic migration of microglia induced by Aß. At first, the effects of gx-50 on the migration of primary cultured microglia to Aß were detected by transwell assay, and the secretion of chemokine CCL5 was measured by ELISA assay. Then, the release of TGF-ß1 was detected by ELISA and quantitative real-time PCR, and the activation of the TGF-ß1-Smad2 pathway was analyzed by Western blotting. The LDH assay revealed that cell viability was not affected by gx-50 at concentrations from 0.01 to 100 µM; thus, combined with our previous studies, 1 µM was chosen as the treatment concentration. The cell transwell measurement demonstrated that gx-50 suppressed the chemotactic migration of microglia by nearly 50% and inhibited the increase in CCL5 triggered by Aß. Moreover, the analysis of the TGF-ß1-Smad2 pathway revealed that gx-50 can antagonize Aß-induced down-regulation of TGF-ß1 at both the mRNA and protein levels and stimulate the signal pathway activation. Simultaneously, gx-50 pretreatment also significantly enhanced the phosphorylation of glycogen synthase kinase-3ß (GSK-3ß), which correlated closely with the migration of microglia. In conclusion, in the presence of Aß, gx-50 pretreatment inhibited the excessive chemotactic migration of microglia.

The alteration of protein profile induced by cigarette smoking via oxidative stress in mice epididymis.

Smoking is associated with a declining quality of semen. The aim of this study was to screen and investigate the differential expression of proteins extracted from the epididymis of mice exposed daily with cigarette smoke. Using MALDI-TOF-MS analysis, we found that the protein profile of the mouse epididymis was altered by cigarette smoking and identified 27 proteins from the most abundant and differentially expressed spots in the 2-DE gels of epididymal samples. These proteins were classified into groups according to their functions such as energy metabolism, reproduction and structural molecule activity. Through pathway analysis, these proteins were associated with the glutathione metabolism and protein processing in the endoplasmic reticulum. These results showed that the epididymis may experience oxidative stress following cigarette smoke exposure, which was confirmed using immunohistochemistry. We determine that cigarette smoking can induce oxidative stress in the mouse epididymis, which may cause protein profile altering, thereby impairing epididymis function, and leading to a decline in semen quality.

NF-κB pathway mediates vascular smooth muscle response to nicotine.

Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) play important roles in nicotine-induced cardiovascular disease. To elucidate the mechanism underlying the abnormal SMC behavioral response to nicotine, we investigated the activation of the NF-κB signal transduction pathway and cell adhesion molecular (CAM) expression on SMCs. Also we used different cell culture manner of SMC sole and EC-SMC co-culture with a 0.4 or a 3 µm membrane pore, to observe whether there is a crosstalk between EC/SMC involved in the process of NF-κB pathway activation. Nicotine-induced effects were observed in SMCs by both monoculture and co-culture with the 3 µm-pore size, including the phosphorylation of IKK and IκB, the shift of transcription factor NF-κB, and the enhancement of SMC cytoskeleton protein expression and migration ability, but none were observed by co-culture with the 0.4 µm-pore size. All of the actions could be distinctly blocked by α-bungarotoxin (α7 nicotinic receptor inhibitor) or PDTC (NF-κB suppressor). Flow cytometry analysis showed that the adhesion molecules ICAM-1 and LFA-1 and VCAM-1 and VLA-4 were better expressed similarly on the surface of SMCs in the monoculture and 3 µm-pore size co-culture system vs. the 0.4 µm co-culture way. The results imply that nicotine induces SMC cytoskeleton protein up-expression and migration via the NF-κB signaling pathway and that EC-SMC crosstalk via CAM facilitates its response to nicotine.

A novel drug candidate for Alzheimer's disease treatment: gx-50 derived from Zanthoxylum bungeanum.

This study focused on a promising drug candidate, N-[2-(3,4-dimethoxyphenyl)ethyl]-3-phenyl-acrylamide (gx-50), a compound extracted from Sichuan pepper (Zanthoxylum Bungeanum), to determine whether it would be an effective therapeutic for Alzheimer's disease (AD) via biological experiments. In vivo, we determined the pharmacokinetic profile of gx-50 and evaluated the effect of gx-50 on the cognitive abilities of amyloid-ß protein precursor transgenic (AßPP-Tg) mice by Morris water maze testing. In addition, we examined the effects of gx-50 on amyloid-ß (Aß) oligomers in the brains of AßPP-Tg mice by immunohistochemistry. In vitro, we observed a direct effect of gx-50 on Aß oligomers by atomic force microscopy, detected the neuroprotective effects of gx-50 by western blotting and cell apoptosis assays, and measured its effects on intracellular calcium currents by laser confocal microscopy. Experiments in vivo showed that gx-50 could penetrate the blood brain barrier and improve the cognitive abilities of mice. Moreover, gx-50 treatment decreased the accumulation of Aß oligomers in the cerebral cortex. The results in vitro demonstrated that gx-50 could disassemble Aß oligomers, inhibit Aß-induced neuronal apoptosis and apoptotic gene expression, and reduce neuronal calcium toxicity. These results strongly suggest that gx-50 is a potential candidate drug for treating AD.