Effects of Probiotics on Colitis-Induced Exacerbation of Alzheimer's Disease in AppNL-G-F Mice.

Alzheimer's disease (AD) is characterized by progressive cognitive decline and is a leading cause of death in the United States. Neuroinflammation has been implicated in the progression of AD, and several recent studies suggest that peripheral immune dysfunction may influence the disease. Continuing evidence indicates that intestinal dysbiosis is an attribute of AD, and inflammatory bowel disease (IBD) has been shown to aggravate cognitive impairment. Previously, we separately demonstrated that an IBD-like condition exacerbates AD-related changes in the brains of the AppNL-G-F mouse model of AD, while probiotic intervention has an attenuating effect. In this study, we investigated the combination of a dietary probiotic and an IBD-like condition for effects on the brains of mice. Male C57BL/6 wild type (WT) and AppNL-G-F mice were randomly divided into four groups: vehicle control, oral probiotic, dextran sulfate sodium (DSS), and DSS given with probiotics. As anticipated, probiotic treatment attenuated the DSS-induced colitis disease activity index in WT and AppNL-G-F mice. Although probiotic feeding significantly attenuated the DSS-mediated increase in WT colonic lipocalin levels, it was less protective in the AppNL-G-F DSS-treated group. In parallel with the intestinal changes, combined probiotic and DSS treatment increased microglial, neutrophil elastase, and 5hmC immunoreactivity while decreasing c-Fos staining compared to DSS treatment alone in the brains of WT mice. Although less abundant, probiotic combined with DSS treatment demonstrated a few similar changes in AppNL-G-F brains with increased microglial and decreased c-Fos immunoreactivity in addition to a slight increase in Aß plaque staining. Both probiotic and DSS treatment also altered the levels of several cytokines in WT and AppNL-G-F brains, with a unique increase in the levels of TNFα and IL-2 being observed in only AppNL-G-F mice following combined DSS and probiotic treatment. Our data indicate that, while dietary probiotic intervention provides protection against the colitis-like condition, it also influences numerous glial, cytokine, and neuronal changes in the brain that may regulate brain function and the progression of AD.

IL-6 Deficiency Exacerbates Allergic Asthma and Abrogates the Protective Effect of Allergic Inflammation against Streptococcus pneumoniae Pathogenesis.

Allergic asthma (AA) is characterized as a Th2-biased airway inflammation that can develop lung inflammation and remodeling of the respiratory tract. Streptococcus pneumoniae is a major respiratory pathogen, causing noninvasive (otitis media and pneumonia) and invasive diseases (sepsis) in humans. We sought to determine the role of IL-6 in the regulation of lung inflammation in murine AA caused by Aspergillus fumigatus as well as its consequence on the regulation of airway barrier integrity and S. pneumoniae disease. In an AA model, IL-6 deficiency led to increased lung inflammation, eosinophil recruitment, tissue pathology, and collagen deposition. Additionally, IL-6-deficient asthmatic mice exhibited reduced goblet cell hyperplasia and increased TGF-ß production. These key changes in the lungs of IL-6-deficient asthmatic mice resulted in dysregulated tight junction proteins and increased lung permeability. Whereas the host response to AA protected against S. pneumoniae lung disease, the IL-6 deficiency abrogated the protective effect of allergic inflammation against S. pneumoniae pathogenesis. Consistent with in vivo data, IL-6 knockdown by small interfering RNA or the blockade of IL-6R signaling exacerbated the TGF-ß-induced dysregulation of tight junction proteins, E-cadherin and N-cadherin expression, and STAT3 phosphorylation in MLE-12 epithelial cells. Our findings demonstrate a previously unrecognized role of host IL-6 response in the regulation of lung inflammation during AA and the control of S. pneumoniae bacterial disease. A better understanding of the interactions between lung inflammation and barrier framework could lead to the development of therapies to control asthma inflammation and preserve barrier integrity.

Development of Biodegradable GQDs-hMSNs for Fluorescence Imaging and Dual Cancer Treatment via Photodynamic Therapy and Drug Delivery.

Recently, nano-based cancer therapeutics have been researched and developed, with some nanomaterials showing anticancer properties. When it comes to cancer treatment, graphene quantum dots (GQDs) contain the ability to generate 1O2, a reactive oxidative species (ROS), allowing for the synergistic imaging and photodynamic therapy (PDT) of cancer. However, due to their small particle size, GQDs struggle to remain in the target area for long periods of time in addition to being poor drug carriers. To address this limitation of GQDs, hollow mesoporous silica nanoparticles (hMSNs) have been extensively researched for drug delivery applications. This project investigates the utilization and combination of biomass-derived GQDs and Stöber silica hMSNs to make graphene quantum dots-hollow mesoporous silica nanoparticles (GQDs-hMSNs) for fluorescent imaging and dual treatment of cancer via drug delivery and photodynamic therapy (PDT). Although the addition of hMSNs made the newly synthesized nanoparticles slightly more toxic at higher concentrations, the GQDs-hMSNs displayed excellent drug delivery using fluorescein (FITC) as a mock drug, and PDT treatment by using the GQDs as a photosensitizer (PS). Additionally, the GQDs retained their fluorescence through the surface binding to hMSNs, allowing them to still be used for cell-labeling applications.

Getting to the Heart of Alzheimer Disease.

In a somewhat narrow diagnostic lens, Alzheimer disease (AD) has been considered a brain-specific disease characterized by the presence of Aß (ß-amyloid) plaques and tau neural fibrillary tangles and neural inflammation; these pathologies lead to neuronal death and consequently clinical symptoms, such as memory loss, confusion, and impaired cognitive function. However, for decades, researchers have noticed a link between various cardiovascular abnormalities and AD-such as heart failure, coronary artery disease, atrial fibrillation, and vasculopathy. A considerable volume of work has pointed at this head to heart connection, focusing mainly on associations between cerebral hypoperfusion and neuronal degradation. However, new evidence of a possible systemic or metastatic profile to AD calls for further analysis of this connection. Aß aggregations-biochemically and structurally akin to those found in the typical AD pathology-are now known to be present in the hearts of individuals with idiopathic dilated cardiomyopathy, as well as the hearts of patients with AD. These findings suggest a potential systemic profile of proteinopathies and a new hypothesis for the link between peripheral and central symptoms of heart failure and AD. Herein, we provide an overview of the cardiovascular links to Alzheimer disease.

Liver Bile Acid Changes in Mouse Models of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive impairment. It is hypothesized to develop due to the dysfunction of two major proteins, amyloid-ß (Aß) and microtubule-associated protein, tau. Evidence supports the involvement of cholesterol changes in both the generation and deposition of Aß. This study was performed to better understand the role of liver cholesterol and bile acid metabolism in the pathophysiology of AD. We used male and female wild-type control (C57BL/6J) mice to compare to two well-characterized amyloidosis models of AD, APP/PS1, and AppNL-G-F. Both conjugated and unconjugated primary and secondary bile acids were quantified using UPLC-MS/MS from livers of control and AD mice. We also measured cholesterol and its metabolites and identified changes in levels of proteins associated with bile acid synthesis and signaling. We observed sex differences in liver cholesterol levels accompanied by differences in levels of synthesis intermediates and conjugated and unconjugated liver primary bile acids in both APP/PS1 and AppNL-G-F mice when compared to controls. Our data revealed fundamental deficiencies in cholesterol metabolism and bile acid synthesis in the livers of two different AD mouse lines. These findings strengthen the involvement of liver metabolism in the pathophysiology of AD.

Ablation of amyloid precursor protein increases insulin-degrading enzyme levels and activity in brain and peripheral tissues.

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein widely studied for its role as the source of ß-amyloid peptide, accumulation of which is causal in at least some cases of Alzheimer's disease (AD). APP is expressed ubiquitously and is involved in diverse biological processes. Growing bodies of evidence indicate connections between AD and somatic metabolic disorders related to type 2 diabetes, and App-/- mice show alterations in glycemic regulation. We find that App-/- mice have higher levels of insulin-degrading enzyme (IDE) mRNA, protein, and activity compared with wild-type controls. This regulation of IDE by APP was widespread across numerous tissues, including liver, skeletal muscle, and brain as well as cell types within neural tissue, including neurons, astrocytes, and microglia. RNA interference-mediated knockdown of APP in the SIM-A9 microglia cell line elevated IDE levels. Fasting levels of blood insulin were lower in App-/- than App+/+ mice, but the former showed a larger increase in response to glucose. These low basal levels may enhance peripheral insulin sensitivity, as App-/- mice failed to develop impairment of glucose tolerance on a high-fat, high-sucrose ("Western") diet. Insulin levels and insulin signaling were also lower in the App-/- brain; synaptosomes prepared from App-/- hippocampus showed diminished insulin receptor phosphorylation compared with App+/+ mice when stimulated ex vivo. These findings represent a new molecular link connecting APP to metabolic homeostasis and demonstrate a novel role for APP as an upstream regulator of IDE in vivo.

Inflammatory mechanisms in neurodegeneration.

This review discusses the profound connection between microglia, neuroinflammation, and Alzheimer's disease (AD). Theories have been postulated, tested, and modified over several decades. The findings have further bolstered the belief that microglia-mediated inflammation is both a product and contributor to AD pathology and progression. Distinct microglia phenotypes and their function, microglial recognition and response to protein aggregates in AD, and the overall role of microglia in AD are areas that have received considerable research attention and yielded significant results. The following article provides a historical perspective of microglia, a detailed discussion of multiple microglia phenotypes including dark microglia, and a review of a number of areas where microglia intersect with AD and other pathological neurological processes. The overall breadth of important discoveries achieved in these areas significantly strengthens the hypothesis that neuroinflammation plays a key role in AD. Future determination of the exact mechanisms by which microglia respond to, and attempt to mitigate, protein aggregation in AD may lead to new therapeutic strategies.

Oral sensitization to whey proteins induces age- and sex-dependent behavioral abnormality and neuroinflammatory responses in a mouse model of food allergy: a potential role of mast cells.

BACKGROUND: Growing evidence has strengthened the association of food allergy with neuropsychiatric symptoms such as depression, anxiety, and autism. However, underlying mechanisms by which peripheral allergic responses lead to behavioral dysfunction are yet to be determined. Allergen-activated mast cells may serve as mediators by releasing histamine and other inflammatory factors that could adversely affect brain function. We hypothesized that eliciting food allergy in experimental animals would result in behavioral changes accompanied by mast cell accumulation in the brain. Our hypothesis was tested in a mouse model of milk allergy using bovine milk whey proteins (WP) as the allergen. METHODS: Male and female C57BL/6 mice at 4 weeks (young) and 10 months (old) of age underwent 5-week WP sensitization with weekly intragastric administration of 20 mg WP and 10 µg cholera toxin as an adjuvant. Age-matched sham animals were given the vehicle containing only the adjuvant. All animals were orally challenged with 50 mg WP in week 6 and their intrinsic digging behavior was assessed the next day. Animals were sacrificed 3 days after the challenge, and WP-specific serum IgE, intestinal and brain mast cells, glial activation, and epigenetic DNA modification in the brain were examined. RESULTS: WP-sensitized males showed significantly less digging activity than the sham males in both age groups while no apparent difference was observed in females. Mast cells and their activities were evident in the intestines in an age- and sex-dependent manner. Brain mast cells were predominantly located in the region between the lateral midbrain and medial hippocampus, and their number increased in the WP-sensitized young, but not old, male brains. Noticeable differences in for 5-hydroxymethylcytosine immunoreactivity were observed in WP mice of both age groups in the amygdala, suggesting epigenetic regulation. Increased microglial Iba1 immunoreactivity and perivascular astrocytes hypertrophy were also observed in the WP-sensitized old male mice. CONCLUSIONS: Our results demonstrated that food allergy induced behavioral abnormality, increases in the number of mast cells, epigenetic DNA modification in the brain, microgliosis, and astrocyte hypertrophy in a sex- and age-dependent manner, providing a potential mechanism by which peripheral allergic responses evoke behavioral dysfunction.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease.

UNLABELLED: Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid ß (Aß)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aß was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aß to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aß produced less microgliosis in mAPP(-/-) mice compared with wild-type mice. The mAPP(-/-) mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aß plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT: A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid ß (Aß) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aß stimulation of microglial activation is one source of brain inflammatory changes during disease. Aß is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aß are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aß production to drive the microgliosis associated with AD brains.

The conformational epitope for a new Aß42 protofibril-selective antibody partially overlaps with the peptide N-terminal region.

Aggregation and accumulation of amyloid-ß peptide (Aß) is a key component of Alzheimer's disease (AD). While monomeric Aß appears to be benign, oligomers adopt a biologically detrimental structure. These soluble structures can be detected in AD brain tissue by antibodies that demonstrate selectivity for aggregated Aß. Protofibrils are a subset of soluble oligomeric Aß species and are described as small (< 100 nm) curvilinear assemblies enriched in ß-sheet structure. Our own in vitro studies demonstrate that microglial cells are much more sensitive to soluble Aß42 protofibrils compared to Aß42 monomer or insoluble Aß42 fibrils. Protofibrils interact with microglia, trigger Toll-like receptor signaling, elicit cytokine transcription and expression, and are rapidly taken up by the cells. Because of the importance of this Aß species, we sought to develop an antibody that selectively recognizes protofibrils over other Aß species. Immunization of rabbits with isolated Aß42 protofibrils generated a high-titer anti serum with a strong affinity for Aß42 protofibrils. The antiserum, termed AbSL, was selective for Aß42 protofibrils over Aß42 monomers and Aß42 fibrils. AbSL did not react with amyloid precursor protein and recognized distinct pathological features in AD transgenic mouse brain slices. Competition studies with an Aß antibody that targets residues 1-16 indicated that the conformational epitope for AbSL involved the N-terminal region of protofibrils in some manner. The newly developed antibody may have potential diagnostic and therapeutic uses in AD tissue and patients, and targeting of protofibrils in AD may have beneficial effects. Read the Editorial Highlight for this article on page 621. Cover Image for this issue: doi. 10.1111/jnc.13827.

Activation of neurotensin receptor 1 facilitates neuronal excitability and spatial learning and memory in the entorhinal cortex: beneficial actions in an Alzheimer's disease model.

Neurotensin (NT) is a tridecapeptide distributed in the CNS, including the entorhinal cortex (EC), a structure that is crucial for learning and memory and undergoes the earliest pathological alterations in Alzheimer's disease (AD). Whereas NT has been implicated in modulating cognition, the cellular and molecular mechanisms by which NT modifies cognitive processes and the potential therapeutic roles of NT in AD have not been determined. Here we examined the effects of NT on neuronal excitability and spatial learning in the EC, which expresses high density of NT receptors. Brief application of NT induced persistent increases in action potential firing frequency, which could last for at least 1 h. NT-induced facilitation of neuronal excitability was mediated by downregulation of TREK-2 K(+) channels and required the functions of NTS1, phospholipase C, and protein kinase C. Microinjection of NT or NTS1 agonist, PD149163, into the EC increased spatial learning as assessed by the Barnes Maze Test. Activation of NTS1 receptors also induced persistent increases in action potential firing frequency and significantly improved the memory status in APP/PS1 mice, an animal model of AD. Our study identifies a cellular substrate underlying learning and memory and suggests that NTS1 agonists may exert beneficial actions in an animal model of AD.

Are GLP-1 receptor agonists useful against traumatic brain injury?

This Editorial highlights a study by Li et al. (2015) in the current issue of J. Neurochem. The image depicts the hypothesized neuroprotective pathway that is proposed by the authors. Using a combination of SH-SY5Y and primary rat neuron cultures the GLP-1R agonist, Liraglutide, was shown to increase SH-SY5Y proliferation and CREB phosphorylation correlating with reduced toxicity, preservation of Bcl2 protein levels, and decreased caspase 3 activity following glutamate or H2 O2 stimulations. These in vitro observations correlated with a Liraglutide-dependent improvement in memory performance in mice subjected to a mild TBI. Bcl2, B-cell lymphoma 2; CREB, cAMP-response element binding protein; GLP-1R, glucagon-like peptide 1 receptor; TBI, traumatic brain injury; PKA, protein kinase A.

Attenuation of microglial activation in a mouse model of Alzheimer's disease via NFAT inhibition.

BACKGROUND: Amyloid ß (Aß) peptide is hypothesized to stimulate microglia to acquire their characteristic proinflammatory phenotype in Alzheimer's disease (AD) brains. The specific mechanisms by which Aß leads to microglial activation remain an area of interest for identifying attractive molecular targets for intervention. Based upon the fact that microglia express the proinflammatory transcription factor, nuclear factor of activated T cells (NFAT), we hypothesized that NFAT activity is required for the Aß-stimulated microgliosis that occurs during disease. METHODS: Primary murine microglia cultures were stimulated with Aß in the absence or presence of NFAT inhibitors, FK506 and tat-VIVIT peptide, to quantify secretion of cytokines, neurotoxins, or Aß phagocytosis. A transgenic mouse model of AD, APP/PS1, was treated subcutaneously via mini-osmotic pumps with FK506 or tat-VIVIT to quantify effects on cytokines, microgliosis, plaque load, and memory. RESULTS: Expression of various NFAT isoforms was verified in primary murine microglia through Western blot analysis. Microglial cultures were stimulated with Aß fibrils in the absence or presence of the NFAT inhibitors, FK506 and tat-VIVIT, to demonstrate that NFAT activity regulated Aß phagocytosis, neurotoxin secretion, and cytokine secretion. Delivery of FK506 and tat-VIVIT to transgenic APP/PS1 mice attenuated spleen but not brain cytokine levels. However, FK506 and tat-VIVIT significantly attenuated both microgliosis and Aß plaque load in treated mice compared to controls. Surprisingly, this did not correlate with changes in memory performance via T-maze testing. CONCLUSIONS: Our findings suggest that development of specific NFAT inhibitors may offer promise as an effective strategy for attenuating the microgliosis and Aß plaque deposition that occur in AD.

Brain changes associated with thromboxane receptor antagonist SQ 29,548 treatment in a mouse model.

The purpose of this study was to characterize behavioral and physiological effects of a selective thromboxane (TP) receptor antagonist, SQ 29,548, in the C57Bl/6 mouse model. At 6 months of age, male mice were given either sham or drug i.p. injections for 3 days at a dose of 2 mg/kg each day. On the day after the final injection, mice were subjected to behavioral testing before brain collection. Left hemisphere hippocampi were collected from all mice for protein analysis via Western blot. Right brain hemispheres were fixed and embedded in gelatin and then serially sectioned. The sections were immunostained with anti-c-Fos antibodies. Prostaglandin analysis was performed from remaining homogenized brain samples, minus the hippocampi. Injection of SQ 29,548 decreased selective brain prostaglandin levels compared with sham controls. This correlated with robust increases in limbic-region c-Fos immunoreactivity in the SQ 29,548-injected mice. However, drug-treated mice demonstrated no significant changes in relevant hippocampal protein levels compared with sham treatments, as determined from Western blots. Surprisingly, injection of SQ 29,548 caused mixed changes in parameters of depression and anxiety-like behavior in the mice. In conclusion, the results indicate that administration of peripheral TP receptor antagonists alters brain levels of prostanoids and influences neuronal activity, with only minimal alterations of behavior. Whether the drug affects neurons directly or through a secondary pathway involving endothelium or other tissues remains unclear.

Pomegranate polyphenols and extract inhibit nuclear factor of activated T-cell activity and microglial activation in vitro and in a transgenic mouse model of Alzheimer disease.

Alzheimer disease (AD) brain is characterized by extracellular plaques of amyloid ß (Aß) peptide with reactive microglia. This study aimed to determine whether a dietary intervention could attenuate microgliosis. Memory was assessed in 12-mo-old male amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice via Barnes maze testing followed by division into either a control-fed group provided free access to normal chow and water or a treatment group provided free access to normal chow and drinking water supplemented with pomegranate extract (6.25 mL/L) for 3 mo followed by repeat Barnes maze testing for both groups. Three months of pomegranate feeding decreased the path length to escape of mice compared with their initial 12-mo values (P < 0.05) and their control-fed counterparts (P < 0.05). Brains of the 3-mo study pomegranate-fed mice had lower tumor necrosis factor α (TNF-α) concentrations (P < 0.05) and lower nuclear factor of activated T-cell (NFAT) transcriptional activity (P < 0.05) compared with controls. Brains of the 3-mo pomegranate or control mice were also compared with an additional control group of 12-mo-old mice for histologic analysis. Immunocytochemistry showed that pomegranate- but not control-fed mice had attenuated microgliosis (P < 0.05) and Aß plaque deposition (P < 0.05) compared with 12-mo-old mice. An additional behavioral study again used 12-mo-old male APP/PS1 mice tested by T-maze followed by division into a control group provided with free access to normal chow and sugar supplemented drinking water or a treatment group provided with normal chow and pomegranate extract-supplemented drinking water (6.25 mL/L) for 1 mo followed by repeat T-maze testing in both groups. One month of pomegranate feeding increased spontaneous alternations versus control-fed mice (P < 0.05). Cell culture experiments verified that 2 polyphenol components of pomegranate extract, punicalagin and ellagic acid, attenuated NFAT activity in a reporter cell line (P < 0.05) and decreased Aß-stimulated TNF-α secretion by murine microglia (P < 0.05). These data indicate that dietary pomegranate produces brain antiinflammatory effects that may attenuate AD progression.

Concurrent Brain Subregion Microgliosis in an HLA-II Mouse Model of Group A Streptococcal Skin Infection.

The broad range of clinical manifestations and life-threatening infections caused by the Gram-positive bacterium, Streptococcus pyogenes or Group A Streptococcus (GAS), remains a significant concern to public health, with a subset of individuals developing neurological complications. Here, we examined the concurrent neuroimmune effects of subcutaneous GAS infections in an HLA-Class II (HLA) transgenic mouse model of subcutaneous GAS infection. To investigate changes in the skin-brain axis, HLA-DQ8 (DQA1*0301/DQB1*0302) mice (DQ8) were randomly divided into three groups: uninfected controls (No Inf), GAS infected and untreated (No Tx), and GAS infected with a resolution by clindamycin (CLN) treatment (CLN Tx) (10 mg/kg/5 days) and were monitored for 16 days post-infection. While the skin GAS burden was significantly reduced by CLN, the cortical and hippocampal GAS burden in the male DQ8 mice was not significantly reduced with CLN. Immunoreactivity to anti-GAS antibody revealed the presence of GAS bacteria in the vicinity of the neuronal nucleus in the neocortex of both No Tx and CLN Tx male DQ8 mice. GAS infection-mediated cortical cytokine changes were modest; however, compared to No Inf or No Tx groups, a significant increase in IL-2, IL-13, IL-22, and IL-10 levels was observed in CLN Tx females despite the lack of GAS burden. Western blot analysis of cortical and hippocampal homogenates showed significantly higher ionized calcium-binding adaptor-1 (Iba-1, microglia marker) protein levels in No Tx females and males and CLN Tx males compared to the No Inf group. Immunohistochemical analysis showed that Iba-1 immunoreactivity in the hippocampal CA3 and CA1 subregions was significantly higher in the CLN Tx males compared to the No Tx group. Our data support the possibility that the subcutaneous GAS infection communicates to the brain and is characterized by intraneuronal GAS sequestration, brain cytokine changes, Iba-1 protein levels, and concurrent CA3 and CA1 subregion-specific microgliosis, even without bacteremia.

HLA-II-Dependent Neuroimmune Changes in Group A Streptococcal Necrotizing Fasciitis.

Streptococcus pyogenes (Group A Streptococcus, GAS) bacteria cause a spectrum of human diseases ranging from self-limiting pharyngitis and mild, uncomplicated skin infections (impetigo, erysipelas, and cellulitis) to highly morbid and rapidly invasive, life-threatening infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (NF). HLA class II allelic polymorphisms are linked with differential outcomes and severity of GAS infections. The dysregulated immune response and peripheral cytokine storm elicited due to invasive GAS infections increase the risk for toxic shock and multiple organ failure in genetically susceptible individuals. We hypothesized that, while the host immune mediators regulate the immune responses against peripheral GAS infections, these interactions may simultaneously trigger neuropathology and, in some cases, induce persistent alterations in the glial phenotypes. Here, we studied the consequences of peripheral GAS skin infection on the brain in an HLA-II transgenic mouse model of GAS NF with and without treatment with an antibiotic, clindamycin (CLN). Mice expressing the human HLA-II DR3 (DR3) or the HLA-II DR4 (DR4) allele were divided into three groups: (i) uninfected controls, (ii) subcutaneously infected with a clinical GAS strain isolated from a patient with GAS NF, and (iii) GAS-infected with CLN treatment (10 mg/kg/5 days, intraperitoneal). The groups were monitored for 15 days post-infection. Skin GAS burden and lesion area, splenic and hippocampal mRNA levels of inflammatory markers, and immunohistochemical changes in hippocampal GFAP and Iba-1 immunoreactivity were assessed. Skin GAS burden and hippocampal mRNA levels of the inflammatory markers S100A8/A9, IL-1ß, IL-33, inflammasome-related caspase-1 (Casp1), and NLRP6 were elevated in infected DR3 but not DR4 mice. The levels of these markers were significantly reduced following CLN treatment in DR3 mice. Although GAS was not detectable in the brain, astrocyte (GFAP) and microglia (Iba-1) activation were evident from increased GFAP and Iba-1 mRNA levels in DR3 and DR4 mice. However, CLN treatment significantly reduced GFAP mRNA levels in DR3 mice, not DR4 mice. Our data suggest a skin-brain axis during GAS NF, demonstrating that peripherally induced pathological conditions regulate neuroimmune changes and gliotic events in the brain.

Streamlined synthesis of potential dual-emissive fluorescent silicon quantum dots (SiQDs) for cell imaging.

One of the current challenges of working with nanomaterials in bioapplications is having a tool that is biocompatible (non-toxic) and produces stable, intense fluorescence for bioimaging. To address these challenges, we have developed a streamlined and one-pot synthetic route for silicon-based quantum dots (SiQDs) using a hydrothermal method. Part of our unique approach for designing the SiQDs was to incorporate (3-aminopropyl) triethoxysilane (APTES), which is an amphipathic molecule with hydroxyl and amine functional groups available for modification. In order to reduce the toxicity of APTES, we chose glucose as a reducing agent for the reaction. The resulting SiQDs produced potent, stable, potential dual-emissive fluorescence emission peaks in the visible and near-infrared (NIR) ranges. Both peaks could be used as distinguishing fluorescence signals for bioimaging, separately or in combination. The physical and optical properties of the SiQDs were determined under a range of environmental conditions. The morphology, surface composition, and electronic structure of the SiQDs were characterized using high resolution-transmission electronic microscopy (HR-TEM), energy dispersive X-ray spectroscopy (EDS), Fourier-transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). The stability of the SiQDs was evaluated under a wide range of pHs. The biocompatibility and imaging potential of the SiQDs were tested in microvascular endothelial cells (MVEC), neural stem cells (NSC), and RAW 264.7 macrophage cells. The images obtained revealed different subcellular localizations, particularly during cell division, with distinct fluorescence intensities. The results demonstrated that SiQDs are a promising, non-toxic labeling tool for a variety of cell types, with the added advantage of having dual emission peaks both in visible and NIR ranges for bioimaging.

Acetate reduces microglia inflammatory signaling in vitro.

Acetate supplementation increases brain acetyl-CoA and histone acetylation and reduces lipopolysaccharide (LPS)-induced neuroglial activation and interleukin (IL)-1ß expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen-activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor-kappa B (NF-κB) in primary and BV-2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS-induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL-6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor-ß1 (TGF-ß1) and both IL-4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2-4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS-induced elevation of NF-κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non-histone protein acetylation.

Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer's disease.

BACKGROUND: Microglial activation is an important histologic characteristic of the pathology of Alzheimer's disease (AD). One hypothesis is that amyloid beta (Aß) peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aß stimulation of microglia may prove to be an important therapeutic strategy for AD. METHODS: Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aß1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aß stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aß plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis. RESULTS: Aß fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aß plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals. CONCLUSIONS: These data suggest that amyloid dependent microgliosis may be Src kinase dependent in vitro and in vivo. This study defines a role for Src kinase in the microgliosis characteristic of diseased brains and suggests that particular tyrosine kinase inhibition may be a valid anti-inflammatory approach to disease. Dasatinib is an FDA-approved drug for treating chronic myeloid leukemia cancer with a reported ability to cross the blood-brain barrier. Therefore, this suggests a novel use for this drug as well as similar acting molecules.