Is Multiple System Atrophy a Prion-like Disorder?

Multiple system atrophy (MSA) is a rapidly progressive, fatal neurodegenerative disease of uncertain aetiology that belongs to the family of α-synucleinopathies. It clinically presents with parkinsonism, cerebellar, autonomic, and motor impairment in variable combinations. Pathological hallmarks are fibrillary α-synuclein (αSyn)-rich glial cytoplasmic inclusions (GCIs) mainly involving oligodendroglia and to a lesser extent neurons, inducing a multisystem neurodegeneration, glial activation, and widespread demyelinization. The neuronal αSyn pathology of MSA has molecular properties different from Lewy bodies in Parkinson’s disease (PD), both of which could serve as a pool of αSyn (prion) seeds that could initiate and drive the pathogenesis of synucleinopathies. The molecular cascade leading to the “prion-like” transfer of “strains” of aggregated αSyn contributing to the progression of the disease is poorly understood, while some presented evidence that MSA is a prion disease. However, this hypothesis is difficult to reconcile with postmortem analysis of human brains and the fact that MSA-like pathology was induced by intracerebral inoculation of human MSA brain homogenates only in homozygous mutant 53T mice, without production of disease-specific GCIs, or with replication of MSA prions in primary astrocyte cultures from transgenic mice expressing human αSyn. Whereas recent intrastriatal injection of Lewy body-derived or synthetic human αSyn fibrils induced PD-like pathology including neuronal αSyn aggregates in macaques, no such transmission of αSyn pathology in non-human primates by MSA brain lysate has been reported until now. Given the similarities between αSyn and prions, there is a considerable debate whether they should be referred to as “prions”, “prion-like”, “prionoids”, or something else. Here, the findings supporting the proposed nature of αSyn as a prion and its self-propagation through seeding as well as the transmissibility of neurodegenerative disorders are discussed. The proof of disease causation rests on the concordance of scientific evidence, none of which has provided convincing evidence for the classification of MSA as a prion disease or its human transmission until now.     

In Vitro Assays to Identify Metabolism-Disrupting Chemicals with Diabetogenic Activity in a Human Pancreatic β-Cell Model

There is a need to develop identification tests for Metabolism Disrupting Chemicals (MDCs) with diabetogenic activity. Here we used the human EndoC-βH1 β-cell line, the rat β-cell line INS-1E and dispersed mouse islet cells to assess the effects of endocrine disruptors on cell viability and glucose-stimulated insulin secretion (GSIS). We tested six chemicals at concentrations within human exposure (from 0.1 pM to 1 µM). Bisphenol-A (BPA) and tributyltin (TBT) were used as controls while four other chemicals, namely perfluorooctanoic acid (PFOA), triphenylphosphate (TPP), triclosan (TCS) and dichlorodiphenyldichloroethylene (DDE), were used as “unknowns”. Regarding cell viability, BPA and TBT increased cell death as previously observed. Their mode of action involved the activation of estrogen receptors and PPARγ, respectively. ROS production was a consistent key event in BPA-and TBT-treated cells. None of the other MDCs tested modified viability or ROS production. Concerning GSIS, TBT increased insulin secretion while BPA produced no effects. PFOA decreased GSIS, suggesting that this chemical could be a “new” diabetogenic agent. Our results indicate that the EndoC-βH1 cell line is a suitable human β-cell model for testing diabetogenic MDCs. Optimization of the test methods proposed here could be incorporated into a set of protocols for the identification of MDCs.     

Secondary Metabolic Profile as a Tool for Distinction and Characterization of Cultivars of Black Pepper (Piper nigrum L.) Cultivated in Pará State,Brazil

This study evaluated the chemical compositions of the leaves and fruits of eight black pepper cultivars cultivated in Pará State (Amazon, Brazil). Hydrodistillation and gas chromatography–mass spectrometry were employed to extract and analyze the volatile compounds, respectively. Sesquiterpene hydrocarbons were predominant (58.5–90.9%) in the cultivars “Cingapura”, “Equador”, “Guajarina”, “Iaçará”, and “Kottanadan”, and “Bragantina”, “Clonada”, and “Uthirankota” displayed oxygenated sesquiterpenoids (50.6–75.0%). The multivariate statistical analysis applied using volatile composition grouped the samples into four groups: γ-Elemene, curzerene, and δ-elemene (“Equador”/“Guajarina”, I); δ-elemene (“Iaçará”/“Kottanadan”/“Cingapura”, II); elemol (“Clonada”/“Uthirankota”, III) and α-muurolol, bicyclogermacrene, and cubebol (“Bragantina”, IV). The major compounds in all fruit samples were monoterpene hydrocarbons such as α-pinene, β-pinene, and limonene. Among the cultivar leaves, phenolics content (44.75–140.53 mg GAE·g⁻¹ FW), the enzymatic activity of phenylalanine-ammonia lyase (20.19–57.22 µU·mL⁻¹), and carotenoids (0.21–2.31 µg·mL⁻¹) displayed significant variations. Due to black pepper’s susceptibility to Fusarium infection, a molecular docking analysis was carried out on Fusarium protein targets using each cultivar’s volatile components. F. oxysporum endoglucanase was identified as the preferential protein target of the compounds. These results can be used to identify chemical markers related to the susceptibility degree of black pepper cultivars to plant diseases prevalent in Pará State.     

Oxidative Stress in Diabetes: Implications for Vascular and Other Complications

In recent decades, oxidative stress has become a focus of interest in most biomedical disciplines and many types of clinical research. Increasing evidence shows that oxidative stress is associated with the pathogenesis of diabetes, obesity, cancer, ageing, inflammation, neurodegenerative disorders, hypertension, apoptosis, cardiovascular diseases, and heart failure. Based on these studies, an emerging concept is that oxidative stress is the “final common pathway” through which the risk factors for several diseases exert their deleterious effects. Oxidative stress causes a complex dysregulation of cell metabolism and cell–cell homeostasis; in particular, oxidative stress plays a key role in the pathogenesis of insulin resistance and β-cell dysfunction. These are the two most relevant mechanisms in the pathophysiology of type 2 diabetes and its vascular complications, the leading cause of death in diabetic patients.     

Convergence: Lactosylceramide-Centric Signaling Pathways Induce Inflammation,Oxidative Stress,and Other Phenotypic Outcomes

Lactosylceramide (LacCer), also known as CD17/CDw17, is a member of a large family of small molecular weight compounds known as glycosphingolipids. It plays a pivotal role in the biosynthesis of glycosphingolipids, primarily by way of serving as a precursor to the majority of its higher homolog sub-families such as gangliosides, sulfatides, fucosylated-glycosphingolipids and complex neutral glycosphingolipids—some of which confer “second-messenger” and receptor functions. LacCer is an integral component of the “lipid rafts,” serving as a conduit to transduce external stimuli into multiple phenotypes, which may contribute to mortality and morbidity in man and in mouse models of human disease. LacCer is synthesized by the action of LacCer synthase (β-1,4 galactosyltransferase), which transfers galactose from uridine diphosphate galactose (UDP-galactose) to glucosylceramide (GlcCer). The convergence of multiple physiologically relevant external stimuli/agonists—platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), stress, cigarette smoke/nicotine, tumor necrosis factor-α (TNF-α), and in particular, oxidized low-density lipoprotein (ox-LDL)—on β-1,4 galactosyltransferase results in its phosphorylation or activation, via a “turn-key” reaction, generating LacCer. This newly synthesized LacCer activates NADPH (nicotinamide adenine dihydrogen phosphate) oxidase to generate reactive oxygen species (ROS) and a highly “oxidative stress” environment, which trigger a cascade of signaling molecules and pathways and initiate diverse phenotypes like inflammation and atherosclerosis. For instance, LacCer activates an enzyme, cytosolic phospholipase A2 (cPLA2), which cleaves arachidonic acid from phosphatidylcholine. In turn, arachidonic acid serves as a precursor to eicosanoids and prostaglandin, which transduce a cascade of reactions leading to inflammation—a major phenotype underscoring the initiation and progression of several debilitating diseases such as atherosclerosis and cancer. Our aim here is to present an updated account of studies made in the field of LacCer metabolism and signaling using multiple animal models of human disease, human tissue, and cell-based studies. These advancements have led us to propose that previously unrelated phenotypes converge in a LacCer-centric manner. This LacCer synthase/LacCer-induced “oxidative stress” environment contributes to inflammation, atherosclerosis, skin conditions, hair greying, cardiovascular disease, and diabetes due to mitochondrial dysfunction. Thus, targeting LacCer synthase may well be the answer to remedy these pathologies.     

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8β1 and α11β1, Induced Specifically on Activated Stellate Cells

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvβ1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFβ activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, “disease specificity” has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbβ3 on platelets, α4β1, α4β7 and αLβ2 on leukocytes. Herein, “disease specific” integrins would serve as attractive targets. α8β1 and α11β1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather “pathology specific” nature of these new integrins. The monoclonal antibodies against α8β1 and α11β1 in preclinical examinations may illuminate the road to the first medical agents.     

Glucocorticoid Receptor β (GRβ): Beyond Its Dominant-Negative Function

Glucocorticoids (GCs) act via the GC receptor (GR), a receptor ubiquitously expressed in the body where it drives a broad spectrum of responses within distinct cell types and tissues, which vary in strength and specificity. The variability of GR-mediated cell responses is further extended by the existence of GR isoforms, such as GRα and GRβ, generated through alternative splicing mechanisms. While GRα is the classic receptor responsible for GC actions, GRβ has been implicated in the impairment of GRα-mediated activities. Interestingly, in contrast to the popular belief that GRβ actions are restricted to its dominant-negative effects on GRα-mediated responses, GRβ has been shown to have intrinsic activities and “directly” regulates a plethora of genes related to inflammatory process, cell communication, migration, and malignancy, each in a GRα-independent manner. Furthermore, GRβ has been associated with increased cell migration, growth, and reduced sensitivity to GC-induced apoptosis. We will summarize the current knowledge of GRβ-mediated responses, with a focus on the GRα-independent/intrinsic effects of GRβ and the associated non-canonical signaling pathways. Where appropriate, potential links to airway inflammatory diseases will be highlighted.     

Effects of Different Basal Cell Culture Media upon the Osteogenic Response of hMSCs Evaluated by 99mTc-HDP Labeling

The osteogenic differentiation of mesenchymal stem cells is now a standard procedure in modern bone tissue engineering. As this is a promising field for future clinical applications, many cell culture media exist to promote osteogenic differentiation. Prior to differentiation, cells must be expanded to obtain sufficient numbers for experiments. Little evidence is available regarding the optimal media combination for expansion and differentiation to maximize the osteogenic response. Therefore, human BM-MSCs (n = 6) were expanded in parallel in DMEM (Dulbecco’s Modified Eagle Medium) LG (Low Glucose) and α-MEM (Minimum Essential Media alpha-modification), followed by simultaneous monolayer differentiation toward the osteogenic lineage in: 1. DMEM LG (Low Glucose), 2. DMEM HG (High Glucose), 3. α-MEM, 4. “Bernese medium”, and 5. “Verfaillie medium”, with a corresponding negative control (total 20 groups). As a marker for osteogenic differentiation, hydroxyapatite was accessed using radioactive ^99mTc-HDP labeling and quantitative alizarin red staining. The results indicate that all media except “Bernese medium” are suitable for osteogenic differentiation, while there was evidence that DMEM LG is partly superior when used for expansion and differentiation of BM-hMSCs. Using “Verfaillie medium” after DMEM LG expansion led to the highest grade of osteogenic differentiation. Nevertheless, the difference was not significant. Therefore, we recommend using DMEM LG for robust osteogenic differentiation, as it is highly suitable for that purpose, economical compared to other media, and requires little preparation time.     

Changes in the Number of Double-Strand DNA Breaks in Chinese Hamster V79 Cells Exposed to γ-Radiation with Different Dose Rates

A comparative investigation of the induction of double-strand DNA breaks (DSBs) in the Chinese hamster V79 cells by γ-radiation at dose rates of 1, 10 and 400 mGy/min (doses ranged from 0.36 to 4.32 Gy) was performed. The acute radiation exposure at a dose rate of 400 mGy/min resulted in the linear dose-dependent increase of the γ-H2AX foci formation. The dose-response curve for the acute exposure was well described by a linear function y = 1.22 + 19.7x, where “y” is an average number of γ-H2AX foci per a cell and “x” is the absorbed dose (Gy). The dose rate reduction down to 10 mGy/min lead to a decreased number of γ-H2AX foci, as well as to a change of the dose-response relationship. Thus, the foci number up to 1.44 Gy increased and reached the “plateau” area between 1.44 and 4.32 Gy. There was only a slight increase of the γ-H2AX foci number (up to 7) in cells after the protracted exposure (up to 72 h) to ionizing radiation at a dose rate of 1 mGy/min. Similar effects of the varying dose rates were obtained when DNA damage was assessed using the comet assay. In general, our results show that the reduction of the radiation dose rate resulted in a significant decrease of DSBs per cell per an absorbed dose.     

Role of Peptides in Diagnostics

The specificity of a diagnostic assay depends upon the purity of the biomolecules used as a probe. To get specific and accurate information of a disease, the use of synthetic peptides in diagnostics have increased in the last few decades, because of their high purity profile and ability to get modified chemically. The discovered peptide probes are used either in imaging diagnostics or in non-imaging diagnostics. In non-imaging diagnostics, techniques such as Enzyme-Linked Immunosorbent Assay (ELISA), lateral flow devices (i.e., point-of-care testing), or microarray or LC-MS/MS are used for direct analysis of biofluids. Among all, peptide-based ELISA is considered to be the most preferred technology platform. Similarly, peptides can also be used as probes for imaging techniques, such as single-photon emission computed tomography (SPECT) and positron emission tomography (PET). The role of radiolabeled peptides, such as somatostatin receptors, interleukin 2 receptor, prostate specific membrane antigen, αβ3 integrin receptor, gastrin-releasing peptide, chemokine receptor 4, and urokinase-type plasminogen receptor, are well established tools for targeted molecular imaging ortumor receptor imaging. Low molecular weight peptides allow a rapid clearance from the blood and result in favorable target-to-non-target ratios. It also displays a good tissue penetration and non-immunogenicity. The only drawback of using peptides is their potential low metabolic stability. In this review article, we have discussed and evaluated the role of peptides in imaging and non-imaging diagnostics. The most popular non-imaging and imaging diagnostic platforms are discussed, categorized, and ranked, as per their scientific contribution on PUBMED. Moreover, the applicability of peptide-based diagnostics in deadly diseases, mainly COVID-19 and cancer, is also discussed in detail.     

Endothelial Dysfunction: An Intermediate Clinical Feature between Urolithiasis and Cardiovascular Diseases

An epidemiological relationship between urolithiasis and cardiovascular diseases has extensively been reported. Endothelial dysfunction is an early pathogenic event in cardiovascular diseases and has been associated with oxidative stress and low chronic inflammation in hypertension, coronary heart disease, stroke or the vascular complications of diabetes and obesity. The aim of this study is to summarize the current knowledge about the pathogenic mechanisms of urolithiasis in relation to the development of endothelial dysfunction and cardiovascular morbidities. Methods: A non-systematic review has been performed mixing the terms “urolithiasis”, “kidney stone” or “nephrolithiasis” with “cardiovascular disease”, “myocardial infarction”, “stroke”, or “endothelial dysfunction”. Results: Patients with nephrolithiasis develop a higher incidence of cardiovascular disease with a relative risk estimated between 1.20 and 1.24 and also develop a higher vascular disease risk scores. Analyses of subgroups have rendered inconclusive results regarding gender or age. Endothelial dysfunction has also been strongly associated with urolithiasis in clinical studies, although no systemic serum markers of endothelial dysfunction, inflammation or oxidative stress could be clearly related. Analysis of urine composition of lithiasic patients also detected a higher expression of proteins related to cardiovascular disease. Experimental models of hyperoxaluria have also found elevation of serum endothelial dysfunction markers. Conclusions: Endothelial dysfunction has been strongly associated with urolithiasis and based on the experimental evidence, should be considered as an intermediate and changeable feature between urolithiasis and cardiovascular diseases. Oxidative stress, a key pathogenic factor in the development of endothelial dysfunction has been also pointed out as an important factor of lithogenesis. Special attention must be paid to cardiovascular morbidities associated with urolithiasis in order to take advantage of pleiotropic effects of statins, angiotensin receptor blockers and allopurinol.     

The RARγ Oncogene: An Achilles Heel for Some Cancers

Cancer “stem cells” (CSCs) sustain the hierarchies of dividing cells that characterize cancer. The main causes of cancer-related mortality are metastatic disease and relapse, both of which originate primarily from CSCs, so their eradication may provide a bona fide curative strategy, though there maybe also the need to kill the bulk cancer cells. While classic anti-cancer chemotherapy is effective against the dividing progeny of CSCs, non-dividing or quiescent CSCs are often spared. Improved anti-cancer therapies therefore require approaches that target non-dividing CSCs, which must be underpinned by a better understanding of factors that permit these cells to maintain a stem cell-like state. During hematopoiesis, retinoic acid receptor (RAR) γ is selectively expressed by stem cells and their immediate progeny. It is overexpressed in, and is an oncogene for, many cancers including colorectal, renal and hepatocellular carcinoma, cholangiocarcinomas and some cases of acute myeloid leukemia that harbor RARγ fusion proteins. In vitro studies suggest that RARγ-selective and pan-RAR antagonists provoke the death of CSCs by necroptosis and point to antagonism of RARγ as a potential strategy to treat metastatic disease and relapse, and perhaps provide a cure for some cancers.     

Biochemical and Cellular Characterization of New Radio-Resistant Cell Lines Reveals a Role of Natural Flavonoids to Bypass Senescence

Cancer is one of the main causes of death worldwide, and, among the most frequent cancer types, osteosarcoma accounts for 56% of bone neoplasms observed in children and colorectal cancer for 10.2% of tumors diagnosed in the adult population. A common and frequent hurdle in cancer treatment is the emergence of resistance to chemo- and radiotherapy whose biological causes are largely unknown. In the present work, human osteosarcoma (SAOS) and colorectal adenocarcinoma (HT29) cell lines were γ-irradiated at doses mimicking the sub-lethal irradiation in clinical settings to obtain two radio-resistant cellular sub-populations named SAOS400 and HT500, respectively. Since “therapy-induced senescence” (TIS) is often associated with tumor response to radiotherapy in cancer cells, we measured specific cellular and biochemical markers of senescence in SAOS400 and HT500 cells. In detail, both cell lines were characterized by a higher level of expression of cyclin-dependent kinase inhibitors p16^INK4 and p21^CIP1 and increased positivity to SAβ-gal (senescence-associated β-galactosidase) with respect to parental cells. Moreover, the intracellular levels of reactive oxygen species in the resistant cells were significantly lower compared to the parental counterparts. Subsequently, we demonstrated that senolytic agents were able to sensitize SAOS400 and HT500 to cell death induced by γ-irradiation. Employing two natural flavonoids, fisetin and quercetin, and a BH3-mimetic, ABT-263/navitoclax, we observed that their association with γ-irradiation significantly reduced the expression of p16^INK4, p21^CIP1 and synergistically (combination index < 1) increased cell death compared to radiation mono-alone treatments. The present results reinforce the potential role of senolytics as adjuvant agents in cancer therapy.     

Investigating Immune Responses to the scAAV9-HEXM Gene Therapy Treatment in Tay–Sachs Disease and Sandhoff Disease Mouse Models

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme β-hexosaminidase A (HexA). HexA consists of an α- and β-subunit; a deficiency in either subunit results in Tay–Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and “non-self”-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed “non-self” proteins, and potentially improve treatment efficacy.     

A Cell-Based Systematic Review on the Role of Annexin A1 in Triple-Negative Breast Cancers

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is often associated with a poorer prognosis and does not respond to hormonal therapy. Increasing evidence highlights the exploitability of Annexin A1 (AnxA1), a calcium dependent protein, as a precision medicine for TNBC. To systematically summarize the role of AnxA1 and its associated mechanisms in TNBC, we performed data mining using three main databases: PubMed, Scopus, and Ovid/Medline. The papers retrieved were based on two different sets of key words such as “Annexin A1” or “Lipocortin 1” and “Breast cancer” or “TNBC”. A total of 388 articles were identified, with 210 chosen for comprehensive screening and 13 papers that met inclusion criteria were included. Current evidence from cell culture studies showed that AnxA1 expression is correlated with NF-κB, which promotes migration by activating ERK phosphorylation. AnxaA1 also activates TGF-β signaling which upregulates MMP-9 and miR196a expression to enhance epithelial-mesenchymal transition and migratory capacity of TNBC cells. AnxA1 can steer the macrophage polarization toward the M2 phenotype to create a pro-tumor immune environment. Existing research suggests a potential role of AnxA1 in the metastasis and immune landscape of TNBC tumors. Preclinical and clinical experiments are warranted to investigate the feasibility and effectiveness of targeting AnxA1 in TNBC.     

[68Ga]Ga-DFO-c(RGDyK): Synthesis and Evaluation of Its Potential for Tumor Imaging in Mice

Angiogenesis has a pivotal role in tumor growth and the metastatic process. Molecular imaging was shown to be useful for imaging of tumor-induced angiogenesis. A great variety of radiolabeled peptides have been developed to target αvβ3 integrin, a target structure involved in the tumor-induced angiogenic process. The presented study aimed to synthesize deferoxamine (DFO)-based c(RGD) peptide conjugate for radiolabeling with gallium-68 and perform its basic preclinical characterization including testing of its tumor-imaging potential. DFO-c(RGDyK) was labeled with gallium-68 with high radiochemical purity. In vitro characterization including stability, partition coefficient, protein binding determination, tumor cell uptake assays, and ex vivo biodistribution as well as PET/CT imaging was performed. [⁶⁸Ga]Ga-DFO-c(RGDyK) showed hydrophilic properties, high stability in PBS and human serum, and specific uptake in U-87 MG and M21 tumor cell lines in vitro and in vivo. We have shown here that [⁶⁸Ga]Ga-DFO-c(RGDyK) can be used for αvβ3 integrin targeting, allowing imaging of tumor-induced angiogenesis by positron emission tomography.     

Immunohistochemical Study of ASC Expression and Distribution in the Hippocampus of an Aged Murine Model of Alzheimer’s Disease

Neuroinflammation is involved in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD), and is notably dependent on age. One important inflammatory pathway exerted by innate immune cells of the nervous system in response to danger signals is mediated by inflammasomes (IF) and leads to the generation of potent pro-inflammatory cytokines. The protein “apoptosis-associated speck-like protein containing a caspase recruitment domain” (ASC) modulates IF activation but has also other functions which are crucial in AD. We intended to characterize immunohistochemically ASC and pattern recognition receptors (PRR) of IF in the hippocampus (HP) of the transgenic mouse model Tg2576 (APP), in which amyloid-beta (Aβ) pathology is directly dependent on age. We show in old-aged APP a significant amount of ASC in microglia and astrocytes associated withAβ plaques, in the absence of PRR described by others in glial cells. In addition, APP developed foci with clusters of extracellular ASC granules not spatiallyrelated to Aβ plaques, which density correlated with the advanced age of mice and AD development. Clusters were associated withspecific astrocytes characterized by their enlarged ring-shaped process terminals, ASC content, and frequent perivascular location. Their possible implication in ASC clearance and propagation of inflammation is discussed.     

Early Gβγ-GRK2 Inhibition Ameliorates Osteoarthritis Development by Simultaneous Anti-Inflammatory and Chondroprotective Effects

The G-protein-coupled receptor kinase 2 (GRK2) is an important regulator of inflammation and pathological macrophage phenotype in a variety of diseases. We hypothesize that Gβγ-GRK2 signaling promotes the early inflammatory response and chondrocyte loss in osteoarthritis (OA). Using the destabilization of the medial meniscus (DMM) model in 12-week-old male C57BL/6 mice, we determined the role of Gβγ-GRK2 signaling in synovitis, macrophage activation, and OA development. We achieved Gβγ-GRK2 inhibition at the time of DMM by administering the Gβγ inhibitor “gallein” and the GRK2 inhibitor “paroxetine” daily, starting from 2 days before DMM surgery, for a duration of 1 or 12 weeks. Synovial and cartilage structural changes were evaluated by histomorphometry, and molecular events and macrophage activation were examined. We studied the direct role of Gβγ-GRK2 in synovitis and macrophage activation in vitro using SW982 and THP1 cells. Continuous Gβγ-GRK2 inhibition initiated at the time of DMM attenuated OA development and decreased chondrocyte loss more effectively than delayed treatment. GRK2 expression and the M1 macrophage phenotype were elevated in the inflamed synovium, while early gallein and paroxetine treatment for 1 and 12 weeks following DMM resulted in their reduction and an upregulated M2 macrophage phenotype. In vitro experiments showed that Gβγ-GRK2 inhibition attenuated synoviocyte inflammation and the M1 phenotype. We show that early Gβγ-GRK2 inhibition is of higher therapeutic efficacy in OA than delayed inhibition, as it prevents OA development by inhibiting the early inflammatory response.