Accelerated accumulation of retinal α-synuclein (pSer129) and tau, neuroinflammation, and autophagic dysregulation in a seeded mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by accumulation of misfolded α-synuclein within the central nervous system (CNS). Visual problems in PD patients are common, although retinal pathology associated with PD is not well understood. The purpose of this study was to investigate retinal pathology in a transgenic mouse model (TgM83) expressing the human A53T α-synuclein mutation and assess the effect of α-synuclein "seeding" on the development of retinal pathology. Two-month-old TgM83 mice were intracerebrally inoculated with brain homogenate from old (12-18 months) TgM83 mice. Retinas were then analyzed at 5 months of age. We analyzed retinas from 5-month-old and 8-month-old uninoculated healthy TgM83 mice, and old (12-18 months) mice that were euthanized following the development of clinical signs. Retinas of B6C3H mice (genetic background of the TgM83 mouse) served as control. We used immunohistochemistry and western blot analysis to detect accumulation of α-synuclein, pTauThr231, inflammation, changes in macroautophagy, and cell death. Raman spectroscopy was used to test the potential to differentiate between retinal tissues of healthy mice and diseased mice. This work demonstrates retinal changes associated with the A53T mutation. Retinas of non-inoculated TgM83 mice had accumulation of α-synuclein, "pre-tangle" tau, activation of retinal glial cells, and photoreceptor cell loss by 8 months of age. The development of these changes is accelerated by inoculation with brain homogenate from clinically ill TgM83 mice. Compared to non-inoculated 5-month-old TgM83 mice, retinas of inoculated 5-month-old mice had increased accumulation of α-synuclein (pSer129) and pTauThr231 proteins, upregulated microglial activation, and dysregulated macroautophagy. Raman spectroscopic analysis was able to discriminate between healthy and diseased mice. This study describes retinal pathology resulting from the A53T mutation. We show that seeding with brain homogenates from old TgM83 mice accelerates retinal pathology. We demonstrate that Raman spectroscopy can be used to accurately identify a diseased retina based on its biochemical profile, and that α-synuclein accumulation may contribute to accumulation of pTauThr231 proteins, neuroinflammation, metabolic dysregulation, and photoreceptor cell death. Our work provides insight into retinal changes associated with Parkinson's disease, and may contribute to a better understanding of visual symptoms experienced by patients.

Targeting the HSP60/10 chaperonin systems of Trypanosoma brucei as a strategy for treating African sleeping sickness.

Trypanosoma brucei are protozoan parasites that cause African sleeping sickness in humans (also known as Human African Trypanosomiasis-HAT). Without treatment, T. brucei infections are fatal. There is an urgent need for new therapeutic strategies as current drugs are toxic, have complex treatment regimens, and are becoming less effective owing to rising antibiotic resistance in parasites. We hypothesize that targeting the HSP60/10 chaperonin systems in T. brucei is a viable anti-trypanosomal strategy as parasites rely on these stress response elements for their development and survival. We recently discovered several hundred inhibitors of the prototypical HSP60/10 chaperonin system from Escherichia coli, termed GroEL/ES. One of the most potent GroEL/ES inhibitors we discovered was compound 1. While examining the PubChem database, we found that a related analog, 2e-p, exhibited cytotoxicity to Leishmania major promastigotes, which are trypanosomatids highly related to Trypanosoma brucei. Through initial counter-screening, we found that compounds 1 and 2e-p were also cytotoxic to Trypanosoma brucei parasites (EC50=7.9 and 3.1µM, respectively). These encouraging initial results prompted us to develop a library of inhibitor analogs and examine their anti-parasitic potential in vitro. Of the 49 new chaperonin inhibitors developed, 39% exhibit greater cytotoxicity to T. brucei parasites than parent compound 1. While many analogs exhibit moderate cytotoxicity to human liver and kidney cells, we identified molecular substructures to pursue for further medicinal chemistry optimization to increase the therapeutic windows of this novel class of chaperonin-targeting anti-parasitic candidates. An intriguing finding from this study is that suramin, the first-line drug for treating early stage T. brucei infections, is also a potent inhibitor of GroEL/ES and HSP60/10 chaperonin systems.

GroEL/ES inhibitors as potential antibiotics.

We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett.2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-µM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.

Two hours of heat stress induces MAP-kinase signaling and autophagasome accumulation in C2C12 myotubes.

Short bouts of heat can induce a hormetic stress response, whereas prolonged or excessive exposure can elicit detrimental effects. We previously demonstrated an increase in autophagic signaling in C2C12 myotubes in response to 1 h of heat at 40 °C. In opposition, longer durations of heat exposure (e.g., 12 and 24 h) lead to an accumulation of autophagasomes and elevations in markers of cellular inflammation, oxidative stress, and apoptosis. Whether a longer, yet moderate, duration of 2 h of heat further enhances autophagic flux and attenuates stress and inflammatory signaling, or transitions the cell toward a dysregulation of autophagy is unclear. In this study, C2C12 myotubes were maintained at 37 °C or exposed to 40 °C (HT) for 2 h, and harvested immediately or following 2, 8, or 24 h of recovery. Two hours of HT immediately increased pAMPK (T172; p = 0.001), and subsequently increased pULK1 (S555) at 2 h of recovery (p = 0.028). LC3 II was increased at 8 h (p = 0.043) and 24 h (p = 0.015) of recovery, whereas p62 was elevated at 2 h (p = 0.002) and 8 h (p < 0.001) of recovery, but returned to baseline by 24 h. In Bafilomycin A1 treated cells, p62 was further increased immediately following HT (p = 0.041). There was also a significant elevation in p-p38 (Thr180/Try182), pJNK (Thr183/Tyr185), and pNFκB (Ser536). These findings suggest that as short as 2 h of heat exposure contributes to cell stress and accumulation of autophagasomes in skeletal muscle.

Acute Heat Exposure Alters Autophagy Signaling in C2C12 Myotubes.

Autophagy is a major intracellular degradation process that is essential for the clearance of unnecessary proteins/organelles and the maintenance of cellular homeostasis. The inhibition of autophagy results in cellular consequences associated with many skeletal muscle pathologies, and therapies designed to elevate autophagic activity may provide protection from such pathologies. Acute exposure to low levels of heat has therapeutic effects; however, the impact of heat on skeletal muscle autophagy remains unclear. In the present study, C2C12 myotubes were maintained at 37°C thermoneutral (TN) or heated at 40°C heat treatment (HT) for 1 h. Myotubes were harvested immediately after heating, or returned to 37°C for recovery of 2 or 24 h. HT resulted in an elevation in pAMPK (T172), Beclin-1, and LC3 II, a marker for autophagosome formation, but no change in p62. In the context of autophagy inhibition with Bafilomycin A1, HT resulted in lower LC3 II compared to TN. The applied heat load induced the heat shock response, as evidenced by immediate upregulation of HSF1 and Hsp70. Hsp70 continued to increase during recovery, whereas pHsp27 was downregulated acutely in response to HT, but retuned to TN levels by 2 h of recovery. HT also reduced the phosphorylation of the MAP-kinases p38 and JNK. These findings suggest that an acute, short bout of mild heat may be beneficial to skeletal muscle by increasing AMPK activity, markers of autophagasome formation, and the heat shock response.

Short-term heat stress altered metabolism and insulin signaling in skeletal muscle.

Heat-related complications continue to be a major health concern for humans and animals and lead to potentially life-threatening conditions. Heat stress (HS) alters metabolic parameters and may alter glucose metabolism and insulin signaling. Therefore, the purpose of this investigation was to determine the extent to which 12 h of HS-altered energetic metabolism in oxidative skeletal muscle. To address this, crossbred gilts (n = 8/group) were assigned to one of three environmental treatments for 12 h: thermoneutral (TN; 21 °C), HS (37 °C), or pair-fed to HS counterparts but housed in TN conditions (PFTN). Following treatment, animals were euthanized and the semitendinosus red (STR) was recovered. Despite increased relative protein abundance of the insulin receptor, insulin receptor substrate (IRS1) phosphorylation was increased (P = 0.0005) at S307, an inhibitory site, and phosphorylated protein kinase B (AKT) (S473) was decreased (P = 0.03) likely serving to impair insulin signaling following 12 h of HS. Further, HS increased phosphorylated protein kinase C (PKC) ζ/λ (P = 0.02) and phosphorylated PKCδ/θ protein abundance (P = 0.02), which are known to regulate inhibitory serine phosphorylation of IRS1 (S307). Sarcolemmal glucose transporter 4 (Glut4) was decreased (P = 0.04) in the membrane fraction of HS skeletal muscle suggesting diminished glucose uptake capacity. HS-mediated increases (P = 0.04) in mechanistic target of rapamycin (mTOR) were not accompanied by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1). HS decreased (P = 0.0006) glycogen synthase (GS) and increased (P = 0.02) phosphorylated GS suggesting impaired glycogen synthesis. In addition, HS altered fatty acid metabolic signaling by increasing (P = 0.02) Acetyl-CoA carboxylase (ACC), decreasing (P = 0.005) phosphorylated ATP-citrate lyase (pATPCL) and fatty acid synthase (P = 0.01) (FAS). These data suggest that 12 h of HS blunted insulin signaling, decreased protein synthesis, and altered glycogen and fatty acid metabolism.

Effect of a single bout of aerobic exercise on high-fat meal-induced inflammation.

BACKGROUND AND AIMS: Chronic low-grade inflammation is involved in the development of metabolic disorders including atherosclerosis, type 2 diabetes (T2D) and metabolic syndrome. Aerobic exercise has been shown to be anti-inflammatory and attenuate postprandial blood lipids, however, the effect of exercise on postprandial inflammation remains unclear. The aim of this study was to determine the protective effect of a single bout of aerobic exercise against postprandial lipemia and peripheral blood mononuclear cell (PBMC) inflammation and to evaluate associations with changes in the energy-sensing enzyme, AMP-activated protein kinase (AMPK). MATERIALS AND METHODS: Healthy male subjects (n=12, age=23±2, %Fat=19±2) reported to the laboratory following an overnight fast (12-14h) on two separate occasions for consumption of a high-fat meal (HFM). Participants completed an acute bout of aerobic exercise the afternoon prior to one of the HFM visits. RESULTS AND CONCLUSION: Results indicate that the single bout of moderate aerobic exercise increased AMPK signaling in PBMCs, as shown by increased phosphorylated acetyl-CoA carboxylase (p-ACC). This may be due to decreases in the AMPK inhibitory kinases PKD and GSK3ß. Additionally, prior moderate intensity exercise decreased postprandial lipemia (PPL) and some mediators of the inflammatory pathway, such as p-NF-κB. These findings that acute aerobic exercise improves AMPK and NF-κB signaling in human PBMCs contribute support to the anti-inflammatory roles of exercise.

Heat stress causes dysfunctional autophagy in oxidative skeletal muscle.

We have previously established that 24 h of environmental hyperthermia causes oxidative stress and have implicated mitochondria as likely contributors to this process. Given this, we hypothesized that heat stress would lead to increased autophagy/mitophagy and a reduction in mitochondrial content. To address this hypothesis pigs were housed in thermoneutral (TN; 20°C) or heat stress (35°C) conditions for 1- (HS1) or 3- (HS3) days and the red and white portions of the semitendinosus collected. We did not detect differences in glycolytic muscle. Counter to our hypothesis, upstream activation of autophagy was largely similar between groups as were markers of autophagosome nucleation and elongation. LC3A/B-I increased 1.6-fold in HS1 and HS3 compared to TN (P < 0.05), LC3A/B-II was increased 4.1-fold in HS1 and 4.8-fold in HS3 relative to TN, (P < 0.05) and the LC3A/B-II/I ratio was increased 3-fold in HS1 and HS3 compared to TN suggesting an accumulation of autophagosomes. p62 was dramatically increased in HS1 and HS3 compared to TN Heat stress decreased mitophagy markers PINK1 7.0-fold in HS1 (P < 0.05) and numerically by 2.4-fold in HS3 compared to TN and BNIP3L/NIX by 2.5-fold (P < 0.05) in HS1 and HS3. Markers of mitochondrial content were largely increased without activation of PGC-1α signaling. In total, these data suggest heat-stress-mediated suppression of activation of autophagy and autophagosomal degradation, which may enable the persistence of damaged mitochondria in muscle cells and promote a dysfunctional intracellular environment.