Neuroprotective Effects of Metformin Through AMPK Activation in a Neurotoxin-Based Model of Cerebellar Ataxia.

Cerebellar ataxia is a heterogeneous group of neural disorders clinically characterized by cerebellar dysfunction. The diagnosis of patients with progressive cerebellar ataxia is complex due to the direct correlation with other neuron diseases. Although there is still no cure for this pathological condition, some metabolic, hereditary, inflammatory, and immunological factors affecting cerebellar ataxia are being studied and may become therapeutic targets. Advances in studying the neuroanatomy, pathophysiology, and molecular biology of the cerebellum (CE) contribute to a better understanding of the mechanisms behind the development of this disorder. In this study, Wistar rats aged 30 to 35 days were injected intraperitoneally with 3-acetylpyridine (3-AP) and/or metformin (for AMP-activated protein kinase (AMPK) enzyme activation) and euthanized in 24 hours and 4 days after injection. We analyzed the neuromodulatory role of the AMPK on cerebellar ataxia induced by the neurotoxin 3-AP in the brain stem (BS) and CE, after pre-treatment for 7 and 15 days with metformin, a pharmacological indirect activator of AMPK. The results shown here suggest that AMPK activation in the BS and CE leads to a significant reduction in neuroinflammation in these regions. AMPK was able to restore the changes in fatty acid composition and pro-inflammatory cytokines caused by 3-AP, suggesting that the action of AMPK seems to result in a possible neuroprotection on the cerebellar ataxia model.

Endoplasmic Stress Affects the Coinfection of Leishmania Amazonensis and the Phlebovirus (Bunyaviridae) Icoaraci.

Viral coinfections can modulate the severity of parasitic diseases, such as human cutaneous leishmaniasis. Leishmania parasites infect thousands of people worldwide and cause from single cutaneous self-healing lesions to massive mucosal destructive lesions. The transmission to vertebrates requires the bite of Phlebotomine sandflies, which can also transmit Phlebovirus. We have demonstrated that Leishmania infection requires and triggers the Endoplasmic stress (ER stress) response in infected macrophages. In the present paper, we tested the hypothesis that ER stress is increased and required for the aggravation of Leishmania infection due to coinfection with Phlebovirus. We demonstrated that Phlebovirus Icoaraci induces the ER stress program in macrophages mediated by the branches IRE/XBP1 and PERK/ATF4. The coinfection with L. amazonensis potentiates and sustains the ER stress, and the inhibition of IRE1α or PERK results in poor viral replication and decreased parasite load in macrophages. Importantly, we observed an increase in viral replication during the coinfection with Leishmania. Our results demonstrated the role of ER stress branches IRE1/XBP1 and PERK/ATF4 in the synergic effect on the Leishmania increased load during Phlebovirus coinfection and suggests that Leishmania infection can also increase the replication of Phlebovirus in macrophages.

Toll-Like Receptor 3 (TLR3) Is Engaged in the Intracellular Survival of the Protozoan Parasite Leishmania (Leishmania) amazonensis.

The protozoan parasite Leishmania (L.) amazonensis infects and replicates inside host macrophages due to subversion of the innate host cell response. In the present study, we demonstrate that TLR3 is required for the intracellular growth of L. (L.) amazonensis. We observed restricted intracellular infection of TLR3-/- mouse macrophages, reduced levels of IFN1ß and IL-10, and increased levels of IL-12 upon L. (L.) amazonensis infection, compared with their wild-type counterparts. Accordingly, in vivo infection of TLR3-/- mice with L. (L.) amazonensis displayed a significant reduction in lesion size. Leishmania (L.) amazonensis infection induced TLR3 proteolytic cleavage, which is a process required for TLR3 signaling. The chemical inhibition of TLR3 cleavage or infection by CPB-deficient mutant L. (L.) mexicana resulted in reduced parasite load and restricted the expression of IFN1ß and IL-10. Furthermore, we show that the dsRNA sensor molecule PKR (dsRNA-activated protein kinase) cooperates with TLR3 signaling to potentiate the expression of IL-10 and IFN1ß and parasite survival. Altogether, our results show that TLR3 signaling is engaged during L. (L.) amazonensis infection and this component of innate immunity modulates the host cell response.

Insulin receptor deficiency reduces lipid synthesis and reproductive function in the insect Rhodnius prolixus.

Rhodnius prolixus, a vector of Chagas disease, is a hematophagous insect that feeds exclusively on blood. Each blood meal is digested within the first fourteen days after feeding, providing substrates for lipid synthesis for storage and egg production. These events are precisely regulated and emerging evidence points to a key function of insulin-like peptides (ILPs) in this control. Here we investigated the role of insulin receptor in the regulation of nutrient metabolism in fed adult females. The expression of insulin receptor (RhoprIR) gene was determined in adult organs, and it was highest in ovaries and previtellogenic follicles. We generated insects with RNAi-mediated knockdown of RhoprIR to address the physiological role of this receptor. RhoprIR deficiency improved longevity and reduced triacylglycerol storage in the fat body, whereas blood digestion remained unchanged for seven days after blood meal. The lower lipid content was attributable to decreased de novo lipogenesis as well as reduced incorporation of hemolymph-derived fatty acids into newly synthesized lipids within this organ. Consistent with that, fat bodies from RhoprIR-deficient insects exhibited decreased gene expression levels of lipophorin receptor (RhoprLpR), glycerol-3-phosphate acyltransferase 1 and 4 (RhoprGpat1 and RhoprGpat4), and carnitine palmitoyltransferase 1 (RhoprCpt1). Although hemolymph lipid profile was not affected by RhoprIR disruption, the concentration of circulating vitellogenin was increased. In line with these changes, RhoprIR-deficient females exhibited smaller ovaries and a marked reduction in oviposition. Taken together, these findings support a key role of insulin receptor in nutrient homeostasis, lipid synthesis and egg production following a blood meal.

Emerging Role for the PERK/eIF2α/ATF4 in Human Cutaneous Leishmaniasis.

Leishmania parasites utilize adaptive evasion mechanisms in infected macrophages to overcome host defenses and proliferate. We report here that the PERK/eIF2α/ATF4 signaling branch of the integrated endoplasmic reticulum stress response (IERSR) is activated by Leishmania and this pathway is important for Leishmania amazonensis infection. Knocking down PERK or ATF4 expression or inhibiting PERK kinase activity diminished L. amazonensis infection. Knocking down ATF4 decreased NRF2 expression and its nuclear translocation, reduced HO-1 expression and increased nitric oxide production. Meanwhile, the increased expression of ATF4 and HO-1 mRNAs were observed in lesions derived from patients infected with the prevalent related species L.(V.) braziliensis. Our data demonstrates that Leishmania parasites activate the PERK/eIF2α/ATF-4 pathway in cultured macrophages and infected human tissue and that this pathway is important for parasite survival and progression of the infection.

Adipokinetic hormone receptor gene identification and its role in triacylglycerol metabolism in the blood-sucking insect Rhodnius prolixus.

Adipokinetic hormone (AKH) has been associated with the control of energy metabolism in a large number of arthropod species due to its role on the stimulation of lipid, carbohydrate and amino acid mobilization/release. In the insect Rhodnius prolixus, a vector of Chagas' disease, triacylglycerol (TAG) stores must be mobilized to sustain the metabolic requirements during moments of exercise or starvation. Besides the recent identification of the R. prolixus AKH peptide, other components required for the AKH signaling cascade and its mode of action remain uncharacterized in this insect. In the present study, we identified and investigated the expression profile of the gene encoding the AKH receptor of R. prolixus (RhoprAkhr). This gene is highly conserved in comparison to other sequences already described and its transcript is abundant in the fat body and the flight muscle of the kissing bug. Moreover, RhoprAkhr expression is induced in the fat body at moments of increased TAG mobilization; the knockdown of this gene resulted in TAG accumulation both in fat body and flight muscle after starvation. The inhibition of Rhopr-AKHR transcription as well as the treatment of insects with the peptide Rhopr-AKH in its synthetic form altered the transcript levels of two genes involved in lipid metabolism, the acyl-CoA-binding protein-1 (RhoprAcbp1) and the mitochondrial glycerol-3-phosphate acyltransferase-1 (RhoprGpat1). These results indicate that the AKH receptor is regulated at transcriptional level and is required for TAG mobilization under starvation. In addition to the classical view of AKH as a direct regulator of enzymatic activity, we propose here that AKH signaling may account for the regulation of nutrient metabolism by affecting the expression profile of target genes.