SERPINA1 modulates expression of amyloidogenic transthyretin.

Hereditary transthyretin amyloidosis (ATTR) is caused by amyloid deposition of misfolded transthyretin (TTR) in various tissues. Recently, reduction of circulating serum TTR, achieved via silencing oligonucleotides, was introduced as therapy of ATTR amyloidosis. We explored the impact of Serpin Family A Member 1 (SERPINA1) on TTR mRNA and protein expression. Oncostatin M (OSM) induced SERPINA1 in hepatoma cells and mice, while concomitantly TTR expression was significantly reduced. SERPINA1 knockdown resulted in specific elevated TTR expression in hepatoma cells; however other genes belonging to the group of acute phase proteins were unaffected. In mice, serum TTR was elevated after mSERPINA1 knockdown throughout antisense treatment. Following SERPINA1 knockdown, TTR deposition in several tissues, including dorsal root ganglia and intestine, was found to be increased, however numbers did not exceed significance levels. The data suggest that SERPINA1 is a co-factor of TTR expression. Our findings provide novel insight in the regulation of TTR and reveal a role of SERPINA1 in the pathogenesis of ATTR amyloidosis.

In Vitro and In Vivo Effects of SerpinA1 on the Modulation of Transthyretin Proteolysis.

Transthyretin (TTR) proteolysis has been recognized as a complementary mechanism contributing to transthyretin-related amyloidosis (ATTR amyloidosis). Accordingly, amyloid deposits can be composed mainly of full-length TTR or contain a mixture of both cleaved and full-length TTR, particularly in the heart. The fragmentation pattern at Lys48 suggests the involvement of a serine protease, such as plasmin. The most common TTR variant, TTR V30M, is susceptible to plasmin-mediated proteolysis, and the presence of TTR fragments facilitates TTR amyloidogenesis. Recent studies revealed that the serine protease inhibitor, SerpinA1, was differentially expressed in hepatocyte-like cells (HLCs) from ATTR patients. In this work, we evaluated the effects of SerpinA1 on in vitro and in vivo modulation of TTR V30M proteolysis, aggregation, and deposition. We found that plasmin-mediated TTR proteolysis and aggregation are partially inhibited by SerpinA1. Furthermore, in vivo downregulation of SerpinA1 increased TTR levels in mice plasma and deposition in the cardiac tissue of older animals. The presence of TTR fragments was observed in the heart of young and old mice but not in other tissues following SerpinA1 knockdown. Increased proteolytic activity, particularly plasmin activity, was detected in mice plasmas. Overall, our results indicate that SerpinA1 modulates TTR proteolysis and aggregation in vitro and in vivo.

Monocarboxylate transporter 1 is a key player in glioma-endothelial cell crosstalk.

Glioblastoma (GBM) is one of the most glycolytic and angiogenic human tumors, characteristics that contribute to the poor prognosis associated with this type of tumor. A lactate shuttle has been described between tumor cells and endothelial cells (ECs), with the monocarboxylate transporters (MCTs) acting as important players in this tumor-EC communication. In this study, we aimed to understand how the tumor microenvironment modulates EC metabolism, and to characterize the role of MCTs in the glioma-brain EC crosstalk. Exposure of human brain microvascular ECs (HBMEC) to GBM cell-conditioned media increased the expression of MCT1, which corresponded to activation of oxidative metabolism and an increase in angiogenic capacity, as determined by increased proliferation, migration, and vessel assembly. Lactate depletion from the microenvironment or inhibition of lactate uptake in HBMEC induced an increase in lactate production and a decrease in proliferation, migration, and vessel assembly. Moreover, addition of lactate to HBMEC media promoted activation of AKT and AMPK pathways and increased expression in NFκB, HIF-1α, and the lactate receptor GPR81. Here, we demonstrate a role for MCT1 as a mediator of lactate signaling between glioma cells and brain ECs. Our results suggest that MCT1 can mediate EC metabolic reprograming, proliferation, and vessel sprouting in response to tumor signaling. Thus, targeting MCT1 in both tumor cells and brain EC may be a promising therapeutic strategy for the treatment of GBM.

Immune response in the adipose tissue of lean mice infected with the protozoan parasite Neospora caninum.

The adipose tissue can make important contributions to immune function. Nevertheless, only a limited number of reports have investigated in lean hosts the immune response elicited in this tissue upon infection. Previous studies suggested that the intracellular protozoan Neospora caninum might affect adipose tissue physiology. Therefore, we investigated in mice challenged with this protozoan if immune cell populations within adipose tissue of different anatomical locations could be differently affected. Early in infection, parasites were detected in the adipose tissue and by 7 days of infection increased numbers of macrophages, regulatory T (Treg) cells and T-bet(+) cells were observed in gonadal, mesenteric, omental and subcutaneous adipose tissue. Increased expression of interferon-γ was also detected in gonadal adipose tissue of infected mice. Two months after infection, parasite DNA was no longer detected in these tissues, but T helper type 1 (Th1) cell numbers remained above control levels in the infected mice. Moreover, the Th1/Treg cell ratio was higher than that of controls in the mesenteric and subcutaneous adipose tissue. Interestingly, chronically infected mice presented a marked increase of serum leptin, a molecule that plays a role in energy balance regulation as well as in promoting Th1-type immune responses. Altogether, we show that an apicomplexa parasitic infection influences immune cellular composition of adipose tissue throughout the body as well as adipokine production, still noticed at a chronic phase of infection when parasites were already cleared from that particular tissue. This strengthens the emerging view that infections can have long-term consequences for the physiology of adipose tissue.

PITB: A high affinity transthyretin aggregation inhibitor with optimal pharmacokinetic properties.

The aggregation of wild-type transthyretin (TTR) and over 130 genetic TTR variants underlies a group of lethal disorders named TTR amyloidosis (ATTR). TTR chemical chaperones are molecules that hold great promise to modify the course of ATTR progression. In previous studies, we combined rational design and molecular dynamics simulations to generate a series of TTR selective kinetic stabilizers displaying exceptionally high affinities. In an effort to endorse the previously developed molecules with optimal pharmacokinetic properties, we conducted structural design optimization, leading to the development of PITB. PITB binds with high affinity to TTR, effectively inhibiting tetramer dissociation and aggregation of both the wild-type protein and the two most prevalent disease-associated TTR variants. Importantly, PITB selectively binds and stabilizes TTR in plasma, outperforming tolcapone, a drug currently undergoing clinical trials for ATTR. Pharmacokinetic studies conducted on mice confirmed that PITB exhibits encouraging pharmacokinetic properties, as originally intended. Furthermore, PITB demonstrates excellent oral bioavailability and lack of toxicity. These combined attributes position PITB as a lead compound for future clinical trials as a disease-modifying therapy for ATTR.

Development of a Highly Potent Transthyretin Amyloidogenesis Inhibitor: Design, Synthesis, and Evaluation.

Transthyretin amyloidosis (ATTR) is a group of fatal diseases described by the misfolding and amyloid deposition of transthyretin (TTR). Discovering small molecules that bind and stabilize the TTR tetramer, preventing its dissociation and subsequent aggregation, is a therapeutic strategy for these pathologies. Departing from the crystal structure of TTR in complex with tolcapone, a potent binder in clinical trials for ATTR, we combined rational design and molecular dynamics (MD) simulations to generate a series of novel halogenated kinetic stabilizers. Among them, M-23 displays one of the highest affinities for TTR described so far. The TTR/M-23 crystal structure confirmed the formation of unprecedented protein-ligand contacts, as predicted by MD simulations, leading to an enhanced tetramer stability both in vitro and in whole serum. We demonstrate that MD-assisted design of TTR ligands constitutes a new avenue for discovering molecules that, like M-23, hold the potential to become highly potent drugs to treat ATTR.

Modulation of the Mechanisms Driving Transthyretin Amyloidosis.

Transthyretin (TTR) amyloidoses are systemic diseases associated with TTR aggregation and extracellular deposition in tissues as amyloid. The most frequent and severe forms of the disease are hereditary and associated with amino acid substitutions in the protein due to single point mutations in the TTR gene (ATTRv amyloidosis). However, the wild type TTR (TTR wt) has an intrinsic amyloidogenic potential that, in particular altered physiologic conditions and aging, leads to TTR aggregation in people over 80 years old being responsible for the non-hereditary ATTRwt amyloidosis. In normal physiologic conditions TTR wt occurs as a tetramer of identical subunits forming a central hydrophobic channel where small molecules can bind as is the case of the natural ligand thyroxine (T4). However, the TTR amyloidogenic variants present decreased stability, and in particular conditions, dissociate into partially misfolded monomers that aggregate and polymerize as amyloid fibrils. Therefore, therapeutic strategies for these amyloidoses may target different steps in the disease process such as decrease of variant TTR (TTRv) in plasma, stabilization of TTR, inhibition of TTR aggregation and polymerization or disruption of the preformed fibrils. While strategies aiming decrease of the mutated TTR involve mainly genetic approaches, either by liver transplant or the more recent technologies using specific oligonucleotides or silencing RNA, the other steps of the amyloidogenic cascade might be impaired by pharmacologic compounds, namely, TTR stabilizers, inhibitors of aggregation and amyloid disruptors. Modulation of different steps involved in the mechanism of ATTR amyloidosis and compounds proposed as pharmacologic agents to treat TTR amyloidosis will be reviewed and discussed.

Enrichment of IFN-γ producing cells in different murine adipose tissue depots upon infection with an apicomplexan parasite.

Here we report that lean mice infected with the intracellular parasite Neospora caninum show a fast but sustained increase in the frequency of IFN-γ-producing cells noticeable in distinct adipose tissue depots. Moreover, IFN-γ-mediated immune memory could be evoked in vitro in parasite antigen-stimulated adipose tissue stromal vascular fraction cells collected from mice infected one year before. Innate or innate-like cells such as NK, NK T and TCRγδ(+) cells, but also CD4(+) and CD8(+) TCRß(+) lymphocytes contributed to the IFN-γ production observed since day one of infection. This early cytokine production was largely abrogated in IL-12/IL23 p40-deficient mice. Moreover, production of IFN-γ by stromal vascular fraction cells isolated from these mice was markedly lower than that of wild-type counterparts upon stimulation with parasite antigen. In wild-type mice the increased IFN-γ production was concomitant with up-regulated expression of genes encoding interferon-inducible GTPases and nitric oxide synthase, which are important effector molecules in controlling intracellular parasite growth. This increased gene expression was markedly impaired in the p40-deficient mice. Overall, these results show that NK cells but also diverse T cell populations mediate a prompt and widespread production of IFN-γ in the adipose tissue of N. caninum infected mice.