Cellular environment of TTR deposits in an animal model of ATTR-Cardiomyopathy.

Introduction: Cardiac amyloidoses are the most fatal manifestation of systemic amyloidoses. It is believed the number of cases to be greatly underestimated mostly due to misdiagnosis. Particularly, the involvement of TTR V30M in the heart of ATTRV30M amyloidosis has not been completely understood specifically in terms of implicated cellular pathways, heart function and cardiac physiology. In the present work we proposed to characterize TTR V30M cardiac involvement particularly at the tissue cellular level in a mouse model. Methods: HSF ± hTTR V30M mice, a model that expresses human TTRV30M in a Ttr null background, widely used for the characterization and modulation of neurological features of ATTRV30M amyloidosis was used. SDS-PAGE of cardiac homogenates followed by Western blot was performed. Immunohistochemistry and double immunofluorescence analyses were carried out to determine TTR deposition pattern and sub-localization. Results: Western blots were able to detect TTR in its monomeric state at ∼14 kDa. Immunofluorescent images showed TTR was found mostly in the intercellular spaces. Blood contamination was excluded by CD31 staining. Tissues were Congo Red negative. Upon TTR and macrophages (CD68) staining in the cardiac tissue a clear tendency of macrophage convergence to the tissue regions where TTR was more abundant was observed. Moreover, in some instances it was possible to detect co-localization of both fluorophores. Cardiac fibroblasts were stained with PDGFr-alpha, and here the co-localization was not so evident although there was some degree of co-occurrence. The hearts of transgenic mice revealed higher content of Galectin-3. Conclusion: This animal model and associated features observed as result of cardiac TTR deposition provide a promising and invaluable research tool for a better understanding of the implicated pathways that lead to the lethality associated to TTR cardiac amyloidosis. New therapeutic strategies can be tested and ultimately this will lead to improved treatment alternatives capable of increasing patient's quality of life and life expectancy and, hopefully to eradicate a condition that is silently spreading worldwide.

Decreased expression of S100A8/A9 in V30M related ATTRv amyloidosis.

INTRODUCTION: Hereditary Transthyretin Amyloidosis is a rare, progressive and life-threatening systemic disease with predominant peripheral and autonomic nervous system involvement caused by mutation of the transthyretin protein. The most common TTR mutation regarding to ATTRv is a substitution of a Methionine for a Valine at position 30 that predisposes TTR to form aggregates and fibrils. METHODS: S100A8 protein levels were measured in plasma samples from ATTRV30M patients and healthy donors. Additionally, S100A8/9 levels were measured in Schwann cells after incubation with human WT or V30M TTR. Moreover, bone marrow derived macrophages of either genetic background were generated and the expression of S100A8/9 was measured in response to toll like receptors agonists. RESULTS: S100A8/A9 mRNA levels are decreased in HSF V30M mice as compared with the WT. Moreover, S100A8 protein levels were found downregulated in plasma samples from ATTRV30M patients. Furthermore, we provide evidence for a dysregulated S100 expression by Schwann cells in response to TTRV30M and by mutated macrophages in response to toll like receptors agonists. CONCLUSION: The presence of TTRV30M impacts S100 expression, possibly contributing to the impaired immune activation of Schwann cells in nerves from ATTRV30M patients. This may be linked to the diminished immune cellular infiltration in these nerves, contributing in this way for the neuronal dysfunction present in the disease.

TLR2 and 4 signaling pathways are altered in macrophages from V30M TTR mice with down-regulated expression of chemokines.

Hereditary amyloid transthyretin (ATTRv) amyloidosis is a fatal neurodegenerative disorder, first identified in Portugal. The most common transthyretin (TTR) mutation in ATTRv results from an exchange of a methionine for a valine at position 30 (V30M). ATTRv is characterized by the extracellular deposition of aggregates and fibrils of mutant forms of TTR, particularly in the nerves and ganglia of the peripheral nervous system (PNS). This phenotype is often accompanied by the lack of inflammatory infiltrates, despite the importance of macrophages in removal of TTR deposits in ATTRv patients. The mechanisms underlying this impairment of inflammatory responses in ATTRv patients are poorly understood. Here, we show a significant down-regulation in the expression of several chemokines by bone marrow-derived macrophages (BMDM) generated from V30M TTR mice upon stimulation with toll-like receptor 4 (TLR4) and TLR2 agonists. The phosphorylation of the MAP kinase p38, important for TLR4 and TLR2 signaling pathways, was also down-regulated in V30M macrophages, as compared with wild-type (WT) ones. The present study contributes with new insights to unravel the molecular mechanisms underlying the lack of inflammatory immune responses observed in ATTRv patients and may help in the development of new immune therapeutic strategies for the disease.

Posttranslational modifications of proteins are key features in the identification of CSF biomarkers of multiple sclerosis.

BACKGROUND: Multiple sclerosis is an inflammatory and degenerative disease of the central nervous system (CNS) characterized by demyelination and concomitant axonal loss. The lack of a single specific test, and the similarity to other inflammatory diseases of the central nervous system, makes it difficult to have a clear diagnosis of multiple sclerosis. Therefore, laboratory tests that allows a clear and definite diagnosis, as well as to predict the different clinical courses of the disease are of utmost importance. Herein, we compared the cerebrospinal fluid (CSF) proteome of patients with multiple sclerosis (in the relapse-remitting phase of the disease) and other diseases of the CNS (inflammatory and non-inflammatory) aiming at identifying reliable biomarkers of multiple sclerosis. METHODS: CSF samples from the discovery group were resolved by 2D-gel electrophoresis followed by identification of the protein spots by mass spectrometry. The results were analyzed using univariate (Student's t test) and multivariate (Hierarchical Cluster Analysis, Principal Component Analysis, Linear Discriminant Analysis) statistical and numerical techniques, to identify a set of protein spots that were differentially expressed in CSF samples from patients with multiple sclerosis when compared with other two groups. Validation of the results was performed in samples from a different set of patients using quantitative (e.g., ELISA) and semi-quantitative (e.g., Western Blot) experimental approaches. RESULTS: Analysis of the 2D-gels showed 13 protein spots that were differentially expressed in the three groups of patients: Alpha-1-antichymotrypsin, Prostaglandin-H2-isomerase, Retinol binding protein 4, Transthyretin (TTR), Apolipoprotein E, Gelsolin, Angiotensinogen, Agrin, Serum albumin, Myosin-15, Apolipoprotein B-100 and EF-hand calcium-binding domain-containing protein. ELISA experiments allowed validating part of the results obtained in the proteomics analysis and showed that some of the alterations in the CSF proteome are also mirrored in serum samples from multiple sclerosis patients. CSF of multiple sclerosis patients was characterized by TTR oligomerization, thus highlighting the importance of analyzing posttranslational modifications of the proteome in the identification of novel biomarkers of the disease. CONCLUSIONS: The model built based on the results obtained upon analysis of the 2D-gels and in the validation phase attained an accuracy of about 80% in distinguishing multiple sclerosis patients and the other two groups.

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.

The Expression of Chemokines Is Downregulated in a Pre-Clinical Model of TTR V30M Amyloidosis.

Inflammation is a hallmark of several neurodegenerative disorders including hereditary amyloidogenic transthyretin amyloidosis (ATTRv). ATTRv is an autosomal dominant neurodegenerative disorder with extracellular deposition of mutant transthyretin (TTR) aggregates and fibrils, particularly in nerves and ganglia of the peripheral nervous system. Nerve biopsies from ATTRv patients show increased cytokine production, but interestingly no immune inflammatory cellular infiltrate is observed around TTR aggregates. Here we show that as compared to Wild Type (WT) animals, the expression of several chemokines is highly downregulated in the peripheral nervous system of a mouse model of the disease. Interestingly, we found that stimulation of mouse Schwann cells (SCs) with WT TTR results in the secretion of several chemokines, a process that is mediated by toll-like receptor 4 (TLR4). In contrast, the secretion of all tested chemokines is compromised upon stimulation of SCs with mutant TTR (V30M), suggesting that V30M TTR fails to activate TLR4 signaling. Altogether, our data shed light into a previously unappreciated mechanism linking TTR activation of SCs and possibly underlying the lack of inflammatory response observed in the peripheral nervous system of ATTRv patients.

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.

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.

Correction: Transthyretin expression in the postischemic brain.

[This corrects the article DOI: 10.1371/journal.pone.0221555.].

Plasma neurofilament light chain: an early biomarker for hereditary ATTR amyloid polyneuropathy.

Background: Transthyretin amyloidosis due to V30M mutation (ATTR-V30M) is the most frequent hereditary ATTR amyloidosis. Besides neurophysiological measures, there are no biomarkers to detect preclinical disease or monitor disease progression. CSF or plasma neurofilament light chain (pNfL) have recently been considered sensitive biomarkers to quantitate neuro-axonal damage in several disorders of the peripheral and central nervous system.Objective: Characterise plasma NfL levels in a series of untreated ATTR-V30M patients stratified by clinical severity using a cross-sectional retrospective study design.Methods: Sixty ATTR-V30M patients and 16 controls from 2 independent cohorts were analysed for pNfL by single-molecule array assay (SIMOA) technique. Disease severity was assessed with Polyneuropathy Disability Score.Results: pNfL is elevated in ATTR-V30M patients as a function of disease severity in both cohorts. Moreover, pNfL discriminates asymptomatic mutation carriers from early symptomatic patients (AUC = 0.97; p < .001) with high sensitivity (92.3%) and specificity (93.8%). pNfL elevation (>66.9 pg/mL) also discriminates patients with sensory neuropathy from patients with motor neuropathy (AUC = 0.91; p < .01) with a sensitivity of 61.5% and a specificity of 92.3%.Conclusion: pNfL is an easily accessible biomarker to establish ATTR-V30M disease conversion and to monitor disease progression. pNfL could be used as efficacy measure of disease-oriented therapies in clinical and pre-clinical trials.

Transthyretin expression in the postischemic brain.

The unknown role of the carrier protein transthyretin (TTR) in mechanisms of functional recovery in the postischemic brain prompted us to study its expression following experimental stroke. Male C57/B6 mice (age 9 to 10 weeks) were subjected to permanent focal ischemia induced by photothrombosis (PT) and brain tissues were analyzed for ttr expression and TTR levels at 24 hours, 48 hours, 7 days and 14 days following the insult by RT-PCR, Western blot and immunohistochemistry. Fourteen days after PT, non-specific TTR-like immunoreactive globules were found in the ischemic core and surrounding peri-infarct region by immunohistochemistry that could not be allocated to DAPI positive cells. No TTR immunoreactivity was found when stainings were performed with markers for neurons (Neuronal Nuclei, NeuN), reactive astrocytes (glial fibrillary acidic protein, GFAP) or microglia (cluster of differentiation 68, CD68). In addition, we could not find TTR by immunoblotting in protein extracts obtained from the ischemic territory nor ttr expression by RT-PCR at all time points following PT. In all experiments, ttr expression in the choroid plexus and TTR in the mouse serum served as positive controls and recombinant legumain peptide as negative control. Together, our results indicate that TTR is not synthesized in brain resident cells in the ischemic infarct core and adjacent peri-infarct area. Thus, it seems unlikely that in situ synthesized TTR is involved in mechanisms of tissue reorganization during the first 14 days following PT.

Uncovering the Neuroprotective Mechanisms of Curcumin on Transthyretin Amyloidosis.

Transthyretin (TTR) amyloidoses (ATTR amyloidosis) are diseases associated with transthyretin (TTR) misfolding, aggregation and extracellular deposition in tissues as amyloid. Clinical manifestations of the disease are variable and include mainly polyneuropathy and/or cardiomyopathy. The reasons why TTR forms aggregates and amyloid are related with amino acid substitutions in the protein due to mutations, or with environmental alterations associated with aging, that make the protein more unstable and prone to aggregation. According to this model, several therapeutic approaches have been proposed for the diseases that range from stabilization of TTR, using chemical chaperones, to clearance of the aggregated protein deposited in tissues in the form of oligomers or small aggregates, by the action of disruptors or by activation of the immune system. Interestingly, different studies revealed that curcumin presents anti-amyloid properties, targeting multiple steps in the ATTR amyloidogenic cascade. The effects of curcumin on ATTR amyloidosis will be reviewed and discussed in the current work in order to contribute to knowledge of the molecular mechanisms involved in TTR amyloidosis and propose more efficient drugs for therapy.

MMP-14 overexpression correlates with the neurodegenerative process in familial amyloidotic polyneuropathy.

Levels of matrix metalloproteases (MMPs) can be differentially regulated in response to injury or neurological diseases. For instance, it is known that selective and short-term inhibition of MMP-14, a membrane-type 1 MMP, accelerates axon regeneration. Because axon growth and regeneration is impaired in familial amyloidotic polyneuropathy (FAP), a neurodegenerative disorder characterized by misfolding and deposition of mutant transthyretin (TTR) in the peripheral nervous system (PNS), we presently investigated the expression levels and the potential role for MMP-14 in this condition. By using cell culture studies, a mouse model of disease and human clinical samples, we observed that MMP-14: (i) is overexpressed in FAP nerves, correlating with TTR deposition; (ii) is upregulated in sciatic nerves from a preclinical transgenic mouse model, increasing with TTR deposition; (iii) levels in the PNS and plasma are rescued upon treatment of mice with anakinra or TTR siRNA, drugs acting over the IL-1 signaling pathway or TTR liver synthesis, respectively; (iv) increases in Schwann cells upon incubation with amyloid-like aggregates; and, finally, (v) is increased in plasma of FAP patients, correlating with disease progression. These results highlight the relevance of MMP-14 in the pathophysiology of FAP, suggesting not only a potential role for this molecule as a novel biomarker for therapy follow up, but also as a new potential therapeutic target.

Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting.

AIM: Gadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses. MATERIALS & METHODS: The ability of the functionalized nanoparticles to target amyloid fibrils made of ß-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy. RESULTS: Nanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with KD values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles. Moreover, they allowed the detection of amyloid deposits on pathological tissues. CONCLUSION: Such functionalized nanoparticles could represent promising flexible and multimodal imaging tools for the early diagnostic of amyloid diseases, in other words, Alzheimer's disease, Type 2 diabetes mellitus and the familial amyloidotic polyneuropathy.

Differential expression of Cathepsin E in transthyretin amyloidosis: from neuropathology to the immune system.

BACKGROUND: Increasing evidence supports a key role for inflammation in the neurodegenerative process of familial amyloidotic polyneuropathy (FAP). While there seems to be an overactivation of the neuronal interleukin-1 signaling pathway, the immune response is apparently compromised in FAP. Accordingly, little immune cell infiltration is observed around pre-fibrillar or fibrillar amyloid deposits, with the underlying mechanism for this phenomenon remaining poorly understood. Cathepsin E (CtsE) is an important intermediate for antigen presentation and chemotaxis, but its role in the pathogenesis of FAP disease remains unknown. METHODS: In this study, we used both mouse primary macrophages and in vivo studies based on transgenic models of FAP and human samples to characterize CtsE expression in different physiological systems. RESULTS: We show that CtsE is critically decreased in bone marrow-derived macrophages from a FAP mouse model, possibly contributing for cell function impairment. Compromised levels of CtsE were also found in injured nerves of transgenic mice and, most importantly, in naïve peripheral nerves, sensory ganglia, murine stomach, and sural nerve biopsies derived from FAP patients. Expression of CtsE in tissues was associated with transthyretin (TTR) deposition and differentially regulated accordingly with the physiological system under study. Preventing deposition with a TTR small interfering RNA rescued CtsE in the peripheral nervous system (PNS). In contrast, the expression of CtsE increased in splenic cells (mainly monocytes) or peritoneal macrophages, indicating a differential macrophage phenotype. CONCLUSION: Altogether, our data highlights the potential of CtsE as a novel FAP biomarker and a possible modulator for innate immune cell chemotaxis to the disease most affected tissues-the peripheral nerve and the gastrointestinal tract.

C1q ablation exacerbates amyloid deposition: A study in a transgenic mouse model of ATTRV30M amyloid neuropathy.

ATTRV30M amyloid neuropathy is a lethal autosomal dominant sensorimotor and autonomic neuropathy, caused by deposition of amyloid fibrils composed of aberrant transthyretin (TTR). Ages of onset and penetrance exhibit great variability and genetic factors have been implicated. Complement activation co-localizes with amyloid deposits in amyloidotic neuropathy and is possibly involved in the kinetics of amyloidogenesis. A candidate gene approach has recently identified C1q polymorphisms to correlate with disease onset in a Cypriot cohort of patients with ATTRV30M amyloid neuropathy. In the current study we use a double transgenic mouse model of ATTRV30M amyloid neuropathy in which C1q is ablated to elucidate further a possible modifier role for C1q. Amyloid deposition is found to be increased by 60% in the absence of C1q. Significant up regulation is also recorded in apoptotic and cellular stress markers reflecting extracellular toxicity of pre-fibrillar and fibrillar TTR. Our data further indicate that in the absence of C1q there is marked reduction of macrophages in association with amyloid deposits and thus less effective phagocytosis of TTR.

Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation.

More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T4) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies.