Structural and thermodynamic characterization of a highly amyloidogenic dimer of transthyretin involved in a severe cardiomyopathy.

Transthyretin (TTR) is a homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy (FAC). Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure (HHP). Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of sub-denaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.

Targeting Biomolecular Condensation and Protein Aggregation against Cancer.

Biomolecular condensates, membrane-less entities arising from liquid-liquid phase separation, hold dichotomous roles in health and disease. Alongside their physiological functions, these condensates can transition to a solid phase, producing amyloid-like structures implicated in degenerative diseases and cancer. This review thoroughly examines the dual nature of biomolecular condensates, spotlighting their role in cancer, particularly concerning the p53 tumor suppressor. Given that over half of the malignant tumors possess mutations in the TP53 gene, this topic carries profound implications for future cancer treatment strategies. Notably, p53 not only misfolds but also forms biomolecular condensates and aggregates analogous to other protein-based amyloids, thus significantly influencing cancer progression through loss-of-function, negative dominance, and gain-of-function pathways. The exact molecular mechanisms underpinning the gain-of-function in mutant p53 remain elusive. However, cofactors like nucleic acids and glycosaminoglycans are known to be critical players in this intersection between diseases. Importantly, we reveal that molecules capable of inhibiting mutant p53 aggregation can curtail tumor proliferation and migration. Hence, targeting phase transitions to solid-like amorphous and amyloid-like states of mutant p53 offers a promising direction for innovative cancer diagnostics and therapeutics.

A onda pró-ciência em tempos de negacionismo: percepção da sociedade brasileira sobre ciência, cientistas e universidades na pandemia da COVID-19 / The pro-science wave in times of denialism: Brazilian society's perception of science, scientists, and universities during the COVID-19 pandemic

Resumo O artigo apresenta resultados de pesquisas sobre percepção pública da ciência na pandemia no Brasil, realizadas no Centro de Estudos SoU_Ciência, sediado na Universidade Federal de São Paulo (Unifesp). Com o intuito de responder à pergunta: "A pandemia da COVID-19 alterou a percepção da sociedade brasileira sobre ciência, cientistas e universidades?", realizamos estudos quantitativos e qualitativos entre agosto/2021 e julho/2022. Em levantamentos quantitativos nacionais de opinião pública, coletamos dados exclusivos de série histórica de enquetes sobre o tema no Brasil, e em grupos focais, aprofundamos estudos sobre percepção e posicionamento político de diferentes segmentos sociais. Em meio ao quadro de crescimento do negacionismo científico, retrocessos políticos e sociais, desmonte de políticas públicas, especificamente científicas e tecnológicas, decorrentes do impeachment de 2016 e da eleição de Bolsonaro em 2018, as pesquisas indicam, aparentemente contrariando a tendência política obscurantista, uma expressiva ampliação do interesse público pela ciência na pandemia no país. Este trabalho analisa a emergência de uma "onda pró-ciência" na opinião pública no Brasil, os fatores que propiciaram seu surgimento na pandemia e suas perspectivas na atualidade.

Abstract This article presents the results of our study on the public perception of science during the COVID-19 pandemic in Brazil, carried out at the Centro de Estudos SoU_Ciência, from Universidade Federal de São Paulo (UNIFESP). To answer the question: "Has the COVID-19 pandemic changed the perception of Brazilian society about science, scientists, and universities?", quantitative and qualitative studies were conducted between August 2021 and July 2022. In national quantitative public opinion surveys, we collected exclusive data from a historical series of polls on the subject in Brazil, and in focus groups, we deepened studies on the perception and political position of different social segments. Amid the growth of scientific denialism; political and social setbacks; and the dismantling of public policies, specifically scientific and technological, resulting from the impeachment of 2016 and the election of Bolsonaro in 2018, research indicates, apparently contrary to an obscurantist political tendency, a significant expansion of public interest in science during the pandemic in the country. This paper analyzes the emergence of a "pro-science wave" in public opinion in Brazil, the factors that led to its emergence during the pandemic, and its current prospects.

JM-20, a Benzodiazepine-Dihydropyridine Hybrid Molecule, Inhibits the Formation of Alpha-Synuclein-Aggregated Species.

Studies showed that JM-20, a benzodiazepine-dihydropyridine hybrid molecule, protects against rotenone and 6-hydroxydopamine neurotoxicity. However, its protective effects against cytotoxicity induced by endogenous neurotoxins involved in Parkinson's disease (PD) pathogenesis have never been investigated. In this study, we evaluated the ability of JM-20 to inhibit alpha-synuclein (aSyn) aggregation. We also evaluated the interactions of JM-20 with aSyn by molecular docking and molecular dynamics and assessed the protective effect of JM-20 against aminochrome cytotoxicity. We demonstrated that JM-20 induced the formation of heterogeneous amyloid fibrils, which were innocuous to primary cultures of mesencephalic cells. Moreover, JM-20 reduced the average size of aSyn positive inclusions in H4 cells transfected with SynT wild-type and synphilin-1-V5, but not in HEK cells transfected with synphilin-1-GFP. In silico studies showed the interaction between JM-20 and the aSyn-binding site. Additionally, we showed that JM-20 protects SH-SY5Y cells against aminochrome cytotoxicity. These results reinforce the potential of JM-20 as a neuroprotective compound for PD and suggest aSyn as a molecular target for JM-20.

Oligomeric α-Synuclein induces skin degeneration in reconstructed human epidermis.

Aged and photoaged skin exhibit fine wrinkles that are signs of epidermal inflammation and degeneration. It has been shown that healthy elderly skin expresses amyloidogenic proteins, including α-Synuclein, which are known to oligomerize and trigger inflammation and neurodegeneration. However, little is known about their putative role in skin physiology and sensitivity. To unravel this possible role, we investigated the impact of oligomeric α-Synuclein (Oα-Syn) in 2D and 3D keratinocyte human models. Exogenous Oα-Syn caused degeneration of reconstructed human epidermis (RHE) by diminishing proliferation and thickness of the stratum basale. Oα-Syn also increased NF-kB nuclear translocation in keratinocytes and triggered inflammation in the RHE, by increasing expression of interleukin-1ß and tumor necrosis factor-alpha, and the release of tumor necrosis factor-alpha in a time-dependent manner. Dexamethasone and an IL-1ß inhibitor partially diminished RHE degeneration caused by Oα-Syn. These findings suggest that Oα-Syn induces epidermal inflammation and decreases keratinocyte proliferation, and therefore might contribute to epidermal degeneration observed in human skin aging.

Peptides derived from gp43, the most antigenic protein from Paracoccidioides brasiliensis, form amyloid fibrils in vitro: implications for vaccine development.

Fungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (mainly P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181-195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that can form amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.

Bioactive Compounds from Kefir and Their Potential Benefits on Health: A Systematic Review and Meta-Analysis.

Despite evidence of health benefits from kefir administration, a systematic review with meta-analysis on bioactive compounds associated with these benefits is still absent in the literature. Kefir is fermented milk resulting from the metabolism of a complex microbiota in symbiosis. Recent researches have investigated the bioactive compounds responsible for the preventive and therapeutic effects attributed to kefir. However, differences in functional potential between industrial and artisanal kefir are still controversial. Firstly, we identified differences in the microbial composition among both types of kefir. Available evidence concerning the action of different bioactive compounds from kefir on health, both from in vitro and in vivo studies, was subsequently summarized to draw a primary conclusion of the dose and the intervention time for effect, the producer microorganisms, the precursor in the milk, and the action mechanism. Meta-analysis was performed to investigate the statistically significant differences (P < 0.05) between intervention and control and between both types of kefir for each health effect studied. In summary, the bioactive compounds more commonly reported were exopolysaccharides, including kefiran, bioactive peptides, and organic acids, especially lactic acid. Kefir bioactive compounds presented antimicrobial, anticancer, and immune-modulatory activities corroborated by the meta-analysis. However, clinical evidence is urgently needed to strengthen the practical applicability of these bioactive compounds. The mechanisms of their action were diverse, indicating that they can act by different signaling pathways. Still, industrial and artisanal kefir may differ regarding functional potential-OR of 8.56 (95% CI: 2.27-32.21, P ≤ .001)-according to the observed health effect, which can be associated with differences in the microbial composition between both types of kefir.

Green Tea Polyphenol Epigallocatechin-Gallate in Amyloid Aggregation and Neurodegenerative Diseases.

The accumulation of protein aggregates in human tissues is a hallmark of more than 40 diseases called amyloidoses. In seven of these disorders, the aggregation is associated with neurodegenerative processes in the central nervous system such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The aggregation occurs when certain soluble proteins lose their physiological function and become toxic amyloid species. The amyloid assembly consists of protein filament interactions, which can form fibrillar structures rich in ß-sheets. Despite the frequent incidence of these diseases among the elderly, the available treatments are limited and at best palliative, and new therapeutic approaches are needed. Among the many natural compounds that have been evaluated for their ability to prevent or delay the amyloidogenic process is epigallocatechin-3-gallate (EGCG), an abundant and potent polyphenolic molecule present in green tea that has extensive biological activity. There is evidence for EGCG's ability to inhibit the aggregation of α-synuclein, amyloid-ß, and huntingtin proteins, respectively associated with PD, AD, and HD. It prevents fibrillogenesis (in vitro and in vivo), reduces amyloid cytotoxicity, and remodels fibrils to form non-toxic amorphous species that lack seed propagation. Although it is an antioxidant, EGCG in an oxidized state can promote fibrils' remodeling through formation of Schiff bases and crosslinking the fibrils. Moreover, microparticles to drug delivery were synthesized from oxidized EGCG and loaded with a second anti-amyloidogenic molecule, obtaining a synergistic therapeutic effect. Here, we describe several pre-clinical and clinical studies involving EGCG and neurodegenerative diseases and their related mechanisms.

New Cardiomyokine Reduces Myocardial Ischemia/Reperfusion Injury by PI3K-AKT Pathway Via a Putative KDEL-Receptor Binding.

Background CDNF (cerebral dopamine neurotrophic factor) belongs to a new family of neurotrophic factors that exert systemic beneficial effects beyond the brain. Little is known about the role of CDNF in the cardiac context. Herein we investigated the effects of CDNF under endoplasmic reticulum-stress conditions using cardiomyocytes (humans and mice) and isolated rat hearts, as well as in rats subjected to ischemia/reperfusion (I/R). Methods and Results We showed that CDNF is secreted by cardiomyocytes stressed by thapsigargin and by isolated hearts subjected to I/R. Recombinant CDNF (exoCDNF) protected human and mouse cardiomyocytes against endoplasmic reticulum stress and restored the calcium transient. In isolated hearts subjected to I/R, exoCDNF avoided mitochondrial impairment and reduced the infarct area to 19% when administered before ischemia and to 25% when administered at the beginning of reperfusion, compared with an infarct area of 42% in the untreated I/R group. This protection was completely abrogated by AKT (protein kinase B) inhibitor. Heptapeptides containing the KDEL sequence, which binds to the KDEL-R (KDEL receptor), abolished exoCDNF beneficial effects, suggesting the participation of KDEL-R in this cardioprotection. CDNF administered intraperitoneally to rats decreased the infarct area in an in vivo model of I/R (from an infarct area of ≈44% in the I/R group to an infarct area of ≈27%). Moreover, a shorter version of CDNF, which lacks the last 4 residues (CDNF-ΔKTEL) and thus allows CDNF binding to KDEL-R, presented no cardioprotective activity in isolated hearts. Conclusions This is the first study to propose CDNF as a new cardiomyokine that induces cardioprotection via KDEL receptor binding and PI3K/AKT activation.

Neutrophil extracellular trap-enriched supernatants carry microRNAs able to modulate TNF-α production by macrophages.

Neutrophil extracellular traps (NETs) emerge from the cell as a DNA scaffold associated with cytoplasmic and granular proteins, able to immobilize and kill pathogens. This association occurs following nuclear and granular membrane disintegration, allowing contact with the decondensed chromatin. Thus, it is reasonable to speculate that the DNA can also mix with miRNAs and carry them in NETs. Here, we report for the first time the presence of the miRNA carriers associated with NETs and miRNAs present in NET-enriched supernatants (NET-miRs), thus adding a novel class of molecules and new proteins that can be released and transported in the NET platform. We observed that the majority of NET-miRs were common to all four stimuli used (PMA, interleukin-8, amyloid fibrils and Leishmania), and that miRNA-142-3p carried by NETs down-modulates protein kinase Cα and regulates TNF-α production in macrophages upon NET interaction with these cells. Our findings unveil a novel role for NETs in the cell communication processes, allowing the conveyance of miRNA from neutrophils to neighboring cells.

Green Tea Polyphenol Microparticles Based on the Oxidative Coupling of EGCG Inhibit Amyloid Aggregation/Cytotoxicity and Serve as a Platform for Drug Delivery.

The accumulation of cross-ß-sheet amyloid fibrils is a hallmark of all human amyloid diseases. The compound epigallocatechin-3-gallate (EGCG), the main polyphenol present in green tea, has been described to have beneficial effects in several pathologies, including amyloidogenic diseases. This polyphenol blocks amyloidogenesis and disaggregates a broad range of amyloidogenic peptides comprising amyloid fibrils in vitro. The mechanism by which EGCG acts in the context of amyloid aggregation is not clear. Most of the biological effects of EGCG are attributable to its antioxidant activity. However, EGCG-oxidized products appear to be sufficient for the majority of EGCG amyloid remodeling observed against some polypeptides. If controlled, EGCG oxidation can afford homogenous microparticles (MPs) and can serve as drug delivery agents. Herein, we produced EGCG MPs by oxidative coupling and analyzed their activity during the aggregation of the protein α-synuclein (α-syn), the main protein related to Parkinson's disease. The MPs modestly remodeled mature amyloid fibrils and efficiently inhibited the amyloidogenic aggregation of α-syn. The MPs showed low cytotoxicity against both dopaminergic cells and microglial cells. The MPs reduced the cytotoxic effects of α-syn oligomers. Interestingly, the MPs were loaded with another antiamyloidogenic compound, increasing their activity against amyloid aggregation. We propose the use of EGCG MPs as a bifunctional strategy, blocking amyloid aggregation directly and carrying a molecule that can act synergistically to alleviate the symptoms caused by the amyloidogenic pathway.

Inflammatory profiling of patients with familial amyloid polyneuropathy.

BACKGROUND: Familial amyloid polyneuropathy (FAP) or ATTRv (amyloid TTR variant) amyloidosis is a fatal hereditary disease characterized by the deposition of amyloid fibrils composed of transthyretin (TTR). The current diagnosis of ATTRv relies on genetic identification of TTR mutations and on Congo Red-positive amyloid deposits, which are absent in most ATTRv patients that are asymptomatic or early symptomatic, supporting the need for novel biomarkers to identify patients in earlier disease phases allowing disease control. METHODS: In an effort to search for new markers for ATTRv, our group searched for nine inflammation markers in ATTRv serum from a cohort of 28 Brazilian ATTRv patients. RESULTS: We found that the levels of six markers were increased (TNF-α, IL-1ß, IL-8, IL-33, IFN-ß and IL-10), one had decreased levels (IL-12) and two of them were unchanged (IL-6 and cortisol). Interestingly, asymptomatic patients already presented high levels of IL-33, IL-1ß and IL-10, suggesting that inflammation may take place before fibril deposition. CONCLUSIONS: Our findings shed light on a new, previously unidentified aspect of ATTRv, which might help define new criteria for disease management, as well as provide additional understanding of ATTRv aggressiveness.

Baseline disease characteristics in Brazilian patients enrolled in Transthyretin Amyloidosis Outcome Survey (THAOS).

Transthyretin amyloidosis (ATTR) is characterized by the deposit of mutant or wild-type transthyretin that forms amyloid fibrils, which are extracellularly deposited within tissues and organs. Clinical manifestations of familial amyloid polyneuropathy vary according to the mutation, age at onset and geographical location. This study aimed to describe baseline disease characteristics of Brazilian patients with transthyretin familial amyloid polyneuropathy (ATTR-FAP) enrolled in the Transthyretin Amyloidosis Outcome Survey (THAOS). METHODS: The THAOS is an international, noninterventional, longitudinal, observational, web-based registry designed to characterize ATTR. The outcome measures included demographics (age at symptom onset, gender, time from onset of symptoms to diagnosis, family history), genotype, and clinical characteristics (presence of amyloid deposit, frequency of misdiagnosis, presenting symptomatology). The analysis was conducted in a dataset from Brazilian patients (from November 2008 to January 2016). RESULTS: One hundred and sixty participants (52.5% male) were included in the analysis. The majority of participants (90.6%) reported a positive family history of ATTR-FAP Median age at symptom onset was 32.5 years. Val30Met mutation was found in 91.9%. Misdiagnosis was observed in 26.6% of symptomatic patients. Over one-third (35.3%) of the misdiagnosed patients experienced a delay of more than one year before receiving a correct diagnosis. At presentation, 79.7% of the patients had motor, 87.5% sensory and 93.8% autonomic symptoms. CONCLUSION: ATTR-FAP in Brazil starts early, has a strong family history and the majority has Val30Met mutation. Misdiagnosis is common and the most common presentation is of a sensorimotor and autonomic neuropathy.

Baseline disease characteristics in Brazilian patients enrolled in Transthyretin Amyloidosis Outcome Survey (THAOS) / Características da polineuropatia amiloidotica familiar ligada à transtirretina em pacientes brasileiros participantes do "Transthyretin Amyloidosis Outcome Survey (THAOS)"

ABSTRACT Transthyretin amyloidosis (ATTR) is characterized by the deposit of mutant or wild-type transthyretin that forms amyloid fibrils, which are extracellularly deposited within tissues and organs. Clinical manifestations of familial amyloid polyneuropathy vary according to the mutation, age at onset and geographical location. This study aimed to describe baseline disease characteristics of Brazilian patients with transthyretin familial amyloid polyneuropathy (ATTR-FAP) enrolled in the Transthyretin Amyloidosis Outcome Survey (THAOS). Methods: The THAOS is an international, noninterventional, longitudinal, observational, web-based registry designed to characterize ATTR. The outcome measures included demographics (age at symptom onset, gender, time from onset of symptoms to diagnosis, family history), genotype, and clinical characteristics (presence of amyloid deposit, frequency of misdiagnosis, presenting symptomatology). The analysis was conducted in a dataset from Brazilian patients (from November 2008 to January 2016). Results: One hundred and sixty participants (52.5% male) were included in the analysis. The majority of participants (90.6%) reported a positive family history of ATTR-FAP Median age at symptom onset was 32.5 years. Val30Met mutation was found in 91.9%. Misdiagnosis was observed in 26.6% of symptomatic patients. Over one-third (35.3%) of the misdiagnosed patients experienced a delay of more than one year before receiving a correct diagnosis. At presentation, 79.7% of the patients had motor, 87.5% sensory and 93.8% autonomic symptoms. Conclusion: ATTR-FAP in Brazil starts early, has a strong family history and the majority has Val30Met mutation. Misdiagnosis is common and the most common presentation is of a sensorimotor and autonomic neuropathy.

RESUMO Amiloidose ligada à transtirretina (ATTR) é caracterizada por depósito de transtirretina que forma fibrilas amiloides, que são depositadas extracelularmente dentro de tecidos e órgãos. As manifestações clínicas de polineuropatia amiloidótica familiar (ATTR-PAF) variam de acordo com a mutação, idade de início e localização geográfica. Este estudo tem como objetivo descrever as características dos pacientes com ATTR no Brasil, com base nos dados coletados no THAOS. Métodos: THAOS é um registro internacional longitudinal observacional desenhado para caracterizar ATTR. As medidas de desfecho incluíram dados demográficos (idade do início dos sintomas, gênero, tempo do início dos sintomas até diagnóstico, histórico familiar), genotipagem e características clínicas (presença de depósito amiloide, frequências de diagnósticos errôneos, sintomatologia presente). Esta analise foi conduzida com dados de pacientes brasileiros registrados no THAOS de Novembro 2008 a Janeiro de 2016. Resultado: Cento e sessenta pacientes (52,5% homens) foram incluídos na análise. Na maioria dos casos (90,6%) observou-se história familiar positiva de ATTR-FAP A idade média de inicio dos sintomas foi 32,5 anos. A mutação Val30Met foi encontrada em 91,9%. Erros diagnósticos foram observados em 26,6% dos casos sintomáticos. Aproximadamente um terço dos pacientes diagnosticados erroneamente tiveram atraso de mais de um ano para receber um diagnostico correto. No momento do diagnóstico 79,7% dos pacientes possuíam sintomas motores, 87,5% sintomas sensitivos e 93,8% sintomas autonômicos. Conclusão: No brasil a ATTR-FAP tem início precoce, historia familiar fortemente positiva e em sua maioria são portadores da mutação Val30Met. Erros diagnósticos são comuns e a apresentação mais comum é polineuropatia sensitivo-motora com disautonomia.

Brain infusion of α-synuclein oligomers induces motor and non-motor Parkinson's disease-like symptoms in mice.

Parkinson's disease (PD) is characterized by motor dysfunction, which is preceded by a number of non-motor symptoms including olfactory deficits. Aggregation of α-synuclein (α-syn) gives rise to Lewy bodies in dopaminergic neurons and is thought to play a central role in PD pathology. However, whether amyloid fibrils or soluble oligomers of α-syn are the main neurotoxic species in PD remains controversial. Here, we performed a single intracerebroventricular (i.c.v.) infusion of α-syn oligomers (α-SYOs) in mice and evaluated motor and non-motor symptoms. Familiar bedding and vanillin essence discrimination tasks showed that α-SYOs impaired olfactory performance of mice, and decreased TH and dopamine levels in the olfactory bulb early after infusion. The olfactory deficit persisted until 45days post-infusion (dpi). α- SYO-infused mice behaved normally in the object recognition and forced swim tests, but showed increased anxiety-like behavior in the open field and elevated plus maze tests 20 dpi. Finally, administration of α-SYOs induced late motor impairment in the pole test and rotarod paradigms, along with reduced TH and dopamine content in the caudate putamen, 45 dpi. Reduced number of TH-positive cells was also seen in the substantia nigra of α-SYO-injected mice compared to control. In conclusion, i.c.v. infusion of α-SYOs recapitulated some of PD-associated non-motor symptoms, such as increased anxiety and olfactory dysfunction, but failed to recapitulate memory impairment and depressive-like behavior typical of the disease. Moreover, α-SYOs i.c.v. administration induced motor deficits and loss of TH and dopamine levels, key features of PD. Results point to α-syn oligomers as the proximal neurotoxins responsible for early non-motor and motor deficits in PD and suggest that the i.c.v. infusion model characterized here may comprise a useful tool for identification of PD novel therapeutic targets and drug screening.

Applying an artificial neural network model for developing a severity score for patients with hereditary amyloid polyneuropathy.

Hereditary (familial) amyloid polyneuropathy (FAP) is a systemic disease that includes a sensorimotor polyneuropathy related to transthyretin (TTR) mutations. So far, a scale designed to classify the severity of this disease has not yet been validated. This work proposes the implementation of an artificial neural network (ANN) in order to develop a severity scale for monitoring the disease progression in FAP patients. In order to achieve this goal, relevant symptoms and laboratory findings were collected from 98 Brazilian patients included in THAOS - the Transthyretin Amyloidosis Outcomes Survey. Ninety-three percent of them bore Val30Met, the most prevalent variant of TTR worldwide; 63 were symptomatic and 35 were asymptomatic. These data were numerically codified for the purpose of constructing a Self-Organizing Map (SOM), which maps data onto a grid of artificial neurons. Mapped data could be clustered by similarity into five groups, based on increasing FAP severity (from Groups 1 to 5). Most symptoms were virtually absent from patients who mapped to Group 1, which also includes the asymptomatic patients. Group 2 encompasses the patients bearing symptoms considered to be initial markers of FAP, such as first signs of walking disabilities and lack of sensitivity to temperature and pain. Interestingly, the patients with cardiac symptoms, which also carry cardiac-associated mutations of the TTR gene (such as Val112Ile and Ala19Asp), were concentrated in Group 3. Symptoms such as urinary and fecal incontinence and diarrhea characterized particularly Groups 4 and 5. Renal impairment was found almost exclusively in Group 5. Model validation was accomplished by considering the symptoms from a sample with 48 additional Brazilian patients. The severity scores proposed here not only identify the current stage of a patient's disease but also offer to the physician an easy-to-read, 2D map that makes it possible to track disease progression.

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.

Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's Amyloid-ß Oligomers in Mice.

Considerable clinical and epidemiological evidence links Alzheimer's disease (AD) and depression. However, the molecular mechanisms underlying this connection are largely unknown. We reported recently that soluble Aß oligomers (AßOs), toxins that accumulate in AD brains and are thought to instigate synapse damage and memory loss, induce depressive-like behavior in mice. Here, we report that the mechanism underlying this action involves AßO-induced microglial activation, aberrant TNF-α signaling, and decreased brain serotonin levels. Inactivation or ablation of microglia blocked the increase in brain TNF-α and abolished depressive-like behavior induced by AßOs. Significantly, we identified serotonin as a negative regulator of microglial activation. Finally, AßOs failed to induce depressive-like behavior in Toll-like receptor 4-deficient mice and in mice harboring a nonfunctional TLR4 variant in myeloid cells. Results establish that AßOs trigger depressive-like behavior via a double impact on brain serotonin levels and microglial activation, unveiling a cross talk between brain innate immunity and serotonergic signaling as a key player in mood alterations in AD. SIGNIFICANCE STATEMENT: Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the main cause of dementia in the world. Brain accumulation of amyloid-ß oligomers (AßOs) is a major feature in the pathogenesis of AD. Although clinical and epidemiological data suggest a strong connection between AD and depression, the underlying mechanisms linking these two disorders remain largely unknown. Here, we report that aberrant activation of the brain innate immunity and decreased serotonergic tonus in the brain are key players in AßO-induced depressive-like behavior in mice. Our findings may open up new possibilities for the development of effective therapeutics for AD and depression aimed at modulating microglial function.