mHTT Seeding Activity: A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease.

Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.

The polyphenol EGCG directly targets intracellular amyloid-ß aggregates and promotes their lysosomal degradation.

The accumulation of amyloidogenic protein aggregates in neurons is a pathogenic hallmark of a large number of neurodegenerative diseases including Alzheimer's disease (AD). Small molecules targeting such structures and promoting their degradation may have therapeutic potential for the treatment of AD. Here, we searched for natural chemical compounds that decrease the abundance of stable, proteotoxic ß-sheet-rich amyloid-ß (Aß) aggregates in cells. We found that the polyphenol (-)-epigallocatechin gallate (EGCG) functions as a potent chemical aggregate degrader in SH-EP cells. We further demonstrate that a novel, fluorescently labeled EGCG derivative (EGC-dihydroxybenzoate (DHB)-Rhodamine) also shows cellular activity. It directly targets intracellular Aß42 aggregates and competes with EGCG for Aß42 aggregate binding in vitro. Mechanistic investigations indicated a lysosomal accumulation of Aß42 aggregates in SH-EP cells and showed that lysosomal cathepsin activity is critical for efficient EGCG-mediated aggregate clearance. In fact, EGCG treatment leads to an increased abundance of active cathepsin B isoforms and increased enzymatic activity in our SH-EP cell model. Our findings suggest that intracellular Aß42 aggregates are cleared through the endo-lysosomal system. We show that EGCG directly targets intracellular Aß42 aggregates and facilitates their lysosomal degradation. Small molecules, which bind to protein aggregates and increase their lysosomal degradation could have therapeutic potential for the treatment of amyloid diseases.

LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells.

Information on protein-protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double-readout bioluminescence-based two-hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence-based co-precipitation (LuC). The double-readout procedure detects interactions with higher sensitivity than traditional single-readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease-causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult-onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease-causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease-associated mutations impair protein activity in biological systems.

Ação da estimulação elétrica nervosa transcutânea sobre o limiar de dor induzido por pressão / Transcutaneous electrical nerve stimulation action on pressure-induced pain threshold

JUSTIFICATIVA E OBJETIVOS: A estimulação elétrica nervosa transcutânea (TENS) é uma correntede baixa frequência, utilizada para tratamento de dor, contudo há lacunas com respeito aos parâmetros de uso, bem como do modo de ação. O objetivo deste estudo foi investigar os efeitos da TENS, na forma burst, sobre ador induzida por pressão.MÉTODO: Estudo duplamente encoberto e cruzado, com 23 indivíduos, de ambos os sexos, alocados em grupo TENS (GT) e grupo placebo (GP). A TENS burst, com duração de fase de 200 µs e com intensidade regulada até o limiar motor foi aplicada em duas regiões dos nervos mediano e ulnar, ambos na região supracondiliana, por 15 minutos. Para avaliação do limiar doloroso foi aplicado um dolorímetro de pressão sobre as regiões tenar e hipotenar nos seguintes períodos:antes e imediatamente após a estimulação, 20 minutos e 1 hora após o término da eletroestimulação.RESULTADOS: Por meio da avaliação do limiar doloroso da região tenar e hipotenar, observou-se que não houve diferença significativa quando comparados intragrupo e entre os grupos, para todos os momentos da avaliação.CONCLUSÃO: O uso de TENS na forma burst não foi eficaz para elevar o limiar na dor induzida por pressão.

BACKGROUND AND OBJECTIVES: Transcutaneous electrical nerve stimulation (TENS) is a low frequency current used to manage pain, however, there are gaps with regard to its usage parameters and way of action. This study aimed at investigating burst TENS effects on pressure-induced pain.METHOD: Double blind crossed study with 23 individuals of both genders, divided into TENS (GT) and placebo (GP) groups. Burst TENS, with 200 µs phaseduration and intensity regulated until motor threshold, was applied in two median and ulnar nerves regions,both in the supracondilar region, for 15 minutes. Pressure dolorimeter was applied to tenar and hypotenar regions to evaluate pain threshold in the following periods: before and immediately after stimulation, 20 minutes and one hour after electrical stimulation.RESULTS: Evaluating the pain threshold of the tenar and hypotenar region, there has been no significant difference when compared intragroup and between groups, for all evaluated moments.CONCLUSION: Burst TENS was not effective to increase pressure-induced pain threshold.