Loss of TDP-43 oligomerization or RNA binding elicits distinct aggregation patterns.

Aggregation of the RNA-binding protein TAR DNA-binding protein 43 (TDP-43) is the key neuropathological feature of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In physiological conditions, TDP-43 is predominantly nuclear, forms oligomers, and is contained in biomolecular condensates assembled by liquid-liquid phase separation (LLPS). In disease, TDP-43 forms cytoplasmic or intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Using a variety of cellular systems to express structure-based TDP-43 variants, including human neurons and cell lines with near-physiological expression levels, we show that oligomerization and RNA binding govern TDP-43 stability, splicing functionality, LLPS, and subcellular localization. Importantly, our data reveal that TDP-43 oligomerization is modulated by RNA binding. By mimicking the impaired proteasomal activity observed in ALS/FTLD patients, we found that monomeric TDP-43 forms inclusions in the cytoplasm, whereas its RNA binding-deficient counterpart aggregated in the nucleus. These differentially localized aggregates emerged via distinct pathways: LLPS-driven aggregation in the nucleus and aggresome-dependent inclusion formation in the cytoplasm. Therefore, our work unravels the origins of heterogeneous pathological species reminiscent of those occurring in TDP-43 proteinopathy patients.

ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.

Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3ß decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.

Acetylation targets mutant huntingtin to autophagosomes for degradation.

Huntington's disease (HD) is an incurable neurodegenerative disease caused by neuronal accumulation of the mutant protein huntingtin. Improving clearance of the mutant protein is expected to prevent cellular dysfunction and neurodegeneration in HD. We report here that such clearance can be achieved by posttranslational modification of the mutant Huntingtin (Htt) by acetylation at lysine residue 444 (K444). Increased acetylation at K444 facilitates trafficking of mutant Htt into autophagosomes, significantly improves clearance of the mutant protein by macroautophagy, and reverses the toxic effects of mutant huntingtin in primary striatal and cortical neurons and in a transgenic C. elegans model of HD. In contrast, mutant Htt that is rendered resistant to acetylation dramatically accumulates and leads to neurodegeneration in cultured neurons and in mouse brain. These studies identify acetylation as a mechanism for removing accumulated protein in HD, and more broadly for actively targeting proteins for degradation by autophagy.

ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport.

Maintenance of cellular proteostasis relies on efficient clearance of defective gene products. For misfolded secretory proteins, this involves dislocation from the endoplasmic reticulum (ER) into the cytosol followed by proteasomal degradation. However, polypeptide aggregation prevents cytosolic dislocation and instead activates ill-defined lysosomal catabolic pathways. Here, we describe an ER-to-lysosome-associated degradation pathway (ERLAD) for proteasome-resistant polymers of alpha1-antitrypsin Z (ATZ). ERLAD involves the ER-chaperone calnexin (CNX) and the engagement of the LC3 lipidation machinery by the ER-resident ER-phagy receptor FAM134B, echoing the initiation of starvation-induced, receptor-mediated ER-phagy. However, in striking contrast to ER-phagy, ATZ polymer delivery from the ER lumen to LAMP1/RAB7-positive endolysosomes for clearance does not require ER capture within autophagosomes. Rather, it relies on vesicular transport where single-membrane, ER-derived, ATZ-containing vesicles release their luminal content within endolysosomes upon membrane:membrane fusion events mediated by the ER-resident SNARE STX17 and the endolysosomal SNARE VAMP8. These results may help explain the lack of benefits of pharmacologic macroautophagy enhancement that has been reported for some luminal aggregopathies.

Epithelial growth factor receptor expression influences 5-ALA induced glioblastoma fluorescence.

The extent of 5-aminolevulinic acid (5-ALA) guided tumor resection has a determining impact in high-grade glioma and glioblastoma surgery. Yet the intensity of the 5-ALA induced fluorescence may vary within the tumor. We aimed to correlate 5-ALA induced fluorescence with the expression of epithelial growth factor receptor (EGFR) and its constitutively active version EGFRvIII in different glioblastoma (GBM) cell lines. To elucidate the role of EGFR in the metabolism of 5-ALA in GBM cell lines with variable EGFR expression status, we analyzed the activation of EGFR by its primary ligand EGF, and its downstream effect on Heme oxygenase-1 (HO-1), a key enzyme regulating the metabolism of Protoporphyrin IX (PpIX), the fluorescent metabolite of 5-ALA. Effects of direct pharmacological inhibition by Tin(IV)-Protoporphyrin (SnPP) or gene knockdown by small interfering RNA (siRNA) on HO-1 enzyme were analyzed in respect to 5-ALA induced fluorescence. Furthermore, inhibition of EGFR by Gefitinib was tested. A significant difference in 5-ALA induced fluorescence was obtained in U87MG (low EGFR expression) and LN229EGFR cells (EGFR overexpression) compared to BS153 (EGFR overexpression/EGFRvIII+). Treatment of U87MG and LN229EGFR cells with EGF significantly reduced cellular fluorescence, by promoting HO-1 transcription and expression in a concentration-dependent manner. This effect could be reversed by EGFR-specific siRNA treatment, which reduced protein expression of about 80% in U87MG. Remarkably, inhibition of HO-1 activity by SnPP or reduction of HO-1 protein levels by siHO-1 treatment restored fluorescence in all cell lines, independently of EGFR quantitative and qualitative expression. Gefitinib treatment was able to restore fluorescence after EGF stimulation in U87MG cells but not in BS153 cells, overexpressing EGFR/EGFRvIII. In GBM cell lines, 5-ALA induced fluorescence is variable and influenced by EGF-induced downstream activation of HO-1. HO-1 protein expression was identified as a negative regulator of 5-ALA induced fluorescence in GBM cells. We further propose that co-expression of EGFRvIII but not quantitative EGFR expression influence HO-1 activity and therefore cellular fluorescence.

Synthesis and structure-activity relationship of 2,6-disubstituted pyridine derivatives as inhibitors of ß-amyloid-42 aggregation.

It is assumed that amyloid-ß aggregation is a crucial event in the pathogenesis of Alzheimer's disease. Novel 2,6-disubstituted pyridine derivatives were designed to interact with the ß-sheet conformation of Aß via donor-acceptor-donor hydrogen bond formation. A series of pyridine derivatives were synthesized and tested regarding their potential to inhibit the aggregation of Aß. The 2,6-diaminopyridine moiety was identified as a key component to inhibit Aß aggregation. Overall, compounds having three 2,6-disubstituted pyridine units separated by at least one C2- or C3-linker displayed the most potent inhibition of Aß aggregation.

Human genome-guided identification of memory-modulating drugs.

In the last decade there has been an exponential increase in knowledge about the genetic basis of complex human traits, including neuropsychiatric disorders. It is not clear, however, to what extent this knowledge can be used as a starting point for drug identification, one of the central hopes of the human genome project. The aim of the present study was to identify memory-modulating compounds through the use of human genetic information. We performed a multinational collaborative study, which included assessment of aversive memory--a trait central to posttraumatic stress disorder--and a gene-set analysis in healthy individuals. We identified 20 potential drug target genes in two genomewide-corrected gene sets: the neuroactive ligand-receptor interaction and the long-term depression gene set. In a subsequent double-blind, placebo-controlled study in healthy volunteers, we aimed at providing a proof of concept for the genome-guided identification of memory modulating compounds. Pharmacological intervention at the neuroactive ligand-receptor interaction gene set led to significant reduction of aversive memory. The findings demonstrate that genome information, along with appropriate data mining methodology, can be used as a starting point for the identification of memory-modulating compounds.

A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein.

Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.

Discovery and structure activity relationship of small molecule inhibitors of toxic ß-amyloid-42 fibril formation.

Increasing evidence implicates Aß peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aß aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aß42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the ß-sheet conformation of Aß42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aß42. The efficacy of these compounds on inhibiting Aß fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aß42 leading to decreased cell toxicity.

Suppression of protein aggregation by chaperone modification of high molecular weight complexes.

Protein misfolding and aggregation are associated with many neurodegenerative diseases, including Huntington's disease. The cellular machinery for maintaining proteostasis includes molecular chaperones that facilitate protein folding and reduce proteotoxicity. Increasing the protein folding capacity of cells through manipulation of DNAJ chaperones has been shown to suppress aggregation and ameliorate polyglutamine toxicity in cells and flies. However, to date these promising findings have not been translated to mammalian models of disease. To address this issue, we developed transgenic mice that over-express the neuronal chaperone HSJ1a (DNAJB2a) and crossed them with the R6/2 mouse model of Huntington's disease. Over-expression of HSJ1a significantly reduced mutant huntingtin aggregation and enhanced solubility. Surprisingly, this was mediated through specific association with K63 ubiquitylated, detergent insoluble, higher order mutant huntingtin assemblies that decreased their ability to nucleate further aggregation. This was dependent on HSJ1a client binding ability, ubiquitin interaction and functional co-operation with HSP70. Importantly, these changes in mutant huntingtin solubility and aggregation led to improved neurological performance in R6/2 mice. These data reveal that prevention of further aggregation of detergent insoluble mutant huntingtin is an additional level of quality control for late stage chaperone-mediated neuroprotection. Furthermore, our findings represent an important proof of principle that DNAJ manipulation is a valid therapeutic approach for intervention in Huntington's disease.

Cancer-specific association between Tau (MAPT) and cellular pathways, clinical outcome, and drug response.

Tau (MAPT) is a microtubule-associated protein causing common neurodegenerative diseases or rare inherited frontotemporal lobar degenerations. Emerging evidence for non-canonical functions of Tau in DNA repair and P53 regulation suggests its involvement in cancer. To bring new evidence for a relevant role of Tau in cancer, we carried out an in-silico pan-cancer analysis of MAPT transcriptomic profile in over 10000 clinical samples from 32 cancer types and over 1300 pre-clinical samples from 28 cancer types provided by the TCGA and the DEPMAP datasets respectively. MAPT expression associated with key cancer hallmarks including inflammation, proliferation, and epithelial to mesenchymal transition, showing cancer-specific patterns. In some cancer types, MAPT functional networks were affected by P53 mutational status. We identified new associations of MAPT with clinical outcomes and drug response in a context-specific manner. Overall, our findings indicate that the MAPT gene is a potential major player in multiple types of cancer. Importantly, the impact of Tau on cancer seems to be heavily influenced by the specific cellular environment.

Tau accumulation in degradative organelles is associated to lysosomal stress.

Neurodegenerative disorders are characterized by the brain deposition of insoluble amyloidogenic proteins, such as α-synuclein or Tau, and the concomitant deterioration of cell functions such as the autophagy-lysosomal pathway (ALP). The ALP is involved in the degradation of intracellular macromolecules including protein aggregates. ALP dysfunction due to inherited defects in lysosomal or non-lysosomal proteins causes a group of diseases called lysosomal storage disorders (LSD) because of abnormal accumulation of lysosomal degradation substrates. Supporting the contribution of ALP defects in neurodegenerative diseases, deposition of amyloidogenic proteins occurs in LSD. Moreover, heterozygous mutations of several ALP genes represent risk factors for Parkinson's disease. The reciprocal contribution of α-synuclein accumulation and lysosomal dysfunction have been extensively studied. However, whether this adverse crosstalk also embraces Tau pathology needs more investigation. Here, we show in human primary fibroblasts that Tau seeds isolated from the brain of Alzheimer's disease induce Tau accumulation in acidic degradative organelles and lysosomal stress. Furthermore, inhibition of glucocerebrosidase, a lysosomal enzyme mutated in Gaucher's disease and a main risk for Parkinson's disease, causes lysosomal dysfunction in primary fibroblasts and contributes to the accumulation of Tau. Considering the presence of Tau lesions in Parkinson's disease as well as in multiple neurodegenerative disorders including Alzheimer's disease, our data call for further studies on strategies to alleviate ALP dysfunction as new therapeutic opportunity for neurodegenerative diseases and LSD.

Tau protein modulates an epigenetic mechanism of cellular senescence in human SH-SY5Y neuroblastoma cells.

Introduction: Progressive Tau deposition in neurofibrillary tangles and neuropil threads is the hallmark of tauopathies, a disorder group that includes Alzheimer's disease. Since Tau is a microtubule-associated protein, a prevalent concept to explain the pathogenesis of tauopathies is that abnormal Tau modification contributes to dissociation from microtubules, assembly into multimeric ß-sheets, proteotoxicity, neuronal dysfunction and cell loss. Tau also localizes in the cell nucleus and evidence supports an emerging function of Tau in DNA stability and epigenetic modulation. Methods: To better characterize the possible role of Tau in regulation of chromatin compaction and subsequent gene expression, we performed a bioinformatics analysis of transcriptome data obtained from Tau-depleted human neuroblastoma cells. Results: Among the transcripts deregulated in a Tau-dependent manner, we found an enrichment of target genes for the polycomb repressive complex 2. We further describe decreased cellular amounts of the core components of the polycomb repressive complex 2 and lower histone 3 trimethylation in Tau deficient cells. Among the de-repressed polycomb repressive complex 2 target gene products, IGFBP3 protein was found to be linked to increased senescence induction in Tau-deficient cells. Discussion: Our findings propose a mechanism for Tau-dependent epigenetic modulation of cell senescence, a key event in pathologic aging.

Tau protein binds to the P53 E3 ubiquitin ligase MDM2.

Tau gene mutations cause a progressive dementia and neurotoxic Tau forms deposited in neurofibrillary tangles are hallmarks of neurodegenerative tauopathies. Loss of non-canonical Tau functions may contribute to disease. In fact, Tau depletion affects the cellular response to DNA damage and tauopathies exhibit the accumulation of DNA lesions. Moreover, Tau modulates P53 activity and cell fate. Considering that MDM2 is the main antagonist of P53, we investigated, using orthogonal assays, if Tau interacts with MDM2. We report the existence in cells and brain of a Tau-MDM2 complex that, in vitro, exhibits reduced P53 ubiquitination activity in a manner sensitive to a Tau mutation. The Tau-MDM2 interaction involves the microtubule-binding domain of Tau and the acidic domain of MDM2, reminiscent of the binding of Tau to negatively charged microtubules. Notably, MDM2 accumulates aberrantly in neurofibrillary tangles. Aging-associated insults may expose a novel loss-of-function of Tau in neurodegeneration and cancer.

Whole-brain microscopy reveals distinct temporal and spatial efficacy of anti-Aß therapies.

Many efforts targeting amyloid-ß (Aß) plaques for the treatment of Alzheimer's Disease thus far have resulted in failures during clinical trials. Regional and temporal heterogeneity of efficacy and dependence on plaque maturity may have contributed to these disappointing outcomes. In this study, we mapped the regional and temporal specificity of various anti-Aß treatments through high-resolution light-sheet imaging of electrophoretically cleared brains. We assessed the effect on amyloid plaque formation and growth in Thy1-APP/PS1 mice subjected to ß-secretase inhibitors, polythiophenes, or anti-Aß antibodies. Each treatment showed unique spatiotemporal Aß clearance, with polythiophenes emerging as a potent anti-Aß compound. Furthermore, aligning with a spatial-transcriptomic atlas revealed transcripts that correlate with the efficacy of each Aß therapy. As observed in this study, there is a striking dependence of specific treatments on the location and maturity of Aß plaques. This may also contribute to the clinical trial failures of Aß-therapies, suggesting that combinatorial regimens may be significantly more effective in clearing amyloid deposition.

Identical oligomeric and fibrillar structures captured from the brains of R6/2 and knock-in mouse models of Huntington's disease.

Huntington's disease (HD) is a late-onset neurodegenerative disorder that is characterized neuropathologically by the presence of neuropil aggregates and nuclear inclusions. However, the profile of aggregate structures that are present in the brains of HD patients or of HD mouse models and the relative contribution of specific aggregate structures to disease pathogenesis is unknown. We have used the Seprion ligand to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA)-based method for quantifying aggregated polyglutamine in tissues from HD mouse models. We used a combination of electron microscopy, atomic force microscopy (AFM) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to investigate the aggregate structures isolated by the ligand. We found that the oligomeric, proto-fibrillar and fibrillar aggregates extracted from the brains of R6/2 and HdhQ150 knock-in mice were remarkably similar. Using AFM, we determined that the nanometre globular oligomers isolated from the brains of both mouse models have dimensions identical to those generated from recombinant huntingtin exon 1 proteins. Finally, antibodies that detect exon 1 Htt epitopes differentially recognize the ligand-captured material on SDS-PAGE gels. The Seprion-ligand ELISA provides an assay with good statistical power for use in preclinical pharmacodynamic therapeutic trials or to assess the effects of the genetic manipulation of potential therapeutic targets on aggregate load. This, together with the ability to identify a spectrum of aggregate species in HD mouse tissues, will contribute to our understanding of how these structures relate to the pathogenesis of HD and whether their formation can be manipulated for therapeutic benefit.

Full-length huntingtin levels modulate body weight by influencing insulin-like growth factor 1 expression.

Levels of full-length huntingtin (FL htt) influence organ and body weight, independent of polyglutamine length. The growth hormone-insulin like growth factor-1 (GH-IGF-1) axis is well established as a regulator of organ growth and body weight. In this study, we investigate the involvement of the IGF-1 pathway in mediating the effect of htt on body weight. IGF-1 expression was examined in transgenic mouse lines expressing different levels of FL wild-type (WT) htt (YAC18 mice), FL mutant htt (YAC128 and BACHD mice) and truncated mutant htt (shortstop mice). We demonstrate that htt influences body weight by modulating the IGF-1 pathway. Plasma IGF-1 levels correlate with body weight and htt levels in the transgenic YAC mice expressing human htt. The effect of htt on IGF-1 expression is independent of CAG size. No effect on body weight is observed in transgenic YAC mice expressing a truncated N-terminal htt fragment (shortstop), indicating that FL htt is required for the modulation of IGF-1 expression. Treatment with 17beta-estradiol (17beta-ED) lowers the levels of circulating IGF-1 in mammals. Treatment of YAC128 with 17beta-ED, but not placebo, reduces plasma IGF-1 levels and decreases the body weight of YAC128 animals to WT levels. Furthermore, given the ubiquitous expression of IGF-1 within the central nervous system, we also examined the impact of FL htt levels on IGF-1 expression in different regions of the brain, including the striatum, cerebellum of YAC18, YAC128 and littermate WT mice. We demonstrate that the levels of FL htt influence IGF-1 expression in striatal tissues. Our data identify a novel function for FL htt in influencing IGF-1 expression.

Proteolysis of mutant huntingtin produces an exon 1 fragment that accumulates as an aggregated protein in neuronal nuclei in Huntington disease.

Huntingtin proteolysis has been implicated in the molecular pathogenesis of Huntington disease (HD). Despite an intense effort, the identity of the pathogenic smallest N-terminal fragment has not been determined. Using a panel of anti-huntingtin antibodies, we employed an unbiased approach to generate proteolytic cleavage maps of mutant and wild-type huntingtin in the HdhQ150 knock-in mouse model of HD. We identified 14 prominent N-terminal fragments, which, in addition to the full-length protein, can be readily detected in cytoplasmic but not nuclear fractions. These fragments were detected at all ages and are not a consequence of the pathogenic process. We demonstrated that the smallest fragment is an exon 1 huntingtin protein, known to contain a potent nuclear export signal. Prior to the onset of behavioral phenotypes, the exon 1 protein, and possibly other small fragments, accumulate in neuronal nuclei in the form of a detergent insoluble complex, visualized as diffuse granular nuclear staining in tissue sections. This methodology can be used to validate the inhibition of specific proteases as therapeutic targets for HD by pharmacological or genetic approaches.

Induction of autophagy with catalytic mTOR inhibitors reduces huntingtin aggregates in a neuronal cell model.

Huntington's disease is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. This expansion produces a mutant form of the huntingtin protein, which contains an elongated polyglutamine stretch at its amino-terminus. Mutant huntingtin may adopt an aberrant, aggregation-prone conformation predicted to start the pathogenic process leading to neuronal dysfunction and cell death. Thus, strategies reducing mutant huntingtin may lead to disease-modifying therapies. We investigated the mechanisms and molecular targets regulating huntingtin degradation in a neuronal cell model. We first found that mutant and wild-type huntingtin displayed strikingly diverse turn-over kinetics and sensitivity to proteasome inhibition. Then, we show that autophagy induction led to accelerate degradation of mutant huntingtin aggregates. In our neuronal cell model, allosteric inhibition of mTORC1 by everolimus, a rapamycin analogue, did not induce autophagy or affect aggregate degradation. In contrast, this occurred in the presence of catalytic inhibitors of both mTOR complexes mTORC1 and mTORC2. Our data demonstrate the existence of an mTOR-dependent but everolimus-independent mechanism regulating autophagy and huntingtin-aggregate degradation in cells of neuronal origin.

Microtiter plate quantification of mutant and wild-type huntingtin normalized to cell count.

Huntington's disease is caused by a gain-of-function neurotoxic mutation in normally neuroprotective huntingtin. Sensitive assays are required to discriminate mutant huntingtin from wild-type huntingtin. We have developed a normalized 384-plate assay for determination of mutant and wild-type huntingtin. Based on a single pipetting step, the sensitive assay uses two antibody pairs for simultaneous mutant and wild-type huntingtin time-resolved fluorescence resonance energy transfer detection combined with PicoGreen quantification of double-stranded DNA. The assay can be used for discovery of drugs reducing mutant huntingtin over wild-type huntingtin and for assessing the value of huntingtin as a disease progression marker, and it is adaptable to other proteins of interest.