An Expanded Polyproline Domain Maintains Mutant Huntingtin Soluble in vivo and During Aging.

Huntington's disease is a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat, encoding for the amino acid glutamine (Q), present in the first exon of the protein huntingtin. Over the threshold of Q39 HTT exon 1 (HTTEx1) tends to misfold and aggregate into large intracellular structures, but whether these end-stage aggregates or their on-pathway intermediates are responsible for cytotoxicity is still debated. HTTEx1 can be separated into three domains: an N-terminal 17 amino acid region, the polyglutamine (polyQ) expansion and a C-terminal proline rich domain (PRD). Alongside the expanded polyQ, these flanking domains influence the aggregation propensity of HTTEx1: with the N17 initiating and promoting aggregation, and the PRD modulating it. In this study we focus on the first 11 amino acids of the PRD, a stretch of pure prolines, which are an evolutionary recent addition to the expanding polyQ region. We hypothesize that this proline region is expanding alongside the polyQ to counteract its ability to misfold and cause toxicity, and that expanding this proline region would be overall beneficial. We generated HTTEx1 mutants lacking both flanking domains singularly, missing the first 11 prolines of the PRD, or with this stretch of prolines expanded. We then followed their aggregation landscape in vitro with a battery of biochemical assays, and in vivo in novel models of C. elegans expressing the HTTEx1 mutants pan-neuronally. Employing fluorescence lifetime imaging we could observe the aggregation propensity of all HTTEx1 mutants during aging and correlate this with toxicity via various phenotypic assays. We found that the presence of an expanded proline stretch is beneficial in maintaining HTTEx1 soluble over time, regardless of polyQ length. However, the expanded prolines were only advantageous in promoting the survival and fitness of an organism carrying a pathogenic stretch of Q48 but were extremely deleterious to the nematode expressing a physiological stretch of Q23. Our results reveal the unique importance of the prolines which have and still are evolving alongside expanding glutamines to promote the function of HTTEx1 and avoid pathology.

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2.

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is key to understanding not only its life cycle, but also to uncover imbalances in the proteostasis network. This method shows how to track the degradation of the disease-causing protein huntingtin. Two versions of huntingtin fused to Dendra2 are expressed in the C. elegans nervous system: a physiological version or one with an expanded and pathogenic stretch of glutamines. Dendra2 is a photoconvertible fluorescent protein; upon a short ultraviolet (UV) irradiation pulse, Dendra2 switches its excitation/emission spectra from green to red. Similar to a pulse-chase experiment, the turnover of the converted red-Dendra2 can be monitored and quantified, regardless of the interference from newly synthesized green-Dendra2. Using confocal-based microscopy and due to the optical transparency of C. elegans, it is possible to monitor and quantify the degradation of huntingtin-Dendra2 in a living, aging organism. Neuronal huntingtin-Dendra2 is partially degraded soon after conversion and cleared further over time. The systems controlling degradation are deficient in the presence of mutant huntingtin and are further impaired with aging. Neuronal subtypes within the same nervous system exhibit different turnover capacities for huntingtin-Dendra2. Overall, monitoring any protein of interest fused to Dendra2 can provide important information not only on its degradation and the players of the proteostasis network involved, but also on its location, trafficking, and transport.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington's, Parkinson's, or Alzheimer's disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

PolyQ Tract Toxicity in SCA1 is Length Dependent in the Absence of CAG Repeat Interruption.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by an expansion of a polyglutamine tract within the ATXN1 gene. Normal alleles have been reported to range from 6 to 35 repeats, intermediate alleles from 36 to 38 repeats and fully penetrant pathogenic alleles have at least 39 repeats. This distribution was based on relatively few samples and the narrow intermediate range makes the accuracy of the repeat sizing crucial for interpreting and reporting diagnostic tests, which can vary between laboratories. Here, we examine the distribution of 6378 SCA1 chromosomes and identify a very late onset SCA1 family with a fully penetrant uninterrupted pathogenic allele containing 38 repeats. This finding supports the theory that polyQ toxicity is related to the increase of the length of the inherited tracts and not as previously hypothesized to the structural transition occurring above a specific threshold. In addition, the threshold of toxicity shifts to a shorter polyQ length with the increase of the lifespan in SCA1. Furthermore, we show that SCA1 intermediate alleles have a different behavior compared to the other polyglutamine disorders as they do not show reduced penetrance when uninterrupted. Therefore, the pathogenic mechanism in SCA1 is distinct from other cytosine-adenine-guanine (CAG) repeat disorders. Accurately sizing repeats is paramount in precision medicine and can be challenging particularly with borderline alleles. We examined plasmids containing cloned CAG repeat tracts alongside a triplet repeat primed polymerase chain reaction (TP PCR) CAG repeat ladder to improve accuracy in repeat sizing by fragment analysis. This method accurately sizes the repeats irrespective of repeat composition or length. We also improved the model for calculating repeat length from fragment analysis sizing by fragment analyzing 100 cloned repeats of known size. Therefore, we recommend these methods for accurately sizing repeat lengths and restriction enzyme digestion to identify interruptions for interpretation of a given allele's pathogenicity.

Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex.

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (HTT). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using in vitro and in vivo assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J-protein) that completely suppresses fibrilization of HttExon1Q48 The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient-derived neural cells and on an organismal level in Caenorhabditis elegans Among the proteins in this chaperone complex, the J-protein is the concentration-limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q97 aggregation and represents a target of future therapeutic avenues for HD.