Biology subjects

Aeschbach, L.

Publications and source records attributed to Aeschbach, L..

5 recordsLinked to original sources

bioRxiv2022

N-terminal mutant Huntingtin deposition correlates with CAG repeat length and disease onset, but not neuronal loss in Huntington's disease

Huntingtons disease (HD) is caused by a CAG repeat expansion mutation in the gene encoding the huntingtin (Htt) protein, with mutant Htt protein subsequently forming aggregates within the brain. Mutant Htt is a current target for novel therapeutic strategies for HD, however, the lack of translation from preclinical research to disease-modifying treatments highlights the need to improve our understanding of the role of Htt protein in the human brain. This study aims to undertake a high-throughput screen of 12 candidate antibodies against various sequences along the Htt protein to characterize Htt distribution and expression in post-mortem human brain tissue microarrays (TMAs). Immunohistochemistry was performed on middle temporal gyrus TMAs comprising of up to 28 HD and 27 age-matched control cases, using 12 antibodies specific to various sequences along the Htt protein. From this study, six antibodies directed to the Htt N-terminus successfully immunolabelled human brain tissue. The Htt aggregates and Htt protein expression levels for the six successful antibodies were subsequently quantified with high-throughput analysis. Htt aggregates were detected in HD cases using antibodies MAB5374, MW1, and EPR5526, despite no change in overall Htt protein expression compared to control cases, suggesting a redistribution of Htt into aggregates in HD. Significant associations were found between the number of Htt aggregates and both age of disease onset, and CAG repeat length in HD. However, the number of Htt aggregates did not correlate with the degree of striatal degeneration or the degree of cortical neuron loss. Together, these results suggest that longer CAG repeat lengths correlate with Htt aggregation in the HD human brain, and Htt cortical aggregate deposition is associated with the onset of clinical symptoms. This study also reinforces that antibodies MAB5492, MW8, and 2B7 which have been utilized to characterize Htt in animal models of HD are not specific for Htt in human brain tissue, thereby highlighting the need for validated means of Htt detection to support drug development for HD.

neuroscience↗

bioRxiv2022

Comparative analysis of total alpha-synuclein (αSYN) immunoassays reveals that they do not capture the diversity of modified αSYN proteoforms

BackgroundThe development of therapeutics for Parkinsons disease (PD) requires the establishment of biomarker assays to enable stratifying patients, monitoring disease progression and assessing target engagement. Attempts to develop diagnostic assays based on detecting levels of the -synuclein (SYN) protein, a central player in the pathogenesis of PD, have yielded inconsistent results. ObjectiveTo determine whether the three commercial kits that have been extensively used for total SYN quantification in human biological fluids (from Euroimmun, MSD, and Biolegend) are capable of capturing the diversity and complexity of relevant SYN proteoforms. MethodsWe investigated and compared the ability of the different assays to detect the diversity of SYN proteoform using a library of SYN proteins that compromise the majority of disease-relevant SYN variants and post-translational modification. ResultsOur findings showed that none of the three tested immunoassays accurately capture the totality of relevant SYN species and are unable to recognize most disease-associated C-terminally truncated variants of SYN. Moreover, several N-terminal truncations and phosphorylation/nitration differentially modify the level of SYN detection and recovery by different immunoassays, and a CSF matrix effect was observed for most of the SYN proteoforms analyzed by the three immunoassays. ConclusionsOur results showed that these immunoassays do not capture the totality of the relevant SYN species and therefore may not be appropriate tools to provide an accurate measure of total SYN levels in samples containing modified forms of the protein. This highlights the need for next-generation SYN immunoassays that capture the diversity of SYN proteoforms.

neuroscience↗

bioRxiv2022

Repeat Detector: accurate, efficient, and flexible sizing of expanded CAG/CTG repeats from targeted DNA sequencing

Targeted DNA sequencing approaches will improve how the size of short tandem repeats is measured for diagnostic tests and pre-clinical studies. The expansion of these sequences causes dozens of disorders, with longer tracts generally leading to a more severe disease. Interrupted alleles are sometimes present within repeats and can alter disease manifestation. Determining repeat size mosaicism and identifying interruptions in targeted sequencing datasets remains a major challenge. This is in part because standard alignment tools are ill-suited for repetitive and unstable sequences. To address this, we have developed Repeat Detector (RD), a deterministic profile weighting algorithm for counting repeats in targeted sequencing data. We tested RD using blood-derived DNA samples from Huntingtons disease and Fuchs endothelial corneal dystrophy patients sequenced using either Illumina MiSeq or Pacific Biosciences single-molecule, real-time sequencing platforms. RD was highly accurate in determining repeat sizes of 609 blood-derived samples from Huntingtons disease individuals and did not require prior knowledge of the flanking sequences. Furthermore, RD can be used to identify alleles with interruptions and provide a measure of repeat instability within an individual. RD is therefore highly versatile and may find applications in the diagnosis of expanded repeat disorders and the development of novel therapies.

neuroscience↗

bioRxiv2018

Uncovering the interplay between epigenome editing efficiency and sequence context using a novel inducible targeting system

Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Using PInT, we found no evidence that the histone deacetylase HDAC5 or the DNA methyltransferase DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by HDAC5 or DNMT1 targeting. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.

molecular biology↗

bioRxiv2017

Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

Expanded CAG/CTG repeats underlie the aetiology of 14 neurological and neuromuscular disorders. The size of the repeat tract determines in large part the severity of these disorders with longer tracts causing more severe phenotypes. Expanded CAG/CTG repeats are also unstable in somatic tissues, which is thought to modify disease progression. Routine molecular biology applications involving these repeats, including quantifying their instability, are plagued by low PCR yields. This leads to the need for setting up more PCRs of the same locus, thereby increasing the risk of carry-over contamination. Here we aimed to reduce this risk by pre-treating the samples with a Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCRs. We successfully applied this method to the PCR amplification of expanded CAG/CTG repeats, their sequencing, and their molecular cloning. In addition, we optimized the gold-standard method for measuring repeat instability, small-pool PCR, such that it can be used together with Ung and dUTP-containing PCRs, without compromising data quality. We expect that the protocols herein to be applicable for molecular diagnostics of expanded repeat disorders and to manipulate other tandem repeats.

molecular biology↗