Dario Besusso’s research while affiliated with National Institute of Molecular Genetics (INGM) and other places

The accurate characterization of triplet repeats, especially the overrepresented CAG repeats, is increasingly relevant for several reasons. First, germline expansion of CAG repeats above a gene-specific threshold causes multiple neurodegenerative disorders; for instance, Huntington’s disease (HD) is triggered by >36 CAG repeats in the huntingtin (HTT) gene. Second, extreme expansions up to 800 CAG repeats have been found in specific cell types affected by the disease. Third, synonymous single nucleotide variants within the CAG repeat stretch influence the age of disease onset. Thus, new sequencing-based protocols that profile both the length and the exact nucleotide sequence of triplet repeats are crucial. Various strategies to enrich the target gene over the background, along with sequencing platforms and bioinformatic pipelines, are under development. This review discusses the concepts, challenges, and methodological opportunities for analyzing triplet repeats, using HD as a case study. Starting with traditional approaches, we will explore how sequencing-based methods have evolved to meet increasing scientific demands. We will also highlight experimental and bioinformatic challenges, aiming to provide a guide for accurate triplet repeat characterization for diagnostic and therapeutic purposes.

Trinucleotide repeats in DNA exhibit a dual nature due to their inherent instability. While their rapid expansion can diversify gene expression during evolution, exceeding a certain threshold can lead to diseases such as Huntington’s disease (HD), a neurodegenerative condition, triggered by >36 C–A–G repeats in exon 1 of the Huntingtin gene. Notably, the discovery of somatic instability (SI) of the tract allows these mutations, inherited from an affected parent, to further expand throughout the patient’s lifetime, resulting in a mosaic brain with specific neurons exhibiting variable and often extreme CAG lengths, ultimately leading to their death. Genome-wide association studies have identified genetic variants—both cis and trans, including mismatch repair modifiers—that modulate SI, as shown in blood cells, and influence HD’s age of onset. This review will explore the evidence for SI in HD and its role in disease pathogenesis, as well as the therapeutic implications of these findings. We conclude by emphasizing the urgent need for reliable methods to quantify SI for diagnostic and prognostic purposes.

Background It is well known that HD patients with similar CAG length show a wide range of variability in motor onset that can account for up to two decades. One possible explanation resides in the fact that the inherited CAG repeats may expand in somatic tissues, especially in post-mitotic neurons, giving rise to a HTT mosaicism that results in longer than inherited CAG tracts in affected tissues, such as the striatum and cortex. This expansion may continue during the lifetime of the individual and contribute to exacerbate neuronal toxicity and selective neuronal degeneration. More recently, trans- and cis- modifiers of age of onset (AOO) have been identified. However, if and how they cause the progressive accumulation of CAG instability is still unclear. Aim To identify new cis and trans modifiers of CAG instability, we aimed to establish an isogenic human stem cell platform that, combined with third generation long-read sequencing, allows to monitor HTT CAG size over time, both during mitotic cell replication and in post-mitotic neurons. Methods Starting from H9 human embryonic stem cell (hES) line, we inserted a monoallelic Recombinant Mediated Exchange Cassette within HTT exon 1, which can be subsequently exchanged with any exon1 variant in an efficient way. We generated a wide variety of exon 1 modified cell lines, which we refer to as the CAGinSTEM platform. Results Our data show that the CAGinSTEM platform is technically robust as for each genotype we have multiple cell lines which have been quality checked. By exploiting the properties of the CAGinSTEM platform, we are testing how CAG length and composition impact on CAG instability in terminally differentiated medium spiny neurons and in active proliferating hES cells. Conclusions The CAGinSTEM platform offers a distinctive biological model system designed to explore genotype-phenotype correlations and investigate the mechanisms underlying CAG instability accumulation in postmitotic human neurons and other cell types.Funded by an ERC Advanced Grant from the European Commission.

Background Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding for the huntingtin protein. Recently, Genome Wide Association Studies (GWAS) have identified genomic variations, occurring both at the HTT locus and in genes mostly involved in mismatch repair pathways, to be associated with disease onset and progression. Such mutations correlate with modulations of somatic instability, which is now considered as the main driver of pathogenesis. Accordingly, the set-up of reliable sequencing-based methods for assessing somatic instability is of paramount importance. Aims In this work, we aimed to compare workflows for assessing somatic instability, by identifying strengths and drawbacks of each enrichment, sequencing and analysis method. Methods Based on research studies focusing on Short Tandem Repeats (STR) characterization available in the literature, we compared multiple enrichment methods coupled to various sequencing platforms and data analysis tools. We then performed a preliminary comparison on internal data, and discussed their accuracy in CAG sizing and sensitivity for rare alleles detection. Results We reported much higher enrichment for PCR-based methods, compared to CRISPR/Cas9 enrichment and adaptive sampling. Moreover, unlike long-read sequencing platforms, we described a decrease in sequencing quality at cycles above 300 for Illumina MiSeq. As a last point, we showed a preliminary comparison among sequencing-based methods on internal data, showing the impact of each step towards the most accurate STR characterization. Conclusions We anticipate that long-read sequencing of PCR amplicons incorporating UMIs may represent a valid alternative to PCR-free enrichment methods, providing highly accurate performances in terms of repeat length and rare alleles detection. Nonetheless, we recommend accurate optimization of PCR primers and conditions, as suboptimal number of PCR cycles may result in off-target products and inadequate number of PCR duplicates.

Synaptic dysfunction is an early pathogenic event leading to cognitive decline in Huntington’s disease (HD). We previously reported that the active ADAM10 level is increased in the HD cortex and striatum, causing excessive proteolysis of the synaptic cell adhesion protein N-Cadherin. Conversely, ADAM10 inhibition is neuroprotective and prevents cognitive decline in HD mice. Although the breakdown of cortico-striatal connection has been historically linked to cognitive deterioration in HD, dendritic spine loss and long-term potentiation (LTP) defects identified in the HD hippocampus are also thought to contribute to the cognitive symptoms of the disease. The aim of this study is to investigate the contribution of ADAM10 to spine pathology and LTP defects of the HD hippocampus. We provide evidence that active ADAM10 is increased in the hippocampus of two mouse models of HD, leading to extensive proteolysis of N-Cadherin, which has a widely recognized role in spine morphology and synaptic plasticity. Importantly, the conditional heterozygous deletion of ADAM10 in the forebrain of HD mice resulted in the recovery of spine loss and ultrastructural synaptic defects in CA1 pyramidal neurons. Meanwhile, normalization of the active ADAM10 level increased the pool of synaptic BDNF protein and activated ERK neuroprotective signaling in the HD hippocampus. We also show that the ADAM10 inhibitor GI254023X restored LTP defects and increased the density of mushroom spines enriched with GluA1-AMPA receptors in HD hippocampal neurons. Notably, we report that administration of the TrkB antagonist ANA12 to HD hippocampal neurons reduced the beneficial effect of GI254023X, indicating that the BDNF receptor TrkB contributes to mediate the neuroprotective activity exerted by ADAM10 inhibition in HD. Collectively, these findings indicate that ADAM10 inhibition coupled with TrkB signaling represents an efficacious strategy to prevent hippocampal synaptic plasticity defects and cognitive dysfunction in HD.

Huntington’s disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.

Background Huntington’s disease (HD) is a motor and cognitive neurodegenerative disorder due to prominent loss of striatal medium spiny neurons (MSNs). Cell replacement using human embryonic stem cells (hESCs) derivatives may offer new therapeutic opportunities to replace degenerated neurons and repair damaged circuits. Methods With the aim to develop effective cell replacement for HD, we assessed the long-term therapeutic value of hESC-derived striatal progenitors by grafting the cells into the striatum of a preclinical model of HD [i.e., adult immunodeficient rats in which the striatum was lesioned by monolateral injection of quinolinic acid (QA)]. We examined the survival, maturation, self-organization and integration of the graft as well as its impact on lesion-dependent motor alterations up to 6 months post-graft. Moreover, we tested whether exposing a cohort of QA-lesioned animals to environmental enrichment (EE) could improve graft integration and function. Results Human striatal progenitors survived up to 6 months after transplantation and showed morphological and neurochemical features typical of human MSNs. Donor-derived interneurons were also detected. Grafts wired in both local and long-range striatal circuits, formed domains suggestive of distinct ganglionic eminence territories and displayed emerging striosome features. Moreover, over time grafts improved complex motor performances affected by QA. EE selectively increased cell differentiation into MSN phenotype and promoted host-to-graft connectivity. However, when combined to the graft, the EE paradigm used in this study was insufficient to produce an additive effect on task execution. Conclusions The data support the long-term therapeutic potential of ESC-derived human striatal progenitor grafts for the replacement of degenerated striatal neurons in HD and suggest that EE can effectively accelerate the maturation and promote the integration of human striatal cells.

Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivo in terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington’s disease (HD).