Elena Cattaneo’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 Over the past 25 years, our laboratory has gathered compelling evidence indicating a reduction in brain cholesterol levels and biosynthesis in Huntington’s disease (HD) mice. This finding has significant implications for understanding HD pathophysiology and for developing potential therapeutic strategies. Consistent with this, all strategies developed so far to increase cholesterol levels in the HD mouse brain have resulted in motor and cognitive improvements, as well as in significant reduction in aggregates and improvement in synaptic parameters. Aim Given the variety of functional and structural roles of cholesterol in many aspects of synaptic physiology, our current focus is to identify the synaptic mechanisms restored upon cholesterol administration in HD. Methods Wild-type and HD primary neuronal monocultures obtained from the striatum and cortex, along with a two-compartment microfluidic device, were employed to reconstruct functional neuronal networks in vitro. Cholesterol was administered in acute and chronic paradigms. Excitatory and inhibitory synapses were identified by immunolabeling pre-synaptic and post-synaptic markers, and synapse connectivity was evaluated following cholesterol administration. Results A time-course characterization of synapse dysfunction in vitro revealed an early loss of synaptic contacts in HD neuronal cultures, as evidenced by the decreased colocalization of pre-synaptic and post-synaptic marker puncta. Notably, cholesterol administration to symptomatic primary HD neurons rescued the synaptic phenotype. Conclusion These results indicate that direct cholesterol administration acts at the synapse to increase connectivity in an HD neuronal network reconstructed in vitro.Funded by the HDF (Leslie Gehry Prize for Innovation in Science) and partially by NSC-Reconstruct (European Union, # 874758).

Background Cortico-striatal dysfunction is an early pathogenic event contributing to cognitive decline in Huntington’s disease (HD). A Disintegrin and Metalloproteinase Domain-Containing Protein 10 (ADAM10) is enriched in the brain and governs excitatory synapse structure and function by shedding neuronal trans-synaptic proteins. We have previously linked ADAM10 hyperactivity and enhanced proteolysis of N-Cadherin to HD synaptic dysfunction and cognitive decline. Aim Our aim is to evaluate whether the ADAM10 inhibitor GI254023X alleviates pre- and postsynaptic defects in the HD cortico-striatal circuitry. Methods A two-compartment microfluidic system was used, together with mouse primary cortical and striatal neurons from wild-type and HD mice, to reconstruct the cortico-striatal circuitry in vitro. Immunolabelling of pre- and post-synaptic proteins was used to identify excitatory synaptic contacts. Furthermore, live-imaging microscopy was employed to monitor Brain-Derived Neurotrophic Factor (BDNF) transport. Cortical and striatal neurons were exposed to GI254023X in acute and chronic paradigms. Results We recreated the wild-type and HD cortico-striatal circuitry in microfluidic chambers. We developed an automated pipeline to quantitatively evaluate excitatory synapse density. We set up conditions to study transport dynamics of BDNF-containing vesicles in the cortical afferents. We identified GI254023X doses and exposure times that conferred neuroprotection to HD neurons. Currently, we are testing if GI254023X restores BDNF transport in HD cortical neurons. Conclusion Our findings suggest that pharmacological inhibition of ADAM10 attenuates synaptic phenotypes in the HD cortico-striatal circuitry. Future studies will evaluate the therapeutic potential of GI254023X in rescuing synaptic and cognitive abnormalities in HD mice.Funded by Telethon Foundation (#GGP20067)

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.

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.

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.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.