Beyond Exon 1: Exploring the Pathogenic Role of Novel Huntingtin Aggregates in vivo.

Mutations within exon 1 of the huntingtin (HTT) protein promote its aggregation and accumulation in the brain and periphery, ultimately driving neuronal dysfunction and the clinical manifestation of Huntington’s disease (HD). Although HTT is a large protein composed of more than 3,000 amino acids, exon 1 represents less than 3% of its total length. For decades, this small N-terminal fragment has been viewed as the principal driver of pathology. Yet HTT is far more than exon 1, and regions outside this domain can also undergo proteolytic processing, potentially generating toxic species whose contribution to disease remains poorly understood. To explore this possibility, the Cicchetti lab combined computational predictions, in vitro assays, and analyses of human post-mortem tissue. In silico screening revealed 110 aggregation-prone regions outside the polyQ tract, suggesting that aggregation competence is not confined to exon 1. Eight of these sequences were synthesized and shown to rapidly assemble into oligomers and amyloid fibrils in vitro. Importantly, aggregation was not restricted to short fragments. Four full-length HTT proteoforms—including HTT lacking exon1 and three polyQ variants (Q23, Q36, Q54)—were all capable of forming aggregates, indicating that polyQ expansion is not strictly required for this process.

Consistent with these findings, mass spectrometry of ex vivo aggregates purified from HD patient brains identified various non-polyQ HTT fragments, together covering nearly half of the protein sequence. The next step undertaken by the Cicchetti lab was to determine whether these newly identified aggregates could recapitulate key pathological hallmarks in vivo, similar to those driven by exon 1. To address this, the peptide tP1—previously identified through in silico analyses, validated for aggregation in vitro, and shown to induce cell death in iPSC-derived neurons—was stereotaxically injected into the brains of wild-type mice. Strikingly, tP1 induced cognitive behavioral alterations as early as four months post-injection, preceding the effects observed with Q48 fibrils, which were used as a positive control. Neuropathological assessment revealed marked cell death in the cortex and striatum, along with the accumulation of protein aggregates, indicating that non-exon 1 fragments are capable of driving HD-like pathology in vivo.

As a summer intern, I will investigate the pathological effects of these newly identified HTT aggregates in vivo using mouse model. As the animal protocol is already underway, my arrival will coincide with a key experimental phase, allowing me to contribute to the post-mortem analyses. I will perform histological and biochemical assessments, including immunohistochemistry, western blotting, and fluorescence imaging, to evaluate core hallmarks of HD-related pathology. These analyses will focus on markers of cell death (e.g., cleaved caspase-3), neurodegeneration, aggregate deposition, and region-specific vulnerability in the cortex and striatum. Through this internship, I aim to develop practical expertise in animal experimentation and neuropathological analysis, while strengthening my ability to interpret in vivo data and understand how HTT-derived aggregates drive disease mechanisms at the tissue level.