Harnessing neuronal extracellular vesicles for Huntington disease biomarkers - PROJECT SUMMARY Huntington disease (HD) is a devastating neurodegenerative disorder with no effective treatment. The disease- causing trinucleotide repeat expansion causes production of mutant HTT (mtHTT), which disrupts many cellular processes. Somatic expansion of the repeat may be a precipitating event in disease onset. Thus, two primary therapeutic strategies are HTT lowering and reduction of somatic expansion. To precisely evaluate these therapies, it is essential to identify sensitive and robust biomarkers of brain HTT that are minimally invasive. For HTT lowering therapy, quantification of mtHTT in cerebrospinal fluid (CSF) can assess target engagement, though this has not been successful for quantifying wtHTT. The current approach of immunodepleting mtHTT and then measuring total HTT is non-specific and incomplete, confounding assessment of allele-selective HTT lowering therapies. Moreover, CSF biomarkers are invasive, and less suitable for trials employing oral or intracranial delivery routes. While some HTT in blood originates from the brain, it does not correlate with CSF mtHTT or HD symptom severity, likely due to contributions from other tissues and modulation by circulating autoantibodies. Thus, blood HTT is not a proxy for brain HTT. However, extracellular vesicles (EVs) containing HTT protein and transcripts are released by brain neurons, cross the blood brain barrier, and enter circulation protecting their cargoes from degradation. EVs contain surface markers and biological cargo specific to their tissue of origin, and we have validated ATP1A3 as a marker for isolating brain neuron-derived EVs from peripheral blood. While most EV studies have focused on exosomes due to the ease of isolation, purification, and characterization, we have found that the bulk of EV HTT is found in the larger ectosomes, which bud from the plasma membrane, more closely representing cytosolic contents. In preliminary work, we have also determined that the quick-freezing methods used to preserve biological samples for biobanking do not maximally preserve EVs, and that the methods used for preserving live cultured cells with intact membranes, freezing slowly at 1oC/minute, are preferred for sensitive quantitative studies. To determine whether HTT protein or transcript instability in brain-derived EVs are responsive to experimental therapies, we will use antisense oligonucleotides to lower either HTT or MSH3 in the CNS of HD mice followed by quantification of HTT protein or transcript instability in brain-derived EVs. Concurrently, to determine the relationship between disease and brain-derived EV HTT protein and transcript instability, we will pursue de novo collection of HD patient and control plasma and CSF at the time of UHDRS evaluation. Samples will be divided and both quick and slow frozen prior to isolation of exosomes and ectosomes by differential ultracentrifugation and further isolation of brain-derived EVs by APT1A3 IP. We will compare mt and wtHTT protein as well as HTT transcript instability to HD symptom severity and to CSF mtHTT to identify procedures and EV subtypes most relevant to disease. Together, this work will provide novel peripheral blood biomarkers of brain HTT and somatic expansion for ongoing HD clinical trials.
