The Role of Huntingtin in Endolysosomal Trafficking and Huntington’s Disease Pathogenesis

Author

• Stephen M. Farmer Jr, The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical SciencesFollow

Author ORCID Identifier

0000-0002-4289-1257

Date of Graduation

8-2026

Document Type

Dissertation (PhD)

Program Affiliation

Molecular and Translational Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Sheng Zhang

Committee Member

Hugo J Bellen

Committee Member

Guangwei Du

Committee Member

Jun Wang

Committee Member

Travis I Moore

Committee Member

Kuang-Lei Tsai

Abstract

With no effective treatments or cures, aging-related neurodegenerative diseases (NDs) are a growing threat to our society. As one of the most extensively studied NDs, autosomal-dominant Huntington’s disease (HD) is caused by an abnormal expansion of the polyglutamine (polyQ) tract at the N-terminus of the Huntingtin (HTT) protein, leading to gained toxicities as well as loss and neomorphic effects on HTT’s normal physiological functions, which together contribute to the selective neuropathology of HD. However, HTT’s normal physiological functions and its regulation remain murky, a major knowledge gap and limiting factor in identifying effective therapies for HD. After its synthesis, apo-HTT interacts with its obligate partner, HAP40, forming a highly stable globular complex, although the functional significance of this interaction was also unclear.

My dissertation has focused on the above questions by employing a multidisciplinary approach, including powerful genetic tools in the invertebrate Drosophila model, live/super-resolution imaging, biochemical assays in cultured mammalian cells, computational modeling, and phenotypic characterization in mouse brains. I found that HTT/HAP40 is structurally and functionally conserved from flies to humans, and identified 10 conserved amino acid pairs required for complex formation. Further, HTT/HAP40 loss-of-function (LOF) phenotypes implicated the complex in endolysosomal trafficking, a membrane network that uptakes, sorts, recycles, and degrades cargo. Consistent with this role, HTT/HAP40 LOF exhibited smaller, more acidic Rab7-positive endolysosomes. Mechanistically, HTT associated with endosomal membranes through HAP40’s conserved N-terminal BΦ motif. Together with the observation that simultaneous HTT/HAP40 overexpression, but not either alone, induced strong BΦ-dependent gain-of-function (GOF) effects, further supports the BΦ motif as a regulatory domain through which HAP40 controls HTT’s in vivo activity and endolysosomal engagement.

Further characterization in Drosophila and cultured mammalian cells established that HTT/HAP40 functions in a novel, conserved endolysosomal process that couples endosomal recycling with degradation to promote the turnover of select cargoes. By completing three independent, unbiased whole-genome screens for modifiers of HTT/HAP40-associated GOF and LOF phenotypes in Drosophila, I identified multiple novel players and potential mechanisms underlying this pathway, including the endosomal regulator Rab5 and its effector Rabankyrin, a potential feed-forward mechanism involving Rab geranylgeranyltransferase (RabGGTb), and a disinhibition mechanism involving TBC1D16.

Finally, to investigate HAP40’s physiological roles in the mammalian brain, I characterized two complementary HAP40 LOF mouse models: 1) targeted deletion of HAP40 in HD-vulnerable GABAergic neurons (VGAT-Hap40^cKO), which surprisingly showed normal aging and physiology, despite prominent thalamic calcium deposition in aged brains, mirroring phenotypes of adult-onset, global HTT depletion; 2) broad deletion of HAP40 in all neural progenitors during early embryogenesis (Nestin-Hap40^cKO), which caused growth deficits, microcephaly, gliosis and postnatal lethality. Most importantly, HAP40 loss led to ~70% depletion of endogenous HTT, revealing a critical role for HAP40 in maintaining HTT stability in mammals and underscoring HAP40 as a promising target for graded, ‘HTT-lowering’ strategies in HD.

In summary, my dissertation identifies HAP40 as a highly conserved regulator of HTT, defines a conserved HTT/HAP40-dependent pathway that controls endolysosomal trafficking, establishes a mechanistic framework for this pathway, and supports HAP40 as a potentially safer target for graded HTT lowering in HD.

Recommended Citation

Farmer, Stephen M. Jr, "The Role of Huntingtin in Endolysosomal Trafficking and Huntington’s Disease Pathogenesis" (2026). Dissertations and Theses (Open Access). 1584.
https://digitalcommons.library.tmc.edu/utgsbs_dissertations/1584

Keywords

HTT, HAP40, Huntington's Disease, Endolysosomal Regulation, Rab GTPases, Drosophila model