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

Stephen M. Farmer, MS (Advisor: Sheng Zhang, PhD)

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 neo-morphic effects on HTT’s normal physiological functions, which together contribute to selective neuropathology. However, HTT’s normal physiological functions and regulation remain poorly understood, a major barrier to the development of safe therapeutic strategies.

After synthesis, apo-HTT interacts with its obligate partner, HAP40, forming a stable globular HTT/HAP40 complex whose function remained unclear. To address this gap, my dissertation used Drosophila genetics, live-cell/super-resolution imaging, mammalian-cell biochemistry, structural modeling, and mouse brain phenotyping to define HTT/HAP40 function. 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) broad HAP40 deletion in neuronal precursors during early embryogenesis (Nestin-Cre/HAP40-cKO), which caused growth deficits, neuronal loss, gliosis, and postnatal lethality; and 2) targeted deletion of HAP40 in HD-vulnerable GABAergic neurons (Vgat-Cre/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. HAP40 loss led to ~70% depletion of endogenous HTT in mouse brains, 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 reveals HAP40 as a highly conserved and critical regulator of HTT, establishes HTT/HAP40 as part of a conserved endolysosomal network, and supports HAP40 as a safer target for HTT-lowering therapies. Given converging evidence that endolysosomal defects contribute broadly to NDs, these findings provide new insight into HD pathogenesis and related brain degenerative disorders.

Advisory Committee:

• Sheng Zhang, PhD, Chair
• Hugo Bellen, DVM, PhD
• Guangwei Du, PhD
• Travis Moore, PhD
• Kuang-Lei Tsai, PhD
• Jun Wang, PhD

Join via Zoom (Please contact Mr. Farmer for his Zoom meeting info.)