Investigating the neuroprotective role of carboxyl-terminus of Hsc70-interacting protein (CHIP) in healthy aging

Abstract

Throughout the past several decades, the elderly population has increased substantially. Yet, in spite of this enormous shift in age-related demographic characteristics, the cellular and physiological mechanisms underlying aging itself remain ill-defined. Their intrinsic complexity is further exacerbated by the difficulty inherent in segregating processes associated with pathological aging from those related to the normal, healthy aging process. Given that protein homeostasis is a major contributor to both normal and malignant aging, the ubiquitin-proteasome system is a promising candidate for teasing apart the pathways driving both healthy and pathological processes.

E3 ubiquitin ligases catalyse the final step of an enzymatic cascade priming proteins for degradation through the ubiquitin-proteasome system and thus are crucial to protein quality control. Carboxyl terminus of Hsp70-interacting protein (CHIP), one such ligase, was initially found to facilitate the polyubiquitination and eventual degradation of aberrantly folded and/or aggregated client proteins in a complex with Hsp70, an essential component of the chaperone system. More recent studies, however, have identified additional, chaperone-independent functions of CHIP in mediating the activity, localization, and steady-state levels of a targeted protein cohort essential for cellular and organismal well-being, including several involved in interferon and insulin signaling as well as membrane integrity. In animal models, deletion of CHIP imparts an early aging phenotype, along with impaired cognitive function and neurodegeneration. CHIP deletion also predisposed animals to increased cell death, mitochondrial dysfunction, and oxidative stress. The functions of CHIP are also context-dependent, with CHIP being rerouted from mediating insulin receptor degradation in normal cellular conditions to protein quality control pathways under conditions of proteotoxic stress, thus stabilizing the insulin receptor and decreasing lifespan.

While its multifaceted role in neuroprotection makes CHIP a promising subject for the study of healthy aging, little is known about the interplay between its functions and their mechanistic underpinnings. Here, I aim to delineate the cellular pathways mediating the neuroprotective effects of CHIP through the proteomic investigation of a whole-animal model system, C. elegans. The objectives of this study are (1) to characterize a methodological approach for the experimental conditions relevant to processing C. elegans samples for proteomic analyses, (2) to explore the effects of chn-1 ablation on the proteomic landscape of nematodes, the simplest whole-animal model systems for the study of aging, and (3) to identify and validate proteomic biomarkers found to be implicated in CHIP-mediated processes including cellular transport, protein quality control, and longevity.

SWATH-MS was performed on chn-1(by155) and chn-1(tm2692) deletion mutants, in addition to wild-type controls, processed by either SDS or CHAPS/urea-based lysis protocols. Between-buffer analyses revealed 1 092 substantially altered proteins, with methodologically contingent trends in protein expression patterns substantially overshadowing differences between independently-derived strains. Core proteins altered in response to chn-1 ablation included the transcription factor SMA-9, a key modulator of the TGF-β signaling cascade, as well as mitochondrial pyruvate carrier 1 (MPC1) and FIS-2, suggesting CHIP-associated mitochondrial dysfunction in chn-1-null animals. Membrane proteins as well as proteins associated with the regulation of trafficking activity, and more specifically retrograde transport, during which proteins and lipids are shuttled between endosomes and the trans-Golgi network, were also preferentially altered in response to chn-1 ablation. In contrast to the broad spectrum of proteins affected by different lysis methods, chn-1 ablation itself impacted a more focused subset of proteins, with only 33 and 46 proteins altered across both chn-1-null strains in CHAPS/urea- and SDS-processed samples, respectively. These findings suggest a tight substrate specificity, and lend further support to the noncanonical function of CHIP as a chaperone-independent/docking-dependent ubiquitin ligase.

Subsequent validation experiments involving lifespan assays on chn-1(tm2692); sma-9 double-knock-out nematodes revealed a less-than-additive epistatic double-mutant phenotype, suggesting that chn-1 and sma-9 interact in a manner that is overlapping but not entirely redundant. Immunofluorescence and Western blotting experiments were also performed in a SH-SY5Y neuroblastoma cell line to further verify the proteomics results. Specifically, Golgi fragmentation was assessed in both chn-1 wild-type and knock-out nematodes expressing a Golgi marker, as well as wild-type and CHIP knock-out cells stained with GM130. Western blotting was performed on several proteins involved in retrograde transport pathways, including COG complex as well as Rab proteins. In CHIP-null cells and animals, aberrant protein expression was correlated with increased Golgi fragmentation, suggesting CHIP loss-of-function may be linked to this early marker of neurodegeneration. Finally, rescue experiments in C. elegans using the human and nematode orthologs of CHIP were created using Gateway cloning, and subsequent proteomics experiments performed to identify a focused cohort of CHIP-associated proteins corresponding specifically to lifespan extension.

In addition to elucidating novel methodological considerations for C. elegans-based proteomics experiments, these findings provide evidence for CHIP-mediated molecular processes underpinning Golgi integrity, protein transport, mitochondrial dysfunction, and longevity, and provide a foundation for future mechanistic studies to further shed light on these CHIP-associated pathways, processes, and signaling cascades.