Molecular Strategies to Target Protein Aggregation in Huntington's Disease

Abstract

Huntington's disease (HD) is a neurodegenerative disorder caused by the aggregation of the mutant huntingtin (mHTT) protein in nerve cells. mHTT self-aggregates to form soluble oligomers and insoluble fibrils, which interfere in a number of key cellular functions. This leads to cell quiescence and ultimately cell death. There are currently still no treatments available for HD, but approaches targeting the HTT levels offer systematic, mechanism-driven routes towards curing HD and other neurodegenerative diseases. This review summarizes the current state of knowledge of the mRNA targeting approaches such as antisense oligonucleotides and RNAi system; and the novel methods targeting mHTT and aggregates for degradation via the ubiquitin proteasome or the autophagy-lysosomal systems. These methods include the proteolysis-targeting chimera, Trim-Away, autophagosome-tethering compound, autophagy-targeting chimera, lysosome-targeting chimera and approach targeting mHTT for chaperone-mediated autophagy. These molecular strategies provide a knowledge-based approach to target HD and other neurodegenerative diseases at the origin.

Keywords: aggregation; huntingtin (HTT); huntington’s disease; mRNA degradation; protein degradation; protein fibrils; protein quality control; proteostasis.

FIGURE 1

Huntington’s disease. (A) Symptoms of Huntington’s disease (HD); the main triad of symptoms is highlighted in bold. (B) The part of the human brain predominantly affected in HD (brown), the striatum. (C) Schematic presentation of the domain organisation of huntingtin (HTT) protein, with the polyglutamine region (PolyQ) (yellow) and HEAT domains (light blue). The repeat numbers indicate the risk of developing HD. Image adapted from Guo et al., 2018. N and C indicate the protein N- and C-terminus; numbers indicate the amino acid length of HTT. (D) Schematic presentation of a HD hallmark—aggregates and inclusion bodies in the cytoplasm of a nerve cell.

FIGURE 2

mHTT exon 1 structure and aggregation model. (A) Schematic presentation of primary and secondary structure of wild-type HTT (wtHTT) and mutant HTT (mHTT) exon 1, showing the HTT^NT (dark blue; α-helix), polyQ domain (yellow; intrinsically disordered) and proline-rich domain (PRD) (lighter blue; polyproline II helices). mHTT contains an expanded polyQ domain compared to wtHTT. N and C indicate the protein N- and C-terminus. (B) The amino acid sequence of the exon 1 mHTT protein. Highlighted are the identified regions of HTT^NT, PolyQ and PRD. The protein sequence is represented by one letter amino acid code. (C) Model of mHTT exon 1 HTT fibril architecture: mHTT interacts through the anti-parallel β-sheet (orange), with flanking HTT^NT and PRD domains (blue) overhanging at the sides of the fibril; single monomer highlighted in darker colours. (adapted from Boatz et al., 2020).

FIGURE 3

Potential outcomes of antisense oligonucleotide (ASO) approach targeting mHTT mRNA. Schematic presentation of possible outcomes of ASO (dark blue) binding to mHTT pre-mRNA (brown indicates introns; dark blue, yellow and light blue indicate exons), all preventing the formation of the mHTT (brown). The outcomes include (1) degradation via the RNase H1 endonuclease (green) mediated degradation; (2) translational arrest of the ribosome (light green) on the mHTT mRNA resulting in incomplete translation of mHTT; (3) alternative splicing caused by masked splicing sequences, and thus prevention of toxic mHTT expression, but restricted only to an altered non-toxic HTT protein version.

FIGURE 4

Potential mHTT mRNA targeting approaches using the RNA interference (RNAi) system. The RNAi based technologies use the endogenous system and target mHTT through an artificially introduced siRNA (small interfering) (dark blue) (A) or siRNA encoded on a plasmid and expressed in a short hairpin RNA (shRNA) (dark blue) (B) or artificial micro RNA (miRNA) (light blue) (C) scaffold. (A) The artificial siRNA requires no processing and, once inside the cell, binds to the RNA-induced silencing complex (RISC) (green). Driven by the siRNA binding, RISC attaches to mHTT mRNA (brown) and induces mRNA degradation, preventing the expression of mHTT. (B–C) The shRNA and artificial miRNA scaffolds enter the cell on a DNA vector (yellow with dark or light blue fragments for shRNA and artificial miRNA, respectively). The shRNA and miRNA are expressed in the nucleus, processed into siRNA and exported into the cytoplasm. There generated siRNA bind to the RISC (green) complex and induces mHTT mRNA (brown) degradation. The shRNA-derived siRNA has a sequence matching perfectly the mRNA, whereas miRNA derived has imperfect matching.

FIGURE 5

Potential mHTT protein degradation approaches using the ubiquitin proteasome system. (A) The ubiquitin activation cascade involving three ligase enzymes, E1, E2, and E3. E1 (dark blue) activates the ubiquitin molecule (pink), which is then transferred to E2 (light blue) conjugating enzyme. Once conjugated to ubiquitin, E2 can bind to E3 ligase enzyme (blue). The E3 ligase is then ready to bind to the target protein to ubiquitinate it. (B) Schematic of a proteolysis-targeting chimera (PROTAC) targeting cytoplasmic mHTT for degradation. PROTAC binds to mHTT (brown) via the protein binding domain (yellow). PROTAC then interacts with ready E3 ligase (blue) with activated ubiquitin (pink) via the E3 ligase interacting domain (light blue). Close proximity of E3 ligase induces ubiquitination of mHTT, and process repeats leading to K48 polyubiquitination of mHTT. mHTT is targeted for proteasomal degradation, while PROTAC and E3 detach. (C) Schematic of Trim-Away approach targeting cytoplasmic mHTT for degradation. Antibody (light blue; Ab) enters the cell and binds to mHTT (brown). TRIM21 (dark blue) attaches to the Ab and triggers self-ubiquitination (pink). Together the complex undergoes proteasomal degradation.

FIGURE 6

Potential mHTT protein degradation approaches using the autophagy-lysosomal pathway. (A) Schematic overview of the autophagy-lysosomal pathway. Lunate-shaped phagophore membrane (blue) forms, and as it elongates it engulfs proteins and organelles (light and dark brown) destined for degradation. The phagophore closes off and becomes an autophagosome vesicle. The autophagosome fuses with the lysosome (dark blue), and the lysosomal enzymes (light blue) degrade the proteins and organelles inside. The same elements are relevant in (B,C), but are not shown in figures. (B) Schematic of an autophagosome-tethering compound (ATTEC) targeting cytoplasmic mHTT for degradation. ATTEC (orange) binds to mHTT (brown), and tethers mHTT to LC3 autophagy protein (green). This leads to mHTT being engulfed into the autophagosome (blue). Upon the fusion with the lysosome (dark blue), mHTT is degraded by the lysosomal enzymes (light blue). (C) Schematic of an autophagy-targeting chimera (AUTAC) targeting mHTT for degradation. AUTAC attaches to mHTT (brown) via the protein binding domain (yellow). The guanine derivative domain of AUTAC (dark green) is recognized by E3 ubiquitin ligase (dark blue), and close proximity to mHTT, induces mHTT ubiquitination. Multiple rounds of ubiquitination lead to K63 polyubiquitination (pink) of mHTT. The polyubiquitination is recognized by an autophagy reporter p62 (light green), which then tethers mHTT to the phagophore and autophagosome via LC3 (green). Upon fusion with the lysosome (dark blue), mHTT in the phagosome is degraded by the lysosomal enzymes (light blue).

FIGURE 7

Potential mHTT protein degradation approach using the chaperone-mediated autophagy (CMA) pathway. The CMA adapter molecule (green) binds to cytoplasmic mHTT (brown). The HSC70 chaperone (light orange), together with co-chaperones (darker orange), binds to the adapter molecule. HSC70 interacts with LAMP2A (dark blue), found on the lysosomal membrane (blue), and induces LAMP2A dimerization. The chaperone complex unfolds mHTT protein and together with mHTT passes through the LAMP2A formed channel into the lysosome, where all is degraded by the lysosomal enzymes (light blue).

FIGURE 8

Potential mHTT protein degradation approach using the endosomal/lysosomal pathway. Schematic presentation of a lysosome-targeting chimera (LYTAC) targeting extracellular mHTT for degradation. LYTAC binds to mHTT via the protein-binding domain (light orange) and then interacts with lysosome targeting receptors (LTR) (green) via the LTR motif domain (orange). Subsequently, mHTT bound to LTR via LYTAC is then internalised into the cell, through an endosome (blue). mHTT with LYTAC is then released form LTR, and LTR are recycled back to the plasma membrane. mHTT is degraded by the lysosomal enzymes (light blue), and upon endosome and lysosome (dark blue) fusion.