A Specific Mini‐Intrabody Mediates Lysosome Degradation of Mutant Huntingtin

Abstract Accumulation of misfolded proteins leads to many neurodegenerative diseases that can be treated by lowering or removing mutant proteins. Huntington's disease (HD) is characterized by the intracellular accumulation of mutant huntingtin (mHTT) that can be soluble and aggregated in the central nervous system and causes neuronal damage and death. Here, an intracellular antibody (intrabody) fragment is generated that can specifically bind mHTT and link to the lysosome for degradation. It is found that delivery of this peptide by either brain injection or intravenous administration can efficiently clear the soluble and aggregated mHTT by activating the lysosomal degradation pathway, resulting in amelioration of gliosis and dyskinesia in HD knock‐in (KI‐140Q) mice. These findings suggest that the small intrabody peptide linked to lysosomes can effectively lower mutant proteins and provide a new approach for treating neurodegenerative diseases that are caused by the accumulation of mutant proteins.


Figure S1 .
Figure S1.Comparison of N-terminal HTT-23Q and -150Q binding to SM3. a) Western blotting analysis of the transfected 293T cells and HTT N171-23Q or -150Q with two anti-HTT antibodies.b) Quantification of the ratios of HTT recognized by HTT antibodies (mEM48 and EPR5526) to vinculin on the western blots.The data were obtained from four independent experiments (n = 4).Data were analyzed by Student's t test and presented as mean ± SEM. ***P=0.0003(mEM48); ***P=0.0006(EPR5526).

Figure S2 .
Figure S2.Analysis of the effect of SM3 on the full-length HTT-120Q transfected 293 cells.a, b) Western blotting analysis of the full-length HTT-120Q stably expressed 293T cells, which were transfected with SM3 or vector expressing HA only.Full-length mHTT (arrow pointed) were detected with 1C2 or mEM48 antibodies.c, d) Quantitation of the ratios of mHTT detected by 1C2 or mEM48 to vinculin on the western blots.The data were obtained from four independent experiments (n = 4).Data are analyzed by two-tailed Student's t test and presented as mean ± SEM; **P = 0.0058 (1C2); **P = 0.0081(mEM48).e) Quantification of human HTT using ELISA.n = 4 per group.Data were analyzed by two-tailed Student's t test and presented as mean ± SEM. **P=0.0058.f) Western blot analysis of HTT-120Q 293 cells transfected with SM3 or the vector expressing HA only.The

Figure S3 .
Figure S3.Analysis of the HD KI-140Q mouse brain injected with SM3.a) Western blotting analysis of the expression of neuronal (NeuN) and glial (GFAP, Iba1) proteins in the striatum of HD KI-140Q and wild type (WT) mice injected with GFP (control) or SM3 at 7 months of age.b) Quantification of the ratios of NeuN, GFAP, and Iba1 to vinculin on the western blots.The data were obtained from four independent experiments (n = 4).Data were analyzed by one-way ANOVA with Dunnett's multiple comparisons test and are presented as mean ± SEM.GFAP: *P =0.0249; ***P = 0.0002.Iba1: **P = 0.0044; ***P = 0.0003.

Figure S4 .
Figure S4.Sagittal images of HA antibody immunofluorescent staining of SM3 in the HD KI-140Q mouse brain.

Figure S5 .
Figure S5.Analysis of HD KI-140Q mouse brain after intravenous injection of SM3.Immunostaining and western blotting of HD KI-140Q mouse striatum one month after intravenous injection of SM3.a-c) Representative immunofluorescent fluorescent images of the striatum from GFP or SM3 injected HD KI-140Q mice.Antibodies for NeuN, GFAP and Iba1 were used.All the sections were stained with mEM48 and anti-GFP as well as DAPI.GFP injection was used as control.Note that the numbers of NeuN-positive neurons did not change in HD KI-140Q mice in all treatments.SM3 injection significantly reduced gliosis

Figure S6 .
Figure S6.RNA-seq analysis of HD-KI mice after SM3 treatment.Total RNAs from WT and HD KI-140Q treated with GFP or SM3 were examined.The striatum from three animals of each group were examined.The mice at 6 months of age were stereotaxically injected with AAV and then their striatal tissues were isolated for RNA-seq analysis one month after injection.The heat map of the RNA-seq analysis reveals the alterations in the transcripts related to immune and inflammatory responses in HD KI mouse brain.

Figure
Figure S7.A proposed model of how SM3 induces mHTT degradation.A small peptide derived from HTT intrabody and linked with the lysosomal targeting signal can selectively bind mHTT and bring it to the lysosome for degradation.