Amplification of neurotoxic HTTex1 assemblies in human neurons

Abstract

Huntington's disease (HD) is a genetically inherited neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) repeat in the exon-1 of huntingtin protein (HTT). The expanded polyQ enhances the amyloidogenic propensity of HTT exon 1 (HTTex1), which forms a heterogeneous mixture of assemblies with a broad neurotoxicity spectrum. While predominantly intracellular, monomeric and aggregated mutant HTT species are also present in the cerebrospinal fluids of HD patients, however, their biological properties are not well understood. To explore the role of extracellular mutant HTT in aggregation and toxicity, we investigated the uptake and amplification of recombinant HTTex1 assemblies in cell culture models. We find that small HTTex1 fibrils preferentially enter human neurons and trigger the amplification of neurotoxic assemblies; astrocytes or epithelial cells are not permissive. The amplification of HTTex1 in neurons depletes endogenous HTT protein with non-pathogenic polyQ repeat, activates apoptotic caspase-3 pathway and induces nuclear fragmentation. Using a panel of novel monoclonal antibodies and genetic mutation, we identified epitopes within the N-terminal 17 amino acids and proline-rich domain of HTTex1 to be critical in neural uptake and amplification. Synaptosome preparations from the brain homogenates of HD mice also contain mutant HTT species, which enter neurons and behave similar to small recombinant HTTex1 fibrils. These studies suggest that amyloidogenic extracellular mutant HTTex1 assemblies may preferentially enter neurons, propagate and promote neurodegeneration.

Keywords: Huntingtin; Huntingtin exon1; Huntington's disease; Neurotoxicity; Seeding.

Figure 1.

HTTex1 assemblies enter and amplify in neurons. (A) Human neurons derived from MESC2.10 embryonic stem cell line was treated with mutant HTTex1 fibrils or sonicated fibrils (seeds), incubated for the indicated time points and processed for ICC using anti-PRD PHP1 antibody (HTT). Insert in the bottom right panel is a magnified seeded neuron. (B) Quantification of seeded neurons at different time points based on reactivity to anti-HTT antibody (PHP1) (C). Examination of the lysates of seeded neurons 24 hr post-treatment by SDD-AGE detected by PHP1. HTTex1 seeds were adjusted to the amounts in the lysates loaded to determine the extent of amplification. (D & E) HTTx1 seeds and its amplified products in MESC 2.10 derived neurons are partially resistant to proteinase K (PK) digestion. Seeds (D) or neural lysates from seeded neurons (E) were treated with an increasing concentration of PK and examined by SDD-AGE and WBs using PHP2 antibody. Data are mean ± SEM; **P<0.01, ***P<0.001.

Figure 2.

HTTex1 seeds accumulate in the nuclei of neurons. (A&B) Fluorophore-labelled (Alexa 555) HTTex1 seeds were added to human neurons for the indicated time points and processed for ICC using PHP1 antibody. Confocal images in the left panels show the labelled HTTex1 seeds and in the right panels are merged images of labelled HTTex1 and amplified species detected by PHP1 antibody. Part B is magnified neurons showing the nuclear localization of labelled HTTex1 seeds. (C) Quantification of neurons with the labelled HTTex1 in part A (left columns) and neurons labelled with anti-HTT antibody (PHP1) (right columns). (D&E) Human neurons from IPSC-derived neuronal progenitors (left panels) or IPSC-derived astrocytes (right panels) were treated with HTTex1 fibrils or sonicated fibrils for 24 hr and processed similar to part (A). Neurons were stained with PHP1 and anti-Tuj-1 and astrocytes were stained with PHP1 and anti-GFAP. Data show mean ± SEM; **P<0.01, ***P<0.001.

Figure 3.

Amplification of HTTex1 seeds causes nuclear damage and caspase-3 activation. (A) For better contrast, black and white images of DAPI stained neurons treated with HTTex1 fibrils or sonicated seeds for the indicated time points are shown to display the progressive nuclear damage. (B) Quantification of DNA damage in neurons seeded with HTTex1 fibrils or seeds (C) Confocal images of neurons with active caspase-3, which was induced in neurons treated with the sonicated HTTex1 seeds. Graphs in D and E show quantification of neurons with HTT aggregates and active caspase-3, respectively. Data are expressed as mean ± SEM; ***P<0.001.

Figure 4.

HTTex1 seeds recruit endogenous HTT species to form assemblies. (A) SDD-AGE analysis followed by WBs of control neuronal lysates (C) or lysates of neurons treated with HTTex1 seeds (SD) probed with the indicated anti-HTT antibodies. Part B is SDS-PAGE of similar lysates probed with antibodies reactive to soluble HTT. Arrowheads indicate depletion of endogenous full-length and N-terminal fragments of HTT in the lysates of seeded neurons. (C) Seeding of recombinant WT HTTex1 monomers by the HTTex1 seeds (S). Products were examined by SDD-AGE and WB using PHP1 antibody. (D) HTTex1 seeds amplify in neural lysates. Lane 1 is seed alone and lane 2 is seeded neuronal lysate. The indicated antibodies below each lane were used to deplete the endogenous HTT species before seeding (lanes 3–6). The binding epitopes of PHP antibodies are shown in supplementary fig. 4A.

Figure 5.

Antibodies to N17 and PRD of HTTex1 block neural seeding. (A) HTTex1 seeds were pre-incubated with the antibodies indicated on top of each panel, subsequently added to neurons and incubated for 24 hr. Samples were processed for ICC, stained with PHP1 and examined by a confocal microscope. Panel B is the quantification of fragmented nuclei in each condition. (C) HTTex1 seeds or those generated from HTTex1 with mutated PRD (HTTx1mL17) were added to neurons and processed as in A. Anti-N17 antibody PHP5 was used to detect the amplified products. Panel D is the quantification of fragmented nuclei in each condition. (E) MESC2.10 neurons engineered to secret control or PHP2 recombinant antibodies (Lenti-PHP2) were treated with the HTTex1 seeds, incubated for 24 hr, processed for ICC using PHP1 antibody and examined by a confocal microscope. Panel F is the quantification of fragmented nuclei in each condition. Data show mean ± SEM; ***P<0.001.

Figure 6.

Mutant HTT seeds accumulate in the brains of ZQ175 HD mice. (A) Fractionation of mouse brains on sucrose gradients and examination by SDD-AGE and WB. HTT was detected by PHP1 antibody. (B) Dialyzed aliquots of each fraction (50 μg) were sonicated and added into human neurons derived from MESC2.10 and incubated for 24 hr. Neurons were processed for ICC and stained with PHP1 antibody. Part C is similar fractions as in A from different cohorts of mice treated with proteinase K (PK) or untreated and processed similarly as in part B. Images were captured with a confocal microscope. (D) Quantification of seeded neurons with fragmented nuclei in each condition is shown. Data show mean ± SEM; ***P<0.001.

Figure 7.

Schematic representation of the extracellular HTTex1 journey in neurons. HTTex1 seeds potentially bind to a surface receptor to enter neurons and ultimately accumulate in the nucleus. Subsequently, the seeds may recruit the endogenous HTT fragments and other neural proteins to amplify. Amplified products induce neurotoxicity potentially by caspase-3 activation and depletion of HTT.