Rac1 Activity Is Modulated by Huntingtin and Dysregulated in Models of Huntington's Disease

Abstract

Background: Previous studies suggest that Huntingtin, the protein mutated in Huntington's disease (HD), is required for actin based changes in cell morphology, and undergoes stimulus induced targeting to plasma membranes where it interacts with phospholipids involved in cell signaling. The small GTPase Rac1 is a downstream target of growth factor stimulation and PI 3-kinase activity and is critical for actin dependent membrane remodeling.

Objective: To determine if Rac1 activity is impaired in HD or regulated by normal Huntingtin.

Methods: Analyses were performed in differentiated control and HD human stem cells and HD Q140/Q140 knock-in mice. Biochemical methods included SDS-PAGE, western blot, immunoprecipitation, affinity chromatography, and ELISA based Rac activity assays.

Results: Basal Rac1 activity increased following depletion of Huntingtin with Huntingtin specific siRNA in human primary fibroblasts and in human control neuron cultures. Human cells (fibroblasts, neural stem cells, and neurons) with the HD mutation failed to increase Rac1 activity in response to growth factors. Rac1 activity levels were elevated in striatum of 1.5-month-old HD Q140/Q140 mice and in primary embryonic cortical neurons from HD mice. Affinity chromatography analysis of striatal lysates showed that Huntingtin is in a complex with Rac1, p85α subunit of PI 3-kinase, and the actin bundling protein α-actinin and interacts preferentially with the GTP bound form of Rac1. The HD mutation reduced Huntingtin interaction with p85α.

Conclusions: These findings suggest that Huntingtin regulates Rac1 activity as part of a coordinated response to growth factor signaling and this function is impaired early in HD.

Keywords: Actin; GTPase; HTT; PI 3-kinase; growth factor; signaling; “Ras-Related C3 Botulinum Toxin Substrate 1”.

Fig.1

Rac (1, 2, 3) activation is altered in human HD fibroblasts and by loss of Huntingtin. (a) Bar graph shows mean±SD of relative Rac (1, 2, 3) activity (OD490) in primary human control and HD fibroblasts. CAG repeat length for both alleles is indicated under the x-axis. PDGF treatment significantly increased relative Rac (1, 2, 3) activity in control fibroblasts but not in three HD cell lines (ANOVA and Tukey’s HSD posthoc tests, F = 5.03, *p < 0.01, n = 6 technical replicates except HD17/69 – PDGF which is n = 4). (b) Huntingtin lowering by siRNA (E1–4). Bar graph shows mean±SD for Huntingtin pixel intensity quantification standardized to tubulin in control fibroblasts as percent of mock by western blot (*p < 0.05, n = 3 biological replicates, unpaired t-test compared to GFP). Western blots are shown in Supplementary Figure 1e. (c) Relative Rac (1, 2, 3) activity (OD490) was measured in lysates from cells transfected with HTT siRNA compared to GFP siRNA. Bar graph shows mean±SD of relative Rac (1, 2, 3) activity with siRNA for GFP or Huntingtin. In the Huntingtin (E1–4) treated cells, there is a significant increase in the basal level of Rac (1, 2, 3) activation per microgram protein (*p < 0.005, n = 6 technical replicates, paired t-test). (d) Huntingtin interacts with PI 3 kinase p85α. Primary human fibroblasts were serum-starved for 48 h then stimulated with PDGF added directly to the medium for 7 minutes (100 ng/ml final concentration). The CAG repeat length for each allele of the HD gene is indicated in parenthesis at top. Anti-Huntingtin antibody MAB2166 was used to isolate Huntingtin from control and HD fibroblasts. Control lanes are B (buffer alone with protein G and primary antibody), and no ab control (protein G incubated with input lysates without primary antibody). Western blots probed with anti-Huntingtin Ab1 then re-probed with anti-p85 PI 3-kinase. β-tubulin was used as a loading control. Molecular mass markers are on the left. Black lines indicated where lanes were removed from the same gel. (e, f) Pixel intensity quantification of immunoprecipitation (IP) results for p85 PI 3-kinase standardized to signal for Huntingtin (e) and inputs standardized to β-tubulin (f) shows a significant increase in level of p85 PI 3-kinase in Huntingtin-immunoprecipitates with PDGF treatment for control fibroblasts but not HD fibroblasts and PI 3-kinase p85α levels were significantly reduced in HD cells compared to the normal control in the inputs. Bar graph shows mean ratio ± SD, *p < 0.05, n = 3 experiments, unpaired t-test.

Fig.2

Rac1 activity in human NSCs and neurons and effects of Huntingtin lowering. (a) Bar graph shows mean±SD of relative Rac1 activity (Luminescence RLUx10⁶) for human control NSCs (WT54 and WT97) and HD NSCs (HD48, HD51, and HD4), without and with BDNF treatment. In WT cells Rac1 activity is increased with BDNF treatment as expected compared to unstimulated WT cells (ANOVA and Tukey’s HSD posthoc tests *p < 0.01; n = 3 technical replicates). In non-stimulated conditions (– BDNF), relative Rac1 activity is increased in HD NSCs compared to WT NSCs (⁺p < 0.01). (b) Graph shows mean±SE of relative Rac1 activity (Luminescence RLU×10⁶) in NSCs from line HD116c (genetically corrected HD4 line) and HD4. Genetic correction significantly decreases relative Rac activity levels (*p < 0.01, n = 12 wells from 3 biological replicates, unpaired t-test). (c) Basal Rac1 activity in human HD (HD51) neurons is increased compared to WT (WT54) neurons. Graph shows mean±SD of relative Rac1 activity at DIV35 (*p < 0.05, unpaired t-test, n = 4 technical replicates). (d-g) Lowering Huntingtin increases Rac1 activity in WT neurons and reduces Rac1 activity to normal levels in HD neurons. WT97 and HD51 were treated with siRNA HTT10150 targeting HTT mRNA or with a non-targeting control (NTC) and harvested for Huntingtin detection by western blot with antibody directed to htt1–17 (Ab1) (d and e) and for Rac1 activity (f and g). Bar graphs show results for mean Huntingtin signal intensity based on pixel intensity quantification as percent of untreated control and relative Rac1 activity in siRNA HTT10150 (HTT) treated and non-targeting control (NTC) treated cells (*p < 0.05, ANOVA with Bonferroni’s Multiple Comparison test, n = 3 technical replicates). Black line in f and g shows Rac1 levels in the untreated cells measured on the same ELISA plate.

Fig.3

Rac1, 2, 3 and Rac1 activity in WT and HD mouse primary neurons, striatum and cortex. (a) Primary embryonic cortical neurons from knock-in Q140/Q140 HD mice have increased basal levels of Rac1, 2, 3 activity compared to WT neurons (*p < 0.01, unpaired t-test, n = 3–4). Reported before in a previous paper retracted for problems with other data. (b) Basal Rac1 activity in the striatum and cortex of WT and Q140/Q140 HD mice. Graphs show mean relative Rac1 activity±SD (ANOVA and Tukey’s HSD posthoc tests, F = 20.36 in “striatum” and 27.32 in “cortex”; *p < 0.001 **p < 0.05, n = 4 mice per genotype at 1.5 months and 4.5 months). In the striatum, relative Rac1 activity is increased in HD compared to WT at 1.5 months, but decreased at 4.5 months. In the cortex relative Rac1 activation is increased in WT and HD mice at 4.5 months compared to 1.5 months. (c) Western blot analysis of lysates from striatum of WT and HD mice at 1.5 and 4.5 months. Top blots probed with antibody to Huntingtin to verify genotype (10μg protein separated on 3–8% Tris-acetate SDS-PAGE). Middle and bottom blots probed with antibody to Rac1 and re-probed with antibody to GAPDH as a loading control (10μg protein separated on 4–12% Bis-tris SDS-PAGE). Space indicates separate gels. Pixel intensity quantification results at right show less Rac1 protein in HD mice at 1.5 months. Bars are mean±SD, *indicates p < 0.05, n = 4 mice, unpaired t-test.

Fig.4

Rac and p85α PI 3-kinase both co-immunoprecipitate with endogenous Huntingtin. (a) Western blot analysis of Huntingtin (Htt) immunoprecipitated from wild-type (WT) and Q140/Q140 (HD) adult mouse brain using anti-Huntingtin antibody MAB2166. Eluates were analyzed using two gel formats. Blot from a 3–8% SDS-PAGE gel was probed with polyclonal anti-Huntingtin Ab1 to confirm presence of full-length Huntingtin (∼350 kDa) (top). Blot from 4–12% SDS-PAGE gel was probed for p85 regulatory subunit of PI 3-kinase (middle blot, arrow) and Rac1 (bottom blot, arrow). The mouse IgG light chains (indicated) are recognized by the secondary antibody on the Rac1 probe. Vertical lines indicate where lanes were removed from same gel.

Fig.5

Affinity chromatography isolates a protein complex with GST-Rac1[GTPγS] including Huntingtin, p85α, and α-actinin-2. Affinity chromatography using GST-Rac1[GSTγS] or GST-Rac1[GDP] with lysates from wild-type (WT) or HD (Q140/Q140) mouse striatum at 1.5 months. Lanes (applies for a-f): P1, 2000 g pellet fraction; S1, 2000 g supernatant which was used as input for column; FT GTP and FT GDP, flow-through fractions; GTP E1–E5, elutions 1–5 from GST-Rac1[GSTγS] column. GDP E1–E5, elutions 1–5 from GST-Rac1[GDP] column; lanes at far right loaded with pure GST-Rac1 protein at indicated concentrations to identify fusion protein on the gels. (a, b) Western blots of fractions indicated at top of blots were probed for Huntingtin (Htt) with Ab1 targeting aa1–17 of Huntingtin, α-actinin-2, PI 3-kinase p85α regulatory subunit, and for Rac1 as indicated at right of blots. Relative molecular mass is indicated in kilodalton at left. Black lines indicate removal of marker lanes from samples that were run on the same gel exactly as in c and d. (c, d) Silver stained gels of the same samples shown in a and b. Molecular weight markers indicated on left or within gel lane at left. Marker 1 (EZ Run), Marker 2 (SeeBlue). Silver stains were overdeveloped to visualize high molecular weight proteins (Huntingtin). (e, f) Western blot and silver stained gel of affinity chromatography input, flow through and elutions using GST alone with lysates from wild-type (WT) mouse striatum at 1.5 months as a control. Low background binding was observed with α-actinin-2, but binding to GST-Rac1 was significantly higher. Vertical lines in a,b, and e indicate where lanes were removed from the same gel.