Suppression of mutant Huntingtin aggregate formation by Cdk5/p35 through the effect on microtubule stability

Abstract

Huntington's disease (HD) is a polyglutamine [poly(Q)] disease with an expanded poly(Q) stretch in the N terminus of the huntingtin protein (htt). A major pathological feature of HD neurons is inclusion bodies, detergent-insoluble aggregates composed of poly(Q)-expanded mutant htt (mhtt). Misfolding of mhtt is thought to confer a toxic property via formation of aggregates. Although toxic molecular species are still debated, it is important to clarify the aggregation mechanism to understand the pathogenesis of mhtt. We show Cdk5/p35 suppresses the formation of mhtt inclusion bodies in cell lines and primary neurons. Although we expressed the N-terminal exon 1 fragment of htt lacking phosphorylation sites for Cdk5 in COS-7 cells, the kinase activity of Cdk5 was required for the suppression. Furthermore, Cdk5/p35 suppressed inclusion formation of atrophin-1, another poly(Q) protein, raising the possibility that Cdk5/p35 generally suppresses inclusion formation of poly(Q) proteins. Microtubules (MTs) were a downstream component of Cdk5/p35 in the suppression of inclusion formation; Cdk5/p35 disrupted MTs, which were required for the formation of inclusions. Moreover, stabilization of MTs by Taxol induced inclusions even with overexpression of Cdk5/p35. The formation of inclusions was also regulated by manipulating the Cdk5/p35 activity in primary rat or mouse cortical neuron cultures. These results indicate that Cdk5-dependent regulation of MT organization is involved in the development of aggregate formation and subsequent pathogenesis of poly(Q) diseases. This Cdk5 inhibition of htt aggregates is a novel mechanism different from htt phosphorylation and interaction with Cdk5 reported previously (Luo et al., 2005; Anne et al., 2007).

Figure 1.

Suppression of the formation of mhtt inclusions by Cdk5/p35. A, Fluorescence microscopic images of tNhtt-16Q, 50Q, or 90Q-EGFP expressed alone (top panels) or coexpressed with Cdk5/p35 (bottom panels) in COS-7 cells (left panels). Cells were analyzed for inclusion formation 24 h after transfection. Scale bar, 10 μm. Quantification of cells containing EGFP inclusions is shown in the right panel (mean ± SEM; **p < 0.001). B, Detection of SDS-insoluble aggregates in COS-7 cells expressing 50Q or 90Q but not 16Q by anti-GFP immunoblotting. A bracket indicates aggregates in the stacking gel. C, Inhibition of tNhtt-90Q-EGFP inclusion formation by Cdk5/p35 when cotransfected in different plasmid ratios. The amount of plasmid DNA (in micrograms) used is indicated below the blot. A bracket indicates SDS-insoluble aggregates. D, Inclusion formation of tNhtt-16Q, 50Q, 90Q-EGFP and suppression by Cdk5/p35 in Neuro2a cells. The number of cells containing EGFP-aggregates was counted and expressed as the percentage ratio to total EGFP-positive cells transfected in the presence (+) or absence (−) of Cdk5/p35 (mean ± SEM; **p < 0.001).

Figure 2.

Requirement of Cdk5 kinase activity for the suppression of mhtt aggregate formation. A, tNhtt-16Q, 50Q, or 90Q-EGFP was cotransfected into COS-7 cells with either Cdk5, p35, or both Cdk5 and p35. Cells bearing EGFP inclusions were counted and expressed as the percentage ratio to total EGFP- and Cdk5- (or p35-) positive cells (mean ± SEM; **p < 0.001). B, tNhtt-16Q or 90Q-EGFP was cotransfected into COS-7 cells with either Cdk5 and p25 or Cdk5 and p39. Cells displaying EGFP inclusions were counted and expressed as the percentage ratio to total EGFP- or EGFP/Cdk5-positive cells (mean ± SEM; **p < 0.001). C, tNhtt-16Q, 50Q, or 90Q-EGFP was cotransfected into COS-7 cells with either Cdk5 and p35, Cdk5-K33T and p35 (K33T), or Cdk5-D144N and p35 (D144N). Cdk5-D144N and Cdk5-K33T are distinct kinase-negative mutants. Cells containing EGFP inclusions were counted and expressed as the percentage ratio to total EGFP- or EGFP/Cdk5-positive cells (mean ± SEM; **p < 0.001). D, Atrophin-1 with 19Q or 81Q was cotransfected into COS-7 cells alone or with Cdk5 and p35 and cells containing EGFP inclusions were counted and expressed as the percentage ratio to total EGFP- or EGFP/Cdk5-positive cells (mean ± SEM; **p < 0.001).

Figure 3.

Inclusion formation is dependent on microtubule organization. A, COS-7 cells expressing tNhtt-90Q-EGFP were treated with 100 ng/ml nocodazole for 18 h starting 6 h after transfection or for 6 h starting 18 h after transfection. At the end of treatment, cells were analyzed for inclusion formation. Typical EGFP fluorescence images are shown. Cells containing EGFP inclusions were either 0 or 9.3 ± 0.4% of the total EGFP-positive cells when treated with nocodazole or not, respectively. B, COS-7 cells expressing tNhtt-50Q or 90Q-EGFP were treated with 20 μm Taxol for 18 h starting 6 h after transfection. At the end of treatment, cells were observed for inclusion formation. Cells containing EGFP inclusions were either 17.5 or 8.5% of the total EGFP-positive cells when treated with Taxol or not, respectively. C, tNhtt-16Q or 90Q-EGFP was cotransfected into COS-7 cells with tau. Cells were analyzed for inclusion formation 24 h after transfection. Cells containing EGFP inclusions were either 11.7 ± 0.7 or 9.3 ± 0.4% of the total 90Q EGFP-positive cells when cotransfected with tau or not, respectively. Scale bars, 10 μm.

Figure 4.

The MT network in COS-7 cells expressing Cdk5, p35, and/or mhtt. A, MTs in COS-7 cells expressing Cdk5 and p35 were observed by immunofluorescence staining with anti-β-tubulin antibody 24 h after transfection (top). Bottom, Immunoblots showing the amount of β-tubulin in the pellet (p) and supernatant (s) in control cells (−), cells treated with nocodazole for 18 h (Noc), transfected with Cdk5/p35 (Cdk5/p35), or treated with Taxol for 18 h (Tax). Polymeric and protomeric tubulin are expressed as the percentage ratio to total β-tubulin under the each lane. B, MT distribution in COS-7 cells expressing tNhtt-16Q, 50Q, or 90Q-EGFP. MTs were stained with anti-β-tubulin antibody (red), and tNhtt-poly(Q)-EGFP was detected by EGFP fluorescence (green). The merged image is shown (bottom). The arrows indicate the discrete tubulin staining observed in the cytoplasm and at the cell periphery. C, Confocal microscopic images of tNhtt-90Q-EGFP (green; top), anti-β-tubulin staining (red; middle), and merged images (bottom) in COS-7 cells expressing Cdk5/p35 or not (vector). D, tNhtt-90Q-EGFP was transfected into COS-7 cells alone or with Cdk5 and p35. Cells were treated with 20 μm Taxol for 18 h starting 6 h after transfection. At the end of treatment, cells were observed for inclusions (left). Cells containing EGFP inclusions were counted and expressed as the percentage ratio to total EGFP-positive cells (right; mean ± SEM; **p < 0.001). Scale bars, 10 μm.

Figure 5.

Modulation of mhtt inclusion formation by manipulating Cdk5/p35 activity in primary cultured neurons. A, Rat brain cortical neurons were transfected with different amounts of tNhtt-166Q-EGFP plasmid DNA (in micrograms), and 18 h after transfection the number of neurons containing EGFP inclusions was counted and expressed as the percentage ratio to total EGFP-positive cells (mean ± SEM; **p < 0.001). B, Immunoblots showing the expression of FLAG-tagged p35 (p35-FLAG), Cdk5, and tNhtt-90Q-EGFP in primary neurons. Endogenous p35 was detected as a lower band in both uninfected (lane 1) and infected (lane 2) neurons. A bracket indicates SDS-insoluble aggregates in the stacking gel. C, Fluorescence images of EGFP in rat cortical primary neurons expressing tNhtt-90Q-EGFP with either p35-FLAG (p35) or not (control). D, Immunoblot showing downregulation of Cdk5 protein in primary cultured neurons treated with Cdk5 siRNA (lane 2) or control siRNA (lane 3). Actin is a loading control. E, Fluorescence images of EGFP in rat cortical primary neurons transfected with tNhtt-90Q-EGFP and either Cdk5 siRNA or control siRNA. Scale bars, 10 μm.

Figure 6.

mhtt inclusion formation in primary cultured neurons of Cdk5^−/− mouse. A, Fluorescence images of EGFP in Cdk5^+/+ or Cdk5^−/− mouse cortical primary neurons transfected with tNhtt-166Q-EGFP. Scale bar, 10 μm. B, Neurons containing EGFP inclusions were counted and expressed as the percentage ratio to total EGFP-positive cells (mean ± SEM; **p < 0.001). C, Immunoblot showing the expression of β-tubulin in control and Cdk5^−/− neurons. D, Immunoblot showing the amount of β-tubulin in the MT polymers (P) and protomeric soluble pool (S) in Cdk5^−/− and control neurons.