IKKβ slows Huntington's disease progression in R6/1 mice

Abstract

Neuroinflammation is an important contributor to neuronal pathology and death in neurodegenerative diseases and neuronal injury. Therapeutic interventions blocking the activity of the inflammatory kinase IKKβ, a key regulator of neuroinflammatory pathways, is protective in several animal models of neurodegenerative disease and neuronal injury. In Huntington's disease (HD), however, significant questions exist as to the impact of blocking or diminishing the activity of IKKβ on HD pathology given its potential role in Huntingtin (HTT) degradation. In cell culture, IKKβ phosphorylates HTT serine (S) 13 and activates HTT degradation, a process that becomes impaired with polyQ expansion. To investigate the in vivo relationship of IKKβ to HTT S13 phosphorylation and HD progression, we crossed conditional tamoxifen-inducible IKKβ knockout mice with R6/1 HD mice. Behavioral assays in these mice showed a significant worsening of HD pathological phenotypes. The increased behavioral pathology correlated with reduced levels of endogenous mouse full-length phospho-S13 HTT, supporting the importance of IKKβ in the phosphorylation of HTT S13 in vivo. Notably, many striatal autophagy genes were up-regulated in HD vs. control mice; however, IKKβ knockout partially reduced this up-regulation in HD, increased striatal neurodegeneration, and enhanced an activated microglial response. We propose that IKKβ is protective in striatal neurons early in HD progression via phosphorylation of HTT S13. As IKKβ is also required for up-regulation of some autophagy genes and HTT is a scaffold for selective autophagy, IKKβ may influence autophagy through multiple mechanisms to maintain healthy striatal function, thereby reducing neuronal degeneration to slow HD onset.

Keywords: Huntington’s disease; IκB kinase; autophagy; huntingtin; neurodegeneration.

Fig. 1.

Effects of IKKβ knockout on behavior in male R6/1 mice. Pole test (8, 12, and 16 wk) and rotarod (8, 13, and 15 wk) are shown for tamoxifen (TAM)- vs. oil-treated R6/1 (HD) and NT controls (n = NT-Oil, 12; NT-TAM, 12; HD-Oil, 9; HD-TAM, 12 at week 8, and NT-Oil, 12; NT-TAM, 11; HD-Oil: 9, HD-TAM, 10 following injections). HD mice performed significantly worse on 12-wk pole test (A) and on weeks 13 and 15 rotarod (B) with TAM-induced IKKβ knockout than oil-treated HD control mice. No significant effect was observed in NT control mice with TAM-induced IKKβ knockout. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 values represent means ± SEM. Statistical significance was determined by one-way ANOVA with Bonferroni posttesting.

Fig. 2.

Levels of S13-phosphorylated HTT are reduced while total full-length (∼350 kDa) mouse HTT abundance is increased with IKKβ knockout in striatum, demonstrating that IKKβ is a relevant striatal HTT S13 kinase in vivo. Male R6/1 (HD) and NT WT controls, both containing the tamoxifen-inducible Cre and floxed alleles of IKKβ, were treated with tamoxifen or oil vehicle control during week 10 and killed at week 16. IKKβ normalized to loading control α-tubulin was significantly reduced in 16-wk striatum of HD and NT mice with tamoxifen treatment over oil control in whole-cell lysate (A–C). Anti-HTT phosphoserine 13 (pS13) antibody was used to immunoprecipitate phosphorylated full-length mouse HTT which was then detected by Western blot with anti-total HTT antibody. Levels of pS13-HTT were significantly reduced relative to total HTT with IKKβ knockout in HD and NT controls, while levels of total HTT normalized to α-tubulin were significantly increased (A–C). Western images (A and B) were quantitated using Scion software (C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 values represent means ± SEM. Statistical significance was determined by paired t test.

Fig. 3.

IKKβ is required for efficient S13 phosphorylation of ∼350-kDa full-length mouse HTT in liver. Male R6/1 (HD) and NT WT controls, both containing the tamoxifen-inducible Cre and floxed alleles of IKKβ, were treated with tamoxifen or oil vehicle control at week 10 and tissue was taken at week 16 at the completion of the study. IKKβ normalized to loading control ERK1/2 was significantly reduced in liver of HD and NT mice with tamoxifen treatment over oil control in whole-cell lysate (A–C). Anti-HTT phosphoserine 13 (pS13) antibody was used to immunoprecipitate phosphorylated full-length mouse HTT which was then detected by Western blot with anti-total HTT antibody. Levels of pS13-HTT were significantly reduced relative to total HTT with IKKβ knockout in HD and NT controls, while levels of total HTT normalized to ERK1/2 were not affected (A–C). Western images (A and B) were quantitated using Scion software (C). *P < 0.05, ****P < 0.0001 values represent means ± SEM. Statistical significance was determined by paired t test.

Fig. 4.

IKKβ knockout and mutant HTT exon 1 protein expression impact levels of autophagy proteins in vivo. R6/1 (HD) or NT WT control male mice containing the tamoxifen-inducible Cre and floxed alleles of IKKβ were treated with tamoxifen or oil vehicle control for 1 wk starting at 10 wk to knock out IKKβ in striatum and liver. At the termination of the study at 16 wk, Western analysis of NT and HD soluble fractions was used to examine levels of autophagy proteins LC3 and LAMP-2A relative to loading controls α-tubulin (striatum) or ERK1/2 (liver). LC3 I was detected in striatum, while LC3 I and II were observed in liver. LC3 I was quantitated for both tissues and was found in striatum, but not in liver, to be significantly increased with IKKβ knockout or with transgene expression. LAMP-2A levels were unchanged in striatum but were significantly increased in HD mouse liver. Western images, shown, were quantitated; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 values represent means ± SEM. Statistical significance was determined by one-way ANOVA with Bonferroni posttesting.

Fig. 5.

IKKβ knockout increases neurodegeneration and microglial activation in striatum. Male R6/1 (HD) mice containing the tamoxifen-inducible Cre and floxed alleles of IKKβ were treated with tamoxifen or oil vehicle control for 1 wk starting at 10 wk. At the termination of the study at 16 wk, consecutive coronal brain sections containing striatum were stained against Fluoro-Jade B, a neuronal death marker (A) or Iba1, a microglia marker (B). Images (20×) show that R6/1 tamoxifen-treated mice had significant increases in both Fluoro-Jade B- (A) and Iba-1– (B) positive cells in the striatum compared with R6/1 (HD) oil-treated mice (representative images shown). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 values represent means ± SEM. Cells were counted using Bitplane’s Imaris microscopy image analysis software and number of microglia per field of vision (FOV) graphed. Statistical comparisons of results were performed by performing one-way ANOVA analysis followed by Bonferroni’s multiple comparison tests. n = 4 per treatment. (Scale bars, 100 μm.)

Fig. 6.

A model for the progression of HD pathogenesis dependent on the autophagic balance of the patient. In premanifest HD (A), mutant HTT exon 1 protein is expressed in striatum due to incomplete transcription of the mutant HTT gene, and accumulation of mutant HTT exon 1 protein (mHTTex1p) causes a cellular stress, which activates the IKK complex. IKKβ activates autophagy gene expression and increases phosphorylation of HTT S13 to induce HTT’s role as an autophagic scaffold protein. This results in autophagic clearance of mHTTex1p, ultimately reducing inflammation, and the cell survives. In manifest HD (B), levels of HTT and other autophagy proteins have declined with age and the ability of the lysosome to degrade autophagic cargos is reduced. With polyQ expansion, mutant HTT is less well phosphorylated reducing its function as an autophagic scaffold. mHTTex1p expressed from incomplete transcription of the mutant HTT gene accumulates and is not cleared by the lysosome, causing an up-regulation of IKK that is unable to further activate autophagy. This results in a robust microglial activation, massive inflammatory pathway activation, cellular dysfunction, and neurodegeneration. Therapies to treat HD need to be designed with the autophagic balance of the patient in mind (C). Early therapies to activate autophagy and IKKβ to degrade mHttex1p and reduce cellular stress may be protective as long as lysosomal function is still intact, but later therapies to inhibit autophagy and block IKKβ may be useful if the aging lysosome can no longer degrade autophagic cargo.