Modulation of mTOR and CREB pathways following mGluR5 blockade contribute to improved Huntington's pathology in zQ175 mice

Abstract

Huntington's disease (HD) is a neurodegenerative disorder caused by a genetic abnormality in the huntingtin gene that leads to a polyglutamine repeat expansion of the huntingtin protein. The cleaved polyglutamine expansion of mutant huntingtin (mHTT) protein can form aggregates strongly correlated with HD progression. We have previously shown that the inhibition of mGluR5 using CTEP, a selective negative allosteric mGluR5 modulator, can delay disease progression and reduce in mHTT aggregates in the zQ175 mouse model of HD. This was paralleled by enhanced catalytic activity of Unc-51-like kinase 1 (ULK1), a kinase modulated by mammalian target of rapamycin (mTOR) and key regulator of autophagy initiation. In the present study, we show that CTEP can correct aberrant phosphoinositide 3-kinase (PI3K)/Akt/mTOR signaling detected in zQ175 mice that may underlie the enhanced ULK1 activity and activation of autophagy. We also show that CTEP can facilitate cAMP response element-binding protein (CREB)-mediated expression of brain-derived neurotrophic factor (BDNF) to foster neuronal survival and reduce apoptosis. Taken together, our findings provide the molecular evidence for how targeting mGluR5 using a well-tolerated selective NAM can mitigate two critical mechanisms of neurodegeneration, autophagy and apoptosis.

Keywords: BDNF; CTEP; Huntington’s disease; ULK1; autophagy; mGluR5; mHTT; mTOR; zQ175.

Fig. 1

CTEP normalizes enhanced mTOR activity in zQ175 mice. a Representative western blots and mean ± SEM of mTOR-pS2448 and (b) p70S6K-pT389 in brain lysates from homozygous zQ175 and wildtype (WT) mice after chronic treatment with either vehicle or CTEP (2 mg/kg). Values are expressed as a fraction of the vehicle-treated WT. mTOR-pS2448 was normalized to total mTOR expression and p70S6K-pT389 was normalized to vinculin expression (n = 5–6 for each group). * P < 0.05 significantly different from vehicle-treated WT mice

Fig. 2

CTEP reverses abnormal PI3K/Akt signaling in zQ175 mice. a Representative western blots and mean ± SEM of phosphoinositide-dependent kinase-1 (PDK) PDK1-pS241 and (b) Akt-pS473 in brain lysates from homozygous zQ175 and wildtype (WT) mice after chronic treatment with either vehicle or CTEP (2 mg/kg). Values are expressed as a fraction of the vehicle-treated WT. PDK1-pS241 was normalized to total PDK1 expression and Akt-pS473 was normalized to total Akt expression (n = 5–6 for each group). * P < 0.05 significantly different from vehicle-treated WT mice

Fig. 3

CTEP facilitates CREB-mediated expression of BDNF in zQ175 mice. a Representative western blots and mean ± SEM of CREB-pS133 (b) cFos and (c) BDNF in brain lysates from homozygous zQ175 and wildtype (WT) mice after chronic treatment with either vehicle or CTEP (2 mg/kg). Values are expressed as a fraction of the vehicle-treated WT. CREB-pS133 was normalized to total CREB, cFos was normalized to vinculin expression and BDNF was normalized to actin expression (n = 5–6 for each group). Representatives for each panel were obtained from the same blot. P < 0.05 * significantly different from vehicle-treated zQ175 mice and ^# P < 0.05 significantly different from vehicle-treated WT mice

Fig. 4

Schematic representation for how mGluR5 antagonism modulates mTOR and CREB signaling in zQ175 mice. The pharmacological inhibition of mGluR5 with CTEP in zQ175 mice abolishes the enhanced signaling of mammalian target of rapamycin (mTOR) by reducing the phosphorylation of phosphoinositide-dependent kinase-1 (PDK1), Akt and mTOR. Reduced mTOR signaling was confirmed by a reduction in the phosphorylation of downstream p70S6K and was associated with decreased inhibitory phosphorylation of ULK1 at S757 leading to activation of autophagy. Activation of autophagy and reduction in mHTT load can facilitate the binding of phosphorylated cAMP response element-binding protein (CREB) activity to cAMP response element (CRE) in the nucleus. Activation of CREB-mediated gene expression of cFos and brain-derived neurotrophic factor (BDNF) synthesis can contribute to neuronal survival and reduced apoptosis in zQ175 mice