Metformin treatment reduces motor and neuropsychiatric phenotypes in the zQ175 mouse model of Huntington disease

Sanchis et al. Experimental & Molecular Medicine (2019) 51:65 https://doi.org/10.1038/s12276-019-0264-9 Experimental & Molecular Medicine ARTICLE Open Access Metformin treatment reduces motor and neuropsychiatric phenotypes in the zQ175 mouse model of Huntington disease 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Ana Sanchis1, María Adelaida García-Gimeno2, Antonio José Cañada-Martínez José María Millán1,4, Pascual Sanz4,5 and Rafael P. Vázquez-Manrique 1,4 3 , María Dolores Sequedo1,4, Abstract Huntington disease is a neurodegenerative condition for which there is no cure to date. Activation of AMP-activated protein kinase has previously been shown to be beneficial in in vitro and in vivo models of Huntington’s disease. Moreover, a recent cross-sectional study demonstrated that treatment with metformin, a well-known activator of this enzyme, is associated with better cognitive scores in patients with this disease. We performed a preclinical study using metformin to treat phenotypes of the zQ175 mouse model of Huntington disease. We evaluated behavior (motor and neuropsychiatric function) and molecular phenotypes (aggregation of mutant huntingtin, levels of brain-derived neurotrophic factor, neuronal inflammation, etc.). We also used two models of polyglutamine toxicity in Caenorhabditis elegans to further explore potential mechanisms of metformin action. Our results provide strong evidence that metformin alleviates motor and neuropsychiatric phenotypes in zQ175 mice. Moreover, metformin intake reduces the number of nuclear aggregates of mutant huntingtin in the striatum. The expression of brain-derived neurotrophic factor, which is reduced in mutant animals, is partially restored in metformin-treated mice, and glial activation in mutant mice is reduced in metformin-treated animals. In addition, using worm models of polyglutamine toxicity, we demonstrate that metformin reduces polyglutamine aggregates and restores neuronal function through mechanisms involving AMP-activated protein kinase and lysosomal function. Our data indicate that metformin alleviates the progression of the disease and further supports AMP-activated protein kinase as a druggable target against Huntington’s disease. Introduction Huntington disease (HD) is a dominant, inherited neurodegenerative disorder that leads to impaired motor coordination associated with chorea and progressive deterioration of cognitive function. Patients with HD have Correspondence: Pascual Sanz () or Rafael P. VázquezManrique () 1 Research Group in Molecular, Cellular and Genomic Biomedicine, Health Research Institute La Fe (Hospital Universitario y Politécnico La Fe), València, Spain 2 Department of Biotechnology, Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural (ETSIAMN), Universitat Politécnica de València, València, Spain Full list of author information is available at the end of the article. These authors contributed equally: Pascual Sanz, Rafael P. Vázquez-Manrique an abnormal CAG expansion within the first exon of the huntingtin gene, HTT. This gene encodes a cytosolic protein, huntingtin (Htt), the function of which is unclear. When HTT has 35 or more CAG triplet repeats, the protein contains abnormally long glutamine tracts (polyQ), resulting in a mutant huntingtin (mHtt), which shows toxic gain-of-function properties. mHtt is prone to improper folding and to forming aggregates, thereby perturbing a range of essential cellular functions1 and impairing cell viability with particularly severe effects in neurons of the striatum2. Neurons respond to toxic mHtt by activating pathways of protein clearance, such as autophagy3 or the proteasome4. Among the molecules that can induce autophagy, © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Korean Society for Biochemistry and Molecular Biology Sanchis et al. Experimental & Molecular Medicine (2019) 51:65 AMP-activated protein kinase (AMPK), the master regulator of energy homeostasis in eukaryotic cells, plays a key role5,6. This enzyme is a heterotrimer (AMPKα is catalytic and AMPKβ and AMPKγ are regulatory subunits7,8) that is activated when ATP levels are reduced. Activated AMPK coordinates metabolism, cell growth, and autophagy, and through the autophagy pathway, AMPK activation is able to reduce the levels of mHtt3. Activation of AMPK by genetic and pharmacological means also reduces neuronal polyQ-induced toxicity in Caenorhabditis elegans9 and reduces cell death in a mammalian in vitro model of HD9, which is accompanied by a reduction in mHtt aggregates. Moreover, overexpression of the AMPKγ gain-of-function mutant reduces neuronal loss in a mouse model of HD9. Therefore, this enzyme has been proposed as a druggable target against HD9. Reducing mHtt expression in models of HD reverses phenotypes associated with the disease10,11. One potential substance that may induce mHtt lowering is metformin, a well-known AMPK activator12,13. Metformin is used worldwide to treat type 2 diabetes mellitus14. Interestingly, researchers discovered that patients suffering from this disease who are chronically taking metformin experience unexpected side benefits, such as protection against cancer15 and nephrotic symptoms16, among others. Metformin demonstrates cell protection properties in polyQ-expressing C. elegans and in vitro models of HD9. In addition, metformin treatment also increases lifespan, among other effects, in males of the R6/2 mouse model of HD17. Finally, a statistical analysis of participants in the Enroll-HD database, a worldwide observational study on HD, showed that metformin intake is associated with better cognitive function in HD patients18, strongly pointing to metformin as a putative treatment for HD. In this study, C. elegans was used to illustrate that metformin is able to reduce the aggregation of polyQs and neuronal impairment in worms in an AMPK- and lysosomal-dependent manner. We also performed a protocol to test the beneficial effects of metformin in the zQ175 mouse model of HD at 3 months of age (i.e., early stages of HD). After 3 months of treatment, untreated heterozyg (...truncated)