Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

Abstract

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a G⇔A transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology. The cerebellum is a coordination center whose function is to fine-tune vertebrates' gait and balance; and for this reason, alterations or damage affecting this structure result in a complex syndrome, called ataxia, with neurological signs that are easily recognized. In the mouse, many mutations producing ataxia have been identified and characterized. They have contributed to a better understanding of the genetics of cerebellum development, physiology, and pathology. The present study identifies the recessive allele responsible for the progressive and massive degeneration of the Purkinje cells observed in mutant mice previously named tambaleante. The mutation leads to a single amino acid substitution in a highly conserved domain (RCC1-like) of the giant protein HERC1. This protein belongs to the families HECT (E3 ubiquitin ligases) and RCC1 (GTPases regulators). While a variety of mutations have been reported in several members of these families, leading to sterility, growth retardation, retinitis pigmentosa, amyotrophic lateral sclerosis, or cancer, no mutation had ever been reported so far in the mouse Herc1 gene. This report demonstrates the crucial and unexpected role of HERC1 in Purkinje cell physiology that could be considered helpful in the development of new therapeutic strategies for neurodegenerative disorders.