Loss of dopaminergic neurons and resulting behavioural deficits in mouse model of Angelman syndrome

Abstract

E6 associated protein is an E3 ubiquitin ligase encoded by the gene Ube3a. Deletion or loss of function of the maternally inherited allele of Ube3a leads to Angelman syndrome. In the present study, we show that maternal loss of Ube3a (Ube3a^m−/p+) in the mouse model leads to motor deficits that could be attributed to the dysfunction of the nigrostriatal pathway. The number of tyrosine hydroxylase positive neurons in the substantia nigra was significantly reduced in Ube3a^m−/p+ mice as compared to the wild type counterparts. The Ube3a^m−/p+ mice performed poorly in behavioural paradigms sensitive to nigrostriatal dysfunction. Even though the tyrosine hydroxylase staining was apparently the same in the striatum of both genotypes, the presynaptic and postsynaptic proteins were significantly reduced in Ube3a^m−/p+ mice. These findings suggest that the abnormality in the nigrostriatal pathway along with the cerebellum produces the observed motor dysfunctions in Ube3a^m−/p+ mice.

Introduction

The gene product of Ube3a called E6 associated protein (E6-AP) belongs to the HECT (Homologous to E6-AP C-terminus) domain family of E3 ubiquitin ligases. E6-AP, the best characterized protein in this family, tags ubiquitin molecules to proteins that are destined to be degraded through the proteasome (Mishra et al., 2009, Mishra and Jana, 2008, Scheffner et al., 1993, Wang and Pickart, 2005). Loss of function mutations or deletions of maternal Ube3a is known to cause Angelman syndrome (AS) (Fang et al., 1999, Kishino et al., 1997). Characteristics of the syndrome include motor dysfunction, seizures and mental retardation (Clayton-Smith and Laan, 2003). In the brain, the maternal allele of Ube3a is predominantly expressed as a result of tissue specific imprinting. Mature neurons exhibit maternal allele-specific expression (Albrecht et al., 1997, Rougeulle et al., 1997) although traces of paternal allele-specific expression are also detected. Biallelic expression is restricted to GFAP positive cells lining the ventricles and absent from GFAP positive astrocytes in other regions of the brain. In the brain, Ube3a predominantly expresses in the cerebellar Purkinje cells, neurons in the hippocampus, the cortex and substantia nigra (Dindot et al., 2008, Gustin et al., 2010). At the cellular level, E6-AP is localized in the nucleus as well as in the cytoplasm. Expression of E6-AP was also found in both presynaptic and postsynaptic compartments in cultured hippocampal neurons (Dindot et al., 2008). Ube3a-maternal deficient mice (Ube3a^m−/p+) exhibit learning and memory deficits as well as motor abnormalities (Heck et al., 2008, Jiang et al., 1998). The motor abnormalities in these Ube3a^m−/p+ mice are so far been shown due to dysfunction of the cerebellum. Paternal deficient Ube3a mice (Ube3a^m+/p−) fail to show these typical characteristics (Jiang et al., 1998).

The protective effect of Ube3a/E6-AP in neurodegenerative diseases was first showed by Cummings et al. (1999). Spinocerebellar ataxia 1 (SCA1) is a polyglutamine disease in which the protein ataxin 1 undergoes aggregation and accumulates mostly in the nucleus. Loss of E6-AP in SCA1 transgenic mice remarkably limited the formation of nuclear inclusions in Purkinje cells of the cerebellum. However the pathology is exacerbated due to the toxic substrates of E6-AP that are accumulated in its absence (Cummings et al., 1999). Later, various studies showed that E6-AP degrades a number of misfolded proteins like polyglutamine, CFTR and α synuclein (Mishra et al., 2008, Mishra et al., 2009, Mulherkar et al., 2009) indicating that E6-AP probably functions as cellular quality control ubiquitin ligase.

Patients with AS have been shown to manifest typical features of Parkinson disease (PD) like tremors, cogwheel rigidity and bradykinesia. This condition was responsive to levodopa, which is widely used for the symptomatic treatment of PD (Harbord, 2001). Recently, we have demonstrated that E6-AP localizes to the Lewy bodies in PD brain and enhances the degradation of α synuclein, which is the main component of the Lewy bodies (Mulherkar et al., 2009). All these findings suggest a role of E6-AP in proper functioning of the dopaminergic system in the brain. In the present study, we report the effect of maternal loss of Ube3a on dopaminergic neurons in the substantia nigra. We observed that Ube3a^m−/p+ mice showed reduced number of dopaminergic neurons in the substantia nigra accompanied by poor performance in behavioural paradigms sensitive to nigrostriatal dysfunction.