Loss of dopaminergic neurons and resulting behavioural deficits in mouse model of Angelman syndrome
Research Highlights
►Loss of dopaminergic neurons in Ube3a-maternal deficient mice. ►The Ube3am−/p+ mice exhibits motor deficits due to nigrostriatal dysfunction. ►The synaptophysin and PSD95 were reduced in Ube3am−/p+ mice striatum.
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 (Ube3am−/p+) exhibit learning and memory deficits as well as motor abnormalities (Heck et al., 2008, Jiang et al., 1998). The motor abnormalities in these Ube3am−/p+ mice are so far been shown due to dysfunction of the cerebellum. Paternal deficient Ube3a mice (Ube3am+/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 Ube3am−/p+ mice showed reduced number of dopaminergic neurons in the substantia nigra accompanied by poor performance in behavioural paradigms sensitive to nigrostriatal dysfunction.
Section snippets
Materials
Mouse monoclonal anti-E6-AP antibody was purchased from BD Biosciences (San Jose, CA, USA). Rabbit polyclonal anti-E6-AP, anti-p53 and anti-GAPDH were purchased from Santa Cruz Biotechnologies (CA, USA). Rabbit polyclonal anti-tyrosine hydroxylase (TH) and anti-dopamine transporter (DAT) were purchased from Chemicon (Temecula, California, USA). Mouse monoclonal anti-synaptophysin from Sigma (St. Louis, MO, USA) and anti-PSD-95 from Stressgen (Victoria, Canada) were used. Anti-phospho Thr286
Motor impairments in Ube3am−/p+ mice
In order to check for the motor deficits caused due to loss of maternal Ube3a, we performed various behavioural tests on 7–8 months old mice. The gait analysis of mice revealed significantly longer hind-limb stride length in the Ube3am−/p+ mice compared to the wild type (Ube3am+/p+) (Fig. 1A). There was no significant difference in the hind-base width between the two genotypes. Our findings are very similar with the observations made by Heck et al. (2008), however, hind-limb stride length seems
Discussion
The well-studied dysfunctions in the motor behaviour of Ube3am−/p+ mice are until now reasoned to be due to loss of Ube3a in the cerebellum. We subjected these mice to a number of behavioural tests to further study these motor deficits. In the footprint gait analysis the increased stride length and wide hind-base width indicate ataxic gait. The rotarod test showed that the Ube3am−/p+ mice performed poorly as compared to the wild type mice (Ube3am+/p−). These motor deficits seem to be more
Acknowledgments
This work was supported by the Department of Biotechnology, Government of India. S. M. was supported by research fellowship from the Council of Scientific and Industrial Research, Government of India. We thank Mr. M. Singh, Mr. Partha Dey and Mr. Narender for technical assistance. We thank Dr. Narender Dhingra from NBRC, India for anti-synaptophysin and anti-PSD95 antibodies and Dr. Shiv Kumar Sharma from NBRC, India for anti-phospho Thr 286 αCaMKII and anti-total αCaMKII antibodies.
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