Analysis of cerebellar function in Ube3a-deficient mice reveals novel genotype-specific behaviors

Detlef H. Heck 0 Yu Zhao 1 Snigdha Roy 0 Mark S. LeDoux 1 Lawrence T. Reiter 1 0 Department of Anatomy and Neurobiology , UTHSC, Memphis, TN, USA 1 Department of Neurology Angelman syndrome (AS) is a childhood-onset neurogenetic disorder characterized by functionally severe developmental delay with mental retardation, deficits in expressive language, ataxia, appendicular action tremors and unique behaviors such as inappropriate laughter and stimulus-sensitive hyperexcitibility. Most cases of AS are caused by mutations which disrupt expression of maternal UBE3A. Although some progress has been made in understanding hippocampal-related memory and learning aspects of the disorder using Ube3a deficient mice, the numerous motoric abnormalities associated with AS (ataxia, action tremor, dysarthria, dysphagia, sialorrhea and excessive chewing/mouthing behaviors) have not been fully explored with mouse models. Here we use a novel quantifiable analysis of fluid consumption and licking behavior along with a battery of motor tests to examine cerebellar and other motor system defects in Ube3a deficient mice. Mice with a maternally inherited Ube3a deficiency (Ube3am2/p1) show defects in fluid consumption behavior which are different from Ube3am2/p2 mice. The rhythm of fluid licking and number of licks per visit were significantly different among the three groups (m2/p2, m2/p1, m1/p1) and indicate that not only was fluid consumption dependent on Ube3a expression in the cerebellum, but may also depend on low levels of Ube3a expression in other brain regions. Additional neurological testing revealed defects in both Ube3am2/p1 and Ube3am2/p2 mice in rope climbing, grip strength, gait and a raised-beam task. Longterm observation of fluid consumption behavior is the first phenotype reported that differentiates between mice with a maternal loss of function versus complete loss of Ube3a in the brain. The neuronal and molecular mechanisms underlying mouse fluid consumption defects specifically associated with maternally inherited Ube3a deficiency may reveal important new insights into the pathobiology of AS in humans. - Angelman syndrome (AS; MIM 105830) is a severe neurodevelopment disorder with an incidence of 1/20 000 and characterized by profound mental, motor and behavioral abnormalities. The molecular lesion in most AS patients is a defect in expression of the maternal copy of the E3 ubiquitin ligase gene UBE3A. Maternal deficiency or loss-of-function mutations in UBE3A are sufficient to cause AS (1,2). The UBE3A gene exhibits maternal allele-specific expression in the Purkinje cell layer of the cerebellum and the cell bodies of CA1 CA2 neurons of the hippocampus in mice and humans (3 5). Essentially, this means that the loss of function allele (be it a point mutation, imprinting center mutation or the more common large 15q11.2 q13 deletion) must be maternally derived to result in an AS phenotype or, in some cases, paternal uniparental disomy (UPD) can prevent the maternal copy of UBE3A from being expressed (reviewed in 6). Recent data also suggest that maternal imprinting is not restricted to neurons in the hippocampus and cerebellum but rather extends to all mature neurons in the brain. Furthermore, these data suggest that the paternal Ube3a allele may not be completely off in all neurons as previously reported (4). A great deal of research has been focused on understanding this complex imprinted regulation of UBE3A in the context of the molecular defects that cause AS (7). Patients with AS consistently exhibit a movement disorder that can be ascribed, in large part, to cerebellar dysfunction (7). In affected individuals, truncal ataxia contributes to postural instability whereas appendicular ataxia is often associated with an action tremor. In AS, postural maintenance is associated with the abnormal rhythmic bursts of muscle activity (8). Cerebellar defects in AS individuals clearly contribute to the motoric aspects of the disease. Furthermore, based on modern views of cerebellar function, cerebellar dysfunction could play a role in the various behavioral and cognitive deficits seen in AS (9 11). However, little progress has been made in the analysis of cerebellar function in mouse models of AS and how cerebellar defects in the mouse model relate to cerebellar motor and possibly cognitive defects present in AS individuals. To date, two Ube3a loss of function mouse models of AS have been generated (12,13). The Ube3a deficient mice generated by Jiang et al. (12) via knock out of exon 2 of the Ube3a gene were analyzed for gross cerebellar defects via accelerating rotarod tests. Mice generated by Miura et al. (13) were constructed by a knock-in strategy resulting in the replacement of exons 15 and 16 of the Ube3a gene with a b-geo reporter gene. These mice show similar rotarod defects and have been used to map regions of the brain that are subject to imprinted expression through the detection of the Ube3a-b-geo reporter gene. Although Ube3a shows clear maternal-specific expression in the cytoplasm of Purkinje neurons, Muria et al. also reported that Ube3ab-geo expression occurred in neurons of the granular cell layer. The subsequent generation of a Ube3aYFP knock-in mouse has verified that maternal-specific expression of Ube3a is not restricted to Purkinje cell bodies, but can be detected in cerebellar neurons of both the molecular and granular cell layers (4). In fact, maternal expression and even low levels of paternal expression of Ube3a have been detected in other regions of the brain including the cortex, thalamus and olfactory bulb (4). The only region where biallelic expression could be detected was the GFAP-positive cells that line the ventricles. Previous results from rotarod and balance beam testing suggest cerebellar-related movement defects and electrophysiological studies have shown that cerebellar neuronal activity is abnormal in Ube3a deficient mice (14). Recently, it has been shown that the rotarod defects in Ube3a deficient mice can be rescued by a mutation in the auto-phosphorylation domain of the a-CaMKII protein (15). However, accelerating rotarod testing may not be the most informative and sensitive behavioral assessment to identify primarily cerebellar defects since it does not reliably differentiate among defects in muscle, nerve, upper motor neuron and cerebellar motor control. A quantifiable test with output metrics that are closely tied to cerebellar activity and outside of the context of locomotion is needed which, ideally, can be combined with electrophysiological measurements to understand the neuronal aspects of cerebellar defects in these animals. Here we propose that fluid-licking behavior in mice is a sensitive behavioral test for both motor and autonomic aspects of cerebellar function. Fluid licking is a naturally occurring behavior that is easy to analyze in the home-cage environment and we have previously shown that it can be combined wi (...truncated)