Haploinsufficiency of the autism candidate gene Neurobeachin induces autism-like behaviors and affects cellular and molecular processes of synaptic plasticity in mice
Highlights
► Neurobeachin haploinsufficiency leads to autistiform behaviors in mice. ► Heterozygous Neurobeachin mice have an enhanced induction of long-term potentiation ► Altered neuronal activity is concomitant with increased phosphorylation of CREB ► Increase in active CREB leads to increased hippocampal BDNF expression.
Introduction
With a prevalence of 0.6%, autism spectrum disorders (ASDs) are among the most common neurodevelopmental disorders. ASD is characterized by various pathognomonic behavioral alterations including impaired social interaction and communication as well as frequent intellectual disability and anxiety (Fombonne, 2009). It is generally accepted that there is a strong genetic basis for ASD that probably involves multiple genes (Abrahams and Geschwind, 2008). Recently, Neurobeachin (NBEA) has been suggested as an ASD candidate gene following the description of a patient with de novo balanced reciprocal translocation t(5;13)(q12.1;q13.2) with a breakpoint in intron 1 of NBEA (Volders et al., 2011). In addition, three patients with a monoallelic NBEA deletion were reported. Initially, a woman with autism was described with a deletion involving 13q13 and portions of q12 and q14. This deletion was not present in an autistic maternal cousin (Ritvo et al., 1988). In the second patient, a paternally derived deletion encompassed a 9 Mb fragment in the 13q12–q13 region. The father was not further investigated for autistic features (Smith et al., 2002). A de novo deletion of 13q13.2–q14.1 was detected in a boy with autism in a screen for the incidence of cytogenetic abnormalities in a population of autism patients (Reddy, 2005). The NBEA gene is located in a 19 cM region that was identified as a candidate region for ASD by linkage study and encompasses a low-frequency common fragile site (FRA13A) linked to ASD (Volders et al., 2011).
Little is known about the function of NBEA, but it has been shown to be a brain-enriched multidomain scaffolding protein located at tubulovesicular endomembranes near the trans-Golgi network (TGN) in neuronal soma and dendrites. A small fraction of NBEA is present at the postsynaptic membrane of a minority of synapses (Wang et al., 2000). The C-terminal part of NBEA contains a pleckstrin homology — beige and Chediak-Higashi (BEACH) — WD40 domain module, generally implicated in vesicle trafficking (Gebauer et al., 2004, Jogl et al., 2002, Wang et al., 2000). The N-terminal region possesses a concanavalin A-like lectin domain flanked by Armadillo repeats suggested to play a role in intracellular trafficking (Breidenbach and Brunger, 2005, Burgess et al., 2009). Distal from these regions, an A kinase anchoring protein (AKAP) domain is present, recruiting cAMP-dependent protein kinase A (PKA) to endomembranes near the TGN by high affinity binding to its RIIα subunit. NBEA has been shown to be involved in post-Golgi membrane trafficking and knockdown of Nbea in a neuroendocrine cell line led to enhanced secretion of dense-core secretory granules (DCSGs), the neuroendocrine counterpart of large dense-core vesicles (LDCVs) (Castermans et al., 2010). Furthermore, blood platelets of a patient haploinsufficient for NBEA showed abnormal morphology of dense granules (Castermans et al., 2010). Knockout Nbea in two independent mouse models resulted in perinatal lethality due to a complete block of evoked neuromuscular synaptic transmission (Medrihan et al., 2009, Su et al., 2004).
These findings suggest that Nbea haploinsufficiency may affect neural functioning in a subtle way, which could in fact explain the behavioral alterations observed in haploinsufficient patients. To test this hypothesis, we investigated whether Nbea haploinsufficiency in laboratory mice leads to changes in behavioral domains related to the diagnostic hallmarks of ASD.
We included tests of social behavior, since they relate to the core symptoms of ASD and only few ASD mouse models display such symptoms (Robertson and Feng, 2011), as well as tests for repetitive behavior, neuromotor performance, exploratory and general cage activity, sensitivity to auditory stimuli and sensorimotor gating, and learning and memory. Brain volume and regional neuronal activity were determined to examine whether Nbea haploinsufficiency affects gross brain morphology and function. As these analyses revealed changes in hippocampal activity, we assessed hippocampal (CA1) synaptic plasticity as well as some crucial plasticity-related molecular processes such as cAMP response element-binding protein (CREB) phosphorylation and levels of its target gene product brain-derived neurotrophic factor (BDNF), which might be at the basis of autistiform and neurocognitive defects.
Section snippets
Animals
The GH240B transgenic line (Su et al., 2004) was backcrossed for at least 10 generations with C57BL/6JRj mice (Janvier). Female mice of 20 weeks of age (17 Nbea+/+ and 18 Nbea+/− mice) at the beginning of testing, were housed in groups in standard cages (wood-shaving bedding) under conventional laboratory conditions (12/12 h light/dark cycle, lights on at 8 am; 22 °C) and were allowed food and water ad libitum. Group-housed females were used to avoid territorial aggression and fighting that
Increased relative brain volume in Nbea+/− mice
The body weight of Nbea+/− mice was significantly lower than Nbea+/+ mice (RM-AN0VA: main effect of genotype: F(1,32) = 39.8, P < 0.001) and this difference remained constant during behavioral testing (Fig. S1A). Further investigation in 12 week old female mice revealed reduced body and brain weight in Nbea+/− mice compared to Nbea+/+ mice (t = 9.80; P < 0.001 and t = 5.27; P < 0.001 respectively) resulting in an elevated brain to body weight ratio (t = − 8.60; P < 0.001) (Figs. S1B–D). Since Nbea+/− mice are
Discussion
Nbea+/− mice displayed several phenotypic changes directly or indirectly related to ASD symptoms. Haploinsufficiency of Nbea increased self-grooming behavior, decreased sociability read-outs, enhanced conditioned fear, and impaired spatial learning and memory. These findings coincided with increased relative brain volume and alterations in DMS and hippocampus activation (based on zif268 ISH) and enhanced hippocampal LTP. At the molecular level, Nbea knockdown in βTC3 cells resulted in increased
Acknowledgments
We thank Sandra Meulemans and Leen Van Aerschot the technical assistance. This work was supported by the “agentschap voor innovatie door wetenschap en technologie Vlaanderen (IWT-Vlaanderen)”, “steunfonds Marguerite-Marie Delacroix”, KU Leuven [IDO/08/013, GOA/12/008], Methusalem grant of the Flemish Government and MEMOSAD [F2-2007-200611] of the European Union. The funding sources had no involvement in the study design; the collection, analysis and interpretation of data; in writing the report
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