Mice Lacking Glypican 4 Display Juvenile Hyperactivity and Adult Social Interaction Deficits
Total Page：16
File Type：pdf, Size：1020Kb
Brain Plasticity 4 (2018) 197–209 197 DOI 10.3233/BPL-180079 IOS Press Research Report Mice Lacking Glypican 4 Display Juvenile Hyperactivity and Adult Social Interaction Deficits Cari Dowling and Nicola J. Allen∗ Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA Accepted: 14 December 2018 Published: 20 December 2018 Abstract. Glypican 4 (Gpc4) is a heparan sulfate proteoglycan that regulates glutamatergic synapse formation and function in the developing brain. Gpc4 KO mice have been shown to have decreased excitatory synapse number and less synaptic GluA1 AMPA receptors, leading to decreased synaptic transmission. Further, decreased expression of Gpc4 has been linked to autism spectrum disorder (ASD). Gpc4 is expressed by both astrocytes and neurons during postnatal development, with astrocyte expression higher in juvenile stages, and neuronal expression increasing with maturation. We therefore asked if mice lacking Gpc4 display behavioral alterations that are consistent with loss of GluA1 or ASD, and if so if they occur at juvenile ages when astrocyte Gpc4 is high, or at adult ages when both astrocytes and neurons express Gpc4. We found that juvenile (P14) Gpc4 KO mice display hyperactivity in the open field, which is corrected in adult mice (3 month). Adult Gpc4 KO mice show deficient behavior in social novelty, whilst non-social behaviors such as working memory and anxiety are unaffected. Thus, Gpc4 KO mice show age-specific behavioral alterations that are consistent with altered synaptic levels of GluA1 and behaviors associated with ASD. Keywords: Astrocyte, synapse, AMPA, neurodevelopment INTRODUCTION gic synapses via the release of glypican 4 (Gpc4), and do so by increasing synaptic levels of the GluA1 Alterations in neuronal synaptic development and subunit of the AMPA glutamate receptor (AMPAR) plasticity can have profound impact on circuit func- [4]. As the development of the appropriate synaptic tion and behavioral performance. In development the connections, and the ability to alter synaptic levels formation of synapses is regulated by interactions of AMPARs, are both important factors underlying between pre and post-synaptic neurons, for example behavioral performance, we set out to determine if adhesion molecules at the synaptic site, as well as mice lacking Gpc4 show any deficits in a series of by extrinsic cues from neighboring astrocytes [1, 2]. behavioral tasks. Astrocytes increase both the number of synapses that Glypicans are a family of heparan sulfate proteo- neurons form, and the function of those synapses, via glycans (HSPGs), of which there are six members the secretion of multiple signals [3]. Weidentified that in mammals [5]. Each glypican consists of a com- astrocytes induce formation of immature glutamater- pact core protein of ∼60kDa, along with multiple heparan sulfate chains attached near the C-terminus. ∗ Correspondence to: Nicola J. Allen, Molecular Neurobiology Glypicans are attached to the extracellular membrane Laboratory, Salk Institute for Biological Studies, 10010 North Tor- of the cell via a GPI-anchor (for which they earn their rey Pines Rd, La Jolla, CA, 92037, USA. Tel.: +1 858 453 4100 /Ext 2129; E-mail: [email protected]. name), and exist in this cell-attached form, or undergo ISSN 2213-6304/18/$35.00 © 2018 – IOS Press and the authors. All rights reserved This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License (CC BY-NC 4.0). 198 C. Dowling and N.J. Allen / Behavioral Phenotype of Glypican 4 Null Mice cleavage near the C-terminus to release a soluble form colliculus at P6 [13]. Further, astrocyte-specific Gpc4 to the extracellular space. Glypicans have important KO mice have the same phenotype as global Gpc4 roles in the development of many tissues, including KO mice in relation to impaired neuronal secretion of the brain [6, 7]. A well-established role of glypicans NP1 at P6, highlighting the important role of astro- is as modulators of growth factor signaling, for exam- cyte Gpc4 in early stages of synaptic development ple binding fibroblast growth factor (FGF) [8]. The [13]. outcome of this binding depends on the location of Alterations in glypican family members have the glypican - if it is in the cleaved form in the extra- been linked to a number of different neurologi- cellular space it will sequester the growth factor and cal disorders, including autism spectrum disorder stop it signaling, whereas if it is in the membrane (ASD; Gpc4,6), schizophrenia (Gpc4,5,6), neu- attached form it can enhance signaling by localizing roticism (Gpc6) and attention-deficit hyperactivity the ligand close to its receptor. disorder (ADHD; Gpc6) [15–18]. For example, Recently glypicans, in particular Gpc4, have been decreased expression of Gpc4 has been identified in demonstrated to play important roles in neuronal rare sporadic cases of ASD, caused by point muta- synapse formation and function [4, 9, 10]. Glypicans tions in regions that regulate Gpc4 expression [19]. are expressed by both astrocytes and neurons during Given the role for Gpc4 in synaptic development and postnatal development [4, 11, 12]. In the mouse brain, function, and the link to ASD, we set out to ask if including in the hippocampus, astrocyte expression absence of Gpc4 has any effect on behavioral per- of Gpc4 mRNA is high in the first two postnatal formance in mice, focusing on those tasks that are weeks and decreases after this, a time period when known to be altered in GluA1 KO mice, and are fur- synapses are forming and being remodeled [4, 11]. ther commonly used to assess mutations linked to Subsets of neurons e.g. dentate granule neurons in ASD. the hippocampus, upregulate Gpc4 in the second to third postnatal week [4, 11]. We identified that astro- cytes secrete Gpc4, and this soluble Gpc4 induces MATERIALS AND METHODS nascent synapse formation by increasing levels of GluA1 AMPARs on the postsynaptic dendrite [4]. Mice We further identified that Gpc4 does this by signal- ing through presynaptic protein tyrosine phosphatase All mouse procedures were approved by the Salk receptor delta (PTPRd) to upregulate secretion of Institute IACUC (Institutional Animal Care and Use the AMPAR clustering factor neuronal pentraxin 1 Committee). Mice were group housed with their lit- (NP1), which then clusters GluA1 AMPA receptors termates at weaning at P21, and kept on a 12 hour on the postsynaptic dendrite [13]. Neuronal Gpc4 is light: 12 hour dark cycle, with ad libitum access membrane attached and present in presynaptic ter- to food and water. Mice remained group housed minals, where it interacts with postsynaptic Lrrtm4 throughout the testing period. to induce synapse formation [9, 10], as well as with The mouse strain used for this research project, presynaptic PTPRs [14]. Thus, the cell-type expres- B6;129S5-Gpc4tm1Lex/Mmucd, identification num- sion, and synaptogenic mode of interaction of Gpc4, ber 032331-UCD, was obtained from the Mutant change across development. Mouse Regional Resource Center, a NCRR-NIH Gpc4 KO mice are viable and born at expected funded strain repository, and was donated to the Mendelian ratios [4, 13]. Analysis of synapse forma- MMRRC by Genentech, Inc., and are as previously tion and function in Gpc4 KO mice revealed a deficit described and validated for loss of Gpc4 mRNA and in synaptic strength in CA1 hippocampal neurons at protein [4, 13]. The presence of the mutant Gpc4 postnatal day (P) 12, manifest as a 30% decrease allele was determined by PCR genotyping. Mice in the average amplitude of individual excitatory were maintained on a C57Bl6/J background (wild synaptic events (mEPSCs), with a smaller but still type mice obtained from Jackson Labs, C57Bl6/J; significant decrease present at P24 [4]. This decrease 000664), or back-crossed for 6 generations to in mEPSC amplitude is correlated with a reduction FVB (wild type mice obtained from Jackson Labs, in GluA1 AMPARs at the postsynaptic density. The FVB/NJ; 001800). The back-crossing to FVB was effects on synapse formation and synaptic GluA1 are initiated as our previous experience working with not restricted to the hippocampus. For example, in the the Gpc4 KO line suggested there may be peri- developing visual system there is a 25% decrease in natal lethality when the mice were maintained on synapse number and synaptic GluA1 in the superior a pure C57Bl6/J background [4], an effect we no C. Dowling and N.J. Allen / Behavioral Phenotype of Glypican 4 Null Mice 199 longer observed when the KO mice were rederived, air puff. Ear Twitch: the tip of a pen was used to suggesting a facility-specific effect. We performed tickle the hair inside the ear. The score was recorded behavioral testing on mice from both genetic back- by how quickly and vigorously the mouse responded grounds, as the background can impact behavioral to the stimulus. Surface grab: the mouse was held performance [20]. by the tail and lowered toward an object, and scored Gpc4 is on the X chromosome, so to obtain mice based on distance from the object the mouse began for experiments breeding was performed as Gpc4 + /- to reach for it. Pupil constriction: in a dark room, female to Gpc4 + /y male, and male mice that were a flashlight was shone directly into the eye of the Gpc4 + /y (WT) or Gpc4-/y (KO) were compared. mouse, and the score recorded based on how quickly It is not possible to obtain littermate WT and Gpc4 the pupil dilated/constricted due to the light source. KO female mice, and female heterozygous mice are Olfactory response: chocolate was used to stimu- mosaic for Gpc4 so not a valid control, so female late an olfactory response, and response recorded mice were not analyzed in this study. based on the reaction time and intensity of reaction.