Densin Regulates Body Growth During A Critical Early Postnatal Period

Autism spectrum disorder（ASD）is a neurodevelopmental disorder characterized by concomitant social interaction disorders,restrictive,persistent and stereotyped behaviors,and abnormal communication skills.Researchers has used whole-genome sequencing and found that the LRRC7 frameshift mutation caused the deletion of Densin,which is tightly related to ASD.In our previous study,we found that Nestin-cre;Lrrc7^fl/fl,the Densin knockout mice,exhibited obvious smaller body size in the early postnatal age.They almost stopped growth during the period of postnatal 14 to 24 days（P14-P24）.They began to regain body weight at P24-P26 and accelerated their recovery after P30.Their body weight was close to that of the control mice until 2-months old.In order to explore how Densin affects the growth of the body,we crossed Lrrc7^fl/fl with three kinds of cre lines,including Emx1-cre,h GFAP-cre and VGAT-cre which target different brain regions and different neural cell types.We found that VGAT-cre;Lrrc7^fl/flhave the same growth arrest phenotype as Nestin-cre;Lrrc7^fl/flmice during P14-P24.This result indicates that the function of Densin in inhibitory neurons regulates the body growth during the critical period.Through enzyme-linked immunosorbent assay,we found that the growth hormone（GH）in the blood of VGAT-cre;Lrrc7^fl/fland Nestin-cre;Lrrc7^fl/flmice decreased significantly.HE staining revealed no significant difference about the adenohypophysis cells between knockout mice and littermate controls.However,we found GH,growth hormone releasing hormone（GHRH）and somatostatin（SST）decreased in the hypothalamus of knockout mice at P9-P30 through real-time fluorescence quantitative PCR and Western blotting.The results indicate that the GH synthesis and release system in the central nervous system is impaired.Densin in the hypothalamus of VGAT-cre;Lrrc7^fl/fl knockout mice was significantly reduced during P9-P30 as examined by real-time fluorescent quantitative PCR and Western blotting,suggesting that Densin is mainly expressed in inhibitory neurons in the hypothalamus.Densin is an abundant post-synaptic protein.Our previous work has found that Densin is an important molecule that regulates the structure and transmission of excitatory synapses.We found that Glu N1,Glu A1 and Glu A2 were all significantly reduced in the hypothalamus of VGAT-cre;Lrrc7^fl/fl mice at P9.They are the important functional proteins of excitatory synapses.Glu N1 is one of the NMDAR receptor subunits.Glu A1 and Glu A2 are AMPAR receptor subunits.Meanwhile,we found thatδ-catenin,β-catenin and N-cadherin were also significantly reduced in the hypothalamus of VGAT-cre;Lrrc7^fl/fl mice at P9.Densin interacts with theδ-catenin that forms adhesion junction complexes withβ-catenin and N-cadherin.δ-catenin,β-catenin and N-cadherin have been reported to be involved in the regulation of excitatory synaptic transmission and the expression and localization of glutamate receptors.These preliminary results suggest that the first to the third postnatal week is a critical period for the hypothalamus to start to control body growth.Densin in hypothalamic inhibitory neurons in mice at around P9 may regulate excitatory synaptic transmission through theδ-catenin/β-catenin/N-cadherin complex that interacts with it.This event may have an important impact on the development and function of neurons involved in regulating the synthesis and release of GH in pituitary gland.Through the analysis of the targeting specificity in hypothalamic nucleus and neurons of different Cre mice,we preliminarily determined that the body growth during the early postnatal stage is regulated by inhibitory neurons located in the arcuate nucleus of hypothalamus in VGAT-cre;Lrrc7^fl/flmice.Next,we will study whether Densin affects the synaptic transmission and excitability of inhibitory neurons in the arcuate nucleus at P9,and whether Densin regulates the development and functional maturation of GHRH neurons in the arcuate nucleus and SST neurons in the paraventricular nucleus of the hypothalamus during the critical period.