Resistance to change and vulnerability to stress: autistic‐like features of GAP43‐deficient mice

There is an urgent need for animal models of autism spectrum disorder (ASD) to understand the underlying pathology and facilitate development and testing of new treatments. The synaptic growth‐associated protein‐43 (GAP43) has recently been identified as an autism candidate gene of interest. Our previous studies show many brain abnormalities in mice lacking one allele for GAP43 [GAP43 (+/?)] that are consistent with the disordered connectivity theory of ASD. Thus, we hypothesized that GAP43 (+/?) mice would show at least some autistic‐like behaviors. We found that GAP43 (+/?) mice, relative to wild‐type (+/+) littermates, displayed resistance to change, consistent with one of the diagnostic criteria for ASD. GAP43 (+/?) mice also displayed stress‐induced behavioral withdrawal and anxiety, as seen in many autistic individuals. In addition, both GAP43 (+/?) mice and (+/+) littermates showed low social approach and lack of preference for social novelty, consistent with another diagnostic criterion for ASD. This low sociability is likely because of the mixed C57BL/6J 129S3/SvImJ background. We conclude that GAP43 deficiency leads to the development of a subset of autistic‐like behaviors. As these behaviors occur in a mouse that displays disordered connectivity, we propose that future anatomical and functional studies in this mouse may help uncover underlying mechanisms for these specific behaviors. Strain‐specific low sociability may be advantageous in these studies, creating a more autistic‐like environment for study of the GAP43‐mediated deficits of resistance to change and vulnerability to stress.     

Neuroprotective Role of Astrocytic Gap Junctions in Ischemic Stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/?)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43^{fl/ fl}/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43^fl/fl] (here designated as Cre(?) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/?) and Cre(+) mice compared to Cx43(+/+) and Cre(?) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/?) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.     

Growth‐associated protein 43 promotes thyroid cancer cell lines progression via epithelial‐mesenchymal transition

Thyroid cancer is maintaining at a high incidence level and its carcinogenesis is mainly affected by a complex gene interaction. By analysis of the next‐generation resequencing of paired papillary thyroid cancer (PTC) and adjacent thyroid tissues, we found that Growth Associated Protein 43 (GAP43), a phosphoprotein activated by protein kinase C, might be novel markers associated with PTC. However, its function in thyroid carcinoma has been poorly understood. We discovered that GAP43 was significantly overexpressed in thyroid carcinoma and these results were consistent with that in The Cancer Genome Atlas (TCGA) cohort. In addition, some clinicopathological features of GAP43 in TCGA database showed that up‐regulated GAP43 is significantly connected to lymph node metastasis (P < 0.001) and tumour size (P = 0.038). In vitro experiments, loss of function experiments was performed to investigate GAP43 in PTC cell lines (TPC‐1 and BCPAP). The results proved that GAP43 knockdown in PTC cell significantly decreased the function of cell proliferation, colony formation, migration, and invasion and induced cell apoptosis. Furthermore, we also indicated that GAP43 could modulate the expression of epithelial‐mesenchymal transition‐related proteins, which could influence invasion and migration. Put those results together, GAP43 is a gene which was associated with PTC and might be a potential therapeutic target.     

Structural abnormalities in the primary somatosensory cortex and a normal behavioral profile in Contactin-5 deficient mice

Contactin-5 (Cntn5) is an immunoglobulin cell adhesion molecule that is exclusively expressed in the central nervous system. In view of its association with neurodevelopmental disorders, particularly autism spectrum disorder (ASD), this study focused on Cntn5-positive areas in the forebrain and aimed to explore the morphological and behavioral phenotypes of the Cntn5 null mutant (Cntn5^?/?) mouse in relation to these areas and ASD symptomatology. A newly generated antibody enabled us to elaborately describe the spatial expression pattern of Cntn5 in P7 wild type (Cntn5^+/+) mice. The Cntn5 expression pattern included strong expression in the cerebral cortex, hippocampus and mammillary bodies in addition to described previously brain nuclei of the auditory pathway and the dorsal thalamus. Thinning of the primary somatosensory (S1) cortex was found in Cntn5^?/? mice and ascribed to a misplacement of Cntn5-ablated cells. This phenotype was accompanied by a reduction in the barrel/septa ratio of the S1 barrel field. The structure and morphology of the hippocampus was intact in Cntn5^?/? mice. A set of behavioral experiments including social, exploratory and repetitive behaviors showed that these were unaffected in Cntn5^?/? mice. Taken together, these data demonstrate a selective role of Cntn5 in development of the cerebral cortex without overt behavioral phenotypes.     

A HuD‐ZBP1 ribonucleoprotein complex localizes GAP‐43 mRNA into axons through its 3′ untranslated region AU‐rich regulatory element

Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β‐actin and GAP‐43 mRNAs. β‐actin 3′UTR has a defined element for interaction with ZBP1, but GAP‐43 mRNA shows no homology to this RNA sequence. Here, we show that an AU‐rich regulatory element (ARE) in GAP‐43′s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP‐43 mRNA levels increase after in vivo injury, and GAP‐43 mRNA shows an increased half‐life in regenerating axons. GAP‐43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co‐immunoprecipitate in an RNA‐dependent fashion. Reporter mRNA with the GAP‐43 ARE competes with endogenous β‐actin mRNA for axonal localization and decreases axon length and branching similar to the β‐actin 3′UTR competing with endogenous GAP‐43 mRNA. Conversely, over‐expressing GAP‐43 coding sequence with its 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP‐43′s 3′UTR.

     

Effects of sensory deprivation on the development of asymmetrical synapses in mouse barrels

Alterations in the numerical density and structure of asymmetrical synapses were examined in thin sections through barrel D4 in six CD/1 mice, including three controls and three sensory deprived animals. Sensory deprivation was effected by once daily trimming of all large mystacial vibrissae on the contralateral side of the snout from P0. The mice were perfuse-fixed at P20, several days following the termination of rapid synaptic growth during barrel development (White et al., Somatosens Mot Res 14: 34-55, 1997). Cerebral hemispheres contralateral to the deprived side were osmicated, sectioned at 40 microm and embedded in plastic for thin sectioning. Sterio's (J Microsc 134: 127-136, 1984) procedure combined with serial thin section analysis (Braendgaard and Gundersen, J Neurosci Meth 18: 39-78, 1986), was applied blindly to systematic random samples of neuropil in barrel hollows and septa. No significant difference in the numerical density, estimated total number, or in the proportion of perforated postsynaptic densities was observed. However, a significant decrease in the diameters of asymmetrical postsynaptic densities was observed in hollow (P < 0.05) and septal (P < 0.05) neuropil of deprived animals. These results demonstrate a significant morphological alteration in asymmetrical synapses of a type consistent with a reduction in synaptic activity consequent to sensory deprivation.     

Common genetic variants in GAL,GAP43 and NRSN1 and interaction networks confer susceptibility to Hirschsprung disease

Hirschsprung disease (HSCR) is a severe multifactorial genetic disorder. Microarray studies indicated GAL, GAP43 and NRSN1 might contribute to the altered risk in HSCR. Thus, we focused on genetic variations in GAL, GAP43 and NRSN1, and the gene‐gene interactions involved in HSCR susceptibility. We recruited a strategy combining case‐control study and MassArray system with interaction network analysis. For GAL, GAP43 and NRSN1, a total of 18 polymorphisms were assessed in 104 subjects with sporadic HSCR and 151 controls of Han Chinese origin. We found statistically significant differences between HSCR and control groups at 5 genetic variants. For each gene, the haplotypes combining all polymorphisms were the most significant. Based on SNPsyn, MDR and GeneMANIA analyses, we observed significant gene‐gene interactions among GAL, GAP43, NRSN1 and our previous identified RELN, GABRG2 and PTCH1. Our study for the first time indicates that genetic variants within GAL, GAP43 and NRSN1 and related gene‐gene interaction networks might be involved in the altered susceptibility to HSCR in the Han Chinese population, which might shed more light on HSCR pathogenesis.     

Organization of myelin in the mouse somatosensory barrel cortex and the effects of sensory deprivation

In rodents, the barrel cortex is a specialized area within the somatosensory cortex that processes signals from the mystacial whiskers. We investigated the normal development of myelination in the barrel cortex of mice, as well as the effects of sensory deprivation on this pattern. Deprivation was achieved by trimming the whiskers on one side of the face every other day from birth. In control mice, myelin was not present until postnatal day 14 and did not show prominence until postnatal day 30; adult levels of myelination were reached by the end of the second postnatal month. Unbiased stereology was used to estimate axon density in the interbarrel septal region and barrel walls as well as the barrel centers. Myelin was significantly more concentrated in the interbarrel septa/barrel walls than in the barrel centers in both control and sensory‐deprived conditions. Sensory deprivation did not impact the onset of myelination but resulted in a significant decrease in myelinated axons in the barrel region and decreased the amount of myelin ensheathing each axon. Visualization of the oligodendrocyte nuclear marker Olig2 revealed a similar pattern of myelin as seen using histochemistry, but with no significant changes in Olig2+ nuclei following sensory deprivation. Consistent with the anatomical results showing less myelination, local field potentials revealed slower rise times following trimming. Our results suggest that myelination develops relatively late and can be influenced by sensory experience. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Sim2基因对PC12细胞分化的影响

[摘要] 目的 探讨位于Down综合征关键位点的Sim2基因对PC12细胞分化的影响及其机制。 方法 以pcDNA3-mSim2真核表达载体稳定转染PC12细胞,以倒置相差显微镜镜观察PC12细胞神经突起的变化;以RT-PCR方法检测神经元分化相关基因GAP43和Synapsin I mRNA表达水平的变化;流式细胞仪检测GAP43蛋白的表达。 结果 RT-PCR结果显示, pcDNA3-mSim2转染后,mSim2 mRNA表达明显上调;与对照组相比,转染mSim2的PC12细胞突起数量显著减少,长度明显变短;GAP43和Synapsin I mRNA表达水平明显降低（P<0.05）;流式细胞仪检测发现,转染mSim2的PC12细胞GAP43蛋白表达水平显著降低（P<0.05）。 结论 Sim2基因可通过影响神经元的分化参与Down综合征的发生。     

Multiple subcellular mRNA distribution patterns in neurons: A nonisotopic in situ hybridization analysis

Previous studies have established that most of the mRNAs that neurons express are localized in the cell body and very proximal dendrites, whereas a small subset of mRNAs is present at relatively high levels in dendrites. It is not clear, however, whether particular mRNAs have the same subcellular distribution in different types of neurons or whether different types of neurons sort mRNAs in different ways. The present study was undertaken to address these questions. Nonisotopic in situ hybridization techniques were used to define the subcellular localization of representative mRNAs including β-tubulin, low-molecular-weight neurofilament protein (NF-68), high-molecular-weight microtubule-associated protein (MAP2), growth-associated protein 43 (F1/GAP43), the alpha subunit of calcium/calmodulin-dependent protein kinase II (αCaMII kinase), and poly(A⁺) mRNA. The mRNAs for β-tubulin, neurofilament 68, and F1/GAP43 were restricted to the region of the cell body and very proximal dendrites in most neurons. In some neuron types, however, labeling for NF-68 extended for considerable distances into dendrites. In some neurons that express MAP2, the mRNA was present at the highest levels in the proximal third to half of the dendritic arbor, whereas in other neurons the highest levels of labeling were in the cell body. In most neurons that express αCaMII kinase, the highest levels of the mRNA were in the cell body, but labeling was also present throughout dendrites. However, in a few types of neurons, αCaMII kinase mRNA was largely restricted to the cell body. The fact that there are no general rules for mRNA localization that apply to all neuron types implies the existence of neuron type-specific mechanisms that regulate mRNA distribution. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 473–493, 1997     

Effects of sensory deprivation on the development of asymmetrical synapses in mouse barrels

Alterations in the numerical density and structure of asymmetrical synapses were examined in thin sections through barrel D4 in six CD/1 mice, including three controls and three sensory deprived animals. Sensory deprivation was effected by once daily trimming of all large mystacial vibrissae on the contralateral side of the snout from P0. The mice were perfuse-fixed at P20, several days following the termination of rapid synaptic growth during barrel development (White et al. , Somatosens Mot Res 14 : 34-55, 1997). Cerebral hemispheres contralateral to the deprived side were osmicated, sectioned at 40 mum and embedded in plastic for thin sectioning. Sterio's ( J Microsc 134 : 127-136, 1984) procedure combined with serial thin section analysis (Braendgaard and Gundersen, J Neurosci Meth 18 : 39-78, 1986), was applied blindly to systematic random samples of neuropil in barrel hollows and septa. No significant difference in the numerical density, estimated total number, or in the proportion of perforated postsynaptic densities was observed. However, a significant decrease in the diameters of asymmetrical postsynaptic densities was observed in hollow (P < 0.05) and septal (P < 0.05) neuropil of deprived animals. These results demonstrate a significant morphological alteration in asymmetrical synapses of a type consistent with a reduction in synaptic activity consequent to sensory deprivation.     

Ankyrin Repeat‐rich Membrane Spanning/Kidins220 protein regulates dendritic branching and spine stability in vivo

The development of nervous system connectivity depends upon the arborization of dendritic fields and the stabilization of dendritic spine synapses. It is well established that neuronal activity and the neurotrophin BDNF modulate these correlated processes. However, the downstream mechanisms by which these extrinsic signals regulate dendritic development and spine stabilization are less well known. Here we report that a substrate of BDNF signaling, the Ankyrin Repeat‐rich Membrane Spanning (ARMS) protein or Kidins220, plays a critical role in the branching of cortical and hippocampal dendrites and in the turnover of cortical spines. In the barrel somatosensory cortex and the dentate gyrus, regions where ARMS/Kidins220 is highly expressed, no difference in the complexity of dendritic arbors was observed in 1‐month‐old adolescent ARMS/Kidins220^+/? mice compared to wild‐type littermates. However, at 3 months of age, young adult ARMS/Kidins220^+/? mice exhibited decreased dendritic complexity. This suggests that ARMS/Kidins220 does not play a significant role in the initial formation of dendrites but, rather, is involved in the refinement or stabilization of the arbors later in development. In addition, at 1 month of age, the rate of spine elimination was higher in ARMS/Kidins220^+/? mice than in wild‐type mice, suggesting that ARMS/Kidins220^+/? levels regulate spine stability. Taken together, these data suggest that ARMS/Kidins220 is important for the growth of dendritic arbors and spine stability during an activity‐ and BDNF‐dependent period of development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Increases in the Numerical Density of GAT-1 Positive Puncta in the Barrel Cortex of Adult Mice after Fear Conditioning

Three days of fear conditioning that combines tactile stimulation of a row of facial vibrissae (conditioned stimulus, CS) with a tail shock (unconditioned stimulus, UCS) expands the representation of “trained” vibrissae, which can be demonstrated by labeling with 2-deoxyglucose in layer IV of the barrel cortex. We have also shown that functional reorganization of the primary somatosensory cortex (S1) increases GABAergic markers in the hollows of “trained” barrels of the adult mouse. This study investigated how whisker-shock conditioning (CS+UCS) affected the expression of puncta of a high-affinity GABA plasma membrane transporter GAT-1 in the barrel cortex of mice 24 h after associative learning paradigm. We found that whisker-shock conditioning (CS+UCS) led to increase expression of neuronal and astroglial GAT-1 puncta in the “trained” row compared to controls: Pseudoconditioned, CS-only, UCS-only and Naïve animals. These findings suggest that fear conditioning specifically induces activation of systems regulating cellular levels of the inhibitory neurotransmitter GABA.     

Synaptic localization of growth‐associated protein 43 in cultured hippocampal neurons during synaptogenesis

Growth‐associated protein 43 (GAP‐43), a novel axonal phosphoprotein, is originally identified as a growth‐cone‐specific protein of developing neurons in vitro. The expression of GAP‐43 is also shown to be up‐regulated concomitant with increased synaptic plasticity in the brains in vivo, but how GAP‐43 is concerned with synaptic plasticity is not well understood. In the present study, therefore, we aimed to elucidate subcellular localization of GAP‐43 as culture development of rat hippocampal neurons. Western blotting showed that the expression of GAP‐43 in the cerebral and hippocampal tissues was prominently high at postnatal days 14 and 21 or the active period of synaptogenesis. Double‐labelling immunohistochemistry with an axonal marker Tau revealed that the immunoreactivity of GAP‐43 was seen throughout axons of cultured hippocampal neurons but stronger at axonal puncta of developing neurons than axonal processes. Double‐labelling immunohistochemistry with presynaptic terminal markers of synapsin and synaptotagmin revealed that the immunoreactivity of GAP‐43 was observed mostly at weak synapsin‐ and synaptotagmin‐positive puncta rather than strong ones. The quantitative analysis of immunofluorescent intensity showed a clear inverse correlation between GAP‐43 and either synapsin or synaptotagmin expression. These data indicate that GAP‐43 is highly expressed at immature growing axonal terminals and its expression is decreased along with the maturation of synaptogenesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Immunohistochemical comparison of whisker pad cutaneous innervation in Swiss Webster and hairless mice

To establish the mouse mutant, hairless (Hr), as a useful model for future analyses of target–ending interactions, we assessed the cutaneous innervation in the whisker pad after loss of primary hair targets. Postnatal (P) development of fur in Hr begins similarly to that of “normal” Swiss Webster (SW) mice. Around P10, hairs are shed and the follicles rendered permanently incompetent. Hair loss progresses rostrocaudally until the entire skin is denuded. Substantial alterations in the distribution and density of sensory and autonomic endings in the mystacial pad vibrissal and intervibrissal fur innervation were discovered. Pilo-neural complexes innervating fur hairs were dismantled in Hr. Epidermal innervation in SW was rich; only a few endings expressed growth-associated protein-43?kdal (GAP), suggesting limited changes in axonal elongation. Innervation in Hr formed a dense layer passing upward through the thickened epidermis, with substantial increases among all types of endings. Vibrissal follicle–sinus complexes were also hyperinnervated. Endings in Hr vibrissae and fur were strongly GAP-positive, suggesting reorganization of innervation. Dermal and vascular autonomic innervation in both strains co-localized tyrosine hydroxylase and neuropeptide Y, but only in Hr did neuropeptide Y co-localize calcitonin gene-related peptide (CGRP) and express GAP immunolabeling. Stereological quantitation of trigeminal ganglia revealed no differences in neuron number between Hr and SW, although there were small increases in cell volume in Hr trigeminal ganglion cells. These results suggested that a form of collateral sprouting was active in Hr mystacial pads, not in response to local injury, but as a result of loss of primary target tissues.     

A survey of morphogenesis during the early postnatal period in PMBSF barrels of mouse SmI cortex with emphasis on barrel D4

The long term goal of this work is to understand synaptogenesis in homologous regions of the cerebral cortex, i.e. a whisker barrel. Hemispheres of aldehyde perfused mice, at various ages from P6 to P65 (DOB = P0; three each), were osmicated and sectioned at 40mm parallel to the pia. Barrels were identified, mapped and measured in sections through mid-level layer IV, and then embedded for electron microscopy. The main findings were: (1) Cell bodies and large diameter dendrites thin out in barrel hollows from P6 to P8. (2) Degeneration occurs primarily from P6 to P11, peaking on P8. (3) Single synapses from narrow, tubular axons predominate before P14; afterwards, multiple synapses from bag-like terminals increase in number. (4) The number of spines increases dramatically between P9 and P12. (5) Asymmetrical and symmetrical synapses occur at all ages studied; their junction lengths are not significantly different at any age. (6) Asymmetrical synapse density increases rapidly from P6 to P8, slowly from P9 to P 12, sharply between P13 and P14 along with patterned whisking, slowly to P20 and drops in adults. (7) Synapses onto spiny and non-spiny stellate cell bodies increase markedly from P10 to P20. (8) Changes in density of asymmetrical synapses in neuropil and of symmetrical synapses on spiny stellate cell bodies follow similar sequences but the sequence in neuropil is 72 h earlier. (9) When barrel size is taken into account, synaptogenesis is monotonic, increasing sharply in the second postnatal week followed by a slower increase into adulthood.     

触觉剥夺后豚鼠桶状皮质DCX 阳性细胞的实验研究*

目的:观察豚鼠单侧触觉剥夺后豚鼠两侧桶状皮质的DCX阳性细胞数量的差别,探讨触觉剥夺对豚鼠桶状皮质神经发生的影响。方法:12只健康豚鼠随机分为2组,每组6只,制作豚鼠单侧(右侧)触觉(胡须)剥夺模型,之后常规饲养1月和2月灌注取材,用免疫组化和免疫荧光双标法观察同一只豚鼠两侧桶状皮质的DCX阳性细胞情况并比较其数量差异。结果:DCX免疫组织化学染色显示两实验组大脑皮层barrel区DCX阳性细胞数实验侧均明显多于对照侧;NeuN免疫组织化学染色显示两实验组动物大脑皮层barrel区两侧NeuN阳性细胞数差别无统计学意义;DCX和NeuN免疫荧光双标染色显示两实验组大脑皮层barrel区均可见双标细胞存在。结论:触觉剥夺后豚鼠两侧桶状皮质的DCX阳性细胞数具有明显差异性,可能是神经再生的表现。     

GAP‐43 mRNA in growth cones is associated with HuD and ribosomes

The neuron‐specific ELAV/Hu family member, HuD, interacts with and stabilizes GAP‐43 mRNA in developing neurons, and leads to increased levels of GAP‐43 protein. As GAP‐43 protein is enriched in growth cones, it is of interest to determine if HuD and GAP‐43 mRNA are associated in developing growth cones. HuD granules in growth cones are found in the central domain that is rich in microtubules and ribosomes, in the peripheral domain with its actin network, and in filopodia. This distribution of HuD granules in growth cones is dependent on actin filaments but not on microtubules. GAP‐43 mRNA is localized in granules found in both the central and peripheral domains, but not in filopodia. Ribosomes were extensively colocalized with HuD and GAP‐43 mRNA granules in the central domain, consistent with a role in the control of GAP‐43 mRNA stability in the growth cone. Together, these results demonstrate that many of the components necessary for GAP‐43 mRNA translation/stabilization are present within growth cones. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2004     

Circuit analysis of experience-dependent plasticity in the developing rat barrel cortex

Sensory deprivation during a critical period reduces spine motility and disrupts receptive field structure of layer 2/3 neurons in rat barrel cortex. To determine the locus of plasticity, we used laser scanning photostimulation, allowing us to rapidly map intracortical synaptic connectivity in brain slices. Layer 2/3 neurons differed in their spatial distributions of presynaptic partners: neurons directly above barrels received, on average, significantly more layer 4 input than those above the septa separating barrels. Complementary connectivity was found in deprived cortex: neurons above septa were now strongly coupled to septal regions, while connectivity between barrel regions and layer 2/3 was reduced. These results reveal competitive interactions between barrel and septal circuits in the establishment of precise intracortical circuits.