Volume 13 Issue 3
Mar.  2022
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Chaojuan Yang, Yonglu Tian, Feng Su, Yangzhen Wang, Mengna Liu, Hongyi Wang, Yaxuan Cui, Peijiang Yuan, Xiangning Li, Anan Li, Hui Gong, Qingming Luo, Desheng Zhu, Peng Cao, Yunbo Liu, Xunli Wang, Min-hua Luo, Fuqiang Xu, Wei Xiong, Liecheng Wang, Xiang-yao Li, Chen Zhang. Restoration of FMRP expression in adult V1 neurons rescues visual deficits in a mouse model of fragile X syndrome[J]. Protein&Cell, 2022, 13(3): 203-219. doi: 10.1007/s13238-021-00878-z
Citation: Chaojuan Yang, Yonglu Tian, Feng Su, Yangzhen Wang, Mengna Liu, Hongyi Wang, Yaxuan Cui, Peijiang Yuan, Xiangning Li, Anan Li, Hui Gong, Qingming Luo, Desheng Zhu, Peng Cao, Yunbo Liu, Xunli Wang, Min-hua Luo, Fuqiang Xu, Wei Xiong, Liecheng Wang, Xiang-yao Li, Chen Zhang. Restoration of FMRP expression in adult V1 neurons rescues visual deficits in a mouse model of fragile X syndrome[J]. Protein&Cell, 2022, 13(3): 203-219. doi: 10.1007/s13238-021-00878-z

Restoration of FMRP expression in adult V1 neurons rescues visual deficits in a mouse model of fragile X syndrome

doi: 10.1007/s13238-021-00878-z
  • Received Date: 2021-05-16
  • Many people affected by fragile X syndrome (FXS) and autism spectrum disorders have sensory processing deficits, such as hypersensitivity to auditory, tactile, and visual stimuli. Like FXS in humans, loss of Fmr1 in rodents also cause sensory, behavioral, and cognitive deficits. However, the neural mechanisms underlying sensory impairment, especially vision impairment, remain unclear. It remains elusive whether the visual processing deficits originate from corrupted inputs, impaired perception in the primary sensory cortex, or altered integration in the higher cortex, and there is no effective treatment. In this study, we used a genetic knockout mouse model (Fmr1KO), in vivo imaging, and behavioral measurements to show that the loss of Fmr1 impaired signal processing in the primary visual cortex (V1). Specifically, Fmr1KO mice showed enhanced responses to low-intensity stimuli but normal responses to high-intensity stimuli. This abnormality was accompanied by enhancements in local network connectivity in V1 microcircuits and increased dendritic complexity of V1 neurons. These effects were ameliorated by the acute application of GABAA receptor activators, which enhanced the activity of inhibitory neurons, or by reintroducing Fmr1 gene expression in knockout V1 neurons in both juvenile and young-adult mice. Overall, V1 plays an important role in the visual abnormalities of Fmr1KO mice and it could be possible to rescue the sensory disturbances in developed FXS and autism patients.
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  • [1]
    Aoki S, Kagitani-Shimono K, Matsuzaki J, Hanaie R, Nakanishi M, Tominaga K, Nagai Y, Mohri I, Taniike M (2019) Lesser suppression of response to bright visual stimuli and visual abnormality in children with autism spectrum disorder:a magnetoencephalographic study. J Neurodev Disord 11:9
    [2]
    Armando B, Laurent M, Patricia J, Jocelyn F (2005) Enhanced and diminished visuo-spatial information processing in autism depends on stimulus complexity. Brain 128:2430
    [3]
    Arsenault J, Gholizadeh S, Niibori Y, Pacey LK, Halder SK, Koxhioni E, Konno A, Hirai H, Hampson DR (2016) FMRP expression levels in mouse central nervous system neurons determine behavioral phenotype. Hum Gene Ther 27:982-996
    [4]
    Atapour N, Majka P, Wolkowicz IH, Malamanova D, Worthy KH, Rosa MGP (2018) Neuronal distribution across the cerebral cortex of the marmoset monkey (Callithrix jacchus). Cereb Cortex 29(9):3836-63
    [5]
    Berman RF, Murray KD, Arque G, Hunsaker MR, Wenzel HJ (2012) Abnormal dendrite and spine morphology in primary visual cortex in the CGG knock-in mouse model of the fragile X premutation. Epilepsia 53(Suppl 1):150-160
    [6]
    Berzhanskaya J, Phillips MA, Shen J, Colonnese MT (2016) Sensory hypo-excitability in a rat model of fetal development in Fragile X Syndrome. Sci Rep 6:30769
    [7]
    Churchill JD, Grossman AW, Irwin SA, Galvez R, Klintsova AY, Weiler IJ, Greenough WT (2002) A converging-methods approach to fragile X syndrome. Dev Psychobiol 40:323-338
    [8]
    Colman RS, Frankel F, Ritvo E, Freeman BJ (1976) The effects of fluorescent and incandescent illumination upon repetitive behaviors in autistic children. J Autism Child Schizophr 6:157-162
    [9]
    Cooke SF, Komorowski RW, Kaplan ES, Gavornik JP, Bear MF (2015) Visual recognition memory, manifested as long-term habituation, requires synaptic plasticity in V1. 18
    [10]
    Corbetta D, Snapp-Childs W (2009) Seeing and touching:the role of sensory-motor experience on the development of infant reaching. Infant Behav Dev 32:44-58
    [11]
    Cornish KM, Munir F, Cross G (1999) Spatial cognition in males with Fragile-X syndrome:evidence for a neuropsychological phenotype. Cortex 35:263-271
    [12]
    Coulter RA (2009) Understanding the visual symptoms of individuals with autism spectrum disorder (ASD). Optom Vis Dev
    [13]
    Dakin S, Frith U (2005) Vagaries of visual perception in autism. Neuron 48:497-507
    [14]
    Dolan BM, Duron SG, Campbell DA, Vollrath B, Shankaranarayana Rao BS, Ko H-Y, Lin GG, Govindarajan A, Choi S-Y, Tonegawa S (2013) Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by the small-molecule PAK inhibitor FRAX486. Proc Natl Acad Sci USA 110:5671-5676
    [15]
    Dölen G, Osterweil E, Shankaranarayana Rao BS, Smith GB, Auerbach BD, Chattarji S, Bear MF (2007) Correction of Fragile X syndrome in mice. Neuron 56:955-962
    [16]
    Farzin F, Whitney D, Hagerman RJ, Rivera SM (2008) Contrast detection in infants with fragile X syndrome. Vis Res 48:1471-1478
    [17]
    Farzin F, Rivera SM, Whitney D (2011) Resolution of spatial and temporal visual attention in infants with fragile X syndrome. Brain 134:3355-3368
    [18]
    Ferreri MC, Gutiérrez ML, Gravielle MC (2015) Tolerance to the sedative and anxiolytic effects of diazepam is associated with different alterations of GABAA receptors in rat cerebral cortex. Neuroscience 310:152-162
    [19]
    Frank T (2003) Primary visual cortex and visual awareness. Nat Rev Neurosci 4:219-229
    [20]
    Gholizadeh S, Tharmalingam S, Macaldaz ME, Hampson DR (2013) Transduction of the central nervous system after intracerebroventricular injection of Adeno-associated viral vectors in neonatal and Juvenile Mice. Hum Gene Ther Methods 24:205-213
    [21]
    Gibbs JW, Sombati S, Delorenzo RJ, Coulter DA (1997) Physiological and pharmacological alterations in postsynaptic GABA(A) receptor function in a hippocampal culture model of chronic spontaneous seizures. J Neurophysiol 77:2139
    [22]
    Goel A, Cantu DA, Guilfoyle J, Chaudhari GR, Newadkar A, Todisco B, de Alba D, Kourdougli N, Schmitt LM, Pedapati E et al (2018) Impaired perceptual learning in a mouse model of Fragile X syndrome is mediated by parvalbumin neuron dysfunction and is reversible. Nat Neurosci 21:1404-1411
    [23]
    Golshani P, Gonçalves JT, Khoshkhoo S, Mostany R, Smirnakis S, Portera-Cailliau C (2009) Internally mediated developmental desynchronization of neocortical network activity. J Neurosci 29:10890-10899
    [24]
    Haberl MG, Zerbi V, Veltien A, Ginger M, Heerschap A, Frick A (2015) Structural-functional connectivity deficits of neocortical circuits in the Fmr1 (-/y) mouse model of autism. Sci Adv 1:e1500775
    [25]
    He CX, Portera-Cailliau C (2013) The trouble with spines in fragile X syndrome:density, maturity and plasticity. Neuroscience 251:120-128
    [26]
    Ingrid B, Shepherd Gordon MG, Karel S (2008) Circuit and plasticity defects in the developing somatosensory cortex of FMR1 knock-out mice. J Neurosci off J Soc Neurosci 28:5178
    [27]
    Irwin SA, Galvez R, Greenough WT (2000) Dendritic spine structural anomalies in fragile-X mental retardation syndrome. Cereb Cortex 10:1038
    [28]
    Irwin SA, Patel B, Idupulapati M, Harris JB, Crisostomo RA, Larsen BP, Kooy F, Willems PJ, Cras P, Kozlowski PB et al (2001) Abnormal dendritic spine characteristics in the temporal and visual cortices of patients with fragile-X syndrome:a quantitative examination. Am J Med Genet 98:161-167
    [29]
    Keown CL, Shih P, Nair A, Peterson N, Mulvey ME, Müller R-A (2013) Local functional overconnectivity in posterior brain regions is associated with symptom severity in autism spectrum disorders. Cell Rep 5:567-572
    [30]
    Kim JY, Grunke SD, Levites Y, Golde TE, Jankowsky JL (2014) Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. J vis Exp Jove 19:51863
    [31]
    Knoth IS, Vannasing P, Major P, Michaud JL, Lippe S (2014) Alterations of visual and auditory evoked potentials in fragile X syndrome. Int J Dev Neurosci 36:90-97
    [32]
    Kogan CS, Turk J, Hagerman RJ, Cornish KM (2008) Impact of the Fragile X mental retardation 1 (FMR1) gene premutation on neuropsychiatric functioning in adult males without fragile X-associated Tremor/Ataxia syndrome:a controlled study. Am J Med Genet B 147B:859-872
    [33]
    Li A, Gong H, Zhang B, Wang Q, Yan C, Wu J, Liu Q, Zeng S, Luo Q (2010) Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain. Science 330:1404-1408
    [34]
    Merenstein SA, Sobesky WE, Taylor AK, Riddle JE, Tran HX, Hagerman RJ (1996) Molecular-clinical correlations in males with an expanded FMR1 mutation. Am J Med Genet 64:388-394
    [35]
    Michalon A, Sidorov M, Ballard TM, Ozmen L, Spooren W, Wettstein JG, Jaeschke G, Bear MF, Lindemann L (2012) Chronic pharmacological mGlu5 inhibition corrects fragile X in adult mice. Neuron 74:49-56
    [36]
    Mottron L, Dawson M, Soulieres I, Hubert B, Burack J (2006) Enhanced perceptual functioning in autism:an update, and eight principles of autistic perception. J Autism Dev Disord 36:27
    [37]
    Musumeci SA, Bosco P, Calabrese G, Bakker C, De Sarro GB, Elia M, Ferri R, Oostra BA (2000) Audiogenic seizures susceptibility in transgenic mice with Fragile X syndrome. Epilepsia 41:19-23
    [38]
    O'Riordan MA, Plaisted KC, Driver J, Baron-Cohen S (2001) Superior visual search in autism. J Exp Psychol Hum Percept Perform 27:719-730
    [39]
    Pereira AM, Campos BM, Coan AC, Pegoraro LF, de Rezende TJR, Obeso I, Dalgalarrondo P, da Costa JC, Dreher J-C, Cendes F (2018) Differences in cortical structure and functional MRI connectivity in high functioning autism. Front Neurol 9:539
    [40]
    Pfeiffer Brad E, Huber KM (2007) Fragile X mental retardation protein induces synapse loss through acute postsynaptic translational regulation. J Neurosci 27:3120-3130
    [41]
    Pfeiffer Brad E, Huber KM (2009) The state of synapses in fragile X syndrome. Neurosci. A Rev. J. Bringing Neurobiol. Neurol. Psychiatry 15:549-567
    [42]
    Plaisted K, O'Riordan M, Baron-Cohen S (1998a) Enhanced visual search for a conjunctive target in autism:a research note. J Child Psychol Psychiatry 39:777-783
    [43]
    Plaisted K, O'Riordan M, Baron-Cohen S (1998b) Enhanced discrimination of novel, highly similar stimuli by adults with autism during a perceptual learning task. J Child Psychol Psychiatry 39:765-775
    [44]
    Pop AS, Gomez-Mancilla B, Neri G, Willemsen R, Gasparini F (2014) Fragile X syndrome:a preclinical review on metabotropic glutamate receptor 5 (mGluR5) antagonists and drug development. Psychopharmacol 231:1217-1226
    [45]
    Radzicki D, Pollema-Mays SL, Sanz-Clemente A, Martina M (2017) Loss of M1 receptor dependent cholinergic excitation contributes to mPFC deactivation in neuropathic pain. J Neurosci off J Soc Neurosci 37:1516-1553
    [46]
    Rais M, Binder DK, Razak KA, Ethell IM (2018) Sensory processing phenotypes in Fragile X syndrome. ASN Neuro 10:1759091418801092
    [47]
    Robertson CE, Thomas C, Kravitz DJ, Wallace GL, Baron-Cohen S, Martin A, Baker CI (2014) Global motion perception deficits in autism are reflected as early as primary visual cortex. Brain 137:2588-2599
    [48]
    Rossignol R, Ranchon-Cole I, Pâris A, Herzine A, Perche A, Laurenceau D, Bertrand P, Cercy C, Pichon J, Mortaud S et al (2014) Visual sensorial impairments in neurodevelopmental disorders:evidence for a retinal phenotype in Fragile X Syndrome. PLoS ONE 9:e105996
    [49]
    Rudie JD, Dapretto M (2013) Convergent evidence of brain overconnectivity in children with autism? Cell Rep 5:565-566
    [50]
    Scerif G, Cornish K, Wilding J, Driver J, Karmiloff-Smith A (2004) Visual search in typically developing toddlers and toddlers with Fragile X or Williams syndrome. Dev Sci 7:116-130
    [51]
    Shah A, Frith U (1983) An islet of ability in autistic children:a research note. J Child Psychol Psychiatry 24:613-620
    [52]
    Sidorov MS, Krueger DD, Taylor M, Gisin E, Osterweil EK, Bear MF (2014) Extinction of an instrumental response:a cognitive behavioral assay in Fmr1 knockout mice. Genes Brain Behav 13:451-458
    [53]
    Sinclair D, Oranje B, Razak KA, Siegel SJ, Schmid S (2017) Sensory processing in autism spectrum disorders and Fragile X syndrome-From the clinic to animal models. Neurosci Biobehav Rev 76:235-253
    [54]
    Sun MK, Hongpaisan J, Alkon DL (2016) Rescue of synaptic phenotypes and spatial memory in young Fragile X Mice. J Pharmacol Exp Ther 357:300-310
    [55]
    Tang G, Gudsnuk K, Kuo S-H, Cotrina ML, Rosoklija G, Sosunov A, Sonders MS, Kanter E, Castagna C, Yamamoto A et al (2014) Loss of mTOR-dependent macroautophagy causes autistic-like synaptic pruning deficits. Neuron 83:1131-1143
    [56]
    Tian Y, Yang C, Cui Y, Su F, Wang Y, Wang Y, Yuan P, Shang S, Li H, Zhao J et al (2018) An excitatory neural assembly encodes short-term memory in the prefrontal cortex. Cell Rep 22:1734-1744
    [57]
    Uddin LQ, Supekar K, Lynch CJ, Khouzam A, Phillips J, Feinstein C, Ryali S, Menon V (2013) Salience network-based classification and prediction of symptom severity in children with autism. JAMA Psychiat 70:869-879
    [58]
    Van der Molen MJW, Van der Molen MW, Ridderinkhof KR, Hamel BCJ, Curfs LMG, Ramakers GJA (2012a) Auditory and visual cortical activity during selective attention in fragile X syndrome:a cascade of processing deficiencies. Clin Neurophysiol off J Int Fed Clin Neurophysiol 123:720-729
    [59]
    Van der Molen MJW, Van der Molen MW, Ridderinkhof KR, Hamel BCJ, Curfs LMG, Ramakers GJA (2012b) Auditory change detection in fragile X syndrome males:a brain potential study. Clin Neurophysiol 123:1309-1318
    [60]
    Wang Y, Su F, Wang S (2019) Efficient implementation of convolutional neural networks in the data processing of two-photon in vivo imaging. Bioinformatics
    [61]
    Ye Z, Yu X, Houston CM, Aboukhalil Z, Franks NP, Wisden W, Brickley SG (2017) Fast and Slow Inhibition in the Visual Thalamus Is Influenced by Allocating GABAAReceptors with Different γ Subunits. Front Cell Neurosci 11:95
    [62]
    Zeier Z, Kumar A, Bodhinathan K, Feller JA, Foster TC, Bloom DC (2009) Fragile X mental retardation protein replacement restores hippocampal synaptic function in a mouse model of fragile X syndrome. Gene Ther 16:1122-1129
    [63]
    Zhang H, Berg AC, Maire M, Malik J (2006) SVM-KNN:discriminative nearest neighbor classification for visual category recognition. Proc IEEE Comput Soc Conf Comput Vis Pattern Recognit. https://doi.org/10.1109/CVPR.2006.301
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