Copy number variation (CNV) at the 15q11.2 region has been identified as a significant risk locus for neurological and neuropsychiatric conditions such as schizophrenia (SCZ) and autism spectrum disorder (ASD). However, the individual roles for genes at this locus in nervous system development, function and connectivity remain poorly understood. Haploinsufficiency of one gene in this region, Cyfip1, may provide a model for 15q11.2 CNV-associated neuropsychiatric phenotypes. Here we show that altering CYFIP1 expression levels in neurons both in vitro and in vivo influences dendritic complexity, spine morphology, spine actin dynamics and synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor lateral diffusion. CYFIP1 is highly enriched at synapses and its overexpression in vitro leads to increased dendritic complexity. Neurons derived from Cyfip1 heterozygous animals on the other hand, possess reduced dendritic complexity, increased mobile F-actin and enhanced GluA2-containing AMPA receptor mobility at synapses. Interestingly, Cyfip1 overexpression or haploinsufficiency increased immature spine number, whereas activity-dependent changes in spine volume were occluded in Cyfip1 haploinsufficient neurons. In vivo, Cyfip1 heterozygous animals exhibited deficits in dendritic complexity as well as an altered ratio of immature-to-mature spines in hippocampal CA1 neurons. In summary, we provide evidence that dysregulation of CYFIP1 expression levels leads to pathological changes in CNS maturation and neuronal connectivity, both of which may contribute to the development of the neurological symptoms seen in ASD and SCZ.
Copy number variations of the 15q11.2 region of the human genome are implicated in the development of neurological and neuropsychiatric conditions such as autism spectrum disorder (ASD), epilepsy, intellectual disability (ID) and schizophrenia (SCZ).1, 2, 3, 4, 5, 6, 7 Copy number variation of one gene within 15q11.2, coding for the cytoplasmic FMRP-interacting protein 1 (CYFIP1), has been linked to both ASD and SCZ.5,8,9 Furthermore, genome-wide expression profiling of patients with duplication of 15q11–q13 has specifically demonstrated upregulated Cyfip1 mRNA in those that suffer from ASD.10 Changes in CYFIP1 levels may thus lead to the neuropsychiatric and cognitive phenotypes associated with copy number variation at 15q11.2. However, the exact role that CYFIP1 has in normal nervous system function and development, and the consequences of its dysregulation, remain poorly understood.
Dendritic morphogenesis, synaptogenesis and continued activity-dependent remodelling of synaptic connections are critical for the development and maintenance of functional neuronal networks.11, 12, 13, 14, 15 A disruption in any of these processes produces network-wide deficits in neuronal connectivity and may help explain why changes in morphological complexity, dendritic spine number, shape and plasticity are associated with several neuropsychiatric and neurological disorders, including SCZ, ASD, ID and epilepsy.16, 17, 18, 19 Actin cytoskeletal dynamics have a key role in the establishment and maintenance of both dendritic arborizations and spines, and are also critical for the structural alterations in spine shape important for cellular forms of synaptic plasticity, such as long-term potentiation and long-term depression.20, 21, 22, 23 Moreover, alterations in the regulation of actin at dendritic spines, by neurodevelopmental disease-associated proteins such as DISC1, DTNBP1, SHANK3, PAK3, Oligophrenin and SRGAP2, have been shown to produce deficits in both synaptic plasticity and the formation and consolidation of long-term memories.23, 24, 25, 26, 27, 28, 29
One critical modulator of cellular actin dynamics is the ubiquitous heteropentameric WAVE regulatory complex consisting of the proteins WAVE, Abi, Nap1, HSPC300 and CYFIP1 (also known as SRA-1) or CYFIP2.30 The main role of CYFIP1 in this complex is to maintain the WAVE regulatory complex in an inhibited state until the small GTPase Rac1, once activated, binds CYFIP1. Rac1 binding results in dissociation of CYFIP1 from the WAVE regulatory complex, allowing WAVE to activate the actin nucleator Arp2/3 and subsequently enabling de novo F-actin assembly.30,31 In this way, CYFIP1 functions as a key, conserved regulator of actin nucleation in cells.30 Furthermore, CYFIP1 couples actin dynamics to other cellular processes, such as carrier vesicle biogenesis in the trans-Golgi network, and, via interactions with the FRMP (Fragile X mental retardation protein), regulates synaptic mRNA translation in an activity-dependent manner.32,33 However, still very little is known about CYFIP1’s role in establishing or maintaining neuronal connectivity, specifically its involvement in the regulation of dendritic morphology, synaptic structural plasticity and neurotransmitter receptor mobility. Furthermore, how both deletions and duplications affecting Cyfip1 expression produce similar neuropsychiatric phenotypes has also not yet been addressed.34,35
Here we show that both CYFIP1 and CYFIP2 are highly enriched at excitatory synapses in dendritic spines. In dissociated neurons, overexpression of CYFIP1 or CYFIP2 leads to increased dendritic complexity and altered spine morphology whereas conversely, reducing CYFIP1 levels causes decreased dendritic complexity and an increase in immature dendritic spines. CYFIP1 deficiency results in increased F-actin assembly within dendritic spines, occludes N-methyl-D-aspartate (NMDA) receptor-induced spine shrinkage and leads to alterations in the surface dynamics and synaptic mobility of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In vivo, Cyfip1 deletion in the mouse leads to delayed embryonic development and early embryonic death whereas Cyfip1 haploinsufficiency causes reduced dendritic complexity and an increase in immature spines in the hippocampus. Our results support a critical role for CYFIP1 in development, dendritic morphogenesis and synaptic connectivity and provide further support for the idea that dysregulated CYFIP1 mediates the molecular aetiology of 15q11.2 Copy number variation-associated neuropsychiatric conditions such as ASD, SCZ and epilepsy.
Materials and Methods
Cell culture and transfection
Hippocampal cultures were obtained from E16 mouse embryos (produced via Cyfip1+/+xCyfip1+/− crosses) or E18 rat embryos as previously described and transfected using Lipofectamine-2000 (Life Technologies, Paisley, UK).36
Microscopy, quantitative analysis of neuronal and dendritic spine morphology, and fluorescence recovery after photobleaching (FRAP)
Confocal images were acquired on a Zeiss LSM700 upright confocal microscope using a 63X oil objective (NA: 1.4) unless otherwise stated (Carl Zeiss, Welwyn Garden City, UK). Image stacks for dendritic morphological and spine analysis had voxel dimensions of 0.39 μm × 0.39 μm × 0.54 μm and 0.19 μm × 0.19 μm × 0.57 μm, respectively. Neuronal arbours were reconstructed and subjected to Sholl analysis, using either Imaris (Bitplane AG, Zurich, Switzerland) or NeuronStudio and ImageJ (NIH, Bethesda, MD, USA).37 Spines were manually identified on 100–200 μm long secondary dendrites and analyzed for morphological classification or volume measurements in Imaris software (Bitplane). For spine classification custom parameters were used and spines were classified into stubby, mushroom, long and thin, and filopodia categories using a ratio of spine head and neck diameters to spine length. Classification was entirely automated until the final step where errors in classification were removed.
Single-plane confocal images were acquired for localization experiments in vitro. To quantify CYFIP1 and CYFIP2 enrichment at synaptic sites, the fluorescence intensity of CYFIP1 or CYFIP2 was measured as the average intensity within the labelled synaptic puncta and normalized to the average intensity of the total process including synaptic puncta using Metamorph software (Molecular Devices, Sunnyvale, CA, USA).
Dendritic morphology and spines in P55–60 mice were analysed using the FD Rapid Golgi Stain kit (FD NeuroTechnologies, Baltimore, MD, USA) and Neurolucida (MBF Bioscience, Williston, VT, USA). Golgi-impregnated brains were sliced at 150 μm using a vibratome (Leica Microsystems, Heerbrugg, Switzerland). Well-isolated hippocampal CA1 neurons were imaged at 60X using the Neurolucida software system and an upright light microscope with a motorized stage (MBF Bioscience). The entire extent (apical and basal) of the dendritic tree was traced and reconstructed. For spine analysis, two apical oblique and two basal dendrites were quantified per cell. Three males were analysed for each condition with a minimum of three cells traced per animal. When using animals from separate litters, each genotype was equally represented from each litter.
Chemical long-term depression and fluorescence recovery after photobleaching (FRAP) on actin in dendritic spines was performed as described previously.38,39 FRAP movies were analysed in ImageJ (NIH) using a custom-built plugin, curves were fitted using Mathematica (Wolfram Research, Champaign, IL, USA). For further details see Supplementary Methods.
Quantum dot (QD) imaging
A total of 12–16DIV hippocampal cells transfected with GluA2SEP were live imaged under perfusion in ACSF at 37 °C and quantum dot (QD) movies were recorded at 8.5 Hz. Labelling of GluA2SEP with QDs was performed at RT using a mouse anti-GFP antibody (10 μg ml−1, Neuromab, N38) and an anti-mouse 605-nm QD (0.5 nM, Life Technologies). Analysis of QD receptor dynamics was performed as described (see Supplementary Methods).40
CYFIP1 and CYFIP2 are enriched at excitatory synapses
To explore the localization of CYFIP1 and CYFIP2 in neurons CYFIP1GFP or CYFIP2GFP (Supplementary Figure 1) were co-expressed in mature hippocampal neurons with DsRed as a marker of neuronal morphology and analysed using confocal microscopy. Compared with the localization of DsRed, which uniformly filled both dendritic processes and spines, CYFIP1GFP and CYFIP2GFP exhibited a nonuniform distribution appearing to be selectively targeted to punctate clusters in spines and along dendrites (Figures 1a and b). A similar enrichment in spines was observed for endogenous CYFIP1 using a CYFIP1-specific antibody and actinGFP to label neuronal morphology (Figure 1c; Supplementary Figure 1). In addition, CYFIP1 is detected within the post synaptic density fraction by western blotting (Supplementary Figure 4). Co-expression experiments with CYFIP1GFP and CYFIP2mCherry revealed that the majority of CYFIP1GFP co-localized with CYFIP2mCherry and overlapping puncta were found both in dendritic processes and spines (Supplementary Figure 2A).
Using immunofluorescence and confocal microscopy, both CYFIP1GFP and CYFIP2GFP levels were found to be ~90% enriched at Homer and VGLUT-positive excitatory synaptic sites compared with total dendritic processes (CYFIP1, 91.4±9.8% increase, P<0.001; CYFIP2, 85.9±12.6% increase, P<0.001; Figures 1d and e,Supplementary Figure 2B). In addition, endogenous CYFIP1 was also highly enriched at excitatory synapses and co-localized with the excitatory marker PSD-95 (Figure 1f). Thus endogenous CYFIP1, along with CYFIP1GFP and CYFIP2GFP, are found at excitatory synapses and exhibit high levels of expression at sites of F-actin accumulation, like dendritic spines.
Overexpression of CYFIP1 and CYFIP2 enhances dendritic complexity and alters dendritic spine morphology
Microduplication of the CYFIP1-encoding region at 15q11.2 is associated with increased neuropsychiatric disease burden, and increased expression of CYFIP1 has specifically been linked to ASD.1,3,10 Overexpression of CYFIP1GFP or CYFIP2GFP resulted in a significant increase in dendritic complexity as a function of distance from the soma compared with control neurons expressing GFP, measured using either number of intersections or number of branch points as indicators of morphological complexity (Figures 2a–c). In addition, total dendritic length and the total number of branch points per cell were also increased in the CYFIP1 and CYFIP2 overexpressors (dendritic length: CYFIP1, 37% increase, 2876±174 μm, P<0.01; CYFIP2, 33% increase, 2787±152 μm, P<0.01; GFP, 2099±163 μm; branch points: CYFIP1, 48% increase, 66.5±7.0, P<0.05; CYFIP2, 101% increase, 90.1±5.6, P<0.001; GFP 44.8±7.7; Figures 2d and e).
To examine whether changes in dendritic complexity were accompanied by changes in excitatory spine number or shape, we measured spines in CYFIP1mCherry+actinGFP-expressing neurons at 21DIV. Compared with DsRed+actinGFP transfected neurons, CYFIP1mCherry+actinGFP-expressing cells displayed no significant difference in spine density (data not shown). However, CYFIP1 overexpressors had significantly fewer stubby spines (30% decrease in stubby spines, DsRed+actinGFP: 0.32±0.01 spines μm−1, CYFIP1mCherry+actinGFP: 0.24±0.01 spines μm−1, P<0.01) and increased numbers of long, thin spines and filopodia (24% increase in long, thin spines, DsRed+actinGFP: 0.14±0.01 spines μm−1, CYFIP1mCherry+actinGFP: 0.17±0.01 spines μm−1, P<0.05; 145% increase in filopodia, DsRed+actinGFP: 0.006±0.001 spines μm−1, CYFIP1mCherry+actinGFP: 0.016±0.003 spines μm−1, P<0.01; Figures 2f and g). Furthermore, spines were significantly longer in CYFIP1 overexpressors regardless of subtype classification (DsRed+actinGFP: 1.13±0.005 μm, CYFIP1mCherry+actinGFP: 1.29±0.005 μm, P<0.0001, Figure 2h). CYFIP1 overexpression also significantly decreased spine diameter, whereas spine volume showed an overall increase due to the dominating effect of the increase in spine length (Supplementary Figures 6C and D). These results demonstrate that increasing CYFIP1 expression levels above baseline enhances dendritic complexity and alters the ratio of mature versus immature spines in neurons, thereby changing network connectivity.
CYFIP1 is essential for embryonic development and its loss leads to marked developmental delay
To study the effects of decreased Cyfip1 levels in vivo, we characterized a Cyfip1 KO mouse41 and found that deletion was embryonically lethal. However, fertilized KO oocytes from Cyfip1+/− × Cyfip1+/− crosses did persist up to the blastocyst stage in vitro (not shown), and in pregnant dams, they were detectable until embryonic day 8.5 (E8.5) postcoitum (Supplementary Table 1). At this stage, Cyfip1 KO embryos were significantly reduced in size and displayed delayed development compared with their heterozygous (Cyfip1+/−) or wild-type (WT) counterparts (Supplementary Figures 3A–C). Further examination revealed complete developmental failure resulting in reduced body elongation and patterning. Cyfip1+/− heterozygous animals on the other hand, were viable until adulthood, were fertile and did not show any gross morphological abnormalities in brain structure when compared with WT (Supplementary Figure 3H). These data suggest that adequate levels of CYFIP1 are indispensable for proper development through early stages of embryogenesis.
Cyfip1 haploinsufficiency results in decreased dendritic complexity and increased immature spine number
Microdeletion at 15q11.2 renders people haploinsufficient for Cyfip1 and is associated with ASD and SCZ.4,6,7 We therefore investigated the effects of decreased CYFIP1 levels by measuring dendritic and spine morphologies in Cyfip1+/− hippocampal neurons (Supplementary Figures 3F and G). Sholl analysis of 14DIV, actinGFP-labelled neurons revealed that reduced CYFIP1 levels had the opposite effect on dendritic complexity to that of CYFIP1 overexpression, leading to a marked decrease in dendritic complexity between 30 and 110 μm away from the soma (Figures 3a–c). Furthermore, both total dendritic length and the total number of branch points were reduced in Cyfip1+/− neurons compared with control (15% decrease in total dendritic length in Cyfip1+/− neurons, WT: 2704±96.8 μm, Cyfip1+/−: 2298±80.2 μm, P<0.01; 20% decrease in number of branch points in Cyfip1+/− neurons, WT: 76.7±4.1, Cyfip1+/−: 61.4±3.6, P<0.01; Figures 3d and e). Spine density was unchanged in Cyfip1+/− neurons compared with WT controls; however, when spines were further classified into the different morphological subtypes, long and thin spines and filopodia were significantly increased (30% increase in long, thin spines, WT: 0.15±0.01 spines μm−1, Cyfip1+/−: 0.20±0.01 spines μm−1, P<0.05; 125% increase in filopodia, WT: 0.007±0.001 spines μm−1, Cyfip1+/−: 0.016±0.003 spines μm−1, P<0.05, respectively; Figures 3f–h). In addition, spines were significantly longer in the Cyfip1+/− neurons irrespective of subtype classification (WT: 1.14±0.003 μm, Cyfip1+/−: 1.27±0.003 μm, P<0.0001, Figure 3i). However, no change in spine diameter or volume was observed in the Cyfip1 haploinsufficient neurons (Supplementary Figures 6A and B). These experiments demonstrate that Cyfip1 haploinsufficiency results in decreased dendritic complexity and produces alterations in spine morphology.
CYFIP1 regulates actin assembly at dendritic spines
CYFIP1 negatively regulates local actin filament assembly, suggesting that actin dynamics may be altered in Cyfip1+/− dendritic spines. FRAP of actinGFP in spines of Cyfip1+/− neurons revealed a significant difference in amount of fluorescence recovery compared with WT controls. Fluorescence intensity within the spine head of WT neurons returned to only 64.1±5.2% of the pre-bleached fluorescence by 40 s compared with 80.0±4.1% in the Cyfip1+/− neurons (P<0.05; Figures 4a and b). Moreover, the fluorescence recovery of Cyfip1+/− neurons plateaued at a greater intensity than WT neurons, which was confirmed by a significant increase in the total mobile fraction (i.e. the proportion of final recovered fluorescence compared with the total bleached fluorescence, 16% increase, WT: 68.4±5.46%, Cyfip1+/−: 84.4±3.97%, P<0.05; Figure 4c). However, there was no change in the recovery rate constant between Cyfip1+/− and WT neurons. Similar results were also obtained using LifeactGFP, an F-actin-specific fluorescent probe suitable for FRAP experiments.42 Photobleaching spines expressing LifeactGFP produced a significantly greater amount of recovered fluorescence in Cyfip1+/− neurons compared with WT again by 40 s (WT: 67.9±4.8%, Cyfip1+/−: 85.5±7.0%, P<0.05; Figure 4d) resulting in a significant increase in the total mobile fraction (9.8% increase, WT: 70.1±3.03%, Cyfip1+/−: 79.9±3.12%, P<0.05; Figure 4e).42,43 Taken together, the increased recovery of actinGFP and LifeactGFP suggests an increase in F-actin assembly in the Cyfip1+/− spines.
Cyfip1 haploinsufficiency occludes NMDA receptor-dependent structural plasticity
As Cyfip1 haploinsufficiency changes actin availability in spines and alters the ratio of mature versus immature spines at steady-state, we hypothesised that stimulus-dependent structural plasticity would also be altered in Cyfip1+/− spines. To study the role that CYFIP1 has in spines during structural plasticity, we utilized an established chemical long-term depression protocol.38 Brief bath application of 20 μM NMDA plus 20 μM glycine induces a 15–20% spine shrinkage within a timeframe of 40 min, which is measured as a decrease in spine volume without a concomitant change in either spine diameter or length (Figures 4f and g; Supplementary Figures 6E and F). Using this stimulus, we were able to induce spine shrinkage in WT neurons in response to NMDA treatment (Figures 4f and g). Cumulative frequency curves of spine volume show a leftward shift following NMDA application in WT neurons, indicating a reduction of spine volume across the population (Figure 4h). Cyfip1+/− spines showed no change in spine volume following NMDA application and spine volume remained unchanged from its basally low levels (16% decrease in WT spine volume following NMDA treatment, no change in Cyfip1+/− spine volume following NMDA treatment, WT saline: 0.30±0.003 μm3, WT NMDA: 0.25±0.001 μm3, Cyfip1+/− saline: 0.26±0.001 μm3, Cyfip1+/− NMDA: 0.26±0.002 μm3, P<0.0001; Figures 4f–h). These data reveal a role for CYFIP1 in mediating structural reorganization during synaptic plasticity, possibly through dynamic regulation of the local actin cytoskeleton in spines.
Altered surface AMPA receptor mobility due to Cyfip1 haploinsufficiency
AMPA receptors mediate fast excitatory synaptic transmission within the CNS.44,45 Actin dynamics in spines have a major role in coordinating the delivery and removal of AMPA receptors and the strength of synapses.45 To address whether CYFIP1-mediated alterations in spine morphology and actin dynamics also impinge on the stability or mobility of AMPA receptors within synapses, we investigated whether surface GluA2-containing receptor dynamics were different from control in Cyfip1+/− neurons. Initial observations suggested that total protein levels of the AMPA receptor subunit GluA2 are unchanged in WT and Cyfip1+/− neurons (Supplementary Figures 5A and B). Therefore, we expressed pH-sensitive superecliptic pHluorin (SEP)-tagged GluA2 in neurons to visualize GluA2-containing synaptic clusters in dendrites. Single GluA2SEP-containing receptors were labelled with 605 nm QDs to follow the diffusion of individual receptors within synaptic clusters.40 QD-labelled GluA2SEP receptors were less mobile when inside synaptic clusters (Din=0.023 μm2 s−1, Dout=0.057 μm2 s−1 where D is the median diffusion coefficient, P<0.0001, Supplementary Figures 5C and D), indicating confined motion at synapses, as previously shown.46 In Cyfip1+/− neurons, however, GluA2SEP-containing receptors exhibited an increased synaptic mobility within clusters when compared with WT neurons, (WT: Din=0.023 μm2 s−1, Cyfip1+/−: Din=0.034 μm2 s−1, P<0.01; Figures 4i and j) as well as a reduced confinement at synapses (Supplementary Figure 5E). GluA2SEP receptor mobility outside synapses remained unchanged between WT and Cyfip1+/− neurons (WT: Dout=0.057 μm2 s−1, Cyfip1+/−: Dout=0.062 μm2 s−1, Figure 4k). These observations suggest that the restricted mobility of GluA2 receptors at synapses is significantly diminished in Cyfip1+/− spines.
CYFIP1 regulates neuronal development and connectivity in vivo
To confirm that Cyfip1 haploinsufficiency produces similar effects on dendritic spines in the intact animal, we investigated dendritic complexity and spine morphology in the hippocampi of WT versus Cyfip1+/− mice using Golgi staining. Cyfip1 haploinsufficiency in postnatal day 55–60 (P55–60) animals was confirmed by western blotting (46.8% CYFIP1 protein decrease, WT control 100±15.4%, Cyfip1+/− 53.2±7.8%, P<0.05; Supplementary Figures 3D and E). Compared with WT male littermate controls, CA1 neurons in Cyfip1+/− animals possessed a significantly decreased dendritic complexity within 100 μm from the soma (Figures 5a and b). Furthermore, spine density was unchanged, but long and thin spines were increased in the apical oblique dendrites in Cyfip1+/− animals (40% increase, WT: 0.56±0.066 spines μm−1, Cyfip1+/−: 0.79±0.078 spines μm−1, P<0.05; Figures 5c and d). These results indicate that genetic dysregulation of Cyfip1 produces penetrant effects on pyramidal neuron dendritic complexity and spine morphology in vivo.
Altered levels of CYFIP1 are associated with several neurological disorders including SCZ and ASD.4,5,10 However, it remains unclear what role CYFIP1 has in the establishment or maintenance of neuronal connectivity in the mammalian CNS, and how changes in its expression contribute to neurological dysfunction. Here we report that CYFIP1 regulates the maturation of neuronal dendrites and dendritic spines, controls the availability of F-actin within spines and impacts both spine structural plasticity and mobility of AMPA receptors at synapses. Furthermore, modelling SCZ- and ASD-associated Cyfip1 haploinsufficiency in mice produces deficits in dendritic complexity and spine morphology in hippocampal pyramidal neurons in vivo.
The activity of Rho GTPases (including RhoA, Rac1 and Cdc42) is associated with increased dendritic branch dynamics and extension.20,21,47,48 Activation of Rac1 releases the inhibitory action of CYFIP1 on WAVE, potentiating Arp2/3-mediated actin polymerization suggesting that changes in CYFIP1 levels could alter signalling events downstream of Rac1.31 Here we demonstrate that CYFIP1 expression levels are important for dendritic arborization and neuronal morphological complexity. Overexpression of CYFIP1 in hippocampal neurons in vitro leads to an increase in dendritic length and complexity whereas conversely, neurons from Cyfip1+/− animals have smaller, less complex dendrites both in vitro and in vivo. Interestingly, in addition to the effects on dendritic morphogenesis that we report here, both WAVE1 and CYFIP1 have previously been implicated in axonal outgrowth.49,50
A strict spatiotemporal balance between actin polymerization and turnover, mediated by Rho GTPases, is also important for spine formation, maturation and stability.20,21 We report that CYFIP1 is highly enriched in dendritic spines where it co-localizes with actin. Thus, CYFIP1 is critically placed, as a key Rac1 effector, to locally regulate WAVE in spines, and variations in its expression levels are likely to impact on Arp2/3-dependent spine actin dynamics downstream of Rac1. In agreement with this, we find that downregulating CYFIP1 expression levels in vitro or in vivo drives an increase in the ratio of immature-to-mature spines suggesting that CYFIP1 is important for actin-dependent regulation of spine maturation. Furthermore, we show that dendritic spines from Cyfip1+/− haploinsufficient neurons display enhanced F-actin availability suggesting that spatiotemporal control over actin polymerization is altered at Cyfip1+/− postsynaptic sites. A role for CYFIP1 in the regulation of Rac1-dependent F-actin assembly in Drosophila NMJ presynaptic terminals was also recently demonstrated, where it was shown to be important for synaptogenesis and presynaptic quantal content.51,52 In addition to previous functions described for CYFIP1 in the regulation of mRNA translation in dendrites and mGluR-long-term depression, our results suggest that CYFIP1 also has a vital role in the local regulation of spine actin dynamics via WAVE and Arp2/3.32,35 This is in agreement with a recent study showing that CYFIP1 translocates between the FMRP-containing protein synthesis machinery and the actin-regulatory complex, and that disrupting CYFIP1’s interaction with members of either complex negatively impacted spine maturation in neurons.34
The surface mobility of neurotransmitter receptors is dynamically regulated to control synaptic activity and is emerging as another key component underlying plasticity at nerve terminals.40,53,54 The trapping of mobile AMPA receptors within the postsynaptic domain is thought to be critical for maintaining and strengthening synaptic communication, and a pathological impairment in this process could have significant implications for neurotransmission.55 The enrichment of CYFIP1 at excitatory synapses implies that CYFIP1 can locally regulate actin cytoskeletal dynamics in the postsynaptic domain. This fine-tuning of actin turnover has previously been demonstrated to be an important regulator of synaptic glutamate receptor mobility. Indeed, we find that the disruption of spine maturation due to decreased CYFIP1 levels also correlates with an increase in AMPA receptor mobility within the spine postsynaptic density. Consequently, CYFIP1 expression levels appear to be critical not just for the maturation of dendritic spines but may also be important for regulating the subsynaptic cytoskeletal architecture essential for the stabilization of postsynaptic AMPA receptors.
Dynamic regulation of F-actin assembly is repeatedly implicated in mechanisms underlying spine structural plasticity.21,56, 57, 58, 59, 60, 61 We observe that NMDA receptor-induced spine remodelling no longer occurs in Cyfip1+/− neurons, suggesting that when CYFIP1 levels are reduced, F-actin assembly is basally overactive and cannot induce further spine remodelling in response to NMDA. CYFIP1 has been previously shown to couple local actin polymerization to membrane budding at subcellular compartments such as the trans-Golgi network.33,62 Therefore, it will be interesting in the future to determine if the coupling of activity-dependent AMPA receptor trafficking (e.g., at the Golgi or in the endocytic network) to alterations in spine volume is also disrupted upon CYFIP1 depletion.63
Our results suggest that CYFIP1 is part of a growing number of mental illness-associated actin-regulatory molecules, including DISC1, DTNBP1, SHANK3, SRGAP2, Oligophrenin and the Abi1-WAVE2 complex that act in Rac1-dependent pathways to regulate dendritic development and spine dynamics.24, 25, 26, 27, 28, 29 As we observe an increase in immature spines upon CYFIP1 overexpression or haploinsufficiency in vitro, it is interesting to note that several other proteins known to signal via actin-regulatory pathways have been shown to drive similar effects on spine structure when either up- or downregulated, including VCP/neurofibromin, NESH/Abi-3 and Cofilin.47,48,57,64, 65, 66, 67 Thus, the precise tuning of CYFIP1 expression levels may be crucial for stabilising synaptic contacts, and skewing its levels above or below a critical threshold may force spines into a structurally unstable state. This may explain why both deletions and duplications of CYFIP1 have been linked to major mental illness.
Recent work has highlighted that rare complete gene knockouts in humans have a significant role in ASD and major mental illness.68 Although both deletions and duplications of CYFIP1 have been identified, total loss of CYFIP1 in humans seems unlikely as we report here that complete CYFIP1 deletion in mice leads to a failure in early embryonic development with Cyfip1-null embryos unable to survive beyond E8.5. Similar developmental abnormalities, resulting in death during early embryogenesis, have been observed in mice lacking other critical actin-regulatory genes such as Rac1 and the WAVE complex component Nap1. Deletion of these genes in the mouse cause death during gastrulation at E7.5 and E9, respectively.69, 70, 71 Therefore, throughout early mouse development, CYFIP1 signalling events, probably via its core conserved actin-regulatory functions, are likely critical for gastrulation and normal patterning of embryonic structure in addition to later roles in neuronal development and plasticity.72 The embryonic lethality observed in the CYFIP1 knockout suggests that CYFIP2 cannot compensate for all CYFIP1 functions. Intriguingly, we also observed CYFIP2 to be enriched in dendritic spines and its overexpression could drive increased dendritogenesis. This suggests that it will be interesting to further explore whether CYFIP2 is a candidate susceptibility gene for major mental illness.
Our results provide important new insights into the role CYFIP1 has in the development of the nervous system and how Cyfip1 dysregulation can alter CNS function to contribute to the development of neuropsychiatric illness. Changes in the structure and function of excitatory synapses, and alterations in the local versus long-range dendritic connectivity of neurons, have both been associated with the development of neuropsychiatric conditions involving imbalances of excitation and inhibition, such as ASD and SCZ.73,74 The roles for CYFIP1 described here may help inform how altered levels of this molecule gives rise to network-level dysfunction that define these pathologies.
Van der Zwaag B, Staal WG, Hochstenbach R, Poot M, Spierenburg HA, de Jonge MV et al. A co-segregating microduplication of chromosome 15q11.2 pinpoints two risk genes for autism spectrum disorder. Am J Med Genet B Neuropsychiatr Genet 2010; 153B: 960–966.
Bittel DC, Kibiryeva N, Butler MG . Expression of 4 genes between chromosome 15 breakpoints 1 and 2 and behavioral outcomes in Prader-Willi syndrome. Pediatrics 2006; 118: e1276–e1283.
Marini C, Cecconi A, Contini E, Pantaleo M, Metitieri T, Guarducci S et al. Clinical and genetic study of a family with a paternally inherited 15q11-q13 duplication. Am J Med Genet A 2013; 161: 1459–1464.
Doornbos M, Sikkema-Raddatz B, Ruijvenkamp CA, Dijkhuizen T, Bijlsma EK, Gijsbers AC et al. Nine patients with a microdeletion 15q11.2 between breakpoints 1 and 2 of the Prader-Willi critical region, possibly associated with behavioural disturbances. Eur J Med Genet 2009; 52: 108–115.
Stefansson H, Rujescu D, Cichon S, Pietiläinen OP, Ingason A, Steinberg S et al. Large recurrent microdeletions associated with schizophrenia. Nature 2008; 455: 232–236.
Levy D, Ronemus M, Yamrom B, Lee Y, Leotta A, Kendall J et al. Rare de novo and transmitted copy-number variation in autistic spectrum disorders. Neuron 2011; 70: 886–897.
Pinto D, Pagnamenta AT, Klei L, Anney R, Merico D, Regan R et al. Functional impact of global rare copy number variation in autism spectrum disorders. Nature 2010; 466: 368–372.
Leblond CS, Heinrich J, Delorme R, Proepper C, Betancur C, Huguet G et al. Genetic and functional analyses of SHANK2 mutations suggest a multiple hit model of autism spectrum disorders. PLoS Genet 2012; 8: e1002521.
Tam GW, van de Lagemaat LN, Redon R, Strathdee KE, Croning DE, Malloy MP et al. Confirmed rare copy number variants implicate novel genes in schizophrenia. Biochem Soc Trans 2010; 38: 445–451.
Nishimura Y, Martin CL, Vazquez-Lopez A, Spence SJ, Alvarez-Retuerto AI, Sigman M et al. Genome-wide expression profiling of lymphoblastoid cell lines distinguishes different forms of autism and reveals shared pathways. Hum Mol Genet 2007; 16: 1682–1698.
Jan Y-N, Jan LY . Branching out: mechanisms of dendritic arborization. Nat Rev Neurosci 2010; 11: 316–328.
Waites CL, Craig AM, Garner CC . Mechanisms of vertebrate synaptogenesis. Annu Rev Neurosci 2005; 28: 251–274.
Alvarez VA, Sabatini BL . Anatomical and physiological plasticity of dendritic spines. Annu Rev Neurosci 2007; 30: 79–97.
Pozo K, Goda Y . Unraveling mechanisms of homeostatic synaptic plasticity. Neuron 2010; 66: 337–351.
Hering H, Sheng M . Dendritic spines: structure, dynamics and regulation. Nat Rev Neurosci 2001; 2: 880–888.
Kulkarni VA, Firestein BL . The dendritic tree and brain disorders. Mol Cell Neurosci 2012; 50: 10–20.
Penzes P, Cahill ME, Jones KA, VanLeeuwen J-E, Woolfrey KM . Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 2011; 14: 285–293.
Ebert DH, Greenberg ME . Activity-dependent neuronal signalling and autism spectrum disorder. Nature 2013; 493: 327–337.
Van Spronsen M, Hoogenraad CC . Synapse pathology in psychiatric and neurologic disease. Curr Neurol Neurosci Rep 2010; 10: 207–214.
Luo L . Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu Rev Cell Dev Biol 2002; 18: 601–635.
Cingolani LA, Goda Y . Actin in action: the interplay between the actin cytoskeleton and synaptic efficacy. Nat Rev Neurosci 2008; 9: 344–356.
Hotulainen P, Hoogenraad CC . Actin in dendritic spines: connecting dynamics to function. J Cell Biol 2010; 189: 619–629.
Kim IH, Racz B, Wang H, Burianek L, Weinberg R, Yasuda R et al. Disruption of Arp2/3 results in asymmetric structural plasticity of dendritic spines and progressive synaptic and behavioral abnormalities. J Neurosci 2013; 33: 6081–6092.
Hayashi-Takagi A, Takaki M, Graziane N, Seshadri S, Murdoch H, Dunlop AJ et al. Disrupted-in-Schizophrenia 1 (DISC1) regulates spines of the glutamate synapse via Rac1. Nat. Neurosci 2010; 13: 327–332.
Ito H, Morishita R, Shinoda T, Iwamoto I, Sudo K, Okamoto K et al. Dysbindin-1, WAVE2 and Abi-1 form a complex that regulates dendritic spine formation. Mol Psychiatry 2010; 15: 976–986.
Hayashi ML, Choi S-Y, Rao BS, Jung H-Y, Lee H-K, Zhang D et al. Altered cortical synaptic morphology and impaired memory consolidation in forebrain- specific dominant-negative PAK transgenic mice. Neuron 2004; 42: 773–787.
Govek E-E, Newey SE, Akerman CJ, Cross JR, Van der Veken L, Van Aelst L . The X-linked mental retardation protein oligophrenin-1 is required for dendritic spine morphogenesis. Nat Neurosci 2004; 7: 364–372.
Charrier C, Joshi K, Coutinho-Budd J, Kim J-E, Lambert N, de Marchena J et al. Inhibition of SRGAP2 function by its human-specific paralogs induces neoteny during spine maturation. Cell 2012; 149: 923–935.
Durand CM, Perroy J, Loll F, Perrais D, Fagni L, Bourgeron T et al. SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism. Mol Psychiatry 2012; 17: 71–84.
Takenawa T, Suetsugu S . The WASP-WAVE protein network: connecting the membrane to the cytoskeleton. Nat Rev Mol Cell Biol 2007; 8: 37–48.
Chen Z, Borek D, Padrick SB, Gomez TS, Metlagel Z, Ismail AM et al. Structure and control of the actin regulatory WAVE complex. Nature 2010; 468: 533–538.
Napoli I, Mercaldo V, Boyl PP, Eleuteri B, Zalfa F, De Rubeis S et al. The fragile X syndrome protein represses activity-dependent translation through CYFIP1, a new 4E-BP. Cell 2008; 134: 1042–1054.
Anitei M, Stange C, Parshina I, Baust T, Schenck A, Raposo G et al. Protein complexes containing CYFIP/Sra/PIR121 coordinate Arf1 and Rac1 signalling during clathrin-AP-1-coated carrier biogenesis at the TGN. Nat Cell Biol 2010; 12: 330–340.
De Rubeis S, Bagni C . Regulation of molecular pathways in the Fragile X Syndrome: insights into Autism Spectrum Disorders. J Neurodev Disord 2011; 3: 257–269.
Bozdagi O, Sakurai T, Dorr N, Pilorge M, Takahashi N, Buxbaum JD . Haploinsufficiency of Cyfip1 produces fragile X-like phenotypes in mice. Dawson TM, editor. PLoS One 2012; 7: e42422.
Arancibia-Carcamo IL, Yuen EY, Muir J, Lumb MJ, Michels G, Saliba RS et al. Ubiquitin-dependent lysosomal targeting of GABAA receptors regulates neuronal inhibition. Proc Natl Acad Sci 2009; 106: 17552–17557.
Wearne SL, Rodriguez A, Ehlenberger DB, Rocher AB, Henderson SC, Hof PR . New techniques for imaging, digitization and analysis of three-dimensional neural morphology on multiple scales. Neuroscience 2005; 136: 661–680.
Nakamura Y, Wood CL, Patton AP, Jaafari N, Henley JM, Mellor JR et al. PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity. EMBO J 2011; 30: 719–730.
Koskinen M, Bertling E, Hotulainen P . Methods to measure actin treadmilling rate in dendritic spines. Methods Enzymol 2012; 505: 47–58.
Muir J, Arancibia-Carcamo IL, MacAskill AF, Smith KR, Griffin LD, Kittler JT . NMDA receptors regulate GABAA receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit. Proc Natl Acad Sci USA 2010; 107: 16679–16684.
Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W, Iyer V et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 2011; 474: 337–342.
Rocca DL, Amici M, Antoniou A, Suarez EB, Halemani N, Murk K et al. The small GTPase Arf1 modulates Arp2/3-mediated actin polymerization via PICK1 to regulate synaptic plasticity. Neuron 2013; 79: 293–307.
Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M et al. Lifeact: a versatile marker to visualize F-actin. Nat Methods 2008; 5: 605–607.
Shepherd JD, Huganir RL . The cell biology of synaptic plasticity: AMPA receptor trafficking. Annu Rev Cell Dev Biol 2007; 23: 613–643.
Anggono V, Huganir RL . Regulation of AMPA receptor trafficking and synaptic plasticity. Curr Opin Neurobiol 2012; 22: 461–469.
Bats C, Groc L, Choquet D . The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking. Neuron 2007; 53: 719–734.
Wear MA, Schafer DA, Cooper JA . Actin dynamics: assembly and disassembly of actin networks. Curr Biol 2013; 10: R891–R895.
Condeelis J . How is actin polymerization nucleated in vivo? Trends Cell Biol 2001; 11: 288–293.
Luo L, Hensch TK, Ackerman L, Barbel S, Jan LY, Jan YN . Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines. Nature 379: 837–840.
Kawano Y, Yoshimura T, Tsuboi D, Kawabata S, Kaneko-Kawano T, Shirataki H et al. CRMP-2 is involved in kinesin-1-dependent transport of the Sra-1/WAVE1 complex and axon formation. Mol Cell Biol 2005; 25: 9920–9935.
Zhao L, Wang D, Wang Q, Rodal AA, Zhang YQ . Drosophila cyfip regulates synaptic development and endocytosis by suppressing filamentous actin assembly. PLoS Genet 2013; 9: e1003450.
Schenck A, Bardoni B, Langmann C, Harden N, Mandel JL, Giangrande A . CYFIP/Sra-1 controls neuronal connectivity in Drosophila and links the Rac1 GTPase pathway to the fragile X protein. Neuron 2003; 38: 887–898.
Triller A, Choquet D . New concepts in synaptic biology derived from single-molecule imaging. Neuron 2008; 59: 359–374.
Pinaud F, Clarke S, Sittner A, Dahan M . Probing cellular events, one quantum dot at a time. Nat Methods 2010; 7: 275–285.
Opazo P, Sainlos M, Choquet D . Regulation of AMPA receptor surface diffusion by PSD-95 slots. Curr Opin Neurobiol 2012; 22: 453–460.
Okamoto K-I, Nagai T, Miyawaki A, Hayashi Y . Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nat Neurosci 2004; 7: 1104–1112.
Fukazawa Y, Saitoh Y, Ozawa F, Ohta Y, Mizuno K, Inokuchi K . Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is essential for late LTP maintenance in vivo. Neuron 2003; 38: 447–460.
Matsuzaki M, Honkura N, Ellis-Davies GC, Kasai H . Structural basis of long-term potentiation in single dendritic spines. Nature 2004; 429: 761–766.
Zhou Q, Xiao M, Nicoll RA . Contribution of cytoskeleton to the internalization of AMPA receptors. Proc Natl Acad Sci USA 2001; 98: 1261–1266.
Wang X, Yang Y, Zhou Q . Independent expression of synaptic and morphological plasticity associated with long-term depression. J Neurosci 2007; 27: 12419–12429.
Gu J, Lee CW, Fan Y, Komlos D, Tang X, Sun C et al. ADF/cofilin-mediated actin dynamics regulate AMPA receptor trafficking during synaptic plasticity. Nat Neurosci 2010; 13: 1208–1215.
Castets M, Schaeffer C, Bechara E, Schenck A, Khandjian EW, Luche S et al. FMRP interferes with the Rac1 pathway and controls actin cytoskeleton dynamics in murine fibroblasts. Hum Mol Genet 2005; 14: 835–844.
Hanley JG, Henley JM . PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking. EMBO J 2005; 24: 3266–3278.
Bae J, Sung BH, Cho IH, Kim S-M, Song WK . NESH regulates dendritic spine morphology and synapse formation. PLoS One 2012; 7: e34677.
Meng Y, Zhang Y, Tregoubov V, Janus C, Cruz L, Jackson M et al. Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice. Neuron 2002; 35: 121–133.
Hotulainen P, Llano O, Smirnov S, Tanhuanpää K, Faix J, Rivera C et al. Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis. J Cell Biol 2009; 185: 323–339.
Wang H-F, Shih Y-T, Chen C-Y, Chao H-W, Lee M-J, Hsueh Y-P . Valosin-containing protein and neurofibromin interact to regulate dendritic spine density. J Clin Invest 2011; 121: 4820–4837.
Lim ET, Raychaudhuri S, Sanders SJ, Stevens C, Sabo A, MacArthur DG et al. Rare complete knockouts in humans: population distribution and significant role in autism spectrum disorders. Neuron 2013; 77: 235–242.
Yan C, Martinez-Quiles N, Eden S, Shibata T, Takeshima F, Shinkura R et al. WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility. EMBO J 2003; 22: 3602–3612.
Migeotte I, Omelchenko T, Hall A, Anderson KV . Rac1-dependent collective cell migration is required for specification of the anterior-posterior body axis of the mouse. PLoS Biol 2010; 8: e1000442.
Rakeman AS, Anderson KV . Axis specification and morphogenesis in the mouse embryo require Nap1, a regulator of WAVE-mediated actin branching. Development 2006; 133: 3075–3083.
Liu D, Bei D, Parmar H, Matus A . Activity-regulated, cytoskeleton-associated protein (Arc) is essential for visceral endoderm organization during early embryogenesis. Mech Dev 2000; 92: 207–215.
Casanova MF, Buxhoeveden DP, Switala AE, Roy E . Minicolumnar pathology in autism. Neurology 2002; 58: 428–432.
Rubenstein JL, Merzenich MM . Model of autism: increased ratio of excitation/inhibition in key neural systems. Genes Brain Behav 2003; 2: 255–267.
We thank Professor M Häusser for kindly providing access to the Neurolucida system and Dr B Clark and Dr A Roth for their help and advice on using the Neurolucida system. We thank Kittler lab members for constructive discussions. This work was supported by the United Kingdom Medical Research Council Senior Non-Clinical Fellowship (MRC; GO802377; JTK). ECD is an MRC-funded student on the MRC Laboratory for Molecular Cell Biology 4-year graduate programme. DFS is in the University College London (UCL) Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (COMPLEX) PhD Programme.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Translational Psychiatry website
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Pathania, M., Davenport, E., Muir, J. et al. The autism and schizophrenia associated gene CYFIP1 is critical for the maintenance of dendritic complexity and the stabilization of mature spines. Transl Psychiatry 4, e374 (2014). https://doi.org/10.1038/tp.2014.16
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