Haploinsufficiency of the schizophrenia and autism risk gene Cyfip1 causes abnormal postnatal hippocampal neurogenesis through microglial and Arp2/3 mediated actin dependent mechanisms

Genetic risk factors can significantly increase chances of developing psychiatric disorders, but the underlying biological processes through which this risk is effected remain largely unknown. Here we show that haploinsufficiency of Cyfip1, a candidate risk gene present in the pathogenic 15q11.2(BP1–BP2) deletion may impact on psychopathology via abnormalities in cell survival and migration of newborn neurons during postnatal hippocampal neurogenesis. We demonstrate that haploinsufficiency of Cyfip1 leads to increased numbers of adult-born hippocampal neurons due to reduced apoptosis, without altering proliferation. We show this is due to a cell autonomous failure of microglia to induce apoptosis through the secretion of the appropriate factors, a previously undescribed mechanism. Furthermore, we show an abnormal migration of adult-born neurons due to altered Arp2/3 mediated actin dynamics. Together, our findings throw new light on how the genetic risk candidate Cyfip1 may influence the hippocampus, a brain region with strong evidence for involvement in psychopathology.


Introduction
Many psychiatric conditions show high heritability. Recent genetic studies in schizophrenia for example have identified up to 160 loci that increase risk for the disease 1,2 enriched within synaptic 3 , histone modifying and immune system genes 4 . CYFIP1 is a genetic risk factor for schizophrenia, autism and developmental delay, by virtue of its presence in the penetrant 15q11.2(BP1-BP2) copy number deletion 5 . Loss of one copy of this interval leads to substantially increased risk for disorder in both Western 6,7 and Han Chinese populations 8 . CYFIP1 haploinsufficiency is likely a major contributor to the 15q11.2 (BP1-BP2) psychiatric phenotype due to evidence of CYFIP1's involvement in a range of synaptic functions, including key roles in dendritic spine morphology and branching [9][10][11] . CYFIP1 protein influences synaptic function in two main ways. CYFIP1 interacts with fragile X mental retardation 1 (FMRP), the protein gene product of FMR1, to suppress translation of up to 800 target transcripts 12 . Secondly, it interacts with Wiskott-Aldrich syndrome protein family member 1 (WAVE1), a key mediator of cytoskeleton dynamics, to modulate ARP2/3 dependant actin branching 13 .
In the present work we focus on microglia and adult hippocampal neurogenesis (AHN) as important sites of CYFIP1 action and as potential contributors to psychiatric phenotypes arising from 15q11.2(BP1-BP2) copy number deletion. Adult-born hippocampal neurons, up to 700 of which are estimated to be born per day in humans 14 , develop through a well-defined series of cellular events, starting from proliferative stem cells through intermediate progenitors, migratory neuroblasts and immature neurons, before eventually forming mature granule cells, which distribute through the dentate gyrus 15 . In rodent models, adult-born hippocampal neurons have been shown to be involved in a number of behavioural processes, spanning pattern separation 16,17 , spatial navigation 18,19 , memory turnover 20 , and acquisition 21 and extinction 22 of fear memory. There behaviours are affected in psychiatric disorders including schizophrenia [23][24][25][26] . Antipsychotic drugs, specifically atypical antipsychotics, also impact AHN 27 and importantly, a number of functionally diverse schizophrenia risk genes, including DISC1 28 , SREB2/GPR85 29 , CACNA1C 30,31 DGCR8 32 and miR137 33 , converge in their ability to modify AHN. Additionally, post-mortem findings from schizophrenia patients reveal changes in cell proliferation and expression of immature neuronal markers in the hippocampus [34][35][36] .
Immune changes are another common hallmark of several psychiatric disorders, including schizophrenia, where evidence from epidemiological data 37,38 and findings showing increased inflammatory factors in the circulation 39 , and in post-mortem brain 40 of schizophrenia patients, implicate altered immune function in the development of disease symptoms. Mechanistic insights linking immune changes to risk for psychopathology are however largely lacking. Recent interest has focused on microglia, the resident macrophage-like immune cells of the central nervous system, with data suggesting possible alterations in microglial functioning in both those at high risk and those diagnosed with schizophrenia [41][42][43][44] . The causal mechanism(s) by which microglia might contribute to pathogenesis remain unclear, though there is increasing evidence that, in addition to canonical immune functions, microglia also modify neuronal networks through synaptic pruning during development 45 and into adulthood 46 . This microglial-mediated pruning is important for normal brain and behavioural functioning 47 . Additionally, microglia can modulate neuronal firing through direct cell-cell contacts 48 , and have been shown to be capable of phagocytosing both developing 49 and adult 50 neurons.
In this work we used a translationally relevant haploinsufficient mouse model to reveal hitherto undescribed effects of manipulating Cyfip1 on the survival and migration of newborn neurons in dentate gyrus hippocampus, key cellular processes in the development and function of the adult hippocampus. We also reveal the mechanisms mediating these effects, highlighting CYFIP1 effects on microglia mediated apoptosis and Arp2/3 dependent actin dynamics, respectively. We suggest these findings may have relevance to how Cyfip1 haploinsufficiency can influence hippocampal function in the context of the markedly increased risk for psychiatric disorder arising from the 15q11.2(BP1-BP2) copy number deletion.

Materials and methods
For full Materials and methods, please see Supplementary Information.
Cyfip1 heterozygous knockout mice Cyfip1 tm2a(EUCOMM)Wtsi animals (MGI:5002986, referred to hereafter as Cyfip1 +/− ) were maintained heterozygously on a C57/BL6J background, for genotyping examples, see Fig. S1. Animals used were of mixed sex and 8-12 weeks old. Cyfip1 +/− and wild-type littermates were kept in conventional cages with 12 h light-dark cycle and ad libitum access to water and food at all time. All experiments were performed in accordance with the Animal (Scientific Procedures) Act 1986, and approved by the local animal welfare committee.

Tissue harvesting and immunohistochemistry
Paraformaldehyde fixed brains from 8 to 12-week-old Cyfip1 +/− and wild-type littermates were prepared as previously described 51 . Free floating 40 μm cryostat sections were used for immunohistochemistry. Immunohistochemistry was performed as previously described 51 , with a 1:12 stereotactic sampling rate. Detection of cleaved-caspase 3 used a modified protocol, where primary antibodies were incubated for 48 h in blocking solution at 4°C. Primary and secondary antibodies are described in Table S1.

Primary hippocampal progenitor culture
Hippocampal progenitors were isolated from P7 to P8 animals. Isolated hippocampi were dissociated in papain and progenitors were enriched through Optiprep density gradient centrifugation. Cells were cultured on poly-Llysine and laminin in the presence of EGF and FGF2.

Primary microglial culture
Microglia were isolated from P7 to P8 whole brain mixed glia through the shake-off method.

Immunocytochemistry
Fixed cultures were blocked and permeabilized with 5% donkey serum and 0.1% Triton X100 in PBS (PBST) for 30 min at room temperature. Primary antibodies were applied in the same solution overnight at 4°C. Antibodies are detailed in Table S1. Following three washes in PBST, relevant secondary antibodies were applied in PBST for 2 h at room temperature. For identification of microglia, Alexa 568 conjugated IB4 (ThermoFisher I21412) was added to secondary antibody solutions at 2.5 µg/ml. Cells were washed, counterstained with DAPI, and mounted for microscopy.

Conditioned medium
Microglia conditioned medium was prepared by incubating 5 × 10 5 microglia per ml in progenitor medium for 24 h. 25% conditioned medium was added to hippocampal cultures.

Time-lapse imaging and cell tracking
Primary hippocampal progenitors were plated on poly-D-lysine and laminin coated 24 well plates, and cultured for 5 days. At 5 DIV, cultures were imaged on an inverted Leica DMI600B microscope, at 37°C, with 5% CO 2 in air. Cells were imaged for 2 h, with image acquisition every 5 min. Total distance covered was determined through manual cell tracking using the MTrackJ plugin in Ima-geJ 52 . A minimum of 50 cells ware analysed per condition per animal.

Arp2/3 inhibition
Cells were treated with 50 μM CK-548 or 250 μM CK-666 in DMSO, or vehicle, for 2 h prior to time-lapse imaging. Drugs were present during imaging.

Increased neuronal numbers in Cyfip1 haploinsufficiency is due to decreased apoptosis
To investigate the cell-intrinsic effects of Cyfip1 haploinsufficiency independent of niche effects we used primary hippocampal progenitor cultures. The in vitro data corroborated the ex vivo finding, showing an increase in the fraction of DCX immunoreactive cells in the cultures prepared from Cyfip1 +/− animals (5.5 ± 0.5% vs. 8.8 ± 1.4%, t (16) = 2.78, p = 0.014, Fig. 2A This suggested an apototic mechanism. Indeed, we found significantly fewer cleaved-caspase-3 immunoreactive cells in vivo in the dentate gyrus of Cyfip1 +/− animals (2.1 ± 0.2 vs. 1.2 ± 0.1 cells/mm 2 , t (23) = 3.01, p = 0.006, Fig. 2I, J). To confirm this, we investigated apoptosis specifically in the DCX+ populations in our cultures. We found a marked reduction in the fraction of DCX+/nestin − cells positive for cleaved-caspase-3 (25.9 ± 5.5% vs. 7.2 ± 2.5%, t (16) = 2.32, p = 0.034, Fig. 2K, L), again demonstrating a high level of concordance between effects in cell culture and intact brain tissue. We found no sex effects in any of the phenotypes investigated in the primary cultures (see Supplementary Results and Fig. S4). Together, these data suggested Cyfip1 haploinsufficiency impaired a normal homoeostatic apoptotic mechanism for modulating newborn neuron number, leading to a greater number surviving to go on to maturity.

Evidence that apoptosis of newly born neurons is mediated by microglia secreted factors
Given the known roles microglia play in earlier stages of neurogenesis 53 , we speculated that microglia may play a role in the survival and death of adult newborn immature neurons. First, we established that there were no genotype differences in density of Iba1+ microglia in hippocampus ex vivo or in vitro (Fig. S5, in either sex). To investigate the role of microglia in neuronal apoptosis, we depleted microglia from wild-type primary hippocampal progenitor cultures using the specific microglia toxin Mac-1-SAP, targeting CD11b expressing cells. We established the efficacy of the toxin against primary mouse microglia (Fig. 3A).
Depletion of microglia from hippocampal cultures led to an increase in the proportion of DCX+ cells (7.5 ± 1.6% vs. 12.7 ± 0.3%, t (7) = 3.19, p = 0.019, Fig. 3B), and a specific decrease in the fraction of apoptotic DCX+ cells (13.1 ± 3.1% vs. 2.7 ± 1.7%, t (7) = 2.93, p = 0.026, Fig. 3C),   2 Primary hippocampal progenitor cultures prepared from P7 to P8 brain confirm ex vivo findings and indicate that increased neuronal numbers in Cyfip1 +/− animals is due to reduced apoptosis. A Immunoreactivity for immature neuron marker DCX and hippocampal marker Prox1 confirm cultures contain hippocampus-derived developing neurons. B Cyfip1 +/− animals showed a significantly higher proportion of DCX + cells in the total DAPI+ population (n = 17). C Representative staining for progenitor markers GFAP and nestin showed cultures contained a mixed population of progenitors at differing stages of development. D There was no significant difference in the proportion of early GFAP+/nestin+ cells (n = 36). E The proportion of later GFAP−/nestin+ cells (n = 36) was also unaffected. F Live imaging showed cultures are largely comprised of healthy, MitoTracker+ cells, with some dying PI+ cells. G A small but significant increase in the proportion of MitoTracker+ cells was observed in the Cyfip1 +/− cultures (n = 15). H Conversely, a significant decrease in the proportion of PI+ cells was seen in the Cyfip1 +/− cultures (n = 14). I Examples of apoptotic cells in the dentate gyrus, as marked by immunoreactivity for cleaved-caspase 3. J Cyfip1 +/− animals (n = 13) have significantly fewer apoptotic cells than wild-type animals (n = 12). All data are shown as mean ± SEM. K Immunoreactivity for apoptotic marker cleaved-caspase with DCX and nestin showed specific neuronal apoptosis in primary cultures. Arrowheads indicate an apoptotic neuron. K The fraction of immature neurons undergoing apoptosis was significantly lower in the Cyfip1 +/− cultures (n = 17). L Representative image of apoptotic cells in the dentate gyrus.
without any associated changes in the nestin+ population (50.7 ± 2.7% vs. 53.8 ± 1.0%, t (7) = 1.07, p = 0.32, Fig. 3D), phenocopying the Cyfip1 +/− cultures, and showing a significant role for microglia in regulating DCX numbers. Conversely, addition of exogenous microglia on a semipermeable membrane, allowing only for soluble factors to influence neuronal apoptosis, had opposite effects. Under these conditions we observed, a decreased proportion of DCX+ cells (5.9 ± 0.8% vs. 1.9 ± 0.5%, t (15) = 4.36, p = 6.5 × 10 −4 , Fig. 3F), increased apoptosis in the DCX+ population (14.8 ± 4.7% vs. 51.7 ± 9.0%, t (15) = 3.27, p = 0.006, Fig. 3G) and no effect on the nestin+ population Haploinsufficiency of Cyfip1 inhibits the ability of microglia to support apoptosis of newborn immature hippocampal neurons Having established a novel microglial dependent mechanism for regulation of neuronal survival, we next tested whether effects on this process could underlie the effects of Cyfip1 haploinsufficiency on neuronal survival. To directly address the potential role the induction of apoptosis by microglial secreted factors in an unbiased way, we used a cross-genotype conditioned medium approach. Medium obtained from isolated wild-type and Cyfip1 +/− microglia preparations was added to both the wild-type and Cyfip1 +/− hippocampal progenitor enriched cultures (see Fig. 3I, M for schematic of design) and cell proportions and levels of apoptosis were determined. In the wild-type progenitor cultures, adding conditioned medium from wild-type microglia resulted in a significant reduction in the proportion of DCX+ cells, compared to unstimulated cells, whereas Cyfip1 +/− conditioned medium had no effect (5.4 ± 0.9% vs. 2.9 ± 0.2% vs. 5.4 ± 0.3% for unstimulated, wild-type stimulated and Cyfip1 +/− stimulated respectively, main effects of genotype F (2,9) = 6.31, p = 0.019, Fig. 3J). Post hoc testing confirmed significant differences between controls and wild-type medium (p = 0.031) and between those stimulated with wild-type and Cyfip1 +/− medium (p = 0.033). This pattern of effects was mirrored inversely in the fraction of DCX+ cells that were also immunopositive for cleaved-caspase-3, with only conditioned medium from wild-type microglia cultures increasing apoptosis (16.1 ± 3.6% vs. 34.5 ± 3.5% vs. 14.8 ± 2.5%, main effects of genotype F (2,9) = 11.57, p = 0.003, Fig. 3K). Again, post hoc testing showed the wildtype stimulated condition was significantly different from controls and Cyfip1 +/− stimulated condition (p = 0.007 and p = 0.005, respectively). Consistent with the conclusion that these effects were limited to the DCX+ immature neuron population of cells, the proportion of cultures that was nestin+ was unchanged across all conditions (65.0 ± 5.2% vs. 60.7 ± 4.4% vs. 57.1 ± 4.1%, F (2,9) = 0.75, p = 0.50, Fig. 3L).
Importantly, identical patterns of effects were seen in the Cyfip1 +/− hippocampal progenitor cultures. Using an identical experimental design (Fig. 3M), we found that conditioned medium from wild-type microglia, but not from Cyfip1 +/− microglia, decreased the proportion of DCX+ cells (8.1 ± 0.6% vs. 5.1 ± 0.2% vs. 9.0 ± 0.7%, main effects of genotype F (2,9) = 13.82, p = 0.002, Fig. 3N) with post hoc analysis confirming wild-type microglia medium stimulated conditions were significantly different from control and Cyfip1 +/stimulated conditions (p = 0.008 and p = 0.002, respectively). Similarly, only conditioned medium from wild-type microglia was associated with a significant increase in the proportion of apoptotic DCX+ cells (12.8 ± 2.3% vs. 28.3 ± 5.0% vs. 13.0 ± 0.9%, main effects of genotype F (2,9) = 6.12, p = 0.021, Fig. 3O) with post hoc analysis confirming the wild-type stimulated condition was significantly different from the unconditioned medium controls and Cyfip1 +/− stimulated condition (p = 0.007 and p = 0.005, respectively). As before, no effect of any of the medium conditions was seen on the nestin-positive population (64.7 ± 2.6% vs. 64.0 ± 2.3% vs. 57.5 ± 3.3%, F (2,9) = 2.13, p = 0.18, Fig. 3P). These data provide converging evidence for induction of neuronal (see figure on previous page) Fig. 3 Cyfip1 haploinsufficiency affects the ability if microglia to regulate number and apoptosis rates of immature neurons. A Experimental setup, Mac-1-Sap was present in wild-type hippocampal progenitor cultures from 2 h after isolation until 3 days in vitro. Dose-response curves of microglial death after Mac-1-SAP administration show near-complete depletion of Iba1+ microglia was achieved within 48 h (n = 6). B Cultures where microglia were depleted (n = 4) showed a higher proportion of DCX+ cells than controls (n = 4). C Correspondingly, the fraction of DCX cells undergoing apoptosis, as marked by cleaved-caspase 3 staining, was significantly decreased in the absence of microglia. D The proportion of nestin+ cells was unaffected by microglia depletion. E Experimental setup, membrane inserts containing wild-type microglia prepared from P7-8 brain were added to cultures 2 h after progenitor isolation and were present throughout the remainder of the experiment. Due to the experimental conditions, no cell-cell contact between microglia and progenitors was possible, but secreted factors were free to diffuse. F The presence of microgliacontaining inserts (n = 8) significantly decreased the proportion of DCX expressing cells compared to controls (n = 8). G Conversely, the added microglia significantly increased apoptosis in DCX+ cells. H No effect was seen in nestin expressing cells. I Experimental design, wild-type hippocampal progenitors were exposed to conditioned medium from either wild-type or Cyfip1 +/− microglia. J In wild-type cultures, conditioned medium from wild-type microglia induced a significant reduction in the proportion of DCX+ cells, whereas medium from Cyfip1 +/− microglia was unable to do this (n = 4 in all conditions). K An inverse pattern was seen in neuronal apoptosis where wild-type microglia medium increased apoptosis in DCX+ immature neurons but Cyfip1 +/medium did not. L Nestin+ cells were not significantly affected by the presence of microglial factors. M Experimental design, Cyfip1 +/− hippocampal progenitors were exposed to conditioned medium from either wild-type or Cyfip1 +/− microglia. N As seen in the wild-type cultures, a significant decrease in DCX+ cells was observed only after stimulation with medium from wild-type microglia (n = 4 in all conditions). O Similarly, medium from Cyfip1 +/− microglia was unable to induce apoptosis in Cyfip1 +/− progenitors. P Again, the nestin+ population was unaffected. All data are shown as mean ± SEM. apoptosis through factors secreted by microglia a process which is disrupted in Cyfip1 +/− animals.
Proliferative cells are abnormally positioned in the Cyfip1 +/− hippocampus As well as having to survive, newborn cells have to migrate to the correct location to functionally integrate into existing hippocampal circuitry. We noted that the gross positioning of newborn cells appeared different in the Cyfip1 +/− animals. Cells undergoing proliferation are normally largely limited to the subgranular zone (SGZ) of the dentate gyrus, apart from some radially migrating neuroblasts undergoing their final divisions. Indeed, the majority of Ki67+ cells were found in the SGZ, however a significantly larger fraction was found in the SGZ in the Cyfip1 +/− animals (Fig. 4A, B, 80.4 ± 1.1% vs. 88.2 ± 0.6%, t (14) = 6.001, p = 3.2 × 10 −5 ). As well as an increased fraction remaining in the SGZ, Cyfip1 +/− Ki67+ cells were also found significantly less far out into the granular zone (Fig. 4C, 17.0 ± 1.4 µm vs. 11.9 ± 1.0 µm, t (14) = 2.999, p = 0.009). These data suggested a migration deficit in Cyfip1 +/− animals.

Cyfip1 +/− adult-born neurons show impaired migration into the granular layer
In order to gain direct evidence of abnormal migration in Cyfip1 +/− animals we tracked the final positioning of newly born neurons using a 30 day BrdU pulse-chase paradigm followed by staining for BrdU/Neun. Neurons born in the SGZ normally undergo a radial migration outwards into the granular layer. Consistent with a migration deficit in the Cyfip1 +/− animals we found a greater proportion of BrdU+NeuN+ cells had remained in the SGZ in (Fig. 4D, E, 52.2 ± 3.6% vs. 66.7 ± 2.8%, t (12) = 3.197, p = 0.008) and those cells that had migrated had moved a smaller distance into the granular zone (Fig.  4F, 13.6 ± 2.3 µm vs. 6.0 ± 0.7 µm, t (12) = 3.549, p = 0.004). When we further analysed the location of the BrdU+ /NeuN+ cells that had left the SGZ by dividing the dentate gyrus into four quartiles mapping on to the route taken by migrating cells, we found that the distribution of cells over these quartiles was significantly different (Fig. S6, χ 2 = 25.7, p = 1.1 × 10 −5 ) and, as would be anticipated with a migration deficit, BrdU+/NeuN+ cells in the Cyfip1 +/− animals were skewed towards the two most proximal quartiles (i.e. those closest to the starting point in the SGZ). Finally, we examined the putative migration phenotype observed in vivo by performing time-lapse imaging in primary hippocampal cultures. Over the 2 h imaging period, Cyfip1 +/− cells covered significantly less distance than wild-type cells (Fig. 4G, H, 133.0 ± 4.7 vs. 90.7 ± 12.0 µm, t (10) = 3.993, p = 0.003). Both the in vivo and in vitro findings were consistent with a migration deficit due to Cyfip1 haploinsufficiency.

Actin dynamics are altered in Cyfip1 +/− cells and inhibition of Arp2/3 activity rescues migration deficits in vitro
As CYFIP1 contributes to the regulation of actin branching and polymerisation through the WAVE1 complex, a function crucial for cytoskeletal reorganisation and cell migration, we speculated that the Cyfip1 +/− related migration phenotype was related to alterations in actin dynamics. A main determinant of actin function in the context of cytoskeleton reorganisation is the ratio of filamentous (F) to globular (G) actin. We first checked this ratio in primary hippocampal cultures, staining with fluorescently labelled phalloidin for F-actin and DNAse1 for G-actin, and found a significant increase in F-G-actin ratio in Cyfip1 +/− cells (Fig. 5A, B, 0.67 ± 0.3 vs. 1.48 ± 0.28, t (5) = 2.904, p = 0.034). An increase in F-G-actin ratio would be consistent with concurrent increased activity in Arp2/3 brought about by an expected reduction of CYFIP1-WAVE1 complexing under conditions of Cyfip1 haploinsufficiency. Hence, we further speculated that treatment with Arp2/3 inhibitors would influence, and potentially rescue, the abnormal cell migration phenotypes in Cyfip1 +/− cultures. To do this we used two drugs; CK-548, which induces a conformation change inhibiting actin binding and CK-666, which stabilises the inactive form of Arp2/3.

Discussion
We have shown changes in the survival and migration of newly born neurons in hippocampus in a model of Cyfip1 haploinsufficiency, recapitulating the lowered gene dosage found in the pathogenic 15q11.2(BP1-BP2) copy number deletion. We report that Cyfip1 +/− animals have larger numbers of surviving newly born neurons in hippocampus, in the absence of increases in numbers of stem cells or rate of proliferation, and provide evidence that this is the result of a specific failure in microglia-induced apoptosis (see summary of proposed mechanism in Fig. S7). These newly born neurons also show an Arp2/3 dependent deficit in migration, resulting in a failure to reach their appropriate locations within the dentate gyrus.
Our study is the first to our knowledge to demonstrate a direct effect of microglial soluble factors on the survival of immature neurons as a homoeostatic mechanism. In contrast to earlier findings examining the effect of microglia on adult neurogenesis 54-56 , we show this occurs in the absence of overt inflammatory disease or microglial activation. Our data indicate this is a mechanism susceptible to pathogenic changes, as evidenced by the effects of Cyfip1 haploinsufficiency on the ability of microglia to regulate apoptosis of immature neurons. The effects of Cyfip1 haploinsufficiency were highly cell-type specific and appeared limited to the stage of cell maturity where neuronal progenitors are fully committed to a neuronal phenotype, a stage of development characterised by high levels of apoptosis. Whilst we have demonstrated that soluble factors from microglia are involved in controlling numbers of immature neurons during adult neurogenesis, and that haploinsufficiency of Cyfip1 leads to reduced apoptosis via an effect on factors secreted from microglia, the soluble factor(s) responsible remain to be identified. Future proteomic and pharmacological work will serve to identify and validate the identity of the responsible medatior(s). Cyfip1 has pleotropic roles but the potential links between CYFIP1, actin remodelling. and cytokine secretion are especially interesting 57 . A focus for CYFIP1 action in microglia gains further evidence from a recent preprint by showing the effect of cell specific deletion of Cyfip1 on microglia morphology, movement and function 58 .
In parallel, we have shown that adult-born neurons in Cyfip1 +/− animals show a significantly impaired migration phenotype. Here our data point to a key role for CYFIP1 effects on actin dynamics mediated by Arp2/3 activity. We hypothesise that lowered dosage will lead to reduced engagement of CYFIP protein with the CYFIP-WAVE1-Arp2/3 pathway resulting in reduced suppression of Arp2/3 activity and abnormal actin cytoskeleton remodelling required for migration. We show evidence to support this hypothesis though, as predicted, an increase in F-G-actin ratio and rescue of the migration phenotype by pharmacological inhibition of Arp2/3 activity. The activation of the WAVE-Arp2/3 system is complex and, can take place in at least two different Rac1 mediated pathways 59,60 . It is unlikely that CYFIP1 is the sole Fig. 5 Disruption of Arp2/3 mediated actin dynamics is responsible for migration deficit. A Fluorescently labelled phalloidin and DNAse show the amount of filamentous and globular actin, respectively. B Quantification of fluorescence intensity shows Cyfip1 +/− cells have an increased F-to Gactin ratio. C Treatment with Arp2/3 inhibitors CK-548 or CK-666 in wild-type cells significantly reduces the distance moved by cells during live imagine, whereas treatment of Cyfip1 +/− cells restores distances to wild-type levels.
regulator of this activation, but our data point towards a significant role in this process.
The combined effects of failure to undergo homoeostatic apoptosis and failure to migrate to the correct location is likely to have profound effects on the functionality of adult-born neurons in hippocampus.. We observed lasting effects up to 30 days post division, when adult-born neurons will already have local connections 61 . The prolonged survival and expression of mature markers of these ectopic cells makes it likely these cells will be active, indeed, it is known that synaptic NMADR signalling is required for neuronal survival prior to this developmental timepoint 62 , making it likely these cells are integrated in some form in the local circuitry. The exact functional consequences on a network level are difficult to predict and will require further electrophysiological study, both on the individual cell and network level. Haploinsufficiency of Cyfip1 has been shown to have disease relevant behavioural consequences in both mice 63,64 and rat 65 models, but to date, much of the neurobiology and behaviour in these models has remained unexplored. Our data would indicate a focus on hippocampal circuitry would prove of interest. Neuronal migration in the hippocampus has, to our knowledge, not been examined in patient populations. However, as AHN does take place in humans, and the biochemical pathways relevant to this study are well conserved, it is likely similar deficits would be observed in 15q11.2BP1-BP2 carriers.
The 15q11.2(BP1-BP2) deletion is not solely associated with schizophrenia, it also markedly increases risk for autism spectrum disorders 66 , ADHD and developmental delay 67 and epilepsy 68 . Whether altered microglia functioning due to Cyfip1 haploinsufficiency, impacting on AHN, also plays a part in these disorders is an open question, but all have been associated with alterations in immune system and microglia functioning 69,70 . Likewise, alterations in migration would have equally profound consequences in other disorders.
As noted previously, a number of other schizophrenia risk genes have also been shown to modify AHN in model systems. Whilst the effects are distinct from those of Cyfip1, each risk gene impacting on neurogenesis in its own way, this convergence of function may be indicative of a common pathogenic route for a number of diverse psychiatric risk genes.