Mutation of the Human Circadian Clock Gene CRY1 in Familial Delayed Sleep Phase Disorder

Highlights

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  A human subject with DSPD with a variation in CRY1 has altered circadian rhythms
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  Proband kindred and unrelated carrier families display aberrant sleep patterns
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  The allele alters circadian molecular rhythms
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  The genetic variation enhances CRY1 function as a transcriptional inhibitor

• Summary

  Patterns of daily human activity are controlled by an intrinsic circadian clock that promotes ∼24 hr rhythms in many behavioral and physiological processes. This system is altered in delayed sleep phase disorder (DSPD), a common form of insomnia in which sleep episodes are shifted to later times misaligned with the societal norm. Here, we report a hereditary form of DSPD associated with a dominant coding variation in the core circadian clock gene CRY1, which creates a transcriptional inhibitor with enhanced affinity for circadian activator proteins Clock and Bmal1. This gain-of-function CRY1 variant causes reduced expression of key transcriptional targets and lengthens the period of circadian molecular rhythms, providing a mechanistic link to DSPD symptoms. The allele has a frequency of up to 0.6%, and reverse phenotyping of unrelated families corroborates late and/or fragmented sleep patterns in carriers, suggesting that it affects sleep behavior in a sizeable portion of the human population.
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  Introduction

  The circadian clock is an internal self-sustained oscillator that operates in organisms’ tissues and cells to align recurrent daily changes in physiology and behavior with 24-hr environmental cycles. In humans, dysfunction or misalignment of the circadian clock with environmental cues alters the timing of the sleep-wake cycle, leading to a variety of circadian rhythm sleep disorders (American Academy of Sleep Medicine, 2005). Delayed sleep phase disorder (DSPD), which is characterized by a persistent and intractable delay of sleep onset and offset times relative to the societal norm, represents the most commonly diagnosed type of circadian rhythm sleep disorder, with an estimated prevalence of 0.2%–10% in the general population (Zee et al., 2013). The wide range of prevalence estimates reflects heterogeneity in the manifestation of the disorder as well as variation in the stringency with which clinical diagnosis criteria are applied (Sack et al., 2007, Weitzman et al., 1981). The pathophysiology of DSPD remains obscure, with suspected causes including a differential susceptibility of an individual’s circadian clock to environmental entrainment cues such as the light/dark cycle and altered properties of the oscillator itself that affect its period length (Aoki et al., 2001, Campbell and Murphy, 2007, Chang et al., 2009, Duffy et al., 2001, Micic et al., 2013).

  The circadian clock is genetically encoded and susceptible to modification by spontaneous or targeted mutation of the respective factors in animal models (Crane and Young, 2014, Lowrey and Takahashi, 2011). In humans, rare genetic variations that shorten circadian period are linked to familial advanced sleep phase disorder (FASPD), a type of circadian rhythm sleep disorder with habitual sleep times earlier than the societal norm (Hirano et al., 2016, Toh et al., 2001, Xu et al., 2005, Xu et al., 2007). No comparable evidence has yet emerged for DSPD and the association of proposed genetic polymorphisms with late chronotype, and DSPD has remained controversial (Kripke et al., 2014). Yet, many classical twin studies have found a strong hereditary component to chronotype preference in the range of 40%–50%, arguing for an important role of genetic predisposition to DSPD etiology (Barclay et al., 2010, Hur et al., 1998, Koskenvuo et al., 2007, Vink et al., 2001). Here, we report a case of familial DSPD linked to a dominant coding variation in cryptochrome circadian clock 1 (CRY1). This association is maintained in unrelated carrier families of the CRY1 variant. The studied allele encodes a CRY1 protein with an internal deletion, affecting its function as a transcriptional inhibitor and causing lengthening of the circadian period.

  Results

  
  

  Characterization of Intrinsic Circadian Rhythmicity in the DSPD Proband

  The clinical diagnosis of DSPD in the proband, subject “TAU11” (female, aged 46), was based on a sleep history and diagnostic interview, chronotype questionnaires, and actigraphy combined with a sleep log (Figure 1A). To better characterize the intrinsic circadian behavior, the subject completed an in-laboratory study during which sleep and core body temperature were continuously monitored (Figure 1B). The protocol consisted of a 2-day entrainment period with habitual sleep times derived from the sleep log. Entrained phase was determined by salivary dim light melatonin onset (DLMO) on the second entrainment night. This was followed by 2 days of enforced time in bed from 23:00 to 7:00. At the end of the 4-day entrainment interval, the subject entered a 14-day period of time isolation during which sleep was permitted whenever so inclined (free-run).

  Compared to a control subject of normal chronotype undergoing the same protocol, several circadian abnormalities were apparent in the proband: consistent with a phase delay, entrained DLMO occurred at 2:32, well after the time expected in a subject of normal chronotype (typically between 20:00 and 22:00) and closer to the time of habitual sleep onset (Figure 1C) (Chang et al., 2009, Molina and Burgess, 2011). Sleep during the free-run was highly variable both in the timing and the duration of major sleep periods, consistent with at-home actigraphy and sleep-log records (Figures 1A and 1C). The resulting gross sleep/wake rhythm had a period of 24.5 hr with noticeably dampened amplitude (Figure 1D). By contrast, the 24.2-hr period length of a control subject undergoing the same protocol matches the intrinsic period length reported for normal human subjects (Czeisler et al., 1999). Aberrant rhythmicity in the sleep behavior of TAU11 was mirrored by the pattern of core body temperature oscillations in which a long-period rhythm of 24.8 hr and diminished amplitude were even more pronounced (Figures 2A–2C and S1). The phenotypic concordance of the different circadian measures strongly argues for the presence of an intrinsic circadian rhythm disorder in the proband.

  Identification of CRY1 c.1657+3A>C as a Candidate DSPD Allele

  To identify the cause of circadian dysfunction in the proband, we performed candidate sequencing of genes that form the circadian clock in mammals. The core molecular clock consists of a negative-feedback loop in which the activity of the transcription factors Clock and Bmal1 (called ARNTL in humans) is repressed by the products of its target genes of the Per and Cry family, creating a cycle that takes ∼24 hr to complete (Figure 3A). In this complex process also involving regulation of post-translational modification and nuclear translocation, Cry1 is commonly recognized as the main transcriptional repressor of Clock and Bmal1 (Anand et al., 2013, Griffin et al., 1999, Kume et al., 1999, Oster et al., 2002, van der Horst et al., 1999, Vitaterna et al., 1999, Ye et al., 2014). By contrast, the mechanism of action of the Per proteins appears to be more variable, ranging from indirect repression through recruitment of generic chromatin modifiers to in fact promoting transcriptional de-repression (Chiou et al., 2016, Duong et al., 2011, Duong and Weitz, 2014). Our candidate gene sequencing identified an adenine-to-cytosine transversion within the 5′ splice site following exon 11 in one allele of the proband’s CRY1 gene (Figures 3B and 3C). Given usual conservation of the +3 position as a purine, this change is expected to cause splice site disruption and exon skipping (King et al., 1997). To test for a resulting coding change, we amplified part of the CRY1 cDNA encompassing exon 11 from a primary dermal fibroblast cell line derived from the proband. Indeed, an additional product corresponding to the expected Δ11 size was present in the proband’s sample, but not in those derived from 18 other unrelated subjects (Figure 3D). With a size of 72 base pairs, exon 11 skipping is predicted to cause an in-frame deletion of 24 residues in the C-terminal region of the CRY1 protein, and a matching, higher-mobility band was specifically detected in protein extracts from the proband cell line (Figure 3E).

  Given the prominent role of CRY1 in the mammalian clock, we postulated that the circadian abnormalities in the proband were related to the observed modification of CRY1. To test this hypothesis, we obtained information on sleep patterns from members of the proband’s family and genotyped them for presence or absence of the candidate allele. Delayed sleep behavior was found to be common among male and female family members and across several generations, consistent with an autosomal-dominant inheritance pattern (Figures 4A and S2; Table S1). Presence of the CRY1 c.1657+3A>C allele segregated with delayed sleep timing, with the exception of one carrier (TAUX08), who reported a history of persistent sleep problems but was complaint free at the time of study, on an occupationally required very early routine that was purposely maintained on free days (see Table S1 for details).

  In a complementary approach, we also performed an unbiased search for genetic variants co-segregating with aberrant sleep behavior in the proband kindred through whole exome sequencing of additional family members (three affected, one unaffected). Among variants with minor allele frequencies below 1%, which are common to all affected subjects, but not the unaffected, and which are predicted to affect protein coding, the candidate CRY1 allele was the only variant affecting a gene with a known or implicated role in the regulation of sleep or circadian rhythmicity (Table S2). Also, although some additional more common clock-gene variants were also present in the original proband TAU11, none of these segregated with sleep behavior in the family (see Methods Details). These results point to the CRY1 c.1657+3A>C allele as a strong candidate-genetic variant for familial DSPD.

  Reverse Phenotyping of Sleep Behavior in Heterozygous and Homozygous Carriers of the CRY1 c.1657+3A>C Allele from an Unrelated Population

  In databases of human genetic variation, the candidate CRY1 allele has a frequency of up to 0.6% (rs184039278: minor allele frequency 0.0012 in 1000 Genomes, 0.004335 in ExAC total with 0.006537 in non-Finnish Europeans). This frequency lies within the reported range of DSPD prevalence (Zee et al., 2013) and is high enough to attempt the identification of additional carriers consenting to a characterization of their sleep behavior through a reverse-phenotyping approach (zçelik and Onat, 201). In genomic databases of the Turkish population, we identified 28 carriers of the CRY1 c.1657+3A>C allele, including one homozygous individual. Of these, investigation of sleep behavior through questionnaires and personal interview was possible in six unrelated families (DSPD-1, -2, -4, -6, -7, -9, and -14) totaling 70 subjects (8 homozygous carriers, 31 heterozygous carriers, 31 non-carriers) (Figure 5 and Table S1). Subjects also provided a DNA sample to determine the CRY1 allele status. Aberrant sleep behavior was reported by 38 carriers, but not by their non-carrier relatives or spouses, indicating a very high penetrance of CRY1-related sleep disturbance consistent with the original proband family. In addition to late sleep times, a subset of carriers reported a pattern of fragmented sleep consisting of a brief sleep period early in the night and extended naps during the day. Fragmented sleep was particularly prevalent among those carriers for whom early rising was a necessity due to cultural or social obligations. Of note, no difference in sleep behavior was observed between heterozygous and homozygous carriers of the CRY1 allele, consistent with an autosomal-dominant mode of inheritance. The one carrier with reported conventional sleep times (DSPD-6 16-068) was subject to work-imposed strong light exposure, raising the possibility that the CRY1-mediated disposition can be modifiable given adequate environmental conditions. Nevertheless, there was a very strong association between CRY1 allele status and sleep behavior in the reverse-phenotyped families and the original proband kindred (Fisher’s exact p < 0.0001, odds ratio = 1,928, 95% confidence interval 76–48,904).

  CRY1 Exon 11 Deletion Affects Circadian Clock Cycling and CRY1 Molecular Function

  To directly test whether the deletion of exon 11 of CRY1 affects the circadian clock, we created cell lines differing only in the expressed CRY1 form. Human full-length or CRY1 Δ11 variants were expressed in CRY1/2 double-deficient mouse embryonic fibroblasts (DKO MEFs) using regulatory elements previously characterized to recapitulate endogenous CRY1 oscillation (Ukai-Tadenuma et al., 2011). As expected, CRY1 expression restored circadian cycling of a Bmal-luciferase reporter in previously arrhythmic DKO MEFs, albeit with a long period, as previously described for this experimental system (Khan et al., 2012) (Figure 4B). Compared to full-length CRY1, expression of the Δ11 form increased circadian period by approximately half an hour, similar to the phenotype observed in the proband. The effect was not due to differences in the amounts of the ectopically expressed CRY1 forms (Figure 4B). In contrast to CRY1, expression of CRY2 in CRY DKO MEFs did not restore their circadian rhythmicity, consistent with previous reports (Khan et al., 2012), and the differential period length between the two CRY1 forms was still observed in its presence (Figure S3A). These results demonstrate a direct effect of CRY1 exon 11 deletion on circadian period length, which matches DSPD symptoms.

  The Cry1 protein consists of a conserved photolyase homology region, which mediates transcriptional repression of Clock/Bmal1, a C-terminal helix previously described as a predicted coiled coil, which interacts with Per2 and Fbxl3 in a mutually exclusive manner and a C-terminal extension also referred to as the “tail” (Figures 3B and S3B) (Chaves et al., 2011, Merbitz-Zahradnik and Wolf, 2015). The Cry1 tail region represents the most poorly conserved and least functionally and structurally characterized region of the protein. It has been shown to affect Cry1 nuclear translocation, to interact with the Bmal1 transactivation domain possibly in an acetylation-dependent fashion, and to be phosphorylated in a manner that involves regulation by DNA-PK (Chaves et al., 2006, Czarna et al., 2011, Gao et al., 2013, Hirayama et al., 2007, Xu et al., 2015). Interestingly, the tail is not essential to Cry1’s ability to restore circadian cycling to arrhythmic DKO MEFs but does modulate the period length and amplitude of the resulting oscillation (Khan et al., 2012, Li et al., 2016). Overall, current evidence points to a regulatory role of the Cry1 tail in the transcriptional repression complex involving Clock, Bmal1, and possibly other factors at various stages of the circadian cycle. Deletion of exon 11 results in the removal of 24 residues from the CRY1 C-terminal tail. In accordance with previous functional characterizations of the Cry1 protein regions, we did not observe a difference in the capacity of CRY1 Δ11 to inhibit Clock/Bmal1-dependent transcription of an E-box-driven luciferase reporter plasmid in heterologous cell-based assays, which do not require the Cry1 tail (Chaves et al., 2006, Khan et al., 2012) (Figures S3C and S3D). Further, although some modifications within the tail region can affect the half-life of the Cry1 protein under certain conditions (Gao et al., 2013), we did not observe gross differences in the stability of CRY1 Δ11 versus the full-length form in the subject’s primary fibroblasts (Figure S3E), and luciferase fusion proteins with the respective CRY1 forms decayed at a similar rate (Figure S3F).

  The existence of a nuclear localization signal in the Cry1 tail, albeit C-terminal to the exon 11 region, prompted us to assess the subcellular distribution of the different CRY1 forms. Unexpectedly, deletion of exon 11 increased CRY1 abundance in the nuclear fraction of the proband’s fibroblasts throughout the circadian cycle (Figures 6A and S4A). This increased abundance was not caused by potential additional variations in the proband’s cells but represents an intrinsic property of the modified CRY1 protein, as enhanced CRY1 Δ11 nuclear localization was also observed in DKO MEFs engineered to express both CRY1 forms (CRY1 fl/Δ MEFs) (Figures 6B and S4B).

  CRY1 Δ11 Shows Enhanced Interactions with Clock and Bmal1 Proteins

  Preferential nuclear localization of CRY1 Δ11 led us to assess its binding to its target transcription factors Clock and Bmal1. Although both CRY1 forms present in the subject’s fibroblasts were found to be capable of interaction, the fraction of CRY1 immunoprecipitating with ARNTL or Clock was enriched for the Δ11 form (Figures 6C and S4C). This is not solely a reflection of differential subcellular distribution as ARNTL or Clock immunoprecipitated from purified nuclear extracts still bound more Δ11 than full-length CRY1. Enhanced interaction with the CRY1 Δ11 form was replicated in CRY1 fl/Δ MEFs independent of circadian phase (Figures 6D and S4D). Interestingly, although exon 11 partially overlaps with a region in the Cry1 tail that has been identified as a binding site for the Bmal1 transactivation domain acetylated at lysine 538, we still observed preferential binding of CRY1 Δ11 to acetylated Bmal1. We also consistently detected higher overall levels of acetyl-Bmal1 in control DKO MEFs, which only received empty vector and remained devoid of cryptochromes, potentially indicating a more complex role of Bmal1 acetylation than currently suggested. Selective expression of either the full-length or the CRY1 Δ11 form in DKO MEFs allowed us to assess CRY1 binding to its interaction partners in reciprocal immunoprecipitations of the respective CRY1 form. Consistent with our other findings, more Clock, acetyl-Bmal1, and total Bmal1 immunoprecipitated with CRY1 Δ11 than with the full-length protein (Figures 6E and S4E). At the same time, the levels of CRY1-associated Per2 remained similar between the two CRY1 forms, suggesting the presence of separate CRY1-containing protein complexes with differential susceptibility to exon 11 deletion. Together, these results demonstrate that, rather than disabling CRY1, deletion of exon 11 enhances its presence in the nucleus and the binding to its target transcription factors, properties that are expected to promote its function as a transcriptional inhibitor.

  CRY1 Δ11 Strengthens Transcriptional Inhibition

  To directly test whether CRY1 Δ11 acts as a more potent transcriptional inhibitor during the intact clock cycle, we compared the expression of selected target genes in our engineered cell lines expressing either full length or CRY1 Δ11. As expected, cyclic CRY1 expression restored the circadian oscillation of pre-Bmal1, pre-Per2, pre-Per1, and pre-Dbp mRNAs with a long-period rhythm, although the sampling interval impeded an accurate determination of period length, as previously achieved by the high-resolution luciferase assay (Figure 6F). Compared to CRY1 full-length cells, the levels of pre-Per2, pre-Per1, and pre-Dbp mRNAs were reduced in CRY1 Δ11 cells, demonstrating stronger repression of Clock/Bmal1-mediated transcription by CRY1 Δ11 consistent with its other properties. In contrast, expression of pre-Bmal1, which is controlled by a different set of regulatory elements (Preitner et al., 2002, Ueda et al., 2002), remained unaffected by the CRY1 modification, as did the levels of a non-circadian control gene.

  Given enhanced association with the target transcription factors as well as reduced expression of the relevant transcripts, we wondered whether exon 11 deletion affected CRY1 occupancy at its target gene promoters. In the circadian transcriptional feedback loop, repression can occur by blocking of the DNA-bound transcription factors or by their displacement and sequestration away from DNA (Menet et al., 2010). Cry1-dependent inhibition of gene expression has been shown to involve both of these modes (Ye et al., 2014). Using our cell lines engineered to selectively express full length or CRY1 Δ11, we measured the binding of CRY1, Bmal1, and Clock to target regions in the Per2 and Dbp promoters by chromatin immunoprecipitation (Figures 7A–B). At the time of high Bmal1/low Per2 expression, reduced promoter association of CRY1, Bmal1, and Clock was observed in cells expressing CRY1 Δ11 compared to the full-length form, while the association of the control histone 3 trimethylated at lysine 4 (H3K4me3) remained unaltered. As expected, in control reactions measuring a non-circadian promoter, only H3K4me3 binding was observed while the amounts of CRY1, Bmal1, and Clock were at or near the background levels of the assay (Figure 7C). These results demonstrate that CRY1 exon 11 deletion specifically reduces the presence of clock gene proteins at target gene promoters, consistent with Cry1-mediated transcriptional regulation through displacement of Clock and Bmal1.

  Discussion

  As the major transcriptional inhibitor in the negative feedback loop that constitutes the core molecular clock, Cry1 represents a critical regulator of circadian period length. In general, there is a positive correlation between the amount of Cry1 and period length, although exceptions to this rule can occur upon manipulation of selected protein regions (Busino et al., 2007, Godinho et al., 2007, Hirota et al., 2012, Ode et al., 2016, Oshima et al., 2015, Siepka et al., 2007, van der Horst et al., 1999, Vitaterna et al., 1999, Zhang et al., 2009). Moreover, period length has been shown to correlate with the affinity of Cry1 to Bmal1 (Xu et al., 2015).

  Our results show that the CRY1 DSPD allele represents a gain-of-function mutation with deletion of exon 11 leading to increased CRY1 nuclear localization, enhanced interaction with the transcription factors Clock and Bmal1, their displacement from chromatin, and heightened inhibition of their target genes (Figure S5). Expression of this more potent CRY1 form (CRY1 Δ11) is associated with a lengthened period of molecular circadian rhythms in cells. A human carrier of CRY1 Δ11 studied in temporal isolation displayed corresponding, long-period behavioral and body-temperature rhythms with diminished amplitudes. These phenotypic changes are consistent with the established positive correlation of period length with CRY1 availability and affinity to its target transcription factors, thus providing a mechanistic explanation for the development of DSPD in carriers of the CRY1 Δ11 allele.

  The stronger inhibitory function of the CRY1 Δ11 variant is only observed in the context of an intact clock cycle, raising interesting questions regarding the mechanism by which the CRY1 protein tail influences Clock/Bmal1 transcriptional activity. While currently available structural characterizations of the mammalian cryptochrome proteins have been insightful regarding their binding to Per2 and Fbxl3, the interaction with their target transcription factors has yet to be visualized, and none of the structures includes the Cry1 tail region (Merbitz-Zahradnik and Wolf, 2015). It is conceivable that the tail could affect transcription factor/repressor interaction through regulated binding to the CRY1 photolyase homology region or Clock/Bmal1, causing conformational changes to the complex. Such an event could be temporally controlled through recruitment or loss of additional complex components, through inducible post-translational modification of any of the proteins, or through changes to the CRY1 protein such as its redox state or the presence of cofactors, including flavin adenine dinucleotide or zinc ions. While dispensable for basic repression, the CRY1 tail could thus exert the capacity to modulate transcriptional inhibition at defined stages of the circadian cycle.

  In our analyses of cellular circadian rhythms, the CRY1 Δ11 allele consistently lengthened the period of molecular oscillations by approximately half an hour. Earlier work has demonstrated a strong relationship between circadian period, entrained phase, and sleep timing in humans, such that moderate changes in period are associated with much larger shifts in the relative phases of bedtime and the evening increase in serum melatonin (Gronfier et al., 2007, Wright et al., 2005). Accordingly, a half-hour change in the period of the human circadian clock is expected to change the relationship of sleep timing and evening melatonin onset (DLMO) by ∼2–2.5 hr. These predictions agree well with the behavioral and physiological findings we have presented.

  Databases of human genetic variation report a frequency between 0.1% and 0.6% for the CRY1 c.1657+3A>C allele, such that up to 1 in 75 members of certain populations could carry the dominant CRY1 variant. Our analyses of the original proband family as well as a large number of subjects from unrelated families of completely different ethnicity show that both homo- and heterozygous CRY1 c.1657+3A>C carrier status is strongly associated with late sleep times and sleep fragmentation. Possibly, the latter behavior may be a manifestation of carrier allele status under environmental conditions that do not accommodate late sleep times, as can often be the case due to cultural, social, or professional obligations. Alternatively, inter-individual differences in genetic background or exposure to environmental entrainment signals may affect the nature and penetrance of sleep disturbances in CRY1 Δ11 allele carriers, and similar phenomena have been observed in both human and animal studies of circadian rhythmicity (Azzi et al., 2014, Pittendrigh and Daan, 1976, Shimomura et al., 2013, Toh et al., 2001). The CRY1 Δ11 variant may thus lead to a broader range of sleep-disorder phenotypes with delay being the most common manifestation.

  Author Contributions

  A.P., P.J.M., S.S.C., and M.W.Y. conceived of the project. P.J.M. and S.S.C. designed experiments and collected data for Figures 1, 2, 4A, S1, and S2. A.P. and M.W.Y. analyzed data for Figures 1, 2, 4A, S1, and S2 with input from P.J.M., A.C.K. and S.S.C. A.P. designed and performed experiments in Figures 3, 4B, 6, 7, S3, and S4. O.E.O. and T.Ö. collected data for Figure 5. A.P., O.E.O., T.Ö., and M.W.Y. analyzed data for Figure 5. A.P. prepared all figures and wrote the manuscript with input from all authors. A.P., T.Ö., S.S.C., and M.W.Y. secured funding.

  Acknowledgments

  We thank the human study participants; the technical staff of the Laboratory of Human Chronobiology; Adam Savitz for conducting physical exams; Mary Morton for obtaining skin biopsies; Melanie Roberts for recruiting and obtaining data from the proband’s family; Boris Dubrovsky for scoring polysomnography records; Nazlı Başak, Ali Dursun, Uğur Özbek, Köksal Özgül, and Bülent Yıldız for establishing initial contact to Turkish DSPD families; Hiroki Ueda, Steve Kay, and Steven Reppert for reagents; Avinash Abhyankar and the New York Genome Center for help with whole exome sequencing; Jeffrey Friedman for discussion and help with subject identification; Philip Kidd for help with processing of raw core body temperature data; the Friedman and Tarakhovsky laboratories for generously sharing equipment; and Cori Bargmann, Joseph Gleeson, André Hoelz, and Leslie Vosshall for comments on the manuscript. This work was supported by NIH grant RO1 NS052495 (S.S.C.), a sub-award #12081164 of NS052495 provided by Weill Cornell Medical College (M.W.Y.), Calico Life Sciences LLC (M.W.Y.), The Rockefeller University Center for Clinical and Translational Science grants UL1 TR000043 and UL1 TR001866 (A.P.), the Turkish Academy of Sciences-TÜBA (T.Ö.), The Rockefeller University Women & Science Postdoctoral Fellowship program (A.P.), and a NARSAD Young Investigator Grant #21131 from the Brain & Behavior Research Foundation (A.P.). P.J.M is currently employed by Eisai Inc.
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