What genetic mutation is associated with autism?
KATNAL2
Microtubule severing enzymes implement a diverse range of tissue-specific molecular functions throughout development and into adulthood. Although microtubule severing is fundamental to many dynamic neural processes, little is known regarding the role of the family member Katanin p60 subunit A-like 1, KATNAL1, in central nervous system CNS function. Recent studies reporting that microdeletions incorporating the KATNAL1 locus in humans result in intellectual disability and microcephaly suggest that KATNAL1 may play a prominent role in the CNS; however, such associations lack the functional data required to highlight potential mechanisms which link the gene to disease symptoms. Here we identify and characterise a mouse line carrying a loss of function allele in Katnal1. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. We show that mutants express behavioural deficits including in circadian rhythms, sleep, anxiety and learning/memory. Furthermore, in the brains of Katnal1 mutant mice we reveal numerous morphological abnormalities and defects in neuronal migration and morphology. Furthermore we demonstrate defects in the motile cilia of the ventricular ependymal cells of mutants, suggesting a role for Katnal1 in the development of ciliary function. We believe the data we present here are the first to associate KATNAL1 with such phenotypes, demonstrating that the protein plays keys roles in a number of processes integral to the development of neuronal function and behaviour. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. Text: Microtubule severing enzymes are a family of AAA-ATPase proteins that participate in fundamental cellular processes such as mitosis, ciliary biogenesis and growth cone motility. In neurons this family is known to control such processes as axonal elongation 1 and synaptic development. 2 In addition, mutations in microtubule severing enzyme genes SPG4, KATNB1 and KATNAL2 are associated with hereditary spastic paraplegia, cerebral malformations and autism, respectively,  and mutations in Fign cause a range of phenotypes in mice. 7 Currently the microtubule severing enzyme KATNAL1 is poorly characterised and it is not yet understood how the enzyme functions in the nervous system. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. Recent evidence from genetic characterisation of human patients suggests that haploinsufficiency of KATNAL1 is linked with a number of symptoms including intellectual disability ID and craniofacial dysmorphologies. 8, 9 It is also notable that a very rare KATNAL1 mutation has been associated with schizophrenia 10 book.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and that Peters syndrome and autism have both been associated with the chromosomal region containing the KATNAL1 locus. 11, 12 Although such association studies strongly suggest that KATNAL1 plays a fundamental role in the central nervous system CNS , additional studies using cellular or animals models are required to understand how the gene may be causative for disease. Here we present the first study describing neural and behavioural deficits associated with a loss of function allele of Katnal1 in the mouse. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. This mutant mouse line was independently identified in two parallel phenotyping screens, which demonstrated that mutant mice showed both male sterility and circadian phenotypes. Subsequent behavioural investigations demonstrated that this mutation is associated with anxiety and memory deficits. Underlying these behavioural phenotypes, we identified histopathological abnormalities in the brains of Katnal1 1H/1H mutants, including disordered cellular layers in the hippocampus and cortex and substantially larger ventricles. Further investigations demonstrated that Katnal1 1H/1H mice show neuronal migration and ciliary function deficits suggesting KATNAL1 plays an essential role in these processes. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. These findings are the first to our knowledge to conclusively show that mutations in Katnal1 lead to behavioural and neuronal disturbances and provide insight regarding the clinical associations that have been linked to the gene. performed on mouse cohorts that were partially or completely congenic on the C57BL/6 J background. Circadian wheel running was performed as previously described. 14 Sleep assessment by electroencephalography and electromyography Electroencephalography and electromyography was performed as previously described. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. 15 Behavioural phenotyping Spontaneous alternation. Mice were placed in a walled T-maze black polyvinyl chloride, lined with sawdust; stem = 88 × 13 cm; arms = 32 × 13 cm and allowed to enter a goal arm of their choice. The mouse was confined in the goal arm for 30 s, before being allowed a second free choice of goal arm. An alternation was recorded if the second choice differed from that of the first. One trial was performed per day for 10 days. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Open field behaviour. Mice were placed into a walled arena grey polyvinyl chloride; 45 × 45 cm and allowed to explore for 20 min. Animals were monitored by EthoVision XT analysis software Noldus, Wageningen, Netherlands . Video tracking in the home cage. Activity in the home cage was recorded by video tracking as previously described. 16 Morris water maze and ultrasonic vocalisation. These tests were performed as previously described. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. 17 Brain histology and immunofluorescence Brains were mounted in OCT VWR and 12 μm coronal sections taken. Sections were stained with hematoxylin and eosin, or immunolabelled following standard protocols. In vivo neuronal migration assessment was performed as previously described 18 using embryos at either E13 or E15 three mothers per age group and pups at P9. Cell counts were performed using ImageJ NIH, Bethesda, MD, USA . In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. In vitro neuronal migration assessment was performed using a Boyden chamber migration protocol as previously described. 19 Micro-computed tomography scanning Micro-computed tomography was performed using a Skyscan 1172 at 90 kV, 112 μA using an aluminium and copper filter, a rotation step of 0.250 degrees and a pixel size of 4.96 μm. Segmentation, volume calculation and 3D modelling was performed using ITK-SNAP version 3.0.0 ref. 20 and 3DSlicer. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. 21 Golgi-Cox staining of neurons Golgi-Cox neuronal staining was performed using the FD Rapid GolgiStain Kit FD NeuroTechnologies, Columbia, MD, USA . Neurons were analysed using ImageJ. Brains from P2 mice were dissected, and the dorsal cerebral half was sectioned 250 μm through the floor of the lateral and 3rd ventricle using a vibratome. Ciliary beat frequency and pattern was analysed as previously described. 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . 22 Electron microscopy For Scanning Electron Microscopy the ependymal lining of the lateral ventricle was fixed in 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, incubated in 2% osmium tetroxide, and dehydrated through ethanol solutions. Samples were critical point dried using an Emitech K850 Quorum Technologies, East Sussex, UK , coated with platinum using a Quorom Q150R S sputter coater Quorum Technologies . and visualised using a JEOL LSM-6010 scanning electron microscope Jeol, Herts, UK . Transmission electron microscopy was performed as previously described. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. 22 Statistical analysis Data was analysed using two-tailed students T test or AVOVA using SPSS IBM or GraphPad Prism 5.0 GraphPad Software, La Jolla, CA, USA . Significance level for all analysis was set at Po 0.05. All graphs are presented showing mean ± s.e.m. Additional and more detailed methods can be found in supplementary information. Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . Identification and cloning of the Katnal1 1H mutation To identify novel gene mutations affecting circadian behaviour we undertook a circadian running wheel screen of pedigrees of N-ethyl-N-nitrosourea mutagenised mice. 13 In one pedigree 17.65% of animals showed a short circadian period in constant darkness o 23 h observed in 12 out of 68 animals screened . An outcross using an affected female produced no affected animals 33 animals screened . In subsequent intercross screens 15.5% of animals were affected 53 out of 342 animals screened , suggesting that the pedigree carries a mutation causing a recessive circadian phenotype which is 60% penetrant. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. We found no gender bias in affected animals proportion of affected animals: male = 47.2%; female = 52.8% . Concurrently a male sterility phenotype was identified within the same pedigree. 23 Genome-wide SNP linkage analysis mapped the circadian and sterility phenotypes to the same region on chromosome 5 and subsequent sequencing identified the causative mutation as a T to G single point mutation within exon seven of the Katnal1 gene. For full details of mapping and identification of the mutation see reference 23. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. This mutant allele was designated Katnal1 1H , and results in a leucine to valine substitution at residue 286 of the protein. In vitro functional analysis demonstrated that the mutation is a recessive loss-offunction allele. 23 3D modelling of the protein suggests that this loss of function is due to hydrophobic changes in the AAA domain of the enzyme Supplementary Figure S1 . Genotyping confirmed that the mutation was homozygous in affected circadian animals and wild type or heterozygous in unaffected animals, confirming that Katnal1 1H was causative for the circadian phenotype. Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Circadian and sleep anomalies in Katnal1 1H/1H mice More extensive circadian phenotyping conducted on Katnal1 homozygotes Katnal1 1H/1H and wild-type littermates Katnal1 +/+ confirmed that Katnal1 1H/1H mice had a shorter free-running circadian period Figures 1a-c and furthermore revealed that Katnal1 1H/1H animals were more active in the light phase of the light/dark cycle Figure 1d , showed increased anticipation of light to dark transitions and greater shift in activity onset when released from light/dark cycles to constant darkness Figure 1e . Data and cohort details are given in Supplementary Table S1 . Bioluminescence recordings performed using PER2::LUCIFERASE reporter mice carrying the Katnal1 1H mutation revealed that these circadian changes were not due to changes to the core molecular clock of the suprachiasmatic nucleus the site of the master circadian clock in the brain; Supplementary Figure S2 . Circadian disruptions are often associated with deficits in sleep homeostasis. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Therefore to complement our circadian studies we conducted wireless electroencephalography recordings over a baseline period of 24 h and following a 6 h period of sleep deprivation. A detailed summary of electroencephalography analysis is given in Supplementary Table S1. Compared to wildtype littermates, the non-REM delta power of Katnal1 1H/1H mice was higher in the dark phase of baseline sleep mixed ANOVA, interaction factors 'genotype X time, F 1,88 = 8.91, P = 0.0175 Figure 1f and in both the light and dark phases of recovery sleep mixed ANOVA, interaction factors 'genotype X time', F 1,136 = 11.93, P = 0.0086; Figure 1g . All other sleep parameters were unaffected in Katnal1 1H/1H animals. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Katnal1 1H/1H mice display a spectrum of behavioural deficits Human patients carrying a heterozygous deletion incorporating the Katnal1 locus show a number of cognitive deficits including ID and a delay in language acquisition. 8, 9 We therefore investigated whether these deficits were modelled in Katnal1 1H/1H mice by subjecting animal cohorts to a battery of behavioural tests. Data and cohort details are given in Supplementary Table S2 . Both working memory and spatial memory were significantly poorer in Katnal1 1H/1H mice, as evidenced by reduced spontaneous alternations in a T-maze Figure 2a and in the Morris water maze where mutants take longer to find the platform in acquisition trials Figure 2b Compared to wild-type littermates, Katnal1 1H/1H animals have a shorter period c , are more active in the light phase of the light/dark cycle d and show an earlier onset of activity in light/dark transitions and in the transition from light/dark cycles to constant darkness e . In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. In EEG recordings during sleep, Katnal1 1H/1H mice show increased non-REM delta power in the dark phase of the light/dark cycle f and following sleep deprivation g . *P ⩽ 0.05; **P ⩽ 0.01; ***P ⩽ 0.001. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle. type = 164 ± 12 m, Katnal1 1H/1H = 243 ± 20 m, P = 0.02; distance travelled in periphery of open field: wild type = 4.3 ± 0.2 m, Katnal1 1H/1H = 6 ± 0.3 m, P = 0.004 . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . Conversely when mouse activity was recorded in the home cage, we found no difference between genotypes distance travelled over 24 h: wild type = 399 ± 77 m, Katnal1 1H/1H = 418 ± 41 m, P = 0.833 suggesting that the former activity differences were due to the novel environment of the open field rather than generalised hyperactivity in Katnal1 1H/1H animals. Finally, in certain conditions such as maternal separation mice emit ultrasonic vocalisations USVs . To test whether Katnal1 1H/1H animals vocalised differently to wild types, we separated pups at postnatal days 7-8 the age at which mice show peak of USV emission 24 and recorded their USVs. In these tests, compared to wild types, Katnal1 1H/1H pups produced fewer Figure 2g and shorter Figure 2h vocalisations, containing fewer phrases Figure 2i . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Gross brain morphological abnormalities in Katnal1 1H/1H mice Since we observed a number of behavioural phenotypes in Katnal1 1H/1H mice, we performed histological analysis to ascertain whether differences in brain histology underlied these behaviours. Data and cohort details are given in Supplementary Table S3 . Analysis of hematoxylin and eosin stained brain sections revealed that, compared to wildtype littermates, Katnal1 1H/1H animals had less tightly packed pyramidal cell layers in the hippocampus Figures 3a and b and a narrower cortical layer 1 and wider cortical layer 6 Figures 3c-e . To confirm these cortical layer differences, immunofluorescence was performed using the Figures 3l and m . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Quantification of fluorescence intensity demonstrated that in Katnal1 1H/1H cortex both calbindin and CUX1 labelling was more intense closer to the cortical surface, which is consistent with the reduction in the size of layer 1 two-way analysis of variance ANOVA , interaction factors 'genotype X distance of fluorescence from cortical surface', calbindin: F 75,988 = 16.8, P o 0.0005; CUX1: F 93,372 = 2.17, P = 0.001; Figures 3h and k . Similar quantification revealed that FOXP2 labelling extended further from layer 6b as labelled by CTGF in the Katnal1 1H/1H cortex, which is consistent with an increase in the size of layer 6 two-way ANOVA, interaction factors 'genotype X distance of fluorescence from CTGF labelling:' F 93,372 = 1.32, P = 0.038; Figure 3n . Finally, three dimensional models of the ventricular system were constructed from brain micro-computed tomography scans Figures 3o and p . Volumetric analysis revealed that Katnal1 1H/1H mice had substantially larger ventricles than wild types Figure 3q . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . Neuronal migration and morphology defects in Katnal1 1H/1H brains The histological phenotypes of Katnal1 1H/1H mouse brains described above are suggestive of neuronal migration defects. 18 We therefore investigated whether Katnal1 1H/1H mice showed abnormal neuronal migration using BrdU labelling of E13 and E15 embryos and quantified labelled cells in the cortex of P9 pups described in reference 18 . At both ages Katnal1 1H/1H animals had greater numbers of labelled neurons in bins close to the cortical surface neurons positioned closer to the cortical surface compared to wild type. To confirm these results we used a Boyden chamber 19 and performed in vitro neuronal migration analysis in E13.5 primary cortical neuronal cultures. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Here we found that a greater proportion of Katnal1 1H/1H cortical neurons migrated to the base of the cell culture insert compared to wildtype controls Supplementary Figure S3 . Since in both BrdU labelling and the Boyden assay neurons from Katnal1 1H/1H animals migrated further than those of wild-type littermates, these results suggest that Katnal1 1H/1H cortical neurons show defects in the termination of cortical neuronal migration. Given its role in cytoskeletal organisation, we also hypothesised that neuronal morphology is modulated by Katnal1. Analysis of golgi stained neurons from layers 2-3 of the cortex Figures 4g and i demonstrated that, compared to wild-type littermates, Katnal1 1H/1H neurons had larger soma Figure 4k , and shorter and thinner axons Figures 4l and m data and cohort details are given in Supplementary Table S3 . Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. Furthermore, analysis at higher magnification Figures 4h and j , demonstrated that the number of synaptic spines on Katnal1 1H/1H neurons was significantly reduced compared to wild type Figure 4n . Recent studies have demonstrated that mutations in some microtubule severing enzymes can cause defects in cilia. 5 Since such ciliary defects could underlie the phenotypes described above we studied the motile cilia of the ependymal lining of the lateral ventricle in sections of postnatal day 2 mouse brains from both Katnal1 1H/1H n = 4 and wild-type animals n = 3 . We found that the ciliary beat frequency CBF of Katnal1 1H/1H animals was significantly attenuated compared to wild-type CBF: wildtype = 22.39 ± 0.94 Hz, Katnal1 1H/1H = 14.25 ± 0.92 Hz, P = 0.0001; Figure 5a , Supplementary Movies S1 . This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. This reduction in CBF in Katnal1 1H/1H animals was also associated with an increased proportion of cilia with an abnormal beat pattern ciliary dyskinesia proportion of dyskinetic cilia: wild type = 17%, Katnal1 1H/1H = 75% Figure 5b and Supplementary Movies S1 . Visual inspection of the cilia identified a number of ciliary abnormalities such as a swollen ciliary tip Supplementary Movie S3 or extremely long cilia Supplementary Movie S4 scattered throughout the field of cilia in Katnal1 1H/1H ventricles. These abnormalities were observed in approximately 25% of Katnal1 1H/1H brain slices. The abnormal cilia always showed a dyskinetic beat pattern and lower beat frequency. To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . To further investigate ciliary morphology we performed scanning electron microscopy upon the ependymal lining of the lateral ventricles of both Katnal1 1H/1H n = 3 and wild-type animals n = 3; Figures 5c and d . Cilia measurements showed no significant differences in average cilia length between genotypes average cilia length: wild type = 6.22 ± 0.86 μm, Katnal1 1H/1H = 6.54 ± 0.94 Hz, P = 0.303 . However in Katnal1 1H/1H samples we noted the presence of both long and short cilia Figures 5e and f ; defined as two standard deviations longer or shorter than the average cilia length that were not present in wild-type samples. In addition, inspection of Katnal1 1H/1H cilia identified ciliary abnormalities including bifurcated cilia Figure 5g , abnormal kinks and bends in the cilia Figure 5h and swellings along the length of the cilia Figure 5i . Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Transmission electron microscopy of ependymal cilia found that vesicular aggre- Katnal1 disruption affects CNS functions G Banks et al gates were present within the ciliary swellings described above Figure 5j . Although these abnormalities were present in only a small proportion o1% of Katnal1 1H/1H cilia, they were notably absent from wild-type cilia. Microtubule severing enzymes play diverse roles in the nervous system. 1, 2 However, at present the microtubule severing enzyme Katnal1 is poorly defined in the context of CNS development and function. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Here we present a detailed phenotypic analysis of Katnal1 1H and show that the mutation is associated with changes in circadian rhythms, sleep and behaviour. Furthermore we demonstrate that defects in brain histopathology, neuronal migration and neuronal morphology underlie these phenotypes. Finally we also demonstrate that Katnal1 1H causes a range of defects in the motile cilia of ventricular ependymal cells. The data we present here are the first to associate KATNAL1 with such dysfunctions with important implications for clinical association studies. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. The Katnal1 1H mutation was initially identified with a circadian deficit including a short free-running period and advanced activity onset. However subsequent ex vivo experiments using SCN slices of animals carrying the PER2::LUC reporter gene demonstrated no defects in SCN cellular rhythms, suggesting that the core circadian clock was unperturbed by the mutation. Phenotypes in circadian running wheel rhythms that are not associated with changes to the core clock mechanism have also been reported in mouse models of schizophrenia. 25 Here it has been suggested that the wheel running changes observed are the result in defects in output pathways from the SCN circadian clock. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. Similarly, in Katnal1 1H/1H mice we hypothesise that the defects we demonstrate in neuronal anatomy and neuronal morphology may disrupt output signals from the SCN. Alternatively given that various neuropeptides such as oxytocin are secreted in a circadian manner from ependymal cells lining the third ventricle of the brain, 26 altered ventricular morphology and ciliary function in Katnal1 1H/1H mice may disrupt the circulation of factors secreted by the ciliated ventricular ependymal cells and contribute to the disruption of the behavioural rhythms observed. The behavioural consequences of microtubule severing enzyme dysfunction in mouse models have been poorly characterised. Currently the phenotypes described are limited to motor dysfunction in mice lacking the Spg4 gene 27 and head shaking and circling in the Fign mutant. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 7, 28, 29 In contrast, here we demonstrate that loss of function of Katnal1 is associated with a range of behavioural phenotypes, including changes in circadian activity, poor learning and memory, hyperactivity in a novel environment the open field and deficits in USVs. Notably the learning and memory, anxiety and vocalisation phenotypes reprise the clinical symptoms of ID, increased anxiety in novel situations and delays in language acquisition reported in human patients who carry microdeletions incorporating haploinsufficiency of KATNAL1. 8, 9 While it is also worth noting that mutant mice spend more time the centre of the open field than wild types implying that Katnal1 1H/1H animals show reduced anxiety , we suggest that this result is confounded by the hyperactivity in novel environments phenotype we also describe in mutant mice. This observation is backed up by the fact that mutant animals showed increased activity in all regions of the open field rather than just the anxiolytic periphery. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. Here we also highlight defects in Katnal1 1H/1H mice such as compromised neuronal migration and morphology which may underpin such phenotypes. In Drosophila, the homologue of Katnal1 kat-60L1 has been demonstrated to play a critical role in neuronal morphology during development, 30 however the data that we present here is the first to demonstrate a similar phenotype in mammals and furthermore suggests how subtle perturbations to KATNAL1 function may contribute to specific neural and behavioural conditions. For example, defects in neuronal migration, synaptic spines and neuronal morphology such as those we have demonstrated here, have been suggested to underpin ID in conditions such as lissencephaly, 18 Down's syndrome 31 and Rett syndrome. 32 While we are not suggesting that Katnal1 is causative for these conditions, similarities in symptoms and neuronal phenotypes between these conditions and those linked to Katnal1 dysfunction should be appreciated. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Furthermore a rare mutation in KATNAL1 has been associated with schizophrenia 10 book/genebook.cgi?user = guest&cmd = verb-gene&tbox = KATNAL1 and KATNAL1 has been shown to interact with the schizophrenia associated gene DISC1. 33 In line with these observations we note that increases in ventricular volume and reductions in synaptic spines have been reported in schizophrenic patients 34, 35 and our data demonstrates the same phenotypes in Katnal1 1H/1H mice. Thus the range of phenotypes associated with defects in the function of Katnal1 strongly suggests that the gene should be considered in the pathology of disorders such as ID and schizophrenia. We do note one key genetic difference between the human patients and Katnal1 1H/1H animals. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. While the human patients were all heterozygous for the Katnal1 deletion, we found no phenotype in heterozygous mutant mice data not shown suggesting that while haploinsufficiency is causative for phenotypes in humans, mice require complete loss of KATNAL1 function to show similar effects. A similar discrepancy between humans and mice has also been noted for the intellectual disability candidate gene CTNNB1. 17 While heterozygous loss of function mutations in CTNNB1 are causative for intellectual disability in humans, conditional knock outs for CTNNB1 have no reported behavioural or craniofacial phenotypes. 36, 37 These differences demonstrate that while mouse models of intellectual disability are of great use in our understanding of the causative mechanisms which underlie the condition, there are still genetic and neurodevelopmental differences between species which also must be taken into account. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. We also note that while the Katnal1 1H mutation shows a loss of catalytic function in both HEK293 cells and Sertoli cells, 23 this loss of function has not been verified in neuronal cells. However, given that our data demonstrates that the Katnal1 1H mutation lies in an essential catalytic domain and that we show neuronal phenotypes in Katnal1 1H/1H mice, we would expect to see the same loss of catalytic function in neurons. The data we present here also demonstrate defects in motile cilia in Katnal1 1H/1H mice. Ciliary disruptions in humans ciliopathies include Bardet-Biedl and Joubert syndrome. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. 38 While there is currently limited data available regarding the behavioural phenotypes of mouse models of ciliopathies, we note that ciliary dysfunction in mice has been linked with learning and memory 39 and vocalisation phenotypes, 40 both of which were disturbed in the Katnal1 1H/1H mice described here. It is also notable that the neuronal migration and enlarged ventricle phenotypes that we describe in Katnal1 1H/1H mice recapitulate features associated with known ciliopathy gene mutations. Furthermore in Bardet-Biedl syndrome mouse models ciliary defects such as reduced CBF 45 and structural defects such as abnormal lengthening and swellings along their length 41 have been described, that are similar to those we describe in Katnal1 1H/1H mice. There is strong evidence that ciliopathy associated genes play a number of roles in neuronal development by affecting processes such as progenitor proliferation or maintenance of the radial glia scaffold. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. 43 However it is also clear that defects in microtubule organisation also affect synaptic structure. 2 At present it is difficult to disentangle the relative contributions of defects in microtubule severing and ciliary abnormalities to the overall phenotypes we observe in Katnal1 1H/1H mice. Further investigations are required to clarify the impacts of these two processes. However it is notable that while defects in cilia structure may contribute to the phenotypes we describe in Katnal1 1H/1H mice, they are far less prominent in Katnal1 1H/1H mice than in other mouse ciliopathy models, 41 suggesting that the ciliary component of KATNAL1 dysfunction may be mild compared to other ciliopathies. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. Similarly while hydrocephalus has been suggested to be a component of some ciliopathy mouse models, 46 Katnal1 1H/1H mice showed only increased ventricle size rather than an increased incidence of hydrocephalus, further suggesting the ciliary defects in these animals are mild compared to other ciliopathies. In summary the data presented here clearly demonstrate that KATNAL1 plays an important role in a variety of neuronal processes including neuronal migration, neuronal morphology and ependymal ciliary function. The downstream effect of these defects leads in turn to a number of behavioural changes including in learning and memory, reaction to anxiogenic situations and circadian rhythms. These data therefore highlight how perturbations in KATNAL1 may play a role in neuronal dysfunction and demonstrates that the enzyme is a novel candidate in the study of behavioural and neurodevelopmental disorders. The authors declare no conflict of interest.