A missense mutation in Katnal1 underlies behavioural, neurological and ciliary anomalies

Abstract

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. 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.

Figure 1

Circadian and sleep anomalies in Katnal1^1H/1H mice. (a and b): Double plotted actograms from wild-type (a) and Katnal1^1H/1H (b) animals. Wheel running activity is represented by vertical black bars, with each horizontal row representing two days of time; shaded regions show time spent in darkness, unshaded regions show time spent in light (see reference for descriptions of double plotted actograms). 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. EEG, electroencephalography; DD, constant darkness; LD, light/dark cycle.

Figure 2

Katnal1^1H/1H mice display a spectrum of abnormal behaviours. Compared to wild-type littermates, Katnal1^1H/1H mice show: reduced spontaneous alternations in a T-maze (a); an increased latency to find the platform in Morris water maze trials (b); reduced improvement to find the platform in the Morris water maze (c); reduced time in the correct quadrant of the Morris water maze (d); increased time in the centre of an open field (e); greater movement in an open field (f); fewer USVs (g); shorter USVs (h); fewer phrases in their USVs (i). *P⩽0.05; **P⩽0.01; ***P⩽0.001. USV, ultrasonic vocalisation.

Figure 3

Aberrant brain histology in Katnal1^1H/1H mice. (a,b): Hippocampal histology in wild-type (a) and Katnal1^1H/1H (b) animals. Inserts (i) show CA1 layer. (c,d): Cortical layers in wild-type (c) and Katnal1^1H/1H (d) animals. (e: Katnal1^1H/1H animals have a narrower cortical layer 1 and a wider layer 6, compared to wild types. (f to n) Immunofluorescence of cortical layers: Calbindin immunofluorescence in cortical sections of wild-type (f) and Katnal1^1H/1H (g) animals. (h): Quantification of calbindin immunofluorescence demonstrates that Katnal1^1H/1H animals have a higher proportion of labelling towards the cortical surface than wild types. CUX1 immunofluorescence in cortical sections of wild-type (i) and Katnal1^1H/1H (j) animals. (k): Quantification of CUX1 immunofluorescence demonstrates that Katnal1^1H/1H animals have a higher proportion of labelling towards the cortical surface than wild types. FOXP2 (green) and CTGF (red) immunofluorescence in cortical sections of wild-type (l) and Katnal1^1H/1H (m) animals. (n): Quantification of FOXP2 immunofluorescence demonstrates that Katnal1^1H/1H animals have a higher proportion of labelling distant from layer 6b than wild-types. (o,p): μCT scans of the ventricular system (yellow) in wildwild-type (o) and Katnal1^1H/1H (p) brains. (q): Quantification of ventricular volume demonstrates that Katnal1^1H/1H mice have larger ventricles than wild types. Scale bars: 500 μm in a and b; 100 μm in d–g. ***P⩽0.001.

Figure 4

Neuronal migration and morphology abnormalities in Katnal1^1H/1H mice. (a,b,d,e): BrdU labelling of cortical neurons following injection of BrdU at either E13 (a,b) or E15 (d,e) in wild-type (a,d) and Katnal1^1H/1H (b,e) animals. Quantification of BrdU immunohistochemistry shows that Katnal1^1H/1H animals have increased numbers of labelled neurons closer to the cortical surface following injection at both E13 (c) and E15 (f). (g–j): Golgi labelling of neurons and dendrites in wild-type (g,h) and Katnal1^1H/1H (i,j) animals. (k–n): Quantification of golgi labelling shows that Katnal1^1H/1H animals have larger soma (k), shorter axons (l), thinner axons (m) and fewer dendritic spines (n) than wild types. **P⩽0.01; ***P⩽0.001.

Figure 5

Ciliary dysfunction in Katnal1^1H/1H mice. (a,b): Ependymal motile cilia in the lateral ventricle of Katnal1^1H/1H animals have a significantly reduced CBF (a) and a higher proportion of ciliary dyskinesia (b) compared to wild-type littermates. (c,d): Scanning electron micrographs (SEM) of the ependymal motile cilia in the lateral ventricles of wiltype (c) and Katnal1^1H/1H (d) animals. (e–i): Structural ciliary abnormalities found in Katnal1^1H/1H animals include abnormally long cilia (e) abnormally short cilia (f) bifurcated cilia (g) kinks in the cilia (h) and swellings along the length of the cilia (i). Transmission Electron Micrographs show vesicular aggregates within ciliary swellings (j). Arrows in (e–i) indicate ciliary abnormalities. Scale bars: c–i=5 μm; j, 500 nm (***P⩽0.001). CBF, ciliary beat frequency.