Loss of Dynein Axonemal Heavy Chain 5 Causes Cortical Development Disorders and CSF Flow Stagnation

doi:10.21203/rs.3.rs-3882997/v1

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Loss of Dynein Axonemal Heavy Chain 5 Causes Cortical Development Disorders and CSF Flow Stagnation

https://doi.org/10.21203/rs.3.rs-3882997/v1

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Background: Dynein axonemal heavy chain 5 (Dnah5) has been identified as a key gene associated with primary ciliary dyskinesia in humans. Studies have demonstrated that mice lacking Dnah5 (Dnah5-/-) develop acute hydrocephalus shortly after birth due to impaired ciliary motility, resulting in cerebrospinal fluid (CSF) stagnation and ultimately death within approximately a month. Notably, such hydrocephalus has not been reported in humansharboring this mutation. We aimed to elucidate the pathogenesis of hydrocephalus in Dnah5-deficient mice and by that improve our understanding of the role these pathogenetic mechanisms play in human hydrocephalus.

Methods: Using CRISPR/Cas9, we targeted exon 2 of the Dnah5 gene on chromosome 15, introducing a 4-base pair deletion to generate Dnah5-/- mice. We analyzed gene expression in the cerebral cortex using microarrays. Subsequently, we performed immunostaining of the cerebral cortex and ventricular wall using specific antibodies against dynein, n-cadherin, and nestin, and determined the gene expression levels and protein quantities through real-time Polymerase Chain Reaction and Western blot analysis.

Results: Hydrocephalus was observed in all Dnah5-/- mice. Electron microscopy images revealed an absence of the axonemal outer dynein arm of the peripheral doublet microtubules. Ventricular size of Dnah5-/- mice was enlarged immediately after birth and it progressed through life. The number of mature neurons in the cerebral motor cortex of Dnah5-/- mice was reduced by approximately 25% compared to wild-type mice. The level ofexpression of the Dynein Cytoplasmic 1 Heavy Chain 1 (Dync1h1) gene was decreased. Cytoplasmic dynein in the cerebral cortex of Dnah5-/- mice showed a 60% decrease compared to the wild-type mice. It was also observed a 32% reduction in nestin and 35% reduction in N-cadherin in the lateral ventricular wall of Dnah5-/- mice.

Conclusion: The reduction of cytoplasmic dynein resulted in the suppression of axonal growth and disrupted neurogenesis, and, consequently, a decrease in cell density in the ventricular wall layers. Hydrocephalus in the Dnah5-/- mouse model may arise from the stagnation of CSF due to impaired motile cilia function, and cortical malformations caused by cytoplasmic dynein deficiency.

Dnah5

motile cilia

neurogenesis

cytoplasmic dynein

Congenital hydrocephalus is one of the most common congenital anomalies. While the precise mechanisms of various types of congenital hydrocephalus are not fully understood, significant genetic anomalies associated with the condition have been identified, such as X-linked hydrocephalus, Walker-Warburg syndrome, primary ciliary dyskinesia (PCD), and hydrocephalus complicated by other ciliopathies [1]. Despite surgical intervention in early postnatal stages, congenital hydrocephalus often leads to impaired cortical development, resulting in a high incidence of moderate-to-severe developmental delays and challenges in social integration [2, 3]. The gene L1-CAM, implicated in X-linked hydrocephalus, functions as a cell adhesion molecule critical for neuronal migration, axonal extension, axon bundle formation, myelination, and synaptic plasticity control [4, 5].

Mutations in ciliary-related genes have been extensively reported and considered to be linked with congenital hydrocephalus [1, 6, 7]. However, only few reports have linked mutations associated with ciliary motility disorders in humans to hydrocephalus to date [8]. Several studies utilizing animal models have explored specific genes related to ciliary motility, revealing abnormalities in ciliary length or number that affect. their motility and cerebrospinal fluid (CSF) flow, subsequently inducing ventricular enlargement [9, 10]. In humans, genes such as Foxj1, Cfap43, and Cwh43 as the causes of Primary Ciliary Dyskinesia (PCD) were shown to be linked to adult-onset chronic hydrocephalus, demonstrating phenotypes similar to those observed in animals [11–14]. The knockout of Dynein axonemal heavy chain 5 (Dnah5) gene, one of the most frequently mutated genes in human PCD, were found to show rapid hydrocephalus progression in mice, leading to death within approximately a month [16, 17]; however, no clear association between Dnah5 and onset of hydrocephalus in humans has been established to date.

Mutations in the causative genes lead to various abnormalities in ciliary structures, resulting in ciliary motility disorders and the development of hydrocephalus with diverse outcomes. Moreover, the same genetic mutation may lead to significant different phenotypes between humans and animal models [11–14, 16–19]. In this study, we aimed to explore the pathogenesis of hydrocephalus in Dnah5 deficient mice and by that provide better understanding of the mechanisms potentially playing role in pathogenesis of hydrocephalus in humans.

Animals

All experimental animals were group-housed with 2–5 animals per cage and maintained in a temperature and humidity-controlled facility (23 ± 1 ℃, 55 ± 5% humidity, 12 h light/12 h dark) at our animal care facility in the Center for Experimental Medicine of Juntendo University, Japan. All procedures involving animals were approved by the Ethics Review Committee for Animal Experimentation of the Juntendo University School of Medicine (approval no. 1337) and performed in accordance with the Principles of Laboratory Animal Care outlined by the National Institutes of Health.

After measuring the body weight of all animals, deep anesthesia was induced using a mixed anesthetic agent (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol) administered intraperitoneally, followed by decapitation for brain dissection, as part of the research protocol. Each brain sample was immediately stored in RNAlater® solution (AM7021; Thermo Fisher Scientific, Waltham, MA, USA) for DNA, RNA and protein extractions.

Generation of Dnah5-/- Mice

Dnah5-/- mice were generated by a CRISPR/Cas9 system in our facility (Wang et al., 2013; Yang et al., 2013). Cas9 Protein (Takara Bio Inc., Shiga, Tokyo) and sgRNA were microinjected into the cytoplasm of fertilized one-cell eggs at the pronuclei stage from C57BL/6J female mice (Charles River Laboratories Japan, Inc., Yokohama, Japan).

Mutations were evaluated using polymerase chain reaction (PCR) followed by T7E1 enzyme assays. The sequence of sgRNA used for Dnah5 KO mouse generation is 5’-CCTGTTTGGACCTGAACAAACCA-3’ (chromosome 15, GRCm38. p4). Genomic DNA was extracted from the tail chips of Dnah5 KO mice using the DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer’s instructions. PCR conditions were 98 ℃ for 10 s, 60 ℃ for 30 s, and 72 ℃ for 1 min for 30 cycles (1st PCR) and for 35 cycles (2nd PCR). The primer sequences used for genotyping of Dnah5 KO mice were as follows: F_1st: 5’-GGCAACGGAGGTCAGCAAT-3’; R_1st: 5’-AGAAGCAGGCATCATCATCAA-3’; F_2nd: 5’-GGTCCCATTAAGCTGCCTGCTA-3’; R_2nd: 5’-ACAAGTACCATCATTCAACCTGGAG-3 The annealed PCR products were treated with T7 endonuclease to evaluate mismatches at the target site. The PCR products were column purified using Amicon Ultra 0.5 ml-30KDa (MERCK KGaA, Darmstadt, Germany), and sequenced to determine the mutations. Consequently, a single strain of Dnak5-/- mice was established.

Genotyping of Dnah5 Knockout Mice

To verify the genotypes of the mice, PCR amplification(107bp) was performed using a PyroMark PCR Kit (catalog no.978703, Qiagen, Hilden, Germany)from 10ng of mouse DNA for 4% gel loading and pyrosequencing. DNAH5 of PCR and Sequence primers were as follows: forward, 5’- ATG GGC GGC ATG ACT ATC − 3’; reverse, 5’- CAC AAA GCT TCC TAC CTG ATT ACC − 3.’ Sequence, 5’-TCT GTT TGC AAT TGT AGC − 3’ Dispensation Order was 5’- GTC ATG TGA CT -3.’

Reverse Transcription PCR

Total RNA was extracted from 12 to 20 mg of the cerebral ventricle and cortex tissues of three-week-old mice using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using the SuperScript™ⅣVILO™Master Mix (Thermo Fisher Scientific), and PCR was performed with ProFlex™ PCR System (Thermo Fisher Scientific). GAPDH was used as the reference gene for Dnah5 expression.

PCR primers were as follows: Dnah5(XM_006520014, 423bp) forward, 5’- CTT CAC ACC GGA CAA CAA GC -3’; reverse, 5’ -TCA CGA ACA CCT TGG GCT TT -3,’ GAPDH(XM_017321385.1,164bp) forward, 5’- TCA CCA CCA TGG AGA AGG C -3’; reverse, 5’- GCT AAG CAT TGG TGG TGC A -3.’

Hematoxylin Eosin (HE) Staining

Brains of wild-type and Dnah5-/- mice on days zero, three, five, eight, and ten were fixed in 4% paraformaldehyde fixative (33111; Muto Pure Chemicals Co., Tokyo, Japan) for at least 1 week, embedded in paraffin, and cut into 5µm sections. Sections were stained with 3,3’-diaminobenzidine and counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted. The sections were viewed under an E800 microscope (Nikon, Tokyo, Japan), and images were captured with an AxioCam 506 color digital camera using AxioVison Rel version 4.7.2.0 image-processing software (Carl Zeiss Microimaging GmbH, Jena, Germany).

Ventricular Injection of Dye (DiI)

Brains of Dnah5-/- mice on day four were fixed in a stereotaxic apparatus (Narishige Scientific Instrument Laboratory, Tokyo, Japan), and a small burr hole was drilled into the cranium on the right side, 1 mm lateral to the bregma. The needle of a micro syringe (MS-E05; Ito Corporation, Shizuoka, Japan) was slowly inserted into the lateral ventricle 1.5 mm from the brain surface through the hole and 1µl of Cell Tracker™ CM-DiI dye (C7001; Thermo Fisher Scientific) was injected at 2µl/min. Ten minutes after injection, mouse brains were harvested, fixed in 4% paraformaldehyde for 72 hours, and then cut into 30µm coronal cryosections (CM3050 S, LEICA Microsystems GmbH, Wetzler, Germany).

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

Brains of wild-type and Dnah5-/- mice on day three were perfused with 4% paraformaldehyde. The materials were immersed in a 2.5% glutaraldehyde solution after being cut into smaller fragments for (trasmission electron microscopy TEM(1x2mm²) and (scaning electron microscopy SEM(3x3mm²). They were washed in Phosphate-buffered saline (PBS), post-fixed in 2% osmium tetroxide for 2 hours at 4 ℃ and dehydrated with graded concentrations of ethanol. For TEM, they were placed in resin for 4 days at 60 ℃. Ultrathin sections were observed under an HT7700 electron microscope (Hitachi High Technologies, Tokyo, Japan).

For the SEM analysis, the samples were freeze-dried. Gold palladium-coated tissues were scanned using an S-4800 scanning electron microscope (Hitachi High Technologies).

Confirmation of Ciliary Motility

To confirm the ciliary movement, we removed the brains of mice on day 10, and created 1 mm brain slices. 10µm microbeads (Polybeads® Polystyrene Black Dyed 10.0 Micron Microspheres, # 24294-2, Polysciences Inc., PA, USA) were diluted 5 times with PBS, and 1µl of this diluted solution was placed into the lateral ventricle after removing the choroid plexus. The movement of the microbeads was recorded for 5 min at a rate of 6 fps using a video microscope (DMI 3000 B, LEICA) and Leica Application Suite ver. 4.2.0 software. The trajectories of the beads were traced using ImageJ ver. 2.1.0 and TrackMate ver. 6.0.1.

Microarray

Gene expression microarray analysis was performed using the hemispheric brain of wild-type and Dnah5-/- mice on day three. Total RNA was extracted using RNeasy Mini Kit (Qiagen) from RNAlater™ Stabilization Solution (Cat.no.AM7021, Thermo Fisher Scientific) following the manufacturer’s instruction. RNA quality and integrity were measured a 4150TapeSation (Agilent Technologies Inc., Santa Clara, CA, USA). For 250ng of total RNA was conducted a WT Expression Kit and GeneChip^TMMouse Exon 1.0 ST Array (Affymetrix, Thermo Fisher Scientific), following the manufacturer’s protocol. We used the Transriptome Analysis Console (TAC) software (version4.0.2.15) and the Robust Multichip Average (RMA) algorithm for normalization. The data of comparative analysis, over and below log2 fold change cut-off values, were entered into the Ingenuity Pathway Analysis (IPA) software (Qiagen). Disease and functional analyses were performed using the IPA.

Real Time Quantitative PCR

The real time quantitative PCR was conducted using brain cortex of three-day-old Dnah5-/- mice. Total RNA (250 ng) was converted into single-stranded cDNA using SuperScript IV VILO™ (SSIV VILO) Master Mix (Cat.no.11756050, Invitrogen, Thermo Fisher Scientific). The ABI 7500 Real-time PCR System (Applied Biosystems, Thermo Fisher Scientific) and TaqMan® Gene Expression Assays (Applied Biosystems, Thermo Fisher Scientific) were used according to the instructions from the manufacturer to quantify gene expression. Assay IDs are listed (Additional File 1 of Table S1). Target gene expression was standardized to that of Actb. The presence of a single PCR amplicon was confirmed using melting curve analysis. The expression of each gene in each sample was analyzed in triplicate. The real-time PCR were quantified using the 2^−ΔΔCt method.

Immunofluorescence Staining

Brains of three-day-old wild-type and Dnah5-/- mice were removed and post-fixed with 4% paraformaldehyde in 0.01-M phosphate buffer (pH 7.2). Paraffin-embedded sections (5µm) were blocked with 5× SEA BLOCK™ blocking buffer (37527; Thermo Fisher Scientific) and 1% donkey serum in Phosphate buffered saline (PBS; AJ9P003; TaKaRa, Shiga, Japan) for 30 min, incubated in primary antibody overnight at 4 ℃, and secondary antibodies for 60 min at room temperature. Primary and secondary antibodies used are listed (Additional File 1: Table S2).

Nuclei in all sections were examined under a confocal scanning microscope (TCS-SP5; Leica Microsystems) using Leica Application SuiteX 3.4.2.18368 (Leica Microsystems, GmbH, Wetzlar, Germany).

Cell Count

The numbers of nuclei of mature neurons were counted in all layers of the selected three-day-old wild-type and Dnah5-/- mice cerebral motor cortex stained with Neuro Trace. Eight slices were selected for each Dnah5-/- mice and wild-type mice, and the width was set to 100 micrometers.

Western Blotting Analysis

Proteins were extracted from the brain cortexes of Dnah5-/- mice on day three and lysed in 200µl of lysis buffer (N-PER; Thermo Fischer Scientific) containing protease inhibitor cocktail (cOmplete ULTRA Mini EDTA-free EASYpack; Roche, Basel, Switzerland). Lysates were segregated by centrifugation at 20,000×g at 4 ℃ for 15 min, and protein concentrations of the resultant supernatants were determined using BCA Protein Assay Kits (Thermo Fischer Scientific). After that 10 µg (Dynein) and 20 µg (N Cadherin, Nestin) of protein were heated at 70 ℃ for 10 min in NuPAGE® LDS Sample Buffer (NP0008; Invitrogen), samples were electrophoresed on 3–8% NuPAGE® Tris-Acetate Mini Gel by NuPAGE® Tris-Acetate SDS Running Buffer (20×) (LA0041; Invitrogen), and then transferred to a polyvinylidene fluoride membrane. The primary antibodies used are listed in Additional File 1 of Table S2. Chemiluminescence signals were detected using a Western Breeze Kit (WB7106; Invitrogen). Immunoreactive bands were detected using ImageLab version 4.1 software (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical Analysis

All experimental results are presented as mean ± standard deviation. The data distribution was evaluated graphically using histograms and Q–Q plots. The Shapiro-Wilk test was used to assess the normality of the distributions. For real-time PCR performed in at least five independent experiments, significant differences among the groups were determined using Welch’s t-test. Statistical differences in the number of neurons in the motor cortex between wild-type and Dnah5-/- mice were assessed using the Mann–Whitney U test. All calculations were performed using IBM SPSS Statistics ver25, with statistical significance set at P < 0.05.

Generation of Dnah5-/- Mice

To investigate the effect of the Dnah5 gene on the brain tissue, mice lacking the Dnah5 gene (Dnah5-/- mice) were generated. DNA sequencing confirmed a 4-base pair (bp) deletion in the targeted Exon 2 of the Dnah5-/- mice on chromosome 15 (Fig. 1A). Consequently, a frameshift occurred, resulting in the 25th codon becoming a TGA stop codon. RT-PCR results also confirming the absence of a 500bp band in Dnah5-/- mice, which was detected in the wild-type mice (Fig. 1B). Pyrosequencing results also confirmed the genotype of the generated Dnah5-/- mice, revealing a 4-bp deletion as well (Fig. 1C). Hydrocephalus and situs inversus occurred in all and 26% of the homozygous individuals among the Dnah5-/- mice, respectively (Fig. 1D).

Comparative analysis of body weight changes at zero-, three-, five-, eight-, and ten-day-old revealed a significant difference on day eight, with no significant differences observed until day five (Additional File 1: Figure S1). Comparison of head morphologies of five-day-old mice showed a distinct round deformation in Dnah5-/- mice compared to that of wild-type mice (Fig. 1D).

Examinations of the ventricular morphology of Dnah5-/- mice using coronal sections stained with Hematoxylin and Eosin (HE) revealed that these mice already displayed enlargement of the lateral ventricles early after birth, with the enlargement becoming more pronounced on subsequent days (Fig. 1E).

Ciliary Structure and CSF Flow

TEM analysis of the cross-section of cilia in Dnah5-/- mice at on day three revealed that the outer dynein arm structure of the peripheral doublet microtubules was completely absent (Fig. 2A). The overall morphology of motile cilia based on scanning electron microscopy (SEM) images also revealed uniformly formed cilia bundles that extended into the ventricular cavity in wild-type mice. In contrast, Dnah5-/- mice exhibited uneven morphology, which appeared to collapse towards the ventricular wall (Fig. 2A).

An experiment involving dye injection was conducted using Dnah5-/- mice on day four (Fig. 2B), by puncturing the right lateral ventricle with a microneedle and injecting DiI (a fluorescent dye). The result revealed no occlusion of the cerebral aqueduct despite ventricular enlargement. Therefore, enlargement of the ventricles was observed prior to the occlusion of the cerebral aqueduct (Fig. 2B).

To confirm ependymal flow by motile cilia, coronal sections were obtained immediately after extraction from live mice. Microbeads were placed in the lateral ventricular cavity of the slice and the flow of microbeads in PBS was recorded and tracked. In wild-type mice, a consistent vortical flow was observed, whereas no flow of cerebrospinal fluid was detected in Dnah5-/- mice (Fig. 2C).

The Effect of Dnah5 Gene Deficiency on Cortex

Coronal sections of the cerebral cortex of brains of Dnah5-/- and wild-type mice on day three were examined. Immunostaining of the motor cortex with Neuro Trace revealed that mature neurons were densely packed and arranged radially in the cerebral cortex of wild-type mice, whereas an uneven arrangement was observed in Dnah5-/- mice (Fig. 3A). The nuclei of mature neurons in the motor cortex were counted throughout the entire cortical layer in 100µm intervals using Neuro Trace as well (Fig. 3A). An approximately 25% reduction in cell count was observed in Dnah5-/- mice, compared to that in wild-type (Fig. 3A).

Microarray analysis was performed using one hemisphere of the Dnah5-/- mouse brain. A total of 1052 out of the 2534 genes selected by TAC were analyzed using Ingenuity® Pathway Analysis (IPA) for "Disease and Function" category. Upon ranking based on Fisher's Exact Test p-values, the top category was found to be "Development of neural cells" (Additional File 1: Table S3). Significant downregulation of NeuroD6 in Dnah5-/- mice, and the downregulation of NeuroD2, which is involved in the same pathway as NeuroD6, was also observed [20] (Additional file 1: Table S4). RT-PCR was also conducted for the NeuroD6, NeuroD2, and Ascl1 genes that are involved in the same pathway. The results indicated decreased expression compared to the control, with relative values for NeuroD6 at 0.357, NeuroD2 at 0.445, and Ascl1 at 0.421 (Additional file 1: Figure S2). This decrease is attributable to the Dnah5 knockout. The expression of the Dynein Cytoplasmic 1 Heavy Chain 1 (Dync1h1), a gene involved in the heavy chain structure of cytoplasmic dynein, was also decreased (Additional file 1: Table S5). Real-time PCR also confirmed a substantial decreased in the expression of Dync1h1 to 0.326, relative to the control (Additional file 1: Figure S3).

Observation of dynein in the cerebral cortex revealed that in wild-type mice, the expression of the anti-dynein antibody was radial in the direction, coinciding with cortical neuronal axons. In contrast, the expression was clearly scarce in Dnah5-/- mice (Fig. 3C). Western blotting also confirmed a 60% decrease in dynein expression.

The Effect on the Ependymal and Subependymal Zone Layers

To observe the ependymal layer of the lateral ventricles in wild-type and Dnah5-/- mice at three-day-old, we performed HE staining (Fig. 4A). The cell density of the ependymal and subependymal zone layers lining the lateral ventricle wall in Dnah5-/- mice was sparser, the layers being coarser than that in wild-type mice. Immunostaining with Neuro Trace of the ependymal and subependymal zone layers of the lateral ventricle wall revealed a reduced cell density in Dnah5-/- mice (Fig. 4B). Nestin antibody staining in the same region showed reduced staining in the ependymal and subependymal layers of Dnah5-/- mice compared to wild-type mice. N-cadherin antibody staining was reduced in Dnah5-/- mice (Fig. 4C). Western blotting confirmed a 32% decrease in nestin and 35% decrease in N-cadherin protein expression levels (Fig. 4D).

Genes Associated with Cilia Motility

A previous case report documented the co-occurrence of hydrocephalus and developmental disorders in a three-generation Jordanian family with autosomal recessive PCD [21]. Additionally, mutations in the genes Dnah2 and Dnah14, which contribute to motile cilia structure, have been associated with primary microcephaly and neurodevelopmental disorders, along with other conditions such as seizures [22, 23]. Dnah2 and Dnah14 are essential for the inner dynein arm structure within the peripheral doublet microtubules of motile cilia [24]. We have previously reported that mutations in Dnah14 are linked to the development of chronic hydrocephalus in humans [25].

As a common hallmark of PCD, hydrocephalus has been linked with PCD in genetically modified animal -in particular mouse models in several studies [26]. Sawamoto et al. used Tg737 mutant mice to demonstrate the involvement of motile cilia in brain neuronal migration [27]. However, a direct causal relationship between cilia-related genes and brain formation has not been established. Therefore, we have used a knockout mouse model of Dnah5, a representative causative gene for PCD, and focused on (i) changes in the cerebral cortex immediately after birth, and (ii) changes in the ventricular wall before the onset of hydrocephalus, to identify the mechanisms underlying hydrocephalus.

Ibanez et al. created a mouse model that stopped at exon 16, as within exon 79 on chromosome 15; there are six P-loops responsible for ATP hydrolysis and a microtubule-binding site from exon 17 onwards, which are involved in ciliary motility. Approximately 20% of mice developed hydrocephalus, with aqueductal obstruction occurring at 3–5 days of age, followed by rapid ventricular enlargement after day 5[16, 17]. Here, we created a mouse model using CRISPR/Cas9 technology, resulting in the complete absence of the outer dynein arms and 100% incidence of hydrocephalus. Our findings revealed mild ventricular enlargement from birth and simultaneous impairment of cerebral cortex formation. Hence, Dnah5 may impact not only on the motility of ependymal cilia, but also on the formation and development of the cerebral cortex.

The Effect on Cerebral Cortex Formation in Dnah5-/- Mice

Mild ventricular enlargement was observed early after birth in Dnah5-/- mice. However, from day five and onwards, weight gain deteriorated upon completion of aqueductal obstruction and appeared obvious ventricular enlargement. When compared to wild-type mice, no significant weight difference was observed before aqueductal obstruction. Therefore, experiments were conducted using the brains of mice from day three, in which the effects of eventual cortical compression due to ventricular enlargement and metabolic disturbances were less pronounced [28, 29]. In the cerebral cortex of Dnah5-/- mice, a reduction in the cell density of mature neurons was observed on day three, as evidenced by Neuro Trace staining.

In both microarray and real-time PCR analyses of Dnah5-/- mice, a decrease in the expression of the NeuroD family was observed. The neuroD family comprises closely related proteins, such as NeuroD1, D2, D4, and D6, which play essential roles as basic helix-loop-helix (bHLH) genes in regulating neurogenesis. Among these, NeuroD2 and D6 are particularly crucial factors for guiding axon navigation and regulating axon tracts in the mouse cerebral cortex [30]. Studies using a double mutant mouse model of NeuroD2 and D6 have shown a decrease in functional glutamatergic synapses in the cerebral cortex, resulting in a reduced excitatory cortical network [31]. The observed reduction in the expression of neuroD family members in the genetic analysis of Dnah5-/- mice suggests a potential impact on neuronal migration and proliferation.

Gene mutation studies in patients with cortical formation abnormalities have identified strong associations between genes, such as Dcx, Lis1, Tuba1a, Flna, Akt3, Pik3ca, Dync1h1, Kif5c, Kif7, Kif1a, and Kif26a [32]. These genes play crucial roles in the formation of the microtubule cytoskeleton within axons during cerebral cortex development. In particular, mutations in Dync1h1 gene, which encodes the heavy chain of cytoplasmic dynein, have been reported to affect cortical development, leading to conditions such as microcephaly and abnormalities in brain structures, such as nodular heterotopia, hypoplasia of the corpus callosum, and dysmorphism of the basal ganglia [33–35].

In the Dnah5-/- mouse model the reduced expression of Dync1h1 lead to the impairment of cytoplasmic dynein. Cytoplasmic dynein and kinesin function in the axonal microtubules of neurons and play significant roles in neuronal cell movement [36, 37]. Decreased Dync1h1 expression is involved in the reduction of cytoplasmic dynein, thereby potentially interfering with neuronal migration and affects cerebral cortex formation.

The Effect on the Structure of the Ventricular Wall in Dnah5-/- Mice

To elucidate the effect of decreased cytoplasmic dynein on primary cilia, we observed not only cilia, but also the ependymal cell and subventricular zone layers.

Cilia are categorized into motile and non-motile types. Motile cilia are further categorized into two subtypes with different structures: 9 + 2 structure cilia with central and peripheral microtubules involved in ciliary movement, and 9 + 0 structure nodal cilia responsible for left-right axis determination [8, 38]. Primary cilia, which are classified as non-motile cilia, lack dynein arms and serve as sensory organs for various signals [38]. Primary cilia play a significant role in the migration of neurons from the subventricular zone (SVZ), and thus contribute to cortical formation [39, 40]. Abnormalities in primary cilia function have been linked to conditions such as Joubert, Meckel-Gruber, and Bardet-Biedl syndromes, highlighting their role in brain development and associated cognitive functions [41–45].

Ependymal cells form a multiciliated monolayer columnar epithelium that lines the surface of the ventricular wall, while SVZ, located beneath, acts as a detector of various signals through primary cilia, regulating neuronal cell division [39, 46–50]. Primary cilia protrude through the intercellular spaces of ependymal cells toward the ventricular surface to detect these signals upon contact with CSF [39, 40, 51]. Radial glia, which extend the primary cilia towards the ventricular surface, play a central role in the proliferation of neural cells, and newly generated neural cells from this region migrate radially to form the cerebral parenchyma [40].

In Dnah5-/- mice on day three, a decrease in cell density was observed in the ependymal and SVZ layers, suggesting reduced neurogenesis in these regions.

Cadherin is a cell adhesion molecule abundant in the radial glia, with a crucial role in the maturation of neural cells [52]. The loss of N-cadherin in radial glia was shown to hinder cortical development processes, including cell proliferation, differentiation, and migration [53–55]. Nestin is a cytoskeletal protein classified as an intermediate filament specific to neural stem cells and also a marker for these cells. Inhibition of neurogenesis from radial glia in the ventricular zone, accompanied by a decrease in cell proliferation, is supported by the reduced expression of N-cadherin and nestin in the ventricular wall.

Cytoplasmic dynein is crucial for the intrafragellar transporter (IFT) of cilia, contributing to the maintenance of primary and motile cilia function [36, 37, 56–58]. The proper functioning of cytoplasmic dynein and kinesin in IFT is thus essential for maintaining cellular homeostasis [59, 60]. Downregulation of Dync1h1 in Dnah5-/- mice resulted in the impairment of cytoplasmic dynein function, leading to dysfunction of primary cilia, and suggesting a decrease in cell density in the ventricular wall due to impaired neurogenesis. Furthermore, primary cilia control the concentration gradient of signals that regulate neural cell migration. Normal beating motion of motile cilia is crucial for this regulation [27]. In Dnah5-/- mice, the significantly deficient cilia motions disrupt the proper mixing of the CSF, and the resulting impaired function of primary cilia prevents the sensing of signals from the ventricular side. As a result, neurogenesis in the radial glia is impaired, leading to a decrease in cell density in the ependymal and SVZ layers.

Dnah5 deficiency reduces Dync1h1 expression levels, which in turn affects cytoplasmic dynein assembly. We report here, for the first time, that impairment of neuronal migration causes cortical malformations due to dysfunctional primary cilia and compromised neurogenesis. We can hypothesize that brain malformations and progressive hydrocephalus could result in severe disabilities during the prenatal period in humans, potentially leading to a presumable non-viable birth.

LIMITATION

We have not directly investigated primary cilia in the ependymal cell layer or neuronal migration. Hence, the mechanisms by which Dnah5 affects cytoplasmic dynein and primary cilia remain unclear. Primary cilia and neuronal migration should be investigated in future studies to clarify these mechanisms.

We generated a genetically modified mouse model of Dnah5, a representative gene associated with PCD, and proposed mechanisms to explain the observed cortical malformations. Hydrocephalus in the Dnah5-/- mouse model was found to arise from the stagnation of cerebrospinal fluid due to impaired motile cilia function, and cortical malformations due to cytoplasmic dynein deficiency.

Ethics approval and consent to participate:

All procedures involving animals were approved by the Ethics Review Committee for Animal Experimentation of the Juntendo University School of Medicine (mice: approval no. 1337) and followed the Principles of Laboratory Animal Care as outlined by the National Institutes of Health.

Consent for publication:

Not applicable.

Availability of data and materials:

Data are available from the corresponding author with reasonable request.

Competing interests:

The authors declare that they have no competing interests.

Funding:

This study was financially supported by the Juntendo Research Branding Project, Health, Labor and Welfare Sciences Research Grants (22GB1002), and the Japan Society for the Promotion of Science under Grants-in-Aid for Scientific Research (Grant Numbers 18H02916, 20K09355, 20K09398, and 22H03675).

Authors' contributions:

MM, MN and KS designed the experiments; KS and IO performed immunohistochemistry, HE-staining and dye injection, analyzed all of the data and wrote the manuscript; IO performed microarray, real-time PCR, western blotting and immunofluorescence, KS performed TEM imaging; KK edited the manuscript; EN and NT generated knockout mice; and MM, MN, KH, RM, CK and AK were responsible for critical review. All authors read and approved the final manuscript.

Acknowledgements:

We gratefully acknowledge the contributions of the Laboratory of Morphology and Image Analysis, Laboratory of Biomedical Research Resources, Laboratory of Proteomics and Biomolecular Science, Laboratory of Molecular and Biochemical Research, Biomedical Research Core Facilities, Graduate School Research Program of Juntendo University, Juntendo University Research Institute for Diseases of Old Age, and the Juntendo University Graduate School of Medicine for their technical assistance.

Authors' information:

1. Department of Neurosurgery, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

2. Department of Neurosurgery, Juntendo Tokyo Koto Geriatric Medical Center, 3‑3‑20 Shinsuna, Koto‑ku, Tokyo 136‑0075, Japan

3. Department of Genetic Analysis Model Laboratory, Juntendo University Graduate School of Medicine, Hongo Bunkyo‑ku, Tokyo 113‑8421, Japan.

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Table 1

The results of the category analysis using IPA
	Diseases or Functions Annotation	p-value	# Molecules
1	Development of neural cells	5.82E-47	198
2	Development of neurons	4.24E-45	189
3	Morphology of nervous system	3.35E-39	203
4	Differentiation of neural cells	4.02E-37	130
5	Locomotion	2.23E-36	105
6	Differentiation of neurons	4.31E-35	110
7	Abnormal morphology of nervous system	4.44E-34	170
8	Development of head	1.61E-33	194
9	Development of body axis	3.48E-32	199
10	Morphology of central nervous system	2.05E-31	133

Table 2

Significant downregulated genes on three-day-old in Dnah5-/- mice
Gene Symbols	Description	Fold Change	Chromosome
NEUROD6	neurogenic differentiation 6	-187.65	chr6
FEZF2	Fez family zinc finger 2	-62.42	chr14
ZBTB18	zinc finger and BTB domain containing 18	-38.09	chr1
MEF2C	myocyte enhancer factor 2C	-32.05	chr13
PTK2B	PTK2 protein tyrosine kinase 2 beta	-27.68	chr14
TBR1	T-box brain gene 1	-21.66	chr2
FOXG1	forkhead box G1	-20.28	chr12
TIAM2	T cell lymphoma invasion and metastasis 2	-18.15	chr17
MET	met proto-oncogene	-17.48	chr6
NEUROD2	neurogenic differentiation 2	-10.44	chr11

Table 3

Significant down regulated genes involved in Dynein structure.
Gene	Description	Fold Change
Dync1h1	Dynein cytoplasmic 1 heavy chain 1	-2.36
Dync1i1	Dynein cytoplasmic 1 intermediate chain 1	-2.58
Dync1i2	Dynein cytoplasmic 1 intermediate chain 1	-1.12
Dync1li1	Dynein cytoplasmic 1 light intermediate chain 1	1.1
Dync1li2	Dynein cytoplasmic 1 light intermediate chain 1	1.19
Dync2h1	Dynein cytoplasmic 1 heavy chain 1	-1.26
Dync2li1	Dynein cytoplasmic 1 intermediate chain 1	-1.07

No competing interests reported.

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Loss of Dynein Axonemal Heavy Chain 5 Causes Cortical Development Disorders and CSF Flow Stagnation

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Animals

Generation of Dnah5-/- Mice

Genotyping of Dnah5 Knockout Mice

Reverse Transcription PCR

Hematoxylin Eosin (HE) Staining

Ventricular Injection of Dye (DiI)

Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

Confirmation of Ciliary Motility

Microarray

Real Time Quantitative PCR

Immunofluorescence Staining

Cell Count

Western Blotting Analysis

Statistical Analysis

RESULTS

Generation of Dnah5-/- Mice

Ciliary Structure and CSF Flow

The Effect of Dnah5 Gene Deficiency on Cortex

The Effect on the Ependymal and Subependymal Zone Layers

DISCUSSION

Genes Associated with Cilia Motility

The Effect on Cerebral Cortex Formation in Dnah5-/- Mice

The Effect on the Structure of the Ventricular Wall in Dnah5-/- Mice

CONCLUSION

Declarations

References

Tables

Additional Declarations

Supplementary Files

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