Heterozygous mutation p.Asp249Asn (Tubb4a^D249N/+) mice were generated using CRISPR-Cas-9 technology by substituting p.Asp249Asn (c.745G > A) mutation in exon 4 of the Tubb4a gene. Known off-target effects include one synonymous mutation in cis at p.Lys244Lys (c.732C > A) with the pathogenic variant at p.Asp249Asn (variant at c.745G > A).

Tubb4a^D249N/+ mice were bred to obtain a homozygous Tubb4a^D249N/D249N mouse colony (Figure 1A). Homozygous mice were studied in parallel with Tubb4a^D249N/+ mice, because in a rat model of Tubb4a mutation (Li et al., 2003), the homozygous animals develop phenotypic manifestations earlier than heterozygous animals. In WT mice, Tubb4a gene expression is highest in the cerebellum, spinal cord and striatum (compared to other CNS areas), which are also typically affected brain regions in H-ABC individuals. However, Tubb4a gene expression in WT, Tubb4a^D249N/+and Tubb4a^D249N/D249N mice are similar in these brain areas (Figure 1B), indicating there is no transcriptional change in the face of the mutation.

The homozygous Tubb4a^D249N/D249N mutant mice appear normal compared to WT and heterozygous Tubb4a^D249N/+ littermates until ~post natal (P) day P8. However, by ~P9, Tubb4a^D249N/D249N mice display tremor (Figure 1—videos 1 and 2), which progressively worsens with age (Figure 1—videos 3 and 4) until mice become severely ataxic and dystonic (~P21). Additionally, Tubb4a^D249N/D249N mice show gradual weight reduction from P35-37 compared to Tubb4a^D249N/+ and WT mice (p<0.001, Figure 1D).

Motor skills were assessed by measuring ambulation, hanging grip strength, rotarod testing and righting reflexes over time. Tubb4a^D249N/D249N mice exhibit developmental delay with persistent asymmetric limb movement and crawling gait at P10 relative to ambulation in WT controls (p<0.05, see Table 1, Figure 1E–F). While Tubb4a^D249N/D249N mice achieve walking by P14, they display tremor, ataxia and abnormal gait. Ambulatory angle is consistently wider in Tubb4a^D249N/D249N mice from P14 to P35 (p<0.001, Figure 1G–H) suggesting gait instability. Additionally, Tubb4a^D249N/D249N mice demonstrate early deficits in hanging grip strength test, where compared to WT mice, they fall at a lesser angle (p<0.001, Figure 1I–J). Rotarod performance further shows a shorter latency to fall in Tubb4a^D249N/D249N mice on an accelerating rotarod, with worsening over time (p<0.001, Figure 1K, Figure 1—video 5). By ~P35-P40, Tubb4a^D249N/D249N mice are unable to feed themselves, with a decreased righting reflex (p<0.001, Figure 1L). At this time, mice reach a compassionate end-point (survival curve, p<0.001, Figure 1M–N) and are considered ‘end-stage’.

Heterozygous Tubb4a^D249N/+ mice transition well from crawling to ambulation and walk with a normal gait reminiscent of WT mice. Rotarod on Tubb4a^D249N/+ mice at 9 months and 1 year of age demonstrates no motor or gait abnormalities even at later timepoints (Figure 1—figure supplement 1A). Further, Tubb4a^D249N/+ mice display similar survival compared to their WT littermates and die because of advanced age (Kaplan-Meier survival curve, Figure 1—figure supplement 1B).

Myelination in mice starts at birth in the brainstem and spinal cord and has a nearly adult pattern by P21 (Baumann and Pham-Dinh, 2001). Profound myelination deficits are observed in Tubb4a^D249N/D249N mice, relative to their WT littermates, in both corpus callosum and cerebellum at P14 and P21 (Eri-C staining in Figure 2D and F and Figure 2—figure supplement 1 and Figure 2—figure supplement 2) with further loss of myelination by end-stage (~P35-P40) (p<0.001, Figure 2C–F). Based on Eri-C staining, myelination also decreases in Tubb4a^D249N/+ mice at 1 year of age relative to WT littermates (p<0.001, Figure 2G–H).

The myelin proteins, PLP and MBP, measured by immunostaining, are also decreased in Tubb4a^D249N/D249N mice relative to Tubb4a^D249N/+ and WT mice (Figure 2W and Figure 2—figure supplement 2, p<0.001), with a significant difference at P21 and end-stage (Figure 2L, P, T and X). This decrease in PLP and MBP protein is further confirmed with immunoblotting in forebrain (Figure 2M–N and U–V) and cerebellum (Figure 2Q–R and Y–Z) of Tubb4a^D249N/D249N mice at P21 (p<0.05) and end-stage (p<0.001). Tubb4a^D249N/+ mice have comparable MBP and PLP immunoreactivity as WT mice at P14, P21 (Figure 2—figure supplements 1 and 2), and ~P35-P40 (Figure 2). However, by 1 year, their myelin protein levels significantly diminish in the corpus callosum compared to WT mice (p<0.05, PLP: Figure 1—figure supplement 1C–D; p<0.05, Figure 2I–J).

To understand the developmental and degenerative abnormalities of myelin formation in Tubb4a^D249N/+ and Tubb4a^D249N/D249N mice, oligodendrocytes (OLs) were assessed using ASPA stain in the corpus callosum at P14, P21 and end-stage (Figure 3C). A dramatic decline occurs in the number of OLs in the Tubb4a^D249N/D249N mice relative to their WT and Tubb4a^D249N/+ littermates at all timepoints (p<0.001, Figure 3D, Figure 3—figure supplement 1A–B). However, upon examining the cells double-labeled with CC1 (mature OL marker) and cleaved caspase 3 (cell death marker); no cell death is detected at end-stage (Figure 3—figure supplement 2A), potentially ruling out apoptosis mediated OL death. To test, if OL numbers are in fact altered due to an effect on the progenitor pool, the total number of NG2 cells as well as NG2+ Cleaved Caspase 3+ cells were studied for cell death. While the total number of NG2 cells are unchanged (Figure 3E–F), a significant number of NG2 cells undergo apoptosis in Tubb4a^D249N/D249N mice compared to WT and Tubb4a^D249N/+ mice at P14, P21 and end-stage (p<0.01, p<0.001, Figure 3G–H and Figure 3—figure supplement 2B–C).

Interestingly, in spite of the ongoing loss, the total number of NG2 and Olig2 populations are stable (Figure 3—figure supplement 1C–E). Loss or damage to OPCs can result in a proliferative response (Hughes et al., 2013), hence the number of dividing NG2 cells was next assessed at P14, P21 and end-stage timepoints. A significant increase is detected in NG2+ cells co-labeled with Ki-67 (proliferation marker) in Tubb4a^D249N/D249N mice at all timepoints compared to Tubb4a^D249N/+ and WT mice (p<0.05, p<0.01; Figure 3I–J, Figure 3—figure supplement 2D–E), possibly contributing to the steady number of NG2+ and Olig2+ cells. In summary, OPCs undergo cell death and are being replaced in the Tubb4a^D249N/D249N mice, though possible defects in OPC differentiation results in fewer mature OLs.

Loss of myelination was examined in epoxy sections of optic nerves from Tubb4a^D249N/D249N mice compared to WT and Tubb4a^D249N/+ mice, at P21 (Figure 4—figure supplement 1) and end-stage (data not shown). Electron microscopy further confirms that end-stage Tubb4a^D249N/D249N optic nerves (n = 3) contain mostly unmyelinated axons and/or axons with myelin sheaths that are inappropriately thin for their axonal caliber (Figure 4A–C and A’-C’). In Tubb4a^D249N/+ spinal cords, myelin sheaths are also noticeably thinner than in WT littermates as seen in the epoxy (Figure 4K–O) and EM sections (Figure 4—figure supplement 2).

Along with dramatically reduced numbers of normally myelinated axons, Tubb4a^D249N/D249N optic nerves exhibit microglia and/or macrophages containing myelin debris and lipid droplets (Figure 4D–D’), dying OLs (Figure 4E) as well as vacuoles (Figure 4G). As seen in taiep rats, we find enlarged astrocytic processes (‘astrogliosis’), axons with abnormal accumulations of lysosomes, degenerating unmyelinated and/or myelinated axons, but not bundles of microtubules in OLs (Duncan et al., 2017; Duncan et al., 1992; O'Connor et al., 2000; Roncagliolo et al., 2006; Wilkins et al., 2010). Many OLs are partially surrounded by unmyelinated axons, some of which are variably surrounded by cytoplasmic invaginations, reminiscent of non-myelinating Schwann cells in peripheral nerves (Figure 4F). Tubb4a^D249N/+ optic nerves, in contrast, look similar to WT optic nerves except for more conspicuous astrocytic processes (Figure 4—figure supplement 1). These findings are even more striking in Tubb4a^D249N/D249N spinal cord at end-stage, where most large axons of the ventral funiculus are unmyelinated with conspicuous vacuoles, along with microglia and/or macrophages comprising of myelin debris, lipid droplets, and other unidentified material (Figure 4—figure supplement 2).

Compared to WT mice, the g-ratio, a measure of myelin thickness, is significantly higher in optic nerves of Tubb4a^D249N/D249N and Tubb4a^D249N/+ mice (Figure 4H, p<0.001). The axon caliber in optic nerves is significantly decreased in Tubb4a^D249N/D249N mice versus WT mice at end-stage (Figure 4I, p<0.05). Further, plotting g-ratio as a function of axon diameter (Figure 4J) demonstrates that intact axons of all calibers in optic nerves have relatively thinner myelin sheaths in Tubb4a^D249N/+ and Tubb4a^D249N/D249N mice. The analysis of the percentage of myelination in the optic nerve reveals that ~65% of the axons are unmyelinated (Figure 4K, p<0.001) and ~30% of axons are thinly dysmyelinated axons in the Tubb4a^D249N/D249N (Figure 4K, p<0.01) compared to ~3% unmyelinated and 13% thinly dysmyelinated axons in WT mice. While about ~56% of axons are normally myelinated in the Tubb4a^D249N/+ mice (Figure 4K, p<0.001),~5% of axons are unmyelinated and about ~39% thinly myelinated axons (Figure 4K, p<0.001). Similar to optic nerves, compared to WT mice, the g-ratio in spinal cords of Tubb4a^D249N/D249N and in Tubb4a^D249N/+ mice are also significantly different (Figure 4L–N and O, p<0.001). Further, plotting g-ratio as a function of axon diameter demonstrates that all axons in spinal cord have relatively thinner myelin sheaths in Tubb4a^D249N/+ and Tubb4a^D249N/D249N mice (Figure 4P).

The survival of neurons was examined in the cerebellum and striatum of WT, Tubb4a^D249N/+ and Tubb4a^D249N/D249N mice with NeuN and Nissl’s stain across developmental time points (Figure 5A). Nissl’s stain on cerebellar sections of Tubb4a^D249N/D249N mice reveals a severe progressive loss of the granular neuron layer from P21 to end-stage and a remarkable decrease in cerebellar volume (Figure 5B–C). Tubb4a^D249N/D249N mice display comparable number of granular neurons at P14 compared to WT mice (Figure 5E and Figure 5—figure supplement 1A), however, a dramatic and progressive granular neuron loss is observed at P21 and end-stage (p<0.001, Figure 5D–E). In addition, a significant increase in the number of Cleaved Caspase 3 positive cells co-localized with NeuN is observed at P21 and end-stage, indicating cellular apoptosis (p<0.001, Figure 5F–G). The loss of other neuronal populations in the cerebellum, such as Purkinje neurons stained with calbindin, do not display any change in the different groups of mice (Figure 5—figure supplement 1D–E).

In the striatum, NeuN counts for Tubb4a^D249N/+ and Tubb4a^D249N/D249N are comparable to WT mice at P14 and P21 time points (Figure 5J and Figure 5—figure supplement 1B). But at end-stage, Tubb4a^D249N/D249N mice demonstrate a significant loss of striatal neurons compared to WT mice (p<0.01, Figure 5I–J).

In order to explore the effect of Tubb4a mutations in OLs and neurons, independent of their in-situ environment, we studied in vitro cultures derived from WT, Tubb4a^D249N/+, and Tubb4a^D249N/D249N mice. OPCs were examined using A2B5 stain and a significant decrease is observed in the number of A2B5+ cells isolated from Tubb4a^D249N/+ and Tubb4a^D249N/D249N mice compared to control WT mice (Figure 6—figure supplement 1A–D; p<0.01). Next, to assess if the decrease in OPCs is due to proliferation or cell death, A2B5 cells were co-labeled with Ki-67 or Caspase3 respectively. A significant decrease was noted in the number of A2B5+ Ki-67+ proliferating OPCs in Tubb4a^D249N (p<0.05) and Tubb4a^D249N/D249N (p<0.01) group compared to the WT mice (Figure 6—figure supplement 1A–C, Figure 6—figure supplement 1E). No changes were observed in the number of A2B5+ Caspase3+ cells between the three groups (Figure 6—figure supplement 1F). The OPCs were then differentiated towards an OL fate and were examined for PLP, a mature OL marker co-localized with Olig2, a pan OL lineage marker. A significant decrease in the number of PLP+ OLs is observed (Figure 6A–C) in Tubb4a^D249N/+ mice (p<0.05) and Tubb4a^D249N/D249N mice (p<0.01) compared to mature OLs derived from WT mice (Figure 6E). However, the total number of Olig2+ cells are similar in all the groups (Figure 6D), resulting in a significant reduction in the proportion of mature OLs from the total cells committed to the OL lineage (PLP+/Olig2 cells, Figure 6F) in Tubb4a^D249N/+ (p<0.01) and Tubb4a^D249N/D249N mice (p<0.01) compared to WT mice. These results overall reflect similar changes in OL development as seen in vivo in the mouse tissues and support a cell-autonomous contribution of Tubb4a^D249N/+ mutation in the development of OL lineage cells.

We also examined cortical neurons using in vitro cultures derived from WT, Tubb4a^D249N/+ and Tubb4a^D249N/D249N mice. We analyzed survival based on the number of neurons labeled with Tuj1 and MAP2 stains at one-week post-plating. Tubb4a^D249N/D249N neurons display a significant decrease in survival relative to WT neurons (Figure 6G–I, p<0.01) and Tubb4a^D249N/+ neurons. We also assessed whether Tubb4a^D249N affects neuronal morphology, by measuring axonal outgrowth and total dendritic length. Axon length is shorter in Tubb4a^D249N/+ neurons (Figure 6J) and significantly shorter in Tubb4a^D249N/D249N neurons than WT neurite length (p<0.05). Total dendritic length is not significantly altered across genotypes (Figure 6K), although Tubb4a^D249N/D249N neurons shows a trend for shorter dendritic arbors. Overall, H-ABC associated Tubba^D249N mutation affects neuronal development, particularly axonal elongation.

We next investigated whether Tubb4a mutation affects microtubules dynamics in the distal axons of cortical neurons expressing mCherry-tagged EB3, a protein that forms comet-like structures at the growing plus-ends of microtubules from WT, Tubb4a^D249N/+ and Tubb4a^D249N/D249N mice (Figure 7A; Stepanova et al., 2003). The average number of EB3 comets is not significantly different in neurons from WT, Tubb4a^D249N/+, or Tubb4a^D249N/D249N mice (Figure 7B). However, we noted that the Tubb4a^D249N/D249N data shows a greater variance (Browne-Forsythe test for equality of variances revealed a significant difference among the conditions, p<0.05) compared to WT and Tubb4a^D249N/+ neurons. Histogram analysis reveals two distinct populations within the Tubb4a^D249N/D249N dataset, with some neurons displaying a lower density of EB3-comets and others a higher density of comets than seen in neurons from either the WT or heterozygous mice (Figure 7C). EB3 comet run-time (Figure 7D) in Tubb4a^D249N/D249N neurons is significantly shorter (p<0.001) compared to WT neurons and Tubb4a^D249N/+ neurons. Similarly, EB3 comet run-length in Tubb4a^D249N/D249N and Tubb4a^D249N/+ neurons is significantly shorter than in WT neurons (p<0.001, Figure 7E). In contrast, EB3 comet velocity is similar across all genotypes (Figure 7—figure supplement 1), indicating that microtubule growth rates were not affected.

Heterozygous Tubb4a^D249N/+ mice were generated using CRISPR-Cas9 technology by inserting the c.745G > A (p.Asp249Asn) mutation in exon 4 of the mouse Tubb4a gene. Methodologies are available in Appendix 1 and gRNA sequences is listed in the key resources table. Sequencing of other areas with high homology to the target sequence was performed to assess that only variants in the Tubb4a gene were retained in the final mouse line. Control (WT) mice were generated from breeding pairs of heterozygous Tubb4a^D249N/+ mice to further provide reassurance of limited of target effects in other homologous genes.

We established one founder line of Tubb4a^D249N/+ mice, which were bred to produce wild type (WT), Tubb4a^D249N/+, and Tubb4a^D249N/D249N mice. The animals were genotyped at all experimental steps and were maintained under a 12 hr (h) light:12 hr dark cycle in a clean facility and given free access to food and water. The methods and study protocols were approved in full by the Institutional Animal Care and Use Committee of the Children’s hospital of Philadelphia and conformed with the revised National Institutes of Health Office of Laboratory Animal Welfare Policy.

Ambulatory angle, ambulation, hanging grip strength, righting reflex (Feather-Schussler and Ferguson, 2016), and rotarod (Shiotsuki et al., 2010) were assessed at defined developmental intervals (Figure 1C). Detailed methods are provided in SI Methods. A cohort of at least 10 animals per genotype were included for the behavioral tests.

To determine the relative expression of Tubb4a in brain, fresh brain areas as cerebellum, cortex, hippocampus, hypothalamus, pre-frontal cortex, striatum and spinal cord were dissected at end-stage (~P35-P40) and snap frozen. RNA extraction, C-DNA and qPCR details are provided in Appendix 1.

Mice were anesthetized based on weight with ketamine and xylazine and transcardially perfused with 4% paraformaldehyde (PFA) after an initial flush with 1X PBS. Brains were collected and post-fixed with 4% PFA in 1X PBS overnight, infiltrated in 30% sucrose in 1X PBS, embedded in optimal cutting temperature compound and sectioned either as coronal or sagittal (50 μm) sections on a cryostat microtome (CM 3050 s, Leica biosystems, USA).

Myelin quantification was performed by Eriochrome Cynanine (Eri-C) stain, PLP and MBP in the densely myelinated region- corpus callosum (Baxi et al., 2017) and cerebellum. Eriochrome Cynanine (Eri-C) and neurofilament staining was according to previously published protocols (Sahinkaya et al., 2014) (Appendix 1). For Nissl staining, frozen sections were stained with 0.1% cresyl violet for 15 min, rinsed with PBS, dehydrated with graded alcohols (from 70–100%), followed by xylene, and mounted with Permount. For immunofluorescence staining, free floating sections were blocked with 2% bovine serum albumin and 0.1% Triton X-100 for 1 hr at room temperature and then sequentially incubated with primary antibodies overnight at 4°C and the fluorescent secondary antibodies for 1 hr at room temperature. Primary antibodies are listed in the Key Resource Table above. AlexaFluor-488 or AlexaFluor-647 conjugated secondary antibodies against rabbit, mouse, or rat (1:1000; Invitrogen) were used, and nuclei were counterstained with DAPI. Image acquisition and image quantification details are provided in the SI.

To determine the relative levels of key myelin proteins in the affected regions of our mouse model and corresponding to areas of disease in H-ABC, immunoblotting was performed on cerebellum and forebrain at post-natal (P)14, P21, and end-stage (~P35-P40). The details of immunoblotting are explained in SI.

A separate cohort of mice were perfused transcardially with 0.9% saline followed by 2% PFA and 2% glutaraldehyde in 0.1M phosphate buffer (PB; pH 7.4) at end-stage (~day 35) (n = 3/group) (Lancaster et al., 2018). The optic nerve and cervical spinal cord were examined as they are highly myelinated and routinely used for studying myelin ultrastructure (Lancaster et al., 2018). The optic nerve and cervical spinal cord were dissected, post-fixed for 24 hr, rinsed in 0.1M PB, transferred to 2% OsO4 in 0.1M PB for 1 hr, then processed for embedding in Epon (Lancaster et al., 2018). Semi-thin sections were cut, stained with alkaline toluidine blue, and visualized using a light microscope (Lecia DMR) interactive software (Leica Application Suite). Ultra-thin sections (70 nm) were cut, stained with lead citrate and uranyl acetate, and imaged using Jeol-1010 transmission electron microscope. The images for EM sections were assessed using Image J software and the inner and outer axonal area was measured for g-ratio analysis and quantified in 50 axons per animal with n = 3 per group as previously published (Lancaster et al., 2018). The axons were classified as normally myelinated, unmyelinated axons (no myelin detected) or thinly myelinated (compared to normal WT tissue) in WT, Tubb4a^D249N and Tubb4a^D249N optic nerves (n = 3/group). The percentage of these three groups were plotted and the data was analyzed.

Primary oligodendrocyte precursor cells (OPCs) were isolated from cerebral cortices of WT, Tubb4a^D249N/+ and Tubb4a^D249N/D249N mice between postnatal day P4-P7 using the Miltenyl anti-O4 microbeads (Flores-Obando et al., 2018) (Appendix 1). O4+ cells were plated at the density of 20,000 OPCs per well of a 24 well plate, allowed to proliferate for 5–7 days, following which they were differentiated in the media without platelet-derived growth factor and basic fibroblast growth factor and with thyroxine T4 (20 μg/ml) for another 5 days. The cells were then fixed with 4% PFA, washed twice with 1X PBS, permeabilized with 0.2% Triton X-100, blocked in 10% normal goat serum solution for 1 hr, and incubated overnight at 4°C with primary antibodies – rabbit anti-Olig2 (1:800; EMD Millipore, Cat: AB9610), rat anti-PLP (1:1), mouse anti-A2B5 (1:1), rabbit anti-Caspase3 (1:200) and rat anti-MBP (1:1) (IDDRC hybridoma, courtesy Dr. Judith Grinspan). The cells were washed the next day in PBS, and incubated with the appropriate secondary fluorescent antibodies (1:500; anti-rat IgG Alexa Fluor 488, anti-rabbit IgG Alexa Fluor 647; 1:500; anti-mouse IgM Alexa Fluor 488), mounted in Prolong gold antifade reagent (Thermo Fisher Scientific), and imaged using a Nikon microscope with 20 × or 40 × objectives for analysis of cellular counts. Each experiment was repeated at least three separate times and based on the litters, 1–3 pups of the same genotype were used with technical replicate of n = 3/ pup for each experiment.

Primary cortical neurons were isolated from E15.5 embryos of WT, Tubb4a^D249N/+, and Tubb4a^D249N/D249N mice as described previously (Guedes-Dias et al., 2019). In brief, the cortex was dissected from each embryo and washed with Hanks' Balanced Salt Solution (HBSS), then 2.5% trypsin was added to each sample and incubated at 37°C for seven mins. The trypsin was removed and washed four times with fresh warm HBSS and then resuspended in attachment medium (described in SI). The cells were dissociated and plated on poly-L-lysine (PLL) coated MatTek dishes at a density of 150,000 cells/plate for imaging and 100,000 cells/well of a 24 well plate. The medium was changed after 4 hr to pre-equilibrated maintenance media (described in SI). After 3 days, 20–30% of the medium was replaced with fresh medium supplemented with the mitotic inhibitor 1 μM Cytosine arabinoside. The neurons were plated at equal densities for all genotypes in 24-well plate were assessed for cell survival, axonal and dendritic length at 1 week post-plating. Neurons were immunostained with anti-microtubule associated protein (MAP2; 1:200; Sigma, Cat: 1406) and anti- Neuron-specific Class III β-tubulin (TuJ1; 1:200; Abcam, Cat: ab18207), labelled by appropriate secondary fluorescent antibodies (1:500; anti-rat IgG Alexa Fluor 488, anti-rabbit IgG Alexa Fluor 647), and imaged using a Nikon microscope with at 20 × or 40 × objective for cellular counts and measuring axonal and dendritic length using the Neurite tracer plugin in FiJi software. Each experiment was repeated at least three separate times and based on the litters, 1–3 pups of the same genotype were used with technical replicate of n = 3/ pup for each experiment.

On day in vitro (DIV) 6, cortical neurons were transfected with end-binding protein 3 (EB3) -mCherry using Lipofectamine 2000 (Invitrogen). 20–24 hr after transfection, maintenance medium was exchanged to low fluorescence Hibernate E imaging medium (BrainBits) supplemented with 2% B27 and 2 mM GlutaMAX. The neurons were imaged in an environmental chamber at 37°C on a PerkinElmer UltraView Vox Spinning Disk Confocal system with a Nikon Eclipse Ti inverted microscope using a Plan Apochromat 60 × 1.40 NA oil immersion objective. Axons were identified by established morphological criteria and images were acquired with a Hamamatsu EMCCD C9100-50 camera driven by Volocity software (PerkinElmer) at a rate of 2 s per frame for 600 s. Quantification of EB3 dynamics was performed as previously described (Guedes-Dias et al., 2019). The ImageJ macro toolset KymoClear was used to generate kymographs (Mangeol et al., 2016). The KymoClear toolset passes a Fourier filter on the original kymograph allowing for automated discrimination of anterograde, retrograde or static components and improves the signal-to-noise ratio of EB3 comets without affecting quantitative analysis of the data. The tracks of individual EB3 comets were manually traced using a custom MATLAB GUI (Kymograph Suite) and used to determine run-length, run-time, and velocity of each comet. The investigator was blinded for the neuronal genotype during both image acquisition and kymograph analysis. Each experiment was repeated at least three separate times and based on the litters, 1–3 pups of the same genotype were used with technical replicate of n = 3/ pup for each experiment.

All graph data are presented as the mean ± standard error mean (SEM). For mouse studies, ‘n’ represents the number of animals used per experiment unless indicated otherwise. Gait abnormalities, righting reflex, rotarod, and weight assessments were analyzed by two-way ANOVA with repeated measures followed by the post-hoc Tukey test. For grip strength and ambulation, one-way ANOVA with post-hoc Tukey test was performed. Survival was analyzed by the Kaplan-Meier method, and the differences between groups were estimated by the Gehan-Breslow-Wilcoxon test. Comparisons in myelin quantification, NeuN, ASPA, NG2, Olig2, Ki-67 and cleaved caspase three counts and fluorescent intensity were analyzed by ordinary two-way ANOVA with multiple comparisons post-hoc Tukey tests. EM analysis for G-ratio and axon diameter was conducted using one-way ANOVA with the post-hoc Tukey test and the % myelin analysis was conducted with two-way ANOVA followed by post-hoc Tukey test. Neuronal survival, axon and dendritic length, and assessment of OL markers examined in vitro were compared using one-way ANOVA with the post-hoc Tukey test. EB3 dynamics in neurons were analyzed using Kruskal-Wallis test and Dunn’s multiple comparisons test. All statistical analyses were performed using Prism 7.0 (GraphPad Software) with p<0.05 considered statistically significant.