Human CFEOM1 mutations attenuate KIF21A autoinhibition and cause oculomotor axon stalling

Abstract

The ocular motility disorder "Congenital fibrosis of the extraocular muscles type 1" (CFEOM1) results from heterozygous mutations altering the motor and third coiled-coil stalk of the anterograde kinesin, KIF21A. We demonstrate that Kif21a knockin mice harboring the most common human mutation develop CFEOM. The developing axons of the oculomotor nerve's superior division stall in the proximal nerve; the growth cones enlarge, extend excessive filopodia, and assume random trajectories. Inferior division axons reach the orbit but branch ectopically. We establish a gain-of-function mechanism and find that human motor or stalk mutations attenuate Kif21a autoinhibition, providing in vivo evidence for mammalian kinesin autoregulation. We identify Map1b as a Kif21a-interacting protein and report that Map1b⁻/⁻ mice develop CFEOM. The interaction between Kif21a and Map1b is likely to play a critical role in the pathogenesis of CFEOM1 and highlights a selective vulnerability of the developing oculomotor nerve to perturbations of the axon cytoskeleton.

Figure 1. Kif21a R954W KI mice recapitulate human CFEOM1

(A) Schematic of KIF21A protein highlighting amino acid substitutions (red) and single residue deletion (purple) resulting from human CFEOM1 mutations. The number of reported unrelated probands indicated within parenthesis if more than one. (B) Alignment of human and mouse KIF21A demonstrates conservation of the motor domain (MT1) and two 3^rd coiled-coil stalk domain (MT2 and MT3) substitutions studied. (C) 129/S1 Kif21a^+/+ mouse with normal eyes. (D, E) 129/S1 Kif21a^KI/KI mice with bilateral (D) and unilateral (E) ptosis and globe retraction. (F) Penetrance of eye phenotype in adult Kif21a^+/KI and Kif21a^KI/KI mice. (G) Bilateral motor neuron cell counts of adult Kif21a^+/+ versus Kif21a^KI/KI mice: OMN 1023±31 vs. 612±54; abducens 137±6 vs. 113±5; facial 1628±75 vs. 1579±40; trochlear 249±25 vs. 239±22; n= 4,4 **p<0.001, ***p<0.0001. (H–K) H&E stained cross-sections of EOMs posterior to the globe from adult Kif21a^+/+ (H, J) and Kif21a^KI/KI (I, K) mice, with LPS and SR at higher magnification in J and K (n=6,6). LSP indicated by arrow. Scale bars (H, I) 200 μm, (J, K) 50 μm. (L–Q) Confocal images of isolated EOM innervation after anterograde lipophilic dye tracer studies of ocular cranial nerves in P0 Kif21a^+/+ (L–N) and Kif21a^KI/KI (O–Q) mice. Note decreased innervation received by LPS (L, O), SR (M, P), and LR (N, Q) in Kif21a^KI/KI compared to Kif21a^+/+ mice (n= 5,5). Dashed lines outline boundary of muscle bodies oriented from origin (top) to insertion (bottom). Scale bars 100 μm. Abbreviations: optic nerve (ON), levator palpebrae superioris (LPS), superior rectus (SR), lateral rectus (LR), medial rectus (MR), inferior rectus (IR). See also Figure S1.

Figure 2. Oculomotor nerve pathology in Kif21a^KI/KI mice

(A–B) Whole mount fluorescent immunostaining with anti-NF (red) and anti-GFP (green) antibodies of E11.5 Kif21a^+/+;Isl^MN:GFP (A) and Kif21a^KI/KI;Isl^MN:GFP (B) embryos (n=22,20) reveal similar trajectories of WT and mutant cranial nerves. Arrowhead in (A) points to VI nerve; arrow in (B) highlights distal thinning of III nerve in the mutant compared to WT. Scale bar 200 μm. (C–H) Images of dorsal view of flat-mounted midbrain tissue from Kif21a^+/+;Isl^MN:GFP (C, E, G) and Kif21a^KI/KI;Isl^MN:GFP (D, F, H) embryos at E10.5 (C, D), E12.5 (E, F), E13.5 (G, H); n=5,5 for each age. Note the normal appearance of mutant III and IV nuclei and exiting nerves. At E12.5 and E13.5, mutant III nerve is thickened following exit into the periphery compared to WT (F, H, arrows). Scale bar 100 μm. (I, J) Confocal images of proximal III nerves oriented with brainstem to the left and orbit to the right from E12.5 Kif21a^+/+;Isl^MN:GFP (I) and Kif21a^KI/KI;Isl^MN:GFP (J) embryos reveal a bulb within the proximal nerve (arrow) followed by distal thinning (arrowhead) in all mutant and no WT nerves (n=20,20). Scale bar 100 μm. (K–N) Confocal images of WT and mutant III nerves stained with anti-NF antibody. (K) and (L) correspond to regions of arrow and arrowhead in (I), while (M) and (N) correspond to regions of arrow and arrowhead in (J) (n=3,3). Scale bars 20 μm. (O–T) Stitched images of multi-color anterograde labeled III axons in Kif21a^+/+ (O, Q, S) and Kif21a^KI/KI (P, R, T) at E12.5. OMNsd (Sd) contralateral neurons are red (Q, R), while both ipsilateral and contralateral neurons are green (O, P). When merged (S, T), Sd axons from the contralateral nucleus appear yellow, while ipsilateral OMNid (Id) axons remain green. Primarily contralateral yellow axons (T) terminate within the bulb (arrow in P, R) and along the nerve, failing to reach the developing eye (white circle) in Kif21a^KI/KI but not Kif21a^+/+ embryos (n=3,3) Scale bar 200 μm. (U–Z) Confocal images highlight normal nuclear formation and normal density of the axons of Sd motor neurons as they cross the midline in mutant versus WT mice (U–X are higher magnification of E–H). Scale bar 200 μm. (AA–AF) Ventricular view of flat-mounted midbrains with motor neurons in the OMN nucleus labeled retrogradely following dye application in the orbit of one eye of E12.5–E14.5 Kif21a^+/+ (AA, AC, AE) or Kif21a^KI/KI (AB, AD, AF) embryos (n=12,11). In WT embryos, dye fills the axons of the Sd motor neurons crossing the midline of Kif21a^+/+ embryos. In contrast, in mutant embryos, fewer Sd axons are retrograde labeled from the orbit in Kif21a^KI/KI embryos, despite similar density by GFP detection (refer to U–Z). Scale bar 30 μm. Abbreviations: III=OMN nerve, IV=trochlear nerve, Vs=sensory branches of the trigeminal nerve, Vm=motor branch of the trigeminal nerve, VI=abducens nerve, VII=facial nerve, IX=glossopharyngeal nerve, XI=accessory nerve, XII=hypoglossal nerve, E=eye, Sd=OMNsd, Id=OMNid. See also Figure S2.

Figure 3. Failure of oculomotor axon development is the primary defect in CFEOM1

(A–J) Confocal images of ocular motor nerves dissected from Kif21a^+/+;Isl^MN:GFP and Kif21a^KI/KI;Isl^MN:GFP mice, with brainstem to the left and eye to the right. III nerve and its branches labeled in red text, IV and VI nerves labeled in yellow text. (A, B) Low magnification of ocular motor nerves from E12.5 WT (A) and mutant (B) mice; n=10, 10. Note the proximal bulb (*) and distal thinning of III with hypoplasia of the distal superior (Sd) and inferior (Id) divisions of the mutant nerve, and thinning of VI (arrowhead) compared to WT. Scale bars 200 μm. (C–J) Distal OMN nerves in WT (C, E, G, I) and mutant (D, F, H, J) mice at E11.5 (C, D), E12.5 (E, F), E13.5 (G, H), and E15.5 (I, J); n=10, 10 for each age. At E11.5, both WT and mutant nerves reach the orbit and appear to explore the local environment, but mutants have fewer exploring axons within the distal decision region (C, D). At E12.5, a small, defasciculated Sd protrudes from the WT nerve, and the Id exhibits an enlarged distal region with a subset of axons pursuing convoluted trajectories (E). In contrast, the E12.5 mutant nerve (F) has thinner Sd and Id, and several aberrant, fasciculated branches (arrows) emerge from the Id exploratory region. At E13.5, the WT nerve (G) has a more substantial Sd with several fasciculated bundles, while the mutant (H) Sd is thin or absent, and the Id exploratory region is small and has aberrant branches (arrow) as well as the normal branch to the IO. At E15.5, WT III (I) appears fully developed, with Sd branches to the SR and LPS. The Id now sends off several branches, presumably to the MR, IR, and IO. In comparison, mutant III (J) Sd is severely hypoplastic, while the Id is thin but sends off several stereotyped branches similar to WT, with no visible aberrant branches. Compared to WT, mutant VI was also hypoplastic compared to WT (arrow head) while mutant IV appeared normal. (K) Onset of Kif21a expression in EOMs during development. Immunostaining with anti-Kif21a antibody (red) and anti-Myo antibody (green) on coronal sections through the eye of E12.5, E13.5 and E14.5 WT mice. Kif21a is detected in the retina and in rootlets of many developing nerves (including III and VI, arrowheads) but is absent from green EOMs (arrows) until E14.5, subsequent to its neuronal expression. (L) Histological sections of the mouse orbit at E14.5 reveal proper size and positioning of EOMs in Kif21a^KI/KI compared to Kif21a^+/+ mice. (M–R) Fluorescent whole mount imaging of developing III nerves stained with anti-NF antibody (red) and anti-Tuj1 antibody (green) at E10.5 (M, N), E11.5 (O, P), and E12.5 (Q, R) from Kif21a^+/+;Isl^MN:GFP WT (M, O, Q) and Kif21a^KI/KI;Isl^MN:GFP mutant (N, P, R) embryos. Anti-Tuj1 staining was omitted from E12.5 because of high background at this age. Arrowheads and arrows indicate proximal and distal OMN nerve at points of measurement in (S). Scale bars 100μm. (S) Quantification of proximal (odd numbered solid bars) and distal (even numbered hatched bars) III nerve diameters in (M–R) from left and right OMN nerves from at least n=3 Kif21a^+/+;Isl^MN:GFP (blue bars 1–6) and Kif21a^KI/KI;Isl^MN:GFP (green bars 7–12) embryos at E10.5–E12.5. *Significant p-value < 0.004. P-values: 1:2<0.001*, 1:3=0.032, 1:5=0.000*, 1:7=0.098, 2:4<0.001*, 2:6<0.001*, 2:8=0.713, 3:4=0.010, 3:5=0.195, 3:9=0.335, 4:6=0.001*, 4:10=0.006, 5:6=0.148, 5:11=0.025, 6:12<0.001*, 7:8=0.007, 7:9=0.541, 7:11=0.004, 8:10=0.056, 8:12=0.037, 9:10<0.001*, 9:11=0.059, 10:12=0.990, 11:12=0.001*. (T) Increased apoptosis of III neurons is observed at E12.5 and 14.5 in Kif21a^KI/KI (right) compared to Kif21a^+/+ (left) mice. Note a similar proportion of Isl1-positive motor neurons crossing the midline (*) in WT and mutant mice, and an increase in Caspase3+ cells in Kif21a^KI/KI embryos both within the III nuclei and in neurons crossing the midline at E14.5. (U) Quantification of Caspase3+ cells in Kif21a^+/+ and Kif21a^KI/KI embryos demonstrates increased apoptosis at E12.5–E14.5 in the III but not the IV nuclei; (n=4,4) at each stage. Caspase3+ neurons in the III nucleus bilaterally of WT versus Kif21a^KI/KI were E11.5, 6±0.5 vs. 7 ±1; E12.5, 8±1 vs. 34±3; E13.5, 28±2 vs. 68±3; E14.5, 43±2 vs. 76±3; E15.5, 43±1 vs. 44±0.5; **p<0.001. Abbreviations: Sd=OMNsd, Id=OMNid, IO=inferior oblique branch, O=optic nerve and as per Figure 2. See also Movies S1 and S2.

Figure 4. Ultrastructural analysis of developing oculomotor nerves in Kif21a^+/+ and Kif21a^KI/KI embryos

(A–E) Stitched electron microscopy (EM) images (4800x) of E12.5 Kif21a^+/+;Isl^MN:GFP (A, B) and E12.5 Kif21a^KI/KI;Isl^MN:GFP (C, D, E) III nerve cross-sections. (A) and (C) are equivalent proximal levels while (B) and (D) are equivalent distal levels, and correspond to cross-sections of the nerve just proximal and distal to the bulb (E) in the mutant nerve. Scale bars 10 μm. (F–M) EM images (13000x) from cross-sections in (A–E). Proximal (F, H) and distal (G, I) sections of WT (F, G) and mutant (H, I) nerves consist primarily of axons running parallel to the nerve trajectory with a few central growth cones (stars) and lamellipodia/filopodia (asterisk). Cross-section of mutant bulb (J–M) contain highly disorganized axons and increased numbers of longitudinal axons (arrows), enlarged central growth cones (stars), lamellipodia/filopodia (asterisks), aggregation of mitochondria, membranes and vesicles (empty arrowheads), and degenerating axons (filled arrowheads). Scale bars 500 nm. (N, O) Stacked graphs showing numbers (N) and average areas (O) of cross-sectional, longitudinal, and degenerating axons, of central growth cones, and of lamellipodia/filopodia from two mutant and two WT III nerves at cross-sectional levels shown in A–E. The number/value for each object is provided within or above each fraction. #1 and #2 = WT nerves; #3 and #4 = mutant nerves; p=proximal; d=distal; b=bulb. See also Figure S3.

Figure 5. Loss of FL Kif21a does not cause CFEOM1

(A–H) OMN anterograde dye tracer studies in Kif21a^+/+ and Kif21a^KOMT/KOMT embryos (n=10, 9), as shown for Kif21a^KI/KI mice in Figure S2O, reveal normal exit, fasciculation, and trajectory of III axons and absence of a bulb or distal thinning at E12.5 (A–D) and E13.5 (E–H). Scale bars 100 μm (A, C, E, G), 20 μm (B, D, F, H). (I, J) Islet1 and Cas3+ immunohistochemistry of E14.5 WT (I) and Kif21a^KOMT/KOMT (J) III nuclei reveal Islet1+ motor neurons crossing the midline and several Cas3+ apoptotic neurons. (K) Quantification of the number of Cas3+ cells in (I) and (J) reveal absence of pathological apoptosis in III nuclei of Kif21a^KOMT/KOMT compared to Kif21a^+/+ embryos at E13.5 (n=6,6, 29±2 vs. 30±2, p=0.206) and E14.5 (n=6,6, 48±3 vs. 46±2, p=0.334). (L) Representative western blot analysis of Kif21a protein levels in E18.5 brain tissues corresponding to genotypes in allelic series (n=4). Note lowest levels of Kif21a protein detected in Kif21a^KI/KOMT mice. (M) Summary of genotypes and phenotypes from allelic analysis of Kif21a mutant mice. Percentages are relative to WT/WT mice. A 2X2 contingency table with Fisher’s exact test was used to determine differences in penetrance between Kif21a^KI/KOMT and Kif21a^KI/KI mice (p=0.0001). (N, O) E18.5 Kif21a^+/+ or Kif21a^KI/KI brain lysates were incubated with polymerized microtubules and AMP-PNP or ATP and microtubule co-sedimentation assays performed. Representative western blot (N) and quantification (O) show significantly increased relative levels of Kif21a in microtubule pellet fraction (P2) for endogenous mutant Kif21a compared to WT Kif21a (n=3). Mean ± SEM. **p<0.01, ***p<0.001, ns=not significant. (P) Names and corresponding schematics of the FL and truncated Kif21a constructs. Kif21a domains are noted above the top left construct. Constructs containing mutant amino acid residues corresponding to MT1, MT2, or MT3 have the amino acid substitution represented in red font. Construct code names begin or end with the tag designation: “G-“ at the start denotes an N-terminal GFP tag, while “-G”, “-mCh”, or “-M” at the end denote a C-terminal GFP, mCherry, or Myc tag, respectively; “m” or “h” prior to the parenthesis denotes mouse or human construct, respectively; the number within the parentheses denotes the amino acid residues contained in the construct, and is followed by MT1, MT2, or MT3 if the construct contains a CFEOM1 amino acid substitution. Mouse and human constructs have slightly different amino acid numbering. See also Figure S4.

Figure 6. CFEOM1 mutations disrupt Kif21a intramolecular interactions between the motor and 3^rdcoiled-coil domains, attenuate autoinhibition, and enhance Kif21a microtubule binding without altering run length or velocity

(A and B) Mouse GFP-fused FL WT, MT1, MT2, or MT3 Kif21a, or stalk-truncated WT or MT1-mutant Kif21a was over-expressed in HEK293 cells, and microtubule co-sedimentation assays performed. Representative western blot (A, n=3) and quantification (B) show significantly increased relative levels in the microtubule pellet fraction (P2) of the three mutant FL Kif21a and both WT and MT1 stalk truncations compared to the WT FL Kif21a. Mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ns=not significant. (C) Representative western blot (n=3) shows that over-expressed GFP-fused motor (G-m(1-417)) domain specifically co-immunoprecipitates with Myc-tagged 3^rd coiled-coil (h(891-1300)-M) domain and not with 1^st and 2^nd coiled-coil (h(385-890)-M) or WD40 (h(1301-1674)-M) domain from HEK293 cell lysates using anti-myc antibody, and 3^rd coiled-coil domain CFEOM1 mutations (MT2 and MT3) disrupt this co-immunoprecipitation. (D) Representative western blot (n=3) shows that introduction of the MT1 mutation into over-expressed GFP-fused motor domain disrupts immunoprecipitation with WT Myc-tagged 3^rd coiled-coil domain from HEK293 cell lysates using anti-myc antibody. (E–J) Representative kymographs showing the displacement on a microtubule over 5 minutes in BRB80 motility buffer of mouse GFP-fused Kif21a: (E) WT FL; (F) WT truncation; (G) MT1-mutant truncation; (H) WT truncation plus 50uM purified WT 3^rd coiled-coil domain; (I) WT truncation plus 50uM purified MT3 3^rd coiled-coil domain; (J) MT1-mutant Kif21a truncation plus 50uM purified WT-3CC. Scale bars: x axis = 5 μm, y axis = 1min. (K) Quantification of (E–J) reveals Kif21a WT {G-m(1-917)} and mutant {G-m(1-917MT1)} truncations have significantly higher microtubule landing frequencies compared to that of Kif21a WT FL {G-m(1-1573)}. Purified WT-3CC inhibits landing frequencies of WT Kif21a truncation, while CFEOM1 mutations (MT1 and MT3) have a reduced inhibitory effect. (L–N) Representative kymographs showing the displacement on a microtubule over 5 minutes in BB30 motility buffer of mouse GFP-fused Kif21a: (L) WT FL; (M) MT3-mutant FL; and (N) MT1-mutant FL. Scale bars as per E–J. (O, P) Quantification of mouse WT FL or mutant (MT3 and MT1) FL Kif21a on microtubules for 10 minutes in BRB30 motility buffer reveals a significant increase in landing frequencies (O) of MT3 and MT1 mutant FL Kif21a compared to WT, but no significant changes in velocity or run length (P). For each construct, data were acquired from two independent experiments, and from three time-lapsed images for each experiment. Velocity: mean ± SD, run length: mean ± SEM. *p<0.05, **p<0.01, ns=not significant. Constructs are per Figure 5P. See also Figure S5 and Movies S3–S9.

Figure 7. Oculomotor growth cones in Kif21a^KI/KI explant cultures have increased growth cone size and number of filopodia

(A) Representative immunofluorescent images showing Thy1:GFP WT and Kif21a^KI/KI OMN explants cultured for 3 days. Scale bars 400 μm. (B) Quantification of axon outgrowth from (A) shows no significant differences in OMN axon outgrowth between WT/WT and KI/KI mice (n=5,3). Mean ± SEM. (C–F) Percentage of growth cones with forward, stationary, or retracted movements (C), forward distance travelled (D), total displacement (E), and percent collapse (F) quantified from 30 minute recordings of Isl^MN:GFP Kif21a^KI/KI and WT OMN explants cultured for 17–20 hours. No significant differences between WT and Kif21a^KI/KI mice were detected (n=9,8). Mean ± SEM. (G) Representative immunofluorescent images of phalloidin-stained WT and Kif21a^KI/KI OMN axon growth cones in explants cultured for 18 hours from the Isl^MN:GFP mice. Scale bars 5 μm. (H, I) Quantification of (G) reveals a significant increase in growth cone area (H) and number of filopodia per growth cone (I) of Kif21a^KI/KI explants compared to WT (n=4 explants and 164 growth cones, n=3 explants and 139 growth cones). Mean ± SEM. *p<0.05. See also Figure S6.

Figure 8. Endogenous Kif21a and MAP1B interact, Map1b^−/− mice have CFEOM, and the penetrance of the oculomotor phenotype is greater in Map1b^+/−

;Kif21a^+/KI compared to single heterozygous embryos. (A) E18.5 WT and Kif21a^KI/KI brain lysates immunoprecipitated with control IgG, anti-Kif21a antibody, or anti-Map1b antibody and subjected to Western blotting using the antibodies indicated. WT and mutant Kif21a interact equally with Map1b in both directions, but do not interact with Map2. (B) Western blot of GST-pull down of E18.5 WT brain lysates by purified GST-motor, GST-Kif21a-3^rd coiled-coil and -WD40 domains. Kif21a interacts with Map1b through its 3^rd coiled-coil and WD40 domains. (C) E18.5 Map1b^+/+ or Map1b^−/− brain lysates were incubated with AMP-PNP or ATP and microtubule co-sedimentation assays were performed. Western blot shows similar relative levels of Kif21a and KHC in the microtubule pellet fraction (P2). (D, E) Map1b^+/+ mice have normal eyes (D), while Map1b^−/− mice have globe retraction and ptosis (E). (F, G) Fluorescent whole-mount imaging of E11.5 III nerve (arrow) from Map1b^+/+ (F) and Map1b^−/− mice (G) stained with anti-NF antibody (red). The nerves have similar trajectories (n=4, 4). (H–S) Confocal images of III, IV, and VI nerves (left column) and higher magnification of the distal III nerve (right column) from E12.5 Isl^MN:GFP embryos, oriented and labeled as described in Figure 3A–3J, with VI nerve (arrowhead), aberrant III nerve branches (arrows), OMN bulb (*). WT (H, I), Map1b^+/− (J, K), Map1b^−/− (L, M), Kif21a^+/KI (N–Q), double heterozygous Kif21a^+/KI:Map1b^+/− mice (R, S); n=10,10 for each genotype. Scale bars 200 μm (H, J, L, N, P, R) and 100 μm (I, K, M, O, Q, S). Abbreviations as per Figure 2.