Human CHN1 Mutations Hyperactivate α2-Chimaerin and Cause Duane's Retraction Syndrome

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Science  08 Aug 2008:
Vol. 321, Issue 5890, pp. 839-843
DOI: 10.1126/science.1156121


Duane's retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1, a gene on chromosome 2q31 that encodes α2-chimaerin, a Rac guanosine triphosphatase–activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase α2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance α2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant α2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that α2-chimaerin has a critical developmental function in ocular motor axon pathfinding.

Ocular motility and binocular vision depend on the precise innervation of six extraocular muscles by the oculomotor, trochlear, and abducens cranial motor neurons (fig. S1A) (1). Disruptions in these developmental processes can cause complex congenital eye movement disorders (2, 3), the most common of which is Duane's retraction syndrome (DRS) with an incidence in the general population of about 0.1%. Individuals with DRS have restricted abduction and in some cases adduction of their eyes, with retraction of the globe upon attempted adduction. Postmortem studies of sporadic DRS reveal absence of the abducens motor neurons and cranial nerve, with anomalous innervation of its target, the lateral rectus muscle, by a branch of the oculomotor nerve (fig. S1B) (4, 5).

Four pedigrees (IJ, UA, JH, and FY; fig. S1D) segregating a DRS variant as a dominant trait are reported to map to the DURS2 locus on chromosome 2q31 (68). Examinations of affected family members established that although some have a phenotype indistinguishable from sporadic DRS, overall they have a higher incidence of bilateral involvement and of vertical movement abnormalities (Fig. 1A) (810). Consistent with these clinical findings, our magnetic resonance (MR) imaging of members of pedigrees FY and JH revealed that, in addition to absent or hypoplastic abducens nerves and aberrant lateral rectus innervation by the oculomotor nerve, some individuals had hypoplastic oculomotor nerves and small oculomotor-innervated muscles (10). Thus, mutations in the DURS2 gene appear to affect primary development of the abducens and, to a lesser degree, the oculomotor nerve (fig. S1C).

Fig. 1.

Duane's retraction syndrome (DRS) and corresponding CHN1 mutations. (A) Affected member of pedigree JH with limited outward gaze (abduction) and narrowing of the palpebral fissure on attempted inward gaze (adduction) most obvious on leftward gaze. He also has bilateral exotropia on downgaze. (B) The seven DURS2-DRS pedigrees and corresponding heterozygous CHN1 mutations. (C) Schematic representation of α1-chimaerin (top, 334 amino acids) and α2-chimaerin (bottom, 459 amino acids) protein. The isoforms contain identical C1 and RacGAP domains; only α2-chimaerin contains an SH2 domain. Mutations alter residues unique to α2-chimaerin or common to both proteins, as indicated by the arrows. No mutations were found in the α1-chimaerin N-terminal sequence (highlighted in black).

To identify the DURS2 gene, we further analyzed the recombination events that defined the published DURS2 critical region (6, 7), reducing it from 9.9 to 4.6 Mb (fig. S2, A and B), and then sequenced 22 positional candidate genes (fig. S2B) in a proband from each of the four published pedigrees. We identified in each a unique heterozygous missense change in CHN1, which encodes two Rac-specific guanosine triphosphatase (GTPase)–activating α-chimaerin isoforms. We then screened 16 smaller pedigrees that segregated DRS in a dominant fashion, and identified three additional heterozygous CHN1 missense changes in pedigrees RF, IS, and AB (Fig. 1B and figs. S1E and S2C). All seven nucleotide substitutions cosegregated with the affected haplotypes, and none were present in online single-nucleotide polymorphism databases or on 788 control chromosomes. Five of the substitutions are predicted to result in nonconservative amino acid substitutions [Leu20 → Phe (L20F), Tyr143 → His (Y143H), Gly228 → Ser (G228S), Pro252 → Gln (P252Q), and Glu313 → Lys (E313K)] and two in conservative amino acid substitutions [Ile126 → Met (I126M) and Ala223 → Val (A223V)] (Fig. 1B). All are predicted to alter amino acids that are conserved in eight different species (fig. S2D).

The Rho family member Rac is a GTPase that is active when GTP-bound; it serves as a regulator of downstream intracellular signaling cascades controlling cytoskeleton dynamics, including the growth and development of dendrites and axons. Rac is inactivated by 12 Rac GTPase-activating proteins (GAPs) in the mammalian genome (11), including α1- and α2-chimaerin (encoded by CHN1) and paralogs β1- and β2-chimaerin (encoded by CHN2). In the rodent brain, α2-chimaerin has been shown to serve as an effector for axon guidance (1216), whereas α1-chimaerin appears to play a later role in dendritic pruning (17, 18).

CHN1 is alternatively spliced, and the α1-chimaerin promoter lies in intronic sequence upstream of α2-chimaerin exon 7 (19). Thus, the two isoforms share a RacGAP domain that interacts with and down-regulates Rac activity, as well as a C1 domain that binds to diacylglycerol (DAG), a membrane-associated phorbol ester signaling lipid. Only α2-chimaerin contains an N-terminal Src homology 2 (SH2) domain (20, 21). Three DURS2 mutations alter amino acids unique to α2-chimaerin, whereas four alter residues shared by α1- and α2-chimaerin (Fig. 1C and table S1). Because we cannot distinguish between the two groups clinically, the DURS2 phenotype most likely results from altered α2-chimaerin function.

In situ studies in rats (20, 21) revealed widespread embryonic neuronal expression of α2-chimaerin mRNA. Expression in the caudal brainstem and cephalic flexure peaked at embryonic day (E) 12.5, whereas mouse embryonic expression peaked overall at E10.5 (fig. S3, A and B), both consistent with expression of α2-chimaerin in developing ocular motor neurons. We found similar widespread expression of α2-chimaerin mRNA during human development, strongest at Carnegie stage (CS) 15 and CS16 in the midbrain and hindbrain (Fig. 2, fig. S3, C to E, and fig. S4). Therefore, although expressed in developing ocular motor neurons, the expression pattern alone does not account for the striking restriction of the DURS2 phenotype.

Fig. 2.

Human developmental expression profile of α2-chimaerin mRNA by in situ hybridization. (A) Transverse section of CS15 human embryo showing α2-chimaerin mRNA expression (purple deposit) in midbrain, hindbrain (rhombomere 2 indicated), and spinal cord. (B) Higher magnification of (A) showing expression in the ventricular layer of rhombomeres 3 and 4. (C) At CS16, expression is also seen in midbrain, hindbrain, spinal cord, and vestibulocochlear (viii) and vagus (x) nuclei. Higher magnifications of (C) show (D) expression in developing oculomotor neurons and (E) in neurons of rhombomeres 5 (developing abducens neurons) and 6. In CS19 sagittal section (F), expression has declined in basal midbrain and hindbrain and is now found in dorsal root ganglia, cerebellum, diencephalon, and telencephalon. At later stages (G), expression is located in specific regions of the cortical plate and the intermediate and ventricular zones of the forebrain (11 weeks post-ovulation). No signal was detected in corresponding sections hybridized with sense probe (fig. S4). Abbreviations: mb, midbrain; hb, hindbrain; rh, rhombomere; sc, spinal cord; o, oculomotor nucleus; drg, dorsal root ganglia; d, digit; cb, cerebellum; dc, diencephalon; t, telencephalon; c, cortical plate; i and v, intermediate and ventricular zones of the forebrain. Scale bars, 1000 μm [(A) and (C)], 100 μm [(B) and (E)], 200 μm (D), 2000 μm (F), 500 μm (G).

All seven amino acids altered by DURS2 mutations are conserved in α2-chimaerin's paralog, β2-chimaerin (fig. S5, A and B). Both molecules are predicted to exist in inactive, closed conformations in the cytoplasm, and to unfold and translocate to the membrane in response to DAG signaling, exposing their RacGAP domains and inactivating Rac (12, 22). β2-Chimaerin crystallization revealed that its inactive conformation is maintained by intramolecular interactions that impede access to the Rac and DAG binding sites (22). Modeling the DURS2 mutations onto the β2-chimaerin structure (fig. S5, C to E) (22) led to several predictions: (i) α2-Chimaerin Leu20 and Ile126 correspond to two of nine residues predicted by Canagarajah et al. to stabilize the β2-chimaerin closed conformation and, when mutated to alanine, were shown to enhance β2-chimaerin translocation to the membrane in vitro. (ii) Tyr143 is predicted to interact with Tyr221, and Ala223 is adjacent to Asn224 (which is predicted to interact with Tyr133); altering either of these residues may also destabilize the α2-chimaerin closed conformation. (iii) α2-Chimaerin Gly228 is the predicted DAG binding site. (iv) Glu313 is adjacent to the predicted Rac binding site. These predictions led us to hypothesize that DURS2 mutations hyperactivate α2-chimaerin RacGAP activity by destabilizing its closed conformation, or by directly altering DAG or Rac binding.

To determine whether DURS2 mutations alter the RacGAP activity of α2-chimaerin, we made full-length wild-type and mutant α2-chimaerin constructs that expressed equally stable proteins in human embryonic kidney (HEK) 293T cells and primary neurons (Fig. 3 and fig. S6A). Consistent with α2-chimaerin function, wild-type overexpression resulted in a reduction in Rac-GTP levels from baseline in HEK 293T cells (Fig. 3A). As predicted, overexpression of each mutant α2-chimaerin protein resulted in a significant further reduction in Rac-GTP levels relative to wild-type protein (Fig. 3, A and B), including when both wild-type and L20F α2-chimaerin were coexpressed together in the presence of the DAG analog, phorbol 12-myristate 13-acetate (PMA) (fig. S6, B and C). We conclude that all seven DURS2 mutations behave as dominant gain-of-function alleles (these and other data for each mutation are summarized in table S1).

Fig. 3.

DURS2-DRS mutations enhance α2-chimaerin function in vitro. (A) Rac-GTP levels were measured in HEK 293T cells transfected with plasmids encoding mycephexin, epitope-tagged V5–empty vector, V5–α2-chimaerin wild-type, or V5–α2-chimaerin mutants. Rac-GTP levels are reduced by overexpression of wild-type α2-chimaerin relative to empty vector, and are further reduced in cells expressing each mutant, but are elevated with overexpression of a guanine nucleotide exchange factor, myc-ephexin (27). (B) Densitometric analysis of Rac-GTP levels normalized to total Rac and V5–α2-chimaerin levels. Values are expressed as percent of wild-type α2-chimaerin (mean ± SEM, n = 6 to 10). The difference between the reduction of Rac-GTP levels for each mutant compared to wild-type α2-chimaerin is significant by one-way analysis of variance (ANOVA) with Dunnett's adjustment (F = 9.89, *P < 0.03, **P < 0.005, ***P < 0.0001). (C) α2-Chimaerin translocation examined by immunoblots of total, soluble, and pellet fraction of wild-type and mutant α2-chimaerin with or without 10 μM PMA stimulation. (D) Graphical representation of translocation after PMA treatment, expressed as percent of α2-chimaerin remaining in the soluble fraction (mean ± SEM, n = 3). Enhanced translocation compared to wild-type is significant for L20F, Y143H, A223V, and P252Q by one-way ANOVA with Dunnett's adjustment (F = 21.00, *P < 0.0001). (E) GFP–α2-chimaerin immunoprecipitates with V5–wild type or V5-L20F α2-chimaerin in the presence of PMA, and minimally in its absence. (F) In the presence of PMA, immunoprecipitation of wild-type α2-chimaerin is enhanced by all mutant α2-chimaerins relative to the wild type except G228S and E313K, which were equivalent to wild-type α2-chimaerin. Results were consistent over at least four independent experiments (see also fig. S6, F and G).

Next, we quantified the amount of wild-type and mutant α2-chimaerin translocated to the HEK 293T cell membrane before and after stimulation with PMA. About 15% of wild-type α2-chimaerin, but significantly greater fractions of L20F, Y143H, A223V, and P252Q α2-chimaerin mutant proteins, translocated to the membrane fraction in a PMA dose–dependent manner (Fig. 3, C and D, and fig. S6, D and E). Thus, these four mutant residues appear to enhance membrane translocation and RacGAP activity by destabilizing the closed conformation of α2-chimaerin in response to PMA.

Individuals with DURS2-DRS harbor one mutant and one wild-type CHN1 allele. Therefore, we performed coimmunoprecipitation experiments to investigate whether mutant hyperactivated α2-chimaerin could interact with the wild-type protein, thus potentially recruiting the wild-type pool to the membrane and further reducing Rac activity in vivo. α2-Chimaerin and each of the seven mutants were precipitated minimally by wild-type α2-chimaerin in the absence of PMA, and to a much greater extent in its presence; these results suggest that α2-chimaerin can complex with itself in a manner partially dependent on the PMA dose (Fig. 3, E and F, and fig. S6F). In addition, in the presence of PMA, the interaction of wild-type α2-chimaerin with all mutants except G228S and E313K was enhanced relative to its interaction with itself (Fig. 3F). Neither wild-type nor L20F α2-chimaerin coimmunoprecipitated with α1-chimaerin (fig. S6G); this result supports a direct or indirect association of α2-chimaerin with itself that may involve its SH2 domain.

On the basis of our findings that DURS2 mutations hyperactivate α2-chimaerin, we hypothesized that overexpressing α2-chimaerin may result in aberrant axon development in vivo. To test this idea, we used the chick in ovo system to overexpress α2-chimaerin in the embryonic oculomotor nucleus. This nucleus is more amenable than the abducens to electroporation, its development in chick has been defined (23), and we previously showed that some DURS2-DRS individuals have clinical and MR findings supporting a primary defect in oculomotor nerve development (810). Similar to rodents and humans, chick α2-chimaerin mRNA is expressed in neuroepithelia at stages of cranial motor neuron development (E4), and specifically in the developing oculomotor nucleus at the stage of axon extension and branching (E6) (Fig. 4, A and B). We electroporated embryonic chick midbrains with green fluorescent protein (GFP)–tagged wild-type and mutant α2-chimaerin (L20F and G228S) and GFP-alone control constructs at E2. These were analyzed between E5.5 (fig. S7), when oculomotor axons have extended along an unbranched trajectory to their distal target, the ventral oblique muscle (vo), and E6, when branching to the other target muscles has ensued (Fig. 4, C to I) (23). All 18 GFP control embryos showed a normal projection pattern in which the oculomotor nerve reached the ventral oblique muscle and branched correctly into other target muscles by E6 (Fig. 4D) (23). In the majority (71 to 87%) of embryos overexpressing wild-type or mutant constructs, the oculomotor nerve stalled and its axons terminated prematurely adjacent to the dorsal rectus muscle (Fig. 4, G to I). In addition, 67% of embryos overexpressing mutant constructs displayed aberrant branching and/or defasciculation of the oculomotor nerve, whereas only 24% of embryos overexpressing wild-type constructs did so (Fig. 4F and fig. S7, A to H). Regardless of the construct we used, the electroporated oculomotor nucleus appeared normal in size and neuron cell bodies displayed normal sorting, including normal migration across the midline (fig. S7, I and J) (23), consistent with a primary defect in axon rather than cell body development. Taken together, these observations suggest that elevated RacGAP activity as a result of hyperactivated mutant or overexpressed wild-type α2-chimaerin results in deregulation of normal oculomotor axon development.

Fig. 4.

α2-Chimaerin overexpression results in stalling of developing chick oculomotor nerves. (A) Transverse section through E4 whole chick embryo, showing wide neuroepithelial expression of α2-chimaerin mRNA including the hindbrain (hb), forebrain (fb), and trigeminal ganglion (tg). (B) Transverse section through E5–6 chick midbrain, showing α2-chimaerin mRNA expression in the oculomotor nuclei (left nucleus circled in white). (C) Tabulated results of electroporated constructs. (D to I) Confocal image montages (white hatches denote image breaks) at E6 of electroporated oculomotor nerves (green) and extraocular muscles (red) labeled with antibody to myosin [(D), (E), and (G) to (I)] or α-bungarotoxin (F); constructs as labeled. All GFP control (D), 28% of wild type (E), and only 5 to 13% of mutant α2-chimaerin electroporated oculomotor nerves extend normally from the midbrain neuroepithelium, at left, past the dorsal rectus muscle (dr), ciliary ganglion (*), and ventral (vr) and medial (mr) recti to innervate the first target, the ventral oblique (vo) muscle. Nerves expressing mutant α2-chimaerin have a higher incidence of aberrant branching [arrow in (F) with higher-magnification inset] and defasciculation than the wild type (fig. S7). Remarkably, 72% of wild-type α2-chimaerin (G), 87% of L20F α2-chimaerin (H), and 71% of G228S α2-chimaerin (I) electroporated nerves stall in the vicinity of the dr muscle. Scale bars, 200 μm; lr, lateral rectus.

Eph receptors and ephrins (24), as well as neuropilin receptors and semaphorins (25), are expressed in developing cranial motor nuclei in the chick and/or rodent. Several recent papers report that α2-chimaerin interacts with the EphA4 receptor and inactivates Rac in response to ephrin/EphA4 signaling (1316). Loss of α2-chimaerin impairs EphA4 forward signaling in vivo and eliminates ephrin-induced growth cone collapse in vitro (1316). α2-Chimaerin has also been implicated in semaphorin 3A–induced growth cone collapse (12). EphA4 receptor stimulation can recruit and activate phospholipase C–γ1, elevating DAG levels (26). Therefore, mutant α2-chimaerin may be hyperactivated in response to chemorepellents such as ephrins or semaphorins, resulting in pathological inactivation of Rac and altered transduction of downstream signals (fig. S8, A to C).

Mice with loss of α2-chimaerin have disrupted ephrin/EphA4 signaling and elevated Rac-GTP levels, with a phenotype limited to a hopping rabbit-like gait resulting from excessive and aberrant midline crossing of corticospinal tract axons and spinal interneuron projections, with no cranial nerve defects reported (1315). We have now identified human α2-chimaerin mutations that enhance its function, reduce Rac-GTP levels, and lead to an ocular motor phenotype as a result of errors in cranial motor neuron development. It is remarkable that the up- and down-regulation of such a widely expressed signaling molecule results in two restricted and apparently non-overlapping phenotypes. It remains to be determined in which signaling pathways α2-chimaerin functions in corticospinal and cranial motor axons and why these different motor circuits are uniquely vulnerable to different perturbations in Rho GTPase activity.

Supporting Online Material

Materials and Methods

Figs. S1 to S8

Table S1


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