Masasuke Araki

The clinical and molecular genetic features of idiopathic infantile periodic alternating nystagmus

Periodic alternating nystagmus consists of involuntary oscillations of the eyes with cyclical changes of nystagmus direction. It can occur during infancy (e.g. idiopathic infantile periodic alternating nystagmus) or later in life. Acquired forms are often associated with cerebellar dysfunction arising due to instability of the optokinetic-vestibular systems. Idiopathic infantile periodic alternating nystagmus can be familial or occur in isolation; however, very little is known about the clinical characteristics, genetic aetiology and neural substrates involved. Five loci (NYS1-5) have been identified for idiopathic infantile nystagmus; three are autosomal (NYS2, NYS3 and NYS4) and two are X-chromosomal (NYS1 and NYS5). We previously identified the FRMD7 gene on chromosome Xq26 (NYS1 locus); mutations of FRMD7 are causative of idiopathic infantile nystagmus influencing neuronal outgrowth and development. It is unclear whether the periodic alternating nystagmus phenotype is linked to NYS1, NYS5 (Xp11.4-p11.3) or a separate locus. From a cohort of 31 X-linked families and 14 singletons (70 patients) with idiopathic infantile nystagmus we identified 10 families and one singleton (21 patients) with periodic alternating nystagmus of which we describe clinical phenotype, genetic aetiology and neural substrates involved. Periodic alternating nystagmus was not detected clinically but only on eye movement recordings. The cycle duration varied from 90 to 280u2009s. Optokinetic reflex was not detectable horizontally. Mutations of the FRMD7 gene were found in all 10 families and the singleton (including three novel mutations). Periodic alternating nystagmus was predominantly associated with missense mutations within the FERM domain. There was significant sibship clustering of the phenotype although in some families not all affected members had periodic alternating nystagmus. In situ hybridization studies during mid-late human embryonic stages in normal tissue showed restricted FRMD7 expression in neuronal tissue with strong hybridization signals within the afferent arms of the vestibulo-ocular reflex consisting of the otic vesicle, cranial nerve VIII and vestibular ganglia. Similarly within the afferent arm of the optokinetic reflex we showed expression in the developing neural retina and ventricular zone of the optic stalk. Strong FRMD7 expression was seen in rhombomeres 1 to 4, which give rise to the cerebellum and the common integrator site for both these reflexes (vestibular nuclei). Based on the expression and phenotypic data, we hypothesize that periodic alternating nystagmus arises from instability of the optokinetic-vestibular systems. This study shows for the first time that mutations in FRMD7 can cause idiopathic infantile periodic alternating nystagmus and may affect neuronal circuits that have been implicated in acquired forms.

Extraocular dorsal signal affects the developmental fate of the optic vesicle and patterns the optic neuroepithelium

Dorsal–ventral (DV) specification in the early optic vesicle plays a crucial role in the proper development of the eye. To address the questions of how DV specification is determined and how it affects fate determination of the optic vesicle, isolated optic vesicles were cultured either in vitro or in ovo. The dorsal and ventral halves of the optic vesicle were fated to develop into retinal pigment epithelium (RPE) and neural retina, respectively, when they were separated from each other and cultured. In optic vesicles treated with collagenase to remove the surrounding tissues, the neuroepithelium gave rise to cRax expression but not Mitf, suggesting that surrounding tissues are necessary for RPE specification. This was also confirmed in in ovo explant cultures. Combination cultures of collagenase‐treated optic vesicles with either the dorsal or ventral part of the head indicated that head‐derived factors have an important role in the fate determination of the optic vesicle: in the optic vesicles co‐cultured with the dorsal part of the head Mitf expression was induced in the neuroepithelium, while the ventral head portion did not have this effect. The dorsal head also suppressed Pax2 expression in the optic vesicle. These observations indicate that factors from the dorsal head portion have important roles in the establishment of DV polarity within the optic vesicle, which in turn induces the patterning and differentiation of the neural retina and pigment epithelium.

Abnormal retinal development associated with FRMD7 mutations

Idiopathic infantile nystagmus (IIN) is a genetically heterogeneous disorder, often associated with FRMD7 mutations. As the appearance of the retina is reported to be normal based on conventional fundus photography, IIN is postulated to arise from abnormal cortical development. To determine whether the afferent visual system is involved in FRMD7 mutations, we performed in situ hybridization studies in human embryonic and fetal stages (35 days post-ovulation to 9 weeks post-conception). We show a dynamic retinal expression pattern of FRMD7 during development. We observe expression within the outer neuroblastic layer, then in the inner neuroblastic layer and at 9 weeks post-conception a bilaminar expression pattern. Expression was also noted within the developing optic stalk and optic disk. We identified a large cohort of IIN patients (n = 100), and performed sequence analysis which revealed 45 patients with FRMD7 mutations. Patients with FRMD7 mutations underwent detailed retinal imaging studies using ultrahigh-resolution optical coherence tomography. The tomograms were compared with a control cohort (n = 60). The foveal pit was significantly shallower in FRMD7 patients (P < 0.0001). The optic nerve head morphology was abnormal with significantly decreased optic disk area, retinal nerve fiber layer thickness, cup area and cup depth in FRMD7 patients (P < 0.0001). This study shows for the first time that abnormal afferent system development is associated with FRMD7 mutations and could be an important etiological factor in the development of nystagmus.

Anteroventrally localized activity in the optic vesicle plays a crucial role in the optic development

The vertebrate eye develops from the optic vesicle (OV), a laterally protrusive structure of the forebrain, by a coordinated interaction with surrounding tissues. The OV then invaginates to form an optic cup, and the lens placode develops to the lens vesicle at the same time. These aspects in the early stage characterize vertebrate eye formation and are controlled by appropriate dorsal-ventral coordination. In the present study, we performed surgical manipulation in the chick OV to remove either the dorsal or ventral half and examined the development of the remaining OV. The results show that the dorsal and ventral halves of the OV have a clearly different developmental pattern. When the dorsal half was removed, the remaining ventral OV developed into an entire eye, while the dorsal OV developed to a pigmented vesicle consisting of retinal pigmented epithelium alone. These results indicate that the ventral part of the OV retains the potency to develop the entire eye structure and plays an essential role in proper eye development. In subsequent manipulations of early chick embryos, it was found that only the anterior ventral quadrant of the OV has the potential to develop the entire eye and that no other part of the OV has a similar activity. Fgf8 expression was localized in this portion and no Fgf8 expression was observed within the OV when the ventral OV was removed. These results suggest that the anterior ventral portion of the OV plays a crucial role in the proper development of the eye, possibly generating the dorsal-ventral gradients of signal proteins within the eye primordium.

Transgenic Xenopus laevis with the ef1-α promoter as an experimental tool for amphibian retinal regeneration study

Complete retinal regeneration occurs after the removal of the whole tissue in mature Xenopus laevis, as well as in the newt. Here, we produced F1 and F2 lines of transgenic X. laevis containing an EGFP gene under a translation elongation factor 1‐α (ef1‐α) promoter and investigated how the gene is reactivated in retinal pigmented epithelial (RPE) cells when the neural retina (NR) is removed. The results showed that EGFP expression is reduced in the adult ocular tissues of nonmanipulated transgenic animals, and EGFP‐expressing cells are occasionally found heterogeneously in the lens, NR and RPE tissues. During retinal regeneration, the EGFP gene is reactivated in the RPE and ciliary marginal cells. Transgenic animals were also used for a transplant study because of the genetic marker of the donor tissue. Transplanted RPE clearly transdifferentiated to regenerate the retina in the ocular chamber. This study is, to our knowledge, the first report of a transgenic study of amphibian retinal regeneration, and the approach is promising for future molecular analyses. genesis 50:642–650, 2012.

Regulatory expression of Neurensin-1 in the spinal motor neurons after mouse sciatic nerve injury.

Axonal regeneration after crush injury of the sciatic nerve has been intensely studied for the elucidation of molecular and cellular mechanisms. Neurite extension factor1 (Nrsn1) is a unique membranous protein that has a microtubule-binding domain and is specifically expressed in neurons. Our studies have shown that Nrsn1 is localized particularly in actively extending neurites, thus playing a role in membrane transport to the growing distal ends of extending neurites. To elucidate the possible role of Nrsn1 during peripheral axonal regeneration, we examined the expression of Nrsn1 mRNA by in situ hybridization and Nrsn1 localization by immunocytochemistry, using a mouse model. The results revealed that during the early phase of axonal regeneration of motor nerves, Nrsn1 mRNA is upregulated in the injured motor neuron. Nrsn1 is localized in the cell bodies of motor neurons and at the growing distal ends of regenerating axons. These results indicate that Nrsn1 plays an active role in axonal regeneration as well as in embryonic development.

Neurensin-1 expression in the mouse retina during postnatal development and in cultured retinal neurons

Neurensin-1/Neuro-p24 (previously named Neuro-p24) is a neuron-specific membrane protein that is localized particularly in neurites. Neurensin-1 is considered to play an essential role in neurite extension during nervous development, regeneration and plasticity. To understand what role Neurensin-1 plays in retinal differentiation, we examined Neurensin-1 distribution and gene expression pattern in the postnatally developing retina of the mouse, because the retina is an excellent model for nervous development. In the postnatal day (PD) 1 retina, intense Neurensin-1 immunoreactivity was found in the optic nerve fiber layer. Faint staining was seen in the ganglion cells, presumptive amacrine and horizontal cells. As the postnatal development proceeded, the optic fibers became more intensely stained in addition to other parts of the retina such as the ganglion cells, inner plexiform layer and horizontal cells. In PD 10 retinas, the horizontal cell processes showed a prominently stained configuration. As the retina developed further to attain maturity, the staining in the retina became less pronounced, although the optic nerves remained positively stained. The distribution of Neurensin-1 mRNA was consistent with these results and confirmed that the ganglion, amacrine and horizontal cells actively synthesize Neurensin-1 in the developing retina. In the retinal cell culture from newborn mice, two types of neural cells were stained for Neurensin-1, one of which showed long processes and appeared presumptive ganglion cells. These results suggest that Neurensin-1 plays a role in the fiber extension of the retinal neurons, as has been observed in other central nervous tissues, and indicate that the developing retina is a suitable experimental model for the analysis of Neurensin function, both in vivo and in vitro.

Localization of Neurensin1 in cerebellar Purkinje cells of the developing chick and its possible function in dendrite formation

Neurensin1 (Nrsn1) gene, highly specific to neurons, has been considered to play a role in neurite growth during neuronal development and regeneration in mice. Intense expression of Nrsn1 was found particularly in projecting neurons like retinal ganglion cells and spinal motor neurons, suggesting that Neurensin1 is needed for active neurite growth. In the present study we cloned chick Nrsn1 gene and produced an antibody against cNrsn1 to examine Nrsn1 localization in the chick brain, since the chick is a suitable animal model for the study of developmental neurobiology. We found that there are neurons intensely stained for Nrsn1 antibody localized in the optic tectum, the cerebellum and the brain stem. These neurons are large in size and considered to be projecting neurons. In the cerebellum, Purkinje cells are the only one type of neurons stained for Nrsn1. During Purkinje cell development the arborized dendrites and axons become intensely stained at stages E17-18. A siRNA gene knock down was applied to the cultured embryonic cerebellar tissues and the result showed that Nrsn1 has an important role in dendrite formation of Purkinje cells. These findings suggest that Neurensin1 is also involved in neural development in the chick brain and that the embryonic chick brain is a good model to disclose the molecular and physiological functions of Nrsn1.

Recent Developments in the Treatment of Ankle and Subtalar Instability

It was nearly a centenary ago that severe ankle sprain was recognized as an injury of the ankle ligament(s). With the recent technological advances and tools in imaging and surgical procedures, the management of ankle sprains - including subtalar injuries - has drastically improved. The repair or reconstruction of ankle ligaments is getting more anatomical and less invasive than previously. More specifically, ligamentous reconstruction with tendon graft has been the gold standard in the management of severely damaged ligament, however, it does not reproduce the original ultrastructure of the ankle ligaments. The anatomical ligament structure of a ligament comprises a ligament with enthesis at both ends and the structure should also exhibit proprioceptive function. To date, it remains impossible to reconstruct a functionally intact and anatomical ligament. Cooperation of the regenerative medicine and surgical technology in expected to improve reconstructions of the ankle ligament, however, we need more time to develop a technology in reproducing the ideal ligament complex.