Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies.

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.

Comparing retinal reflectance changes elicited by transcorneal electrical retinal stimulation with those of optic chiasma stimulation in cats.

PURPOSE: To compare retinal reflectance changes (RCs) elicited by transcorneal electrical stimulation (TES) to those elicited by electrical stimulation of the optic chiasma (OX). METHODS: Two eyes of two cats were studied under general anesthesia. Biphasic electrical pulses at 20 Hz were applied for 5 ms in TES, and monophasic pulses were applied for 50 µs at 100 Hz in OX stimulation. Fundus images observed with near-infrared light (800-880 nm) were recorded every 25 ms for 26 s, beginning 2 s before and ending 20 s after the electrical stimulation. To improve the signal-to-noise ratio, the images of ten consecutive recordings were averaged. Two-dimensional topographic maps of the RCs were constructed by subtracting images before the stimulation from those after the stimulation. The electrically evoked potentials (EEPs) were recorded at the OX. The effect of an intravitreal injection of tetrodotoxin (TTX) on the RCs elicited by electrical stimulation was also determined. RESULTS: After electrical stimulation, RCs were observed at the optic disc, retinal arteries, and retinal veins. The two-dimensional maps of the RCs elicited by both TES and OX stimulation were similar. The latency of the RCs ranged from 2.0 to 4.0 s, and the peak occurred 6 to 9 s after the onset of the ES. The intensity of the RCs was correlated with the amplitude of EEP elicited by TES stimulation. The RCs disappeared after the TTX injection in both TES and OX stimulation. CONCLUSIONS: TES activates principally the retinal ganglion cells, and a change in the blood flow is initiated thereafter.

Optical imaging to evaluate retinal activation by electrical currents using suprachoroidal-transretinal stimulation.

PURPOSE: To determine whether reflectance changes of the retina after electrical suprachoroidal-transretinal stimulation (STS) can be detected with a newly developed optical imaging fundus camera. METHODS: Ten eyes of 10 cats were studied. A small retinal area was focally stimulated with electric currents passing between an active electrode placed in the fenestrated sclera and a reference electrode in the vitreous. Biphasic pulses were applied for 4 seconds with a current up to 500 muA. Images of the fundus illuminated with near-infrared (800-880 nm) light were obtained every 20 msec for 26 seconds between 2 seconds before and 20 seconds after the STS. Twenty images of 20 consecutive experiments were averaged. A two-dimensional map of the reflectance changes was constructed by subtracting the images before the stimulation from those after the stimulation. STS-evoked potentials (EPs) were recorded from the optic chiasma. RESULTS: Approximately 0.5 second after the onset of STS, reflectance changes were observed around the retinal locus, where the stimulating electrodes were positioned. The intensity of the reflectance changes was correlated with the intensity of the stimulus current. The area of the reflectance change increased as the current intensity increased and was correlated with the amplitude of the EPs (R(2) = 0.82). CONCLUSIONS: Reflectance changes after STS were localized to the area around the electrode. The strong correlation between the area of the reflectance changes and the amplitude of the EPs suggested that the reflectance changes reflected the activity of retinal neurons elicited by electrical stimulation.

Effect of transcorneal electrical stimulation in patients with nonarteritic ischemic optic neuropathy or traumatic optic neuropathy.

PURPOSE: To determine whether transcorneal electrical stimulation (TES) can improve the visual function of patients with nonarteritic ischemic optic neuropathy (NAION) or traumatic optic neuropathy (TON). METHODS: Eight consecutive patients at the Osaka University Hospital were studied. TES (600-800 microA, 20 Hz, 30 min) was applied once each to three eyes with NAION and to five eyes with TON, using a contact lens-type stimulating electrode. The primary outcome measurement was the change in visual acuity at 1 to 3 months after TES. An improvement in visual acuity was defined as a change of > or =0.3 log (minimum angle of resolution) (logMAR) units. The side effects of TES were also investigated. RESULTS: After TES application, the visual acuity improved in two patients with NAION and in four patients with TON. Visual acuity did not worsen in any of the eyes. Only a mild superficial punctuate keratopathy was observed in all eyes immediately after TES, and it healed by the next day. CONCLUSIONS: Visual acuity can be improved after TES without major complications in some patients with NAION or TON. These results suggest that TES should be considered as a new treatment for eyes with optic neuropathy.

Evaluation of residual retinal function by pupillary constrictions and phosphenes using transcorneal electrical stimulation in patients with retinal degeneration.

BACKGROUND: To evaluate inner-retinal function by pupillary constrictions and phosphenes evoked by transcorneal electrical stimulation (TES) in patients with hereditary retinal degeneration. METHODS: Consecutive 20 eyes of 20 patients (16 with retinitis pigmentosa (RP); and four with cone-rod dystrophy (CRD)) whose visual acuity was equal to or worse than 20/2000 at Osaka University Hospital and eight eyes of eight healthy subjects were enrolled. TES was performed on with a contact lens stimulating electrode. The electrically evoked pupillary response (EEPR) was recorded by a pupillometer, and the phosphenes by the subjective responses. Three electrical current thresholds were determined: T1, threshold current for initial phosphene; T2, threshold for eliciting a phosphene extending into the central field; and P, threshold for a relative pupillary constriction > or = 3%. The EEPR and phosphene thresholds were compared with the visual acuity or the visual field. RESULTS: All T1, T2 and P were significantly higher in patients than in normals (Mann-Whitney, P<0.001). Both T1 and T2 were not correlated with visual acuity but depended on the area and location of the residual visual field. T1 and T2 in RP eyes with a EEPR was significantly lower than that in RP eyes without an EEPR. During TES, all subjects and patients had no pain, and no complications except for a slight corneal superficial punctuate keratopathy. CONCLUSIONS: The safety and the efficacy of TES to estimate the residual inner-retinal function in patients with retinal degeneration indicate that TES can be used as one of the most important test to select candidates for retinal prostheses.

Wavefront analysis of eyes with cataracts in patients with monocular triplopia.

PURPOSE: To determine whether wavefront analysis using a Hartmann-Shack (H-S) aberrometer can reveal the cause of monocular triplopia in eyes with mild cataracts. METHODS: Six patients (nine eyes; age range 38-58 years; average 49.8 +/- 6.9 years) who complained of monocular triplopia at the Osaka University Hospital between January and December 2003 were examined. Wavefront analyses of ocular and corneal aberrations of the central 4 mm diameter were performed using a H-S aberrometer equipped with a Placido ring videokeratoscope. The ocular and corneal higher-order wavefronts were fitted with a fourth-order Zernike expansion. RESULTS: All nine eyes showed mild nuclear cataract and had a mean spherical refractive error of -10.3 +/- 3.5 D. The visual acuity was > or = 20/40 except in one eye with glaucoma. For the Zernike polynomials, the trefoil aberration (C3-3) and the spherical aberration (C40) were significantly higher than those of age-matched normal controls (p < 0.001). The simulated retinal image of a Landolt C showed that the combination of trefoil aberration and the spherical aberration can cause an image with a triple configuration. CONCLUSIONS: Monocular triplopia was reported by middle-aged patients with mild nuclear cataract and high myopia. Wavefront analyses suggested that the triple configuration was caused by the combined increase of the trefoil and spherical aberration in lenses with mild nuclear cataracts.