Binding of a glaucoma-associated myocilin variant to the αB-crystallin chaperone impedes protein clearance in trabecular meshwork cells.

Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.

Glaucoma - Next Generation Therapeutics: Impossible to Possible.

The future of next generation therapeutics for glaucoma is strong. The recent approval of two novel intraocular pressure (IOP)-lowering drugs with distinct mechanisms of action is the first in over 20 years. However, these are still being administered as topical drops. Efforts are underway to increase patient compliance and greater therapeutic benefits with the development of sustained delivery technologies. Furthermore, innovations from biologics- and gene therapy-based therapeutics are being developed in the context of disease modification, which are expected to lead to more permanent therapies for patients. Neuroprotection, including the preservation of retinal ganglion cells (RGCs) and optic nerve is another area that is actively being explored for therapeutic options. With improvements in imaging technologies and determination of new surrogate clinical endpoints, the therapeutic potential for translation of neuroprotectants is coming close to clinical realization. This review summarizes the aforementioned topics and other related aspects.

A murine glaucoma model induced by rapid in vivo photopolymerization of hyaluronic acid glycidyl methacrylate.

Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) resulting in progressive loss of retinal ganglion cells (RGCs) and optic nerve degeneration, leading to blindness. New therapeutic approaches that better preserve the visual field by promoting survival and health of RGCs are highly needed since RGC death occurs despite good IOP control in glaucoma patients. We have developed a novel approach to reliably induce chronic IOP elevation in mouse using a photopolymerizable biomatrix, hyaluronic acid glycidyl methacrylate. This is achieved by rapid in vivo crosslinking of the biomatrix at the iridocorneal angle by a flash of ultraviolet A (UVA) light to impede the aqueous outflow pathway with a controllable manner. Sustained IOP elevation was induced after a single manipulation and was maintained at ~45% above baseline for >4 weeks. Significant thinning of the inner retina and ~35% reduction in RGCs and axons was noted within one month of IOP elevation. Optic nerve degeneration showed positive correlation with cumulative IOP elevation. Activation of astrocytes and microglia appeared to be an early event in response to IOP elevation preceding detectable RGC and axon loss. Attenuated glial reactivity was noted at later stage where significant RGC/axon loss had occurred suggesting astrocytes and microglia may play different roles over the course of glaucomatous degeneration. This novel murine glaucoma model is reproducible and displays cellular changes that recapitulate several pathophysiological features of glaucoma.

The relationship between nerve fiber layer and perimetry measurements.

PURPOSE: Losses of retinal ganglion cells (RGCs) in glaucoma are the cause of visual field defects and thinning of the retinal nerve fiber layer (RNFL), but methods of correlating these events have not been developed. The present study was conducted to investigate the relationship between standard automated perimetry (SAP) measures of RGCs and optical coherence tomography (OCT) measures of the ganglion cell axons entering the optic nerve from corresponding visual field locations. METHODS: SAP and OCT data from normal monkeys were used to develop methods for estimating neuron counts and mapping SAP visual field locations onto the optic nerve head (ONH). The procedures developed for normal eyes were applied to monkeys with experimental glaucoma. RESULTS: The number of neurons derived from SAP and OCT data for normal eyes were in close agreement. The estimates of the number of RGCs in retinal areas of the Humphrey Field Analyzer 24-2 (Carl Zeiss Meditec, Inc., Dublin, CA) visual field and the axons entering the ONH were both approximately 1.5 million. The neural losses derived from subjective and objective measurements in monkeys with early experimental glaucoma correlated highly, with a mean +/- SD difference of 0.6% +/- 22% between the two estimates in control eyes and 3% +/- 24% in laser-treated eyes. CONCLUSIONS: SAP measures of visual field defects and OCT measures of RNFL defects are correlated measures of glaucomatous neuropathy. The normal intersubject variability and the dynamic ranges of the measurements suggest that RNFL thickness may be a more sensitive measurement for early stages and perimetry a better measure for moderate to advanced stages of glaucoma.

Effects of Spectral Characteristics of Ganzfeld Stimuli on the Photopic Negative Response (PhNR) of the ERG.

PURPOSE: To determine flash and background colors that best isolate the photopic negative response (PhNR) and maximize its amplitude in the primate ERG. METHODS: Photopic full-field flash ERGs were recorded from anesthetized macaque monkeys before and after pharmacologic blockade of Na(+)-dependent spiking activity with tetrodotoxin (TTX, 1 to 2 muM, n = 3), blockade of ionotropic glutamatergic transmission with cis-2,3 piperidine dicarboxylic acid (PDA, 3.3-3.8 mM, n = 3) or laser-induced monocular experimental glaucoma (n = 6), and from six normal human subjects. Photopically matched colored flashes of increasing stimulus strengths were presented on scotopically matched blue, white, or yellow backgrounds of 100 scot cd/m(2) using an LED-based stimulator. RESULTS: PhNRs that could be eliminated by TTX or severe experimental glaucoma were present in responses to brief (<5 ms) and long-duration (200 ms) stimuli of all color combinations. In normal monkey and human eyes for brief low-energy flashes, PhNR amplitudes were highest for red flashes on blue backgrounds and blue flashes on yellow backgrounds. For high-energy flashes, amplitudes were more similar for all color combinations. For long-duration stimuli, the PhNR(on) at light onset in monkeys was larger for red and blue stimuli, regardless of background color, than for spectrally broader flashes, except for stimuli >17.7 cd/m(2) when PhNR(on)s were all of similar amplitude. For red flashes, eliminating the PhNR(on) pharmacologically or by glaucoma removed the slowly recovering negative wave that normally followed the transient b-wave and elevated the whole ON response close to the level of the b-wave peak. However, for white, blue, and green flashes, a lower-amplitude plateau that could be removed by PDA remained. CONCLUSIONS: For weak to moderate flash strengths, the best stimulus for maximizing PhNR amplitude is one that primarily stimulates one cone type, on a background with minimal adaptive effect on cones. For stronger stimuli, differences in amplitude are smaller. For long-duration stimuli, red best isolates the PhNR(on) because it minimizes the overlapping lower-level plateau that originates from the activity of second-order hyperpolarizing retinal neurons.

Oscillatory potentials of the slow-sequence multifocal ERG in primates extracted using the Matching Pursuit method.

This study used the Matching Pursuit (MP) method, a time-frequency analysis, to identify and characterize oscillatory potentials (OPs) in the primate electroretinogram (ERG). When the slow-sequence mfERG from the macular region of the retina was matched with Gabor functions, OPs were identified in two distinct bands: a high-frequency band peaking around 150 Hz that contributes to early OPs, and a low-frequency band peaking around 80 Hz that contributes to both early and late OPs. Pharmacological blockade and experimental glaucoma studies showed that the high-frequency OPs depend upon sodium-dependent spiking activity of retinal ganglion cells, whereas the low-frequency OPs depend primarily upon non-spiking activity of amacrine cells, and more distal retinal activity.

Long-acting protein drugs for the treatment of ocular diseases.

Protein drugs that neutralize vascular endothelial growth factor (VEGF), such as aflibercept or ranibizumab, rescue vision in patients with retinal vascular diseases. Nonetheless, optimal visual outcomes require intraocular injections as frequently as every month. Here we report a method to extend the intravitreal half-life of protein drugs as an alternative to either encapsulation or chemical modifications with polymers. We combine a 97-amino-acid peptide of human origin that binds hyaluronan, a major macromolecular component of the eye's vitreous, with therapeutic antibodies and proteins. When administered to rabbit and monkey eyes, the half-life of the modified proteins is increased ∼3-4-fold relative to unmodified proteins. We further show that prototype long-acting anti-VEGF drugs (LAVAs) that include this peptide attenuate VEGF-induced retinal changes in animal models of neovascular retinal disease ∼3-4-fold longer than unmodified drugs. This approach has the potential to reduce the dosing frequency associated with retinal disease treatments.

Effect of experimental glaucoma in primates on oscillatory potentials of the slow-sequence mfERG.

PURPOSE: To determine the effect of experimental glaucoma in macaque monkeys on oscillatory potentials (OPs) in the slow-sequence multifocal electroretinogram (mfERG). METHODS: Photopic slow-sequence mfERGs were recorded from anesthetized adult macaque monkeys and normal human subjects. The stimulus consisted of 103 equal-sized hexagons within 17 degrees of the fovea. The m-sequence was slowed, with 14 blank frames, approximately 200 ms, interleaved between flashes for monkeys and 7 blank frames, approximately 100 ms, for humans, to produce waveforms similar to the photopic full-field flash ERG. Recordings were made under control conditions (24 monkey eyes, 7 human) and after laser-induced experimental glaucoma in monkeys (n = 8). A Fourier fast transform [FFT] was used to determine the frequency ranges of the major OPs. OP amplitudes were quantified by using root mean square (RMS) for two-frequency bands in five horizontal and four vertical locations. Visual field defects were assessed using behavioral static perimetry. Full-field photopic flash ERGs also were recorded. RESULTS: OPs in two distinct frequency bands were discriminated in the monkey mfERG: fast OPs, with a peak frequency of 143 +/- 20 Hz, and slow OPs, with a peak at 77 +/- 8 Hz. There were similar findings in humans and with the flash ERG in monkeys. The fast OP RMS in monkey control eyes was significantly larger in temporal than nasal retina (P < 0.01) and in superior versus inferior retina (P < 0.05) as reported previously. The slow OP RMS was largest in the foveal region. Experimental glaucoma reduced fast OP RMS in all locations studied, even when visual field defects were moderate (MD = -5 to -10 dB; P < 0.05), whereas the slow OP RMS was reduced significantly primarily in the foveal region when field defects were severe (MD < -10 dB; P < 0.05). The fast OP RMS showed a moderate correlation with local visual field sensitivity and with local ganglion cell density (calculated from visual field sensitivity). For the slow OPs the correlation was much poorer. Consistent with previous studies, the photopic negative response (PhNR) amplitude was significantly reduced when the visual sensitivity was minimally affected. CONCLUSIONS: OPs in the ERG of primates fall in two frequency bands: fast OPs with a peak frequency around 143 Hz and slow OPs, with a peak frequency around 77 Hz. The fast OPs, which rely more on the integrity of retinal ganglion cells and their axons than do the slow OPs, have potential utility for monitoring the progression of glaucoma and the effects of treatment.

Regional variations in local contributions to the primate photopic flash ERG: revealed using the slow-sequence mfERG.

PURPOSE: To determine the variations with eccentricity of the primate photopic ERG and to separate contributions by different retinal cells by using intravitreal pharmacologic agents. METHODS: Slow-sequence multifocal (mf)ERGs were obtained from 19 anesthetized adult rhesus monkeys and 5 normal human subjects. Recordings in monkeys were obtained before and after injections of tetrodotoxin citrate (TTX) to block sodium-dependent spiking; TTX+N-methyl-D-aspartic acid (NMDA)+picrotoxin (PTX) or gamma-aminobutyric acid (GABA) to block all inner retinal activity; L-2 amino-4-phosphonobutyric acid (APB) to block the On-pathway; and cis-2, 3 piperidine dicarboxylic acid (PDA) to block the Off-pathway and the otherwise unblocked inner retinal activity. The stimulus consisted of 103 equal-sized hexagons within 17 degrees of the fovea; every 200 ms (15 frames), each hexagon had a 50% chance of remaining at 20 cd/m(2) or increasing briefly to 4.7 cd-s/m(2). Oscillatory potentials (OPs; 90-300 Hz) were extracted. RESULTS: The slow-sequence mfERG summed over the stimulated area looked similar to a standard photopic, full-field ERG, with a- and b-waves and OPs. OPs in the foveal and temporal retina were larger than in the nasal retina. This nasotemporal asymmetry was removed by TTX, and the OPs were eliminated, either by blocking inner retina activity or by blocking the On-pathway. The summed mfERG waveform, including OPs, was shaped mainly by the more peripheral retinal regions. The foveal b-wave peak occurred about 5 to 6 ms later than in the periphery, with the depolarizing peak of the On-pathway/bipolar contribution occurring earlier than the depolarizing peak of the Off contribution at all eccentricities. The a-wave was composed of a small photoreceptor contribution and postreceptoral portion originating from hyperpolarizing neurons. CONCLUSIONS: The variations in the primate photopic ERG with eccentricity are due to spike-driven oscillatory activity that is more prominent in central and temporal retina than in nasal retina and to the slower timing of all responses in the central, compared with the peripheral, retina. The full-field, photopic ERG most closely resembles the mfERG responses to stimulation of peripheral regions.

Photopic ERGs in patients with optic neuropathies: comparison with primate ERGs after pharmacologic blockade of inner retina.

PURPOSE: To determine whether anterior ischemic optic neuropathy and compressive optic neuropathy in humans alter the photopic flash ERG and to investigate the cellular origins of the waves that are affected by pharmacologic agents in primates. METHODS: Photopic flash ERGs were recorded differentially, with DTL electrodes, between the two eyes of 22 patients with diagnosed optic neuropathy (n = 17, anterior ischemic optic neuropathy [AION]; n = 5, compressive optic neuropathy) and 25 age-matched control subjects and in 17 eyes of 13 monkeys (Macaca mulatta). The stimulus consisted of brief (<5 ms) red (lambda(max) = 660 nm) Ganzfeld flashes (energy range, 0.5-2.0 log td-s) delivered on a rod-saturating blue background of 3.7 log sc td (lambda(max) = 460 nm). An eye of the patient with ischemic changes at the disc was classified as symptomatic if it showed visual field defects with a mean deviation (MD) of P < 2%. Recordings in macaque monkeys were made before and after inner retinal blockade with tetrodotoxin (TTX) (1.2-2.1 microM; n = 7), TTX+N-methyl-d-aspartate (NMDA; 1.4-6.4 mM; n = 7), and cis-2, 3 piperidine dicarboxylic acid (PDA; 3.3-3.8 mM; n = 3). RESULTS: The PhNR amplitude was significantly reduced in both symptomatic (P = 3.4 x 10(-8)) and asymptomatic (P = 0.036) eyes of patients with AION or compressive optic neuropathy (P = 0.0054) compared with control subjects. The PhNR amplitude in the symptomatic eye showed a moderate correlation with field defects (P < 0.05) similar to previous findings in open-angle glaucoma. The a-wave also was reduced significantly in the symptomatic eye (P = 0.0002) of patients with AION. The i-wave, a positive wave on the trailing edge of the b-wave peaking around 50 ms, became more prominent in eyes in which the PhNR was significantly reduced. In monkeys, the PhNR was eliminated by TTX. The a-wave at the peak and later times was reduced by TTX, further reduced by NMDA, and eliminated after PDA in response to the red stimuli. PDA also eliminated the i-wave. CONCLUSIONS: PhNR amplitude is significantly reduced in eyes with open-angle glaucoma, AION, and compressive optic neuropathy. Experiments in primates indicate that this reduction reflects loss of a spike-driven contribution to the photopic ERG. There also are small spike-driven contributions to the a-wave elicited by full-field red stimuli. The i-wave, which becomes more prominent when the PhNR is reduced, has origins in the off-pathway distal to the ganglion cells.

Correlating RNFL thickness by OCT with perimetric sensitivity in glaucoma patients.

PURPOSE: To determine whether a structure-function model developed for normal age-related losses of retinal ganglion cells also models the retinal ganglion cell losses in glaucomatous optic neuropathy. METHODS: The model to relate age-related loss of retinal nerve fiber layer thickness and reduced sensitivity for standard automated perimetry was evaluated with data from 30 glaucoma patients and 40 normal individuals. Perimetry thresholds were translated into separate retinal ganglion cell body estimates for test locations in the superior and inferior visual fields. The retinal nerve fiber layer thickness from optical coherence tomography was also divided into regions representing the superior and inferior hemifields to obtain estimates of the axons in each hemifield. The 2 estimates of retinal ganglion cell populations were compared for corresponding regions. RESULTS: Agreement between neural estimates was good for normal individuals and patients with early glaucomatous damage. Results for individuals with advanced glaucoma showed disparities between neural estimates that were proportional to the stage of disease. A correction factor for the stage of disease was introduced for the derivation of ganglion cell populations from the nerve fiber layer measurements, which produced agreement between the optical coherence tomography and perimetric estimates for all patients. CONCLUSIONS: The modified structure-function model provided well-correlated relationships between the subjective measures of visual sensitivity and the objective measures of retinal nerve fiber layer thickness when parameters for the patient's age and the severity of the disease were included. The results suggest constitutive relationships between structure and function for the full spectrum of normal-to-advanced glaucomatous neuropathy.

A comparison of visual field sensitivity to photoreceptor thickness in retinitis pigmentosa.

PURPOSE: To explore the relationship between visual field sensitivity and photoreceptor layer thickness in patients with retinitis pigmentosa (RP). METHODS: Static automated perimetry (central 30-2 threshold program with spot size III; Humphrey Field Analyzer; Carl Zeiss Meditec, Inc., Dublin, CA) and frequency domain optical coherence tomography (Fd-OCT) scans (Spectralis HRA+OCT; Heidelberg Engineering, Vista, CA) were obtained from 10 age-matched normal control subjects and 20 patients with RP who had retained good central vision (better than 20/32). The outer segment (OS+) thickness (the distance between retinal pigment epithelium [RPE])/Bruch's membrane [BM] to the photoreceptor inner-outer segment junction), outer nuclear layer (ONL), and total retinal thickness were measured at locations corresponding to visual field test loci up to 21 degrees eccentricity. RESULTS: The average OS+ thickness in the control eyes was 63.1 +/- 5.2 microm, varying from approximately 69 microm in the foveal center to 56 microm at 21 degrees eccentricity. In patients with RP, OS+ thickness was below normal limits outside the fovea, and thickness decreased with loss in local field sensitivity, reaching an asymptotic value of 21.5 microm at approximately -10 dB. The ONL thickness also decreased with local field sensitivity loss. Although relative OS thickness was linearly related to visual field loss at all locations examined, a slightly better correlation was found between the product of OS and ONL thickness and visual field loss. CONCLUSIONS: In patients with RP with good foveal sensitivity, the OS thickness and the product of OS thickness and ONL thickness (assumed to represent the number of photoreceptors) decreases linearly with loss of local field sensitivity. In general, in regions where perimetric sensitivity loss is -10 dB or worse, the OS+ thickness approaches the thickness of the RPE/BM complex.

Age-related losses of retinal ganglion cells and axons.

PURPOSE: Age-related losses in retinal nerve fiber layer (RNFL) thickness have been assumed to be the result of an age-dependent reduction of retinal ganglion cells (RGCs), but the published rates differ: age-related losses of RGCs of approximately 0.6%/year compared to 0.2%/year for thinning of the RNFL. An analysis of normative data for standard automated perimetry (SAP) sensitivities and optical coherence tomography (OCT) measures of RNFL thickness showed that the apparent disagreement in age-dependent losses of RGCs and axons in the RNFL can be reconciled by an age-dependent decrease in the proportion of the RNFL thickness that is composed of axons. The purpose of the present study was to determine whether the mechanisms of age-related losses that were suggested by the normative data can be confirmed with data from healthy, normal eyes. METHODS: Data were obtained from visual fields (normal results in a Glaucoma Hemifield Test [GHT] on standard automated perimetry [SAP] 24-2 fields) and RNFL thickness measurements (standard OCT scan) of 55 patients (age range, 18-80 years; mean, 44.5 +/- 17.3). The SAP measures of visual sensitivity and OCT measures of RNFL thickness for one eye of each patient were used to estimate neuron counts by each procedure. RESULTS: The age-related thinning of RNFL was 0.27%/year when a constant axon density was used to derive axon counts from RNFL thickness, compared with 0.50%/year for the age-related loss of RGCs from SAP. In agreement with the model developed with normative clinical data, concordance between losses of axons and soma was achieved by an age-dependent reduction of 0.46%/year in the density of axons in the RNFL. CONCLUSIONS: The results suggest that the proportion of RNFL that is composed of RGC axons is not constant with age; rather, the proportion of the total thickness from non-neuronal tissue increases with age. If a similar compensation occurs in the RNFL thickness with axon loss from glaucoma, then a stage-dependent correction to translate OCT measurements to neuronal components is needed, in addition to the age-dependent correction.

Adaptive optics scanning laser ophthalmoscopy for in vivo imaging of lamina cribrosa.

The lamina cribrosa has been postulated from in vitro studies as an early site of damage in glaucoma. Prior in vivo measures of laminar morphology have been confounded by ocular aberrations. In this study the lamina cribrosa was imaged after correcting for ocular aberrations using the adaptive optics scanning laser ophthalmoscope (AOSLO) in normal and glaucomatous eyes of rhesus monkeys. All measured laminar morphological parameters showed increased magnitudes in glaucomatous eyes relative to fellow control eyes, indicating altered structure. The AOSLO provides high-quality images of the lamina cribrosa and may have potential as a tool for early identification of glaucoma.