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Simon W M John

Publications and source records attributed to Simon W M John.

At least 19 recordsLinked to original sources
PubMed2025

Single-cell profiling of trabecular meshwork identifies mitochondrial dysfunction in a glaucoma model that is protected by vitamin B3 treatment.

Since the trabecular meshwork (TM) is central to intraocular pressure (IOP) regulation and glaucoma, a deeper understanding of its genomic landscape is needed. We present a multimodal, single-cell resolution analysis of mouse limbal cells (includes TM). In total, we sequenced 9,394 wild-type TM cell transcriptomes. We discovered three TM cell subtypes with characteristic signature genes validated by immunofluorescence on tissue sections and whole-mounts. The subtypes are robust, being detected in datasets for two diverse mouse strains and in independent data from two institutions. Results show compartmentalized enrichment of critical pathways in specific TM cell subtypes. Distinctive signatures include increased expression of genes responsible for 1) extracellular matrix structure and metabolism (TM1 subtype), 2) secreted ligand signaling to support Schlemm's canal cells (TM2), and 3) contractile and mitochondrial/metabolic activity (TM3). ATAC-sequencing data identified active transcription factors in TM cells, including LMX1B. Mutations in LMX1B cause high IOP and glaucoma. LMX1B is emerging as a key transcription factor for normal mitochondrial function and its expression is much higher in TM3 cells than other limbal cells. To understand the role of LMX1B in TM function and glaucoma, we single-cell sequenced limbal cells from Lmx1b V265D/+ mutant mice (2,491 TM cells). In V265D/+ mice, TM3 cells were uniquely affected by pronounced mitochondrial pathway changes. Mitochondria in TM cells of V265D/+ mice are swollen with a reduced cristae area, further supporting a role for mitochondrial dysfunction in the initiation of IOP elevation in these mice. Importantly, treatment with vitamin B3 (nicotinamide), to enhance mitochondrial function and metabolic resilience, significantly protected Lmx1b mutant mice from IOP elevation.

Journal Article↗
PubMed2006

Mutant myocilin nonsecretion in vivo is not sufficient to cause glaucoma.

Glaucoma is a leading cause of blindness, affecting over 70 million people worldwide. Vision loss is the result of death of the retinal ganglion cells. The best-known risk factor for glaucoma is an elevated intraocular pressure (IOP); however, factors leading to IOP elevation are poorly understood. Mutations in the MYOC gene are an important cause of open-angle glaucoma. Over 70 MYOC mutations have been identified, and they lead to approximately 5% of all primary open-angle glaucoma cases. Nevertheless, the pathogenic mechanisms by which these mutations elevate IOP are presently unclear. Data suggest that a dominant interfering effect of misfolded mutant MYOC molecules may be pathogenic. To test this hypothesis, we have generated mice carrying a mutant allele of Myoc that is analogous to a human mutation that leads to aggressive glaucoma in patients. We show that mutant MYOC is not secreted into the aqueous humor. Instead of being secreted, mutant MYOC accumulates within the iridocorneal angle of the eye, consistent with the behavior of abnormally folded protein. Surprisingly, the accumulated mutant protein does not activate the unfolded protein response and lead to elevated intraocular pressure or glaucoma in aged mice of different strains. These data suggest that production, apparent misfolding, and nonsecretion of mutant MYOC are not, by themselves, sufficient to cause glaucoma in vivo.

Animals↗
PubMed2006

Genetic context determines susceptibility to intraocular pressure elevation in a mouse pigmentary glaucoma.

BACKGROUND: DBA/2J (D2) mice develop an age-related form of glaucoma. Their eyes progressively develop iris pigment dispersion and iris atrophy followed by increased intraocular pressure (IOP) and glaucomatous optic nerve damage. Mutant alleles of the Gpnmb and Tyrp1 genes are necessary for the iris disease, but it is unknown whether alleles of other D2 gene(s) are necessary for the distinct later stages of disease. We initiated a study of congenic strains to further define the genetic requirements and disease mechanisms of the D2 glaucoma. RESULTS: To further understand D2 glaucoma, we created congenic strains of mice on the C57BL/6J (B6) genetic background. B6 double-congenic mice carrying D2-derived Gpnmb and Tyrp1 mutations develop a D2-like iris disease. B6 single-congenics with only the Gpnmb and Tyrp1 mutations develop milder forms of iris disease. Genetic epistasis experiments introducing a B6 tyrosinase mutation into the congenic strains demonstrated that both the single and double-congenic iris diseases are rescued by interruption of melanin synthesis. Importantly, our experiments analyzing mice at ages up to 27 months indicate that the B6 double-congenic mice are much less prone to IOP elevation and glaucoma than are D2 mice. CONCLUSION: As demonstrated here, the Gpnmb and Tyrp1 iris phenotypes are both individually dependent on tyrosinase function. These results support involvement of abnormal melanosomal events in the diseases caused by each gene. In the context of the inbred D2 mouse strain, the glaucoma phenotype is clearly influenced by more genes than just Gpnmb and Tyrp1. Despite the outward similarity of pigment-dispersing iris disease between D2 and the B6 double-congenic mice, the congenic mice are much less susceptible to developing high IOP and glaucoma. These new congenic strains provide a valuable new resource for further studying the genetic and mechanistic complexity of this form of glaucoma.

Animals↗
PubMed2006

Scanning transmission X-ray microscopic analysis of purified melanosomes of the mouse iris.

Melanosomes are specialized intracellular membrane bound organelles that produce and store melanin pigment. The composition of melanin and distribution of melanosomes determine the color of many mammalian tissues, including the hair, skin, and iris. However, the presence of melanosomes within a tissue carries potentially detrimental risks related to the cytotoxic indole-quinone intermediates produced during melanin synthesis. In order to study melanosomal molecules, including melanin and melanin-related intermediates, we have refined methods allowing spectromicroscopic analysis of purified melanosomes using scanning transmission X-ray microscopy. Here, we present for the first time absorption data for melanosomes at the carbon absorption edge ranging from 284 to 290 eV. High-resolution images of melanosomes at discrete energies demonstrate that fully melanized mature melanosomes are internally non-homogeneous, suggesting the presence of an organized internal sub-structure. Spectra of purified melanosomes are complex, partially described by a predominating absorption band at 288.4 eV with additional contributions from several minor bands. Differences in these spectra were detectable between samples from two strains of inbred mice known to harbor genetically determined melanosomal differences, DBA/2J and C57BL/6J, and are likely to represent signatures arising from biologically relevant and tractable phenomena.

Absorption↗
PubMed2006

Role of COL4A1 in small-vessel disease and hemorrhagic stroke.

Small-vessel diseases of the brain underlie 20 to 30 percent of ischemic strokes and a larger proportion of intracerebral hemorrhages. In this report, we show that a mutation in the mouse Col4a1 gene, encoding procollagen type IV alpha1, predisposes both newborn and adult mice to intracerebral hemorrhage. Surgical delivery of mutant mice alleviated birth-associated trauma and hemorrhage. We identified a COL4A1 mutation in a human family with small-vessel disease. We concluded that mutation of COL4A1 may cause a spectrum of cerebrovascular phenotypes and that persons with COL4A1 mutations may be predisposed to hemorrhage, especially after environmental stress.

Animals↗
PubMed2005

Retinal ganglion cell degeneration is topological but not cell type specific in DBA/2J mice.

Using a variety of double and triple labeling techniques, we have reevaluated the death of retinal neurons in a mouse model of hereditary glaucoma. Cell-specific markers and total neuron counts revealed no cell loss in any retinal neurons other than the ganglion cells. Within the limits of our ability to define cell types, no group of ganglion cells was especially vulnerable or resistant to degeneration. Retrograde labeling and neurofilament staining showed that axonal atrophy, dendritic remodeling, and somal shrinkage (at least of the largest cell types) precedes ganglion cell death in this glaucoma model. Regions of cell death or survival radiated from the optic nerve head in fan-shaped sectors. Collectively, the data suggest axon damage at the optic nerve head as an early lesion, and damage to axon bundles would cause this pattern of degeneration. However, the architecture of the mouse eye seems to preclude a commonly postulated source of mechanical damage within the nerve head.

Amacrine Cells↗
PubMed2005

Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage.

In glaucoma, harmful intraocular pressure often contributes to retinal ganglion cell death. It is not clear, however, if intraocular pressure directly insults the retinal ganglion cell axon, the soma, or both. The pathways that mediate pressure-induced retinal ganglion cell death are poorly defined, and no molecules are known to be required. DBA/2J mice deficient in the proapoptotic molecule BCL2-associated X protein (BAX) were used to investigate the roles of BAX-mediated cell death pathways in glaucoma. Both Bax+/- and Bax-/- mice were protected from retinal ganglion cell death. In contrast, axonal degeneration was not prevented in either Bax+/- or Bax-/- mice. While BAX deficiency did not prevent axonal degeneration, it did slow axonal loss. Additionally, we compared the effects of BAX deficiency on the glaucoma to its effects on retinal ganglion cell death due to two insults that are proposed to participate in glaucoma. As in the glaucoma, BAX deficiency protected retinal ganglion cells after axon injury by optic nerve crush. However, it did not protect retinal ganglion cells from N-methyl-D-aspartate (NMDA)-induced excitotoxicity. BAX is required for retinal ganglion cell death in an inherited glaucoma; however, it is not required for retinal ganglion cell axon degeneration. This indicates that distinct somal and axonal degeneration pathways are active in this glaucoma. Finally, our data support a role for optic nerve injury but not for NMDA receptor-mediated excitotoxicity in this glaucoma. These findings indicate a need to understand axon-specific degeneration pathways in glaucoma, and they suggest that distinct somal and axonal degeneration pathways may need to be targeted to save vision.

Journal Article↗
PubMed2005

Mutations in Col4a1 cause perinatal cerebral hemorrhage and porencephaly.

Porencephaly is a rare neurological disease, typically manifest in infants, which is characterized by the existence of degenerative cavities in the brain. To investigate the molecular pathogenesis of porencephaly, we studied a mouse mutant that develops porencephaly secondary to focal disruptions of vascular basement membranes. Half of the mutant mice died with cerebral hemorrhage within a day of birth, and approximately 18% of survivors had porencephaly. We show that vascular defects are caused by a semidominant mutation in the procollagen type IV alpha 1 gene (Col4a1) in mice, which inhibits the secretion of mutant and normal type IV collagen. We also show that COL4A1 mutations segregate with porencephaly in human families. Because not all mutant mice develop porencephaly, we propose that Col4a1 mutations conspire with environmental trauma in causing the disease.

Alleles↗
PubMed2005

High-dose radiation with bone marrow transfer prevents neurodegeneration in an inherited glaucoma.

Here, we show that high-dose gamma-irradiation accompanied with syngeneic bone marrow transfer can confer complete protection against glaucoma in a mouse model. Because bone marrow genotype was unaltered by this procedure, it was not the causative agent. The neuroprotection is robust and highly reproducible. Glaucoma-prone DBA/2J mice received a single treatment at 5-8 weeks of age and were protected from glaucomatous retinal ganglion cell degeneration out to 14 months of age (oldest assessed). By 12-14 months, retinal ganglion cell degeneration is usually very severe and essentially complete in the majority of untreated DBA/2J mice. To assess reproducibility, three groups of mice were treated at different times, and the results were essentially the same each time. Considering all experiments, the vast majority of treated mice had no detectable glaucomatous neurodegeneration. A beneficial effect of treatment including high-dose radiation is unprecedented, and we are not aware of any other neuroprotective effects this substantial. Because of the robust protective effect, this treatment offers another tool for studying mechanisms of neuroprotection.

Animals↗
PubMed2005

Glaucoma: thinking in new ways-a rôle for autonomous axonal self-destruction and other compartmentalised processes?

Glaucoma is a common neurodegenerative disease that affects retinal ganglion cells (RGCs). Substantial effort is being expended to determine how RGCs die in glaucoma. As in other neurodegenerative diseases, the majority of effort focuses on characterising apoptotic self-destruct pathways. However, apoptosis is not the only self-destruct mechanism that may be activated in neurons. It is now known that neurons have distinct classes of self-destruct programme that are spatially compartmentalised. In addition to the well-described intracellular suicide machinery in the neuronal soma, responsible for apoptosis, there is another, molecularly distinct, self-destruct programme localised in the axon. Evidence also supports the existence of compartmentalised degeneration programmes in synapses and dendrites. RGCs are no exception to this. Recent data, from in vitro studies and from an inherited mouse model of glaucoma, suggest that molecularly distinct degenerative pathways underlie the destruction of RGC somata and RGC axons. In various neurodegenerative diseases, axons, dendrites and synapses often degenerate well before the cells die, and there is increasing evidence that this is important for the production of clinical symptoms and signs. We hypothesise that such compartmentalised and autonomous programmes are of critical importance in the pathophysiology of glaucoma, and we suggest that studies of these processes are essential for a complete understanding of this complex disease.

Axons↗
PubMed2005

Complex genetics of glaucoma susceptibility.

Glaucoma describes a group of diseases that kill retinal ganglion cells. There are different types of glaucoma, and each appears to be genetically heterogeneous. Different glaucoma genes have been identified, but these genes account for only a small proportion of glaucoma. Most glaucoma cases appear to be multifactorial, and are likely affected by multiple interacting loci. A number of genetic susceptibility factors have been suggested to contribute to glaucoma. These factors fit into two broad groups, those affecting intraocular pressure and those important in modulating retinal ganglion cell viability. Defining the complex genetics of glaucoma will require significant further study of the human disease and animal models. Genetic approaches are essential and will be enhanced by recently developed genomic and proteomic technologies. These technologies will provide valuable clues about pathogenesis for subsequent testing. In this review, we focus on endogenous genetic susceptibility factors and on how experimental studies will be valuable for dissecting the multifactorial complexity of their interactions.

Animals↗
PubMed2004

Genetically increasing Myoc expression supports a necessary pathologic role of abnormal proteins in glaucoma.

Despite the importance of MYOC for glaucoma, the protein's normal function(s) and the pathogenic mechanism(s) of MYOC mutations are not clear. Elevated intraocular pressure (IOP) and glaucoma are sometimes induced by corticosteroids, and corticosteroid use can result in substantially increased MYOC expression. It has been suggested, therefore, that steroid-induced MYOC protein levels cause steroid-induced glaucoma and that protein level-increasing mutations in MYOC contribute to glaucoma not associated with steroid use. A causative role of elevated MYOC levels in steroid-induced glaucoma is controversial, however, and it is not clear if elevated MYOC levels can result in IOP elevation. To directly test if increased levels of MYOC can cause IOP elevation and glaucoma, we generated bacterial artificial chromosome transgenic mice that overexpress Myoc at a level similar to that induced by corticosteroid use. These mice do not develop elevated IOP or glaucoma. Our present findings, along with the absence of glaucoma in mice completely lacking MYOC, show that changing the level of MYOC is not pathogenic (from absent to approximately 15 times normal). These findings suggest that noncoding sequence variants are unlikely to influence glaucoma and that disease pathogenesis in primary open-angle glaucoma patients is dependent upon the expression of abnormal mutant proteins. This work does not support a causative role for increased MYOC levels or the MYOC gene in steroid-induced glaucoma.

Adrenal Cortex Hormones↗
PubMed2004

Intraocular pressure in zebrafish: comparison of inbred strains and identification of a reduced melanin mutant with raised IOP.

PURPOSE: The goal was to establish an accurate method for measuring intraocular pressure (IOP) in living adult zebrafish and to determine the average IOPs of genetically different strains and mutants. The overall purpose was to develop the zebrafish system for studies of IOP and glaucoma. Elevated IOP and anterior segment dysgenesis are two known risk factors for glaucoma. Because albino mammals have increased IOP and are prone to anterior segment anomalies, the study was focused on a reduced melanin zebrafish mutant, brass. METHODS: Servo-null electrophysiology was used to measure IOP in anesthetized adult zebrafish. A pulled-glass microelectrode was used to penetrate the cornea into the anterior chamber, and pressure transduction was recorded. IOP, histology, and visual behavioral assays were used to assess the brass mutant. RESULTS: Significant differences in IOP were detected between genetically distinct zebrafish strains. IOP averages were highest in the long fin (LF) strain (20.5 +/- 1.2 mm Hg) and lowest in the Oregon AB (AB) strain (10.8 +/- 0.3 mm Hg). In brass mutants, which show a reduced melanin phenotype, IOPs were elevated and mild iris hypoplasia was noted. No defects were observed within the retina or in visual function. CONCLUSIONS: In the present study, an accurate method was developed for measuring IOP in adult zebrafish. Both the ability to measure IOP in the genetically powerful zebrafish model and the conservation of elevated IOP and anterior segment anomalies between albino mammals and hypopigmented fish are important. They strongly support the use of zebrafish for studying the complex genetics of elevated IOP and glaucoma.

Animals↗
PubMed2004

Anterior segment development relevant to glaucoma.

Development of the ocular anterior segment involves a series of inductive interactions between neural ectoderm, surface ectoderm and periocular mesenchyme. The timing of these events is well established but less is known about the molecular mechanisms involved. Various genes that participate in these processes have been identified. As the roles of more genes are determined, developmental pathways and networks will emerge. Here, we focus on recent advances made using mouse models. We summarize key morphological events in formation of anterior chamber structures, including the aqueous humor drainage structures that are involved in intraocular pressure (IOP) regulation and glaucoma. We discuss the developmental roles of genes that associate with abnormal anterior segment development and elevated IOP or glaucoma (including Bmp4, Cyp1b1, Foxc1, Foxc2, Pitx2, Lmx1b and Tyr ) and how some of these genes may fit into developmental networks.

Animals↗
PubMed2003

By altering ocular immune privilege, bone marrow-derived cells pathogenically contribute to DBA/2J pigmentary glaucoma.

Pigment dispersion syndrome causes iris pigment release and often progresses to elevated intraocular pressure and pigmentary glaucoma (PG). Because melanin pigment can have adjuvant like properties and because the Gpnmb gene, which contributes to pigment dispersion in DBA/2J (D2) mice, is expressed in dendritic cells, we tested the hypothesis that ocular immune abnormalities participate in PG phenotypes. Strikingly, we show that D2 eyes exhibit defects of the normally immunosuppressive ocular microenvironment including inability of aqueous humor to inhibit T cell activation, failure to support anterior chamber (AC)-associated immune deviation, and loss of ocular immune privilege. Histologic analysis demonstrates infiltration of inflammatory leukocytes into the AC and their accumulation within the iris, whereas clinical indications of inflammation are typically very mild to undetectable. Importantly, some of these abnormalities precede clinical indications of pigment dispersal, suggesting an early role in disease etiology. Using bone marrow chimeras, we show that lymphohematopoietic cell lineages largely dictate the progression of pigment dispersion, the ability of the eye to support induction of AC-associated immune deviation, and the integrity of the blood/ocular barrier. These results suggest previously unsuspected roles for bone marrow-derived cells and ocular immune privilege in the pathogenesis of PG.

Animals↗
PubMed2003

Effects of pressure overload on extracellular matrix expression in the heart of the atrial natriuretic peptide-null mouse.

This study tested the hypothesis that atrial natriuretic peptide has direct antihypertrophic actions on the heart by modulating expression of genes involved in cardiac hypertrophy and extracellular matrix production. Hearts of male, atrial natriuretic peptide-null and control wild-type mice that had been subjected to pressure overload after transverse aortic constriction and control unoperated hearts were weighed and subjected to microarray, Northern blot, and immunohistochemical analyses. Microarray and Northern blot analyses were used to identify genes that are regulated differentially in response to stress in the presence and absence of atrial natriuretic peptide. Immunohistochemical analysis was used to identify and localize expression of the protein products of these genes. Atrial natriuretic peptide-null mice demonstrated cardiac hypertrophy at baseline and an exaggerated hypertrophic response to transverse aortic constriction associated with increased expression of the extracellular matrix molecules periostin, osteopontin, collagen I and III, and thrombospondin, as well as the extracellular matrix regulatory proteins, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-3, and the novel growth factor pleiotrophin compared with wild-type controls. These results support the hypothesis that atrial natriuretic peptide protects against pressure overload-induced cardiac hypertrophy and remodeling by negative modulation of genes involved in extracellular matrix deposition.

Animals↗
PubMed2003

Modification of ocular defects in mouse developmental glaucoma models by tyrosinase.

Mutations in the cytochrome P450 family 1, subfamily B, polypeptide 1 (CYP1B1) gene are a common cause of human primary congenital glaucoma (PCG). Here we show that Cyp1b1-/- mice have ocular drainage structure abnormalities resembling those reported in human PCG patients. Using Cyp1b1-/- mice, we identified the tyrosinase gene (Tyr) as a modifier of the drainage structure phenotype, with Tyr deficiency increasing the magnitude of dysgenesis. The severe dysgenesis in eyes lacking both CYP1B1 and TYR was alleviated by administration of the tyrosinase product dihydroxyphenylalanine (l-dopa). Tyr also modified the drainage structure dysgenesis in mice with a mutant Foxc1 gene, which is also involved in PCG. These experiments raise the possibility that a tyrosinase/l-dopa pathway modifies human PCG, which could open new therapeutic avenues.

Albinism, Ocular↗