Xianjun Zhu

Radiation treatment inhibits monocyte entry into the optic nerve head and prevents neuronal damage in a mouse model of glaucoma

Glaucoma is a common ocular disorder that is a leading cause of blindness worldwide. It is characterized by the dysfunction and loss of retinal ganglion cells (RGCs). Although many studies have implicated various molecules in glaucoma, no mechanism has been shown to be responsible for the earliest detectable damage to RGCs and their axons in the optic nerve. Here, we show that the leukocyte transendothelial migration pathway is activated in the optic nerve head at the earliest stages of disease in an inherited mouse model of glaucoma. This resulted in proinflammatory monocytes entering the optic nerve prior to detectable neuronal damage. A 1-time x-ray treatment prevented monocyte entry and subsequent glaucomatous damage. A single x-ray treatment of an individual eye in young mice provided that eye with long-term protection from glaucoma but had no effect on the contralateral eye. Localized radiation treatment prevented detectable neuronal damage and dysfunction in treated eyes, despite the continued presence of other glaucomatous stresses and signaling pathways. Injection of endothelin-2, a damaging mediator produced by the monocytes, into irradiated eyes, combined with the other glaucomatous stresses, restored neural damage with a topography characteristic of glaucoma. Together, these data support a model of glaucomatous damage involving monocyte entry into the optic nerve.

Common variants near ABCA1 and in PMM2 are associated with primary open-angle glaucoma

We performed a genome-wide association study for primary open-angle glaucoma (POAG) in 1,007 cases with high-pressure glaucoma (HPG) and 1,009 controls from southern China. We observed genome-wide significant association at multiple SNPs near ABCA1 at 9q31.1 (rs2487032; P = 1.66 × 10−8) and suggestive evidence of association in PMM2 at 16p13.2 (rs3785176; P = 3.18 × 10−6). We replicated these findings in a set of 525 HPG cases and 912 controls from Singapore and a further set of 1,374 POAG cases and 4,053 controls from China. We observed genome-wide significant association with more than one SNP at the two loci (P = 2.79 × 10−19 for rs2487032 representing ABCA1 and P = 5.77 × 10−10 for rs3785176 representing PMM2). Both ABCA1 and PMM2 are expressed in the trabecular meshwork, optic nerve and other ocular tissues. In addition, ABCA1 is highly expressed in the ganglion cell layer of the retina, a finding consistent with it having a role in the development of glaucoma.

Mutations in a P-Type ATPase Gene Cause Axonal Degeneration

Neuronal loss and axonal degeneration are important pathological features of many neurodegenerative diseases. The molecular mechanisms underlying the majority of axonal degeneration conditions remain unknown. To better understand axonal degeneration, we studied a mouse mutant wabbler-lethal (wl). Wabbler-lethal (wl) mutant mice develop progressive ataxia with pronounced neurodegeneration in the central and peripheral nervous system. Previous studies have led to a debate as to whether myelinopathy or axonopathy is the primary cause of neurodegeneration observed in wl mice. Here we provide clear evidence that wabbler-lethal mutants develop an axonopathy, and that this axonopathy is modulated by Wlds and Bax mutations. In addition, we have identified the gene harboring the disease-causing mutations as Atp8a2. We studied three wl alleles and found that all result from mutations in the Atp8a2 gene. Our analysis shows that ATP8A2 possesses phosphatidylserine translocase activity and is involved in localization of phosphatidylserine to the inner leaflet of the plasma membrane. Atp8a2 is widely expressed in the brain, spinal cord, and retina. We assessed two of the mutant alleles of Atp8a2 and found they are both nonfunctional for the phosphatidylserine translocase activity. Thus, our data demonstrate for the first time that mutation of a mammalian phosphatidylserine translocase causes axon degeneration and neurodegenerative disease.

Drosophila Pipe protein activity in the ovary and the embryonic salivary gland does not require heparan sulfate glycosaminoglycans

The Drosophila pipe gene encodes ten related proteins that exhibit amino acid sequence similarity to vertebrate heparan sulfate 2-O-sulfotransferase. One of the Pipe isoforms, which is expressed in the ventral follicular epithelium, is a key determinant of embryonic dorsoventral polarity, suggesting that Pipe-mediated sulfation of a heparan sulfate proteoglycan provides a spatial cue for dorsoventral axis formation. We used several approaches to investigate this possibility in the work described here. We determined the nucleotide alterations in 11 different pipe alleles. Ten of the mutations specifically affect the pipe isoform that is expressed in the ovary. Among these ten mutations, two alter an amino acid in the putative binding site for 3′-phosphoadenosine 5′-phosphosulfate, the universal sulfate donor. Using Alcian Blue, a histochemical stain that detects sulfated glycans, we observed a novel, pipe-dependent macromolecule in the embryonic salivary glands. Genes known to participate in the formation of heparan sulfate in Drosophila are not required for the production of this material. To investigate whether a heparan sulfate proteoglycan is involved in pipe function in dorsoventral patterning, we generated females carrying follicle cell clones mutant for heparan sulfate synthesis-related genes. Embryos from follicles with mutant clones did not exhibit a dorsalized phenotype. Taken together, our data provide evidence that Pipe acts as a sulfotransferase, but argue against the hypothesis that the target of Pipe is a heparan sulfate glycosaminoglycan.

Phospholipid flippase ATP8A2 is required for normal visual and auditory function and photoreceptor and spiral ganglion cell survival.

ABSTRACT ATP8A2 is a P4-ATPase that is highly expressed in the retina, brain, spinal cord and testes. In the retina, ATP8A2 is localized in photoreceptors where it uses ATP to transport phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the exoplasmic to the cytoplasmic leaflet of membranes. Although mutations in ATP8A2 have been reported to cause mental retardation in humans and degeneration of spinal motor neurons in mice, the role of ATP8A2 in sensory systems has not been investigated. We have analyzed the retina and cochlea of ATP8A2-deficient mice to determine the role of ATP8A2 in visual and auditory systems. ATP8A2-deficient mice have shortened photoreceptor outer segments, a reduction in photoresponses and decreased photoreceptor viability. The ultrastructure and phagocytosis of the photoreceptor outer segment appeared normal, but the PS and PE compositions were altered and the rhodopsin content was decreased. The auditory brainstem response threshold was significantly higher and degeneration of spiral ganglion cells was apparent. Our studies indicate that ATP8A2 plays a crucial role in photoreceptor and spiral ganglion cell function and survival by maintaining phospholipid composition and contributing to vesicle trafficking.

A Genetic Variant in the SKIV2L Gene Is Significantly Associated With Age-Related Macular Degeneration in a Han Chinese Population

PURPOSE Previous studies have shown that genetic variants in the complement component 2 (C2)/complement factor B (BF) gene are associated with AMD in Caucasians, but not in Han Chinese. Recent studies have indicated that genetic variants in the neighboring superkiller viralicidic activity 2-like (SKIV2L) gene showed significant association with AMD. We conducted this study to investigate whether genetic variants in the SKIV2L gene are associated with AMD in a Han Chinese population. METHODS Thirteen single nucleotide polymorphisms (SNPs) in the C2-BF-RDBP-SKIV2L-STK19 region were genotyped by the SNaPshot method in a cohort composed of 449 patients with choriodal neovascularization (CNV) AMD and 1025 healthy controls of Han Chinese descent. RESULTS Among the SNPs genotyped, P values of seven SNPs were less than 0.05; however, only rs429608 was found to be significantly associated with AMD after correction for multiple testing. The minor allele (A) frequency of rs429608 was 0.050 in cases and 0.089 in controls, and the P value was 3.76 × 10(-4) (0.00489 after Bonferroni correction), with an odds ratio of 0.55 (95% confidence interval, 0.40-0.77). The SKIV2L gene was expressed in the human RPE, retina, and D407 (human RPE) cells, and in mouse retinas and RPE. CONCLUSIONS We demonstrated that the rs429608 genetic variant in the SKIV2L gene was significantly associated with AMD in a Han Chinese population. SKIV2L may play an important role in the development of AMD.

Genetic Variants in PVRL2-TOMM40-APOE Region Are Associated with Human Longevity in a Han Chinese Population

Purpose Human longevity results from a number of factors, including genetic background, favorable environmental, social factors and chance. In this study, we aimed to elucidate the association of human longevity with genetic variations in several major candidate genes in a Han Chinese population. Methods A case-control association study of 1015 long-lived individuals (aged 90 years or older) and 1725 younger controls (30–70 years old) was undertaken. Rs2075650 in TOMM40 was firstly genotyped using the ABI SNaPshot method in an initial cohort consisted of 597 unrelated long-lived individuals and 1275 younger controls enrolled from Sichuan. Secondly, eighteen tag single-nucleotide polymorphisms (SNPs) in the PVRL2-TOMM40-APOE locus were genotyped for extensive study in the same cohort. Finally, 5 associated SNPs were genotyped in a replication cohort including 418 older individuals and 450 younger controls. The genotype and allele frequencies were evaluated using the χ2 tests. The linkage disequilibrium (LD) block structure was examined using the program Haploview. Results The case-control study of rs2075650 in TOMM40 showed significant difference in allele frequencies between cases and controls (P = 0.006) in an initial study. Of the 18 SNPs genotyped, rs405509 in APOE and another three SNPs (rs12978931, rs519825 and rs395908) in the PVRL2 gene also showed significant association with human longevity in extensive study in the same cohort. Rs2075650 in TOMM40, rs405509 in APOE and rs519825 in PVRL2 showed a significant association with human longevity in a replication cohort. Conclusion These results suggested that PVRL2, TOMM40 and APOE might be associated with human longevity. However, further research is needed to identify the causal variants and determine which of these genes are involved in the progress of human longevity.

Synthesis of the sulfate donor PAPS in either the Drosophila germline or somatic follicle cells can support embryonic dorsal-ventral axis formation

The establishment of dorsal-ventral (DV) polarity in the Drosophila embryo depends upon a localized signal that is generated in the perivitelline space of the egg through the action of a serine proteolytic cascade. Spatial regulation of this pathway is determined by the expression of the pipe gene in a subpopulation of ventral follicle cells in the developing egg chamber. The Pipe protein exhibits homology to vertebrate glycosaminoglycan sulfotransferases. In a previous study, we demonstrated that embryonic DV polarity depends upon the sulfotransferase activity of Pipe. Surprisingly, however, our results also indicated that formation of the embryonic DV axis does not require the synthesis of the high-energy sulfate donor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS) in the follicle cells in which Pipe is presumed to function. Here, we resolve this apparent paradox by demonstrating that dorsalized embryos are only produced by egg chambers in which both germline and follicle cells lack PAPS synthetase activity. Thus, PAPS produced either in the germline or in the follicular epithelium can support the requirement for Pipe sulfotransferase activity in embryonic DV patterning. This finding indicates the existence of a conduit for the movement of PAPS between the germline and the follicle cells, which highlights a previously unappreciated mechanism of soma/germline cooperation affecting pattern formation.

Early immune responses are independent of RGC dysfunction in glaucoma with complement component C3 being protective

Significance Exactly how high intraocular pressure (IOP) initiates glaucoma is unknown. Immune responses occur early in glaucoma, but whether they are induced by high IOP or occur secondarily to retinal ganglion cell (RGC) dysfunction and molecular changes in neurons and glia remains unknown. This paper addresses these relationships and provides a deeper understanding of this very common neurodegeneration. Overall, our data suggest that early immune responses are independent of RGC dysfunction and thus are triggered as a more direct result of high IOP. Furthermore, early immune responses by astrocytes that involve complement C3 and EGFR signaling are beneficial. Various immune response pathways are altered during early, predegenerative stages of glaucoma; however, whether the early immune responses occur secondarily to or independently of neuronal dysfunction is unclear. To investigate this relationship, we used the Wlds allele, which protects from axon dysfunction. We demonstrate that DBA/2J.Wlds mice develop high intraocular pressure (IOP) but are protected from retinal ganglion cell (RGC) dysfunction and neuroglial changes that otherwise occur early in DBA/2J glaucoma. Despite this, immune pathways are still altered in DBA/2J.Wlds mice. This suggests that immune changes are not secondary to RGC dysfunction or altered neuroglial interactions, but may be directly induced by the increased strain imposed by high IOP. One early immune response following IOP elevation is up-regulation of complement C3 in astrocytes of DBA/2J and DBA/2J.Wlds mice. Unexpectedly, because the disruption of other complement components, such as C1Q, is protective in glaucoma, C3 deficiency significantly increased the number of DBA/2J eyes with nerve damage and RGC loss at an early time point after IOP elevation. Transcriptional profiling of C3-deficient cultured astrocytes implicated EGFR signaling as a hub in C3-dependent responses. Treatment with AG1478, an EGFR inhibitor, also significantly increased the number of DBA/2J eyes with glaucoma at the same early time point. These findings suggest that C3 protects from early glaucomatous damage, a process that may involve EGFR signaling and other immune responses in the optic nerve head. Therefore, therapies that target specific components of the complement cascade, rather than global inhibition, may be more applicable for treating human glaucoma.

Whole-exome Sequencing Analysis Identifies Mutations in the EYS Gene in Retinitis Pigmentosa in the Indian Population

Retinitis pigmentosa (RP) is a rare heterogeneous genetic retinal dystrophy disease, and despite years of research, known genetic mutations can explain only approximately 60% of RP cases. We sought to identify the underlying genetic mutations in a cohort of fourteen Indian autosomal recessive retinitis pigmentosa (arRP) families and 100 Indian sporadic RP cases. Whole-exome sequencing (WES) was performed on the probands of the arRP families and sporadic RP patients, and direct Sanger sequencing was used to confirm the causal mutations identified by WES. We found that the mutations of EYS are likely pathogenic mutations in two arRP families and eight sporadic patients. Specifically, we found a novel pair of compound heterozygous mutations and a novel homozygous mutation in two separate arRP families, and found two novel heterozygous mutations in two sporadic RP patients, whereas we found six novel homozygous mutations in six sporadic RP patients. Of these, one was a frameshift mutation, two were stop-gain mutations, one was a splicing mutation, and the others were missense mutations. In conclusion, our findings expand the spectrum of EYS mutations in RP in the Indian population and provide further support for the role of EYS in the pathogenesis and clinical diagnosis of RP.