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Dr. Shivani Dixit, D17574, Dr. Sushma Tejwani, Dr. Harsha Rao, Dr. Zia SultanPradhan

ABSTRACT:

Title: De-regulated Notch signaling in the lens capsule of eyes with pseudoexfoliation syndrome

Purpose: Notch is a signaling pathway which regulates cell proliferation versus differentiation. It is vital for eye development, both in the lens and the retina. We hypothesized that altered notch signaling in the eye may account for differing phenotypes in pseudoexfoliation. This study aimed to identify the alterations in the expression of Notch pathway molecules in the lens capsules of eyes with pseudoexfoliation deposits.

Methods: Anterior lens capsules were collected from 35patients undergoing cataract surgery. These included patients with pseudoexfoliation syndrome/PXF (n=11), pseudoexfoliation glaucoma/PXG (n=7), primary open-angle glaucoma/POAG (n=8) and controls (n=9). Gene expression profiling for Notch pathway molecules was performed on the tissue using quantitative polymerase chain reaction. The results were confirmed by protein analysis using dot-blot or immune staining techniques.

Results: There was no difference in the demographic characteristics of the 4 groups. There was an increase in the Notch 4 receptor expression (>15 fold) while decrease in Notch 2 expression in the PXF group as compared to the controls. Similarly, the Delta-like 3 and Delta-like 4 ligands were significantly elevated in the PXF group as compared to the controls (p<0.05). Downstream targets HES 3 and HEY 1 expression revealed significantly elevated levels (p<0.001) in PXF lens capsules confirming a higher activity of Notch signaling in this cohort. Immunostaining also corroborated the gene expression profile.

Conclusion: Notch signaling is highly activated in the lens capsule of eyes with PXF, but not in PXG or POAG eyes. This may have an implication in the protective effect of activated Notch signaling in preventing glaucoma in eyes with pseudoexfoliation deposits.

KEYWORDS: Notch signaling pathway, Pseudoexfoliation, pseudoexfoliation glaucoma

INTRODUCTION

The notch signalling pathway plays an important role in determiningcell fate and regulating pattern formation in a wide range of tissues, which isessential for the orderly development of multicellular life.

The core components of Notch pathway in mammalian cells include (shown in Figure 1):

  1. Ligands – Delta-like 1, Delta-like 3, Delta-like 4, Jagged 1, Jagged 2
  2. Receptors – Notch 1, Notch 2, Notch 3 and Notch 4
  3. Transcription factor of CSL family and downstream target genes – HES 1, HES 3, HES 5, HEY 1

Activation of this pathway occurs when a Notch receptor is engaged from a neighbouring cell via the Delta-like (DLL) or Jagged (Jag) ligands, as shown in figure 1. The Notch receptor undergoes proteolyticcleavage which liberates an intracellular domain (NotchIC); this thentranslocates to the nucleus and acts with transcription factors (also known as RBP-Jκ1, CSL, or CBF-1) to activate the transcriptional repressors.1,2 Notch activation generally prevents differentiation and maintains progenitor or stem cell proliferation.3,4 Notch signaling has diverse, outcomes, since it can inhibit, delay or induce differentiation determining the survival and fate of cells.

Low levels of Notch signaling decrease the neuro-protective function of glial cells in the post-natal retina.We hypothesized that altered notch signaling in the eye may account for differing phenotypes in pseudoexfoliation. This study aimed to identify the alterations in the expression of Notch pathway molecules in the lens capsules of eyes with pseudoexfoliation deposits.

METHODS

This was a prospective, observational study conducted at  a tertiary eye care center between January 2016 to July 2017. The methodology adhered to the tenets of the Declaration of Helsinki for research involving human subjects. Written informed consent was obtained from allparticipants and the study was approved by the Institute’s Ethics Committee.

Anterior lens capsules were collected from patients undergoing cataract surgery. These included eyes with pseudoexfoliation syndrome (PXF), pseudoexfoliation glaucoma (PXG), primary open-angle glaucoma (POAG) and controls. Control subjects were patients who had IOP < 21mmHg, normal anterior segment examination apart from a cataract and normal posterior segment examination withnon-glaucomatous optic discs,as assessed by glaucoma experts. PXF patients had clinical features of pseudoexfoliative material deposition in the anterior segment with IOP <21 mmHg, non-glaucomatous optic discs and normal visual fields.PXG patients had clinically evident pseudoexfoliativematerial with IOP>21 mmHg along with glaucomatous changes on optic nerve head examination as documented by glaucoma experts (neuroretinal rim narrowing, notching, and retinal nerve fiber layer defects), and/or visual fields changes.POAG patients had open angles on gonioscopy, IOP>21 mmHgand glaucomatous optic disc or visual field changes. All participants were above 18 years of age. Eyes with a history of trauma or inflammation were excluded. All participants underwent acomprehensive ocular examination, which included a detailed medical history, slit-lamp biomicroscopy, Goldmannapplanationtonometry, gonioscopy,dilated fundus examination, and ocular biometry (axial length and central corneal thickness).

The anterior capsules collected at the time of surgery were stored in 0.5 ml balanced salt solution at -80 ̊C.Gene expression profiling for Notch pathway molecules (ligands, receptors and downstream genes) was performed using quantitative polymerase chain reaction (PCR). Any overexpression of RNA detected was confirmed by performingprotein expression studies (dot-blot or immunostaining). The expression of Notch pathway molecules was compared between the groups using the ANOVA statistical test. Statistical analyses were performed using commercial software (Stata ver. 13.1; StataCorp, College Station Texas, USA). A P value of <0.05 was considered statistically significant.

RESULTS

Thirty-five eyes of 35 patients were included for the initial part of the study. The demographic details are shown in Table 1. The groups were well matched for age, axial length and central corneal thickness. However, comparison of the two glaucomatous groups showed the PXG eyes had significantly higher IOP and more advanced glaucoma (worse MD and thinner RNFL) than the POAG eyes.

The gene expression ligands, notch receptions and downstream genes were studied using PCR. Delta-like 3 (p=0.008) and Delta-like 4 (p=0.006) ligands were significantly elevated in the PXF group as compared to the other groups, as seen in figure 2.

The expression of Notch 2 receptor was significantly reduced while that of Notch 4 receptor was >15-fold higher in PXF eyes, as shown in figure 3.

HES3 & HEY1 expression was significantly elevated in PXF lens capsules (p<0.0001) as compared to the other groups, as shown in figure 4.

These findings were confirmed by analysing protein expression using dot-blot or immunostaining techniques, as shown in figure 5.

DISCUSSION

The present study shows the altered expression of Notch pathway molecules in the lens capsules of eyes with PXF as compared to those in PXG, POAG and controls. The role of notch signaling in the pathogenesis of pseudoexfoliation has not been previously explored.

Notch signaling has been shown to be essential for lens development by Rowan S et al.5They found that Jag1 ligand and Notch1/2 receptor domains overlap significantly in the lens pit, during lens development. This finding has been supported by other studies as well, which have reported expression of Notch1, Notch2, and Jag1 in the developing lens.6-9Regarding the role of downstream targets, Jia et al. propose Hey1 as a major Notch effector gene, while Rowan et al believe in the strong expression of Hes1 throughout lens development.10

Besides the lens, the Notch pathway in the posterior segment has also been evaluated.Ghai K et al showed that low levels of Notch signaling is associated with reduced neuroprotective function of the Muller cells in the mature retina.11

In our study, we found thatin PXF capsules, there was as increase in the expression of molecules of all the three components of Notch pathway, including DLL3 and DLL4 ligands, Notch 4 receptor as well as HES3 and HEY1 target genes. It suggests that Notch signaling is highly activated in the lens capsule of eyes with PXF, but not in PXG or POAG eyes. This may have an implication in the protective effect of activated Notch signaling in preventing glaucoma in eyes with pseudoexfoliation deposits. Further studies are required to elucidate the signaling intricacies in various ocular tissues with pseudoexfoliation.

REFERENCES

  1. Fischer, A., Gessler, M., 2007. Delta–Notch—and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors.Nucleic Acids Res. 35, 4583–4596.
  2. Ilagan, M.X., Kopan, R., 2007. SnapShot: notch signaling pathway. Cell 128, 1246.
  3. Bolós, V., Grego-Bessa, J., de la Pompa, J.L., 2007. Notch signaling in development and cancer. Endocr. Rev. 28, 339–363.
  4. Yoon, K., Gaiano, N., 2005. Notch signaling in the mammalian central nervous system:insights from mouse mutants. Nat. Neurosci. 8, 709–715.
  5. Rowan S et al. Notch signaling regulates growth and differentiation in the mammalian lens. Developmental Biology 2008;321:111-22.
  6. Bao, Z.Z., Cepko, C.L., 1997. The expression and function of Notch pathway genes in the developing rat eye. Journal of Neuroscience 17, 1425–1434.
  7. Bettenhausen, B., de Angelis, M.H., Simon, D., Guenet, J., Gossler, A., 1995. Transient andrestricted expression during mouse embryogenesis of Dll1, a murine gene closelyrelated to Drosophila Delta. Development 121, 2407–2418.
  8. Ishibashi, M., Ang, S.-L., Shiota, K., Nakanishi, S., Kageyama, R., Guillemot, F., 1995. Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helix-loop-helix factors, premature neurogenesis, and severe neural tube defects. Genes Dev. 9, 9136–9148.
  9. Weinmaster, G., Roberts, V.J., Lemke, G., 1991. A homolog of Drosophila Notch expressed during mammalian development. Development 113, 199–205.
  10. Jia, J., Lin, M., Zhang, L., York, J.P., Zhang, P., 2007. The notch signaling pathway controls the size of the ocular lens by directly suppressing p57Kip2  expression. Mol. Cell. Biol. 27, 7236–7247.
  1. Ghai K et al. Notch signaling influences neuroprotection and proliferative properties of mature muller glia. J Neuroscience 2010;30:3101-12.

TABLE

Table 1: Demographic and clinical characteristics of study participants

CONTROL PXF PXG POAG P VALUE
No of Eyes 9 11 7 8
Mean Age (SD)/years 65.9 (9.6) 67.7 (5.9) 69.8 (9.7) 68.4 (3.5) 0.76
Mean IOP (SD)/ mmHg 14.9 (3.5) 16.3 (1.8) 21.6 (5.4) 16.5 (3.7) 0.006
Mean Axial length (SD)/ mm 23.4 (1.1) 22.8 (0.9) 23.0 (0.7) 23.6 (0.7) 0.23
Mean CCT (SD)/ µ 532.5 (40) 524.9 (40) 506.3 (37) 531.8 (28) 0.50
Mean mean deviation (SD)/ dB – – -25.3 (4.06) -4.2 (3.15) <0.001
Mean Avg RNFL thickness (SD)/ µ 95.3 (13) 94.0 (11) 61.8 (8) 85.0 (8) <0.001

 

LEGENDS:

Figure 1: Notch signaling pathway showing Delta-type ligand, the receptor Notch and the CSL transcription factor

Figure 2:A graph comparing the expression of Notch ligands in controls, PXF, PXG and POAG subjects

Figure 3: A graph comparing the expression of Notch receptors in controls, PXF, PXG and POAG subjects

Figure 4: A graph comparing the expression of downstream targets in controls, PXF, PXG and POAG subjects

Figure 5: A slide showing immunostaining for Notch 4 receptors in PXF, PXG and controls

FIGURES:

Figure 1: Notch signaling pathway showing Delta-type ligand, the receptor Notch and the CSL transcription factor

Figure 2: A graph comparing the expression of Notch ligands in controls, PXF, PXG and POAG subjects

Figure 3: A graph comparing showing the expression of Notch receptors in controls, PXF, PXG and POAG subjects

Figure 4: A graph comparing showing the expression of downstream targets in controls, PXF, PXG and POAG subjects

Figure 5: A slide showing immunostaining for Notch 4 receptors in PXF, PXG and controls

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