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other·ophthalmology, glaucoma, neuro-ophthalmology, proteomics, molecular biology·PMC10377047

Glaucoma-Associated CDR1 Peptide Promotes RGC Survival in Retinal Explants through Molecular Interaction with Acidic Leucine Rich Nuclear Phosphoprotein 32A (ANP32A)

Biomolecules · 9 authors, 3 centres

AI SUMMARY

FIDELITY 94%

POPULATIONRetinal explants from house swine (Sus scrofa domestica) with axotomy-induced RGC degeneration

INTERVENTIONSynthetic CDR1 peptide (SCTGTSSDVGGYNYVSWYQ) at 50 µg/mL and 100 µg/mL

COMPARISONUntreated control retinal explants (CTRL)

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TL;DR

A synthetic CDR1 peptide (SCTGTSSDVGGYNYVSWYQ) significantly increased retinal ganglion cell (RGC) survival by up to 31% in an ex vivo glaucoma model. The peptide binds to the leucine-rich repeat domain of ANP32A, altering downstream protein expression related to energy metabolism, protein phosphorylation, and histone acetylation. These findings identify ANP32A as a potential new drug target for neuroprotective glaucoma therapy.

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**Background:** Glaucoma is a leading cause of blindness worldwide, characterized by progressive loss of retinal ganglion cells (RGCs). Current therapies focus on lowering intraocular pressure (IOP), but many patients continue to lose vision, highlighting the need for direct neuroprotective strategies. Antibody-derived peptides, particularly complementarity-determining region (CDR) peptides, have shown promise in promoting RGC survival. This study investigates the neuroprotective potential of a synthetic CDR1 peptide (SCTGTSSDVGGYNYVSWYQ) previously identified as a biomarker in primary open-angle glaucoma (POAG) patients, and aims to characterize its molecular interaction with acidic leucine-rich nuclear phosphoprotein 32A (ANP32A).

**Methods:** Retinal explants from house swine were cultured for 24 h after optic nerve axotomy to induce RGC degeneration. Explants were treated with 50 µg/mL or 100 µg/mL of synthetic CDR1 peptide or left untreated (CTRL). RGC survival was assessed by immunohistochemical staining for Brn3a, a specific RGC marker, and manual counting of Brn3a+ cells. To identify protein interaction partners of CDR1, peptide-based immunoprecipitation was performed using biotinylated CDR1 immobilized on streptavidin magnetic beads, followed by mass spectrometry (MS) analysis. Molecular dynamics (MD) simulations and virtual docking (AutoDock Vina) were used to predict the binding site of CDR1 on ANP32A (PDB: 4XOS). Quantitative proteomic analysis (LC-MS/MS) was performed on retinal explants treated with 100 µg/mL CDR1 versus CTRL (n=3 per group). Differentially expressed proteins were identified using MaxQuant and Perseus (t-test, p<0.05). Western blotting assessed protein phosphorylation (Ser/Thr) and histone acetylation.

**Key Results:** CDR1 treatment significantly increased RGC density in a concentration-dependent manner. At 50 µg/mL, RGC density was 352 ± 62 RGCs/mm² vs. 274 ± 30 in CTRL (p<0.05, ~22% increase). At 100 µg/mL, RGC density reached 399 ± 52 RGCs/mm² vs. 274 ± 30 in CTRL (p<0.001, ~31% increase). Immunoprecipitation-MS identified ANP32A as a highly enriched protein in the CDR1-bound fraction (p<0.001, log2 fold change >3). MD simulations revealed a β-hairpin structure in the central region of CDR1 (residues S6-G11), and docking predicted binding to the N-terminal leucine-rich repeat (LRR) domain of ANP32A (residues F41-K111). Quantitative proteomics of retinal explants identified 25 significantly changed proteins (12 up, 13 down) with CDR1 treatment (p<0.05). Upregulated proteins included pyruvate carboxylase (PC, 2.99-fold), moesin (MSN, 2.88-fold), pyruvate dehydrogenase E1α (PDHA1, 1.31-fold), and endophilin-A1 (SH3GL2, 1.45-fold). Downregulated proteins included vacuolar protein sorting-associated protein 26B (VPS26B, 9.97-fold), ATP synthase subunit gamma (ATP5F1C, 10.30-fold), cell cycle exit and neuronal differentiation protein 1 (CEND1, 11.81-fold), and ANP32A-interacting proteins nucleoside diphosphate kinase A (NME1, 1.99-fold) and serine/threonine-protein phosphatase 2A activator (PPP2R4, 1.56-fold). ANP32A itself showed a slight, non-significant decrease (p=0.105). IPA pathway analysis highlighted Acetyl-CoA Biosynthesis I (p=4.62), Mitochondrial Dysfunction (p=4.47), Granzyme A Signaling (p=3.71), and Oxidative Phosphorylation (p=3.62). Western blotting indicated increased histone acetylation in CDR1-treated retinae, while protein phosphorylation showed a non-significant increase (p=0.18).

**Clinical Implications:** This study demonstrates that a synthetic CDR1 peptide derived from a glaucoma-associated antibody sequence promotes RGC survival ex vivo by specifically interacting with ANP32A. The peptide appears to modulate ANP32A function, leading to changes in energy metabolism (acetyl-CoA biosynthesis), protein phosphorylation (via PPP2R4/PP2A), and histone acetylation. These findings suggest that ANP32A is a promising new drug target for neuroprotective glaucoma therapy. The peptide's ability to enhance RGC survival by up to 31% in a concentration-dependent manner, combined with its specific molecular mechanism, supports further development of CDR1-based therapeutics. However, the required peptide concentration (50-100 µg/mL) is higher than in previous studies targeting HTRA2, likely due to the high abundance of ANP32A in the retina. Future studies should validate the CDR1-ANP32A interaction using techniques such as FRET or SPR, and explore the therapeutic potential of this peptide in in vivo glaucoma models.

PICO

PPOPULATION

Retinal explants from house swine (Sus scrofa domestica) with axotomy-induced RGC degeneration

IINTERVENTION

Synthetic CDR1 peptide (SCTGTSSDVGGYNYVSWYQ) at 50 µg/mL and 100 µg/mL

OOUTCOME

RGC density (Brn3a+ cells/mm²) and proteomic changes

STUDY TYPE

other

SPECIALTY

ophthalmology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

94% · A

ORIGINAL PAPER

PMC10377047

Glaucoma-Associated CDR1 Peptide Promotes RGC Survival in Retinal Explants through Molecular Interaction with Acidic Leucine Rich Nuclear Phosphoprotein 32A (ANP32A)

Biomolecules

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ABSTRACT

Abstract Glaucoma is a complex, multifactorial optic neuropathy mainly characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, resulting in a decline of visual function. The pathogenic molecular mechanism of glaucoma is still not well understood, and therapeutic strategies specifically addressing the neurodegenerative component of this ocular disease are urgently needed. Novel immunotherapeutics might overcome this problem by targeting specific molecular structures in the retina and providing direct neuroprotection via different modes of action. Within the scope of this research, the present study showed for the first time beneficial effects of the synthetic CDR1 peptide SCTGTSSDVGGYNYVSWYQ on the viability of RGCs ex vivo in a concentration-dependent manner compared to untreated control explants (CTRL, 50 µg/mL: p < 0.05 and 100 µg/mL: p < 0.001). Thereby, this specific peptide was identified first as a potential biomarker candidate in the serum of glaucoma patients and was significantly lower expressed in systemic IgG molecules compared to healthy control subjects. Furthermore, MS-based co-immunoprecipitation experiments confirmed the specific interaction of synthetic CDR1 with retinal acidic leucine-rich nuclear phosphoprotein 32A (ANP32A; p < 0.001 and log2 fold change > 3), which is a highly expressed protein in neurological tissues with multifactorial biological functions. In silico binding prediction analysis revealed the N-terminal leucine-rich repeat (LRR) domain of ANP32A as a significant binding site for synthetic CDR1, which was previously reported as an important docking site for protein-protein interactions (PPI). In accordance with these findings, quantitative proteomic analysis of the retinae ± CDR1 treatment resulted in the identification of 25 protein markers, which were significantly differentially distributed between both experimental groups (CTRL and CDR1, p < 0.05). Particularly, acetyl-CoA biosynthesis I-related enzymes (e.g., DLAT and PDHA1), as well as cytoskeleton-regulating proteins (e.g., MSN), were highly expressed by synthetic CDR1 treatment in the retina; on the contrary, direct ANP32A-interacting proteins (e.g., NME1 and PPP2R4), as well as neurodegenerative-related markers (e.g., CEND1), were identified with significant lower abundancy in the CDR1-treated retinae compared to CTRL. Furthermore, retinal protein phosphorylation and histone acetylation were also affected by synthetic CDR1, which are both partially controlled by ANP32A. In conclusion, the synthetic CDR1 peptide provides a great translational potential for the treatment of glaucoma in the future by eliciting its neuroprotective mechanism via specific interaction with ANP32A’s N terminal LRR domain. Keywords: glaucoma, neuroprotection, ANP32A, proteomics, mass spectrometry

CITATION EXPORT

Carsten Schmelter, Kristian Nzogang Fomo, Alina Brueck et al.. Glaucoma-Associated CDR1 Peptide Promotes RGC Survival in Retinal Explants through Molecular Interaction with Acidic Leucine Rich Nuclear Phosphoprotein 32A (ANP32A). Biomolecules. (2023). https://doi.org/10.3390/biom13071161

AUTHORS

9 · 3 CENTRES

Carsten Schmelter, Kristian Nzogang Fomo, Alina Brueck, Natarajan Perumal, Sascha D Markowitsch, Gokul Govind, Thomas Speck, Norbert Pfeiffer, Franz H Grus

1Department of Experimental and Translational Ophthalmology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany; [email protected] (C.S.); [email protected] (K.N.F.); [email protected] (A.B.); [email protected] (N.P.); [email protected] (N.P.)
2Department of Urology and Pediatric Urology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany; [email protected]
3Institute of Physics, Johannes Gutenberg University, 55131 Mainz, Germany; [email protected] (G.G.);