Roshana Vander Wall

Amyloid β accumulation and inner retinal degenerative changes in Alzheimer's disease transgenic mouse.

The APP-PS1ΔE9 mouse model of Alzheimers disease (AD) exhibits age dependent amyloid β (Aβ) plaque formation in their central nervous system due to high expression of mutated human APP and PSEN1 transgenes. Here we evaluated Aβ deposition and changes in soluble Aβ accumulation in the retinas of aged APP-PS1 mice using a combination of immunofluorescence, retinal flat mounts and western blotting techniques. Aβ accumulation in the retina has previously been shown to be associated with retinal ganglion cell apoptosis in animal models of glaucoma. This study investigated changes in the inner retinal function and structure in APP-PS1 mice using electrophysiology and histological approaches respectively. We report for the first time a significant decline in scotopic threshold response (STR) amplitudes which represents inner retinal function in transgenic animals compared to the wild type counterparts (p<0.0001). Thinning of the retina particularly involving inner retinal layers and reduction in axonal density in the optic nerve was also observed. TUNEL staining was performed to examine neuronal apoptosis in the inner retina. Intraocular pressure (IOP) measurements showed that APP-PS1ΔE9 mice had a slightly elevated IOP, but the significance of this finding is not yet known. Together, these results substantiate previous observations and highlight that APP-PS1ΔE9 mice show evidence of molecular, functional and morphological degenerative changes in the inner retina.

One protein, multiple pathologies: multifaceted involvement of amyloid β in neurodegenerative disorders of the brain and retina

Accumulation of amyloid β (Aβ) and its aggregates in the ageing central nervous system is regarded synonymous to Alzheimer’s disease (AD) pathology. Despite unquestionable advances in mechanistic and diagnostic aspects of the disease understanding, the primary cause of Aβ accumulation as well as its in vivo roles remains elusive; nonetheless, the majority of the efforts to address pathological mechanisms for therapeutic development are focused towards moderating Aβ accumulation in the brain. More recently, Aβ deposition has been identified in the eye and is linked with distinct age-related diseases including age-related macular degeneration, glaucoma as well as AD. Awareness of the Aβ accumulation in these markedly different degenerative disorders has led to an increasing body of work exploring overlapping mechanisms, a prospective biomarker role for Aβ and the potential to use retina as a model for brain related neurodegenerative disorders. Here, we present an integrated view of current understanding of the retinal Aβ deposition discussing the accumulation mechanisms, anticipated impacts and outlining ameliorative approaches that can be extrapolated to the retina for potential therapeutic benefits. Further longitudinal investigations in humans and animal models will determine retinal Aβ association as a potential pathognomonic, diagnostic or prognostic biomarker.

Age-related neurodegenerative disease associated pathways identified in retinal and vitreous proteome from human glaucoma eyes

AbstarctGlaucoma is a chronic disease that shares many similarities with other neurodegenerative disorders of the central nervous system. This study was designed to evaluate the association between glaucoma and other neurodegenerative disorders by investigating glaucoma-associated protein changes in the retina and vitreous humour. The multiplexed Tandem Mass Tag based proteomics (TMT-MS3) was carried out on retinal tissue and vitreous humour fluid collected from glaucoma patients and age-matched controls followed by functional pathway and protein network interaction analysis. About 5000 proteins were quantified from retinal tissue and vitreous fluid of glaucoma and control eyes. Of the differentially regulated proteins, 122 were found linked with pathophysiology of Alzheimer’s disease (AD). Pathway analyses of differentially regulated proteins indicate defects in mitochondrial oxidative phosphorylation machinery. The classical complement pathway associated proteins were activated in the glaucoma samples suggesting an innate inflammatory response. The majority of common differentially regulated proteins in both tissues were members of functional protein networks associated brain changes in AD and other chronic degenerative conditions. Identification of previously reported and novel pathways in glaucoma that overlap with other CNS neurodegenerative disorders promises to provide renewed understanding of the aetiology and pathogenesis of age related neurodegenerative diseases.

DBA/2J mouse model for experimental glaucoma: pitfalls and problems

The DBA/2J mouse has been described as a model for congenital experimental glaucoma. It develops anterior segment anomalies with synechiae and pigment dispersion leading to raised intraocular pressure and glaucomatous damage. However, there are serious practical considerations when using this model in longitudinal studies.

Glaucoma is associated with plasmin proteolytic activation mediated through oxidative inactivation of neuroserpin

Neuroserpin is a serine protease inhibitor that regulates the activity of plasmin and its activators in the neuronal tissues. This study provides novel evidence of regulatory effect of the neuroserpin on plasmin proteolytic activity in the retina in glaucoma. Human retinal and vitreous tissues from control and glaucoma subjects as well as retinas from experimental glaucoma rats were analysed to establish changes in plasmin and neuroserpin activity. Neuroserpin undergoes oxidative inactivation in glaucoma which leads to augmentation of plasmin activity. Neuroserpin contains several methionine residues in addition to a conserved reactive site methionine and our study revealed enhanced oxidation of Met residues in the serpin under glaucoma conditions. Met oxidation was associated with loss of neuroserpin inhibitory activity and similar findings were observed in the retinas of superoxide dismutase (SOD) mutant mice that have increased oxidative stress. Treatment of purified neuroserpin with H2O2 further established that Met oxidation inversely correlated with its plasmin inhibitory activity. Dysregulation of the plasmin proteolytic system associated with increased degradation of the extracellular matrix (ECM) proteins in the retina. Collectively, these findings delineate a novel molecular basis of plasmin activation in glaucoma and potentially for other neuronal disorders with implications in disease associated ECM remodelling.

Molecular determinants and interaction data of cyclic peptide inhibitor with the extracellular domain of TrkB receptor

TrkB is a high affinity receptor for the brain derived neurotrophic factor (BDNF) and its phosphorylation stimulates activation of several intracellular signalling pathways linked to cellular growth, differentiation and maintenance. Identification of various activators and inhibitors of the TrkB receptor and greater understanding their binding mechanisms is critical to elucidate the biochemical and pharmacological pathways and analyse various protein crystallization studies. The data presented here is related to the research article entitled “Brain Derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling” [1]. Cyclotraxin B (CTXB) is a disulphide bridge linked cyclic peptide molecule that interacts with TrkB receptor and inhibits the BDNF/TrkB downstream signalling. This article reports for the first time binding mechanism and interaction parameters of CTXB with the TrkB receptor. The molecular model of CTXB has been generated and it’s docking with TrkB domain carried out to determine the critical residues involved in the protein peptide interaction.

188. AAV Mediated Gene Therapy to Modulate Neurotropic Factors in the Retina and in Neuronal Cells in Culture

Neurotrophic factors, particularly Brain Derived Neurotrophic Factor (BDNF) and its high affinity receptor TrkB, play an important role in protecting the retinal ganglion cells (RGCs) in glaucoma. Translation of the immense neuro-protective potential of BDNF/TrkB activation has been largely unsuccessful and gene therapy to modulate BDNF and TrkB have also shown only short-lived protective effects on the RGCs. PTPN11 phosphatase plays a key role in the regulation of BDNF/TrkB signaling in the retina. Here, we investigated the effects of PTPN11 modulation involving both over-expression and knock-down paradigms on the TrkB activation. PTPN11 and PTPN11-shRNA sequences were separately incorporated into the AAV2 vector under cytomegalovirus chicken β actin hybrid promoter linked with green fluorescent protein (GFP). SH-SY5Y cells were transfected with each of the PTPN11 modulating and control GFP constructs. PTPN11 over-expression resulted in significant reduced TrkB phosphorylation and accordingly reduced viability of the SH-SY5Y cells was observed while PTPN11 knockdown did not exhibit any significant effects on either TrkB phosphorylation or cellular survival. PTPN11 over-expression in neuronal cells was also accompanied by elevation of GADD, PERK and XBP-1 endoplasmic reticulum (ER) stress marker proteins as detected using western blotting. The roles of PTPN11 and TrkB in mediating ER stress were evaluated by treating cells with PTPN11 inhibitor (PHPS1) or TrkB agonist (7,8-dihydroxyflavone) independently, both of which ablated the up-regulation of ER stress proteins upon PTPN11 over-expression. The effects of PTPN11 over-expression on the rat retina were evaluated by administering PTPN11 AAV2 construct along with corresponding GFP controls individually into the rat eyes. Briefly, 5µl of AAV2 constructs were injected intravitreally into SD rats (2×1010 vg). Anti-GFP staining in retinal sections demonstrated AAV2 transduction in the RGC layer. Anti-NeuN/ anti-PTPN11 staining confirmed the ganglion cell specific over-expression of PTPN11. Rat retinas were assessed for inner retinal functional changes using scotopic threshold response (STR) measurements. Retinal structural changes were assessed using histological analysis. Results indicate that pSTR amplitude which primarily reflects the function of the RGCs was significantly diminished in the PTPN11 over-expression model. Hematoxylin and eosin staining also revealed degenerative changes primarily associated with the inner retina. Assessment of optic nerve sections using Bielschowskys staining further identified reduced axonal density. Concluding, our findings strongly indicate that PTPN11 genetic modulation holds the promise to regulate neuronal cell survival through its effects on TrkB activation. PTPN11 over-expression exerts detrimental impact on the inner retinal health which has significant implications in glaucoma and other retinal disorders. Further studies involving rescue of the disease phenotype using PTPN11 knockdown in animal models will substantiate these observations and provide mechanistic insights into the roles of neurotrophic factor regulation in retina.