September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
SHP2 (PTPN11) over-expression by AAV gene delivery impairs neuronal cell growth in SH-SY5Y cells and induces neurodegeneration of SD rat retinal ganglion cells
Author Affiliations & Notes
  • Nitin Chitranshi
    Macquarie University, Sydney, New South Wales, Australia
  • Vivek Gupta
    Macquarie University, Sydney, New South Wales, Australia
  • Roshana Vanderwall
    Macquarie University, Sydney, New South Wales, Australia
  • YOGITA DHEER
    Macquarie University, Sydney, New South Wales, Australia
  • Stuart L Graham
    Macquarie University, Sydney, New South Wales, Australia
    University of Sydney, Save Sight Institute, Sydney, New South Wales, Australia
  • Footnotes
    Commercial Relationships   Nitin Chitranshi, None; Vivek Gupta, None; Roshana Vanderwall, None; YOGITA DHEER, None; Stuart Graham, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3997. doi:
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      Nitin Chitranshi, Vivek Gupta, Roshana Vanderwall, YOGITA DHEER, Stuart L Graham; SHP2 (PTPN11) over-expression by AAV gene delivery impairs neuronal cell growth in SH-SY5Y cells and induces neurodegeneration of SD rat retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3997.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : SH2 domain containing phosphatase-2 protein (Shp2) (PTPN11), dephosphorylates tropomyosin receptor kinase B (TrkB) which plays an important role in the retina. We assessed adeno-associated virus (AAV) mediated over expression of Shp2 in neuronal cells in culture and rat RGCs in vivo and evaluated its effects on the neuronal cell viability and retinal ganglion cells (RGCs) in vivo.

Methods : The SH-SY5Y neuronal cell line was transfected using AAV-SHP2 viral vector, which was also injected into the eyes of SD male rats (n=24). The control GFP viral vector without Shp2 (AAV-GFP) was used for both neuronal cells and rat eyes. Gene expressions were evaluated both in vitro and in vivo by immunofluorescence. Retinal function was analysed by electroretinogram (ERG) and scotopic threshold response (STR) and structural changes by OCT (Micron IV) and histology. IOP was measured with Icare-tonometer.

Results : SH-SY5Y cells with Shp2 over-expression showed significantly reduced viability: 95.67+2.24 vs 71.33+2.67%. Anti-GFP antibody staining demonstrated AAV transduction in neuronal cells and in the ganglion cell layer. Anti-NeuN/anti-Shp2 staining confirmed the cell specific over-expression and localization of Shp2. ERG amplitudes remained unaltered but the positive scotopic threshold response (STR) amplitude was reduced: 29.27+2.84 µV vs 18.32+1.84 (P<0.0001, n=12; control vs treated). Retinal fundus morphology appeared grossly intact but H and E staining of retinal sections highlighted a thinner inner retina: 91.83±1.26 vs61.55±2.35 % (P<0.0001, n=6; control vs treated). Optic nerve histological analysis also showed a reduced axonal density (81.38 ±1.91 vs 59.01±0.73 %; p<0.0001). No obvious changes were observed in the IOP 10.2±0.3 vs 9.1±0.4.

Conclusions : Eyes injected with AAV-Shp2 indicated a decline in RGC numbers and reduced STR. These findings indicate that Shp2 over-expression may have harmful effects on the inner retinal health and have implications in glaucoma and other optic nerve and retinal diseases. Further studies in larger cohort of animals will substantiate these observations and provide mechanistic insights into the role of Shp2 in the retina.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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