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. 2013 Jan 2;54(1):25-36.
doi: 10.1167/iovs.12-10861.

Protection of mouse retinal ganglion cell axons and soma from glaucomatous and ischemic injury by cytoplasmic overexpression of Nmnat1

Yanli Zhu  1 , Lihong Zhang, Yo Sasaki, Jeffrey Milbrandt, Jeffrey M Gidday
Affiliations

Protection of mouse retinal ganglion cell axons and soma from glaucomatous and ischemic injury by cytoplasmic overexpression of Nmnat1

Yanli Zhu et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: The Wlds mutation affords protection of retinal ganglion cell (RGC) axons in retinal ischemia and in inducible and hereditary preclinical models of glaucoma. We undertook the present study to determine whether the Nmnat1 portion of the chimeric protein provides axonal and somatic protection of RGCs in models of ischemia and glaucoma, particularly when localized to nonnuclear regions of the cell.

Methods: The survival and integrity of RGC axons and soma from transgenic mice with confirmed cytoplasmic overexpression of Nmnat1 in retina and optic nerve (cytNmnat1-Tg mice) were examined in the retina and postlaminar optic nerve 4 days following acute retinal ischemia, and 3 weeks following the chronic elevation of intraocular pressure.

Results: Ischemia- and glaucoma-induced disruptions of proximal segments of RGC axons that comprise the nerve fiber layer in wild-type mice were both robustly abrogated in cytNmnat1-Tg mice. More distal portions of RGC axons within the optic nerve were also protected from glaucomatous disruption in the transgenic mice. In both disease models, Nmnat1 overexpression in extranuclear locations significantly enhanced the survival of RGC soma.

Conclusions: Overexpression of Nmnat1 in the cytoplasm and axons of RGCs robustly protected against both ischemic and glaucomatous loss of RGC axonal integrity, as well as loss of RGC soma. These findings reflect the more pan-cellular protection of CNS neurons that is realized by cytoplasmic Nmnat1 expression, and thus provide a therapeutic strategy for protecting against retinal neurodegeneration, and perhaps other CNS neurodegenerative diseases as well.

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Conflict of interest statement

Disclosure: Y. Zhu, None; L. Zhang, None; Y. Sasaki, None; J. Milbrandt, None; J.M. Gidday, None

Figures

Figure 1.
Figure 1.
cytNmnat1 (34 kDa) Western blot results, using an anti-6xHis tag antibody, from matched regions of brain, optic nerve (postlaminar, prechiasm), and retina from cytNmnat1-Tg and WT mice. Each band represents pooled tissue from two animals.
Figure 2.
Figure 2.
SMI32-immunostaining of RGCs in retinal flat mounts from ischemic and fellow eyes of WT and cytNmnat1-Tg mice. (A) Representative lower-power montages from ischemic (b, d) and fellow (a, c) eyes of WT (a, b) and cytNmnat1-Tg (c, d) mice. Scale bar = 200 μm. (B) Representative higher-power images of RGC axons and soma, by quadrant, in the midperipheral retinae of both ischemic (right) and fellow (left) eyes of WT (top pair) and cytNmnat1-Tg (bottom pair) mice. Scale bar = 100 μm. (C) Quantification of axonal SMI32 fluorescence for all quadrants, normalized to the fellow eye, is shown for WT (n = 3) and cytNmnat1-Tg (n = 7) mice. Severe damage in many quadrants of the ischemic retinae of many WT mice made overall quantification of SMI32 immunofluorescence impossible (n/a). *P < 0.05 versus fellow retina.
Figure 3.
Figure 3.
SMI32 and GFAP immunohistochemistry in cross sections of postlaminar optic nerve from ischemic and fellow eyes of WT and cytNmnat1-Tg mice. (A) Representative photomicrographs of SMI32- and GFAP-immunostained optic nerve cross-sections from ischemic and fellow eyes of each genotype (with DAPI for nuclear counterstaining). (B, C) Quantification of SMI32 immunofluorescence for RGC axons (B) and GFAP fluorescence for astrocytes (C), normalized to levels in respective fellow eye, showed no differences for either marker between nerves from ischemic and fellow eyes in either the WT (n = 7) and cytNmnat1-Tg (n = 8) mice, indicative of a lack of distal RGC axonal injury at 4 days postischemia. Scale bar in (A) = 200 μm.
Figure 4.
Figure 4.
NeuN-immunostaining in retinal flat mounts from ischemic and fellow eyes of WT and cytNmnat1-Tg mice. (A) Representative images of NeuN+ soma, by quadrant, in the midperipheral retinae of both ischemic (right) and fellow (left) eyes of WT (top pair) and cytNmnat1-Tg (bottom pair) mice. Scale bar = 100 μm. (B) Quantification of NeuN-positive neurons in the ganglion cell layer (GCL) for all quadrants, normalized to the fellow eye, is shown for WT (n = 4) and cytNmnat1-Tg (n = 4) mice. Note, it was not possible (n/a) to quantify NeuN-immunopositive cells in the ischemic retinae of WT mice due to the severity of the ischemic injury in many retinal quadrants across many WT retinae, whereas the overall integrity of NeuN+ soma in the cytNmnat1-Tg mice was similar to that of fellow eyes, with only a mild-to-moderate loss (P = 0.125 versus fellow retina) of NeuN-positive soma.
Figure 5.
Figure 5.
IOP changes in the experimental glaucoma groups. (A) Temporal changes in IOP in response to episcleral vein ligation in experimental eyes (filled symbols) and fellow eyes (open symbols) of WT (circles; n = 9) and cytNmnat1-Tg (squares; n = 9) mice during the 3-week period of intraocular hypertension. (B) Tabulation of baseline IOP (shown at time zero) and mean (from 24 hours to 3 weeks) IOP for the glaucoma and fellow eyes of mice from each genotype. *P < 0.05 versus baseline; #P < 0.05 versus fellow eye at the same time point.
Figure 6.
Figure 6.
SMI32-immunostaining of RGCs in retinal flat mounts from glaucomatous and fellow eyes of WT and cytNmnat1-Tg mice. (A) Lower-power montages from glaucomatous (b, d) and fellow (a, c) eyes of representative WT (a, b) and cytNmnat1-Tg (c, d) mice. Scale bar = 200 μm. (B) Higher-power images of RGC axons and soma, by quadrant, in the peripheral retinae of both glaucomatous (GL; right) and fellow (left) eyes of representative WT (top pair) and cytNmnat1-Tg (bottom pair) mice. Scale bar = 100 μm. (C) Quantification of axonal SMI32 fluorescence for all quadrants, normalized to the fellow eye, is shown for WT (n = 9) and cytNmnat1-Tg (n = 9) mice. Note that the extent of glaucomatous RGC axonal loss in WT was significantly reduced in cytNmnat1-Tg mice. *P < 0.05 versus fellow retina. #P < 0.05 versus GL retina in corresponding WT group.
Figure 7.
Figure 7.
SMI32 and GFAP immunohistochemistry in cross sections of postlaminar optic nerve from glaucomatous and fellow eyes of WT and cytNmnat1-Tg mice. (A) Representative photomicrographs of SMI32- and GFAP-immunostained optic nerve cross-sections from GL and fellow eyes of each genotype (with DAPI for nuclear counterstaining). All scale bars = 20 μm. (B, C) Quantification of SMI32 immunofluorescence for RGC axons (B) and GFAP fluorescence for astrocytes (C) revealed a significant loss of axons, and, concomitantly, a significant increase in astrocytosis, in the GL optic nerves from WT mice (n = 9 and 8, respectively), both of which were abrogated in the glaucomatous nerves from cytNmnat-Tg mice (n = 9 and 8, respectively). *P < 0.05 versus fellow nerve. #P < 0.05 versus GL nerve in corresponding WT group.
Figure 8.
Figure 8.
NeuN-immunostaining in retinal flat mounts from GL and fellow eyes of WT and cytNmnat1-Tg mice. (A) Representative images of NeuN+ soma, by quadrant, in the peripheral retinae of GL and fellow eyes of WT (top pair) and cytNmnat1-Tg (bottom pair) mice. Scale bar = 100 μm. (B) Quantification of NeuN-positive neurons in the GCL, normalized to the fellow eye, is shown for WT (n = 6) and cytNmnat1-Tg (n = 5) mice. Note the significant protection against glaucomatous NeuN+ soma loss in the cytNmnat1-Tg mice relative to WT mice. *P < 0.05 versus fellow retina. #P < 0.05 versus GL retina in WT group.
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References

    1. Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol. 2006; 90: 262–267 - PMC - PubMed
    1. Libby RT, Li Y, Savinova OV, et al. Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage. PLoS Genet. 2005; 1: 17–26 - PMC - PubMed
    1. Nickells RW, Semaan SJ, Schlamp CL. Involvement of the Bcl2 gene family in the signaling and control of retinal ganglion cell death. Prog Brain Res. 2008; 173: 423–435 - PubMed
    1. Almasieh M, Wilson AM, Morquette B, Cueva Vargas JL, Di Polo A. The molecular basis of retinal ganglion cell death in glaucoma. Prog Retin Eye Res. 2012; 31: 152–181 - PubMed
    1. Coleman MP, Freeman MR. Wallerian degeneration, wld(s), and nmnat. Annu Rev Neurosci. 2010; 33: 245–267 - PMC - PubMed

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