Abstract
Axotomised retinal ganglion cells (RGCs) die rapidly by apoptosis and fail to regenerate because of the limited availability of neurotrophic factors and a lack of axogenic stimuli. However, we have recently showed that pigment epithelium-derived factor (PEDF) promotes RGC survival and axon regeneration after optic nerve crush injury. PEDF has multiple fragments of the native peptide that are neuroprotective, anti-angiogenic and anti-inflammatory. Here we investigated the neuroprotective and axogenic properties of a fragment of PEDF, PEDF-34, in retinal neurons in vitro and when delivered by intravitreal injection and eye drops in vivo. We found that PEDF-34 was 43% more neuroprotective and 52% more neuritogenic than PEDF-44 in vitro. Moreover, in vivo, intravitreal delivery of 1.88 nM PEDF-34 was 71% RGC neuroprotective at 21 days after optic nerve crush compared to intact controls, whilst daily eye drops containing 1.88 nM PEDF-34 promoted 87% RGC survival. After topical eye drop delivery, PEDF-34 was detected in the vitreous body within 30 min and attained physiologically relevant concentrations in the retina by 4 h peaking at 1.4 ± 0.05 nM by 14 days. In eye drop- compared to intravitreal-treated PEDF-34 animals, 55% more RGC axons regenerated 250 μm beyond the optic nerve lesion. We conclude that daily topical eye drop application of PEDF-34 is superior to weekly intravitreal injections in promoting RGC survival and axon regeneration through both direct effects on retinal neurons and indirect effects on other retinal cells.
Highlights
ciliary neurotrophic factor (CNTF) promoted the survival of 30–34 ± 6% βIII-tubulin+ neurons after 4 days in cultures. These results show that pigment epithelium-derived factor (PEDF)-34 treatment resulted in a 4.5fold increase in βIII-tubulin+ neuron numbers, whilst survival in CNTF or PEDF-44-treated cultures was 1.9- and 2.4-fold increased, respectively, compared to untreated cultures
PEDF-34 promoted 1.7, 2.2- and 6.3-fold longer neurites and 2.3, 10.5, and 21-fold more βIII-tubulin+ neurons with neurites than in PEDF-44 and CNTF demonstrate that 5.4 pM of PEDF-34 and PEDF-44 optimally promotes neuronal survival. (B) The mean proportion of neurons with neurites and (C) the mean neurite length increased with increasing concentration of PEDF-34 and PEDF-44 with peak values at 5.4 pM and 10.8 pM, respectively. (D) Representative images of βIII-tubulin+ neurons to demonstrate neurite outgrowth. *** = P b 0.0001; * = P b 0.05
We demonstrate that PEDF-34 is both neuroprotective and axogenic in vitro and in vivo for adult rat retinal neurons/retinal ganglion cells (RGCs)
Summary
The 50 kDa neurotrophic factor pigment epithelium-derived factor (PEDF) is a member of the serpin superfamily, first isolated from foetal retinal pigment epithelial cells but expression in normal choroid, ciliary body, corneal epithelium, retinal ganglion cells (RGCs) and photoreceptors has since been reported (Barnstable and Tombran-Tink, 2004; Karakousis et al, 2001; Lange et al, 2008; Ogata et al, 2002; Pang et al, 2007; Tombran-Tink and Barnstable, 2003; Unterlauft et al, 2012). Retinal pathologies, PEDF is secreted by reactive Müller cells and astrocytes and is neuroprotective for RGC and photoreceptors (Cao et al, 2001; Lange et al, 2008; Li et al, 2006; Zhu and Zou, 2012). We confirmed that, after optic nerve crush (ONC), PEDF is RGC neuroprotective and axogenic (Vigneswara et al, 2013), whilst other neuroprotective and anti-angiogenic N-terminal fragments of the native peptide exist (Bilak et al, 2002; Gvritishvili et al, 2010; Li et al, 2006; Mirochnik et al, 2009) including the neuroprotective amino acid residues 78–121, anti-angiogenic residues 16–26, 24–57, 44–77, 60–77 and 78–94 (Alberdi et al, 1999; Bilak et al, 2002; Filleur et al, 2005; Liu et al, 2012) and residues 58–101 which promote the differentiation of PC3 cells into neuro-secretory neurons (Filleur et al, 2005). PEDF-34 was more neuroprotective and axogenic in vivo, after topical eye drop application compared to intravitreal injection
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