Chronic oxidative stress eventually leads to protein aggregation in combination with impaired autophagy, which has been observed in age-related macular degeneration. We have previously shown an effective age-related macular degeneration disease model in mice with nuclear factor-erythroid 2-related factor-2 (NFE2L2) knockout. We have also shown pinosylvin, a polyphenol abundant in bark waste, to increase human retinal pigment epithelium cell viability in vitro. In this work, the effects of commercial natural pinosylvin extract, Retinari™, were studied on the electroretinogram, optical coherence tomogram, autophagic activity, antioxidant capacity, and inflammation markers. Wild-type and NFE2L2 knockout mice were raised until the age of 14.8 ± 3.8 months. They were fed with either regular or Retinari™ chow ( 141 ± 17.0 mg/kg/day of pinosylvin) for 10 weeks before the assays. Retinari™ treatment preserved significant retinal function with significantly preserved a- and b-wave amplitudes in the electroretinogram responses. Additionally, the treatment prevented thinning of the retina in the NFE2L2 knockout mice. The NFE2L2 knockout mice showed reduced ubiquitin-tagged protein accumulation in addition to local upregulation of complement factor H and antioxidant enzymes superoxide dismutase 1 and catalase. Therefore, the treatment in the NFE2L2 KO disease model led to reduced chronic oxidative stress and sustained retinal function and morphology. Our results demonstrate that pinosylvin supplementation could potentially lower the risk of age-related macular degeneration onset and slow down its progression.
Chronic oxidative stress eventually leads to protein aggregation in combination with impaired autophagy, which has been observed in age-related macular degeneration. We have previously shown an effective age-related macular degeneration disease model in mice with nuclear factor-erythroid 2-related factor-2 (NFE2L2) knockout. We have also shown pinosylvin, a polyphenol abundant in bark waste, to increase human retinal pigment epithelium cell viability in vitro. In this work, the effects of commercial natural pinosylvin extract, Retinari™, were studied on the electroretinogram, optical coherence tomogram, autophagic activity, antioxidant capacity, and inflammation markers. Wild-type and NFE2L2 knockout mice were raised until the age of 14:8 ± 3:8 months. They were fed with either regular or Retinari™ chow (141 ± 17:0 mg/kg/day of pinosylvin) for 10 weeks before the assays. Retinari™ treatment preserved significant retinal function with significantly preserved a-and b-wave amplitudes in the electroretinogram responses. Additionally, the treatment prevented thinning of the retina in the NFE2L2 knockout mice. The NFE2L2 knockout mice showed reduced ubiquitin-tagged protein accumulation in addition to local upregulation of complement factor H and antioxidant enzymes superoxide dismutase 1 and catalase. Therefore, the treatment in the NFE2L2 KO disease model led to reduced chronic oxidative stress and sustained retinal function and morphology. Our results demonstrate that pinosylvin supplementation could potentially lower the risk of agerelated macular degeneration onset and slow down its progression.
Introduction: The eye is an excellent target for gene therapy because of its anatomical features. Gene therapy to treat ocular disorders relies on efficient gene delivery and transgene expression in the target cells. The aim of this study was to compare the biodistribution and safety of two different AAV serotypes after intravitreal (IVT) and subretinal injections.Methods: AAV2 (1 × 1012 vg/mL) and AAV9 (5 × 1012 vg/mL) vectors expressing an enhanced green fluorescent protein (EGFP) and an AAV9-empty (6 × 1011 vg/mL) vector were injected intravitreally or subretinally into both eyes of adult C57Bl/OlaHsd mice. The biodistribution of the viral vectors in the eye and off-target tissues was studied using qPCR. GFP expression was studied from cryosections, and GFP transduction efficacy was verified using immunohistostaining for GFP. In addition, electroretinography (ERG) was used to assess the effect of vectors on retinal function.Results: In addition to the eyes, viral vector copies were found in distant off-target tissues such as the liver, especially after AAV9-EGFP IVT and subretinal injections. AAV9-EGFP injections showed more GFP expression throughout the retina compared to AAV2-EGFP. AAV2-EGFP IVT showed transgene expression mainly in the ganglion cell layer, whereas subretinal injection showed GFP expression in the retinal pigment epithelium. In addition, GFP was expressed at a moderate level in the liver after both injection routes of AAV9 and in parts of the brain after all injection groups except AAV9-empty. Lowered a- and b-amplitude values were seen in ERG in both scotopic and photopic experiments after AAV9-EGFP subretinal injection compared to all other groups.Discussion: This study shows that intraocular injection of AAV2 and AAV9 transduces retinal cells. Although the more efficient transduction of the retina, negative effect on the retinal function, and off-target transgene expression of AAV9 makes AAV2 a more suitable gene delivery vector to treat ocular disorders.
Purpose: Chronic oxidative stress, disturbed proteostasis and increased protein aggregation are key hallmarks in Alzheimer's disease and age‐related macular degeneration pathology. In this study, we assessed the protein aggregation and secretory autophagy related biomarker changes in 5xFAD mice model expressing abundant levels of human beta‐amyloid (Aβ42). Methods: The eyes of 6‐ and 12‐month‐old WT (n = 5) and 5xFAD (n = 5) were examined by immunomicroscopy. The paraffin sections were immunostained with the key regulators of secretory autophagy HMGB1, Aβ42, IL‐1β, and ferritin as well as protein aggregation markers ubiquitin protein and SQSTM1/p62. Results: Immunohistochemistry analysis revealed that ubiquitin protein conjugates and Aβ42 were increased in 6 and 12 months old 5xFAD mice. SQSTM1/p62 was decreased in 12 months old 5xFAD mice. In 12‐month‐old time point, they located basolateral side of the retinal pigment epithelial (RPE) cells but also in extracellular space. Concurrently, secretory autophagy markers were elevated in the RPE and extracellular space between RPE and Bruch's membrane. Conclusions: Secretary autophagy may be a novel regulator in drusen biogenesis. 5xFAD mouse is a good model to study protein aggregation and secretory autophagy mechanism in response to degenerative retina processes.
Purpose Circadian clocks in the eye are critical for normal visual function and they relate to periodical renewal of photoreceptor outer segments as well as oxidative stress. Impaired rhythm can thus contribute to the pathogenesis of age‐related macular degeneration (AMD). Chronic oxidative stress leads eventually to protein aggregation in combination with impaired autophagy. We hypothesise that pinosylvin, a polyphenolic compound, slows down the progression of AMD through autophagy induction. Methods We investigated the relative change in electroretinogram (ERG) metrics of mice raised in 12:12 h light‐dark cycle and after a 24 h two‐week dark period in wild type (WT; n = 10), PGC1‐α knock‐out (KO; n = 7) and Nrf2 KO (n = 7) mice aged 9.7 ± 3.4 months. From these, 9 WT, 5 PGC1‐α and 7 Nrf2 KO mice continued to be raised to an age of 17.3 ± 3.8 months and assigned to a treatment and control group and fed for 2 months with pinosylvin‐feed or regular‐feed prior to recording ERG. Results The two‐week dark period resulted in significantly greater b‐wave amplitudes and shorter b‐wave latencies in WT mice in photopic ERG. Nrf2 KO and PGC1‐α/Nrf2 dKO mice exhibited b‐wave latency changes in the opposite. The dKO also had diminished b‐wave amplitudes. Scotopic ERG yielded no significant changes. Pinosylvin treatment in Nrf2 KO and PGC1‐α KO mice caused significantly increased b‐wave amplitudes in photopic ERG. In scotopic ERG, the pinosylvin treated mice had larger a‐ and b‐wave amplitudes in WT and Nrf2 KO mice, but not in PGC1‐α KO mice. Conclusions PGC1‐α KO and Nrf2 KO mice showed different circadian rhythmicity. Pinosylvin treatment showed improved ERG signalling in aged NRf2 KO and WT mice.
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