Microtubule-associated protein 1 light chain 3B, (LC3B) is necessary to maintain lipid-mediated homeostasis in the retinal pigment epithelium
Open accessAbstract
Like other neurons, retinal cells utilize autophagic pathways to maintain cell homeostasis. The mammalian retina relies on heterophagy and selective autophagy to efficiently degrade and metabolize ingested lipids with disruption in autophagy associated degradation contributing to age related retinal disorders. The retinal pigment epithelium (RPE) supports photoreceptor cell renewal by daily phagocytosis of shed photoreceptor outer segments (OS). The daily ingestion of these lipid-rich OS imposes a constant degradative burden on these terminally differentiated cells. These cells rely on Microtubule-Associated Protein 1 Light Chain 3 (LC3) family of proteins for phagocytic clearance of the ingested OS. The LC3 family comprises of three highly homologous members, MAP1LC3A (LC3A), MAP1LC3B (LC3B) and MAP1LC3C (LC3C). The purpose of this study was to determine whether the LC3B isoform plays a specific role in maintaining RPE lipid homeostasis. We examined the RPE and retina of the LC3B-/- mouse as a function of age using in vivo ocular imaging and electroretinography coupled with ex vivo, lipidomic analyses of lipid mediators, assessment of bisretinoids as well as imaging of lipid aggregates. Deletion of LC3B resulted in defects within the RPE including increased phagosome accumulation, decreased fatty acid oxidation and a subsequent increase in RPE and sub-RPE lipid deposits. Age-dependent RPE changes included elevated levels of oxidized cholesterol, deposition of 4-HNE lipid peroxidation products, bisretinoid lipofuscin accumulation, and subretinal migration of microglia, collectively likely contributing to loss of retinal function. These observations are consistent with a critical role for LC3B-dependent processes in the maintenance of normal lipid homeostasis in the aging RPE, and suggest that LC3 isoform specific disruption in autophagic processes contribute to AMD-like pathogenesis.
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TL;DR
In LC3B-/- mice, loss of LC3B impairs RPE phagosome degradation and lipid metabolism, leading to age-dependent lipid deposits, oxidative/pro-inflammatory changes, and reduced retinal function. These findings suggest LC3B-dependent processes maintain RPE lipid homeostasis and that isoform-specific autophagic disruption may contribute to AMD-like pathogenesis.
Key findings
- LC3B-/- RPE showed slowed phagosome clearance, with phagosome numbers elevated before light onset and not returning to WT baseline; β-hydroxybutyrate release was reduced to 6.87 ± 0.71 nmoles vs. 15.0 ± 1.89 nmoles in WT by 9 am.
- Aged LC3B-/- RPE accumulated neutral lipids, with a 1.6-fold increase in BODIPY493/503 intensity and ~185% more lipid puncta than WT; 7-ketocholesterol increased ~5-fold, and 4-HNE adducts increased ~57%.
- Docosanoid mediators were decreased in LC3B-/- RPE, including esterified/free DHA, 17(S)-HDHA, NPD1, 14(S)-HDHA, and Maresin-1; retinal total DHA was lower while free DHA was enhanced.
- Retinal function declined in LC3B-/- mice: dark-adapted a-wave RmP3 was 401.0 ± 29.0 μV vs. 563.8 ± 27.0 μV in WT, b-wave Rmax was 571.9 ± 39.1 μV vs. 724.4 ± 59.1 μV, cone Rmax was 211.1 ± 17.4 μV vs. 283.5 ± 17.1 μV, and the c-wave was reduced to 68.9% of WT and delayed; bisretinoids were over twofold higher.
Methods
- Compared LC3B-/- mice (B6;129P2-Map1Lc3btm1Mrab/J, backcrossed to C57BL6/J) with WT C57BL6/J controls across ages, using in vivo ocular imaging (SLO, SDOCT) and electroretinography.
- Ex vivo analyses included electron microscopy phagosome counts, immunoblotting/immunostaining, BODIPY/filipin lipid staining, β-hydroxybutyrate and citrate synthase assays, lipidomics by LC/MS/MS, bisretinoid UPLC, 4-HNE ELISA, and sterol measurement.
- Statistical comparisons used one-way ANOVA with Fisher LSD, two-way repeated measures ANOVA, Student’s t-tests, and curve fits for ERG luminance-response functions; phagosome counts were performed under masked conditions.
Limitations
- The authors state they used a global LC3B-/- mouse but argue the retinal phenotype is largely due to loss of LC3B in retina/RPE rather than systemic effects, based on prior evidence that these mice develop normally and lack systemic defects.
- The authors note unresolved questions, including whether RUBCN modulates mitophagy and the specific role of LC3A in retina/RPE; they also state future studies are underway to determine the relationship between lipid accumulation and microglia recruitment.
- Evident from the text: findings are from a mouse model, with assays performed at varying ages and often small sample sizes (e.g., N = 3–7 per group), and no human AMD tissue or causal human validation is presented.
Model-written summary. Check it against the paper before relying on it.
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- Findingown data
Age-dependent RPE changes in LC3B-/- mice include elevated levels of oxidized cholesterol.
“Age-dependent RPE changes included elevated levels of oxidized cholesterol”
Quote in the paperSubstantial claim - Findingown data
LC3A mRNA and protein are not significantly upregulated in the RPE or retina of LC3B-/- mice.
“moreover, there was no significant up-regulation of LC3A mRNA or protein in the RPE or retina (Figures 1A–C), consistent with previous studies using mouse embryonic fibroblasts (Cann et al., 2008).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Cann et al., 2008 source
Not checked in the free run.
- Findingown data
Deletion of LC3B results in decreased fatty acid oxidation in the RPE.
“Deletion of LC3B resulted in defects within the RPE including increased phagosome accumulation, decreased fatty acid oxidation and a subsequent increase in RPE and sub-RPE lipid deposits.”
- Also stated in: § Introduction · plain
“We show that loss of LC3B results in decreased RPE-OS phagosome clearance and a concomitant decrease in fatty acid oxidation and ketogenesis.”
Quote in the paperSubstantial claim - Also stated in: § Introduction · plain
- Findingown data
Deletion of LC3B results in decreased RPE-OS phagosome clearance.
“We show that loss of LC3B results in decreased RPE-OS phagosome clearance and a concomitant decrease in fatty acid oxidation and ketogenesis.”
Quote in the paperSubstantial claim - Findingown data
Age-dependent RPE changes in LC3B-/- mice include deposition of 4-HNE lipid peroxidation products.
“deposition of 4-HNE lipid peroxidation products”
Quote in the paperSubstantial claim
- Findingown data
Deletion of LC3B results in decreased ketogenesis in the RPE.
“We show that loss of LC3B results in decreased RPE-OS phagosome clearance and a concomitant decrease in fatty acid oxidation and ketogenesis.”
Quote in the paperSubstantial claim - Findingown data
Deletion of LC3B results in increased phagosome accumulation in the RPE.
“Deletion of LC3B resulted in defects within the RPE including increased phagosome accumulation”
Quote in the paperSubstantial claim - Findingown data
Age-dependent RPE changes in LC3B-/- mice include subretinal migration of microglia.
“subretinal migration of microglia”
Quote in the paperSubstantial claim - Findingown data
Age-dependent RPE changes in LC3B-/- mice include bisretinoid lipofuscin accumulation.
“bisretinoid lipofuscin accumulation”
Quote in the paperSubstantial claim - Findingown data
ATG5 protein levels are not significantly changed in LC3B-/- RPE relative to WT across ages 4, 12, 16, and 20 months.
“No significant change in the levels of ATG5, an essential autophagy related protein and p62, an LC3 adaptor protein involved in selective autophagy (Figure 1C) was detected in L3CB-/- RPE relative to the WT for different age groups analyzed (4, 12, 16, and 20 months).”
Quote in the paperNot a substantial claim - Findingown data
p62 protein levels are not significantly changed in LC3B-/- RPE relative to WT across ages 4, 12, 16, and 20 months.
“No significant change in the levels of ATG5, an essential autophagy related protein and p62, an LC3 adaptor protein involved in selective autophagy (Figure 1C) was detected in L3CB-/- RPE relative to the WT for different age groups analyzed (4, 12, 16, and 20 months).”
Quote in the paperNot a substantial claim - Findingown data
Deletion of LC3B results in increased RPE and sub-RPE lipid deposits.
“a subsequent increase in RPE and sub-RPE lipid deposits”
- Also stated in: § Introduction · hedged
“accumulation of RPE and sub-RPE lipid deposits likely predisposing the RPE to an enhanced pro-inflammatory micro-environment”
Quote in the paperSubstantial claim - Also stated in: § Introduction · hedged
- Findingown data
The decrease in β-HB is not due to altered levels of mitochondrial HMGCS2, since both LC3B-/- and WT RPE express equivalent levels of HMGCS2.
“This decrease is not due to altered levels of mitochondrial HMGCS2, the rate limiting enzyme in ketogenesis, since both LC3B-/- and WT RPE express equivalent levels of HMGCS2 (Supplementary Figures S1B,C).”
Quote in the paperSubstantial claim - Findingown data
At both 12 and 21 months, LC3B-/- mice had hyper-reflective changes that resulted in a significant overall reduction in threshold area relative to controls.
“At both 12 and 21 months, LC3B-/- mice had hyper-reflective changes that resulted in a significant overall reduction in threshold area relative to the controls (Figure 2O), suggesting abnormal retinal pathology near the RPE in the LC3B-/-.mice.”
Quote in the paperSubstantial claim - Findingown data
These atypical retinal features were not found in young LC3B-/- mice (~3.5 months) using either SLO or SDOCT.
“These observations were not found in young mice (∼3.5 months) using either SLO (images not shown) or SDOCT (Figures 2I,J).”
Quote in the paperSubstantial claim - Findingown data
In LC3B-/- mice, the outer retina banding between ONL, IS, OS, RPE and choroid have all undergone localized disruption relative to controls.
“Notably, the outer retina banding between outer nuclear layer (ONL), inner segment (IS), outer segment (OS), RPE and choroid (Ch) have all undergone localized disruption relative to controls.”
Quote in the paperSubstantial claim - Findingown data
LC3B-/- mice exhibit an atypical spatial intensity distribution, with swathes of retina appearing brighter than immediately adjacent normal-appearing regions.
“In contrast to WT controls, L3CB-/- mice exhibited an atypical spatial intensity distribution, as swathes of retina appear brighter than immediately adjacent, normal-appearing regions (Figures 2C,D).”
Quote in the paperSubstantial claim - Findingown data
There was no difference in the number of nuclear layers between young WT and LC3B-/- mice.
“While, there was no difference in the number of nuclear layers between young WT and L3CB-/- mice,”
Quote in the paperNot a substantial claim - Findingcitation
In WT RPE, phagosome numbers reached a sharp peak within an hour of lights on.
“In WT RPE, phagosome numbers reached a sharp peak within an hour of lights on (Figure 4A) (LaVail, 1980; Gibbs et al., 2003; Nandrot et al., 2004).”
Quote in the paperNot a substantial claim0 of 3 sources checked - Findingown data
Atypical retinal features are distinguishable in older LC3B-/- mice compared to age-matched WT controls using both IRDF-SLO and SDOCT imaging modalities.
“Atypical retinal features were distinguishable in older L3CB-/- mice vs. age-matched WT controls using both imaging modalities (Figure 2).”
Quote in the paperSubstantial claim - Findingown data
The hyper-reflective pathology observed in the outer retina of LC3B-/- mice was determined as significant relative to WT at both 12 and 21 months.
“The hyper-reflective pathology observed in the outer retina of L3CB-/- mice was determined as significant relative to WT at both 12 and 21 months (Figure 2P).”
Quote in the paperSubstantial claim - Findingown data
LC3B ablation leads to decline in RPE and retinal cell function.
“as well as decline in RPE and retinal cell function.”
Quote in the paperSubstantial claim - Findingown data
There was a 1.6-fold increase in the intensity of BODIPY493/503 in LC3B-/- mouse RPE compared to WT.
“There was a 1.6-fold increase in the intensity of BODIPYTM493/503 in LC3B-/- mouse RPE compared to WT (Figure 4D),”
Quote in the paperSubstantial claim - Findingown data
No significant differences in IRDF hyper-reflective area were observed between 12 and 21 months in either LC3B-/- or WT controls.
“No significant differences in IRDF hyper-reflective area were observed between 12 and 21 months in either L3CB-/- or WT controls.”
Quote in the paperNot a substantial claim - Findingown data
Elevation in phagosome numbers in LC3B-/- RPE was evident for up to 14 h and never returned to WT baseline values prior to the next burst of OS uptake.
“Elevation in phagosome numbers in LC3B-/- RPE was evident for up to 14 h and never returned to the WT baseline values prior to the next burst of OS uptake (Figure 4A).”
Quote in the paperSubstantial claim - Findingown data
SDOCT shows hyper-reflective changes in the outer retina in LC3B-/- mice compared to age-matched WT controls.
“The qualitative comparison shows hyper-reflective changes in the outer retina in L3CB-/- mice compared to age-matched WT controls (Figures 2L,N vs. 2K,M).”
Quote in the paperSubstantial claim - Findingown data
Such changes were not apparent in a comparison of 3.5 months old WT and LC3B-/- mice.
“In addition, such changes were not apparent in a comparison of 3.5 months old WT and L3CB-/- mice (data not shown).”
Quote in the paperNot a substantial claim - Findingown data
Mitochondrial function based on citrate synthase activity was unaltered in the absence of LC3B.
“Moreover, mitochondrial function based on citrate synthase activity was unaltered in the absence of LC3B (Supplementary Figure S1D).”
Quote in the paperSubstantial claim - Findingown data
IRDF-SLO images from the RPE-choroid complex of LC3B-/- mice show hyper-reflective regions that are on average brighter than the typical homogenous background observed in WT mice.
“Representative IRDF-SLO images collected from the RPE-choroid complex of L3CB-/- mice show hyper-reflective regions (Figures 2C,D) that were on average, brighter than the typical, homogenous background observed in WT mice (Figures 2A,B).”
Quote in the paperSubstantial claim - Findingown data
There was an increase in vesicular binding of filipin, consistent with elevated intracellular cholesterol in LC3B-/- RPE.
“We observed an increase in vesicular binding of filipin, consistent with elevated intracellular cholesterol in LC3B-/- RPE.”
Quote in the paperSubstantial claim- Rests on an assumption: Cholesterol is a major component of neutral lipid droplets.
- Findingown data
The responses of LC3B-/- mice are reduced as compared to WT (P < 0.0001) in cone ERG luminance-response functions.
“The responses of LC3B-/- mice are reduced as compared to WT (P < 0.0001).”
Quote in the paperSubstantial claim - Findingown data
Although the mouse retina and RPE also express LC3A, there appears to be little to no compensatory upregulation in this isoform.
“Although the mouse retina and RPE also express LC3A, there appears to be little to no compensatory upregulation in this isoform (Figure 1).”
Quote in the paperSubstantial claim - Findingown data
The levels of atRALdiPE and A2GPE were over twofold higher in LC3B-/- compared to WT mice.
“The levels of these adducts were over twofold higher in LC3B-/- compared to WT mice (Figures 9A,B).”
Quote in the paperSubstantial claim - Findingown data
The c-wave peak time was also significantly slower in LC3B-/- as compared to WT mice (P < 0.04).
“The c-wave peak time was also significantly slower in LC3B-/- as compared to WT mice (P < 0.04).”
Quote in the paperSubstantial claim - Findingown data
Cholesterol rich vesicles were more abundant in LC3B-/- RPE compared to WT.
“Cholesterol rich vesicles were more abundant in LC3B-/- RPE compared to WT (Supplementary Figure S3B).”
Quote in the paperSubstantial claim - Findingown data
Numbers of lipid puncta were ~185% higher in LC3B-/- RPE compared to WT.
“with numbers of lipid puncta ∼185% higher in LC3B-/- RPE compared to WT (Figure 4D).”
Quote in the paperSubstantial claim - Findingown data
Maresin-1 levels and its stable intermediate derivative 14(S)-HDHA were decreased in LC3B-/- RPE of 18-20 months old mice compared to WT.
“Maresin-1 levels were also decreased as well as its stable intermediate derivative, 14(S)-HDHA in the LC3B-/- RPE of 18–20 months old mice as compared to WT (Figures 5E,F).”
Quote in the paperSubstantial claim - Findingreasoning
There was a concomitant decrease in 17(S)-HDHA and NPD1 in LC3B-/- RPE.
“Consistent with a decrease in DHA, there was also a concomitant decrease in 17(S)-HDHA and NPD1 in LC3B-/- RPE (Figures 5C,D).”
Quote in the paperSubstantial claim- Rests on an assumption: Free DHA serves as a substrate for the synthesis of NPD1, synthesized by 15-lipoxygenase-1 via the short live intermediate 17(S)-HpDHA.
- Findingown data
ERG responses of LC3B-/- mice were reduced compared to age-matched WT animals throughout the stimulus range examined.
“Throughout the stimulus range examined, responses of LC3B-/- mice were reduced as compared to those of age-matched WT animals.”
Quote in the paperSubstantial claim - Findingown data
The c-wave reduction (to 68.9% of WT) is comparable to that of RmP3 (to 71.1% of WT).
“The c-wave reduction (to 68.9% of WT) is comparable to that of RmP3 (to 71.1% of WT).”
Quote in the paperNot a substantial claim - Findingown data
There was a ~57% increase in levels of 4-HNE in LC3B-/- RPE explants compared to controls (WT 24 months, LC3B-/- 21 months p < 0.002).
“There was a ∼57% increase in levels of 4-HNE in the LC3B-/- RPE explants as compared to controls (WT 24 months, LC3B-/- 21 months p < 0.002, Figure 6A).”
Quote in the paperSubstantial claim - Findingown data
Iba1 positive cells were more abundant in the sub-retinal space of LC3B-/- mice compared to WT, with 62 ± 20.19 vs. 2.13 ± 0.55 per 1.62 mm2 in LC3B-/- vs. WT, respectively.
“Interestingly, while there were several Iba1 positive cells in both WT and LC3B-/- retina, these cells were more abundant in the sub-retinal space of LC3B-/- mice (Figures 7A,B), clustered close to the optic nerve with 62 ± 20.19 vs. 2.13 ± 0.55 per 1.62 mm2 in the LC3B-/- vs. WT, respectively (Supplementary Figure S4A).”
Quote in the paperSubstantial claim - Findingcitation
There appears to be an increase in Iba positive cells in the inner retina of LC3B-/- compared to WT.
“There appears to be increase in Iba positive cells in the inner retina of LC3B-/- compared to the WT,”
Quote in the paperNot a substantial claim - Findingown data
In the LC3B-/- mouse, the c-wave was significantly reduced in amplitude (P < 0.02).
“In the LC3B-/- mouse, the c-wave was significantly reduced in amplitude (P < 0.02).”
Quote in the paperSubstantial claim - Findingown data
In LC3B-/- mice, the protective role(s) of autophagy break down over time.
“In the LC3B-/- mouse we observe a breakdown in protective role(s) of autophagy over time”
Quote in the paperSubstantial claim - Findingown data
p62 levels in the LC3B-/- RPE in both young and older mice are unchanged.
“p62 levels in the LC3B-/- RPE in both young and older mice are unchanged”
Quote in the paperSubstantial claim - Findingown data
Citrate synthase activity (mitochondrial TCA cycle) is unaltered in LC3B-/- RPE.
“citrate synthase activity (mitochondrial TCA cycle) is unaltered (Supplementary Figure S1D).”
Quote in the paperSubstantial claim - Findingown data
In LC3B-/- RPE, phagosome levels do not return to baseline.
“phagosome levels do not return to baseline (Figure 4A)”
Quote in the paperSubstantial claim - Findingown data
Phagosome accumulation is observed in the absence of LC3B in the current studies.
“as well as in the absence of LC3B in the current studies (Figure 4A).”
Quote in the paperSubstantial claim - Findingown data
In LC3B-/- RPE, lipid deposits accumulate.
“there is an accumulation of lipid deposits (Figure 4C and Supplementary Figure S3)”
Quote in the paperSubstantial claim - Interpretationown data
LC3B-dependent processes play a critical role in the maintenance of normal lipid homeostasis in the aging RPE.
“These observations are consistent with a critical role for LC3B-dependent processes in the maintenance of normal lipid homeostasis in the aging RPE”
Quote in the paperSubstantial claim - Premisecitation
The RPE relies on LC3 family proteins for phagocytic clearance of ingested OS.
“These cells rely on Microtubule-Associated Protein 1 Light Chain 3 (LC3) family of proteins for phagocytic clearance of the ingested OS.”
- Also stated in: § Introduction · plain
“The RPE relies on LC3 for (1) organelle associated autophagy to maintain mitochondrial and peroxisomal health (Ferrington et al., 2016) and (2) a hybrid autophagy-phagocytosis degradative pathway termed LC3-associated phagocytosis (LAP) for degradation of OS (Kim et al., 2013; Frost et al., 2015; Martinez et al., 2015).”
Quote in the paperSubstantial claim0 of 3 sources checked - Also stated in: § Introduction · plain
- Interpretationreasoning
The age-dependent RPE changes in LC3B-/- mice likely contribute to loss of retinal function.
“collectively likely contributing to loss of retinal function”
Quote in the paperSubstantial claim - Premisecitation
The RPE relies on LC3 for organelle-associated autophagy to maintain mitochondrial and peroxisomal health.
“The RPE relies on LC3 for (1) organelle associated autophagy to maintain mitochondrial and peroxisomal health (Ferrington et al., 2016)”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Ferrington et al., 2016 source
Not checked in the free run.
- Premisecitation
Phagocytosed OS provide the RPE with fatty acids that are oxidized to provide substrate for ketogenesis.
“The protein- and lipid-rich phagocytosed OS provide the RPE with a metabolic bolus of fatty acids which are oxidized providing substrate for ketogenesis (Reyes-Reveles et al., 2017).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Reyes-Reveles et al., 2017 source
Not checked in the free run.
- Premisecitation
The RPE relies on LC3 for LAP-mediated degradation of OS.
“and (2) a hybrid autophagy-phagocytosis degradative pathway termed LC3-associated phagocytosis (LAP) for degradation of OS (Kim et al., 2013; Frost et al., 2015; Martinez et al., 2015).”
Quote in the paperSubstantial claim0 of 3 sources checked - Premisecitation
Loss of LAP results in decreased retinal function.
“with loss of LAP resulting in decreased retinal function (Kim et al., 2013)”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Kim et al., 2013 source
Not checked in the free run.
- Interpretationreasoning
LC3 isoform specific disruption in autophagic processes contributes to AMD-like pathogenesis.
“suggest that LC3 isoform specific disruption in autophagic processes contribute to AMD-like pathogenesis.”
Quote in the paperSubstantial claim - Premisecitation
Loss of LAP results in dysregulated lipid metabolism.
“and dysregulated lipid metabolism (Reyes-Reveles et al., 2017).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Reyes-Reveles et al., 2017 source
Not checked in the free run.
- Premisecitation
OS phagocytosis allows recycling of DHA and visual pigment to photoreceptors to maintain visual function.
“OS phagocytosis also allows for recycling of docosahexaenoic acid (DHA) (Gordon and Bazan, 1990; Rodriguez de Turco et al., 1991) and visual pigment to photoreceptors to maintain visual function (Tang et al., 2013).”
Quote in the paperNot a substantial claim0 of 3 sources checked - Premisecitation
The RPE has adapted LAP to eliminate stressors, including daily ingestion of lipids and proteins in OS tips.
“The RPE has adapted LAP to eliminate stressors, including the daily ingestion of lipids and proteins in the form of OS tips (Kim et al., 2013; Frost et al., 2015; Reyes-Reveles et al., 2017).”
Quote in the paperSubstantial claim0 of 3 sources checked - Interpretationown data
Collectively, these studies suggest age-dependent changes in the LC3B-/- RPE structure and sub-retinal space with relatively intact retina.
“Collectively, these studies suggest age-dependent changes in the L3CB-/- RPE structure and sub-retinal space with relatively intact retina.”
Quote in the paperSubstantial claim - Interpretationreasoning
Accumulation of RPE and sub-RPE lipid deposits likely predisposes the RPE to an enhanced pro-inflammatory micro-environment.
“accumulation of RPE and sub-RPE lipid deposits likely predisposing the RPE to an enhanced pro-inflammatory micro-environment”
Quote in the paperNot a substantial claim - Premisecitation
The LC3B-/- mouse has a null allele due to deletion of exons III and IV of Map1lc3b.
“We examined the role of LC3B in retinal homeostasis using an LC3B-/- mouse, in which deletion of exons III and IV of Map1lc3b results in a null allele (Cann et al., 2008).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Cann et al., 2008 source
Not checked in the free run.
- Interpretationown data
The RPE displays decreased abundance of docosahexaenoyl containing phospholipids reflecting an impairment in the shedding/phagocytosis and/or in the handling of this disk membrane components for proper photoreceptor outer segment renewal.
“The RPE displays decreased abundance of docosahexaenoyl containing phospholipids (Figure 5G) reflecting an impairment in the shedding/phagocytosis and/or in the handling of this disk membrane components for proper photoreceptor outer segment renewal.”
Quote in the paperSubstantial claim- Rests on an assumption: The RPE handles large amounts of lipids, with OSs serving as a major source of cholesterol.
- Interpretationown data
In LC3B-/- mice the c-wave amplitude reduction and delay are consistent with a reduced contribution of the positive signal generated by the RPE apical membrane, although other mechanisms are possible.
“Taken together, in LC3B-/- mice the c-wave amplitude reduction and delay are consistent with a reduced contribution of the positive signal generated by the RPE apical membrane, although other mechanisms are possible.”
Quote in the paperSubstantial claim - Interpretationown data
LC3A does not functionally compensate for LC3B in RPE as it relates to phagosome degradation and lipid metabolism with altered lipid homeostasis over time.
“Nor does LC3A functionally compensate for LC3B in RPE as it relates to phagosome degradation and lipid metabolism with altered lipid homeostasis over time.”
Quote in the paperSubstantial claim - Interpretationown data
The LC3B-/- mouse is the first to show elevated 7KCh in ocular tissue, in vivo.
“These results are particularly significant because to our knowledge, the LC3B-/- mouse is the first to show elevated 7KCh in ocular tissue, in vivo.”
Quote in the paperSubstantial claim - Premisecitation
There is no indication of systemic defects in the four previous studies using LC3B-/- mice.
“There is no indication of systemic defects in the 4 previous studies using this mouse (Cann et al., 2008; Chen et al., 2010; Lee et al., 2011; Reed et al., 2015).”
Quote in the paperSubstantial claim0 of 3 sources checked - Study design and conductown data
Rabbit anti-LC3B (D11, from Cell Signaling) specifically detects LC3B.
“Here we show that a rabbit anti-LC3B (D11, from Cell Signaling), specifically detects LC3B”
Quote in the paperSubstantial claim- Rests on an assumption: A substantial majority of studies using anti-LC3 antibodies presume them to be LC3B specific.
- Interpretationreasoning
The accumulation of 4-HNE-adducts in the RPE and sub-RPE space leads to loss of RPE and retinal function in LC3B-/- mice.
“the accumulation of 4-HNE-adducts both in the RPE and sub-RPE space leading to loss of RPE and retinal function.”
Quote in the paperSubstantial claim - Interpretationreasoning
The retinal phenotype seen in global LC3B-/- mice is largely due to loss of LC3B in retina and RPE and not due to systemic effects.
“we reason that the retinal phenotype seen is largely due to loss of LC3B in retina and RPE and not due to systemic effects.”
Quote in the paperSubstantial claim - Study design and conductown data
LC3B-/- mice are the same weight as age-matched wild type from 2 to 18 months of age.
“LC3B-/- mice are same weight as age-matched wild type from 2 to 18 months of age (Supplementary Figure S4D)”
Quote in the paperNot a substantial claim - Premisecitation
During OS phagocytosis, LC3B associates with ingested phagosomes, targeting them for degradation in LAP.
“During OS phagocytosis, LC3B associates with ingested phagosomes targeting them for degradation in a process known as LAP (Kim et al., 2013; Frost et al., 2014, 2015, 2017; Muniz-Feliciano et al., 2017).”
Quote in the paperSubstantial claim0 of 3 sources checked - Study design and conductown data
Rabbit anti-LC3A (ab62720, Abcam) detects LC3A.
“while, rabbit anti-LC3A (ab62720, Abcam) detects LC3A.”
Quote in the paperSubstantial claim- Rests on an assumption: A substantial majority of studies using anti-LC3 antibodies presume them to be LC3B specific.
- Interpretationcitation
Functional redundancy between LC3B and other Atg8 homologs may explain the normal development of LC3B-/- mice.
“This may be due to functional redundancy between LC3B and other Atg8 homologs (e.g., GABARAP) (Cann et al., 2008; Klionsky et al., 2016).”
Quote in the paperSubstantial claim0 of 2 sources checked - Interpretationown data
Slowed phagosome degradation coupled with the decrease in fatty acid oxidation (as reflected in β-HB release) could result in an accumulation of lipid in the RPE.
“Slowed phagosome degradation coupled with the decrease in fatty acid oxidation (as reflected in β-HB release) could result in an accumulation of lipid in the RPE.”
Quote in the paperSubstantial claim - Premisecitation
Mice with global KOs of Atg5 and Atg7 die within a day after birth.
“mice with global KOs of the other autophagy related genes like Atg5 and Atg7 that die within a day after birth (Kuma et al., 2004; Komatsu et al., 2005; Cann et al., 2008).”
Quote in the paperSubstantial claim0 of 3 sources checked - Interpretationcitation
After the bolus of ingested OS lipids is metabolized, maintenance of damaged mitochondria and/or peroxisomes through selective autophagy predominates.
“after the bolus of ingested OS lipids is metabolized, the maintenance of damaged mitochondria and/or peroxisomes through selective autophagy predominates”
Quote in the paperSubstantial claim - Premisecitation
Free fatty acids generated upon OS digestion serve as substrates for mitochondrial FAO and ketogenesis.
“Free fatty acids generated upon OS digestion serve as substrates for mitochondrial FAO and ketogenesis (Reyes-Reveles et al., 2017).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Reyes-Reveles et al., 2017 source
Not checked in the free run.
- Interpretationcitation
The circadian fluctuation of LC3 levels is likely correlated with RPE and PR physiological function.
“This fluctuation is likely correlated with RPE and PR physiological function”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Muniz-Feliciano et al., 2017 source
Not checked in the free run.
- Rests on an assumption: LC3 levels fluctuate in a circadian pattern in terminally differentiated visual cells.
- Premisecitation
Phagosome accumulation is observed in the absence of MREG.
“Phagosome accumulation is observed in the absence of MREG (Damek-Poprawa et al., 2009)”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Damek-Poprawa et al., 2009 source
Not checked in the free run.
- Interpretationcitation
The cytoprotective role of autophagy under stress and increased p62 likely favors pexophagy and mitophagy.
“thus the cytoprotective role of autophagy under stress and increased p62 likely favors these two LC3-dependent functions (Mitter et al., 2012; Wang et al., 2014; Song et al., 2017).”
Quote in the paperNot a substantial claim0 of 3 sources checked - Interpretationown data
In the absence of LC3B, the inability to degrade ingested OS lipids contributes to RPE abnormalities.
“Our current studies suggest that in the absence of LC3B, it is the inability to degrade ingested OS lipids that contributes to RPE abnormalities”
Quote in the paperSubstantial claim- Rests on an assumption: The metabolic fate of free fatty acids relies on the maintenance of healthy mitochondria and peroxisomes.
- Interpretationown data
The unchanged p62 levels suggest relatively intact selective autophagy in LC3B-/- RPE.
“suggestive of relatively intact selective autophagy.”
Quote in the paperSubstantial claim - Premisecitation
LAP requires the LC3B adapter melanoregulin (MREG) to mediate LC3B association with newly ingested OS-phagosomes.
“This process requires the LC3B adapter, melanoregulin (MREG) to mediate LC3B association with newly ingested OS-phagosomes (Frost et al., 2015).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Frost et al., 2015 source
Not checked in the free run.
- Premisecitation
There is a second LC3 peak 6 h after light onset.
“there is a second LC3 peak 6 h after light onset (Yao et al., 2014; Frost et al., 2015).”
Quote in the paperNot a substantial claim0 of 2 sources checked
- May combine two claims. 44
- Classified as probably not a substantial claim. 28
May combine two claims.
Findingown dataLC3B transcript and protein are absent in the RPE and retina of LC3B-/- mice.
“As expected, LC3B transcript (Figure 1A) and LC3B protein were absent in the LC3B-/- mouse RPE (Figure 1B and Supplementary Figure S1A) and retina (Figure 1C)”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn WT mice, 15.0 ± 1.89 nmoles of β-HB was released by 9 am; in contrast, the LC3B-/- RPE released only 6.87 ± 0.71 nmoles of β-HB.
“In WT mice, 15.0 ± 1.89 nmoles of β-HB was released by 9 am; in contrast, the LC3B-/- RPE released only 6.87 ± 0.71 nmoles of β-HB (Figure 4B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe spatial intensity distribution is uniform for WT and encompasses nearly 30-40% of the entire image, whereas in LC3B-/- mice the uniformity is substantially reduced, corresponding to ~10% of the image.
“The spatial intensity distribution is quite uniform for WT and encompasses nearly 30–40% of the entire image (Figures 2E,F); whereas in L3CB-/- mice the uniformity is substantially reduced, corresponding to a much smaller fraction (∼10%) of the image (Figures 2G,H).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataOlder LC3B-/- mice show hyper- and hypo-pigmented areas in the RPE and migration of cells (possibly microglia) in the subretinal space.
“Besides this, there appear to be several hyper and hypo-pigmented areas in the RPE from older LC3B-/- and migration of cells (possibly microglia) in the subretinal space (Figures 3A,B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataLC3B-/- RPE exhibited an abundance of neutral lipid droplets, detected as BODIPY positive puncta, compared to WT; in LC3B-/- neutral lipid containing structures were found not only in RPE but also at the RPE-choroid interface.
“LC3B-/- RPE exhibited an abundance of neutral lipid droplets, detected as BODIPYTM positive puncta, compared to WT; in the LC3B-/- neutral lipid containing structures were found not only in RPE but also at RPE-choroid interface (Figure 4C, white arrow).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataThere was a slight decrease (albeit not significant) in the number of nuclear layers for aged (16-19 months) LC3B-/- mice relative to control.
“there was a slight decrease (albeit not significant) in the number of nuclear layers for the aged (16–19 months) L3CB-/- mice relative to control (Figure 3C).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn LC3B-/- RPE, phagosome numbers were already elevated before light onset, and then rose only moderately after light onset.
“In the LC3B-/- RPE, phagosome numbers were already elevated before light onset, and then rose only moderately after light onset.”
Quote in the paperSubstantial claim- Rests on an assumption: Retinal pigment epithelium cells must ingest OSs daily, and any inefficiency in daily phagosome degradation contributes to pathogenesis and age-related visual impairment.
May combine two claims.
Findingown dataONL thickness mean differences were insignificant relative to controls at each age group, although LC3B-/- mice tended to have slightly thinner ONL, with one 21-month-old mouse having 30% thinning.
“Although the analysis demonstrated that L3CB-/- mice tended to have slightly thinner ONL, with one 21 months old mouse having 30% thinning, the mean differences were found to be insignificant relative to controls (Figure 2Q) at each age group.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataWhen the cone ERG luminance-response functions were fit by Equation (2), the amplitude parameter Rmax was significantly increased in LC3B-/- (211.1 ± 17.4 μV) as compared to WT (283.5 ± 17.1 μV) mice (p < 0.04) while K did not differ between genotypes.
“When the cone ERG luminance-response functions were fit by Equation (2), the amplitude parameter Rmax was significantly increased in LCB3-/- (211.1 ± 17.4 μV) as compared to WT (283.5 ± 17.1 μV) mice (p < 0.04) while K did not differ between genotypes.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe overall waveform of the cone ERGs obtained from LC3B-/- mice were comparable to those of WT, except for an overall reduction in response amplitude.
“The overall waveform of the cone ERGs obtained from LC3B-/- mice were comparable to those of WT, except for an overall reduction in response amplitude (Figure 8C).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThere is a marked decrease in esterified DHA in membrane phospholipids and free DHA in the LC3B-/- RPE.
“There is a marked decrease in esterified DHA in membrane phospholipids (Figure 5A) and free DHA in the LC3B-/- RPE (Figure 5B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataANOVA analysis showed that the reduction was significant for the a-wave (P < 0.0001) and b-wave (p < 0.0001).
“ANOVA analysis showed that the reduction was significant for the a-wave (P < 0.0001) and b-wave (p < 0.0001).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataWhile cholesterol levels in the LC3B-/- RPE were elevated, there was a ~5-fold increase in 7-ketocholesterol compared to WT RPE.
“While cholesterol levels in the LC3B-/- RPE were elevated, perhaps more striking was the ∼5-fold increase in 7-ketocholesterol (7KCh) compared to WT RPE (Figure 6C).”
- Also stated in: § Consequences of Dysregulated Lipid Homeostasis · hedged
“Biochemical analysis pointed to a ∼5-fold increase in 7KCh in LC3B-/- RPE/Ch compared to WT.”
Quote in the paperSubstantial claim- Rests on an assumption: A consequence of prolonged oxidative stress is the non-enzymatic conversion of cholesterol to the oxy-sterol, 7-ketocholesterol.
- Also stated in: § Consequences of Dysregulated Lipid Homeostasis · hedged
May combine two claims.
Findingown dataIn the retina, the level of total DHA in LC3B-/- mouse is significantly lower than that of WT, but there is an enhancement in the free DHA pool size.
“In the retina, the level of total DHA in LC3B-/- mouse is significantly lower than that of WT (Figure 5G), but there is an enhancement in the free DHA pool size (Figure 5H).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataLipid debris increased as the mice aged, with virtually no BODIPY493/503 positive puncta in RPE of young animals (2 months old) and an accumulation of RPE and sub-RPE lipid deposits by 18 months.
“Lipid debris increased as the mice aged, with virtually no BODIPYTM493/503 positive puncta in RPE of young animals (2 months old) and an accumulation of RPE and sub-RPE lipid deposits by 18 months (Supplementary Figure S3A).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataImmuno-labeling with antibody to 4-HNE-adducts revealed 4-HNE localization to RPE and choroid, and substantial levels of 4-HNE were detected at 24 months for the LC3B-/-.
“Immuno-labeling with antibody to 4-HNE- adducts revealed 4-HNE localization to RPE and choroid (Figure 6B, 20 months WT and LC3B-/-) and substantial levels of 4-HNE were detected at 24 months for the LC3B-/- (Supplementary Figure S3C, 27 months WT, 24 months LC3B-/-).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFor the b-wave, the amplitude parameter Rmax was significantly reduced in LC3B-/- (571.9 ± 39.1 μV) as compared to WT (724.4 ± 59.1 μV) mice (p < 0.04) while K did not differ between genotypes.
“For the b-wave, the amplitude parameter Rmax was significantly reduced in LCB3-/- (571.9 ± 39.1 μV) as compared to WT (724.4 ± 59.1 μV) mice (p < 0.04) while K did not differ between genotypes.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataSub-retinal Iba1 positive cells were observed in both superior and inferior retina, with ~4-fold more Iba positive cells in the superior than inferior retina in the LC3B-/-.
“Sub-retinal Iba1 positive cells were observed in both superior and inferior retina, with ∼4-fold more Iba positive cells in the superior than inferior retina in the LC3B-/- (Figure 7C, Supplementary Figure S4B, and see Supplementary Figure S4C for no anti-Iba1 control).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingcitationβ-HB production due to slowed OS clearance is delayed in the LC3B-/- RPE, with maximal β-HB release seen 6 h after light onset, in contrast to WT, where maximal release is at 2 h after light onset.
“Herein we show that β-HB production due to slowed OS clearance is delayed in the LC3B-/- RPE, with maximal β-HB release seen 6 h after light onset, in contrast to WT, where maximal release is at 2 h after light onset (Reyes-Reveles et al., 2017).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Reyes-Reveles et al., 2017 source
Not checked in the free run.
May combine two claims.
FindingreasoningThe RPE of LC3B-/- mice shows signs of oxidative stress and accumulation of 4-HNE-adducts in the RPE and sub-RPE space.
“the RPE shows signs of oxidative stress, the accumulation of 4-HNE-adducts both in the RPE and sub-RPE space”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationSome studies suggest isoform specificity in selective autophagy.
“some studies suggest isoform specificity in selective autophagy (Shpilka et al., 2011; Maruyama et al., 2014; Koukourakis et al., 2015).”
Quote in the paperSubstantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThree genes encode the highly homologous LC3 protein family members LC3A, LC3B, and LC3C.
“Three genes encode the highly homologous, LC3 protein family members, MAP1LC3A (LC3A), MAP1LC3B (LC3B) and MAP1LC3C (LC3C) (Klionsky et al., 2016).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Klionsky et al., 2016 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationThe daily burst of OS phagocytosis is accompanied by an increase in LC3, levels of which fluctuate over the 12 h light/dark cycle.
“The daily burst of OS phagocytosis is accompanied by an increase in LC3, levels of which fluctuate over the 12 h light/dark cycle (Yao et al., 2014; Frost et al., 2015).”
Quote in the paperNot a substantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationComponents of LAP, specifically RUN and RUBCN, inhibit starvation-induced autophagy in RPE.
“Recent studies in the RPE suggest that like, macrophages and dendritic cells, components of LAP specifically, RUN (RPIP8/UNC-14/NESCA) and cysteine rich domain containing beclin1 interacting protein (RUBCN), inhibit starvation induced autophagy (Martinez et al., 2015; Muniz-Feliciano et al., 2017).”
Quote in the paperNot a substantial claim0 of 2 sources checkedClassified as probably not a substantial claim.
Study design and conductown dataThe LC3B-/- and WT mice were free of the rd8 mutation.
“The LC3B-/- and the WT mice were confirmed to be free of the rd8 mutation by genomic DNA PCR using primers as described in Chang et al. (2013).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Chang et al., 2013 source
Not checked in the free run.
May combine two claims.
PremisecitationLAP plays a critical role in visual pigment regeneration and phagosome degradation.
“LAP plays a critical role in visual pigment regeneration and phagosome degradation (Kim et al., 2013; Frost et al., 2015; Muniz-Feliciano et al., 2017)”
Quote in the paperSubstantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe LC3B-/- mice were backcrossed for at least five generations onto a C57BL6/J background.
“The LC3B-/- mice were backcrossed for at least five generations onto a C57BL6/J background.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenPhagosome counts were performed by two different individuals, each counting 15 different areas per mouse.
“Counts were performed by two different individuals, each counting 15 different areas per mouse.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataPhagosome counting was performed under masked conditions.
“The count was done under masked conditions so that the genotype of the specimen was unknown to the person performing the analysis.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataLoss of LC3B results in phagosome and neutral lipid accumulation.
“Loss of LC3B results in phagosome and neutral lipid accumulation.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S1 (asserted).
InterpretationreasoningThe 50% decrease in β-HB is likely due to the reduced availability of free fatty acids from ingested OS phospholipids.
“Thus, we hypothesize that the 50% decrease in β-HB is likely due to the reduced availability of free fatty acids from ingested OS phospholipids (Reyes-Reveles et al., 2017).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Reyes-Reveles et al., 2017 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationSubretinal injection of 7KCh has been shown to induce migration of Iba1 positive microglia to the outer retina suggesting a role as a chemo-attractant and thus altering the immune environment.
“Subretinal injection of 7KCh has been shown to induce migration of Iba1 positive microglia to the outer retina suggesting a role as a chemo-attractant and thus altering the immune environment (Indaram et al., 2015).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Indaram et al., 2015 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationDysregulated overactive microglia may contribute to prolonged inflammation and chronic disease progression.
“Although microglia are also neuroprotective under physiological conditions (Ramirez et al., 2017), it has been proposed that dysregulated overactive microglia, those clearing oxysterol debris by way of example, may contribute to the prolonged inflammation and chronic disease progression (Gao and Hong, 2008).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites Ramirez et al., 2017 source
Not checked in the free run.
- cites Gao and Hong, 2008
Not checked in the free run.
May combine two claims.
Interpretationown dataThe present study identifies the LC3 isoform, LC3B, as a critical determinant of lipid homeostasis and as essential in regulating the inflammatory state of the retina/RPE.
“The present study identifies the LC3 isoform, LC3B, as a critical determinant of lipid homeostasis thereby as essential in regulating the inflammatory state of the retina/RPE.”
Quote in the paperSubstantial claim- Rests on an assumption: Autophagic processes are protective; ridding cells of defective mitochondria, peroxisomes and aggregated proteins.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationLipid oxidation is a critical factor in the development of AMD.
“Lipid oxidation is a critical factor in the development of AMD (Balaratnasingam et al., 2017; Golestaneh et al., 2017; Handa et al., 2017; Xu et al., 2017).”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
InterpretationcitationThe increased Iba positive cells in the inner retina may represent migrating activated microglia.
“and may represent migrating activated microglia (Kim et al., 2014).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Kim et al., 2014 source
Not checked in the free run.
May combine two claims.
PremisecitationExcessive lipid accumulation and/or formation of lipid-peroxidation adducts in RPE/Ch as observed in the LC3B-/- model has also been described in mice with an RPE specific loss of Atg7 or Atg5 as well as in the KLC1 KO mouse.
“Excessive lipid accumulation and/or formation of lipid-peroxidation adducts in RPE/Ch as observed in the LC3B-/- model has also been described in mice with an RPE specific loss of Atg7 or Atg 5 (Zhang et al., 2017) as well as in the kinesin-1 light chain (KLC1) KO mouse (Jiang et al., 2015).”
Quote in the paperNot a substantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationDysregulation of cholesterol metabolism and oxysterol accumulation are associated with AMD as well as other pathologies involving oxidative stress such as Huntington's disease, atherosclerosis, and cystic fibrosis.
“Dysregulation of cholesterol metabolism and oxysterol accumulation are also associated with AMD as well as other pathologies involving oxidative stress such as Huntington’s disease, atherosclerosis, and cystic fibrosis (Iuliano et al., 2003, 2009; Jenner et al., 2007; Kreilaus et al., 2016).”
Quote in the paperNot a substantial claim0 of 3 sources checkedMay combine two claims.
Interpretationown dataIn the absence of LC3B, the biosynthesis of the docosanoids NPD1 and maresin-1 is attenuated, likely further exacerbating homeostatic disruptions and the pro-inflammatory environment.
“In the absence of LC3B, the biosynthesis of the docosanoids, NPD1 and maresin-1, is attenuated, likely further exacerbating homeostatic disruptions and the pro-inflammatory environment.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationThere is no gene corresponding to MAPILC3C in mouse and in human LC3C is not expressed in the RPE.
“Moreover, there is no gene corresponding to MAPILC3C in mouse and in human LC3C is not expressed in the RPE (Dhingra et al., 2018).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Dhingra et al., 2018 source
Not checked in the free run.
May combine two claims.
Interpretationown dataLoss of LC3B leads to age-dependent lipid accumulation due to a lifetime of delayed phagosome maturation and content degradation.
“Collectively, these studies suggest that loss of LC3B leads to age-dependent lipid accumulation due to a lifetime of delayed phagosome maturation and content degradation, as described below.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationIncrease in number of sub-retinal microglia with age has been reported and is considered as a common hallmark of various retinal degenerative and inflammatory diseases.
“Increase in number of sub-retinal microglia with age has been reported and more importantly is considered as a common hallmark of various retinal degenerative and inflammatory diseases (Karlstetter and Langmann, 2014).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Karlstetter and Langmann, 2014
Not checked in the free run.
May combine two claims.
PremisecitationSpecifying individual LC3 isoform is necessary because the isoforms have different expression patterns and biology.
“Specifying individual LC3 isoform is necessary as the isoforms have different expression patterns and biology (Cann et al., 2008; Koukourakis et al., 2015).”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductreasoningA major limitation for obtaining complete uniformity in IRDF-SLO images is that the retina is a curved entity and obtaining full-field, aberration-free images with uniform intensity is difficult, if not practically impossible as the reflective signal strength falls off radially from the center of the image.
“The major limitation for obtaining “complete” uniformity can be attributed to the fact that the retina is a curved entity and obtaining full-field, aberration-free images with uniform intensity is difficult, if not practically impossible as the reflective signal strength falls off radially from the center of the image.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
InterpretationreasoningThese lung conditions are unlikely to be pertinent to the current study.
“These conditions are unlikely to be pertinent to our current study”
Quote in the paperSubstantial claimClassified as probably not a substantial claim. May combine two claims.
PremisecitationLC3B-/- mice display enhanced indices of pulmonary hypertension after chronic hypoxia but not under normoxic conditions.
“LC3B-/- mice display enhanced indices of pulmonary hypertension after chronic hypoxia but not under normoxic conditions (Lee et al., 2011).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Lee et al., 2011 source
Not checked in the free run.
May combine two claims.
Interpretationown dataIn our own studies, LC3B is protective during aging/increased oxidative stress, as its loss leads to several retinal/RPE abnormalities.
“in our own studies LC3B is protective during aging/increased oxidative stress as its loss leads to several retinal/rpe abnormalities.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationPhotooxidative processes initiated by PR-derived bisretinoids likely contribute to HNE-modifications and trapping of cholesterol within RPE, potentially altering autophagic processes.
“The photooxidative processes initiated by PR derived bisretinoids likely also contribute to HNE-modifications (Zhou et al., 2005) and trapping of cholesterol within RPE, potentially altering autophagic processes (Toops et al., 2015).”
Quote in the paperNot a substantial claim0 of 2 sources checkedMay combine two claims.
Study design and conductown dataLC3B-/- mice live for over 2 years and are overall healthy like WT controls.
“they live for over 2 years, and are overall healthy like WT controls.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationLC3B-/- mice develop normally, reach adulthood, and are fertile.
“LC3B-/- mice develop normally, reach adulthood, are fertile”
Quote in the paperSubstantial claim0 of 3 sources checkedClassified as probably not a substantial claim.
PremisecitationIn response to oxidative stress, autophagy increases to remove oxidatively damaged organelles.
“In response to oxidative stress, autophagy increases in an attempt to remove oxidatively damaged organelles (Lee et al., 2012; Seo et al., 2012).”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationProlonged fasting reduces inflammation, with β-HB selectively suppressing inflammasome activation in response to lipotoxic fatty acids.
“Prolonged fasting reduces inflammation, with β-HB selectively suppressing inflammasome activation in response to lipotoxic fatty acids (Vasconcelos et al., 2014).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Vasconcelos et al., 2014 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationThe traditional view of β-HB as solely an energy carrier has greatly expanded to include β-HB mediated inhibition of inflammasome activation.
“The traditional view of β-HB as solely an energy carrier has greatly expanded to include β-HB mediated inhibition of inflammasome activation (Youm et al., 2015).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Youm et al., 2015 source
Not checked in the free run.
May combine two claims.
Interpretationown dataTotal β-HB release is decreased, suggesting a further loss of protective lipid-derived mediators likely contributing to the RPE and retinal abnormalities observed in aged mice.
“Moreover, total β-HB release is decreased, suggesting a further loss of protective lipid-derived mediators likely contributing to the RPE and retinal abnormalities observed in aged mice.”
Quote in the paperSubstantial claim- Rests on an assumption: The inhibitory effect of β-HB on inflammasome activation is independent of autophagy and TCA cycle dependent oxidation, with other ketogenic intermediates such as acetoacetyl-CoA having no affect.
Probably not a substantial claim: may not pass S3 (load-bearing).
InterpretationreasoningThe increase in bisretinoids observed in LC3B-/- mice may be due to alterations in PR lipid homeostasis in the absence of LC3B.
“the increase in bisretinoids observed may be due to alterations in PR lipid homeostasis in the absence of LC3B.”
Quote in the paperNot a substantial claim- Rests on an assumption: Bisretinoids form in photoreceptor cell outer segments before phagocytosis by RPE.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationLC3B plays a protective role in hypoxia, a pro-pathogenic role in cigarette smoke-induced emphysema, and increased susceptibility to lung pathology during respiratory viral infection.
“it has been shown to play a protective role in hypoxia; pro-pathogenic role in cigarette smoke induced emphysema; increased susceptibility to lung pathology during respiratory viral infection.”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationOS phagocytosis triggers a downregulation of starvation-induced autophagy so that LC3B-associated phagocytosis may proceed.
“recent studies suggest that OS phagocytosis triggers a downregulation of starvation induced autophagy so that LC3B-associated phagocytosis may proceed (Muniz-Feliciano et al., 2017).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Muniz-Feliciano et al., 2017 source
Not checked in the free run.
May combine two claims.
PremisecitationThe RPE utilizes LC3 in LAP to decorate the outer membrane of phagosomes and in mitophagy and pexophagy to remove defective mitochondria and peroxisomes.
“The RPE utilizes LC3 in both processes; in LAP to decorate the outer membrane of phagosomes and in mitophagy and pexophagy to remove defective mitochondria and peroxisomes, respectively (Ferrington et al., 2016).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Ferrington et al., 2016 source
Not checked in the free run.
May combine two claims.
PremisecitationPexophagy and mitophagy both utilize the LC3 adapter p62.
“Pexophagy and mitophagy both utilize the LC3 adapter p62”
Quote in the paperNot a substantial claim0 of 3 sources checkedMay combine two claims.
PremisecitationLC3II/LC3I ratios were lower in RPE from AMD patients, with decreased autophagic flux and mitochondrial accumulation.
“LC3II/LC3I ratios were lower in RPE from AMD patients with decreased autophagic flux and mitochondrial accumulation (suggestive of altered mitophagy) (Golestaneh et al., 2017)”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Golestaneh et al., 2017 source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationDysregulated or dysfunctional autophagy contributes to AMD.
“leading these and other investigators doing in vitro work to conclude that dysregulated or dysfunctional autophagy contributes to AMD.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Golestaneh et al., 2017 source
Not checked in the free run.
- Rests on an assumption: LC3B or LC3A is required for starvation-induced and selective autophagy, including pexophagy, mitophagy, secretory autophagy, LAP, and chaperone-mediated autophagy.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationLAP-mediated increases in RUBCN inhibit starvation-induced autophagy in the RPE.
“recent studies have also shown that LAP-mediated increases in RUBCN inhibit starvation induced autophagy in the RPE (Muniz-Feliciano et al., 2017).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Muniz-Feliciano et al., 2017 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
InterpretationcitationUpon light onset, LAP-dependent pathways predominate, consistent with high MREG-LC3B levels at this time.
“we predict that upon light onset LAP-dependent pathways predominate, consistent with high MREG-LC3B levels at this time (Frost et al., 2015).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Frost et al., 2015 source
Not checked in the free run.
May combine two claims.
PremisecitationRPE cultures derived from human donor eyes of AMD patients exhibited increased intracellular lipid accumulation and oxidative stress.
“RPE cultures derived from human donor eyes (patients with AMD), exhibited increased intracellular lipid accumulation and oxidative stress.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Golestaneh et al., 2017 source
Not checked in the free run.
The paper relies on this without support.
The role of LC3 isoforms in maintaining RPE homeostasis is unknown.
“Whether the LC3 isoforms are functionally redundant or play a specialized role in maintaining RPE homeostasis is completely unknown and is the focus of our studies.”
The paper relies on this without support.
Phagocytic degradation of OS tips is critical for photoreceptor renewal and allows post-mitotic photoreceptor cells to maintain constant length and normal physiological function.
“This phagocytic degradation of OS tips is a critical aspect of photoreceptor renewal and allows the post-mitotic photoreceptor cells to maintain a constant length and normal physiological function.”
The paper relies on this without support.
The daily ingestion of lipid-rich OS imposes a constant degradative burden on RPE cells.
“The daily ingestion of these lipid-rich OS imposes a constant degradative burden on these terminally differentiated cells.”
The paper relies on this without support.
Cholesterol is a major component of neutral lipid droplets.
“Cholesterol is a major component of neutral lipid droplets.”
The paper relies on this without support.
Free DHA serves as a substrate for the synthesis of NPD1, synthesized by 15-lipoxygenase-1 via the short live intermediate 17(S)-HpDHA.
“Free DHA serves as a substrate for the synthesis of NPD1, synthesized by 15-lipoxygenase-1 (Figure 5I) via the short live intermediate 17(S)-HpDHA.”
The paper relies on this without support.
A consequence of prolonged oxidative stress is the non-enzymatic conversion of cholesterol to the oxy-sterol, 7-ketocholesterol.
“A consequence of prolonged oxidative stress is the non-enzymatic conversion of cholesterol to the oxy-sterol, 7-ketocholesterol.”
The paper relies on this without support.
Retinal pigment epithelium cells must ingest OSs daily, and any inefficiency in daily phagosome degradation contributes to pathogenesis and age-related visual impairment.
“Retinal pigment epithelium cells are functionally distinct from most other phagocytes in that they must ingest OSs daily, thus any inefficiency in daily phagosome degradation contributes to pathogenesis and age-related visual impairment.”
The paper relies on this without support.
Autophagic processes are protective; ridding cells of defective mitochondria, peroxisomes and aggregated proteins.
“Autophagic processes are protective; ridding cells of defective mitochondria, peroxisomes and aggregated proteins.”
The paper relies on this without support.
The inhibitory effect of β-HB on inflammasome activation is independent of autophagy and TCA cycle dependent oxidation, with other ketogenic intermediates such as acetoacetyl-CoA having no affect.
“This inhibitory effect is independent of autophagy, and TCA cycle dependent oxidation, with other ketogenic intermediates such as acetoacetyl-CoA having no affect.”
The paper relies on this without support.
The RPE handles large amounts of lipids, with OSs serving as a major source of cholesterol.
“Moreover, given that the RPE handles large amounts of lipids, with OSs serving as a major source of cholesterol, understanding how loss of LC3B contributes to the regulation of cholesterol efflux pathways (Storti et al., 2017) will provide valuable insight into age-related perturbations of lipid homeostasis.”
The paper relies on this without support.
Bisretinoids form in photoreceptor cell outer segments before phagocytosis by RPE.
“Since bisretinoid form in photoreceptor cell outer segments before phagocytosis by RPE”
The paper relies on this without support.
The metabolic fate of free fatty acids relies on the maintenance of healthy mitochondria and peroxisomes.
“The metabolic fate of the free fatty acids relies on the maintenance of healthy mitochondria and peroxisomes, organelles necessary in the oxidation of long chain fatty acids.”
The paper relies on this without support.
LC3B or LC3A is required for starvation-induced and selective autophagy, including pexophagy, mitophagy, secretory autophagy, LAP, and chaperone-mediated autophagy.
“LC3B or LC3A is required for starvation induced as well as selective autophagy which includes, pexophagy, mitophagy, secretory autophagy, LAP as well as chaperone mediated autophagy, among others.”
The paper relies on this without support.
LC3 levels fluctuate in a circadian pattern in terminally differentiated visual cells.
“The visual system is unique in that in these terminally differentiated cells, LC3 levels fluctuate in circadian pattern.”
The paper relies on this without support.
A substantial majority of studies using anti-LC3 antibodies presume them to be LC3B specific.
“A caveat to such studies is that a substantial majority of these studies use anti-LC3 antibodies presumed to be LC3B specific.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The Lc3b−/− mouse shows a decrease in LC3-mediated OS phagosome clearance, with no change in classical autophagy.
“In contrast, the Lc3b−/− mouse shows a decrease in LC3-mediated OS phagosome clearance, with no change in classical autophagy (Dhingra et al., 2018).”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Dhingra et al., 2018
In vitro and in vivo studies suggest that approximately 30-45% of ingested phagosomes are LC3B-positive.
“While LC3B-II-opsin association is clearly observed (Fig. 10), the extent of LC3B-II association with opsin-positive phagosomes remains unclear; in vitro (Frost et al., 2015) and in vivo (Dhingra et al., 2018) studies suggest that ~30–45% of ingested phagosomes are LC3B-positive.”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Dhingra et al., 2018
Loss of LC3B results in age-dependent deposition of lipid-rich structures between the RPE and choroid, an increase in lipid peroxidation adducts, and increases in the pro-inflammatory lipid 7-ketocholesterol.
“Loss of LC3B (Dhingra et al., 2018) results in age-dependent deposition of lipid-rich structures between the RPE and choroid, an increase in lipid peroxidation adducts, and increases in the pro-inflammatory lipid 7-ketocholesterol.”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Dhingra et al., 2018
Fatty acid oxidation and ketogenesis are coupled to phagosome maturation processes.
“thus coupling FAO and ketogenesis to phagosome maturation processes (Reyes-Reveles et al., 2017; Dhingra et al., 2018).”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Dhingra et al., 2018
In Lc3b−/− and Mreg−/− mice, which display delayed phagosome maturation, there is a delay in β-HB production.
“In Lc3b−/− and Mreg−/− mice, that display delayed phagosome maturation, there was also a delay in β-HB production”
From The cell biology of the retinal pigment epithelium (2020) · cites this paper as Dhingra et al., 2018
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
RPE DHA result cites a retina panel
MinorThe discussion cites Figure 5G for decreased RPE phospholipids, but the figure caption identifies panel G as total DHA in the retina; panel A is the RPE esterified-DHA result.
“The RPE displays decreased abundance of docosahexaenoyl containing phospholipids (Figure 5G)”
“In retinal pigment epithelial cells (RPE/choroid): Esterified DHA (total) (A); free DHA (B); 17(S)-HDHA (C); NPD1 (D); 14(S)-HDHA (E), and Maresin-1 (F). In retina total (G) and free (H) DHA.”
Cone ERG Rmax is described as increased despite being lower
MinorThe reported cone ERG Rmax is 211.1 μV in LC3B-/- mice versus 283.5 μV in WT mice, so it is decreased, not increased. This also conflicts with the preceding statement that LC3B-/- cone responses were reduced.
“the amplitude parameter Rmax was significantly increased in LCB3-/- (211.1 ± 17.4 μV) as compared to WT (283.5 ± 17.1 μV) mice (p < 0.04)”
“The responses of LC3B-/- mice are reduced as compared to WT (P < 0.0001).”
The citation lists haven’t been fetched yet
References 97 · Cited by 41
- Authors
- Dhingra, A., Bell, B. A., Peachey, N. S., Daniele, L. L., Reyes-Reveles, J., Sharp, R. C., Jun, B., Bazan, N. G., Sparrow, J. R., Kim, H. J., Philp, N. J., & Boesze-Battaglia, K.
- Affiliations
- University of Pennsylvania (US)
- Cleveland Clinic (US)
- Louisiana State University (US)
- Columbia University Irving Medical Center (US)
- Thomas Jefferson University (US)
- ORCID
- Venue
- Frontiers in Cellular Neuroscience
- Publisher
- Frontiers Media SA
- Published
- 2018-10-08
- Volume
- 12
- Pages
- 351-351
- ISSN
- 1662-5102 (ISSN-L)
- PMC
- PMC6186781
- OpenAlex
- W2897599900
- Semantic Scholar
- b7bea6732bf4edda3f36131141206d88cbae7aca
- Cited by
- 41
- References
- 97
- Citations by year
- 2026: 1 · 2025: 5 · 2024: 5 · 2023: 6 · 2022: 7 · 2021: 7 · 2020: 4 · 2019: 5 · 2018: 1
- FWCI
- 3.32
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Frontiers in Cellular Neuroscience (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- The Medicine Forum (submittedVersion, cc-by)
- Scholarly Commons (University of Pennsylvania) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- U.S. Department of Health and Human Services (EY10420-21, EY026525, EY005121, GM103340, EY025585, EY012951)
- U.S. Department of Veterans Affairs (I01BX002340)
- Topics
- Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
- Health Sciences › Medicine › Ophthalmology › Retinal Diseases and Treatments
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
- Keywords
- LC3B, retinal pigment epithelium, autophagy, lipid homeostasis, photoreceptor outer segment phagocytosis, heterophagy, selective autophagy, age-related macular degeneration, oxysterols, 4-hydroxynonenal, lipid peroxidation, bisretinoids, lipofuscin, fatty acid oxidation, microglial migration, retinal aging
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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