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Age-related macular degeneration (AMD) is a debilitating eye disease that tends to affect people over the age of 55. Lipofuscins are autofluorescent, toxic byproducts of the visual cycle thought to contribute toward the progression of the disease. Targeting the accumulation of lipofuscin through catabolism may serve as a method for the early treatment of AMD. Thus, an enzymatic approach capable of degrading lipofuscin, cycloretinal (all

Protein amyloid-β (Aβ) oligomers with β-sheet-like backbone (β-structured) form extracellular amyloid plaques associated with Alzheimer’s disease (AD). However, the relationship to AD is not known. Some investigations suggest that the toxic Aβ component has α-sheet-like backbone (α-structured) subsequently detoxified by intracellular α-to-β conversion before plaque formation. Our objective is to compare this latter hypothesis with observations made by electron microscopy of thin sections of AD-cerebral cortex. We observe irregular, 200–2,000 nm, intracellular, lipofuscin-like inclusions. Some are light-staining and smooth. Others are dark-staining and made granular by fibers that are usually overlapping and are sometimes individually seen. Aspects unusual for lipofuscin include 1) dark and light inclusions interlocking as though previously one inclusion, 2) dark inclusion-contained 2.6 nm thick sub-fibers that are bent as though α-structured, and 3) presence of inclusions in lysosomes and apparent transfer of dark inclusion material to damaged, nearby lysosomal membranes. These data suggest the following additions to α-structure-based hypotheses: 1) Lipofuscin-associated, α-structured protein toxicity to lysosomal membranes is in the chain of AD causation; 2) α-to-β detoxification of α-structured protein occurs in lipofuscin and causes dark-to-light transition that, when incomplete, is the origin of cell-to-cell transmission essential for development of AD.

In-situ hybridization has been a robust method for detection of mRNA expression within whole-mount samples or tissue sections for more than 50 years. Recent technical advances for in-situ hybridization methods have incorporated oligo-based probes that attain greater tissue penetration and signal amplification steps with restricted localization for visualization of specific mRNAs within single cells. One such method is Third Generation in-situ Hybridization Chain Reaction (V3HCR) developed by Choi et al. that was first described in 2018. Here, we report an optimized protocol for V3HCR detection of gene expression using sectioned frozen tissues from mouse and human on microscope slides. Our methods and modifications for cryo-sectioning, tissue fixation, and processing steps over a three-day V3HCR protocol are detailed along with recommendations for aliquoting and storing V3HCR single-stranded DNA probes and hairpin amplifiers. In addition, we describe a method for blocking background signal from lipofuscin, a highly auto-fluorescent material that is widespread in human neurons and often complicates imaging efforts. After testing multiple strategies for reduction of lipofuscin we determined that application of lipofuscin quencher dye is compatible with V3HCR in contrast to other methods like cupric sulfate quenching or Sudan Black B blocking that cause V3HCR signal loss. This adaptation enables application of V3HCR for in-situ detection of gene expression among human neuronal populations that are otherwise problematic due to lipofuscin background auto-fluorescence. BASIC PROTOCOL 1: Mouse and Human Fresh-Frozen Tissue In-situ Hybridization Chain Reaction on Microscope Slides SUPPORT PROTOCOL 1: Aliquoting HCR Probes and Hairpins from Molecular Instruments

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Oxidative stress plays an important role in the ageing of the retina and in the pathogenesis of retinal diseases such as age-related macular degeneration (ARMD). Hydrogen peroxide is a reactive oxygen species generated by the photo-excited lipofuscin that accumulates during ageing in the retinal pigment epithelium (RPE), and the age-related accumulation of lipofuscin is associated with ARMD. Iron also accumulates with age in the RPE that may contribute to ARMD as an important source of oxidative stress. The aim of this work was to investigate the effects of L-Citrulline (CIT), a naturally occurring amino acid with known antioxidant properties, on oxidative stressed cultured RPE cells. Human RPE (ARPE-19) cells were exposed to hydrogen peroxide (H

Background and Objectives Stargardt disease produces lipofuscin accumulation predisposing to subretinal fibrosis (SRFib) after ocular trauma. Noninvasive imaging techniques allow in vivo assessment. The purpose of this study is to determine the prevalence of SRFib in a cohort of Stargardt patients, the presence of history of ocular trauma, the clinical features and possible genotype-phenotype associations in Stargardt patients with SRFib. Methods We evaluated retrospectively 106 Stargardt patients and analysed the multimodal imaging and the genotype of patients with SRFib. Results Six patients exhibited SRFib, three of them with history of ocular trauma. Multimodal imaging showed extensive SRFib principally in the temporal midperipheral retina with no fluid associated. SRFib was better defined by short wavelength autofluorescence and spectral domain optical coherence tomography and appeared clinically stable over time. There was no particular genotype associated to SRFib. Conclusion SRFib occurs in a significant percentage of patients with Stargardt disease and can be diagnosed through multimodal imaging regardless the history of trauma, further sustaining the importance of an appropriate imaging in such patients. No genotype-phenotype association has been established, supporting the traumatic etiology in half of cases. The remaining cases may be classified as idiopathic or have a minimal trauma occurring early in life that may be not recalled by the patients.

Made available in DSpace on 2022-04-28T19:51:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Background: Autophagy is a natural and evolutionary mechanism that reduces cell toxic components and reutilizes metabolites to provide energy and renew cell function, which is linked to a wide range of age-related diseases, including those that affect the skin. Positive modulation of autophagy is useful to treat skin disorders and new active herbal products are potential candidates as autophagy modulators. Aims: The present study aimed to evaluate the effects of a phytocosmetic formulation containing Myrothamnus flabellifolia leaf and Coffea arabica seed plant extracts (MflCas) on the ubiquitin-proteasome and autophagy markers in human dermal fibroblasts, and investigate its topical skin effects in a randomized, simple-blind, and placebo-controlled trial. Methods: Human dermal fibroblasts were used to determine proteasome activity, protein carbonylation, LC3B protein, and lipofuscin production by luminescence and immune-enzymatic assays, and to determinate gene expression of autophagy biomarkers (Atg5, Atg7, EI24, EIF2A, Park2, foxo1, and mTOR) by RT-PCR. A clinical trial was conducted to evaluate the effects of MflCas on the hand, face, and forearms skin features after treatment by 56 days. Results: Topical treatment with MflCas improved several skin features of volunteers, mainly skin aging and pigmentation signals. On the hand skin, MflCas 2% after 56 days of treatment, reduced the spots length (30.8%), skin contrast (42.2%), and increased skin homogeneity (63.2%) and skin lightening effect (1.4%). On the face skin, topical treatment after 56 days reduced the spots length (21.5%), wrinkles area (8.1%), and wrinkles volume (5.6%) with an increment in face skin homogeneity (59.5%). These effects were related to the ability of MflCas to reduce proteasome activity protein carbonylation, and lipofuscin level, increase LC3B production, downregulate Atg7 and mTOR genes, and upregulate Park2 gene expressions. Conclusions: The phytocosmetic preparation containing Myrothamnus flabellifolia leaf and Coffea arabica seed modulated ubiquitin-proteasome and autophagy process, representing an innovative and safe herbal preparation to improve skin features, mainly acting as skin anti-aging and lightening agent. Research and Development Department Chemyunion Química Ltda Department of Biophysics and Pharmacology Laboratory of Phytomedicines Pharmacology and Biotechnology (PhytoPharmaTech) Institute of Biosciences São Paulo State University (Unesp) Department of Biophysics and Pharmacology Laboratory of Phytomedicines Pharmacology and Biotechnology (PhytoPharmaTech) Institute of Biosciences São Paulo State University (Unesp)

Background The neuronal ceroid lipofuscinosis (NCL) are a group of autosomal recessive neurodegenerative disorders that are characterized by the accumulation of ceroid lipofuscins. The NCLs are categorized into four classes based on the age of onset. Kufs disease is a rare adult-onset NCL caused by mutations in the CLN6 gene, which is rarely observed in the Japanese population. Case We previously reported a case study on a patient with Kufs disease, whose parents had a consanguineous marriage. Later, we observed another unrelated patient with Kufs. Here we present the case and mutational gene report in patients with Kufs disease. Conclusions Gene analysis results of the first patient revealed a homozygous mutation c231C > G, p.Asn77Lys in exon 3 and a homozygous c.297 + 48 A > T mutation in intron 3 in the CLN6 gene. The Asn amino acid is perfectly conserved among species. In silico analysis showed that the mutation is predicted to be probably damaging. Moreover, the second patient with Kufs disease also had the same homozygous mutations. These data suggest that the missense mutation must be pathogenic. Furthermore, the patients had lived in the same district; therefore, they both potentially inherited the founder effect mutations.

To study the ultrastructure of microglia adjacent to oligodendrocytes in white matter of the prefrontal cortex in continuous schizophrenia (CSch) as compared to controls and attack-like schizophrenia (ASch) and to perform correlation analysis between the parameters of microglia and adjacent oligodendrocytes previously detected in both clinical types of schizophrenia.Electron microscopic morphometric study of microglia adjacent to oligodendrocytes was performed in postmortem white matter of the prefrontal cortex (BA10) in 9 cases of CSch, 8 cases of ASch and 20 healthy controls. Group comparisons were made by ANCOVA and Pearson correlation analyses.The reduction of volume fraction (Vv) and the number of mitochondria in microglia was found in elderly subjects (50 y.o.) as compared to young controls (60%,Specific features of CSch as compared to ASch might be associated with the disturbances of mitochondrial and lipid metabolism in microglia, dysfunction of nucleus and accelerated aging of microglia that might lead to alterations of mitochondrial metabolism in oligodendrocytes.Изучение ультраструктуры микроглии, контактирующей с олигодендроцитами, в белом веществе префронтальной коры при непрерывнотекущей шизофрении (НТШ) по сравнению с контролем и приступообразно-прогредиентной шизофренией (ППШ) и анализ корреляционных связей между параметрами микроглии и исследованных ранее олигодендроцитов в контроле и при разных типах течения шизофрении.На аутопсийном материале проведено электронно-микроскопическое морфометрическое исследование микроглии, контактирующей с олигодендроцитами, в белом веществе префронтальной коры (поле 10) в 9 случаях НТШ, в 8 случаях ППШ и 20 контролях без психической патологии. Групповые сравнения проводили с помощью ковариационного и корреляционного анализов.В группе НТШ, в отличие от ППШ, найдены снижение объемной фракции (Vv) в микроглии в подгруппе старше 50 лет по сравнению с контрольной подгруппой моложе 50 лет (60%,Особенности НТШ по сравнению с ППШ могут быть связаны с нарушениями митохондриального и липидного метаболизма в микроглии, дисфункцией ядра и ускоренным старением микроглии, что может приводить к нарушению метаболизма митохондрий в олигодендроцитах.

Age-related macular degeneration (AMD) is the primary cause of central blindness among the elderly. AMD is associated with progressive accumulation of lipofuscin granules in retinal pigment epithelium (RPE) cells. Lipofuscin contains bisretinoid fluorophores, which are photosensitizers and are phototoxic to RPE and neuroretinal cells. In the presence of oxygen, bisretinoids are also oxidized, forming various products, consisting primarily of aldehydes and ketones, which are also potentially cytotoxic. In a prior study, we identified that in AMD, bisretinoid oxidation products are increased in RPE lipofuscin granules. The purpose of the present study was to determine if these products were toxic to cellular structures. The physicochemical characteristics of bisretinoid oxidation products in lipofuscin, which were obtained from healthy donor eyes, were studied. Raman spectroscopy and time-of-flight secondary ion mass spectrometry (ToF–SIMS) analysis identified the presence of free-state aldehydes and ketones within the lipofuscin granules. Together, fluorescence spectroscopy, high-performance liquid chromatography, and mass spectrometry revealed that bisretinoid oxidation products have both hydrophilic and amphiphilic properties, allowing their diffusion through lipofuscin granule membrane into the RPE cell cytoplasm. These products contain cytotoxic carbonyls, which can modify cellular proteins and lipids. Therefore, bisretinoid oxidation products are a likely aggravating factor in the pathogenesis of AMD.

Purpose Macular atrophy (MA) of retinal pigment epithelium (RPE) and photoreceptors leads to vision loss in neovascular age-related macular degeneration (nAMD) despite successful treatment with antiangiogenic agents. To enhance understanding of MA, fortify the cellular basis of fundus autofluorescence (FAF) imaging, and inform management of nAMD, we performed histologic analysis of an eye with multimodal clinical imaging and apparent prior exudation due to nAMD. Design Case study and clinicopathologic correlation. Participant A White woman in whom age-related macular degeneration (AMD) findings of inactive subretinal fibrosis (right eye) were followed for 9 years using FAF and OCT, with no detectable subretinal fluid or other recurrent exudation and no intravitreal injections before her death at age 90 years. Methods The right eye was preserved 6.25 hours after death, postfixed in osmium tannic acid paraphenylenediamine, and prepared for submicrometer epoxy resin sections (n = 115), with 19 matched to clinical OCT B-scans. Main Outcome Measures Light microscopic morphology of a hyperautofluorescent (hyperFAF) area due to prior exudation (“floodplain” hyperFAF), hypoautofluorescent (hypoFAF) spots of MA, and areas of unremarkable FAF. Results Floodplain hyperFAF was visible throughout the 9 years of follow-up, with several hypoFAF atrophic spots expanding within it over time. The hyperFAF pattern corresponded to outer retinal atrophy (ORA) on OCT and photoreceptor loss over dysmorphic yet continuous RPE in histology. The hypoFAF spots inside the floodplain corresponded to complete RPE and outer retinal atrophy (cRORA) on OCT and loss of both photoreceptors and RPE in histology. In contrast, areas of unremarkable FAF showed continuous RPE accompanied by full-length photoreceptors and a thick outer nuclear layer. Conclusions This direct clinicopathologic correlation for FAF imaging is the first for nAMD. Fundus autofluorescence is a projection image that involves optical signal modulation by photoreceptors as well as emission signal sources in RPE. Hyperautofluorescence due to an exudative floodplain signifies loss of photoreceptors over continuous RPE. Hypoautofluorescence in MA signifies loss of both cell layers. For maximal value, fundus autofluorescence imaging should be interpreted with the multilayer perspective provided by OCT. Prevention of exudation in nAMD may preserve photoreceptors.

Introduction Although 18F-FDG PET was originally developed to evaluate benign and malignant tumors, the frequency of detection of benign adrenocortical adenomas showing FDG-PET accumulation has increased. However, the details of FDG-PET-accumulated benign adrenocortical adenomas have not been elucidated. Methods To elucidate the pathophysiology of FDG-PET-positive cortisol-producing adrenal tumors, we performed clinicopathological and genetic analyses of adrenocortical adenomas examing FDG-PET in 30 operated patients with unilateral cortisol-producing adrenal tumors (26 adrenal adenomas and 4 adrenal cancers). Results All adrenocortical carcinomas and 17/26 (65%) benign adrenocortical adenomas showed high FDG accumulation (SUVmax ≥ 3). In adrenocortical adenomas with high FDG accumulation (SUVmax ≥ 3), SUVmax showed a positive correlation with the CT Hounsfield units. A higher SUVmax showed a clear black adenoma appearance with predominantly compact cells, which exhibited high T1 and T2 signals, a lack of signal drop on out-of-phase imaging on MRI, and less accumulation on 131-I adsterol scintigraphy. Furthermore, RNA-sequencing analysis revealed significant increases in the lysosomal and autophagy pathways and metabolic pathways, including glycolysis through glucose transporter (GLUT) 1 and 3, in black adenomas with high-level FDG accumulation. Discussion A black adenoma is blackish due to lipofuscin, which accumulates as a result of damaged mitochondria or proteins that escape lysosomal degradation or autophagy. Since FDG in PET is taken up via GLUTs, alteration of the intracellular metabolic dynamics associated with mitochondrial damage in black adenomas may increase PET accumulation. Conclusion Black adrenal adenomas should be considered with adrenal tumors showing PET accumulation and low lipid contents.

Air pollution exposure is a risk factor for arrhythmia. The atrioventricular (AV) conduction axis is key for the passage of electrical signals to ventricles. We investigated whether environmental nanoparticles (NPs) reach the AV axis and whether they are associated with ultrastructural cell damage. Here, we demonstrate the detection of the shape, size, and composition of NPs by transmission electron microscopy (TEM) and energy-dispersive X-ray spectrometry (EDX) in 10 subjects from Metropolitan Mexico City (MMC) with a mean age of 25.3 ± 5.9 and a 71-year-old subject without cardiac pathology. We found that in every case, Fe, Ti, Al, Hg, Cu, Bi, and/or Si spherical or acicular NPs with a mean size of 36 ± 17 nm were present in the AV axis in situ, freely and as conglomerates, within the mitochondria, sarcomeres, lysosomes, lipofuscin, and/or intercalated disks and gap junctions of Purkinje and transitional cells, telocytes, macrophages, endothelium, and adjacent atrial and ventricular fibers. Erythrocytes were found to transfer NPs to the endothelium. Purkinje fibers with increased lysosomal activity and totally disordered myofilaments and fragmented Z-disks exhibited NP conglomerates in association with gap junctions and intercalated disks. AV conduction axis pathology caused by environmental NPs is a plausible and modifiable risk factor for understanding common arrhythmias and reentrant tachycardia. Anthropogenic, industrial, e-waste, and indoor NPs reach pacemaker regions, thereby increasing potential mechanisms that disrupt the electrical impulse pathways of the heart. The cardiotoxic, oxidative, and abnormal electric performance effects of NPs in pacemaker locations warrant extensive research. Cardiac arrhythmias associated with nanoparticle effects could be preventable.

Fundus autofluorescence (FAF) has been a well-known imaging method for quite some time. However, with developing technologies and novel imaging devices, FAF is being used more often to diagnose and monitor retinal diseases. The density of lipofuscin (LF) and other fluorophores in the retina have a determining role in FAF images. In dry age-related macular degeneration (AMD), hyperautofluorescence is seen in cases of increasing LF in the retina pigment epithelium, whereas hypoautofluorescence is detected in decreasing LF resulting from geographic atrophy. In recent years, studies have shown that FAF images provide prognostic information in patients with AMD. This review aims to highlight the importance of FAF imaging in dry AMD.

Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy, characterized by bilateral progressive central vision loss and subretinal deposition of lipofuscin-like substances. Recent advances in molecular diagnosis and therapeutic options are complemented by the increasing recognition of new multimodal imaging biomarkers that may predict genotype and disease progression. Unique non-invasive imaging features of STDG1 are useful for gene variant interpretation and may even provide insight into the underlying molecular pathophysiology. In addition, pathognomonic imaging features of STGD1 have been used to train neural networks to improve time efficiency in lesion segmentation and disease progression measurements. This review will discuss the role of key imaging modalities, correlate imaging signs across varied STGD1 presentations, and illustrate the use of multimodal imaging as an outcome measure in determining the efficacy of emerging STGD1 specific therapies. This article is protected by copyright. All rights reserved.

Background and Objectives: Age-related macular degeneration is a slow-progressing disease in which lipofuscin accumulates in the retina, causing inflammation and apoptosis of retinal pigment epithelial (RPE) cells. This study aimed to identify N-methyl-D-aspartate (NMDA) signaling as a novel mechanism for scavenging N-retinylidene-N-retinylethanolamine (A2E), a component of ocular lipofuscin, in human RPE cells. Materials and Methods: A2E degradation assays were performed in ARPE-19 cells using fluorescently labeled A2E. The autophagic activity in ARPE-19 cells was measured upon blue light (BL) exposure, after A2E treatment. Autophagy flux was determined by measuring LC3-II formation using immunoblotting and confocal microscopy. To determine whether autophagy via the NMDA receptor is involved in A2E clearance, ATG5-deficient cells were used. Results: Ro 25-6981, an NR2B-selective NMDA receptor antagonist, effectively cleared A2E. Ro 25-6981 reduced A2E accumulation in the lysosomes of ARPE-19 cells at sub-cytotoxic concentrations, while increasing the formation of LC3-II and decreasing p62 protein levels in a concentration-dependent manner. The autophagic flux monitored by RFP-GFP-LC3 and bafilomycin A1 assays was significantly increased by Ro 25-6981. A2E clearance by Ro 25-6981 was abolished in ATG5-depleted ARPE-19 cells, suggesting that A2E degradation by Ro 25-6981 was mediated by autophagy. Furthermore, treatment with other NMDA receptor antagonists, CP-101,606 and AZD6765, showed similar effects on autophagy activation and A2E degradation in ARPE-19 cells. In contrast, glutamate, an NMDA receptor agonist, exhibited a contrasting effect, suggesting that both the activation of autophagy and the degradation of A2E by Ro 25-6981 in ARPE-19 cells occur through inhibition of the NMDA receptor pathway. Conclusions: This study demonstrates that NMDA receptor antagonists degrade lipofuscin via autophagy in human RPE cells and suggests that NMDA receptor antagonists could be promising new therapeutics for retinal degenerative diseases.

Immunodeficient mice are widely used in human stem cell transplantation research. Recombination activating gene 1 (Rag1) deletion results in immunodeficiency and leads to accelerated aging in zebrafish with increased cytosolic accumulation of lipofuscin (LF). Unlike zebrafish, mammals have two homologs, Rag1 and Rag2, that regulate adaptive immunity. Currently, little is known if and how Rag1

Dry age-related macular degeneration (AMD) is marked by the accumulation of extracellular and intracellular lipid-rich deposits within and around the retinal pigment epithelium (RPE). Inducing autophagy, a conserved, intracellular degradative pathway, is a potential treatment strategy to prevent disease by clearing these deposits. However, mTOR inhibition, the major mechanism for inducing autophagy, disrupts core RPE functions. Here, we screened autophagy inducers that do not directly inhibit mTOR for their potential as an AMD therapeutic in primary human RPE culture. Only two out of more than thirty autophagy inducers tested reliably increased autophagy flux in RPE, emphasizing that autophagy induction mechanistically differs across distinct tissues. In contrast to mTOR inhibitors, these compounds preserved RPE health, and one inducer, the FDA-approved compound flubendazole (FLBZ), reduced the secretion of apolipoprotein that contributes to extracellular deposits termed drusen. Simultaneously, FLBZ increased production of the lipid-degradation product β-hydroxybutyrate, which is used by photoreceptor cells as an energy source. FLBZ also reduced the accumulation of intracellular deposits, termed lipofuscin, and alleviated lipofuscin-induced cellular senescence and tight-junction disruption. FLBZ triggered compaction of lipofuscin-like granules into a potentially less toxic form. Thus, induction of RPE autophagy without direct mTOR inhibition is a promising therapeutic approach for dry AMD.

To investigate the influence of age on the thickness of the retinal pigment epithelium (RPE)/Bruch's membrane (BM) complex and the quantitative autofluorescence (qAF) and to study the possible correlation existing between these 2 parameters in a healthy White population.Cross-sectional, observational study.Healthy White volunteers aged 18 to 65 years.All subjects underwent spectral domain OCT (SD-OCT) and qAF imaging with the Heidelberg HRA-Spectralis (Heidelberg Engineering, Heidelberg, Germany). Spectral domain OCT images were analyzed using the in-built graph-based automatic segmentation algorithm for single retinal layer identification to assess RPE/BM complex thickness in vivo. The thickness values of both inner and outer rings of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid, generated by the software using the "RPE" segmentation, were averaged to obtain a single RPE/BM complex thickness value in each eye. Quantitative autofluorescence images were also evaluated using a dedicated software. The qAF values of 8 subfields forming a ring centered onto the fovea were collected and averaged to obtain a single qAF value (qAFThe in vivo correlation between RPE/BM complex thickness and qAF.A total of 105 eyes from 105 subjects (mean age, 42.1 ± 13.9 years; range, 18-65) were included in the analysis. The mean RPE/BM complex thickness significantly increased with age (r = 0.33, P = 0.0006). The values of qAF also positively increased with age (P0.0001). A significant correlation was found between qAF and RPE/BM complex thickness (r = 0.27, P = 0.004). After adjusting for age, iris color, and gender, the correlation remained significant only for subjects aged less than 40 years (P = 0.009).BM complex thickness was significantly co/BM complex thickness increased with age in a healthy White population. A similar increase was found for qAF values. After adjusting for age and iris color, qAF and RPE/BM complex thickness were still correlated in subjects aged less than 40 years. The RPE/BM complex thickness could reflect the lipofuscin/melanolipofuscin accumulation in normal subjects, adding great interest in RPE cell biology.

Lipofuscin is an intracellular aging pigment with fluorescent properties, found in retinal pigment epithelium cells of the eye. It is the main fluorophore used in fundus autofluorescence imaging techniques to diagnose, describe, and follow retinal disease. Lipofuscin forms by incomplete lysosomal degradation of cellular material previously subjected to oxidative changes. A2E is the most studied fluorescent component of lipofuscin, but most of its composition remains unknown. Lipofuscin is photoreactive, generating reactive oxygen species, which may explain its role in disease development. Further knowledge is needed concerning lipofuscin genesis, biochemical composition, fluorescent compounds, and role in pathogenesis of retinal degenerative disease.

To compare efficiency and specific features of transthyretin amyloid staining by different histological dyes and thus to assess their suitability for diagnostic purposes.Samples of left and right heart ventricles were taken from patients over 70 years-old of both genders (n=10) with immunohistochemically verified transthyretin amyloidosis (ATTR). All samples were stained with Congo red, Alcian blue, toluidine blue and methylene violet.Specificity and sensitivity of Congo red staining was comparable to those of immunohistochemical staining. For verification of amyloid presence after Congo red staining one could use fluorescent microscopy instead of polarization microscopy. It allows a more accurate diagnosis of amyloidosis. Confocal microscopy with spectral unmixing improves detection sensitivity of amyloid by elimination of background fluorescence of muscle tissue and autofluorescence of lipofuscin. Alcian blue staining gives the same result as Congo red. In addition, its less labor-intensive and free of false-positive and false-negative results caused by final processing of slide preparation. Toluidine blue and methylene violet develop metachromatic staining upon binding to transthyretin fibrils, likely due to specific biochemical features of these fibrils.The most reliable method for histochemical diagnosis of ATTR is the Congo red staining with subsequent analysis using fluorescence or confocal microscopy. For diagnostic screening, the use of Sodium sulphate-Alcian blue staining method is highly promising. Metachromatic stains are less effective for ATTR diagnosis.Цель. Изучить особенности окраски транстиретинового амилоида разными гистохимическими красителями и оценить эффективность их использования для диагностических целей. Материалы и методы. Материалом для исследования послужили образцы миокарда левого и правого желудочка лиц обоих полов в возрасте старше 70 лет (n=10) с иммуногистохимически верифицированным транстиретиновым амилоидозом. Препараты всех отобранных случаев были окрашены Конго красным, альциановым синим, толуидиновым синим и метиловым фиолетовым. Результаты. В случае транстиретинового амилоидоза миокарда окраска Конго красным по чувствительности и специфичности выявления амилоида сопоставима с иммуногистохимической реакцией. Для верификации присутствия амилоида вместо поляризационной микроскопии может быть использован метод флуоресцентной микроскопии, который дает возможность более надежно определить природу конгофильных скоплений. Применение метода конфокальной микроскопии и функции спектрального разделения позволяет усилить контрастность выявления амилоида за счет элиминирования фоновой флуоресценции мышечной ткани и автофлуоресценции липофусцина. Метод окраски альциановым синим по чувствительности и специфичности обнаружения амилоида сопоставим с Конго красным. При этом он менее трудоемок и позволяет избежать ложноположительных и ложноотрицательных результатов, обусловленных особенностями финальной обработки препаратов. Толуидиновый синий и метиловый фиолетовый проявляют метахроматическое окрашивание при связывании с транстиретиновым амилоидом, что может быть обусловлено биохимическими особенностями амилоида этого типа. Заключение. Наиболее надежным методом гистохимической диагностики транстиретинового амилоидоза является окраска Конго красным с последующим исследованием препаратов методами флуоресцентной или конфокальной микроскопии. В качестве скринингового метода в диагностических целях может быть применена окраска альциановым синим. Метахроматические красители менее эффективны для диагностики транстиретинового амилоидоза миокарда.

Near-infrared autofluorescence (NIA) is a non-invasive retinal imaging technique for examination of the retinal pigment epithelium (RPE) based on the autofluorescence of melanin. Melanin has several functions within the RPE cells, in one of them it serves as a protective antioxidative factor within the RPE cells and is involved in the phagocytosis of photoreceptor outer segments. Disorders that affect the photoreceptor-RPE complex result in alterations of RPE cells which are detectable by alterations of NIA. Therefore, NIA allows to detect early alterations in inherited and acquired chorioretinal disorders, frequently prior to ophthalmoscopical visualisation and often prior to alterations in lipofuscin associated fundus autofluorescence (FAF) or optical coherence tomography (OCT). Although NIA and FAF relate to disorders affecting the RPE, findings between both imaging methods differ and the area involved has been demonstrated to be larger in NIA compared to FAF in several disorders (e.g., age-related macular degeneration, retinitis pigmentosa, ABCA4-gene associated Stargardt disease and cone-rod dystrophy, light damage), indicating that NIA detects earlier alterations compared to FAF. In addition, due to the absence of blue-light filtering which limits foveal visualisation in FAF, foveal alterations can be much better detected using NIA. A reduced subfoveal NIA intensity is the earliest sign of autosomal dominant BEST1-associated disease, when FAF and OCT are still normal. In other disorders, a normal subfoveal NIA intensity is associated with good visual acuity. This review summarizes the present knowledge on NIA and demonstrates biomarkers for various chorioretinal disorders.

Hepatic adenomas occur most commonly in women between the ages of 20 and 40 years, but rarely they occur in older aged persons, including those 60 years of age or older. This group of adenomas, however, has not been systemically examined. Twenty-six hepatic adenomas in persons 60 years of age or older were studied, along with a control group of 50 hepatic adenomas in persons aged 30 to 39. Hepatic adenomas in persons 60 or more years of age were found in 21 women and 5 men, while the control group had 44 women and 6 men. Subtyping the adenomas in persons 60 years or older showed the following results: 18 HNF1A-inactivated adenomas (69%), 4 inflammatory adenomas (15%), and 4 unclassified adenomas (15%). In contrast, the control group showed a significantly different pattern (P=0.003), with a greater percentage of inflammatory adenomas (28, 56%), fewer HNF1A-inactivated adenomas (8, 16%), and more unclassified adenomas (14, 28%). Atypia and malignant transformation within the hepatic adenomas was studied next. Of the hepatic adenomas in persons age 60 or greater, 3 (12%) showed atypical histologic features, and 6 (23%) had a malignant transformation. In contrast, for hepatic adenomas in the control group, only 4 (8%) adenomas showed atypical histologic features, and 3 (6%) had undergone malignant transformation. In addition, the hepatic adenomas that were atypical or showed early malignant transformation were less likely to have beta-catenin activation in patients over 60 (2/9 cases) compared with those between 30 and 39 years (5/7 cases). Myxoid change and heavy lipofuscin deposition were also more common in adenomas in older aged persons. In conclusion, hepatic adenomas in persons 60 years of age or older are enriched for HNF1A-inactivated adenomas and have a higher frequency of malignant transformation. Malignant transformation, however, is less likely to develop through activation of the beta-catenin pathway.

23 Páginas.-- 17 Figuras, PLA2G6-Associated Neurodegeneration (PLAN) is a rare neurodegenerative disease with autosomal recessive inheritance, which belongs to the NBIA (Neurodegeneration with Brain Iron Accumulation) group. Although the pathogenesis of the disease remains largely unclear, lipid peroxidation seems to play a central role in the pathogenesis. Currently, there is no cure for the disease., This work was supported by FIS PI16/00786 and PI19/00377 grants, Instituto de Salud Carlos III, Spain and Fondo Europeo de Desarrollo Regional (FEDR-Unión Europea), Proyectos de Investigación de Excelencia de la Junta de Andalucía CTS-5725 and PY18-850 and by AIDNAI (Association Internationale de Dystrophie Neuro Axonale Infantile), ENACH (Asociación de Enfermos de Neurodegeneración con Acumulación Cerebral de Hierro), AEPMI (Asociación de Enfermos de Patología Mitocondrial), FEDER (Federación Española de Enfermedades Raras) and Fundación MERK Salud. S. Povea-Cabello is a recipient of Ayudas para la Formación del Profesorado Universitario (FPU) from Ministerio de Universidades de España.

Inactivating mutations in the PPT1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) underlie the CLN1 disease, a devastating neurodegenerative lysosomal storage disorder. The mechanism of pathogenesis underlying CLN1 disease has remained elusive. PPT1 is a lysosomal enzyme, which catalyzes the removal of palmitate from S-palmitoylated proteins (constituents of ceroid lipofuscin) facilitating their degradation and clearance by lysosomal hydrolases. Thus, it has been proposed that Ppt1-deficiency leads to lysosomal accumulation of ceroid lipofuscin leading to CLN1 disease. While S-palmitoylation is catalyzed by palmitoyl acyltransferases (called ZDHHCs), palmitoyl-protein thioesterases (PPTs) depalmitoylate these proteins. We sought to determine the mechanism by which Ppt1-deficiency may impair lysosomal degradative function leading to INCL pathogenesis. Here we report that in Ppt1(−/−) mice, which mimic CLN1 disease, low level of inositol 3-phosphate receptor-1 (IP3R1) that mediates Ca(++)-transport from the ER to the lysosome dysregulated lysosomal Ca(++) homeostasis. Intriguingly, the transcription factor NFATC4, which regulates IP3R1-expression, required S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8, which catalyzed S-palmitoylation of NFATC4. Notably, in Ppt1(−/−) mice, reduced ZDHHC4 and ZDHHC8 levels markedly lowered S-palmitoylated NFATC4 (active) in the nucleus, which inhibited IP3R1-expression, thereby, dysregulating lysosomal Ca(++) homeostasis. Consequently, Ca(++)-dependent lysosomal enzyme activities were markedly suppressed. Impaired lysosomal degradative function impaired autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1-overexpression in Ppt1(−/−) mouse fibroblasts ameliorated this defect. Our results reveal a previously unrecognized role of Ppt1 in regulating lysosomal Ca(++)-homeostasis and suggest that this defect contributes to pathogenesis of CLN1 disease.

International audience; Background: The cellular and molecular alterations associated with synapse and neuron loss in Alzheimer's disease (AD) remain unclear. In transgenic mouse models that express mutations responsible for familial AD, neuronal and synaptic losses occur in populations that accumulate fibrillar amyloid-β 42 (Aβ42) intracellularly.Objective: We aimed to study the subcellular localization of these fibrillar accumulations and whether such intraneuronal assemblies could be observed in the human pathology.Methods: We used immunolabeling and various electron microscopy techniques on APP x presenilin1 - knock-in mice and on human cortical biopsies and postmortem samples.Results: We found an accumulation of Aβ fibrils in lipofuscin granule-like organelles in APP x presenilin1 - knock-in mice. Electron microscopy of human cortical biopsies also showed an accumulation of undigested material in enlarged lipofuscin granules in neurons from AD compared to age-matched non-AD patients. However, in those biopsies or in postmortem samples we could not detect intraneuronal accumulations of Aβ fibrils, neither in the lipofuscin granules nor in other intraneuronal compartments.Conclusion: The intralysosomal accumulation of Aβ fibrils in specific neuronal populations in APPxPS1-KI mice likely results from a high concentration of Aβ42 in the endosome-lysosome system due to the high expression of the transgene in these neurons.

Increased oxidative stress has been associated with several neurodegenerative diseases such as Alzheimer's disease, but also with neurological diseases sharing pathophysiological pathways like epilepsy. Lipofuscin is a nondegradable end-product of oxidative stress; its cerebral presence reflects the cumulative amount of oxidative stress the brain has endured. In this study, we have observed prominent autofluorescent particles in the pial arterial wall and in neocortical parenchyma of young, drug-resistant epilepsy patients (18-28 years old) who underwent resective brain surgery (n= 6), as well as in older control patients (n= 3). With fluorescence spectroscopic imaging, brightfield microscopy, histochemistry and fluorescence lifetime imaging, these autofluorescent particles were identified as theage pigmentlipofuscin. An evaluation of these lipofuscin particles using Imaris (c) software allowed robust quantification, while the 3D properties allowed visualization of the complex configuration. We elaborate on the usefulness of lipofuscin as a marker of cumulative oxidative stress in the brain. Furthermore, we speculate on the observed differences in particle size and density that we found between young patients and older controls, which could imply a role for lipofuscin in the pathophysiology of epilepsy and possibly other neurological diseases.

Accumulation of cytotoxic lipofuscin bisretinoids may contribute to atrophic age-related macular degeneration (AMD) pathogenesis. Retinal bisretinoid synthesis depends on the influx of serum all-trans-retinol (1) delivered via a tertiary retinol binding protein 4 (RBP4)–transthyretin (TTR)–retinol complex. We previously identified selective RBP4 antagonists that dissociate circulating RBP4–TTR–retinol complexes, reduce serum RBP4 levels, and inhibit bisretinoid synthesis in models of enhanced retinal lipofuscinogenesis. However, the release of TTR by selective RBP4 antagonists may be associated with TTR tetramer destabilization and, potentially, TTR amyloid formation. We describe herein the identification of bispecific RBP4 antagonist–TTR tetramer kinetic stabilizers. Standout analogue (±)-44 possesses suitable potency for both targets, significantly lowers mouse plasma RBP4 levels, and prevents TTR aggregation in a gel-based assay. This new class of bispecific compounds may be especially important as a therapy for dry AMD patients who have another common age-related comorbidity, senile systemic amyloidosis, a nongenetic disease associated with wild-type TTR misfolding.

The neuronal ceroid lipofuscinoses (NCLs) are at least 13 distinct progressive neurodegenerative disorders unified by the accumulation of lysosomal auto-fluorescent material called lipofuscin. The only form that occurs via autosomal-dominant inheritance exhibits adult onset and is sometimes referred to as Parry type NCL. The manifestations may include behavioral symptoms followed by seizures, ataxia, dementia, and early death. Mutations in the gene DNAJC5 that codes for the presynaptic co-chaperone cysteine string protein-α (CSPα) were recently reported in sporadic adult-onset cases and in families with dominant inheritance. The mutant CSPα protein may lead to disease progression by both loss and gain of function mechanisms. Iron chelation therapy may be considered as a possible pharmaceutical intervention based on our recent mechanism-based proposal of CSPα oligomerization via ectopic Fe-S cluster-binding, summarized in this review.

Intranuclear inclusions present in the brains of patients with Fragile X-associated tremor/ataxia syndrome (FXTAS) have historically been difficult to study due to their location and scarcity. The recent finding that these particles autofluoresce has complicated the use of immunofluorescence techniques, but also offers new opportunities for purification. We have ascertained the features of the autofluorescence, including its excitation/emission spectrum, similarities and differences compared with lipofuscin autofluorescence, and its presence/absence under various fixation, mounting and UV light exposure conditions. Immunofluorescence at various wavelengths was conducted to determine which conditions are ideal for minimizing autofluorescence confounds. We also present a technique for autofluorescence-based sorting of FXTAS inclusions using flow cytometry, which will allow researchers in the field to purify inclusions more successfully for unbiased analyses.

A 16-year-old boy presented with a tumor located in fourth ventricle, which showed histological features of an ependymoma replete with perivascular pseudorosettes and true ependymal rosettes. Interestingly, many of the tumor cells exhibited abundant cytoplasm stuffed with a grayish brown pigment. Histochemical stains showed the pigment to be acid fast and periodic acid–Schiff positive and negative for Masson-Fontana melanin stain. Additionally, the pigment displayed brilliant autofluorescence under ultraviolet light of a fluorescent microscope. Ultrastructure examination of the pigment revealed a non–membrane-bound biphasic structure with an electron-dense core and electron-lucent periphery. Only few similar case reports mention such pigmented ependymomas to contain a mixture of neuromelanin and lipofuscin while others mention it to be melanin itself. Our workup suggests the pigment to represent lipofuscin or its derivative. Generally known to be a pigment of wear and tear, the significance of finding it in a tumor with such abundance remains to be understood and explored.

BACKGROUND: Diagnosis of small choroidal melanoma is mainly based on tumour thickness, subretinal fluid, or lipofuscin pigment. Ultra-wide-field imaging (UWF) allows depiction of choroidal lesions through a red (RC) and a green channel (GC). Aim of the study was to determine the utility of this tool in the detection of small choroidal melanoma. METHODS: Retrospective cross-sectional study of patients with small choroidal pigmented lesions up to 3 mm in thickness. All patients underwent clinical and imaging assessment including UWF. Lesions were subcategorized based on thickness and lesion type. A qualitative assessment ensued using the red and green channels feature. RESULTS: A total of 152 patients were included. Melanotic naevi (76/152,50%) and small choroidal melanomas (55/152,36%) were the predominant types. Thickness was

Stargardt disease (STGD1), known as inherited retinal dystrophy, is caused by ABCA4 mutations. The pigmented Abca4-/- mouse strain only reflects the early stage of STGD1 since it is devoid of retinal degeneration. This blue light-illuminated pigmented Abca4-/- mouse model presented retinal pigment epithelium (RPE) and photoreceptor degeneration which was similar to the advanced STGD1 phenotype. In contrast, wild-type mice showed no RPE degeneration after blue light illumination. In Abca4-/- mice, the acute blue light diminished the mean autofluorescence (AF) intensity in both fundus short-wavelength autofluorescence (SW-AF) and near-infrared autofluorescence (NIR-AF) modalities correlating with reduced levels of bisretinoid-fluorophores. Blue light-induced RPE cellular damage preceded the photoreceptors loss. In late-stage STGD1-like patient and blue light-illuminated Abca4-/- mice, lipofuscin and melanolipofuscin granules were found to contribute to NIR-AF, indicated by the colocalization of lipofuscin-AF and NIR-AF under the fluorescence microscope. In this mouse model, the correlation between in vivo and ex vivo assessments revealed histological characteristics of fundus AF abnormalities. The flecks which are hyper AF in both SW-AF and NIR-AF corresponded to the subretinal macrophages fully packed with pigment granules (lipofuscin, melanin, and melanolipofuscin). This mouse model, which has the phenotype of advanced STGD1, is important to understand the histopathology of Stargardt disease.

Age-related macular degeneration (AMD) is a complex eye disease underlined by the death of photoreceptors and degeneration of retinal pigment epithelium (RPE) and choriocapillaris (CC). The mechanism(s) responsible for massive and progressive retinal degeneration is not completely known. Senescence, a state of permanent inhibition of cell growth, may be induced by many factors important for AMD pathogenesis and results in senescence-associated secretory phenotype (SASP) that releases growth factors, cytokines, chemokines, proteases and other molecules inducing inflammation and other AMD-related effects. These effects can be induced in the affected cell and neighboring cells, leading to progression of AMD phenotype. Senescent cells also release reactive oxygen species that increase SASP propagation. Many other pathways of senescence-related AMD pathogenesis, including autophagy, the cGAS-STING signaling, degeneration of CC by membrane attack complex, can be considered. A2E, a fluorophore present in lipofuscin, amyloid-beta peptide and humanin, a mitochondria-derived peptide, may link AMD with senescence. Further studies on senescence in AMD pathogenesis to check the possibility of opening a perspective of the use of drugs killing senescent cells (senolytics) and terminating SASP bystander effects (senostatics) might be beneficial for AMD that at present is an incurable disease.

Background/aim The role of senescence in defining tumor aggressiveness at a clinical level remains obscure. A novel mixed histochemical/immunohistochemical method (SenTraGor™, STG) detecting lipofuscin accumulation allows the assessment of senescent cells in paraffin-embedded tissue material. Materials and methods STG expression was quantified in 98 surgically resected primary non-small-cell-lung carcinomas (NSCLC). Data were analyzed in parallel with other immunohistochemical markers related to hypoxia and autophagy. Results Strong STG staining was noted in 36/98 cases (36.7%). High STG expression was significantly associated with high HIF1α expression and high expression of glucose (GLUT1) and monocarboxylate (MCT2) transporters, pointing to a link between senescence, hypoxia and glycolysis. High STG expression was also linked with high cytoplasmic accumulation of MAP1-LC3B, TFEB and LAMP2a, suggestive of a blockage of autophagy flux in tumors with intense senescence. Survival analysis showed a direct association with poor survival, independently of stage. Conclusion SenTraGor™ provides a reliable methodology to detect lipofuscin accumulation in cancer cells in paraffin-embedded tissues, opening a new field for translational studies focused on senescence.

The human amygdala is involved in processing of memory, decision-making, and emotional responses. Previous studies suggested that the amygdala may represent a neurogenic niche in mammals. By combining two distinct methodological approaches, lipofuscin quantification and C-14-based retrospective birth dating of neurons, along with mathematical modelling, we here explored whether postnatal neurogenesis exists in the human amygdala. We investigated post-mortem samples of twelve neurologically healthy subjects. The average rate of lipofuscin-negative neurons was 3.4%, representing a substantial proportion of cells substantially younger than the individual. Mass spectrometry analysis of genomic C-14-concentrations in amygdala neurons compared with atmospheric C-14-levels provided evidence for postnatal neuronal exchange. Mathematical modelling identified a best-fitting scenario comprising of a quiescent and a renewing neuronal population with an overall renewal rate of >2.7% per year. In conclusion, we provide evidence for postnatal neurogenesis in the human amygdala with cell turnover rates comparable to the hippocampus. Lipofuscin labeling and (14) C retrospective birth-dating of neurons, along with mathematical modelling, here suggest continued postnatal neurogenesis in the human amygdala, rather than protracted maturation of developmentally generated neurons.

A fast calculation method was used to obtain the spectral optical properties of human normal and pathological (chromophobe renal cell carcinoma) kidney tissues. Using total transmittance, total reflectance and collimated transmittance spectra acquired from ex vivo kidney samples, the spectral optical properties of both tissues, namely the absorption, the scattering and the reduced scattering coefficients, as well as the scattering anisotropy, dispersion and light penetration depth, were calculated between 200 and 1000 nm. Analysis of the mean ab sorption coefficient spectra of the kidney tissues showed that both contain melanin and lipofuscin, and that 83 % of the melanin in the normal kidney converts into lipofuscin in the pathological kidney.

Loss-of-function mutations in progranulin (GRN) are a major autosomal dominant cause of frontotemporal dementia. Most pathogenicGRNmutations result in progranulin haploinsufficiency, which is thought to cause frontotemporal dementia inGRNmutation carriers. Progranulin haploinsufficiency may drive frontotemporal dementia pathogenesis by disrupting lysosomal function, as patients withGRNmutations on both alleles develop the lysosomal storage disorder neuronal ceroid lipofuscinosis, and frontotemporal dementia patients withGRNmutations (FTD-GRN) also accumulate lipofuscin. The specific lysosomal deficits caused by progranulin insufficiency remain unclear, but emerging data indicate that progranulin insufficiency may impair lysosomal sphingolipid-metabolizing enzymes. We investigated the effects of progranulin insufficiency on sphingolipid-metabolizing enzymes in the inferior frontal gyrus of FTD-GRNpatients using fluorogenic activity assays, biochemical profiling of enzyme levels and posttranslational modifications, and quantitative neuropathology. Of the enzymes studied, only β-glucocerebrosidase exhibited impairment in FTD-GRNpatients. Brains from FTD-GRNpatients had lower activity than controls, which was associated with lower levels of mature β-glucocerebrosidase protein and accumulation of insoluble, incompletely glycosylated β-glucocerebrosidase. Immunostaining revealed loss of neuronal β-glucocerebrosidase in FTD-GRNpatients. To investigate the effects of progranulin insufficiency on β-glucocerebrosidase outside of the context of neurodegeneration, we investigated β-glucocerebrosidase activity in progranulin-insufficient mice. Brains fromGrn−/−mice had lower β-glucocerebrosidase activity than wild-type littermates, which was corrected by AAV-progranulin gene therapy. These data show that progranulin insufficiency impairs β-glucocerebrosidase activity in the brain. This effect is strongest in neurons and may be caused by impaired β-glucocerebrosidase processing.

The survival of erythrocytes in the circulating blood depends on their membranes’ structural and functional integrity. One of the mechanisms that may underlie the process of joint degeneration is the imbalance of prooxidants and antioxidants, promoting cellular oxidative stress. The study is aimed at observing the effects of the 21-day general rehabilitation program on the erythrocytes redox status and serum ST2 marker in patients after knee or hip replacement in the course of osteoarthritis. Erythrocytes and serum samples were collected from 36 patients. We analyzed the selected markers of the antioxidant system in the erythrocytes: catalase (CAT), glutathione reductase (glutathione disulfide reductase (GR, GSR)), total superoxide dismutase activity (SOD), glutathione peroxidase (GPx), glutathione transferase (GST) activity, and cholesterol and lipofuscin (LPS) concentration. In serum, we analyzed the concentration of the suppression of tumorigenicity 2 (ST2) marker. After the 21-day general rehabilitation program, the total SOD and GPx activity, measured in the hemolysates, significantly increased ( p < 0.001 ) while LPS, cholesterol, and ST2 levels in serum significantly decreased ( p < 0.001 ). General rehabilitation reduces oxidative stress in patients after knee or hip replacement in the course of osteoarthritis. Individually designed, regular physical activity is the essential element of the postoperative protocol, which improves the redox balance helping patients recover after the s4urgery effectively.

Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE), where its fluorescence properties are used to assess retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in the early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE and lipofuscin-laden cells to visible light, and to determine whether an abundant component of lipofuscin, docosahexaenoate (DHA), can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible light leads to a decrease in its long-wavelength fluorescence at about 610 nm, with a concomitant increase in the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure of lipofuscin-laden cells to light leads to a loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes in fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra, together with quantitation of the intensity of long-wavelength fluorescence, can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, and hence useful for screening the retina for increased oxidative damage and early AMD-related changes.

Autofluorescent granules of various sizes were observed in primary human liver endothelial cells (LSECs) upon laser irradiation using a wide range of wavelengths. Autofluorescence was detected in LAMP-1 positive vesicles, suggesting lysosomal location. Confocal imaging of freshly prepared cultures and imaging flow cytometry of non-cultured cells revealed fluorescence in all channels used. Treatment with a lipofuscin autofluorescence quencher reduced autofluorescence, most efficiently in the near UV-area. These results, combined with the knowledge of the very active blood clearance function of LSECs support the notion that lysosomally located autofluorescent material reflected accumulation of lipofuscin in the intact liver. These results illustrate the importance of careful selection of fluorophores, especially when labelling of live cells where the quencher is not compatible.

Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.

Focal neuronal lipofuscinosis (FNL) is an uncommon epileptic disorder related to an excess of lipofuscin accumulation within dysmorphic-appearing neurons (DANs), whose epileptogenic mechanisms are still poorly understood. It shares some clinical and neuroimaging similarities with focal cortical dysplasia of type IIb (FCDIIb), but it represents a different pathological entity. Here, we identified two patients with FNL among a 10-year cohort of 323 patients who underwent neurosurgery for a focal pharmacoresistant epilepsy. We describe the electroclinical, metabolic and neuropathological features of both patients with FNL who benefited from a comprehensive presurgical investigation. While the previous reports showed frontal lobe localization of the lesion, FNL was identified in the temporal lobe, in one of our patients. EEG investigations in both patients showed striking focal and rich interictal activity resembling that described in FCDIIb. Besides focal intraneuronal lipofuscin accumulation, the neuropathological analysis demonstrated that somata of DANs were surrounded by a large amount of GABAergic presynaptic buttons, suggesting the involvement of interneurons in the epileptogenicity of FNL. To further explore the role of GABAergic transmission in the generation of epileptiform activity in FNL, we performed in vitro multi-electrode array recordings on the post-surgery tissue from one patient. Spontaneous interictal-like discharges (IILDs) were identified only in the restricted area displaying the highest density of lipofuscin-containing DANs, suggesting a close correlation between the density of lipofuscin-containing neurons and epileptogenicity. Moreover, IILDs were blocked by the GABAA receptor antagonist gabazine. All together, these findings showed how GABA signaling may contribute to the generation of interictal-like activity in FNL tissue.

Aspartame (ASP) and sucralose (SUC) are non-nutritive sweeteners which are widely consumed worldwide. They are considered safe for human consumption, but their effects on certain physiological aspects, such as the lifespan or health status, of the organism have not yet been studied in depth and only limited data are available in the literature. The objectives of this study were to evaluate the effects of ASP and SUC on the lifespan and health indexes using

Introduction: The accumulation of lipofuscin is a hallmark in the pathogenesis of Stargardt disease type 1 (STGD1) and geographic atrophy (GA) secondary to age-related macular degeneration. Limiting lipofuscin accumulation by inhibiting the retinol-binding protein 4 (RBP4) is being explored as a potential treatment target for those diseases. In this study, we aimed to establish the concentration of RBP4 in the systemic circulation in different age cohorts of healthy individuals and to check if patients with STGD1 or GA may show abnormal RBP4 levels. Methods: Forty healthy subjects of various age-groups, 15 Stargardt patients, and 15 GA patients were included in the study. We measured RBP4 levels, serum retinol (SR) levels, complete blood count, and blood chemistry including liver function tests. Results: Mean RBP4 for all cohorts was 26,911.40 ± 6,198.61 ng/mL, and mean SR 1.75 ± 0.36 µmol/L. Age was not found to significantly impact levels neither of RBP4 and SR nor of the RBP4-to-SR ratio. Also, the 2 patient groups showed similar blood levels to their age-matched controls. Conclusion: Serum RBP4 and SR do not appear to be affected by age in healthy individuals and remain within normal limits in both STGD1 and GA.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn(−/−) mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn(−/−) brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN – a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn(−/−) phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine and disease pathology in Grn(−/−) CNS, including microgliosis, lipofuscinosis and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Funding Information: Supported by Funda??o para a Ciência e Tecnologia (FCT) ? Portugal co-funded by FEDER under the PT2020 Partnership Agreement (to MCS, including project PTDC/MED-PAT/30385/2017, iNOVA4Health-UIDB/04462/2020, research infrastructure PPBI-POCI-01-0145-FEDER-022122, M-ERA.NET 2/0005/2016), Boehringer Ingelheim (to MCS), Fight for Sight UK (to MCS), Wellcome Trust grant number 212216/Z/18/Z/ (to CEF). MJH was funded by Moor-fields Eye Charity with the Bill Brown 1989 Charitable Trust PhD studentship 538158, MLS was funded by FCT-CEECIND/01536/2018, ACF was funded by FCT PhD studentship (PD/BD/135503/2018). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund) and this article is supported by the LYSOCIL project funded by the European Union?s Horizon 2020 programme under grant agreement No. 811087. Funding Information: Supported by Fundação para a Ciência e Tecnologia (FCT) – Portugal co-funded by FEDER under the PT2020 Partnership Agreement (to MCS, including project PTDC/MED-PAT/30385/2017, iNOVA4Health-UIDB/04462/2020, research infrastructure PPBI-POCI-01-0145-FEDER-022122, M-ERA.NET 2/0005/2016), Boehringer Ingelheim (to MCS), Fight for Sight UK (to MCS), Wellcome Trust grant number 212216/Z/18/Z/ (to CEF). MJH was funded by Moor-fields Eye Charity with the Bill Brown 1989 Charitable Trust PhD studentship 538158, MLS was funded by FCT-CEECIND/01536/2018, ACF was funded by FCT PhD studentship (PD/BD/135503/2018). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund) and this article is supported by the LYSOCIL project funded by the European Union’s Horizon 2020 programme under grant agreement No. 811087. Publisher Copyright: Copyright 2021 The Authors PURPOSE. We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers. METHODS. We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing. RESULTS. AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. CONCLUSIONS. We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation. publishersversion published

Intervertebral disc (IVD) degeneration is considered an important contributor of low back pain, a major age-related disease. Interestingly, an unprecedented high number of senescent cells has been reported in aged and degenerated IVDs, most probably affecting tissue homeostasis. In previous studies classical markers of cellular senescence have been used, such as SA-β-gal staining or p16Ink4a expression. Aim of the presented study was a re-evaluation of the number of senescent IVD cells by using a newly established staining procedure for lipofuscin, based on a Sudan Black-B analogue (GL13), which can be used in fresh, as well as in fixed and embedded tissues. In cultures of senescent rat and human IVD cells both SA-β-gal and GL13 gave similar percentages of senescent cells. Similarly, in fresh tissues from old rats the ratios of senescent cells were high with both detection procedures. Finally, in formalin-fixed and paraffin-embedded tissues from humans, a significant increased number of GL13-positive cells was found in herniated tissues, as compared to apparently normal ones, while similar numbers of p16Ink4a-positive cells were observed. These data confirm the significantly enhanced number of senescent cells in aged and degenerated IVDs, most probably contributing to the degeneration of this tissue.

Mesenchymal stem cells (MSCs) are broadly applied in regenerative therapy to replace cells that are lost or impaired during disease. The low survival rate of MSCs after transplantation is one of the major limitations heavily influencing the success of the therapy. Unfavorable microenvironments with inflammation and oxidative stress in the damaged regions contribute to MSCs loss. Most of the strategies developed to overcome this obstacle are aimed to prevent stress-induced apoptosis, with little attention paid to senescence—another common stress reaction of MSCs. Here, we proposed the strategy to prevent oxidative stress-induced senescence of human endometrial stem cells (hMESCs) based on deferoxamine (DFO) application. DFO prevented DNA damage and stress-induced senescence of hMESCs, as evidenced by reduced levels of reactive oxygen species, lipofuscin, cyclin D1, decreased SA-β-Gal activity, and improved mitochondrial function. Additionally, DFO caused accumulation of HIF-1α, which may contribute to the survival of H2O2-treated cells. Importantly, cells that escaped senescence due to DFO preconditioning preserved all the properties of the initial hMESCs. Therefore, once protecting cells from oxidative damage, DFO did not alter further hMESCs functioning. The data obtained may become the important prerequisite for development of a new strategy in regenerative therapy based on MSCs preconditioning using DFO.