Papers by Kai Kaarniranta
Acta Ophthalmologica, 2013
ABSTRACT Purpose Age-related macular degeneration pathogenesis involves impaired protein degradat... more ABSTRACT Purpose Age-related macular degeneration pathogenesis involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome system and the autophagy pathway are major proteolytic processes in eukaryotic cells. SQSTM1/p62 has been shown as a key player linking the proteasomal and lysosomal clearance systems. The present study investigated the effects of AICAR (AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) with/without MG-132 (proteasome inhibitor) on autophagy regulation in RPE cells. Methods ARPE-19 cells were treated with MG-132 (5µM) and/or AICAR (2mM). p62 and MAP1LC3A/LC3 (LC3II) were analyzed by Western blotting. LC3 lipidation was used to study autophagic flux. Transmission electron microscopy was used to detect protein aggregates and autophagosomes. pDendra2-hLC3 construct was used to detect macroautophagy in confocal microscopy analysis. Results AICAR+MG-132 co-treatment induces autophagy clearance of p62 and increased LC3 lipidation. AICAR is able to completely abolish the MG-132-induced protein aggregation after 24 h treatment; at the same time, the co-treatment increases the number of autophagic vesicles. Cells treated with MG-132+AICAR exhibit a strong reduction of the perinuclear aggregates containing both LC3II and p62 proteins. Conclusion Autophagy is emerging as a novel target of therapies aimed to counteract protein aggregation and improve cell viability. In this way, our findings indicate that AICAR could be useful in the acceleration of protein clearance in RPE cells.
Age-related macular degeneration (AMD) is characterized primarily by degeneration of the macular ... more Age-related macular degeneration (AMD) is characterized primarily by degeneration of the macular retinal pigment epithelium (RPE) that secondarily leads to cell death of photoreceptors and impaired central vision. Hallmarks of AMD are accumulation of lysosomal lipofuscin and extracellular drusen, which indicate impaired proteolysis in RPE cells. Cellular proteostasis is strongly regulated by molecular chaperones such as Hsp70 and proteasomal and autophagic clearance systems. We have recently shown that autophagy receptor SQSTM1/p62 binds irreversibly to proteasome inhibitor-induced perinuclear protein aggregates and undergoes autophagic clearance in RPE cell cultures. Revealing decreased autophagy, SQSTM1/p62 accumulates in macular area of donor AMD patient samples. In this study, we show that Hsp70 binds reversibly to proteasome inhibitor-induced perinuclear protein aggregates and does not become degraded by autophagy in ARPE-19 cells. Our observation reveals new opportunities to u...
Histology and histopathology, Jan 19, 2015
Macular corneal dystrophy is a rare autosomal recessive eye disease affecting primarily the corne... more Macular corneal dystrophy is a rare autosomal recessive eye disease affecting primarily the corneal stroma. Abnormal accumulation of proteoglycan aggregates has been observed intra- and extracellularly in the stromal layer. In addition to the stromal keratocytes and corneal lamellae, deposits are also present in the basal epithelial cells, endothelial cells and Descemet's membrane. Misfolding of proteins has a tendency to gather into aggregating deposits. We studied interaction of molecular chaperones and proteasomal clearance in macular dystrophy human samples and in human corneal HCE-2 epithelial cells. Seven cases of macular corneal dystrophy and four normal corneal buttons collected during corneal transplantation were examined for their expression patterns of heat shock protein 70, ubiquitin protein conjugates and SQSTM1/p62. In response to proteasome inhibition the same proteins were analyzed by western blotting. Slit-lamp examination, in vivo confocal cornea microscopy and...
Acta Ophthalmologica, 2010
The pathogenesis of age-related macular degeneration involves impaired protein degradation in ret... more The pathogenesis of age-related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Prior to proteolysis, heat shock proteins (HSPs) attempt to refold stress-induced misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the functional roles of p62 and HSP70 were evaluated in conjunction with proteasome inhibitor-induced autophagy in human RPE cells . Methods: The p62, HSP70, and ubiquitin protein levels and localization were analyzed by western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. The p62 and HSP70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. Results: Proteasome inhibition evoked the accumulation of perinuclear aggregates that strongly colocalized with p62 and HSP70. The p62 perinuclear accumulation was time-and concentration-dependent after MG-132 proteasome inhibitor loading. The silencing of p62, rather than Hsp70, evoked suppression of autophagy, when related to decreased LC3-II levels after bafilomycin treatment. In addition, the p62 silencing decreased the ubiquitination level of the perinuclear aggregates. Recently, we showed that hsp70 mRNA depletion increased cell death in ARPE-19 cells. Here, we demonstrate that p62 mRNA silencing has similar effects on cellular viability.
Oxidative medicine and cellular longevity, 2015
Numerous studies have highlighted the key roles of oxidative stress and inflammation in aging-rel... more Numerous studies have highlighted the key roles of oxidative stress and inflammation in aging-related diseases such as obesity, type 2 diabetes, age-related macular degeneration (AMD), and Alzheimer's disease (AD). In aging cells, the natural antioxidant capacity decreases and the overall efficiency of reparative systems against cell damage becomes impaired. There is convincing data that stilbene compounds, a diverse group of natural defence phenolics, abundant in grapes, berries, and conifer bark waste, may confer a protective effect against aging-related diseases. This review highlights recent data helping to clarify the molecular mechanisms involved in the stilbene-mediated protection against oxidative stress. The impact of stilbenes on the nuclear factor-erythroid-2-related factor-2 (Nrf2) mediated cellular defence against oxidative stress as well as the potential roles of SQSTM1/p62 protein in Nrf2/Keap1 signaling and autophagy will be summarized. The therapeutic potential ...
Cellular and molecular life sciences : CMLS, Jan 29, 2015
Recent studies have revealed that the members of an ancient family of nonheme Fe(2+)/2-oxoglutara... more Recent studies have revealed that the members of an ancient family of nonheme Fe(2+)/2-oxoglutarate-dependent dioxygenases (2-OGDO) are involved in the functions associated with the aging process. 2-Oxoglutarate and O2 are the obligatory substrates and Fe(2+) a cofactor in the activation of 2-OGDO enzymes, which can induce the hydroxylation of distinct proteins and the demethylation of DNA and histones. For instance, ten-eleven translocation 1-3 (TET1-3) are the demethylases of DNA, whereas Jumonji C domain-containing histone lysine demethylases (KDM2-7) are the major epigenetic regulators of chromatin landscape, known to be altered with aging. The functions of hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD1-3) as well as those of collagen hydroxylases are associated with age-related degeneration. Moreover, the ribosomal hydroxylase OGFOD1 controls mRNA translation, which is known to decline with aging. 2-OGDO enzymes are the sensors of energy metabolism, since the Krebs cy...
The plant Cannabis sativa containing psychoactive chemical compounds known as cannabinoids has be... more The plant Cannabis sativa containing psychoactive chemical compounds known as cannabinoids has been used for thousands of years in the treatment of different diseases and adverse conditions. Analgesic and anti-inflammatory properties of cannabinoids were deciphered experimentally a long time before the discovery of mammalian endocannabinoid system with cannabinoid-sensing receptors CB1 and CB2. Psychoactive effects of cannabinoids are commonly associated with the CB1 receptors, whereas the CB2 receptors primarily mediate anti-inflammatory and immunomodulatory effects. Cannabinoid receptors become upregulated in various pathological conditions and in many cases, their activation results in the alleviation of adverse effects. In this article, we will review the signaling of CB2 receptors with particular emphasis been placed on the associations with inflammation and autophagy. Increased inflammation and decreased autophagy are hallmarks of various age-related diseases which could poten...
Ageing Research Reviews, 2009
Age-related macular degeneration (AMD) is the major diagnosis for severe and irreversible central... more Age-related macular degeneration (AMD) is the major diagnosis for severe and irreversible central loss of vision in elderly people in the developed countries. The loss of vision involves primarily a progressive degeneration and cell death of postmitotic retinal pigment epithelial cells (RPE), which secondarily evokes adverse effects on photoreceptor cells. The RPE cells are exposed to chronic oxidative stress from
Acta Ophthalmologica, 2015
Disease-specific instruments have shown significant gains in measuring health-related quality of ... more Disease-specific instruments have shown significant gains in measuring health-related quality of life (HRQoL) in subjects having cataract surgery. However, the usage of generic instruments has resulted in conflicting evidence. In this prospective study, we have evaluated the impact of cataract surgery on subjects' HRQoL measured with a 15-dimension generic instrument, the 15D. The HRQoL of cataract subjects was compared with that of an age- and gender-standardized sample of the general population in Finland. A total of 152 subjects (mean age 74 years, 66% females) with a first-eye cataract surgery completed the 15D questionnaire both before and 12 months after cataract surgery. When compared with the general population, cataract subjects had much lower HRQoL at baseline (mean difference 0.037 (95% CI: 0.020, 0.054), p < 0.001). At 12 months after cataract surgery, the overall utility index improved from the mean of 0.837 to 0.855 (mean difference 0.018 (95% CI: 0.007, 0.029), p = 0.002). Significant improvement was observed on the following five dimensions: seeing, moving, hearing, usual activities and discomfort/symptoms in the 15D questionnaire. Our data indicate that at 12 months after first-eye cataract surgery, patients' HRQoL is slightly better than that before surgery. However, patients' postoperative HRQoL may remain lower than that of an age-and gender-standardized control population.
Progress in Neurobiology, 2015
The amyloid cascade hypothesis for the pathogenesis of Alzheimer&... more The amyloid cascade hypothesis for the pathogenesis of Alzheimer's disease (AD) was proposed over twenty years ago. However, the mechanisms of neurodegeneration and synaptic loss have remained elusive delaying the effective drug discovery. Recent studies have revealed that amyloid-β peptides as well as phosphorylated and fragmented tau proteins accumulate within mitochondria. This process triggers mitochondrial fission (fragmentation) and disturbs Krebs cycle function e.g. by inhibiting the activity of 2-oxoglutarate dehydrogenase. Oxidative stress, hypoxia and calcium imbalance also disrupt the function of Krebs cycle in AD brains. Recent studies on epigenetic regulation have revealed that Krebs cycle intermediates control DNA and histone methylation as well as histone acetylation and thus they have fundamental roles in gene expression. DNA demethylases (TET1-3) and histone lysine demethylases (KDM2-7) are included in the family of 2-oxoglutarate-dependent oxygenases (2-OGDO). Interestingly, 2-oxoglutarate is the obligatory substrate of 2-OGDO enzymes, whereas succinate and fumarate are the inhibitors of these enzymes. Moreover, citrate can stimulate histone acetylation via acetyl-CoA production. Epigenetic studies have revealed that AD is associated with changes in DNA methylation and histone acetylation patterns. However, the epigenetic results of different studies are inconsistent but one possibility is that they represent both coordinated adaptive responses and uncontrolled stochastic changes, which provoke pathogenesis in affected neurons. Here, we will review the changes observed in mitochondrial dynamics and Krebs cycle function associated with AD, and then clarify the mechanisms through which mitochondrial metabolites can control the epigenetic landscape of chromatin and induce pathological changes in AD.
Journal of biochemical and pharmacological research, 2014
Various preservative-free eye drop formulations for glaucoma treatment have been marketed intendi... more Various preservative-free eye drop formulations for glaucoma treatment have been marketed intending to decrease ocular surface side effects and improve tolerability. However, preservative-free eye drops including different solubilizers to dissolve the antiglaucoma drugs may induce detrimental effects in the eye. In this study, we exposed human corneal epithelial cells (HCE-2) for 1, 6, 12, 24 and 48 hours to the first preservative-free (PF) tafluprost (Taflotan(®)), the recently-launched preservative-free (PF) latanoprost (Monoprost(®)), preservative benzalkonium chloride (BAK) and the excipient macrogolglycerol hydroxystearate 40 (MGHS40) using dilutions 0.1%, 0.3%, 1.0%, 3.0% and 10.0% of the original products. The cells also were exposed to undiluted PF tafluprost and PF latanoprost once a day for 9 days. Cellular morphology was examined by light microscopy and cell proliferation by Ki-67 fluorescent staining with cell viability being determined by erythrosine staining and the re...
Biochimica et biophysica acta, 2014
Retinal pigment epithelium (RPE) plays a major role in the maintenance of photoreceptors, and deg... more Retinal pigment epithelium (RPE) plays a major role in the maintenance of photoreceptors, and degeneration of RPE results in the development of age-related macular degeneration (AMD). Accumulation of intracellular protein aggregates, increased oxidative stress, and chronic inflammation are all factors damaging the functionality of aged RPE cells. Here, we report that inhibition of proteasomal degradation with MG-132 and autophagy with bafilomycin A1 resulted in the release of IL-1β but not that of IL-18 in human ARPE-19 cells. NLRP3 receptor became upregulated, and caspase-1, the functional component of an inflammasome complex, was activated. In addition to accumulating intracellular protein aggregates, inhibition of degradation systems induced oxidative stress which was demonstrated by elevated amounts of intracellular 4-hydroxynonenal (HNE)-protein adducts. Along with IL-1β, exposure to MG-132 and bafilomycin A1 resulted in the secretion of IL-8. A low concentration (1pg/ml) of IL...
Molecular vision, 2014
In this work, we investigated the ability of pinosylvin (PS), 3,5-dihydroxy-trans-stilbene, to mo... more In this work, we investigated the ability of pinosylvin (PS), 3,5-dihydroxy-trans-stilbene, to modulate oxidative stress in human RPE cells. PS, a stilbenoid polyphenol, occurs in high concentrations in bark byproducts and therefore represents an attractive bioactive compound for health-promoting applications. First, we evaluated the toxicity range of PS by exposing ARPE-19 cells to 0.1-200 µM concentrations of PS for 24 h followed by the cell viability test. In the next stage, the ARPE-19 cells were preincubated in PS for 24 h followed by hydroquinone (HQ) exposure without PS for another 24 h. The cell viability test was conducted after HQ exposure. To elucidate the potential mechanisms behind PS-mediated protection against oxidative stress, the ARPE-19 cells were treated with 5 µM PS for 6 h, and mRNA was extracted at four time points (2 h, 6 h, 12 h, 24 h) to determine changes in the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), sequestosome 1 (p62/SQSTM1), he...
Ageing research reviews, 2014
Many aging theories have proposed that mitochondria and energy metabolism have a major role in th... more Many aging theories have proposed that mitochondria and energy metabolism have a major role in the aging process. There are recent studies indicating that Krebs cycle intermediates can shape the epigenetic landscape of chromatin by regulating DNA and histone methylation. A growing evidence indicates that epigenetics plays an important role in the regulation of healthspan but also is involved in the aging process. 2-Oxoglutarate (α-ketoglutarate) is a key metabolite in the Krebs cycle but it is also an obligatory substrate for 2-oxoglutarate-dependent dioxygenases (2-OGDO). The 2-OGDO enzyme family includes the major enzymes of DNA and histone demethylation, i.e. Ten-Eleven Translocation (TETs) and Jumonji C domain containing (JmjC) demethylases. In addition, 2-OGDO members can regulate collagen synthesis and hypoxic responses in a non-epigenetical manner. Interestingly, succinate and fumarate, also Krebs cycle intermediates, are potent inhibitors of 2-OGDO enzymes, i.e. the balance ...
Progress in neurobiology, 2011
Alzheimer's disease is a tauopathy which involves the deposition of microtubule-associated ta... more Alzheimer's disease is a tauopathy which involves the deposition of microtubule-associated tau proteins into neurofibrillary tangles. Post-translational modifications, in particular site-specific phosphorylations, affect the conformation of tau protein which is an intrinsically disordered protein. These structural changes significantly increase the affinity of tau protein for certain molecular chaperones. Hsp90 is a major cellular chaperone which assembles large complexes with a variety of co-chaperones. The main function of Hsp90 complexes is to maintain protein quality control and assist in protein degradation via proteasomal and autophagic-lysosomal pathways. Tau protein is a client protein for these Hsp90 complexes. If the tau protein is in an abnormal or modified form, then it can trigger the recruitment of CHIP protein, a co-chaperone with E3 activity, to the complex which induces the ubiquitination of tau protein and activates its downstream degradation processes. Large i...
Frontiers in bioscience (Elite edition), 2010
The pathogenesis of age-related macular degeneration (AMD) essentially involves chronic oxidative... more The pathogenesis of age-related macular degeneration (AMD) essentially involves chronic oxidative stress, increased accumulation of lipofuscin in retinal pigment epithelial (RPE) cells and extracellular drusen formation, as well as the presence of chronic inflammation. The capacity to prevent the accumulation of cellular cytotoxic protein aggregates is decreased in senescent cells which may evoke lipofuscin accumulation into lysosomes in postmitotic RPE cells. This presence of lipofuscin decreases lysosomal enzyme activity and impairs autophagic clearance of damaged proteins which should be removed from cells. Proteasomes are another crucial proteolytic machine which degrade especially cellular proteins damaged by oxidative stress. This review examines the cross-talk between lysosomes, autophagy and proteasomes in RPE cell protein aggregation, their role as a possible therapeutic target and their involvement in the pathogenesis of AMD.
Age-related macular degeneration (AMD) is the leading cause of severe and irreversible loss of vi... more Age-related macular degeneration (AMD) is the leading cause of severe and irreversible loss of vision in the elderly in developed countries. AMD is a complex chronic neurodegenerative disease associated with many environmental, lifestyle, and genetic factors. Oxidative stress and the production of reactive oxygen species (ROS) seem to play a pivotal role in AMD pathogenesis. It is known that the macula receives the highest blood flow of any tissue in the body when related to size, and anything that can reduce the rich blood supply can cause hypoxia, malfunction, or disease. Oxidative stress can affect both the lipid rich retinal outer segment structure and the light processing in the macula. The response to oxidative stress involves several cellular defense reactions, for example, increases in antioxidant production and proteolysis of damaged proteins. The imbalance between production of damaged cellular components and degradation leads to the accumulation of detrimental products, for example, intracellular lipofuscin and extracellular drusen. Autophagy is a central lysosomal clearance system that may play an important role in AMD development. There are many anatomical changes in retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris in response to chronic oxidative stress, hypoxia, and disturbed autophagy and these are estimated to be crucial components in the pathology of neovascular processes in AMD.
Ageing Research Reviews, 2014
Processing of misfolded proteins is important in order for the cell to maintain its normal functi... more Processing of misfolded proteins is important in order for the cell to maintain its normal functioning and homeostasis. Three systems control the quality of proteins: chaperone-mediated refolding, proteasomal degradation of ubiquitinated proteins, and finally, when the two others fail, aggrephagy, as selective form of autophagy, degrades ubiquitin-labelled aggregated cargos. In this route misfolded proteins gradually form larger aggregates, aggresomes and they eventually become double membrane-wrapped organelles called autophagosomes, which become degraded when they fuse to lysosomes, for reuse by the cell. The stages, the main molecules participating in the process, and the regulation of aggrephagy are discussed here, as is the role of protein aggregation in protein accumulation diseases. In particular, we emphasize that both Alzheimer's disease and age-related macular degeneration, two of the most common pathologies in the aged, are characterized by altered protein clearance and deposits. Based on the hypothesis that manipulations of autophagy may be potentially useful in these and other aggregation-related diseases, we will discuss some promising therapeutic strategies to counteract protein aggregates-induced cellular toxicity.
Redox Biology, 2014
Heat shock proteins (HSPs), originally identified as heat-inducible gene products, are a family o... more Heat shock proteins (HSPs), originally identified as heat-inducible gene products, are a family of highly conserved proteins that respond to a wide variety of stress including oxidative stress. Although both acute and chronic oxidative stress have been well demonstrated to induce HSP responses, little evidence is available whether increased HSP levels provide enhanced protection against oxidative stress under elevated yet sublethal temperatures. We studied relationships between oxidative stress and HSPs in a physiological model by using Garra rufa (doctor fish), a fish species naturally acclimatized to different thermal conditions. We compared fish naturally living in a hot spring with relatively high water temperature (34.4 7 0.6°C) to those living in normal river water temperature (25.4 7 4.7°C), and found that levels of all the studied HSPs (HSP70, HSP60, HSP90, HSC70 and GRP75) were higher in fish living in elevated water temperature compared with normal river water temperature. In contrast, indicators of oxidative stress, including protein carbonyls and lipid hydroperoxides, were decreased in fish living in the elevated temperature, indicating that HSP levels are inversely associated with oxidative stress. The present results provide evidence that physiologically increased HSP levels provide protection against oxidative stress and enhance cytoprotection.
Histology and histopathology, Jan 19, 2015
Macular corneal dystrophy is a rare autosomal recessive eye disease affecting primarily the corne... more Macular corneal dystrophy is a rare autosomal recessive eye disease affecting primarily the corneal stroma. Abnormal accumulation of proteoglycan aggregates has been observed intra- and extracellularly in the stromal layer. In addition to the stromal keratocytes and corneal lamellae, deposits are also present in the basal epithelial cells, endothelial cells and Descemet's membrane. Misfolding of proteins has a tendency to gather into aggregating deposits. We studied interaction of molecular chaperones and proteasomal clearance in macular dystrophy human samples and in human corneal HCE-2 epithelial cells. Seven cases of macular corneal dystrophy and four normal corneal buttons collected during corneal transplantation were examined for their expression patterns of heat shock protein 70, ubiquitin protein conjugates and SQSTM1/p62. In response to proteasome inhibition the same proteins were analyzed by western blotting. Slit-lamp examination, in vivo confocal cornea microscopy and...
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Papers by Kai Kaarniranta