Marialaura Amadio

University of Pavia | UNIPV · Department of Drugs Sciences

Aims/Purpose: The transcription nuclear factor E2‐related factor 2 (Nrf2) pathway is a key antioxidant system in the RPE, protecting it from oxidative stress and inflammation. Its dysfunction is a renowned aspect of Age‐related Macular Degeneration (AMD). As previously documented, RNA‐binding protein HuR/ELAVL1 interacts with Nrf2 mRNA, as well as...

Aims/Purpose: To evaluate the impact of ELAVL1 overexpression on survival and function of retinal ganglion cells (RGC) in rat glaucoma model. Methods: The study included ex vivo and in vivo approaches. Ex vivo, we used rat retinal explants to investigate, whether AAV‐ELAVL1 delivery affects the survival of RGC in the degeneration model induced by a...

Dimethyl fumarate (DMF) is an FDA-approved drug for the treatment of psoriasis and multiple sclerosis. DMF is known to stabilize the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. It has also been shown that DMF influences autophagy and participates in the transcriptional control of inflammato...

Dimethyl fumarate (DMF) is a well-characterized molecule that exhibits immuno-modulatory, anti-inflammatory, and antioxidant properties and that is currently approved for the treatment of psoriasis and multiple sclerosis. Due to its Nrf2-dependent and independent mechanisms of action, DMF has a therapeutic potential much broader than expected. In t...

Introduction ELAVL1/HuR is a keystone regulator of gene expression at the posttranscriptional level, including stress response and homeostasis maintenance. The aim of this study was to evaluate the impact of hur silencing on the age-related degeneration of retinal ganglion cells (RGC), which potentially describes the efficiency of endogenous neurop...

References: Antioxidant systems are essential in many elderly diseases, including age-related macular degeneration (AMD). Oxidative stress, inflammation, protein degradation impairment, are well-described in AMD retinal pigment epithelial (RPE) cells. Nrf2 (NF-E2-related factor 2), master regulator of antioxidant defence, is linked to AMD, autophag...

References: Nrf2 (NF-E2-related factor 2) pathway is a major defensive system orchestrating cellular response to pro-oxidant and pro-inflammatory stress and regulating the expression of many cytoprotective genes. Pharmacologic activation of Nrf2 has emerged as an attractive strategy for many pathologic conditions. The best-known Nrf2 activator is d...

Purpose: To evaluate the impact of HuR gene silencing on the ratio of age-related degeneration of Retinal Ganglion Cells (RGC), which potentially describes the efficiency of endogenous neuroprotection mechanisms, as well as to assess the exogenous neuroprotection capacity of HuR- silenced RGC in rat glaucoma model treated with Metallothionein. Meth...

Dimethyl fumarate (DMF) is a small molecule currently approved and used in the treatment of psoriasis and multiple sclerosis due to its immuno-modulatory, anti-inflammatory, and antioxidant properties. As an Nrf2 activator through Keap1 protein inhibition, DMF unveils a potential therapeutical use that is much broader than expected so far. In this...

Dimethyl fumarate (DMF) is a well-known activator of Nrf2 (NF-E2-related factor 2), used in the treatment of psoriasis and multiple sclerosis. The mechanism of action consists in the modification of the cysteine residues on the Nrf2-inhibitor Keap1, thus leading to the dissociation of these two proteins and the consequent activation of Nrf2. Consid...

Antioxidant systems play key roles in many elderly diseases, including age-related macular degeneration (AMD). Oxidative stress, autophagy impairment and inflammation are well-described in AMD, especially in retinal pigment epithelial (RPE) cells. The master regulator of antioxidant defense Nrf2 has been linked to AMD, autophagy and inflammation. I...

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. In our previous work, we identified in racemate 1–2, based on the 2-benzyl-3-hydroxypropyl ester scaffold, two new potent and promising PKCα and PKCδ ligands, targeting the C1 doma...

Hu proteins are members of the RNA-binding protein (RBP) family and play a pivotal role in the regulation of post-transcriptional processes. Through interaction with selected mRNAs, RBPs regulate their function and stability; as a consequence, RBP dysregulation can cause abnormal translation of key proteins involved in several pathologies. In the p...

Oxidative stress (OS) plays a key role in retinal dysfunctions and acts as a major trigger of inflammatory and neurodegenerative processes in several retinal diseases. To prevent OS-induced retinal damage, approaches based on the use of natural compounds are actively investigated. Recently, structural features from curcumin and diallyl sulfide have...

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monit...

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monit...

the PDF can be download freely on pubmed. https://pubmed.ncbi.nlm.nih.gov/33634751/

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monit...

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monit...

The transcription factor Nrf2 coordinates a multifaceted response to various forms of stress and to inflammatory processes, maintaining a homeostatic intracellular environment. Nrf2 anti-inflammatory activity has been related to the crosstalk with the transcription factor NF-κB, a pivotal mediator of inflammatory responses and of multiple aspects o...

In the last decades, the post-transcriptional control of gene expression has become an area of intense investigation, delineating a complex scenario where several factors (e.g. RNA-binding proteins, coding and non-coding RNAs) orchestrate the fate of a given transcript. An intriguing hypothesis suggests that loss of RNA homeostasis is a central fea...

Age-related macular degeneration (AMD) is a common disease with a multifactorial aetiology, still lacking effective and curative therapies. Among the early events triggering AMD is the deterioration of the retinal pigment epithelium (RPE), whose fundamental functions assure good health of the retina. RPE is physiologically exposed to high levels of...

Silk sericin (SS) is, together with silk fibroin (SF), one of the two proteins forming the silkworm cocoon. SS is ideal ingredient for cosmetic applications in the formulation of specific products for skin care and hair due to its peculiar physical-chemical composition. SS also showed a great potential in different pharmacological and biotechnologi...

Purpose Oxidative stress, inflammation, protein aggregation, are major features of Age‐related Macular Degeneration (AMD). A primary system used by the RPE to neutralize oxidative stress and maintain cellular homeostasis is the transcription nuclear factor E2‐related factor 2 (Nrf2) pathway, whose impairment in AMD is documented. Many Nrf2 targets...

Retinal neurons are not able to undergo spontaneous regeneration in response to damage. A variety of stressors, i.e., UV radiation, high temperature, ischemia, allergens, and others, induce reactive oxygen species production, resulting in consecutive alteration of stress-response gene expression and finally can lead to cell apoptosis. Neurons have...

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress...

RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal...

Effects on beclin-1 expression and cell viability following AICAR + MG132 exposure. (A) Representative Western blotting (upper) and densitometric analysis (lower) of beclin-1 protein levels in the cytoplasm of ARPE-19 cells exposed to either solvent (CTR) or AICAR and MG132 (A + M) for 2 hrs. Optical densities of beclin-1 bands were normalized to α...

AICAR + MG132 induces de novo translation of HuR mRNA. ARPE-19 cells were treated with either solvent (DMSO) or AICAR + MG132 for 2 hrs followed by polysome separation and Real-time qPCR analysis. HuR transcript level quantification was performed in free RNA, monosomes, and polysome of ARPE-19 cells. Relative expression of HuR was normalized to mRN...

Effects of AMPK inhibitor on HuR translocation. (A) Immunocytochemistry of ARPE-19 cells exposed to either solvent (CTR) or AICAR + MG132 alone or together, in the presence or not of AMPK inhibitor (5 μM CC) for 2 hrs. The left panels of immunocytochemistry show HuR staining (red), the right panels show nuclei staining with DAPI (blue), and the mid...

Effects on JNK activation following AICAR + MG132 exposure. Levels of phosphorylated JNK in the cytoplasm of ARPE-19 cells exposed for 2 hrs to either solvent (CTR) or AICAR (A + M) measured by ELISA (n = 4; ∗∗∗ p < 0.0001; Student's t-test).

Effects of cPKC inhibitor on Erk activation. Representative Western blotting (upper) and densitometric analyses (lower) of phospho-Erk1/2 (p-Erk) protein levels in the cytoplasm of ARPE-19 cells exposed to either solvent or AICAR (A + M), in the presence or not of cPKC inhibitor (2 μM Gö6976) for 2 hrs. Optical densities of p-Erk bands were normali...

Evaluation of HuR protein and mRNA levels following AICAR + MG132 exposure. (A) Representative Western blotting (upper) and densitometric analyses (lower) of HuR protein levels in the total homogenates of ARPE-19 cells exposed to either solvent (CTR) or AICAR + MG132 (A + M) for 2 hrs in the presence or not of puromycin (1 μM, PURO). Optical densit...

Effects on Erk1/2 activation following AICAR + MG132 exposure in the presence of Erk1/2 inhibitor. (A) Representative Western blotting (upper) and densitometric analyses (lower) of phospho-Erk1/2 (p-Erk) protein levels in the cytoplasm of ARPE-19 cells exposed to either solvent or AICAR + MG132 (A + M), in the presence or not of Erk1/2 inhibitor (5...

The RNA-binding protein, HuR, modulates mRNA processing and gene expression of several stress response proteins i.e. Hsp70 and p53 that have been postulated to be involved in the pathogenesis of glaucoma, a chronic optic neuropathy leading to irreversible blindness. We evaluated HuR protein expression in retinas and optic nerves of glaucomatous rat...

Purpose Age‐related Macular Degeneration (AMD) is a severe neurodegenerative disease and a major cause of blindness in the elderly worldwide. Oxidative stress is a common feature of AMD pathogenesis, and a positive modulation of Nrf2 activity has been demonstrated to be protective. Natural products are generally a rich source of therapeutics; we re...

RNA-protein interactions are pivotal in the regulation of biological systems and they are implicated in gene expression defect. In fact, RNAs in cells are in complex with RNA-binding proteins (RBPs) which influence every aspect of their biogenesis and function. Recent works have highlighted the involvement of these complexes in various pathological...

RNA-binding proteins play a key role in post-transcriptional processes; among these proteins, Embryonic Lethal Abnormal Vision (ELAV) proteins are among the best described. ELAV proteins predominantly act as positive regulators of gene expression, and their dysregulation is involved in several pathologies, such as cancer, inflammation and neurodege...

In industrialised countries, the elderly population is predicted to increase from % to % by. As a consequence, the functional decline associated with the ageing people creates a challenge to be met by the national healthcare systems. Among the age-related ocular pathologies, the WHO has identiied some irreversible diseases, like age-related macular...

Purpose Cataract is a common age-related ocular disease having as major determinants oxidative stress, accumulation of protein aggregates and inflammation. It has been previously reported that the expression of the RNA-binding HuR/ELAVL1 (Embryonic Lethal Abnormal Vision-Like 1) protein, a master regulator of many cellular functions including cell...

Purpose To evaluate impact of raised intraocular pressure on RNA binding protein ELAVL1/HuR expression in retina. Methods In order to induce glaucomatous damage, intraocular pressure was increased unilaterally using modified rat bead model and maintained for up to 8 weeks. Fellow eye was used as a healthy control. Animals were sacrificed, retina a...

Alzheimer's disease (AD), the most common cause of dementia, is an irreversible and progressive neurodegenerative disorder. It affects predominantly brain areas that are critical for memory and learning and is characterised by two main pathological hallmarks: extracellular amyloid plaques and intracellular neurofibrillary tangles. Protein kinase C...

Neuronal ELAV/Hu (nELAV) are RNA-binding proteins that mainly regulate gene expression by increasing the stability and/or translation rate of target mRNAs bearing ARE (adenine and uracil-rich elements) sequences. Among nELAV target transcripts there is ADAM10, an α-secretase involved in the non-amyloidogenic processing of the amyloid-β protein prec...

We evaluated whether specifically and directly targeting human antigen R (HuR), a member of embryonic lethal abnormal vision (ELAV) proteins family, may represent a new potential therapeutic strategy to manage diabetic retinopathy. Nanosystems loaded with siRNA silencing HuR expression (lipoplexes), consisting of solid lipid nanoparticles (SLN) and...

Purpose The redox milieu, together with reactive oxygen species (ROS) accumulation, may play a role in mediating some biological effects of extremely-low-frequency electromagnetic fields (ELF-EMF). Some of us have recently reported that a pulsed EMF (PEMF) improves the antioxidant response of a drug-sensitive human neuroblastoma SH-SY5Y cell line t...

Roughly ten years ago the FDA approved most of the presently used anti-VEGF drugs for the treatment of neovascular AMD and other eye pathologies characterized by ocular neoangiogenesis. However, the recent findings on the physiologic activities of VEGF isoforms impose to reconsider the inhibitory effects of pan-VEGF antagonists and the concept that...

Purpose The redox milieu, together with reactive oxygen species (ROS) accumulation, may play a role in mediating some biological effects of extremely-low-frequency electromagnetic fields (ELF-EMF). Some of us have recently reported that a pulsed EMF (PEMF) improves the antioxidant response of a drug-sensitive human neuroblastoma SH-SY5Y cell line t...

Roughly ten years ago the FDA approved most of the presently used anti-VEGF drugs for the treatment of neovascular AMD and other eye pathologies characterized by ocular neoangiogenesis. However, the recent findings on the physiologic activities of VEGF isoforms impose to reconsider the inhibitory effects of pan-VEGF antagonists and the concept that...

Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular...

Purpose Age‐related macular degeneration ( AMD ) pathogenesis is characterized by protein degradation impairment in retinal pigment epithelial ( RPE ) cells. We previously found that the expression of autophagy receptor SQSTM 1/p62 is positively regulated by the RNA ‐binding HuR/ ELAV protein. We investigated the effects of AICAR (autophagy inducer...

Purpose To clarify the complex role of impaired autophagy in RPE damage, we analysed mice deficient in both Nrf2 and PGC ‐1 α . Increasing evidence of impaired autophagy as a contributor to AMD has raised the importance of animal models that fundamentally mimic autophagy decline in RPE . Methods We analysed morphological and immunohistochemical ch...

Purpose Neovascular diabetic retinopathy ( DR ) and age‐related macular degeneration ( AMD ) are characterized by increased VEGF signaling. VEGF can be targeted using monoclonal antibody‐based drugs (e.g. ranibizumab, aflibercept) or by modified oligonucleotides (e.g. pegaptanib). The main difference between the two classes is that antibodies recog...

Purpose The cornea is exposed to constant oxidative stress that may lead to protein homeostasis imbalance and tissue damage during aging process. The ubiquitin‐proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems to clean damaged proteins in cells. Our aim was to investigate the role of the Nrf2 and PGC...

Vascular endothelial growth factor (VEGF) A is a master regulator of neovascularization and angiogenesis. VEGFA is potently induced by hypoxia and by pathological conditions including diabetic retinopathy and tumorigenesis. Fine-tuning of VEGFA expression by different stimuli is important for maintaining tissue vascularization and organ homeostasis...

Electromagnetic fields (EMFs) have been linked to increased risk of cancers and neurodegenerative diseases; however, EMFs can also elicit positive effects on biological systems, and redox status seems crucially involved in EMF biological effects. This study aimed to assess whether a short and repeated pulsed EMF (PEMF) could trigger adaptive respon...

Over the past fifteen years, we reported the design and synthesis of different series of compounds targeting the C1 domain of protein kinase C (PKC) that were based on various templates. Out of the pivalate template, 2-[4-(benzyloxy)phenyl]-3-hydroxypropyl pivalate (compound 1) emerged as the most potent and promising PKCα ligand, showing a Ki valu...

Heme oxygenase-1 (HO-1) is an inducible rate-controlling enzyme of heme catabolism. The cytoprotective function of HO-1 activity has been verified in multiple studies, and together with its by-products is considered a key component of the cellular stress response. The transcriptional induction of HO-1 has been largely studied in response to multipl...

In neurogenerative diseases, comprising Alzheimer's (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology....

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-...

Purpose Age‐related macular degeneration (AMD) pathogenesis involves impaired protein degradation in retinal pigment epithelial (RPE) cells. Our recent observation reveals that the expression of autophagy receptor SQSTM1/p62 is positively regulated by HuR protein (mRNA‐stabilizing human Embryonic Lethal Abnormal Vision protein) (Viiri et al. 8(7):e...

Purpose Impaired protein degradation in retinal pigment epithelial (RPE) cells contributes to age‐related macular degeneration (AMD) pathogenesis. SQSTM1/p62 protein is involved in autophagy and proteasome‐mediated proteolysis, and expressed strongly under toxic stimuli. ELAVL1/HuR is a RNA‐binding protein which, in response to various stressors, r...

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 p...

Purpose To explore whether, following direct contact, there is a mutual influence between pericytes (PC) and endothelial cells (EC), and to establish whether PKC activation, a condition associated with hyperglycaemia in diabetic retinopathy (DR), can affect, via the mRNA‐binding ELAVL1/HuR protein, VEGF expression. Methods Retinal PC and EC were cu...

Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and ex...

MG-132 induces ELAVL1/HUR and SQSTM1/p62 accumulation. A densitometric analysis of SQSTM1/p62 (A) and ELAVL1/HuR (B) proteins in the nuclear/perinuclear compartment (left panels) and in the cytoplasm (right panels) of ARPE-19 cells after exposure to MG-132 (5 µM) for 24 h. Control cells were exposed to solvent (DMSO). α-tubulin was used as a loadin...

The 24 h MG-132-mediated upregulation of SQSTM1/p62 mRNA expression is counteracted by the ELAVL1/HuR silencing. Determination of ELAVL1/HuR (A) and SQSTM1/p62 (B) mRNA levels by real-time qPCR in the total homogenates of control (CTR), MG-132-treated cells (MG-132), and MG-132-treated ELAVL1/HuR silenced ARPE-19 cells (ELAVL1/HuR-siRNA+MG-132). EL...

AICAR is well-tolerated and prevents cell death in MG-132 long-exposed ARPE-19 cells. (A): Maximum release of lactate dehydrogenase (LDH) enzyme in ARPE-19 cells treated with MG-132 5 µM and/or AICAR 2 mM for 24 h. Celastrol (10 µM) treatment is reported as a positive control since it induces maximum LDH release. Results are presented as mean optic...

Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel g...

Embryonic Lethal Abnormal Vision (ELAV) proteins are RNA-binding proteins that bind specific Adenine and uridine-Rich Elements mainly located in the 3'-untranslated region of target mRNAs, preventing their otherwise rapid degradation and thus increasing gene expression. Starting from our previous discovery and applying an integrated in vitro/in sil...

Embryonic lethal abnormal vision (ELAV) proteins are RNA‐binding proteins that bind specific adenine and uridine‐rich elements mainly located in the 3′‐untranslated region of target mRNAs, preventing their otherwise rapid degradation and thus increasing gene expression. Starting from our previous discovery and applying an integrated in vitro/in sil...

To explore whether, following direct contact, there is mutual influence between pericytes (PC) and endothelial cells (EC), and to establish whether protein kinase C (PKC) activation, a condition associated with hyperglycemia, can affect, via the mRNA-binding Hu-antigen R (HuR)/ELAV protein, the expression of vascular endothelial growth factor (VEGF...