Marialaura Amadio

Defining a neuron: neuronal ELAV proteins

Abstract.Neuronal cells strongly depend on the control exerted by RNA-binding proteins (RBPs) on gene expression for the establishment and maintenance of their phenotype. Neuronal ELAV (nELAV) proteins are RBPs able to influence virtually every aspect of the postsynthesis fate of bound mRNAs, from polyadenylation, alternative splicing and nuclear export to cytoplasmic localization, stability and translation. They enhance gene expression through the last two, best documented activities, increasing mRNA half-life and promoting protein synthesis by a still-unknown molecular mechanism. Developmentally, nELAV proteins have been shown to act as inducers of the transition between neural stem/progenitor cells and differentiation-committed cells, also assisting these neuroblasts in the completion of their maturation program. In brain physiology, they are also the first RBPs demonstrated to have a pivotal role in memory, where they probably control mRNA availability for translation in subcellular domains, thereby providing a biochemical means for selective increase in synaptic strength.

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

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 extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates SQSTM1/p62 mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.

The PKCβ/HuR/VEGF pathway in diabetic retinopathy

We investigated whether the diabetes-related PKCbeta activation affects VEGF expression through the mRNA-stabilizing human embryonic lethal abnormal vision (ELAV) protein, HuR, in the retina of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in rats by STZ injection. Retinal tissues were processed to detect PKCbetaI, PKCbetaII, VEGF and HuR contents, as well as HuR phosphorylation. Immunoprecipitation coupled to RT-PCR was employed to evaluate HuR binding to VEGF mRNA in RiboNucleoProteic (RNP) complexes. Statistical analysis was performed by ANOVA followed by an appropriate post hoc comparison test. Following experimental diabetes PKCbetaI and PKCbetaII levels were increased compared to sham; there was also a PKC-mediated phosphorylation/activation of HuR. These effects were blunted by the in vivo co-administration of a selective PKCbeta inhibitor. A specific binding between the HuR protein and the VEGF mRNA was also detected. The PKCbeta/HuR activation was accompanied by enhanced VEGF protein expression that was, again, blunted by the PKCbeta inhibitor. These findings first demonstrate the activation, in the retina, of the PKCbeta/HuR/VEGF pathway following experimental diabetes and disclose a new potential pharmacological target to counteract pathologies implicating VEGF deregulation, such as diabetic retinopathy.

Clearance of misfolded and aggregated proteins by aggrephagy and implications for aggregation diseases.

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 Alzheimers 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.

Post-transcriptional regulation of HSP70 expression following oxidative stress in SH-SY5Y cells: the potential involvement of the RNA-binding protein HuR.

Brain aging is associated with a progressive imbalance between intracellular concentration of Reactive Oxygen Species (ROS) and cells ability to activate defensive genes. Heat Shock Protein 70 (HSP70) has been shown to act as a fundamental defensive mechanism for neurons exposed to an oxidant challenge, and its expression decreases during senescence. In the present report we show that the RNA-binding protein ELAV/HuR can affect, post-transcriptionally, the fate of HSP70 mRNA following H(2)O(2)-mediated oxidative stress in SH-SY5Y human neuroblastoma cells. As a consequence of H(2)O(2) treatment (1mM for 30 minutes), HSP70 mRNA accumulates in the ribosomes associated to the cytoskeleton, where parallel Western blotting experiments reveal statistically significant increase for both HuR and HSP70 protein levels. We also confirm the capability of HuR to bind to HSP70 mRNA, and describe how the biological effect of this ELAV protein on the HSP70 mRNA could be due to a direct phosphorylation in serine/threonine residues of HuR itself by the early (10 minutes) H(2)O(2)-mediated activation of PKC alpha. Our findings shed light on the post-transcriptional regulation of HSP70 expression, suggesting the existence of a new molecular cascade -involving PKC/HuR/HSP70- that possibly represents an early event in the cellular response to H(2)O(2)-mediated oxidative stress in SH-SY5Y human neuroblastoma cells. The present results lead us to speculate that an impairment in this regulatory mechanism might directly contribute to the defective cellular response to oxidative stress, thus helping to dissect a potential tool useful to counteract some aspects associated to cerebral senescence.

Targeting VEGF in eye neovascularization: What's new?: A comprehensive review on current therapies and oligonucleotide-based interventions under development

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 to face pathological alterations at ocular level is possible only through the full block of all VEGF isoforms. In fact, although pan-VEGF agents rapidly and effectively contrast ocular neovascularization, vascular leakage, and other pathological changes, in the long-term the inhibition of all VEGF isoforms likely may result in the loss of the physiologic effects exerted by VEGF121 and the anti-angiogenic VEGF165b. Notably, selective inhibitors of VEGF165a, such as pegaptanib, spare these targets. Moreover, preclinical and clinical evidence suggests that also systemic side effects, secondary to intraocular treatment with non-selective anti-VEGF drugs, may be reinterpreted in light of these recent findings, which may be useful to clinicians for the choice of the most appropriate anti-VEGF agent. Another aspect that should be considered is the involvement of VEGF-independent pathways in ocular neovascularization, therefore a combined therapy can represent a more effective pharmacological approach that might help also to counteract tachyphylaxis, an important issue in anti-VEGF treatment. This complex picture and the recent findings on current anti-VEGF drugs should be therefore taken into account to guide the development of novel agents targeting VEGF and/or other key factors involved in the pathogenesis of neovascular ocular diseases along the signaling pathways stimulated by the various isoforms. Accordingly, this review also reports on novel pharmacological molecules targeting VEGF at ocular level and currently under development, with a special attention to oligonucleotide-based interventions.

Senescence of the Brain: Focus on Cognitive Kinases

Ageing is characterized by alterations in brain anatomy and physiology, finally contributing to an impairment in cognitive functions, such as memory. The most relevant observations indicate that senescent-related cognitive decline is not only due to neuronal loss, instead, functional changes occurring over time play a key role. Overall, these modifications are indeed responsible for an altered interneuronal communication that can represent, rather than morphological modifications, the primum movens leading to cognitive decline. Among the age-induced changes underlying alterations in neuronal communication and synaptic plasticity, those related to neurotransmitter/neurotrophin systems and downstream signalling pathways are of great relevance. In particular, considering that protein kinases play a strategic role aimed to convert the extracellular signals into biological responses, functional alterations on kinases may directly contribute to age-dependent neuronal dysfunctions. Within this context, numerous studies point out on several kinases as positive regulators for memory function and suggest that various memory disturbances are the result of a deficit in kinase signalling pathways. Many kinases associated with synaptic function are indeed age-sensitive; in fact, various studies in senescent animals indicate that a reduction in kinases expression/function in some brain areas correlates with ageing and memory decline. In line with these concepts, pharmacological modulation of kinases may lead to neuroprotective effects that can prevent or counteract age-related memory impairment. This review will mainly focus on the age-induced changes on Protein Kinase C (PKC), Protein Kinase A (PKA), Calcium/calmodulin-dependent Protein Kinase (CaMK), Tyrosine Kinase, widely accepted as key actors in signalling pathways associated with memory.

nELAV Proteins Alteration in Alzheimer's Disease Brain: A Novel Putative Target for Amyloid-β Reverberating on AβPP Processing

Neuronal ELAV (nELAV) proteins are RNA-binding proteins which play a physiological role in controlling gene expression in memory formation, and their alteration may contribute to cognitive impairment associated with neurodegenerative pathologies such as Alzheimers disease (AD). Indeed, we found that the content of nELAV proteins is significantly decreased along with clinical dementia progression in the hippocampi of AD brains, where it inversely correlates with the amount of amyloid-beta (Abeta). To check the direct influence of Abeta on nELAV, we performed in vitro experiments using human SH-SY5Y cells, finding that Abeta(1-42) specifically determines nELAV proteins reduction. Since ADAM10 mRNA has the predicted sequences targeted by nELAV, we investigated whether Abeta, through nELAV proteins, could originate a vicious circle affecting amyloid-beta protein precursor (AbetaPP) processing. Immunoprecipitation experiments showed that indeed nELAV proteins bind to ADAM10 mRNA and that this binding is disrupted by Abeta(1-42) exposure, resulting in a decreased ADAM10 protein expression. ADAM10 protein diminution was also found in AD hippocampi. These data show for the first time the involvement of nELAV in AD pathology and suggest that their alteration may affect genes implicated in AbetaPP processing.

NGF and heart: Is there a role in heart disease?

The review emphasizes the role of NGF, the most representative member of the neurotrophins family, in cardiac physiopathology with a particular focus on healing and sprouting processes occurring after tissue damage. Cardiac and circulating NGF levels dramatically increase following myocardial injury (MI). A very early rise of this neurotrophin is indeed observed soon after MI (hours). Such a rise may lead to sympathetic nerve sprouting which may underlie the later genesis of arrhythmias but may also favor the healing process. At later times (months after), when heart failure develops, the opposite is detected and NGF tissue levels are below the normal range, an event that may in turn participate to defective innervation and cardiac failure. Through a careful analysis of preclinical and clinical studies, this review proposes that time is the key variable when studying these opposite changes in NGF expression observed following MI and attempting to interpret and correlate them with cardiac physiopathology. The examination of the results leads to the speculation that NGF modulation may be a pharmacological target for interventions in specific stages of heart dysfunction following MI.

PKCβII/HuR/VEGF: A new molecular cascade in retinal pericytes for the regulation of VEGF gene expression

Vascular endothelial growth factor (VEGF)-induced new vessels formation is a key event in diabetic retinopathy, a severe progressive multistage pathology. Literature data indicate that protein kinase C (PKC) is involved in the control of VEGF expression, but, so far, no data are available on the molecular pathway underlying this process. Within this context, we suggest the existence of a new molecular cascade, operating in retinal bovine pericytes and involving PKCbetaII, the mRNA-stabilizing protein HuR, and VEGF. In particular we show that PKCbetaII activation is responsible, through the RNA-binding protein HuR, for the increase of VEGF protein content and its release in the medium. The specificity of the PKCbetaII involvement is confirmed by experiments performed with the LY379196 compound, a selective PKCbetaII inhibitor. Following acute high-glucose insult this pathway seems still functioning, suggesting that a brief exposure to glucose does not compromise this molecular cascade in pericytes. A better understanding on this new pathway could open novel opportunities for the development of innovative pharmacological therapies useful in pathologies where VEGF plays a key role such as in diabetic retinopathy.