Paola de Candia

Fe65 and the protein network centered around the cytosolic domain of the Alzheimer's β-amyloid precursor protein

A distinctive tract of all the forms of Alzheimers disease is the extracellular deposition of a 40–42/43 amino acid‐long peptide derived from the so‐called β‐amyloid precursor protein (APP). This is a membrane protein of unknown function, whose short cytosolic domain has been recently demonstrated to interact with several proteins. One of these proteins, named Fe65, has the characteristics of an adaptor protein; in fact, it possesses three protein‐protein interaction domains: a WW domain and two PID/PTB domains. The interaction with APP requires the most C‐terminal PID/PTB domain, whereas the WW domain is responsible for the interaction with various proteins, one of which was demonstrated to be the mammalian homolog of the Drosophila enabled protein (Mena), which in turn interacts with the cytoskeleton. The second PID/PTB domain of Fe65 binds to the CP2/LSF/LBP1 protein, which is an already known transcription factor. The other proteins interacting with the cytosolic domain of APP are the Go heterotrimeric protein, APP‐BP1 and X11. The latter interacts with APP through a PID/PTB domain and possesses two other protein‐protein interaction domains. The small size of the APP cytodomain and the overlapping of its regions involved in the binding of Fe65 and X11 suggest the existence of competitive mechanisms regulating the binding of the various ligands to this cytosolic domain. In this short review the possible functional roles of this complex protein network and its involvement in the generation of Alzheimers phenotype are discussed.

Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR

The high-throughput analysis of microRNAs (miRNAs) circulating within the blood of healthy and diseased individuals is an active area of biomarker research. Whereas quantitative real-time reverse transcription polymerase chain reaction (qPCR)-based methods are widely used, it is yet unresolved how the data should be normalized. Here, we show that a combination of different algorithms results in the identification of candidate reference miRNAs that can be exploited as normalizers, in both discovery and validation phases. Using the methodology considered here, we identify normalizers that are able to reduce nonbiological variation in the data and we present several case studies, to illustrate the relevance in the context of physiological or pathological scenarios. In conclusion, the discovery of stable reference miRNAs from high-throughput studies allows appropriate normalization of focused qPCR assays.

Copy number variation of CCL3-like genes affects rate of progression to simian-AIDS in Rhesus Macaques (Macaca mulatta).

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10−6) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se.

Role of microRNAs and long‐non‐coding RNAs in CD4+ T‐cell differentiation

CD4+ T lymphocytes orchestrate adaptive immune responses by differentiating into various subsets of effector T cells such as T‐helper 1 (Th1), Th2, Th17, and regulatory T cells. These subsets have been generally described by master transcription factors that dictate the expression of cytokines and receptors, which ultimately define lymphocyte effector functions. However, the view of T‐lymphocyte subsets as stable and terminally differentiated lineages has been challenged by increasing evidence of functional plasticity within CD4+ T‐cell subsets, which implies flexible programming of effector functions depending on time and space of T‐cell activation. An outstanding question with broad basic and traslational implications relates to the mechanisms, besides transcriptional regulation, which define the plasticity of effector functions. In this study, we discuss the emerging role of regulatory non‐coding RNAs in T‐cell differentiation and plasticity. Not only microRNAs have been proven to be important for CD4+ T‐cell differentiation, but it is also likely that the overall T‐cell functioning is the result of a multilayered network composed by coding RNAs as well as by short and long non‐coding RNAs. The integrated study of all the nodes of this network will provide a comprehensive view of the molecular mechanisms underlying T‐cell functions in health and disease.

A combination of genomic approaches reveals the role of FOXO1a in regulating an oxidative stress response pathway.

Background While many of the phenotypic differences between human and chimpanzee may result from changes in gene regulation, only a handful of functionally important regulatory differences are currently known. As a first step towards identifying transcriptional pathways that have been remodeled in the human lineage, we focused on a transcription factor, FOXO1a, which we had previously found to be up-regulated in the human liver compared to that of three other primate species. We concentrated on this gene because of its known role in the regulation of metabolism and in longevity. Methodology Using a combination of expression profiling following siRNA knockdown and chromatin immunoprecipitation in a human liver cell line, we identified eight novel direct transcriptional targets of FOXO1a. This set includes the gene for thioredoxin-interacting protein (TXNIP), the expression of which is directly repressed by FOXO1a. The thioredoxin-interacting protein is known to inhibit the reducing activity of thioredoxin (TRX), thereby hindering the cellular response to oxidative stress and affecting life span. Conclusions Our results provide an explanation for the repeated observations that differences in the regulation of FOXO transcription factors affect longevity. Moreover, we found that TXNIP is down-regulated in human compared to chimpanzee, consistent with the up-regulation of its direct repressor FOXO1a in humans, and with differences in longevity between the two species.

Intracellular Modulation, Extracellular Disposal and Serum Increase of MiR-150 Mark Lymphocyte Activation

Activated lymphocytes release nano-sized vesicles (exosomes) containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation in vitro, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. In vivo, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation.

Role of metabolism in neurodegenerative disorders

Along with the increase in life expectancy over the last century, the prevalence of age-related disorders, such as neurodegenerative diseases continues to rise. This is the case of Alzheimers, Parkinsons, Huntingtons diseases and Multiple sclerosis, which are chronic disorders characterized by neuronal loss in motor, sensory or cognitive systems. Accumulating evidence has suggested the presence of a strong correlation between metabolic changes and neurodegeneration. Indeed epidemiologic studies have shown strong associations between obesity, metabolic dysfunction, and neurodegeneration, while animal models have provided insights into the complex relationships between these conditions. In this context, hormones such as leptin, ghrelin, insulin and IGF-1 seem to play a key role in the regulation of neuronal damage, toxic insults and several other neurodegenerative processes. This review aims to presenting the most recent evidence supporting the crosstalk linking energy metabolism and neurodegeneration, and will focus on metabolic manipulation as a possible therapeutic tool in the prevention and treatment of neurodegenerative diseases.

Serum microRNAs as Biomarkers of Human Lymphocyte Activation in Health and Disease

Induction of the adaptive immune system is evaluated mostly by assessment of serum antibody titers and T lymphocyte responses in peripheral blood, although T and B cell activation occurs in lymphoid tissues. In recent years, the release of microRNAs (miRNAs) in the extra-cellular environment has been exploited to assess cell functions at distance via measurement of serum miRNAs. Activated lymphocytes release a large amount of nano-sized vesicles (exosomes), containing miRNA, however there are insufficient data to determine whether this phenomenon is reflected in modulation of serum miRNAs. Interestingly, miRNA signatures of CD4+ T cell-derived exosomes are substantially different from intracellular miRNA signatures of the same cells. We have recently identified serum circulating miR-150 as a sensor of general lymphocyte activation and we strongly believe that miRNAs differentially released by specific CD4+ effector T cell subsets (Th1, Th2, Th17, and Treg) may serve as serum biomarkers of their elicitation in lymphoid tissues but also in damaged tissues, potentially providing clinically relevant information about the nature of immune responses in health and disease.

Extracellular RNAs: A Secret Arm of Immune System Regulation

The immune system has evolved to protect multicellular organisms from the attack of a variety of pathogens. To exert this function efficiently, the system has developed the capacity to coordinate the function of different cell types and the ability to down-modulate the response when the foreign attack is over. For decades, immunologists believed that these two characteristics were primarily related to cytokine/chemokine-based communication and cell-to-cell direct contact. More recently, it has been shown that immune cells also communicate by transferring regulatory RNAs, microRNAs in particular, from one cell to the other. Several studies have suggested a functional role of extracellular regulatory RNAs in cell-to-cell communication in different cellular contexts. This minireview focuses on the potential role of extracellular RNA transfer in the regulation of adaptive immune response, also contextualizing it in a broader field of what is known of cell-free RNAs in communication among different organisms in the evolutionary scale.

Proteins Implicated In Alzheimer Disease

Alzheimer disease (AD) is a devastating neurodegenerative disorder which affects several million people in the world. It is characterized by progressive memory loss and cognitive deficits. In the U.S. alone, the direct and indirect costs of AD are exorbitant, at an estimated 90 billion dollars per year. The etiology and the molecular basis of AD are unknown. The apparent heterogeneity of the molecules implicated in the pathogenesis of AD is consistent with the hypothesis that the disease could be due to independent molecular defects. However, these apparently unrelated molecular lesions seem to exert similar effects because there are three pathological features common to all cases of AD: i) The presence of extracellular senile plaques in the brain, ii) The appearance of neuro-fibrillary tangles in neurons, and ultimately, iii) Massive neuronal loss. To date, the majority of AD research has focused on senile plaques, neurofibrillary tangles, and their principal components: the s-amyloid peptide (As), its precursor protein (s-APP), and the tau-protein1,2,3,4,5 The repertoire of proteins which play an etiological role in AD increased when linkage studies led to the identification of two genes encoding membrane proteins named pre-senilin 1 (PS1) and presenilin 2 (PS2). Mutations of PS1 and PS2 were demonstrated to be responsible for familial forms of AD, which account for at least 10% of all AD cases. In addition, clear genetic evidence indicates that a naturally occurring allele of the apolipo-protein E gene (s4) is associated with a high risk of AD.