NDM29, a RNA polymerase III-dependent non coding RNA, promotes amyloidogenic processing of APP and amyloid ß secretion.

Neuroblastoma Differentiation Marker 29 (NDM29) is a RNA polymerase (pol) III-transcribed non-coding (nc) RNA whose synthesis drives neuroblastoma (NB) cell differentiation to a nonmalignant neuron-like phenotype. Since in this process a complex pattern of molecular changes is associated to plasma membrane protein repertoire we hypothesized that the expression of NDM29 might influence also key players of neurodegenerative pathways. In this work we show that the NDM29-dependent cell maturation induces amyloid precursor protein (APP) synthesis, leading to the increase of amyloid ß peptide (Aß) secretion and the concomitant increment of Aß x-42/Aß x-40 ratio. We also demonstrate that the expression of NDM29 RNA, and the consequent increase of Aß formation, can be promoted by inflammatory stimuli (and repressed by anti-inflammatory drugs). Moreover, NDM29 expression was detected in normal human brains although an abnormal increased synthesis of this ncRNA is induced in patients affected by neurodegenerative diseases. Therefore, the complex of events triggered by NDM29 expression induces a condition that favors the formation of Aß peptides in the extracellular space, as it may occur in Alzheimer's Disease (AD). In addition, these data unexpectedly show that a pol III-dependent small RNA can act as key regulator of brain physiology and/or pathology suggesting that a better knowledge of this portion of the human transcriptome might provide hints for neurodegeneration studies.

The Murine PSE/TATA-dependent transcriptome: evidence of functional homologies with its human counterpart.

A series of recent studies demonstrated an unexpectedly high frequency of intronic RNA polymerase (pol) III transcription units spread throughout the human genome. The investigation of a subset of these transcripts revealed their tissue/cell-specific transcription together with the involvement in relevant physiopathological pathways. Despite this evidence, these transcripts did not seem to have murine orthologs, based on their nucleotide sequence, resulting in a limitation of the experimental approaches aimed to study their function. In this work, we have extended our investigation to the murine genome identifying 121 pairs of mouse/human transcripts displaying syntenic subchromosomal localization. The analysis in silico of this set of putative noncoding (nc)RNAs suggest their association with alternative splicing as suggested by recent experimental evidence. The investigation of one of these pairs taken as experimental model in mouse hippocampal neurons provided evidence of a human/mouse functional homology that does not depend on underlying sequence conservation. In this light, the collection of transcriptional units here reported can be considered as a novel source for the identification and the study of novel regulatory elements involved in relevant biological processes.

17A, a novel non-coding RNA, regulates GABA B alternative splicing and signaling in response to inflammatory stimuli and in Alzheimer disease.

Alternative splicing is a central component of human brain complexity; nonetheless, its regulatory mechanisms are still largely unclear. In this work, we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol) III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51, GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells induces the synthesis of an alternative splicing isoform that abolish GABA B2 intracellular signaling (i.e., inhibition of cAMP accumulation and activation of K(+) channels). Indeed, 17A is expressed in human brain, and we report that it is upregulated in cerebral tissues derived from Alzheimer disease patients. We demonstrate that 17A expression in neuroblastoma cells enhances the secretion of amyloid ß peptide (Aß) and the Aß x-42/Αß x-40 peptide ratio and that its synthesis is induced in response to inflammatory stimuli. These data correlate, for the first time, the activity of a novel pol III-dependent ncRNA to alternative splicing events and, possibly, to neurodegeneration induced by abnormal GABA B function. We anticipate that further analysis of pol III-dependent regulation of alternative splicing will disclose novel regulatory pathways associated to brain physiology and/or pathology.

An Alu-like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells.

Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III-transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.

Rolly protein (ROLP)-Epb4.1/3: a potential protein-protein interaction relevant for the maintenance of cell adhesion.

We recently described Rolly Protein (ROLP), a small protein synthesized by substrate-adherent cells in a broad range of tissues. In a first set of experiments performed taking advantage of bone forming tibial cartilage as an experimental model we showed that ROLP transcription is associated to cells in an active proliferation state, whereas its downregulation is observed when cell proliferation decreases. Taking advantage of siRNA technology we also documented the expression modulation of some apoptosis-related genes in ROLP-silenced cells. In this work we search for the possible molecular interactors of ROLP by using both the antibody array approach as well as the co-immunoprecipitation approach. Results suggest the occurrence of an interaction of ROLP with Erythrocyte membrane Protein Band 4.1/3 (Epb4.1/3), an oncosuppressor downregulated in tumor development and in metastatic tissues; in addition we report experimental results that keep in line also with a potential interaction of ROLP with other PDZ-containing proteins. We also present experimental evidences supporting a role played by ROLP in cell adhesion thus supporting the existence of a biologically relevant link between ROLP and Epb4.1/3. We here suggest that ROLP might exert its biological role cooperating with Epb4.1/3, a protein that is involved in biological pathways that are often inhibited in tumor metastasis. Given the role of Epb4.1/3 in contrasting cancerogenesis we think that its cooperation with ROLP might be relevant in cancer studies and deserves further investigation.

Role of peroxisome proliferator-activated receptor gamma in amyloid precursor protein processing and amyloid beta-mediated cell death.

Recent data indicate that PPARgamma (peroxisome proliferator-activated receptor gamma) could be involved in the modulation of the amyloid cascade causing Alzheimer's disease. In the present study we show that PPARgamma overexpression in cultured cells dramatically reduced Abeta (amyloid-beta) secretion, affecting the expression of the APP (Abeta precursor protein) at a post-transcriptional level. APP down-regulation did not involve the pathway of the secretases and correlated with a significant induction of APP ubiquitination. Additionally, we demonstrate that PPARgamma was able to protect the cells from H(2)O(2)-induced necrosis by decreasing Abeta secretion. Taken together, our results indicate a novel mechanism at the basis of the neuroprotection shown by PPARgamma agonists and an additional pathogenic role for Abeta accumulation.

An intronic ncRNA-dependent regulation of SORL1 expression affecting Aß formation is upregulated in post-mortem Alzheimer's disease brain samples.

Recent studies indicated that sortilin-related receptor 1 (SORL1) is a risk gene for late-onset Alzheimer's disease (AD), although its role in the aetiology and/or progression of this disorder is not fully understood. Here, we report the finding of a non-coding (nc) RNA (hereafter referred to as 51A) that maps in antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a splicing shift of SORL1 from the synthesis of the canonical long protein variant A to an alternatively spliced protein form. This process, resulting in a decreased synthesis of SORL1 variant A, is associated with impaired processing of amyloid precursor protein (APP), leading to increased Aß formation. Interestingly, we found that 51A is expressed in human brains, being frequently upregulated in cerebral cortices from individuals with Alzheimer's disease. Altogether, these findings document a novel ncRNA-dependent regulatory pathway that might have relevant implications in neurodegeneration.

RNA polymerase III drives alternative splicing of the potassium channel-interacting protein contributing to brain complexity and neurodegeneration.

Alternative splicing generates protein isoforms that are conditionally or differentially expressed in specific tissues. The discovery of factors that control alternative splicing might clarify the molecular basis of biological and pathological processes. We found that IL1-α-dependent up-regulation of 38A, a small ribonucleic acid (RNA) polymerase III-transcribed RNA, drives the synthesis of an alternatively spliced form of the potassium channel-interacting protein (KCNIP4). The alternative KCNIP4 isoform cannot interact with the γ-secretase complex, resulting in modification of γ-secretase activity, amyloid precursor protein processing, and increased secretion of ß-amyloid enriched in the more toxic Aß x-42 species. Notably, synthesis of the variant KCNIP4 isoform is also detrimental to brain physiology, as it results in the concomitant blockade of the fast kinetics of potassium channels. This alternative splicing shift is observed at high frequency in tissue samples from Alzheimer's disease patients, suggesting that RNA polymerase III cogenes may be upstream determinants of alternative splicing that significantly contribute to homeostasis and pathogenesis in the brain.

Modulation of proteasome activity by vitamin E in THP-1 monocytes.

In THP-1 monocytes, cellular proteasome inhibition by ritonavir or ALLN is associated with increased production of oxidative stress. Both compounds produced comparable amounts of oxidative stress; however, normalization by alpha-tocopherol occurred solely after inhibition by ritonavir, and not by ALLN. Similar to that, alpha-tocopherol could normalize the reduced formation of 3-nitrotyrosine-modified proteins only after ritonavir treatment. In the absence of any proteasome inhibitor, intrinsic cellular proteasome activity was not modulated by alpha-, beta-, and gamma-tocopherols; however, delta-tocopherol, alpha-tocotrienol, and alpha-tocopheryl phosphate could significantly inhibit cellular proteasome activity and increased the level of p27(Kip1) and p53. Since oxidative stress was reduced by alpha-tocopherol only after proteasome inhibition by ritonavir and not by ALLN, it is concluded that, in this experimental system, alpha-tocopherol does not act as an antioxidant but interferes with the inhibitory effect of ritonavir.

Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells.

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.

Evidence against the overexpression of APP in Down syndrome.

Down syndrome (DS) is the most common genetic disorder with mental retardation and is caused by trisomy 21. By the age of 40 years, virtually all adults with DS have sufficient neuropathology for a diagnosis of Alzheimer's disease (AD), which is characterized by accumulation of amyloid-beta in senile plaques and formation of neurofibrillary tangles. Amyloid-beta derives from a longer precursor protein, APP, whose gene maps to chromosome 21. In DS, the early appearance of senile plaques is commonly associated with the presence of a third copy of the APP gene. Here we show DS brains and trisomic fibroblasts in which APP is not overexpressed, compared to euploid controls, challenging the notion that the widespread amyloid-beta deposits, consistently found in DS individuals, result from an extra copy of APP.

Microarray analysis in Alzheimer's disease and normal aging.

The purpose of this study was to investigate gene expression in Alzheimer's disease (AD), the most common form of senile dementia. We utilized the microarray technology to simultaneously compare the expression profile of 12,000 human genes in cerebral cortex of AD and normal aging. To identify gene expression related to neurodegeneration, beside the presence of amyloid deposition, we used control brains with abundant amyloid plaques, derived from cognitively normal elderly subjects. The microarray analysis indicated that 314 genes were differentially expressed in AD cerebral cortex, with differences greater than 5 folds in 25 genes. RT-PCR performed on a selected group of genes confirmed the increased expression of the interferon-induced protein 3 in AD brain. This protein, which is highly inducible by both type I and type II interferons, was not previously associated with the neurodegenerative disease.