Use of the combination of spirometry, arterial blood gas analysis and overnight oximetry to predict the outcomes of patients affected by motor neuron disease: The Milan-Torin respiratory score (Mi-To-RS).

BACKGROUND AND PURPOSE: The use of multiple tests, including spirometry, arterial blood gas (ABG) analysis and overnight oximetry (OvOx), is highly recommended to monitor the respiratory function of patients with motor neuron disease (MND). In this study, we propose a composite score to simplify the respiratory management of MND patients and better stratify their prognosis. MATERIALS AND METHODS: We screened the clinical charts of 471 non-ventilated MND patients referred to the Neuro-rehabilitation Unit of the San Raffaele Scientific Institute of Milan (January 2001-December 2019), collecting spirometric, ABG and OvOx parameters. To evaluate the prognostic role of each measurement, univariate Cox regression for death/tracheostomy was performed, and the variables associated with survival were selected to design a scoring system. Univariate and multivariate Cox regression analyses were then carried out to evaluate the prognostic role of the score. Finally, results were replicated in an independent cohort from the Turin ALS Center. RESULTS: The study population included 450 patients. Six measurements were found to be significantly associated with survival and were selected to design a scoring system (maximum score = 8 points). Kaplan-Meier analysis showed significant stratification of survival and time to non-invasive mechanical ventilation adaptation according to score values, and multivariate analysis confirmed the independent effect of the respiratory score on survival of each cohort. CONCLUSION: Forced vital capacity, ABG and OvOx parameters provide complementary information for the respiratory management and prognosis of MND patients and the combination of these parameters into a single score might help neurologists predict prognosis and guide decisions on the timing of the implementation of different diagnostic or therapeutic approaches.

Unveiling the SOD1-mediated ALS phenotype: insights from a comprehensive meta-analysis.

BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis associated with mutations in SOD1 (SOD1-ALS) might be susceptible to specific treatment. The aim of the study is to outline the clinical features of SOD1-ALS patients by comparing them to patients without ALS major gene variants and patients with variants in other major ALS genes. Defining SOD1-ALS phenotype may assist clinicians in identifying patients who should be prioritized for genetic testing. METHODS: We performed an extensive literature research including original studies which reported the clinical features of SOD1-ALS and at least one of the following patient groups: C9ORF72 hexanucleotide repeat expansion (C9-ALS), TARDBP (TARDBP-ALS), FUS (FUS-ALS) or patients without a positive test for a major-ALS gene (N-ALS). A random effects meta-analytic model was applied to clinical data extracted encompassing sex, site and age of onset. To reconstruct individual patient survival data, the published Kaplan-Meier curves were digitized. Data were measured as odds ratio (OR) or standardized mean difference (SMD) as appropriate. Median survival was compared between groups. RESULTS: Twenty studies met the inclusion criteria. We identified 721 SOD1-ALS, 470 C9-ALS, 183 TARDBP-ALS, 113 FUS-ALS and 2824 N-ALS. SOD1-ALS showed a higher rate of spinal onset compared with N-ALS and C9-ALS (OR = 4.85, 95% CI = 3.04-7.76; OR = 10.47, 95% CI = 4.32-27.87) and an earlier onset compared with N-ALS (SMD = - 0.45, 95% CI = - 0.72 to - 0.18). SOD1-ALS had a similar survival compared with N-ALS (p = 0.14), a longer survival compared with C9-ALS (p < 0.01) and FUS-ALS (p = 0.019) and a shorter survival compared with TARDBP-ALS (p < 0.01). DISCUSSION: This study indicates the presence of a specific SOD1-ALS phenotype. Insights in SOD1-ALS clinical features are important in genetic counseling, disease prognosis and support patients' stratification in clinical trials.

Impact of prostate imaging quality (PI-QUAL) score on the detection of clinically significant prostate cancer at biopsy.

PURPOSE: To investigate the impact of Prostate Imaging Quality (PI-QUAL) scores on the diagnostic performance of multiparametric MRI (mpMRI) in a targeted biopsy cohort. PATIENTS AND METHODS: 300 patients who underwent both mpMRI and biopsy were included. PI-QUAL scores were retrospectively assigned by two radiologists in consensus and were correlated to pre-biopsy PI-RADS scores and biopsy outcomes. Clinically significant prostate cancer (csPCa) was defined as ISUP grade ≥ 2. RESULTS: Image quality was optimal (PI-QUAL ≥ 4) in 249/300 (83%) and suboptimal (PI-QUAL < 4) in 51/300 (17%). The proportion of PI-RADS 3 scores referred for biopsy was higher in scans of suboptimal vs optimal quality (51% vs 33%). For PI-QUAL < 4 scans, the positive predictive value (PPV) was lower compared to PI-QUAL ≥ 4 (35% [95%CI: 22, 48] vs 48% [95%CI: 41, 55]; difference -13% [95%CI: -27, 2]; p 0.090), as was the detection rate of csPCa in both PI-RADS 3 and PI-RADS 4-5 (15% vs 23% and 56 vs 63%, respectively). The overall MRI quality increased over time. CONCLUSIONS: Scan quality may affect the diagnostic performance of prostate mpMRI in patients undergoing MRI-guided biopsy. Scans of suboptimal quality (PI-QUAL < 4) were associated with lower PPV for csPCa.

External validation of a convolutional neural network for the automatic segmentation of intraprostatic tumor lesions on 68Ga-PSMA PET images.

Introduction: State of the art artificial intelligence (AI) models have the potential to become a "one-stop shop" to improve diagnosis and prognosis in several oncological settings. The external validation of AI models on independent cohorts is essential to evaluate their generalization ability, hence their potential utility in clinical practice. In this study we tested on a large, separate cohort a recently proposed state-of-the-art convolutional neural network for the automatic segmentation of intraprostatic cancer lesions on PSMA PET images. Methods: Eighty-five biopsy proven prostate cancer patients who underwent 68Ga PSMA PET for staging purposes were enrolled in this study. Images were acquired with either fully hybrid PET/MRI (N = 46) or PET/CT (N = 39); all participants showed at least one intraprostatic pathological finding on PET images that was independently segmented by two Nuclear Medicine physicians. The trained model was available at https://gitlab.com/dejankostyszyn/prostate-gtv-segmentation and data processing has been done in agreement with the reference work. Results: When compared to the manual contouring, the AI model yielded a median dice score = 0.74, therefore showing a moderately good performance. Results were robust to the modality used to acquire images (PET/CT or PET/MRI) and to the ground truth labels (no significant difference between the model's performance when compared to reader 1 or reader 2 manual contouring). Discussion: In conclusion, this AI model could be used to automatically segment intraprostatic cancer lesions for research purposes, as instance to define the volume of interest for radiomics or deep learning analysis. However, more robust performance is needed for the generation of AI-based decision support technologies to be proposed in clinical practice.

Role of [68Ga]Ga-PSMA-11 PET radiomics to predict post-surgical ISUP grade in primary prostate cancer.

PURPOSE: The aim of this study is to investigate the role of [68Ga]Ga-PSMA-11 PET radiomics for the prediction of post-surgical International Society of Urological Pathology (PSISUP) grade in primary prostate cancer (PCa). METHODS: This retrospective study included 47 PCa patients who underwent [68Ga]Ga-PSMA-11 PET at IRCCS San Raffaele Scientific Institute before radical prostatectomy. The whole prostate was manually contoured on PET images and 103 image biomarker standardization initiative (IBSI)-compliant radiomic features (RFs) were extracted. Features were then selected using the minimum redundancy maximum relevance algorithm and a combination of the 4 most relevant RFs was used to train 12 radiomics machine learning models for the prediction of PSISUP grade: ISUP ≥ 4 vs ISUP < 4. Machine learning models were validated by means of fivefold repeated cross-validation, and two control models were generated to assess that our findings were not surrogates of spurious associations. Balanced accuracy (bACC) was collected for all generated models and compared with Kruskal-Wallis and Mann-Whitney tests. Sensitivity, specificity, and positive and negative predictive values were also reported to provide a complete overview of models' performance. The predictions of the best performing model were compared against ISUP grade at biopsy. RESULTS: ISUP grade at biopsy was upgraded in 9/47 patients after prostatectomy, resulting in a bACC = 85.9%, SN = 71.9%, SP = 100%, PPV = 100%, and NPV = 62.5%, while the best-performing radiomic model yielded a bACC = 87.6%, SN = 88.6%, SP = 86.7%, PPV = 94%, and NPV = 82.5%. All radiomic models trained with at least 2 RFs (GLSZM-Zone Entropy and Shape-Least Axis Length) outperformed the control models. Conversely, no significant differences were found for radiomic models trained with 2 or more RFs (Mann-Whitney p > 0.05). CONCLUSION: These findings support the role of [68Ga]Ga-PSMA-11 PET radiomics for the accurate and non-invasive prediction of PSISUP grade.

Hmga2 protein loss alters nuclear envelope and 3D chromatin structure.

BACKGROUND: The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. RESULTS: We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina. CONCLUSIONS: Overall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions.

YAP and TAZ Mediators at the Crossroad between Metabolic and Cellular Reprogramming.

Cell reprogramming can either refer to a direct conversion of a specialized cell into another or to a reversal of a somatic cell into an induced pluripotent stem cell (iPSC). It implies a peculiar modification of the epigenetic asset and gene regulatory networks needed for a new cell, to better fit the new phenotype of the incoming cell type. Cellular reprogramming also implies a metabolic rearrangement, similar to that observed upon tumorigenesis, with a transition from oxidative phosphorylation to aerobic glycolysis. The induction of a reprogramming process requires a nexus of signaling pathways, mixing a range of local and systemic information, and accumulating evidence points to the crucial role exerted by the Hippo pathway components Yes-Associated Protein (YAP) and Transcriptional Co-activator with PDZ-binding Motif (TAZ). In this review, we will first provide a synopsis of the Hippo pathway and its function during reprogramming and tissue regeneration, then we introduce the latest knowledge on the interplay between YAP/TAZ and metabolism and, finally, we discuss the possible role of YAP/TAZ in the orchestration of the metabolic switch upon cellular reprogramming.

Identification of RNA-binding proteins that partner with Lin28a to regulate Dnmt3a expression.

Lin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryonic development and tumorigenesis. It regulates gene expression through two different post-transcriptional mechanisms. The first one is based on the regulation of miRNA biogenesis, in particular that of the let-7 family, whose expression is suppressed by Lin28. Thus, loss of Lin28 leads to the upregulation of mRNAs that are targets of let-7 species. The second mechanism is based on the direct interaction of Lin28 with a large number of mRNAs, which results in the regulation of their translation. This second mechanism remains poorly understood. To address this issue, we purified high molecular weight complexes containing Lin28a in mouse embryonic stem cells (ESCs). Numerous proteins, co-purified with Lin28a, were identified by proteomic procedures and tested for their possible role in Lin28a-dependent regulation of the mRNA encoding DNA methyltransferase 3a (Dnmt3a). The results show that Lin28a activity is dependent on many proteins, including three helicases and four RNA-binding proteins. The suppression of four of these proteins, namely Ddx3x, Hnrnph1, Hnrnpu or Syncrip, interferes with the binding of Lin28a to the Dnmt3a mRNA, thus suggesting that they are part of an oligomeric ribonucleoprotein complex that is necessary for Lin28a activity.

Bmi1 inhibitor PTC-209 promotes Chemically-induced Direct Cardiac Reprogramming of cardiac fibroblasts into cardiomyocytes.

The development of therapeutic approaches based on direct cardiac reprogramming of fibroblasts into induced-cardiomyocytes (iCM) has emerged as an attractive strategy to repair the injured myocardium. The identification of the mechanisms driving lineage conversion represents a crucial step toward the development of new and more efficient regenerative strategies. To this aim, here we show that pre-treatment with the Bmi1 inhibitor PTC-209 is sufficient to increase the efficiency of Chemical-induced Direct Cardiac Reprogramming both in mouse embryonic fibroblasts and adult cardiac fibroblasts. PTC-209 induces an overall increase of spontaneously beating iCM at end-stage of reprogramming, expressing high levels of late cardiac markers Troponin T and myosin muscle light chain-2v. The inhibition of Bmi1 expression occurring upon PTC-209 pre-treatment was maintained throughout the reprogramming protocol, contributing to a significant gene expression de-regulation. RNA profiling revealed that, upon Bmi1 inhibition a significant down-regulation of genes associated with immune and inflammatory signalling pathways occurred, with repression of different genes involved in interleukin, cytokine and chemokine pathways. Accordingly, we observed the down-regulation of both JAK/STAT3 and MAPK/ERK1-2 pathway activation, highlighting the crucial role of these pathways as a barrier for cardiac reprogramming. These findings have significant implications for the development of new cardiac regenerative therapies.

HMGA Proteins in Stemness and Differentiation of Embryonic and Adult Stem Cells.

HMGA1 and HMGA2 are chromatin architectural proteins that do not have transcriptional activity per se, but are able to modify chromatin structure by interacting with the transcriptional machinery and thus negatively or positively regulate the transcription of several genes. They have been extensively studied in cancer where they are often found to be overexpressed but their functions under physiologic conditions have still not been completely addressed. Hmga1 and Hmga2 are expressed during the early stages of mouse development, whereas they are not detectable in most adult tissues. Hmga overexpression or knockout studies in mouse have pointed to a key function in the development of the embryo and of various tissues. HMGA proteins are expressed in embryonic stem cells and in some adult stem cells and numerous experimental data have indicated that they play a fundamental role in the maintenance of stemness and in the regulation of differentiation. In this review, we discuss available experimental data on HMGA1 and HMGA2 functions in governing embryonic and adult stem cell fate. Moreover, based on the available evidence, we will aim to outline how HMGA expression is regulated in different contexts and how these two proteins contribute to the regulation of gene expression and chromatin architecture in stem cells.

Insights into the pathogenesis of ATP1A1-related CMT disease using patient-specific iPSCs.

The development of patient-specific induced pluripotent stem cells (iPSCs) offered interesting insights in modeling the pathogenesis of Charcot-Marie-Tooth (CMT) disease and thus we decided to explore the phenotypes of iPSCs derived from a single CMT patient carrying a mutant ATP1A1 allele (p.Pro600Ala). iPSCs clones generated from CMT and control fibroblasts, were induced to differentiate into neural precursors and then into post-mitotic neurons. Control iPSCs differentiated into neuronal precursors and then into post-mitotic neurons within 6-8 days. On the contrary, the differentiation of CMT iPSCs was clearly defective. Electrophysiological properties confirmed that post-mitotic neurons were less mature compared to the normal counterpart. The impairment of in vitro differentiation of CMT iPSCs only concerned with the neuronal pathway, because they were able to differentiate into mesendodermal cells and other ectodermal derivatives. ATP1A1 was undetectable in the few neuronal cells derived from CMT iPSCs. ATP1A1 gene mutation (p.Pro600Ala), responsible for a form of axonal CMT disease, is associated in vitro with a dramatic alteration of the differentiation of patient-derived iPSCs into post-mitotic neurons. Thus, the defect in neuronal cell development might lead in vivo to a decreased number of mature neurons in ATP1A1-CMT disease.

Novel mutations in dystonin provide clues to the pathomechanisms of HSAN-VI.

OBJECTIVE: To describe a second hereditary sensory autonomic neuropathy type VI (HSAN-VI) family harboring 2 novel heterozygous mutations in the dystonin (DST) gene and to evaluate their effect on neurons derived from induced pluripotent stem cells (iPSC). METHODS: The family consisted of 3 affected siblings from nonconsanguineous healthy parents. All members underwent clinical and electrophysiologic evaluation and genetic analysis. Two patients underwent quantitative sensory testing (QST), cardiovascular reflexes, dynamic sweat test, and skin biopsy to evaluate somatic and autonomic cutaneous innervation and to get fibroblast cultures for developing iPSC-derived neurons. RESULTS: Onset occurred in the first decade, with painless and progressive mutilating distal ulcerations leading to amputation and joint deformity. Sensation to pain, touch, and vibration was reduced. Autonomic disturbances included hypohidrosis, pupillary abnormalities, and gastrointestinal and sexual dysfunction. Nerve conduction studies showed a severe axonal sensory neuropathy. QST and autonomic functional studies were abnormal. Skin biopsy revealed a lack of sensory and autonomic nerve fibers. Genetic analysis revealed 2 pathogenic mutations in the DST gene affecting exclusively the DST neuronal isoform-a2. Neurons derived from iPSC showed absence or very low levels of DST protein and short and dystrophic neuritis or no projections at all. CONCLUSIONS: Unlike the previous HSAN-VI family, our description indicates that DST mutations may be associated with a nonlethal and nonsyndromic phenotype. Neuronal loss affects large and small sensory nerve fibers as well as autonomic ones. Induced-PSC findings suggest that dystonin defect might alter proper development of the peripheral nerves. Dystonin-a2 plays a major role in the HSAN-VI phenotype.

Nanotechnology-Based Cardiac Targeting and Direct Cardiac Reprogramming: The Betrothed.

Cardiovascular diseases represent the first cause of morbidity in Western countries, and chronic heart failure features a significant health care burden in developed countries. Efforts in the attempt of finding new possible strategies for the treatment of CHF yielded several approaches based on the use of stem cells. The discovery of direct cardiac reprogramming has unveiled a new approach to heart regeneration, allowing, at least in principle, the conversion of one differentiated cell type into another without proceeding through a pluripotent intermediate. First developed for cancer treatment, nanotechnology-based approaches have opened new perspectives in many fields of medical research, including cardiovascular research. Nanotechnology could allow the delivery of molecules with specific biological activity at a sustained and controlled rate in heart tissue, in a cell-specific manner. Potentially, all the mediators and structural molecules involved in the fibrotic process could be selectively targeted by nanocarriers, but to date, only few experiences have been made in cardiac research. This review highlights the most prominent concepts that characterize both the field of cardiac reprogramming and a nanomedicine-based approach to cardiovascular diseases, hypothesizing a possible synergy between these two very promising fields of research in the treatment of heart failure.

Angiotensin receptor I stimulates osteoprogenitor proliferation through TGFß-mediated signaling.

Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti-hypertensive drugs that target angiotensin II (Ang II) activity may also reduce loss of bone mineral density. Here, we identified in a genetic screening the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20-17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20-17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20-17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)-Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor ß (TGFß) expression considerably and in an AT1R-dependent manner. Finally, exogenous TGFß was able to restore high proliferative activity to W20-17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFß and targets proliferation of osteoblast progenitors.

Fe65 matters: new light on an old molecule.

The discovery that the main constituents of amyloid deposits, characteristic of Alzheimer neuropathology, derive from the proteolytic processing of the membrane precursor amyloid precursor protein (APP) is one of the milestones of the research history of this disease. Despite years of intense studies, the functions of APP and of its amyloidogenic processing are still under debate. One focus of these studies was the complex network of protein-protein interactions centered at the cytosolic domain of APP, which suggests the involvement of APP in a lively signaling pathway. Fe65 was the first protein to be demonstrated to interact with the APP cytodomain. Starting from this observation, a large body of data has been gathered, indicating that Fe65 is an adaptor protein, which binds numerous proteins, further than APP. Among these proteins, the crosstalk with Mena, mDab, and Abl suggested the involvement of the Fe65-APP complex in the regulation of cell motility, with a relevant role in differentiation and development. Other partners, like the histone acetyltransferase Tip60, indicated the possibility that the nuclear fraction of Fe65 could be involved in gene regulation and/or DNA repair.

Differentiation of embryonic stem cells 1 (Dies1) is a component of bone morphogenetic protein 4 (BMP4) signaling pathway required for proper differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent cells able to grow indefinitely in culture and to differentiate into all cell types of embryos upon specific stimuli. Molecular mechanisms controlling the unique characteristics of ESCs are still largely unknown. We identified Dies1 (Differentiation of ESCs 1), an unpublished gene, that encodes a type I membrane protein. ESCs stably transfected with Dies1 small hairpin RNAs failed to properly differentiate toward neural and cardiac cell fate upon appropriate stimuli and continued to express markers of undifferentiated cells, such as the membrane-associated alkaline phosphatase, and transcription factors, like Oct3/4 and Nanog, when grown under conditions promoting differentiation. Our results demonstrated that Dies1 is required for BMP4/Smad1 signaling cascade; in undifferentiated ESCs Dies1 knockdown did not affect the expression of leukemia inhibitory factor downstream targets, whereas it resulted in a strong decrease of BMP4 signaling, as demonstrated by the decrease of Id1, -2, and -3 mRNAs, the decreased activity of Id1 gene promoter, and the reduced phospho-Smad1 levels. Dies1 knockdown had no effect in murine ESCs when the expression of the BMP4 receptor Alk3 was suppressed. The phenotype induced by Dies1 suppression in ESCs is due to the indirect activation of the Nodal/Activin pathway, which is a consequence of the BMP4 pathway inhibition and is sufficient to support the mESC undifferentiated state in the absence of leukemia inhibitory factor.

Notch activation induces neurite remodeling and functional modifications in SH-SY5Y neuronal cells.

Notch proteins are definitely recognized as key regulators of the neuronal fate during embryo development, but their function in the adult brain is still largely unknown. We have previously demonstrated that Notch pathway stimulation increases microtubules stability followed by the remodeling of neuronal morphology with neurite varicosities loss, thicker neuritis, and enlarged growth cones. Here we show that the neurite remodeling is a dynamic event, dependent on transcription and translation, and with functional implications. Exposure of differentiated human SH-SY5Y neuroblastoma cells to the Notch ligand Jagged1 induces varicosities loss all along the neurites, accompanied by the redistribution of presynaptic vesicles and the decrease in neurotransmitters release. As evaluated by time lapse digital imaging, dynamic changes in neurite morphology were rapidly reversible and dependent on the activation of the Notch signaling pathway. In fact, it was prevented by the inhibition of the proteolytic gamma-secretase enzyme or the transcription machinery, and was mimicked by the transfection of the intracellular domain of Notch. One hour after treatment with Jagged1, several genes were downregulated. Many of these genes encode proteins that are known to be involved in protein synthesis. These data suggest that in adult neurons, Notch pathway activates a transcriptional program that regulates the equilibrium between varicosities formation and varicosities loss in the neuronal presynaptic compartment involving the expression and redistribution of both structural and functional proteins.

A differential proteomic approach reveals an evolutionary conserved regulation of Nme proteins by Fe65 in C. elegans and mouse.

The function of the APP-Fe65 complex is still not definitively understood. To address this point we studied the phenotype of Fe65 (feh-1) ablation, which results in severe developmental defects in C. elegans, including embryonic and larval arrests. To shed light on the complex phenotype of embryonic arrest, we undertook a systematic approach, aiming at the definition of the altered proteomic profile of feh-1 null worms. We defined a panel of 27 regulated proteins, 16 of which actually participating to embryonic development processes in the nematode. Protein spots corresponding to the products of the F25H2.5 gene, the nematode orthologue of mammalian Nm23/Nme gene family members, were consistently up-regulated in feh-1 -/- embryos. We observed similar up-regulation of Nme1 and Nme2 genes, both at the transcript and the protein levels, in the brain of Fe65 knock-out mice, thus highlighting the occurrence of evolutionary conserved mechanisms of Nme expression in nematodes and mammals.

Receptor- and non-receptor tyrosine kinases induce processing of the amyloid precursor protein: role of the low-density lipoprotein receptor-related protein.

The Alzheimer's beta-amyloid peptides derive from the proteolytic processing of the beta-amyloid precursor protein, APP, by beta- and gamma-secretases. The regulation of this processing is not fully understood. Experimental evidence suggests that the activation of pathways involving protein tyrosine kinases, such as PDGFR and Src, could induce the cleavage of APP and in turn the generation of amyloid peptides. In this paper we addressed the effect of receptor and nonreceptor protein tyrosine kinases on the cleavage of APP and the mechanisms of their action. To this aim, we developed an in vitro system based on the APP-Gal4 fusion protein stably transfected in SHSY5Y neuroblastoma cell line. The cleavage of this molecule, induced by various stimuli, results in the activation of the transcription of the luciferase gene under the control of Gal4 cis-elements. By using this experimental system we demonstrated that, similarly to Src, three tyrosine kinases, TrkA, Ret and EGFR, induced the cleavage of APP-Gal4. We excluded that this effect was mediated by the activation of Ras-MAPK, PI3K-Akt and PLC-gamma pathways. Furthermore, the direct phosphorylation of the APP cytosolic domain does not affect Abeta peptide generation. On the contrary, experiments in cells lacking the LDL-receptor related protein LRP support the hypothesis that the interaction of APP with LRP is required for the induction of APP cleavage by tyrosine kinases.

Essential roles for Fe65, Alzheimer amyloid precursor-binding protein, in the cellular response to DNA damage.

Fe65 interacts with the cytosolic domain of the Alzheimer amyloid precursor protein (APP). The functions of the Fe65 are still unknown. To address this point we generated Fe65 knockout (KO) mice. These mice do not show any obvious phenotype; however, when fibroblasts (mouse embryonic fibroblasts), isolated from Fe65 KO embryos, were exposed to low doses of DNA damaging agents, such as etoposide or H2O2, an increased sensitivity to genotoxic stress, compared with wild type animals, clearly emerged. Accordingly, brain extracts from Fe65 KO mice, exposed to non-lethal doses of ionizing radiations, showed high levels of gamma-H2AX and p53, thus demonstrating a higher sensitivity to X-rays than wild type mice. Nuclear Fe65 is necessary to rescue the observed phenotype, and few minutes after the exposure of MEFs to DNA damaging agents, Fe65 undergoes phosphorylation in the nucleus. With a similar timing, the proteolytic processing of APP is rapidly affected by the genotoxic stress: in fact, the cleavage of the APP COOH-terminal fragments by gamma-secretase is induced soon after the exposure of cells to etoposide, in a Fe65-dependent manner. These results demonstrate that Fe65 plays an essential role in the response of the cells to DNA damage.