Increased Expression of Autophagy-Related Genes in Alzheimer’s Disease—Type 2 Diabetes Mellitus Comorbidity Models in Cells

The association between Alzheimer’s disease (AD) and type 2 diabetes mellitus (T2DM) has been extensively demonstrated, but despite this, the pathophysiological mechanisms underlying it are still unknown. In previous work, we discovered a central role for the autophagy pathway in the common alterations observed between AD and T2DM. In this study, we further investigate the role of genes belonging to this pathway, measuring their mRNA expression and protein levels in 3xTg-AD transgenic mice, an animal model of AD. Moreover, primary mouse cortical neurons derived from this model and the human H4Swe cell line were used as cellular models of insulin resistance in AD brains. Hippocampal mRNA expression showed significantly different levels for Atg16L1, Atg16L2, GabarapL1, GabarapL2, and Sqstm1 genes at different ages of 3xTg-AD mice. Significantly elevated expression of Atg16L1, Atg16L2, and GabarapL1 was also observed in H4Swe cell cultures, in the presence of insulin resistance. Gene expression analysis confirmed that Atg16L1 was significantly increased in cultures from transgenic mice when insulin resistance was induced. Taken together, these results emphasise the association of the autophagy pathway in AD-T2DM co-morbidity, providing new evidence about the pathophysiology of both diseases and their mutual interaction

Glutamatergic neurons induce expression of functional glutamatergic synapses in primary myotubes.

The functioning of the nervous system depends upon the specificity of its synaptic contacts. The mechanisms triggering the expression of the appropriate receptors on postsynaptic membrane and the role of the presynaptic partner in the differentiation of postsynaptic structures are little known.To address these questions we cocultured murine primary muscle cells with several glutamatergic neurons, either cortical, cerebellar or hippocampal. Immunofluorescence and electrophysiology analyses revealed that functional excitatory synaptic contacts were formed between glutamatergic neurons and muscle cells. Moreover, immunoprecipitation and immunofluorescence experiments showed that typical anchoring proteins of central excitatory synapses coimmunoprecipitate and colocalize with rapsyn, the acetylcholine receptor anchoring protein at the neuromuscular junction.These results support an important role of the presynaptic partner in the induction and differentiation of the postsynaptic structures

Proteomic profiling of human amnion for preterm birth biomarker discovery

Spontaneous preterm birth (PTB) complicates about 12% of pregnancies worldwide, remaining the main cause of neonatal morbidity and mortality. Spontaneous preterm birth PTBs is often caused by microbial-induced preterm labor, mediated by an inflammatory process threatening both maternal and newborn health. In search for novel predictive biomarkers of PTB and preterm prelabor rupture of the membranes (pPROM), and to improve understanding of infection related PTB, we performed an untargeted mass spectrometry discovery study on 51 bioptic mid zone amnion samples from premature babies. A total of 6352 proteins were identified. Bioinformatics analyses revealed a ranked core of 159 proteins maximizing the discrimination between the selected clinical stratification groups allowing to distinguish conditions of absent (FIR 0) from maximal Fetal Inflammatory Response (FIR 3) stratified in function of Maternal Inflammatory Response (MIR) grade. Matrix metallopeptidase-9 (MMP-9) was the top differentially expressed protein. Gene Ontology enrichment analysis of the core proteins showed significant changes in the biological pathways associated to inflammation and regulation of immune and infection response. Data suggest that the conditions determining PTB would be a transversal event, secondary to the maternal inflammatory response causing a breakdown in fetal-maternal tolerance, with fetal inflammation being more severe than maternal one. We also highlight matrix metallopeptidase-9 as a potential predictive biomarker of PTB that can be assayed in the maternal serum, for future investigation

Distribution of different isoforms of receptor protein tyrosine phosphatase \u3b3 (Ptprg-RPTP \u3b3) in adult mouse brain: upregulation during neuroinflammation.

The receptor protein tyrosine phosphatase \u3b3 (Ptprg-RPTP\u3b3) is a receptor protein widely expressed in many tissues, including the central nervous system (CNS). Several RPTP\u3b3 isoforms are expressed in the brain during development and in adulthood, but their distribution and role are unknown. In this study, we investigated the distribution of some RPTP\u3b3 isoforms in the adult brain using antibodies against the epitopes localized in the C- and in the N-terminal domains of the full length isoform of RPTP\u3b3. We found a predominant and widespread neuronal positivity throughout the neocortex, hippocampus, striatum and in many nuclei of the brainstem and cerebellum. At least 2 distinct isoforms that can co-exist in various compartments in the same cell are detectable in different neuron types. Immunopositivity for epitopes located in both the N- and C-terminus domains were found in the neuropil of cortical and hippocampal neurons, whereas the N-terminal domain positivity was found in the soma, often without colocalization with its C-terminal counterpart. Among glial cells, some protoplasmic and perivascular astrocytes and the cerebellar Bergmann glia, express RPTP\u3b3. The astrocytic expression of RPTP\u3b3 and putative processing isoforms of 120 and 80 kDa increases during neuroinflammation, in particular 24 h after LPS treatment. Activated astrocytes were found to be strongly positive for RPTP\u3b3 also in a mice model of Alzheimer's disease. Our results confirm previous findings and enrich the current knowledge of RPTP\u3b3 distribution in the CNS, highlighting a role of RPTP\u3b3 during neuroinflammation processes

Rho GTPase-dependent plasticity of dendritic spines in the adult brain.

Brain activity is associated with structural changes in the neural connections. However, in vivo imaging of the outer cortical layers has shown that dendritic spines, on which most excitatory synapses insist, are predominantly stable in adulthood. Changes in dendritic spines are governed by small GTPases of the Rho family through modulation of the actin cytoskeleton. Yet, while there are abundant data about this functional effect of Rho GTPases in vitro, there is little evidence that Rho GTPase signaling in the brain is associated with changes in neuronal morphology. In the present work, both chronic in vivo two-photon imaging and Golgi staining reveal that the activation of Rho GTPases in the adult mouse brain is associated with little change of dendritic spines in the apical dendrites of primary visual cortex pyramidal neurons. On the contrary, considerable increase in spine density is observed i) in the basal dendrites of the same neurons ii) in both basal and apical dendrites of the hippocampal CA1 pyramidal cells. Moreover, functional analysis shows increase in basal glutamatergic neurotrasmission and activity-dependent plasticity only in CA1 neurons. While confirming that Rho-GTPase dependent increase in spine density can be substantial, the study indicates region and dendrite selectivity with relative stability of superficial cortical circuits

Glutamatergic Reinnervation and Assembly of Glutamatergic Synapses in Adult Rat Skeletal Muscle Occurs at Cholinergic Endplates

After denervation of adult rat abdominal muscles, the postsynaptic apparatus of neuromuscular junctions (NMJs) retains its original architecture and clustering of acetylcholine receptors (AChRs). When descending fibers of the spinal cord are surgically diverted to this muscle by a nerve grafting procedure, supraspinal glutamatergic neurons can innervate muscle fibers and restore motor function; the newly formed NMJs switch from a cholinergic to a glutamatergic-type synapse. We show here that regenerating nerve endings contact the fibers in an area occupied by cholinergic endplates. These NMJs are morphologically indistinguishable from those in controls, but they differ in the subunit composition of AChRs. Moreover, by immunofluorescence and immunoelectron microscopy, new NMJs express glutamatergic synapse markers. The \u3b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 partially colocalizes with AChRs, and vesicular glutamate transporter 2 is localized in the presynaptic compartment. Immunoprecipitation analysis of membranes from reinnervated muscle showed that AMPA receptor subunits GluR1 and GluR2 coimmunoprecipitate with rapsyn, the AChR-anchoring protein at the NMJ. Taken together, these results indicate that cholinergic endplates can be targeted by new glutamatergic projections and that the clustering of AMPA receptors occurs there

Distribución profunda, aspectos biológicos y ecológicos de Aristeus Antennatus (Risso, 1816) en el Mediterráneo Occidental y Central

[EN] The object of the DESEAS Project, funded by the EC, was to gather preliminary data on the abundance and maximum depth distribution of the rose shrimp Aristeusantennatus in the Mediterranean Sea. An exploratory survey was therefore designed with that goal in mind and conducted on the R/V García del Cid, sampling the maximum depths in three specific areas in the central and western Mediterranean, one off Ibiza (Balearic Islands), one off Calabria (western Ionian Sea), and one off the southern Peloponnesian Peninsula (Gulf of Kalamata, eastern Ionian Sea). The depths sampled ranged from 600 to 4000 m, with specimens of A. antennatus being collected down to 3300 m. There were three distinct boundaries marking the abundance of this species: 1500 m, relatively low abundance (<100 ind km-2). The known population structure of this shrimp species, with increasing proportions of males and juveniles with depth, was also recorded in the deep-sea regions in other areas of the Mediterranean. No evidence of any differences in gonad development or in the presence of spermatophores carried by females was found in any of the three sampling areas. Lastly, a tendency for the relative proportion of juveniles to increase with depth was also observed[ES] El objetivo del proyecto DESEAS, financiado por la CE, fue obtener datos preliminares de abundancia y distribución de profundidad máxima de la gamba rosada Aristeus antennatus en el Mar Mediterráneo. El diseño de la campaña exploratoria fue realizado con este propósito y se desarrolló a bordo del B/O García del Cid. Se realizaron muestreos en las máximas profundidades de tres áreas específicas en el Mediterráneo occidental y central: una cerca de Menorca (Islas Baleares), otra frente a las costas de Calabria (Iónico occidental) y la última al sur de la península del Peloponeso (en el Golfo de Kalamata, Iónico oriental). Las profundidades muestreadas fueron las comprendidas entre 600 y 4000 m, obteniendo individuos de A. antennatus hasta 3300 m. Se detectaron tres niveles de abundancias diferenciados en esta especie: 1500 m, relativamente poco abundante (< 100 ind km-2). En las tres áreas estudiadas se confirmó la estructura de la población conocida hasta el momento, es decir, aumento de la proporción de machos y juveniles con la profundidad. No se encontraron evidencias de diferencias en el desarrollo gonadal o en la presencia de espermatóforos de las hembras entre áreas. Finalmente se observó la existencia de una tendencia en el aumento de la proporción de juveniles con la profundidadThis work was financially supported by the Directorate General Fisheries of the EC as part of the DESEAS survey programme (DGXIV, Study Contract, 2000/39)Peer reviewe

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis

Electrophysiological evaluation of Cystic Fibrosis Conductance Transmembrane Regulator (CFTR) expression in human monocytes.

BACKGROUND: Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells.METHODS: Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays.RESULTS: We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTRinh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis.CONCLUSIONS: We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF.GENERAL SIGNIFICANCE: Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients

Challenging the diagnosis of Cystic Fibrosis in a patient carrying the 186-8T/C allelic variant in the CF Transmembrane Conductance Regulator gene

BACKGROUND: This report describe for the first time a clinical case with a CFTR allelic variant 186-8T/C (c.54-8 T/C) in intron 1 of CFTR and underline the importance of applying a combination of genetic and functional tests to establish or exclude a diagnosis of Cystic Fibrosis. In this case the diagnostic algorithm proposed for CF has been successfully applied at our Center and previous CF diagnosis assigned in a different Center was not confirmed.Case report: A 38 year-old Italian woman had been treated as affected by CF since 2010, following diagnosis based on sweat tests (reported values of 73 and 57 mEq/L) and a clinical history consistent with CF. No mutations were identified by first level of genetic analysis. Afterwards the patient referred to our center for assessing the relevance of these findings. The genetic variant 186-8T/C (c.54-8 T/C) in intron 1 of the CFTR gene was detected by sequencing. Low-level interstitial-alveolar infiltration was recorded by high-resolution computerized tomography. Lung function was normal and sputum and Broncho Alveolar Lavage cultures resulted bacteriologically negative. Sweat chloride levels was re-assessed and resulted with values of 57 and 35 mEq/L, with a borderline range between 40 and 60 mEq/L. Nasal Potential Difference measurements resulted in three reliable measurements consistent with a non-CF phenotype. Differential diagnosis with ciliary dyskinesia was excluded, as was exon 2 skipping of CFTR gene that might have caused a CFTR functional defect. Furthermore, single cell fluorescence analysis in response to cAMP agonists performed in patient's monocytes overlapped those obtained in healthy donors. CONCLUSION: We concluded that this patient was not affected by CF. This case highlights the need for referrals to highly specialized centers and the importance of combined functional and genetic tests in making a correct diagnosis. Moreover, we confirmed a correlation between NPD tracings and cell depolarization in monocytes providing a rationale for proposing the use of leukocytes as a potential support for CF diagnosis