ARMCX3 Mediates Susceptibility to Hepatic Tumorigenesis Promoted by Dietary Lipotoxicity.

ARMCX3 is encoded by a member of the Armcx gene family and is known to be involved in nervous system development and function. We found that ARMCX3 is markedly upregulated in mouse liver in response to high lipid availability, and that hepatic ARMCX3 is upregulated in patients with NAFLD and hepatocellular carcinoma (HCC). Mice were subjected to ARMCX3 invalidation (inducible ARMCX3 knockout) and then exposed to a high-fat diet and diethylnitrosamine-induced hepatocarcinogenesis. The effects of experimental ARMCX3 knockdown or overexpression in HCC cell lines were also analyzed. ARMCX3 invalidation protected mice against high-fat-diet-induced NAFLD and chemically induced hepatocarcinogenesis. ARMCX3 invalidation promoted apoptotic cell death and macrophage infiltration in livers of diethylnitrosamine-treated mice maintained on a high-fat diet. ARMCX3 downregulation reduced the viability, clonality and migration of HCC cell lines, whereas ARMCX3 overexpression caused the reciprocal effects. SOX9 was found to mediate the effects of ARMCX3 in hepatic cells, with the SOX9 interaction required for the effects of ARMCX3 on hepatic cell proliferation. In conclusion, ARMCX3 is identified as a novel molecular actor in liver physiopathology and carcinogenesis. ARMCX3 downregulation appears to protect against hepatocarcinogenesis, especially under conditions of high dietary lipid-mediated hepatic insult.

Identification of novel Ack1-interacting proteins and Ack1 phosphorylated sites in mouse brain by mass spectrometry.

Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in brain. This kinase contains several protein-protein interaction domains and its action is partially regulated by phosphorylation. As a first step to address the neuronal functions of Ack1, here we screened mouse brain samples to identify proteins that interact with this kinase. Using mass spectrometry analysis, we identified new putative partners for Ack1 including cytoskeletal proteins such as Drebrin or MAP4; adhesion regulators such as NCAM1 and neurabin-2; and synapse mediators such as SynGAP, GRIN1 and GRIN3. In addition, we confirmed that Ack1 and CAMKII both co-immunoprecipitate and co-localize in neurons. We also identified that adult and P5 samples contained the phosphorylated residues Thr 104 and Ser 825, and only P5 samples contained phosphorylated Ser 722, a site linked to cancer and interleukin signaling when phosphorylated. All these findings support the notion that Ack1 could be involved in neuronal plasticity.

New partners and phosphorylation sites of focal adhesion kinase identified by mass spectrometry.

The regulation of focal adhesion kinase (FAK) involves phosphorylation and multiple interactions with other signaling proteins. Some of these pathways are relevant for nervous system functions such as branching, axonal guidance, and plasticity. In this study, we screened mouse brain to identify FAK-interactive proteins and phosphorylatable residues as a first step to address the neuronal functions of this kinase. Using mass spectrometry analysis, we identified new phosphorylated sites (Thr 952, Thr 1048, and Ser 1049), which lie in the FAT domain; and putative new partners for FAK, which include cytoskeletal proteins such as drebrin and MAP 6, adhesion regulators such as neurabin-2 and plakophilin 1, and synapse-associated proteins such as SynGAP and a NMDA receptor subunit. Our findings support the participation of brain-localized FAK in neuronal plasticity.

The diverse roles and multiple forms of focal adhesion kinase in brain.

Although it was originally characterized as a constituent of focal adhesions in fibroblasts, focal adhesion kinase (FAK) is now considered to be not only a mediator of adhesion processes but also a crucial regulator of guidance and a modulator of gene expression. FAK is the main transducer of the integrin signaling required to stabilize the actin cytoskeleton. However, additional activities have been described over the years. In the brain, FAK deserves particular attention as it is found in various alternatively spliced forms - these distributed in multiple subcellular compartments or bound to multiple partners. Moreover, its signaling involves not only phosphorylation but also ubiquitination and proteolysis. Several experimental cell models demonstrate that FAK increases or decreases migration, participates in differentiation and contributes to plasticity events. In addition, this kinase is linked to cell survival in cancer and apoptosis. This review focuses on the diversity of events involving brain-located forms of FAK.

Somatic signature of brain-specific single nucleotide variations in sporadic Alzheimer's disease.

BACKGROUND: Although genome-wide association studies have shown that genetic factors increase the risk of suffering late-onset, sporadic Alzheimer's disease (SAD), the molecular mechanisms responsible remain largely unknown. OBJECTIVE: The aim of the study was to investigate the presence of somatic, brain-specific single nucleotide variations (SNV) in the hippocampus of SAD samples. METHODS: By using bioinformatic tools, we compared whole exome sequences in paired blood and hippocampal genomic DNAs from 17 SAD patients and from 2 controls and 2 vascular dementia patients. RESULTS: We found a remarkable number of SNVs in SAD brains (~575 per patient) that were not detected in blood. Loci with hippocampus-specific (hs)-SNVs were common to several patients, with 38 genes being present in 6 or more patients out of the 17. While some of these SNVs were in genes previously related to SAD (e.g., CSMD1, LRP2), most hs-SNVs occurred in loci previously unrelated to SAD. The most frequent genes with hs-SNVs were associated with neurotransmission, DNA metabolism, neuronal transport, and muscular function. Interestingly, 19 recurrent hs-SNVs were common to 3 SAD patients. Repetitive loci or hs-SNVs were underrepresented in the hippocampus of control or vascular dementia donors, or in the cerebellum of SAD patients. CONCLUSION: Our data suggest that adult blood and brain have different DNA genomic variations, and that somatic genetic mosaicism and brain-specific genome reshaping may contribute to SAD pathogenesis and cognitive differences between individuals.

Hypoxia and P1 receptor activation regulate the high-affinity concentrative adenosine transporter CNT2 in differentiated neuronal PC12 cells.

Under several adverse conditions, such as hypoxia or ischaemia, extracellular levels of adenosine are elevated because of increased energy demands and ATP metabolism. Because extracellular adenosine affects metabolism through G-protein-coupled receptors, its regulation is of high adaptive importance. CNT2 (concentrative nucleoside transporter 2) may play physiological roles beyond nucleoside salvage in brain as it does in other tissues. Even though nucleoside transport in brain has mostly been seen as being of equilibrative-type, in the present study, we prove that the rat phaeochromocytoma cell line PC12 shows a concentrative adenosine transport of CNT2-type when cells are differentiated to a neuronal phenotype by treatment with NGF (nerve growth factor). Differentiation of PC12 cells was also associated with the up-regulation of adenosine A1 receptors. Addition of adenosine receptor agonists to cell cultures increased CNT2-related activity by a mechanism consistent with A1 and A2A receptor activation. The addition of adenosine to the culture medium also induced the phosphorylation of the intracellular regulatory kinase AMPK (AMP-activated protein kinase), with this effect being dependent upon adenosine transport. CNT2-related activity of differentiated PC12 cells was also dramatically down-regulated under hypoxic conditions. Interestingly, the analysis of nucleoside transporter expression after experimental focal ischaemia in rat brain showed that CNT2 expression was down-regulated in the infarcted tissue, with this effect somehow being restricted to other adenosine transporter proteins such as CNT3 and ENT1 (equilibrative nucleoside transporter 1). In summary, CNT2 is likely to modulate extracellular adenosine and cell energy balance in neuronal tissue.

The non-canonical Wnt/PKC pathway regulates mitochondrial dynamics through degradation of the arm-like domain-containing protein Alex3.

The regulation of mitochondrial dynamics is vital in complex cell types, such as neurons, that transport and localize mitochondria in high energy-demanding cell domains. The Armcx3 gene encodes a mitochondrial-targeted protein (Alex3) that contains several arm-like domains. In a previous study we showed that Alex3 protein regulates mitochondrial aggregation and trafficking. Here we studied the contribution of Wnt proteins to the mitochondrial aggregation and dynamics regulated by Alex3. Overexpression of Alex3 in HEK293 cells caused a marked aggregation of mitochondria, which was attenuated by treatment with several Wnts. We also found that this decrease was caused by Alex3 degradation induced by Wnts. While the Wnt canonical pathway did not alter the pattern of mitochondrial aggregation induced by Alex3, we observed that the Wnt/PKC non-canonical pathway regulated both mitochondrial aggregation and Alex3 protein levels, thereby rendering a mitochondrial phenotype and distribution similar to control patterns. Our data suggest that the Wnt pathway regulates mitochondrial distribution and dynamics through Alex3 protein degradation.

The Eutherian Armcx genes regulate mitochondrial trafficking in neurons and interact with Miro and Trak2.

Brain function requires neuronal activity-dependent energy consumption. Neuronal energy supply is controlled by molecular mechanisms that regulate mitochondrial dynamics, including Kinesin motors and Mitofusins, Miro1-2 and Trak2 proteins. Here we show a new protein family that localizes to the mitochondria and controls mitochondrial dynamics. This family of proteins is encoded by an array of armadillo (Arm) repeat-containing genes located on the X chromosome. The Armcx cluster is unique to Eutherian mammals and evolved from a single ancestor gene (Armc10). We show that these genes are highly expressed in the developing and adult nervous system. Furthermore, we demonstrate that Armcx3 expression levels regulate mitochondrial dynamics and trafficking in neurons, and that Alex3 interacts with the Kinesin/Miro/Trak2 complex in a Ca(2+)-dependent manner. Our data provide evidence of a new Eutherian-specific family of mitochondrial proteins that controls mitochondrial dynamics and indicate that this key process is differentially regulated in the brain of higher vertebrates.

Expression of Semaphorin 4F in neurons and brain oligodendrocytes and the regulation of oligodendrocyte precursor migration in the optic nerve.

Semaphorins are secreted or membrane-anchored proteins that play critical roles in neural development and adult brain plasticity. Sema4F is a transmembrane semaphorin found on glutamatergic synapses, in which it is attached to the PSD-95-scaffolding protein. Here we further examined the expression of Sema4F by raising specific antibodies. We show that Sema4F protein is widely expressed by neurons during neural development and in the adult brain. We also demonstrate a preferential localization of this protein in postsynaptic dendrites. Moreover, Sema4F is expressed not only by neurons but also by oligodendrocyte precursors in the optic nerve and along the migratory pathways of oligodendroglial cells, and also by subsets of postnatal oligodendroglial cells in the brain. Finally, in vitro experiments demonstrate that endogenous Sema4F expressed by brain cells of oligodendroglial lineage regulates the outgrowth migration of oligodendrocyte precursors and promotes their differentiation. The present data extend our knowledge about the expression of Sema4F and uncover a novel function in the control of oligodendrocyte precursor migration in the developing brain.

Podocalyxin is a novel polysialylated neural adhesion protein with multiple roles in neural development and synapse formation.

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.

Interleukin-10 and interleukin-10 receptor-I are upregulated in glial cells after an excitotoxic injury to the postnatal rat brain.

Inflammation is an important determinant of the severity and outcome of central nervous system injury. The endogenous anti-inflammatory cytokine interleukin-10 (IL-10) is upregulated in the injured adult central nervous system where it controls and terminates inflammatory processes. The developing brain, however, displays differences in susceptibility to insults and in associated inflammatory responses from the adult brain; the anatomic and temporal patterns of injury-induced IL-10 expression in the immature brain after excitotoxic injury are unknown. We analyzed the spaciotemporal gene and protein expression of IL-10 and its receptor (IL-10RI) in N-methyl-d-aspartate-induced excitotoxic injury in 9-day-old and control rats using quantitative reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. In noninjected control brains, both molecules were expressed mainly in white matter on glial cells and blood vessels; IL-10 was also observed on blood vessels in gray matter and in glial fibrillary acidic protein-positive processes in the hippocampus and near leptomeningeal and ventricle surfaces. In N-methyl-d-aspartate-injected brains, IL-10 gene and protein expression were maximal at 72 hours postinjection; IL-10RI gene and protein expression peaked at 48 hours postinjection. Interleukin-10 and IL-10RI expression in injured areas was mainly found in reactive astrocytes and in microglia/macrophages. The expression patterns of IL-10 and IL-10R suggest possible developmental roles, and their upregulation after injury suggests that this expression may have anti-inflammatory effects in distinct anatomic sites in the immature brain.

The CREB/CREM transcription factors negatively regulate early synaptogenesis and spontaneous network activity.

The family of CREB (cAMP response element-binding protein) transcription factors are involved in a variety of biological processes including the development and plasticity of the nervous system. In the maturing and adult brain, CREB genes are required for activity-dependent processes, including synaptogenesis, refinement of connections and long-term potentiation. Here, we use CREB1(Nescre)CREM(-/-) (cAMP-responsive element modulator) mutants to investigate the role of these genes in stimulus-independent patterns of neural activity at early stages. We show that lack of CREB/CREM genes specifically in neural tissue leads to increased synaptogenesis and to a dramatic increase in the levels of spontaneous network activity at embryonic stages. Thus, the functions of CREB/CREM genes in neural activity differ in distinct periods of neural development.

Regulation of neural migration by the CREB/CREM transcription factors and altered Dab1 levels in CREB/CREM mutants.

The family of CREB transcription factors is involved in a variety of biological processes including the development and plasticity of the nervous system. To gain further insight into the roles of CREB family members in the development of the embryonic brain, we examined the migratory phenotype of CREB1(Nescre)CREM(-/-) mutants. We found that the lack of CREB/CREM genes is accompanied by anatomical defects in specific layers of the olfactory bulb, hippocampus and cerebral cortex. These changes are associated with decreased Dab1 expression in CREB1(Nescre)CREM(-/-) mutants. Our results indicate that the lack of CREB/CREM genes, specifically in neural and glial progenitors, leads to migration abnormalities during brain development, suggesting that unidentified age-dependent factors modulate the role of CREB/CREM genes in neural development.

Enhanced susceptibility of Prnp-deficient mice to kainate-induced seizures, neuronal apoptosis, and death: Role of AMPA/kainate receptors.

Normal physiologic functions of the cellular prion protein (PrPc) are still elusive. This GPI-anchored protein exerts many functions, including roles in neuron proliferation, neuroprotection or redox homeostasis. There are, however, conflicting data concerning its role in synaptic transmission. Although several studies report that PrPc participates in NMDA-mediated neurotransmission, parallel studies describe normal behavior of PrPc-mutant mice. Abnormal axon connections have been described in the dentate gyrus of the hippocampi of PrPc-deficient mice similar to those observed in epilepsy. A study indicates increased susceptibility to kainate (KA) in these mutant mice. We extend the observation of these studies by means of several histologic and biochemical analyses of KA-treated mice. PrPc-deficient mice showed increased sensitivity to KA-induced seizures in vivo and in vitro in organotypic slices. In addition, we show that this sensitivity is cell-specific because interference experiments to abolish PrPc expression increased susceptibility to KA in PrPc-expressing cells. We indicate a correlation of susceptibility to KA in cells lacking PrPc with the differential expression of GluR6 and GluR7 KA receptor subunits using real-time RT-PCR methods. These results indicate that PrPc exerts a neuroprotective role against KA-induced neurotoxicity, probably by regulating the expression of KA receptor subunits.

Semaphorin 6C leads to GSK-3-dependent growth cone collapse and redistributes after entorhino-hippocampal axotomy.

We studied the changes in the distribution of a specific variant of Semaphorin Y/6C (Sema6C) in mouse forebrain after axotomy of the entorhino-hippocampal perforant pathway. We found this isoform to be widely expressed during development, remaining in the adult and showing variations in distribution when the perforant pathway was axotomized. These changes were detected in both the hippocampal and entorhinal cortices. Sema6C1 immunoreactivity (IR) was high in the stratum radiatum of the hippocampus proper and the inner molecular layer of the dentate gyrus; the entorhinal cortex showed Sema6C1 IR in both cell bodies and in fibers of the II/III and V/VI layers. In axotomized animals, the IR of the ipsilateral, but not the contralateral, hemisphere showed that IR had moved into the stratum lacunosum-moleculare, the medial molecular layer of the dentate gyrus and the fibers, but not the cell bodies, of the entorhinal cortex. These results were not reproduced after lateral axotomy of the fimbria fornix, indicating a specific role for Sema6C variants in the generation and/or stability of entorhino-hippocampal synapses. Growth cone collapse of entorhinal and pyramidal neurons, as well as activation of glycogen synthase kinase-3 (GSK-3) through depletion of the inactive pool, induced by diffusible Sema6C1 further supports this view.

Reelin expression and glycosylation patterns are altered in Alzheimer's disease.

Reelin is a glycoprotein that is essential for the correct cytoarchitectonic organization of the developing CNS. Its function in the adult brain is less understood, although it has been proposed that Reelin is involved in signaling pathways linked to neurodegeneration. Here we analyzed Reelin expression in brains and cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients and nondemented controls. We found a 40% increase in the Reelin protein levels in the cortex of AD patients compared with controls. Similar increases were detected at the Reelin mRNA transcriptional level. This expression correlates with parallel increases in CSF but not in plasma samples. Next, we examined whether CSF Reelin levels were also altered in neurological diseases, including frontotemporal dementia, progressive supranuclear palsy, and Parkinson's disease. The Reelin 180-kDa band increased in all of the neurodegenerative disorders analyzed. Moreover, the 180-kDa Reelin levels correlated positively with Tau protein in CSF. Finally, we studied the pattern of Reelin glycosylation by using several lectins and the anti-HNK-1 antibody. Glycosylation differed in plasma and CSF. Furthermore, the pattern of Reelin lectin binding differed between the CSF of controls and in AD. Our results show that Reelin is up-regulated in the brain and CSF in several neurodegenerative diseases and that CSF and plasma Reelin have distinct cellular origins, thereby supporting that Reelin is involved in the pathogenesis of a number of neurodegenerative diseases.

Pattern of expression of the podocalyxin gene in the mouse brain during development.

We studied the expression pattern of the major renal protein Podocalyxin during the development of mouse brain using in situ hybridization. Podocalyxin mRNA was widely expressed at least from E14, the first age we studied, and expression remained high until adulthood. The highest levels of expression were postnatal. Podocalyxin expression was particularly elevated in the cortical plate, the hippocampus and cerebellum, and in several basal forebrain nuclei.

Distribution of CNT2 and ENT1 transcripts in rat brain: selective decrease of CNT2 mRNA in the cerebral cortex of sleep-deprived rats.

Nucleoside transport processes regulate the levels of adenosine available to modulate neurotransmission, vascular tone and other physiological events. However, although equilibrative transporter transcripts or proteins have been mapped in the central nervous system of rats and humans, little is known about the presence and distribution of the complete family of nucleoside transporters in brain. In this study, we analysed the distribution of the transcript encoding the high affinity adenosine-preferring concentrative transporter CNT2 in the rat central nervous system and compared it with that of the equilibrative transporter ENT1. Furthermore, we evaluated the changes in expression of these two transporters in a situation of increased extracellular levels of adenosine, such as sleep deprivation. CNT2 mRNA was widespread in rat brain, although most prevalent in the amygdala, the hippocampus, specific neocortical regions and the cerebellum. The distribution of CNT2 mRNA only partially overlapped that of ENT1. Most of the cells labelled were neurones. Total sleep deprivation dramatically diminished the amounts of CNT2 mRNA, whereas ENT1 mRNA remained unchanged. This specific decrease in CNT2 transcript suggests a new physiological role for the transporter in the modulation of extracellular adenosine levels and the sleep/wakefulness cycle.

Regulation of Nogo and Nogo receptor during the development of the entorhino-hippocampal pathway and after adult hippocampal lesions.

Axonal regeneration in the adult CNS is limited by the presence of several inhibitory proteins associated with myelin. Nogo-A, a myelin-associated inhibitor, is responsible for axonal outgrowth inhibition in vivo and in vitro. Here we study the onset and maturation of Nogo-A and Nogo receptor in the entorhino-hippocampal formation of developing and adult mice. We also provide evidence that Nogo-A does not inhibit embryonic hippocampal neurons, in contrast to other cell types such as cerebellar granule cells. Our results also show that Nogo and Nogo receptor mRNA are expressed in the adult by both principal and local-circuit hippocampal neurons, and that after lesion, Nogo-A is also transiently expressed by a subset of reactive astrocytes. Furthermore, we analyzed their regulation after kainic acid (KA) treatment and in response to the transection of the entorhino-hippocampal connection. We found that Nogo-A and Nogo receptor are differentially regulated after kainic acid or perforant pathway lesions. Lastly, we show that the regenerative potential of lesioned entorhino-hippocampal organotypic slice co-cultures is increased after blockage of Nogo-A with two IN-1 blocking antibodies. In conclusion, our results show that Nogo and its receptor might play key roles during development of hippocampal connections and that they are implicated in neuronal plasticity in the adult.

A collection of cDNAs enriched in upper cortical layers of the embryonic mouse brain.

In an attempt to elucidate the molecular basis of neuronal migration and corticogenesis, we performed subtractive hybridization of mRNAs from the upper cortical layers (layer I and upper cortical plate) against mRNAs from the remaining cerebral cortex at E15-E16. We obtained a collection of subtracted cDNA clones and analyzed their 3' UTR sequences, 47% of which correspond to EST sequences, and may represent novel products. Among the cloned sequences, we identified gene products that have not been reported in brain or in the cerebral cortex before. We examined the expression pattern of 39 subtracted clones, which was enriched in the upper layers of the cerebral cortex at embryonic stages. The expression of most clones is developmentally regulated, and especially high in embryonic and early postnatal stages. Four of the unknown clones were studied in more detail and identified as a new member of the tetraspanin superfamily, a putative RNA binding protein, a specific product of the adult dentate gyrus and a protein containing a beta-catenin repeat. We thus cloned a collection of subtracted cDNAs coding for protein products that may be involved in the development of the cerebral cortex.