Proteomic characterization of spinal cord synaptoneurosomes from Tg-SOD1/G93A mice supports a role for MNK1 and local translation in the early stages of amyotrophic lateral sclerosis.

The isolation of synaptoneurosomes (SNs) represents a useful means to study synaptic events. However, the size and density of synapses varies in different regions of the central nervous system (CNS), and this also depends on the experimental species studied, making it difficult to define a generic protocol for SNs preparation. To characterize synaptic failure in the spinal cord (SC) in the Tg-SOD1/G93A mouse model of amyotrophic lateral sclerosis (ALS), we applied a method we originally designed to isolate cortical and hippocampal SNs to SC tissue. Interestingly, we found that the SC SNs were isolated in a different gradient fraction to the cortical/hippocampal SNs. We compared the relative levels of synaptoneurosomal proteins in wild type (WT) animals, with control (Tg-SOD1) or Tg-SOD1/G93A mice at onset and those that were symptomatic using iTRAQ proteomics. The results obtained suggest that an important regulator of local synaptic translation, MNK1 (MAP kinase interacting serine/threonine kinase 1), might well influence the early stages of ALS.

CPEB1 is overexpressed in neurons derived from Down syndrome IPSCs and in the hippocampus of the mouse model Ts1Cje.

Trisomy 21, also known as Down syndrome (DS), is the most frequent genetic cause of intellectual impairment. In mouse models of DS, deficits in hippocampal synaptic plasticity have been observed, in conjunction with alterations to local dendritic translation that are likely to influence plasticity, learning and memory. Here we show that expression of a local translational regulator, the Cytoplasmic Polyadenylation Element Binding Protein 1 (CPEB1), is enhanced in hippocampal neurons from the Ts1Cje DS mouse model. Interestingly, this protein, which is also involved in dendritic mRNA transport, is overexpressed in dendrites of neurons derived from DS human induced pluripotent stem cells (hIPSCs). Moreover, there is an increase in the mRNA levels of α-Calmodulin Kinase II (α-CaMKII) and Microtubule-associated protein 1B (MAP1B), two dendritic mRNAs, in Ts1Cje synaptoneurosomes. Taking into account the fundamental role of CPEB1 protein and its target mRNAs in synaptic plasticity, these data could be relevant to the intellectual impairment in the context of DS.

Proteomic Analysis of Synaptoneurosomes Highlights the Relevant Role of Local Translation in the Hippocampus.

Several proteomic analyses have been performed on synaptic fractions isolated from cortex or even total brain, resulting in preparations with a high synaptic heterogeneity and complexity. Synaptoneurosomes (SNs) are subcellular membranous elements that contain sealed pre- and post-synaptic components. They are obtained by subcellular fractionation of brain homogenates and serve as a suitable model to study many aspects of the synapse physiology. Here the proteomic content of SNs isolated from hippocampus of adult mice, a brain region involved in memory that presents lower synaptic heterogeneity than cortex, is reported. Interestingly, in addition to pre- and post-synaptic proteins, proteins involved in RNA binding and translation are overrepresented in this preparation. These results validate the protocol previously reported for SNs isolation, and, as reported by other authors, highlight the relevance of local synaptic translation for hippocampal physiology.

Local translation of the Down syndrome cell adhesion molecule (DSCAM) mRNA in the vertebrate central nervous system.

Local translation of synaptic mRNAs is an important process related to key aspects of central nervous system development and physiology, including dendritogenesis, axonal growth cone morphology and guidance and synaptic plasticity. Accordingly, local translation is compromised in several intellectual disabilities, including Fragile X syndrome, tuberous sclerosis and Down syndrome. Down Syndrome Cell Adhesion Molecule (DSCAM) is a gene with ascribed functions in neuronal wiring that belongs to the Down Syndrome Critical Region (DSCR) of chromosome 21. In this review, we discuss the evidence for local translation of the DSCAM mRNA in dendrites and axonal growth cones of mouse hippocampal neurons, as well as the possible functions of the locally translated DSCAM protein.

Rapamycin restores BDNF-LTP and the persistence of long-term memory in a model of Down's syndrome.

Down's syndrome (DS) is the most prevalent genetic intellectual disability. Memory deficits significantly contribute to the cognitive dysfunction in DS. Previously, we discovered that mTOR-dependent local translation, a pivotal process for some forms of synaptic plasticity, is deregulated in a DS mouse model. Here, we report that these mice exhibit deficits in both synaptic plasticity (i.e., BDNF-long term potentiation) and the persistence of spatial long-term memory. Interestingly, these deficits were fully reversible using rapamycin, a Food and Drug Administration-approved specific mTOR inhibitor; therefore, rapamycin may be a novel pharmacotherapy to improve cognition in DS.

An increase in basal BDNF provokes hyperactivation of the Akt-mammalian target of rapamycin pathway and deregulation of local dendritic translation in a mouse model of Down's syndrome.

As in other diseases associated with mental retardation, dendrite morphology and synaptic plasticity are impaired in Down's syndrome (DS). Both these features of neurons are critically influenced by BDNF, which regulates local dendritic translation through phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (mTOR) and Ras-ERK signaling cascades. Here we show that the levels of BDNF and phosphorylated Akt-mTOR (but not Ras-ERK) pathway proteins are augmented in hippocampal dendrites of Ts1Cje mice, a DS model. Consequently, the rate of local dendritic translation is abnormally high and the modulatory effect of exogenous BDNF is lost. Interestingly, rapamycin (a Food and Drug Administration-approved drug) restores normal levels of phosphorylated Akt-mTOR proteins and normal rates of local translation in Ts1Cje neurons, opening new therapeutic perspectives for DS. The NMDAR inhibitors APV, MK-801, and memantine also restore the normal levels of phospho-mTOR in dendrites of Ts1Cje hippocampal neurons. We propose a model to explain how BDNF-mediated regulation of local translation is lost in the Ts1Cje hippocampus through the establishment of a glutamatergic positive-feedback loop. Together, these findings help elucidate the mechanisms underlying altered synaptic plasticity in DS.

NMDA-mediated regulation of DSCAM dendritic local translation is lost in a mouse model of Down's syndrome.

Down's syndrome cell adhesion molecule (DSCAM) belongs to the Down's syndrome critical region of human chromosome 21, and it encodes a cell adhesion molecule involved in dendrite morphology and neuronal wiring. Although the function of DSCAM in the adult brain is unknown, its expression pattern suggests a role in synaptic plasticity. Local mRNA translation is a key process in axonal growth, dendritogenesis, and synaptogenesis during development, and in synaptic plasticity in adulthood. Here, we report the dendritic localization of DSCAM mRNA in the adult mouse hippocampus, where it associates with CPEB1 [cytoplasmic polyadenylation element (CPE) binding protein 1], an important regulator of mRNA transport and local translation. We identified five DSCAM isoforms produced by alternative polyadenylation bearing different combinations of regulatory CPE motifs. Overexpression of DSCAM in hippocampal neurons inhibited dendritic branching. Interestingly, dendritic levels of DSCAM mRNA and protein were increased in hippocampal neurons from Ts1Cje mice, a model of Down's syndrome. Most importantly, DSCAM dendritic translation was rapidly induced by NMDA in wild-type, but not in Ts1Cje neurons. We propose that impairment of the NMDA-mediated regulation of DSCAM translation may contribute to the alterations in dendritic morphology and/or synaptic plasticity in Down's syndrome.

Local translation of dendritic RhoA revealed by an improved synaptoneurosome preparation.

Changes in dendritic spine morphology, a hallmark of synaptic plasticity, involve remodeling of the actin cytoskeleton, a process that is regulated by Rho GTPases. RhoA, a member of this GTPase family, segregates to dendrites in differentiated neurons. Given the emerging role of dendritic mRNA local translation in synaptic plasticity, we have assessed the possible localization and translation of RhoA mRNA at dendrites. At this end, we have developed and describe here in detail an improved method for isolating hippocampal and neocortical mouse synaptoneurosomes. This synaptoneurosomal preparation is much more enriched in synaptic proteins than those obtained in former methods, exhibits bona fide electron microscopy pre- and postsynaptic morphologies, contains abundant dendritic mRNAs, and is competent for activity-regulated protein synthesis. Using this preparation, we have found that RhoA mRNA is dendritically localized and its local translation is enhanced by BDNF stimulation. These findings suggest that some of the known functions of RhoA on spine morphology may be mediated by regulating its local translation.

Prenatal treatment with rapamycin restores enhanced hippocampal mGluR-LTD and mushroom spine size in a Down's syndrome mouse model.

Down syndrome (DS) is the most frequent genetic cause of intellectual disability including hippocampal-dependent memory deficits. We have previously reported hippocampal mTOR (mammalian target of rapamycin) hyperactivation, and related plasticity as well as memory deficits in Ts1Cje mice, a DS experimental model. Here we characterize the proteome of hippocampal synaptoneurosomes (SNs) from these mice, and found a predicted alteration of synaptic plasticity pathways, including long term depression (LTD). Accordingly, mGluR-LTD (metabotropic Glutamate Receptor-LTD) is enhanced in the hippocampus of Ts1Cje mice and this is correlated with an increased proportion of a particular category of mushroom spines in hippocampal pyramidal neurons. Remarkably, prenatal treatment of these mice with rapamycin has a positive pharmacological effect on both phenotypes, supporting the therapeutic potential of rapamycin/rapalogs for DS intellectual disability.

Roles for DSCAM and DSCAML1 in central nervous system development and disease.

DSCAMs (Down syndrome cell adhesion molecules) are a group of immunoglobulin-like transmembrane proteins that contain fibronectin III domains. The founding member of the family was isolated in a positional cloning study that sought to identify genes located on chromosome 21 at the locus 21q22.2-q22.3 that is implicated in the neurological and cardiac phenotypes associated with Down's syndrome. In Drosophila, Dscam proteins are involved in neuronal wiring, while in vertebrates, the role of these cell adhesion molecules in neurogenesis, dendritogenesis, axonal outgrowth, synaptogenesis, and synaptic plasticity is only just beginning to be understood. In this chapter, we will review the functions ascribed to the two paralogous proteins found in humans, DSCAM and DSCAML1 (DSCAM-like 1), based on findings in knockout mice. The signaling pathways downstream of DSCAM activation and the role of DSCAM miss-expression in disease will be also discussed, particularly with regard to the intellectual disability in Down's syndrome.

The Akt-mTOR pathway in Down's syndrome: the potential use of rapamycin/rapalogs for treating cognitive deficits.

An increasing amount of evidence suggests that the dysregulation of the Akt-mTOR (Akt-mammalian Target Of Rapamycin) signaling network is associated with intellectual disabilities, such as fragile X, tuberous sclerosis and Rett's syndrome. The Akt-mTOR pathway is involved in dendrite morphogenesis and synaptic plasticity, and it has been shown to modulate both glutamatergic and GABAergic synaptic transmission. We have recently shown that the AktmTOR pathway is hyperactive in the hippocampus of Ts1Cje mice, a model of Down's syndrome, leading to increased local dendritic translation that could interfere with synaptic plasticity. Rapamycin and rapalogs are specific inhibitors of mTOR, and some of these inhibitors are Food and Drug Administration-approved drugs. In this review, we discuss the molecular basis and consequences of Akt-mTOR hyperactivation in Down's syndrome, paying close attention to alterations in the molecular mechanisms underlying synaptic plasticity. We also analyze the pros and cons of using rapamycin/rapalogs for the treatment of the cognitive impairments associated with this condition.

Deregulated mTOR-mediated translation in intellectual disability.

Local translation of dendritic mRNAs is a key aspect of dendrite and spine morphogenesis and synaptic plasticity, two phenomena generally compromised in intellectual disability disorders. Mammalian target of rapamycin (mTOR) is a protein kinase involved in a plethora of functions including dendritogenesis, plasticity and the regulation of local translation. Hence, this kinase may well be implicated in intellectual disability. Hyperactivation of mTOR has been recently reported in mouse models of Fragile X and tuberous sclerosis, two important causes of intellectual disability. Moreover, local dendritic translation seems to be increased in Fragile X syndrome. Recent findings show that the mTOR pathway is also deregulated in murine models of Rett's syndrome and Down's syndrome. As in Fragile X, local dendritic translation seems to be abnormally active in Down's syndrome mice, while rapamycin, a Food and Drug Administration-approved mTOR inhibitor, restores normal rates of translation. Rapamycin administration in tuberous sclerosis mice rescues deficits in behavior and synaptic plasticity. Indeed, mTOR-dependent deregulation of local translation may be a common trait in different intellectual deficiencies, suggesting that mTOR inhibitors may have significant therapeutic potential for the treatment of diverse forms of cognitive impairment.

Dendritic localization and activity-dependent translation of Engrailed1 transcription factor.

Engrailed1 (En1) is a homeoprotein transcription factor expressed throughout adulthood in several midbrain cells, including the dopaminergic neurons of the substantia nigra. Here we report the presence of Engrailed protein and En1 mRNA in proximal dendrites of these neurons and of En1 mRNA in ventral midbrain synaptoneurosomes. We show that the 3' untranslated region of En1 mRNA contains a functional cytoplasmic polyadenylation element (CPE), suggesting that its dendritic localization is regulated by CPE binding protein (CPEB). In order to evaluate activity-regulated translation, conditions were developed using primary midbrain neurons. With this in vitro model, En1 mRNA translation is increased by depolarization in a polyadenylation dependent manner. Furthermore, En1 translation is prevented by rapamycin, implicating the mTOR pathway, which is known to regulate dendritic translation. Together, these results suggest an activity-dependent role for Engrailed in midbrain dopaminergic neuron physiology.

Recycling and EH domain proteins at the synapse.

In neurons, a network of endocytic proteins accomplishes highly regulated processes such as synaptic vesicle cycling and the timely internalization of intracellular signaling molecules. In this review, we discuss recent advances on molecular networks created through interactions between proteins bearing the Eps15 homology (EH) domain and partner proteins containing the Asn-Pro-Phe (NPF) motif, which participate in important aspects of neuronal function as the synaptic vesicle cycle, the internalization of nerve growth factor (NGF), the determination of neuronal cell fate, the development of synapses and the trafficking of postsynaptic receptors. We discuss novel functional findings on the role of intersectin and synaptojanin and then we focus on the features of an emerging family of EH domain proteins termed EHDs (EH domain proteins), which are important for endocytic recycling of membrane proteins.

ABC-type neutral amino acid permease N-I is required for optimal diazotrophic growth and is repressed in the heterocysts of Anabaena sp. strain PCC 7120.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N2 in differentiated cells called heterocysts. The products of Anabaena open reading frames (ORFs) all1046, all1047, all1284, alr1834 and all2912 were identified as putative elements of a neutral amino acid permease. Anabaena mutants of these ORFs were strongly affected (1-12% of the wild-type activity) in the transport of Pro, Phe, Leu and Gly and also impaired (17-30% of the wild-type activity) in the transport of Ala and Ser. These results identified those ORFs as the nat genes encoding the N-I neutral amino acid permease. According to amino acid sequence homologies, natA (all1046) and natE (all2912) encode ATPases, natC (all1047) and natD (all1284) encode transmembrane proteins, and natB (alr1834) encodes a periplasmic substrate-binding protein of an ABC-type uptake transporter. The natA, natC, natD and natE mutants showed defects in Gln and His uptake that were not observed in the natB mutant suggesting that NatB is not a binding protein for Gln or His. The nat mutants released hydrophobic amino acids to the medium, and amino acid release took place at higher levels in cultures incubated in the absence of combined N than in the presence of nitrate. Alanine was the amino acid released at highest levels, and its release was impaired in a mutant unable to develop heterocysts. The nat mutants were also impaired in diazotrophic growth, with natA, natC, natD and natE mutants showing more severe defects than the natB mutant. Expression of natA and natC, which constitute an operon, natCA, as well as of natB was studied and found to take place in vegetative cells but not in the heterocysts. These results indicate that the N-I permease is necessary for normal growth of Anabaena sp. strain PCC 7120 on N2, and that this permease has a role in the diazotrophic filament specifically in the vegetative cells.

An ABC-type, high-affinity urea permease identified in cyanobacteria.

Urea is an important nitrogen source for many microorganisms, but urea active transporters have not been characterized at a molecular level in any bacterium. Cells of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 exhibited the capacity to take up [14C]-urea from low-concentration (<1 microM) urea solutions. The Ks of Anabaena cells for urea was about 0.11 microM, and the observed uptake activity involved the transport and metabolism of urea. In contrast to urease, which was constitutively ex-pressed, expression of the high-affinity urea uptake activity was subjected to nitrogen control. In an Anabaena ureG (urease-) mutant, a concentrative, active transport of urea could be demonstrated. We found that a mutant of open reading frame (ORF) sll0374 from the Synechocystis genomic sequence lacked urea transport activity. This ORF encoded a conserved component of an ABC-type transporter, but it is not clustered together with any other possible transporter-encoding gene. An Anabaena homologue of sll0374, urtE, was isolated and found to be part of a cluster of genes, urtABCDE, putatively encoding all the elements of an ABC-type permease. Although the longest transcript that we could detect only covered urtABC, the impairment of urea transport by inactivation of urtA, urtB or urtE suggested that the whole gene cluster is expressed producing the urea permease. Expression was induced under nitrogen-limiting conditions, and a complex promoter regulated by the cyanobacterial global nitrogen control transcription factor NtcA was found upstream from urtA. Our work adds urea to the known substrates of the versatile class of ABC-type transporters and suggests the involvement of a transporter of this superfamily in urea scavenging by some bacteria in natural environments.

Joint regulation of the MAP1B promoter by HNF3beta/Foxa2 and Engrailed is the result of a highly conserved mechanism for direct interaction of homeoproteins and Fox transcription factors.

The MAP1B (Mtap1b) promoter presents two evolutionary conserved overlapping homeoproteins and Hepatocyte nuclear factor 3beta (HNF3beta/Foxa2) cognate binding sites (defining putative homeoprotein/Fox sites, HF1 and HF2). Accordingly, the promoter domain containing HF1 and HF2 is recognized by cerebellum nuclear extracts containing Engrailed and Foxa2 and has regulatory functions in primary cultures of embryonic mesmetencephalic nerve cells. Transfection experiments further demonstrate that Engrailed and Foxa2 interact physiologically in a dose-dependent manner: Foxa2 antagonizes the Engrailed-driven regulation of the MAP1B promoter, and vice versa. This led us to investigate if Engrailed and Foxa2 interact directly. Direct interaction was confirmed by pull-down experiments, and the regions participating in this interaction were identified. In Foxa2 the interacting domain is the Forkhead box DNA-binding domain. In Engrailed, two independent interacting domains exist: the homeodomain and a region that includes the Pbx-binding domain. Finally, Foxa2 not only binds Engrailed but also Lim1, Gsc and Hoxa5 homeoproteins and in the four cases Foxa2 binds at least the homeodomain. Based on the involvement of conserved domains in both classes of proteins, it is proposed that the interaction between Forkhead box transcription factors and homeoproteins is a general phenomenon.