Search

Pyramidal neurons exhibit a complex dendritic tree that is decorated by a huge number of spine synapses receiving excitatory input. Synaptic signals not only act locally but are also conveyed to the nucleus of the postsynaptic neuron to regulate gene expression. This raises the question of how the spatio-temporal integration of synaptic inputs is accomplished at the genomic level and which molecular mechanisms are involved. Protein transport from synapse to nucleus has been shown in several studies and has the potential to encode synaptic signals at the site of origin and decode them in the nucleus. In this review, we summarize the knowledge about the properties of the synapto-nuclear messenger protein Jacob with special emphasis on a putative role in hippocampal neuronal plasticity. We will elaborate on the interactome of Jacob, the signals that control synapto-nuclear trafficking, the mechanisms of transport, and the potential nuclear function. In addition, we will address the organization of the Jacob/NSMF gene, its origin and we will summarize the evidence for the existence of splice isoforms and their expression pattern.

Microtubule severing regulates cytoskeletal rearrangement underlying various cellular functions. Katanin, a heterodimer, consisting of catalytic (p60) and regulatory (p80) subunits severs dynamic microtubules to modulate several stages of cell division. The role of p60 katanin in the mammalian brain with respect to embryonic and adult neurogenesis is poorly understood. Here, we generated a Katna1 knockout mouse and found that consistent with a critical role of katanin in mitosis, constitutive homozygous Katna1 depletion is lethal. Katanin p60 haploinsufficiency induced an accumulation of neuronal progenitors in the subventricular zone during corticogenesis, and impaired their proliferation in the adult hippocampus dentate gyrus (DG) subgranular zone. This did not compromise DG plasticity or spatial and contextual learning and memory tasks employed in our study, consistent with the interpretation that adult neurogenesis may be associated with selective forms of hippocampal-dependent cognitive processes. Our data identify a critical role for the microtubule-severing protein katanin p60 in regulating neuronal progenitor proliferation in vivo during embryonic development and adult neurogenesis.

eLife 10, e66165 (1-31) (2021). doi:10.7554/eLife.66165, Members of the SH3- and ankyrin repeat (SHANK) protein family are considered as master scaffolds of the postsynaptic density of glutamatergic synapses. Several missense mutations within the canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this proof-of-principle study, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3 and demonstrate that both mutant proteins indeed show distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and distal structural disturbances result in altered synaptic targeting and changes of protein turnover at synaptic sites in rat primary hippocampal neurons., Published by eLife Sciences Publications, Cambridge

Jacob is a synapto-nuclear messenger protein that couples NMDAR activity to CREB-dependent gene expression. In this study, we investigated the nuclear distribution of Jacob and report a prominent targeting to the nuclear envelope that requires NMDAR activity and nuclear import. Immunogold electron microscopy and proximity ligation assay combined with STED imaging revealed preferential association of Jacob with the inner nuclear membrane where it directly binds to LaminB1, an intermediate filament and core component of the inner nuclear membrane (INM). The association with the INM is transient; it involves a functional nuclear export signal in Jacob and a canonical CRM1-RanGTP-dependent export mechanism that defines the residing time of the protein at the INM. Taken together, the data suggest a stepwise redistribution of Jacob within the nucleus following nuclear import and prior to nuclear export.

The complex morphology of neurons, the specific requirements of synaptic neurotransmission and the accompanying metabolic demands create a unique challenge for proteostasis. The main machineries for neuronal protein synthesis and degradation are localized in the soma, while synaptic junctions are found at vast distances from the cell body. Sophisticated mechanisms must, therefore, ensure efficient delivery of newly synthesized proteins and removal of faulty proteins. These requirements are exacerbated at presynaptic sites, where the demands for protein turnover are especially high due to synaptic vesicle release and recycling that induces protein damage in an intricate molecular machinery, and where replacement of material is hampered by the extreme length of the axon. In this review, we will discuss the contribution of the two major pathways in place, autophagy and the endolysosomal system, to presynaptic protein turnover and presynaptic function. Although clearly different in their biogenesis, both pathways are characterized by cargo collection and transport into distinct membrane-bound organelles that eventually fuse with lysosomes for cargo degradation. We summarize the available evidence with regard to their degradative function, their regulation by presynaptic machinery and the cargo for each pathway. Finally, we will discuss the interplay of both pathways in neurons and very recent findings that suggest non-canonical functions of degradative organelles in synaptic signalling and plasticity.

Aerobic exercise is a potential life-style intervention to delay cognitive decline and neurodegeneration. Elevated serum levels of the anti-aging protein α-Klotho (αKL) are a potential mediating factor of exercise benefits on cognition. Here, we examined in older adults how exercise-related changes of αKL levels in serum relate to changes in cerebral blood flow (CBF), hippocampal volumes and episodic memory. We analyzed data from a previously published intervention study in which forty cognitively healthy subjects were pseudo-randomly assigned to either a cardiovascular exercise group (treadmill training, n=21) or control group (indoor progressive-muscle relaxation/stretching, n=19). 3-Tesla gadolinium perfusion imaging was used to track hippocampal CBF changes and high resolution 7-Tesla-T1-images were used to track hippocampal volume changes. Changes in episodic memory performance were measured using the complex figure test (CFT). Longitudinal changes were compared between groups and analyzed with a multiple linear regression approach. CFT and hippocampal volume changes significantly predicted changes in serum αKL levels. For CFT, this effect was found in the exercise but not the control group. Collectively the data suggest that αKL level increases induced by exercise can be associated with improved hippocampal function in older adults.

© 2019 Elsevier Inc. Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders (“synaptopathies”). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org). The SynGO consortium presents a framework to annotate synaptic protein locations and functions and annotations for 1,112 synaptic genes based on published experimental evidence. SynGO reports exceptional features and disease associations for synaptic genes and provides an online data analysis platform.

SummaryAmphisomes are transient organelles that derive from fusion of autophagosomes with late endosomes. They rapidly transform into degradative autolysosomes, whereas non-degradative roles of the autophagic pathway have been barely described. Here we show that in neurons BDNF/TrkB receptor bearing Rab7 / Light chain 3 (LC3) - positive amphisomes signal at presynaptic boutons during retrograde trafficking to the soma. Local signaling and inward transport essentially require the Rap GTPase-activating (RapGAP) protein SIPA1L2, which directly binds to TrkB and Snapin to connect TrkB-containing amphisomes to dynein. Association with LC3 regulates the RapGAP activity of SIPA1L2 and thereby retrograde trafficking. Following induction of presynaptic plasticity amphisomes dissociate from dynein at boutons, and this enables local signaling and promotes transmitter release. Accordingly,sipa1l2knockout mice show impaired BDNF-dependent presynaptic plasticity. Collectively, the data suggest that TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.

The centrosome is thought to be the major neuronal microtubule‐organizing center (MTOC) in early neuronal development, producing microtubules with a radial organization. In addition, albeit in vitro, recent work showed that isolated centrosomes could serve as an actin‐organizing center, raising the possibility that neuronal development may, in addition, require a centrosome‐based actin radial organization. Here, we report, using super‐resolution microscopy and live‐cell imaging of cultured rodent neurons, F‐actin organization around the centrosome with dynamic F‐actin aster‐like structures with F‐actin fibers extending and retracting actively. Photoactivation/photoconversion experiments and molecular manipulations of F‐actin stability reveal a robust flux of somatic F‐actin toward the cell periphery. Finally, we show that somatic F‐actin intermingles with centrosomal PCM‐1 (pericentriolar material 1 protein) satellites. Knockdown of PCM‐1 and disruption of centrosomal activity not only affect F‐actin dynamics near the centrosome but also in distal growth cones. Collectively, the data show a radial F‐actin organization during early neuronal development, which might be a cellular mechanism for providing peripheral regions with a fast and continuous source of actin polymers, hence sustaining initial neuronal development., During early neuronal development, centrosomes regulate F‐actin organization, thereby ensuring proper growth cone dynamics and neurite length.

Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity., There is growing evidence that autophagy might serve specialized functions in neurons besides its role in protein homeostasis. In this study, authors demonstrate that axonal retrograde transport of BDNF/TrkB in neuronal amphisomes is involved in plasticity-relevant local signaling at presynaptic boutons and that SIPA1L2, a member of the SIPA1L family of neuronal RapGAPs, associates via LC3b to TrkB-containing amphisomes to regulate its motility and signaling at the axon terminals

Yokukansan (YKS) is a traditional Japanese herbal medicine that has been used in humans for the treatment of several neurological conditions, such as age-related anxiety and behavioral and psychological symptoms (BPSD) related to multiple forms of dementia, including Alzheimer’s disease (AD). However, the cellular and molecular mechanisms targeted by YKS in the brain are not completely understood. Here, we compared the efficacy of YKS in ameliorating the age- and early-onset familial AD-related behavioral and cellular defects in two groups of animals: 18- to 22-month-old C57BL6/J wild-type mice and 6- to 9-month-old 5xFAD mice, as a transgenic mouse model of this form of AD. Animals were fed food pellets that contained YKS or vehicle. After 1–2 months of YKS treatment, we evaluated the cognitive improvements in both the aged and 5xFAD transgenic mice, and their brain tissues were further investigated to assess the molecular and cellular changes that occurred following YKS intake. Our results show that both the aged and 5xFAD mice exhibited impaired behavioral performance in novel object recognition and contextual fear conditioning (CFC) tasks, which was significantly improved by YKS. Further analyses of the brain tissue from these animals indicated that in aged mice, this improvement was associated with a reduction in astrogliosis, microglia activation and downregulation of the extracellular matrix (ECM), whereas in 5xFAD mice, none of these mechanisms were evident. These results show the differential action of YKS in healthy aged and 5xFAD mice. However, both aged and 5xFAD YKS-treated mice showed increased neuroprotective signaling through protein kinase B/Akt as the common mode of action. Our data suggest that YKS may impart its beneficial effects through Akt signaling in both 5xFAD mice and aged mice, with multiple additional mechanisms potentially contributing to its beneficial effects in aged animals.

The advances in mass spectrometry based proteomics in the past 15 years have contributed to a deeper appreciation of protein networks and the composition of functional synaptic protein complexes. However, research on protein dynamics underlying core mechanisms of synaptic plasticity in brain lag far behind. In this review, we provide a synopsis on proteomic research addressing various aspects of synaptic function. We discuss the major topics in the study of protein dynamics of the chemical synapse and the limitations of current methodology. We highlight recent developments and the future importance of multidimensional proteomics and metabolic labeling. Finally, emphasis is given on the conceptual framework of modern proteomics and its current shortcomings in the quest to gain a deeper understanding of synaptic plasticity.

Endocannabinoids are important players in neural development and function. They act via receptors, whose activation inhibits cAMP production. The aim of the paper was to look for calcium- and cAMP-signaling cross-talk mediated by cannabinoid CB1 receptors (CB1R) and to assess the relevance of EF-hand CaM-like calcium sensors in this regard. Using a heterologous expression system, we demonstrated that CB1R interacts with calneuron-1 and NCS1 but not with caldendrin. Furthermore, interaction motives were identified in both calcium binding proteins and the receptor, and we showed that the first two sensors competed for binding to the receptor in a Ca2+-dependent manner. Assays in neuronal primary cultures showed that, CB1R-NCS1 complexes predominate at basal Ca2+ levels, whereas in the presence of ionomycin, a calcium ionophore, CB1R-calneuron-1 complexes were more abundant. Signaling assays following forskolin-induced intracellular cAMP levels showed in mouse striatal neurons that binding of CB1R to NCS1 is required for CB1R-mediated signaling, while the binding of CB1R to calneuron-1 completely blocked Gi-mediated signaling in response to a selective receptor agonist, arachidonyl-2-chloroethylamide. Calcium levels and interaction with calcium sensors may even lead to apparent Gs coupling after CB1R agonist challenge.

Altres ajuts: CiberNed's intramural program (Ref. No. PI2016/02); Fundació la Marató de TV3 N -methyl--aspartate receptors (NMDARs) respond to glutamate to allow the influx of calcium ions and the signaling to the mitogen-activated protein kinase (MAPK) cascade. Both MAPK- and Ca 2+ -mediated events are important for both neurotransmission and neural cell function and fate. Using a heterologous expression system, we demonstrate that NMDAR may interact with the EF-hand calcium-binding proteins calmodulin, calneuron-1, and NCS1 but not with caldendrin. NMDARs were present in primary cultures of both neurons and microglia from cortex and hippocampus. Calmodulin in microglia, and calmodulin and NCS1 in neurons, are necessary for NMDA-induced MAP kinase pathway activation. Remarkably, signaling to the MAP kinase pathway was blunted in primary cultures of cortical and hippocampal neurons and microglia from wild-type animals by proteins involved in neurodegenerative diseases: α-synuclein, Tau, and p-Tau. A similar blockade by pathogenic proteins was found using samples from the APP transgenic Alzheimer's disease model. Interestingly, a very marked increase in NMDAR-NCS1 complexes was identified in neurons and a marked increase of both NMDAR-NCS1 and NMDAR-CaM complexes was identified in microglia from the transgenic mice. The results show that α-synuclein, Tau, and p-Tau disrupt the signaling of NMDAR to the MAPK pathway and that calcium sensors are important for NMDAR function both in neurons and microglia. Finally, it should be noted that the expression of receptor-calcium sensor complexes, specially those involving NCS1, is altered in neural cells from APP mouse embryos/pups.

Local signaling events at synapses or axon terminals must be communicated to the nucleus to elicit transcriptional responses. The lengths of neuronal processes pose a significant challenge for such intracellular communication. This challenge is met by mechanisms ranging from rapid signals encoded in calcium waves to slower macromolecular signaling complexes carried by molecular motors. Here we summarize recent findings on macromolecular signaling from the synapse to the nucleus, in comparison to those employed in injury signaling along axons. A number of common themes emerge, including combinatorial signal encoding by post-translational mechanisms such as differential phosphorylation and proteolysis, and conserved roles for importins in coordinating signaling complexes. Neurons may integrate ionic flux with motor-transported signals as a temporal code for synaptic plasticity signaling.

Long-lasting changes in neuronal excitability require activity-dependent gene expression and therefore the transduction of synaptic signals to the nucleus. Synaptic activity is rapidly relayed to the nucleus by membrane depolarization and the propagation of Ca(2+)-waves. However, it is unlikely that Ca(2+)-transients alone can explain the specific genomic response to the plethora of extracellular stimuli that control gene expression. In recent years a steadily growing number of studies report the transport of proteins from synapse to nucleus. Potential mechanisms for active retrograde transport and nuclear targets for these proteins have been identified and recent reports assigned first functions to this type of long-distance signaling. In this review we will discuss how the dissociation of synapto-nuclear protein messenger from synaptic and extrasynaptic sites, their transport, nuclear import and the subsequent genomic response relate to the prevailing concept behind this signaling mechanism, the encoding of signals at their site of origin and their decoding in the nucleus.

SummaryThe activation of N-methyl-D-aspartate-receptors (NMDARs) in synapses provides plasticity and cell survival signals, whereas NMDARs residing in the neuronal membrane outside synapses trigger neurodegeneration. At present, it is unclear how these opposing signals are transduced to and discriminated by the nucleus. In this study, we demonstrate that Jacob is a protein messenger that encodes the origin of synaptic versus extrasynaptic NMDAR signals and delivers them to the nucleus. Exclusively synaptic, but not extrasynaptic, NMDAR activation induces phosphorylation of Jacob at serine-180 by ERK1/2. Long-distance trafficking of Jacob from synaptic, but not extrasynaptic, sites depends on ERK activity, and association with fragments of the intermediate filament α-internexin hinders dephosphorylation of the Jacob/ERK complex during nuclear transit. In the nucleus, the phosphorylation state of Jacob determines whether it induces cell death or promotes cell survival and enhances synaptic plasticity.

Elevated c-Jun levels result in apoptosis and are evident in neurodegenerative disorders such as Alzheimer's disease and dementia and after global cerebral insults including stroke and epilepsy. NMDA receptor (NMDAR) antagonists block c-Jun upregulation and prevent neuronal cell death following excitotoxic insults. However, the molecular mechanisms regulating c-Jun abundance in neurons are poorly understood. Here, we show that the synaptic component Proline rich 7 (PRR7) accumulates in the nucleus of hippocampal neurons following NMDAR activity. We find that PRR7 inhibits the ubiquitination of c-Jun by E3 ligase SCF(FBW) (7) (FBW7), increases c-Jun-dependent transcriptional activity, and promotes neuronal death. Microarray assays show that PRR7 abundance is directly correlated with transcripts associated with cellular viability. Moreover, PRR7 knockdown attenuates NMDAR-mediated excitotoxicity in neuronal cultures in a c-Jun-dependent manner. Our results show that PRR7 links NMDAR activity to c-Jun function and provide new insights into the molecular processes that underlie NMDAR-dependent excitotoxicity.

Synapses and nuclei are connected by bidirectional communication mechanisms that enable information transfer encoded by macromolecules. Here, we identified RNF10 as a novel synaptonuclear protein messenger. RNF10 is activated by calcium signals at the postsynaptic compartment and elicits discrete changes at the transcriptional level. RNF10 is enriched at the excitatory synapse where it associates with the GluN2A subunit of NMDA receptors (NMDARs). Activation of synaptic GluN2A-containing NMDARs and induction of long term potentiation (LTP) lead to the translocation of RNF10 from dendritic segments and dendritic spines to the nucleus. In particular, we provide evidence for importin-dependent long-distance transport from synapto-dendritic compartments to the nucleus. Notably, RNF10 silencing prevents the maintenance of LTP as well as LTP-dependent structural modifications of dendritic spines. DOI: http://dx.doi.org/10.7554/eLife.12430.001, eLife digest Brain activity depends on the communication between neurons. This process takes place at the junctions between neurons, which are known as synapses, and typically involves one of the cells releasing a chemical messenger that binds to receptors on the other cell. The binding triggers a cascade of events inside the recipient cell, including the production of new receptors and their insertion into the cell membrane. These changes strengthen the synapse and are thought to be one of the ways in which the brain establishes and maintains memories. However, in order to induce these changes at the synapse, neurons must be able to activate the genes that encode their component parts. These genes are present inside the cell nucleus, which is located some distance away from the synapse. Studies have shown that signals can be sent from the nucleus to the synapse and vice versa, enabling the two parts of the cell to exchange information. Synapses that communicate using a chemical called glutamate have been particularly well studied; but it still remains unclear how the activation of receptors at these “glutamatergic synapses” is linked to activation of genes inside the nucleus at the molecular level. Dinamarca, Guzzetti et al. have now discovered that this process at glutamatergic synapses involves the movement of a protein messenger to the nucleus. Specifically, activation at synapses of a particularly common subtype of receptor, called NMDA, causes a protein called Ring Finger protein 10 (or RNF10 for short) to move from the synapse to the nucleus. To leave the synapse, RNF10 first has to bind to proteins called importins, which transport RNF10 into the nucleus. Once inside the nucleus, RNF10 binds to another protein that interacts with the DNA to start the production of new synaptic proteins. Further work is required to identify the molecular mechanisms that trigger RNF10 to leave the synapse. In addition, future studies should evaluate the levels and activity of RNF10 in brain disorders in which synapses are known to function abnormally. DOI: http://dx.doi.org/10.7554/eLife.12430.002

Transmembrane adaptor proteins (TRAPs) are important organisers for the transduction of immunoreceptor-mediated signals. Prr7 is a TRAP that regulates T cell receptor (TCR) signalling and potently induces cell death when overexpressed in human Jurkat T cells. Whether endogenous Prr7 has a similar functional role is currently unknown. To address this issue, we analysed the development and function of the immune system in Prr7 knockout mice. We found that loss of Prr7 partially impairs development of single positive CD4+ T cells in the thymus but has no effect on the development of other T cell subpopulations, B cells, NK cells, or NKT cells. Moreover, Prr7 does not affect the TCR signalling pathway as T cells derived from Prr7 knockout and wild-type animals and stimulated in vitro express the same levels of the activation marker CD69, and retain their ability to proliferate and activate induced cell death programs. Importantly, Prr7 knockout mice retained the capacity to mount a protective immune response when challenged with Listeria monocytogenes infection in vivo. In addition, T cell effector functions (activation, migration, and reactivation) were normal following induction of experimental autoimmune encephalomyelitis (EAE) in Prr7 knockout mice. Collectively, our work shows that loss of Prr7 does not result in a major immune system phenotype and suggests that Prr7 has a dispensable function for TCR signalling.

It is widely accepted that signaling between synapses and neuronal nuclei regulates activity-dependent gene transcription that plays a crucial role in synaptic plasticity. However, despite many years of research, it is still essentially unclear how signals from distal synapses are transduced to the nucleus. Several studies in the past decade have proposed mechanisms of activity-dependent transport of synaptic proteins to the nucleus. In this chapter we will discuss these mechanisms and how this transport might couple in particular NMDAR activation to specific aspects of gene transcription.

How do neurons integrate intracellular communication from synapse to nucleus and back? Here we briefly summarize aspects of this topic covered by a symposium at Neuroscience 2011. A rich repertoire of signaling mechanisms link both dendritic terminals and axon tips with neuronal soma and nucleus, using motor-dependent transport machineries to traverse the long intracellular distances along neuronal processes. Activation mechanisms at terminals include localized translation of dendritic or axonal RNA, proteolytic cleavage of receptors or second messengers, and differential phosphorylation of signaling moieties. Signaling complexes may be transported in endosomes, or as non-endosomal complexes associated with importins and dynein. Anterograde transport of RNA granules from the soma to neuronal processes, coupled with retrograde transport of proteins translated locally at terminals or within processes, may fuel ongoing bidirectional communication between soma and synapse to modulate synaptic plasticity as well as neuronal growth and survival decisions.

The regulated local synthesis of PtdIns4 P and PtdIns(4,5) P 2 is crucial for TGN ( trans -Golgi network)–plasma membrane trafficking. The activity of PI4Kβ (phosphoinositide 4-kinase IIIβ) at the Golgi membrane is a first mandatory step in this process. In addition to PI4Kβ activity, elevated Ca2+ levels are also needed for the exit of vesicles from the TGN. The reason for this Ca2+ requirement is at present unclear. In the present review, we discuss the role of neuronal Ca2+-sensor proteins in the regulation of PI4Kβ and suggest that this regulation might impose a need for elevated Ca2+ levels for a late step of vesicle assembly. Abbreviations: ER, endoplasmic reticulum; hGH, human growth hormone; NCS, neuronal Ca2+-sensor protein; PI4Kβ, phosphoinositide 4-kinase IIIβ; PTV, Piccolo–Bassoon transport vesicle; TGN, trans-Golgi network

Y-P30 is a polypeptide survival promoting factor that has significant impact on the survival and differentiation of neurons in the developing brain. To address its potential role in brain injury we tested its neuroprotective effects in the oxygen–glucose deprivation (OGD) model with hippocampal slice cultures as an in vitro assay for ischemia. We could demonstrate that supplementation with Y-P30 leads to a significant neuroprotection at concentrations of 200 nM and 2 μM when it was added to the medium of hippocampal slice cultures 2 h before starting the deprivation of oxygen and glucose. A significant neuroprotective effect was found when the peptide was applied 2 h after injury. Y-P30 oligomerises in large complexes, which might hinder the passage through the culture membranes in our system. We therefore also applied Y-P30 directly on the hippocampal slices, which led to the most robust neuroprotection even at very low concentrations.

In neurons multiple signaling pathways converge in the nucleus to regulate the expression of genes associated with long-term structural changes of synapto-dendritic input. Of pivotal importance for this type of transcriptional regulation is synapse-to-nucleus communication. Several studies suggest that the nuclear transport of proteins from synapses is involved in this signaling process, including evidence that synapses contain proteins with nuclear localization sequences and components of the nuclear import machinery. Here, we review the evidence for synapse-to-nucleus signaling by means of retrograde transport of proteins from distal processes. We discuss the mechanisms involved in their translocation and their role in the control of nuclear gene expression. Finally, we summarize the current thinking regarding the functional implications of nuclear signaling and address open questions in this evolving area of neuroscience.

Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon–DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.

Many studies in recent years suggest that schizophrenia is a synaptic disease that crucially involves a hypofunction of N-methyl-D-aspartate receptor-mediated signaling. However, at present it is unclear how these pathological processes are reflected in the protein content of the synapse. We have employed two-dimensional gel electrophoresis in conjunction with mass spectrometry to characterize and compare the synaptic proteomes of the human left dorsolateral prefrontal cortex in chronic schizophrenia and of the cerebral cortex of rats treated subchronically with ketamine. We found consistent changes in the synaptic proteomes of human schizophrenics and in rats with induced ketamine psychosis compared to controls. However, commonly regulated proteins between both groups were very limited and only prohibitin was found upregulated in both chronic schizophrenia and the rat ketamine model. Prohibitin, however, could be a new potential marker for the synaptic pathology of schizophrenia and might be causally involved in the disease process. © 2008 Nature Publishing Group All rights reserved.

Soluble forms of oligomeric amyloid beta (AβO) are involved in the loss of synaptic plasticity and memory, especially in early phases of Alzheimer's disease. Stimulation of dopamine D1/D5 receptors (D1R/D5R) is known to increase surface expression of synaptic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate subtype glutamate and N-methyl-D-aspartate subtype glutamate receptors and facilitates the induction of the late phase of long-term potentiation (LTP), probably via a related mechanism. In this study, we show that the D1/D5R agonist SKF38393 protects LTP of hippocampal CA1 synapses from the deleterious action of oligomeric amyloid beta. Unexpectedly, the D1R/D5R-mediated recovery of LTP is independent of protein kinase A or phospholipase C pathways. Instead, we found that the inhibition of Src-family tyrosine kinases completely abolished the protective effects of D1R/D5R stimulation in a cellular model of learning and memory.

Calneuron-1 and -2 are members of the neuronal calcium-binding protein family (nCaBP). They are transmembrane Calmodulin-like EF-hand Ca2+-sensors, and a function in the control of Golgi-to-plasma membrane vesicle trafficking has been assigned to both proteins. In this paper, we describe the distribution of Calneuron-1 in rat and human brains. We show that Calneuron-1 is ubiquitously expressed in all brain regions examined. The protein is most abundant in Purkinje cells of the cerebellum and principal neurons of the cortex and limbic brain whereas no expression in glial cells is apparent. In addition, we identify two novel splice isoforms of Calneuron-1 with extended N-termini. These isoforms are particular abundant in the cerebellum. Taken together, these data set grounds for a better understanding of the cellular function of Calneurons.

Synapses are specialized contact sites mediating communication between neurons. Synaptogenesis requires the specific assembly of protein clusters at both sides of the synaptic contact by mechanisms that are barely understood. We studied the synaptic targeting of multi-domain proteins of the ProSAP/Shank family thought to serve as master scaffolding molecules of the postsynaptic density. In contrast to Shank1, expression of green-fluorescent protein (GFP)-tagged ProSAP1/Shank2 and ProSAP2/Shank3 deletion constructs in hippocampal neurons revealed that their postsynaptic localization relies on the integrity of the C-termini. The shortest construct that was perfectly targeted to synaptic sites included the last 417 amino acids of ProSAP1/Shank2 and included the C-terminal sterile alpha motif (SAM) domain. Removal of 54 residues from the N-terminus of this construct resulted in a diffuse distribution in the cytoplasm. Altogether, our data delineate a hitherto unknown targeting signal in both ProSAP1/Shank2 and ProSAP2/Shank3 and provide evidence for an implication of these proteins and their close homologue, Shank1, in distinct molecular pathways.

In this report, we describe the identification of a polypeptide survival-promoting factor that is produced by maternal and early postnatal peripheral blood mononuclear cells (PBMCs) of the immune system in Long-Evans rats and humans. The factor, termed Y-P30, most likely arises from proteolytic processing of a larger precursor protein and accumulates mainly in pyramidal neurons of the developing cortex and hippocampus but not in astrocytes. It was released from neurons grown in culture and substantially promotes survival of cells in explant monocultures of perinatal thalamus from the offspring. Y-P30 mRNA was not detectable in infant or adult brain and was present only in blood cells of pregnant rats and humans but not in nonpregnant controls. However, Y-P30 transcription could be induced in PBMCs of adult animals by a central nervous system lesion (i.e., optic nerve crush), which points to a potential role of the factor not only in neuronal development but also in neuroinflammation after white matter injury.

Shank proteins are scaffolding proteins in the postsynaptic density of excitatory synapses in the mammalian brain. In situ hybridization revealed that Shank1/SSTRIP and Shank2/ProSAP1 mRNAs are widely expressed early in postnatal brain development whereas Shank3/ProSAP2 expression increases during postnatal development especially in the cerebellum and thalamus. Shank1 and Shank3 (but not Shank2) mRNAs are present in the molecular layers of the hippocampus, consistent with a dendritic transcript localization. Shank1 and Shank2 transcripts are detectable in the dendritic fields of Purkinje cells, whereas Shank3 mRNA is restricted to cerebellar granule cells. The appearance of dendritic Shank mRNAs in cerebellar Purkinje cells coincides with the onset of dendrite formation. Expression of reporter transcripts in hippocampal neurons identifies a 200-nucleotide dendritic targeting element (DTE) in the Shank1 mRNA. The widespread presence of Shank mRNAs in dendrites suggests a role for local synthesis of Shanks in response to stimuli that induce alterations in synaptic morphology.

We investigated by immunohistochemistry (IHC) the distribution of caldendrin, the founding member of a novel family of neuronal calcium-binding proteins closely related to calmodulin, in human forebrain. Caldendrin immunoreactivity was unevenly distributed, with prominent staining in the paleo- and neocortex, hippocampus, and hypothalamus. With the exception of the hypothalamus, labeling was restricted to the somatodendritic compartment of neurons. This distribution completely matches that reported in rat, indicating that the cellular function is most likely conserved among species. Therefore, one prerequisite for functional studies in rodent models aimed at elucidation of mechanisms with relevance for humans can be based on the present findings.

Caldendrin defines a novel family of neuronal calcium-sensor proteins, the C-terminal moiety of which displays high similarity to calmodulin. We now report that the protein is recruited to the postsynaptic density (PSD) of cortical and hippocampal neurons in response to kainate-induced epileptic seizures, an animal model of human temporal lobe epilepsy. The translocation of caldendrin to the PSD did not occur in kainate-treated rats that did not develop seizures. The enhanced PSD levels of caldendrin are not due to increased protein synthesis and most likely reflect a recruitment from the soluble caldendrin protein pool. These findings suggest that the transduction of dendritic Ca2+-signals via caldendrin is altered by epileptic seizures and that caldendrin might be involved in the pathophysiology of temporal lobe epilepsy.

Caldendrin is a neuronal calcium-binding protein, which is highly enriched in the postsynaptic density fraction and exhibits a prominent somato-dendritic distribution in brain. Two additional splice variants derive from the caldendrin gene, which have unrelated N-termini and were previously only de- tected in the retina. We now show that these isoforms are present in neurohypophyseal axons and on secretory granules of endocrine cells. In light of the described interaction of the Caldendrin C-terminus with Q-type Cav2.1 calcium channels these data suggest that this interaction takes place in neuro- hypophyseal axons and pituitary cells indicating functions of the short splice variants in triggering Ca 2+ transients to a vesic- ular target interaction. ! 2003 Federation of European Biochemical Societies. Pub- lished by Elsevier Science B.V. All rights reserved.

Proteins of the presynaptic exocytic machinery have been found associated with the acrosome of male germ cells, suggesting that the sperm acrosome reaction and neurotransmission at chemical synapses may share some common mechanisms. To substantiate this hypothesis, we studied the expression and ultrastructural localization of prominent pre- and postsynaptic protein components in rat testis. The presynaptic membrane trafficking proteins SV2 and complexin, the vesicular amino acid transporters VGLUT and VIAAT, the postsynaptic scaffolding protein ProSAP/Shank, and the postsynaptic calcium-sensor protein caldendrin, could be identified in germ line cells. Immunogold electron microscopy revealed an association of these proteins with the acrosome. In addition, evidence was obtained for the expression of the plasmalemmal glutamate transporters GLT1 and GLAST in rat sperm. The novel finding that not only presynaptic proteins, which are believed to be involved in membrane fusion processes, but also postsynaptic elements are present at the acrosome sheds new light on its structural organization. Moreover, our data point to a possible role for neuroactive amino acids in reproductive physiology.

Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.

In a screen to identify genes that are expressed differentially in the retina after partial optic nerve crush, we identified MAP1B as an up-regulated transcript. Western blot analysis of inner retina protein preparations confirmed changes in the protein composition of the microtubule-associated cytoskeleton of crushed vs. uncrushed nerve. MAP1B immunoreactivity and transcript levels were elevated for two weeks after crush. Immunostaining and Western blots with monoclonal antibodies directed against developmentally regulated phosphorylation sites on MAP1B revealed a gradient of MAP1B phosphorylation from the proximal optic nerve stump to the soma of retinal ganglion cells. Most interestingly, using antibodies directed against developmentally regulated phosphorylation sites on MAP1B, we observed that a significant number of crushed optic nerve axons develop MAP1B-immunopositive growth cones, which cross the crush site and migrate along the distal nerve fragment. In parallel, an abnormal distribution of highly phosphorylated neurofilament protein (pNF-H) in the cell soma and dendrites of presumably axotomized retinal ganglion cells was observed following partial nerve crush. This redistribution is present for the period between day 7 and 28 postcrush and is not seen in cells that stay connected to the superior colliculus. Axotomized ganglion cells, which contain pNF-H in soma and dendrites appear to have been disconnected from the colliculus at an early stage but survive axonal trauma for long periods.

Limited optic nerve crush is a model of diffuse mechanical axon injury, the most prevalent cause of secondary neurodegeneration after closed head neurotrauma. In this report, a protocol is presented which allows for the rapid screening of differential gene expression in the inner retina, as well as the optic nerve, in response to partial nerve crush. To prove the reliability of the method, prototypically, the differential expression profiles of three candidate genes (kinesin light chain, ferritin, RYB-A) were verified. The method seems to be suitable to address the question of how differential gene expression contributes to degeneration, survival and axonal repair after partial nerve crush.

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings. The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

The expression of c-fos, c-jun, jun-b, jun-d, srf and pc4 mRNA was examined after partial optic nerve crush in the adult rat retina by in situ hybridization. Optic nerve injury led exclusively to the upregulation of c-jun, with cellular label indicative for c-jun mRNA in the retinal ganglion cell layer after two days, three days and one week post-injury. This expression pattern was in accordance with the appearance of c-Jun immunoreactivity in retinal flat mounts. Injection of an antisense but not a missense oligonucleotide against c-jun after partial crush resulted in a reduced number of connected retinal ganglion cells (RGCs) as shown by retrograde labeling. Prelabeling of RGCs with fluorogold before optic nerve section and subsequent antisense targeting against c-jun, however, led to a slightly higher number of surviving but axotomized RGCs. C-Jun antibody staining of retinal whole mounts pre- or postlabeled after crush by intracollicular administration of fluorogold showed strong c-Jun immunoreactivity in connected RGCs and also in a population of disconnected RGCs. Double labeling with an antibody directed against the transcription factor ATF-2 revealed strong co-expression of c-Jun and ATF-2 in connected RGCs but not in axotomized cells. Taken together these data indicate that both RGCs in continuity and those in discontinuity with the superior colliculus respond both equally to the noxious stimulus with c-Jun expression. Moreover, the co-expression of c-Jun with high levels of ATF-2 appears to be essential for either the continuity or survival of RGCs which remain connected with their target. In disconnected RGCs, however, low levels of ATF-2 and the co-expression of c-Jun may be related to cell death.

Bassoon is a 420-kDa protein specifically localized at the active zone of presynaptic nerve terminals. It is thought to be involved in the structural organization of the neurotransmitter release site. We studied the distribution of Bassoon transcripts and protein in rat brain and assessed which types of presynaptic terminals contain the protein. As shown by in situ hybridization, Bassoon transcripts are widely distributed in the brain and occur primarily in excitatory neurons. In addition, examples of gamma-aminobutyric acid (GABA)-ergic neurons expressing Bassoon are detected. At the light microscopic level, Bassoon immunoreactivity is found in synaptic neuropil regions throughout the brain, with the strongest expression in the hippocampus, the cerebellar cortex, and the olfactory bulb. Immunoelectron microscopy showed that Bassoon immunoreactivity is found in both asymmetric type 1 and symmetric type 2 synapses. Immunopositive asymmetric synapses include mossy fiber boutons and various spine and shaft synapses in the hippocampus and mossy fiber terminals and parallel fiber terminals in the cerebellum. Bassoon-containing symmetric synapses are observed, e.g., between basket and granule cells in the hippocampus, between Golgi cells and granule cells, and between basket cells and Purkinje cells in the cerebellum. Within synaptic terminals, Bassoon appears highly concentrated at sites opposite to postsynaptic densities. In cultured hippocampal neurons, Bassoon was found to colocalize with GABA(A) and glutamate (GluR1) receptors. These data indicate that Bassoon is a component of the presynaptic apparatus of both excitatory glutamatergic and inhibitory GABAergic synapses.

We have investigated time course and characteristics of retinal ganglion cell (RGC) death after partial optic nerve injury. In situ end labeling of DNA fragments with the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine (dUTP)-biotin nick end labeling (TUNEL) method revealed the presence of apoptotic cells on as early as 5 days postcrush with a very high number of TUNEL-positive cells 1 week postinjury. At the ultrastructural level, features of apoptosis were clearly present in the ganglion cell layer at this time point. Moreover, TUNEL-positive cells could be identified as retinal ganglion cells by retrograde labeling with fluorogold. In addition, DNA laddering characteristic for apoptosis was found 1 week postinjury. A considerable number of TUNEL-labeled cells was still found after 2 weeks postinjury. Retinal whole mounts prepared at postlesion days 2-5, however, revealed that many cell bodies with ruptured membranes as evidenced by nucleosomal Sytox staining were present. These cells were also identified as retinal ganglion cells by retrograde labeling with fluorogold. Moreover, at this early stages of RGC degeneration necrotic cellular profiles could be detected by electron microscopic analysis. Thus, evidence is provided that necrosis and apoptosis follow a distinctly different time course after partial optic nerve injury.

Brevican is a member of the aggrecan/versican family of proteoglycans. In contrast to the other family members, brevican occurs both as soluble isoforms secreted into the extracellular space and membrane-bound isoforms which are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety. Expression of both variants, which are encoded by two differentially processed transcripts from the same gene, is confined to the nervous system. In the current study, we have used in situ hybridization to examine the cellular sites of synthesis for both mRNAs during postnatal development of the rat brain. Whereas the 3.6-kb transcript encoding secreted brevican displays a widespread distribution in grey matter structures, including cerebellar and cerebral cortex, hippocampus and thalamic nuclei with silver grains accumulating over neuronal cell bodies, the smaller transcript (3.3 kb) encoding GPI-anchored isoforms appears to be largely confined to white matter tracts and diffusely distributed glial cells. This expression pattern is further confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments with RNA from different glial cell cultures, and by biochemical data demonstrating that the crude membrane fraction from isolated optic nerve contains high amounts of phosphatidylinositol-specific phospholipase C (PI-PLC)-sensitive brevican immunoreactivity. During ontogenetic development, both brevican transcripts are generally up-regulated. However, the expression of glypiated brevican is delayed by about 1 week, compared with the expression of the secreted isoform. This late appearance of GPI-linked brevican, its predominant expression in glial cells and its tight association with brain myelin fractions suggest a functional role in neuroglia.

Recently emerging evidence suggests important roles for inositol polyphosphates and inositol phospholipids in neuronal Ca2+ signalling, membrane vesicle trafficking and cytoskeletal rearrangement. A prerequisite for a detailed physiological characterization of the signalling of both potential second messengers inositol-(1,3,4,5)-tetrakisphosphate (InsP4) and phosphatidylinositol-3,4,5-trisphosphate (PtdInsP3) in the nervous system is the precise cellular localization of their receptors. Based on the cDNA sequence of a recently cloned brain-specific receptor with high affinity for both InsP4 and PtdInsP3 (InsP4-PtdInsP3R), p42IP4/centaurin-alpha, we localized the mRNA and the protein in rat brain. In situ hybridization revealed a widespread expression of the InsP4-PtdInsP3R with prominent labelling in cerebellum, hippocampus, cortex and thalamus, which moreover is developmentally regulated. Using peptide-specific antibodies, the immunoreactivity was localized in the adult brain in the vast majority of neuronal cell types and probably also in some glial cells. Prominent immunoreactivity was found in axonal processes and in cell types characterized by extensive neurites. In the hypothalamus a subpopulation of parvocellular neurons in the peri- and paraventricular nuclei was most heavily labelled. This was confined by strong immunoreactivity in the lamina externa of the median eminence in close proximity to portal plexus blood vessels. Electron microscopy revealed that the InsP4-PtdInsP3R was frequently associated with presynaptic vesicular structures. Further studies should identify the role of the InsP4-PtdInsP3R in cellular neural processes.

When exposed to short-day conditions, hamsters and other long-day breeders undergo gonadal regression. With chronic exposure to short days, however, the animals become photorefractory and gonadal recrudescence occurs. The underlying mechanism for this insensitivity is still unknown. There is growing evidence, however, that specific cells of the pituitary pars tuberalis (PT) mediate these photoperiod/nonphotoperiod-dependent changes as a direct or indirect "Zeitgeber" for the endocrine system. We investigated messenger RNA (mRNA)/protein formation for several hypophyseal hormones (beta-TSH, beta-LH, PRL, common alpha-chain) in the pars distalis (PD) and PT of female Djungarian hamsters in long photoperiod (LP) and after 18, 28, and 38 weeks of short photoperiod (SP). As indicated by gonadal and body weight, the hamsters displayed gonadal regression after 18 and 28 weeks of SP; after 38 weeks of SP, all animals showed recrudescence. At 18 and 28 weeks of SP, only PRL mRNA and protein levels were significantly reduced in the PD and returned to LP values after 38 weeks of SP. The expression of hypothalamic tyrosine hydroxylase in the arcuate nucleus that was determined by immunocytochemistry and by in situ hybridization was also down-regulated in SP18 and SP28 with increasing levels at SP38. Urinary 6-sulfatoxymelatonin levels were elevated in SP with highest levels in the SP18 group. In the PT, beta-TSH mRNA and protein were not detectable in all SP groups compared with the moderate signal intensity in LP. The common alpha-chain mRNA and protein, however, which were also reduced in the animals of the SP18 group, were already elevated after 28 weeks of SP and nearly reached LP-levels after 38 weeks of SP. These results show that, in contrast to LH and TSH, PRL expression in the PD is a sensitive indicator for photoperiod dependent changes of the endocrine system and seems to be tyrosine hydroxylase independent. The increase of common alpha-chain expression in PT-specific cells depending upon duration of SP that precedes the hormonal changes in the PD leads us to speculate that PT-specific cells initiate spontaneous recrudescence via a PT-PD pathway.