Neuronal Cell Surface Research Articles

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (“antibody feeding”) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking ("antibody feeding") to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

Espectro clínico y valor diagnóstico de los anticuerpos contra el complejo proteico asociado a canales de potasio

IntroductionAntibodies against a protein complex that includes voltage-gated potassium channels (VGKC) have been reported in patients with limbic encephalitis, peripheral nerve hyperexcitability, Morvan's syndrome, and a large variety of neurological syndromes. Review summaryIn this article, a review is presented of the syndromes associated with antibodies against VGKC-related proteins and the main antigens of this protein complex, the proteins LGI1 (leucine rich glioma inactivated protein 1) and Caspr2 (contactin-associated protein-like 2). The conceptual problems and clinical implications of the description of antibodies against VGKC-related proteins other than LGI1 and Caspr2 are also discussed.Although initial studies indicated the occurrence of antibodies against VGKC, recent investigations have shown that the main antigens are a neuronal secreted protein known as LGI1 which modulates synaptic excitability, and a protein called Caspr2 located on the cell surface and processes of neurons of different brain regions, and at the juxtaparanodal region of myelinated axons. While antibodies against LGI1 preferentially associate with classical limbic encephalitis, antibodies against Caspr2 associate with a wider spectrum of symptoms, including Morvan's syndrome, peripheral nerve hyperexcitability or neuromyotonia, and limbic or more extensive encephalitis. In addition there are reports of patients with antibodies against VGKC-related proteins that are different from LGI1 or Caspr2. In these cases, the identity and location of the antigens are unknown, the syndrome association is not specific, and the response to treatment uncertain. ConclusionsThe discovery of antigens such as LGI1 and Caspr2 has resulted in a clinical and molecular definition of the broad group of diseases previously attributed to antibodies against VGKC. Considering the literature that describes the presence of antibodies against VGKC other than LGI1 and Caspr2 proteins, we propose a practical algorithm for the diagnosis and treatment of these patients.

Wnt-5a increases NO and modulates NMDA receptor in rat hippocampal neurons

Wnt signaling has a crucial role in synaptic function at the central nervous system. Here we evaluate whether Wnts affect nitric oxide (NO) generation in hippocampal neurons. We found that non-canonical Wnt-5a triggers NO production; however, Wnt-3a a canonical ligand did not exert the same effect. Co-administration of Wnt-5a with the soluble Frizzled related protein-2 (sFRP-2) a Wnt antagonist blocked the NO production. Wnt-5a activates the non-canonical Wnt/Ca2+ signaling through a mechanism that depends on Ca2+ release from Ryanodine-sensitive internal stores. The increase in NO levels evoked by Wnt-5a promotes the insertion of the GluN2B subunit of the NMDA receptor (NMDAR) into the neuronal cell surface. To the best of our knowledge, this is the first time that Wnt-5a signaling is related to NO production, which in turn increases NMDARs trafficking to the cell surface.

Magnetic resonance and optical imaging probes for NMDA receptors on the cell surface of neurons: synthesis and evaluation in cellulo.

A second generation of N-methyl-d-aspartate (NMDA) receptor-targeted MRI contrast agents has been synthesised and evaluated in cellulo, based on established bicyclic NMDA receptor antagonists. Their use as responsive MR imaging probes has been evaluated in suspensions of NSC-34 cells, and one agent exhibited significant enhancements in measured longitudinal and transverse water proton relaxation rates (19 and 38% respectively; 3 T, 298 K). A biotin derivative of the lead compound was prepared and the specificity and reversibility of binding to the NMDA cell surface receptors demonstrated using confocal laser scanning microscopy. Competitive and reversible binding of glutamate to the receptors was also visualised, suggesting that the receptor-targeted approach may allow MRI to be used to monitor neuronal events associated with modulation of local glutamate concentrations.

Autoimmune-Mediated Cognitive Impairment: A Case Report

Autoimmune encephalopathy encompasses a complex groupof diseaseswith diverse clinicalmanifestations and therapeutic outcomes. Central nervous system (CNS) autoimmune inflammation can be triggered in multiple ways, including (1) systemic infections/vaccinations, (2) various cancers (paraneoplastic autoimmune encephalitis), (3) systemic autoimmunedisorders, and (4)without an identifiable cause (e.g., nonparaneoplastic autoimmune encephalitis). The discovery of neuropsychiatric disorders associated with antibodies to neuronal cell surface proteins, which play a crucial role in synaptic transmission and plasticity, has resulted in a paradigm shift in our understanding of CNS autoimmunity. We report a case of a young woman with multiple autoimmune disorders who presented with significant memory problems and relapse of major depressive disorder. Her cognitive problems did not resolve upon treatment of depression and continued to result in significant impairment of her daily functioning. A comprehensive neuropsychiatric and neurologic evaluation helped in establishing and providing a successful treatment. This case underscores the importance of recognizing neuropsychiatric manifestations of autoimmune disorders and the use of a multidisciplinary approach for the evaluation and treatment of neuropsychiatric conditions. Medicine, Baltimore, MD; Department of Neurology, Case Western Reserve University School of Medicine, Cleveland, OH. Send correspondence and reprint requests to Bhargavi Devineni,M.B.B.S., Division of Geriatrics and Neuropsychiatry, Department of Psychiatry, Johns Hopkins University of Medicine, 5300 Alpha Commons Drive 4th Floor, Alpha Commons Building, Baltimore, MD 21224; e-mail: bdevine2@jhmi.edu & 2014TheAcademy of PsychosomaticMedicine. Published by Elsevier Inc. All rights reserved. Case Report

Characterisation of a syndrome of autoimmune adult onset focal epilepsy and encephalitis

We report a series of patients with a clinical syndrome characterised by the explosive onset in adulthood of recurrent focal seizures of frontotemporal onset and features suggestive of autoimmune encephalitis. We propose that this presentation of “autoimmune adult onset focal epilepsy and encephalitis” is a recognisable clinical syndrome, and provide evidence it may be associated with heterogeneous immunological targets. Between 2008 and 2011 we encountered six patients with new-onset epilepsy in whom we suspected an autoimmune aetiology. We first characterised the clinical, electroencephalographic, cerebrospinal fluid (CSF), imaging, and pathological findings of this syndrome. We subsequently tested them for antibodies against both intracellular and neuronal cell surface antigens. All patients presented with recurrent seizures with focal frontotemporal onset, refractory to multiple anticonvulsants. Four had focal T2-weighted hyperintensities on MRI. CSF mononuclear cells were variably elevated with positive oligoclonal bands in four. Brain biopsy in one patient demonstrated perivascular lymphocytic infiltration. Two were treated with immunosuppression and went on to achieve complete seizure control and return to baseline cognition. Three of four patients who received only pulsed steroids or no treatment had ongoing frequent seizures, with two dying of sudden unexpected death in epilepsy. Subsequently, three had antibodies identified against neuronal cell surface antigens including N-methyl-d-aspartate receptor and leucine-rich glioma inactivated 1. We suggest that patients with such a presentation should be carefully evaluated for a suspected autoimmune aetiology targeting cell surface antigens and have a therapeutic trial of immunosuppression as this may improve their long-term outcome.

The Neuroplastin Adhesion Molecules Are Accessory Proteins That Chaperone the Monocarboxylate Transporter MCT2 to the Neuronal Cell Surface

BackgroundThe neuroplastins np65 and np55 are two synapse-enriched immunoglobulin (Ig) superfamily adhesion molecules that contain 3 and 2 Ig domains respectively. Np65 is implicated in long term, activity dependent synaptic plasticity, including LTP. Np65 regulates the surface expression of GluR1 receptor subunits and the localisation of GABAA receptor subtypes in hippocampal neurones. The brain is dependent not only on glucose but on monocarboxylates as sources of energy. The. monocarboxylate transporters (MCTs) 1–4 are responsible for the rapid proton-linked translocation of monocarboxylates including pyruvate and lactate across the plasma membrane and require association with either embigin or basigin, proteins closely related to neuroplastin, for plasma membrane expression and activity. MCT2 plays a key role in providing lactate as an energy source to neurons.Methodology/FindingsHere we use co-transfection of neuroplastins and monocarboxylate transporters into COS-7 cells to demonstrate that neuroplastins can act as ancillary proteins for MCT2. We also show that Xenopus laevis oocytes contain endogenous neuroplastin and its knockdown with antisense RNA reduces the surface expression of MCT2 and associated lactate transport. Immunocytochemical studies show that MCT2 and the neuroplastins are co-localised in rat cerebellum. Strikingly neuroplastin and MCT2 are enriched in the same parasagittal zebrin II-negative stripes.ConclusionsThese data strongly suggest that neuroplastins act as key ancillary proteins for MCT2 cell surface localisation and activity in some neuronal populations, thus playing an important role in facilitating the uptake of lactate for use as a respiratory fuel.

Paraneoplastic and Autoimmune Encephalopathies

Immune-mediated encephalitis is an increasingly recognized cause of neurologic dysfunction including behavioral change, psychosis, movement disorders, seizures, autonomic instability, and coma. Associated antineuronal antibodies are of two main subtypes, those targeting neuronal cell surface antigens, which are pathogenic, and nonpathogenic antibodies targeting intracellular antigens. Antibody identification aids in screening for underlying cancers and prediction of outcome. Cancer is found most commonly with antibodies targeting intracellular neural components. Certain cancers, such as small-cell lung carcinoma, and breast and ovarian cancer are particularly immunogenic. When cancer is detected, oncologic treatment should be followed with immunotherapy. Nonpathogenic antibody disorders respond poorly to treatment, whereas pathogenic antibodies predict a favorable response to immune treatment. If no cancer is identified, then ongoing surveillance is recommended for 5 years after detection of most antineuronal antibodies.

Membrane-tethered monomeric neurexin LNS-domain triggers synapse formation.

Neurexins are presynaptic cell-adhesion molecules that bind to postsynaptic cell-adhesion molecules such as neuroligins and leucine-rich repeat transmembrane proteins (LRRTMs). When neuroligins or LRRTMs are expressed in a nonneuronal cell, cocultured neurons avidly form heterologous synapses onto that cell. Here we show that knockdown of all neurexins in cultured hippocampal mouse neurons did not impair synapse formation between neurons, but blocked heterologous synapse formation induced by neuroligin-1 or LRRTM2. Rescue experiments demonstrated that all neurexins tested restored heterologous synapse formation in neurexin-deficient neurons. Neurexin-deficient neurons exhibited a decrease in the levels of the PDZ-domain protein CASK (a calcium/calmodulin-activated serine/threonine kinase), which binds to neurexins, and mutation of the PDZ-domain binding sequence of neurexin-3β blocked its transport to the neuronal surface and impaired heterologous synapse formation. However, replacement of the C-terminal neurexin sequence with an unrelated PDZ-domain binding sequence that does not bind to CASK fully restored surface transport and heterologous synapse formation in neurexin-deficient neurons, suggesting that no particular PDZ-domain protein is essential for neurexin surface transport or heterologous synapse formation. Further mutagenesis revealed, moreover, that the entire neurexin cytoplasmic tail was dispensable for heterologous synapse formation in neurexin-deficient neurons, as long as the neurexin protein was transported to the neuronal cell surface. Furthermore, the single LNS-domain (for laminin/neurexin/sex hormone-binding globulin-domain) of neurexin-1β or neurexin-3β, when tethered to the presynaptic plasma membrane by a glycosylinositolphosphate anchor, was sufficient for rescuing heterologous synapse formation in neurexin-deficient neurons. Our data suggest that neurexins mediate heterologous synapse formation via an extracellular interaction with presynaptic and postsynaptic ligands without the need for signal transduction by the neurexin cytoplasmic tail.

Neuronal Surface and Glutamic Acid Decarboxylase Autoantibodies in Nonparaneoplastic Stiff Person Syndrome

High titers of autoantibodies to glutamic acid decarboxylase (GAD) are well documented in association with stiff person syndrome (SPS). Glutamic acid decarboxylase is the rate-limiting enzyme in the synthesis of γ-aminobutyric acid (GABA), and impaired function of GABAergic neurons has been implicated in the pathogenesis of SPS. Autoantibodies to GAD might be the causative agent or a disease marker. To investigate the characteristics and potential pathogenicity of GAD autoantibodies in patients with SPS and related disorders. Retrospective cohort study and laboratory investigation. Weatherall Institute of Molecular Medicine, University of Oxford. Twenty-five patients with SPS and related conditions identified from the Neuroimmunology Service. Neurological examination, serological characterization and experimental studies. Characterization of serum GAD antibodies from patients with SPS and evidence for potential pathogenicity. We detected GAD autoantibodies at a very high titer (median, 7500 U/mL) in 19 patients (76%), including all 12 patients with classic SPS. The GAD autoantibodies were high affinity (antibody dissociation constant, 0.06-0.78 nmol) and predominantly IgG1 subclass. The patients’ autoantibodies co-localized with GAD on immunohistochemistry and in permeabilized cultured cerebellar GABAergic neurons, as expected, but they also bound to the cell surface of unpermeabilized GABAergic neurons. Adsorption of the highest titer (700 000 U/mL) serum with recombinant GAD indicated that these neuronal surface antibodies were not directed against GAD itself. Although intraperitoneal injection of IgG purified from the 2 available GAD autoantibody–ositive purified IgG preparations did not produce clinical or pathological evidence of disease, SPS and control IgG were detected in specific regions of the mouse central nervous system, particularly around the lateral and fourth ventricles. Autoantibodies to GAD are associated with antibodies that bind to the surface of GABAergic neurons and that could be pathogenic. Moreover, in mice, human IgG from the periphery gained access to relevant areas in the hippocampus and brainstem. Identification of the target of the non-GAD antibodies and peripheral and intrathecal transfer protocols, combined with adsorption studies, should be used to demonstrate the role of the non-GAD IgG in SPS.

A new locus for familial temporal lobe epilepsy on chromosome 3q

Temporal lobe epilepsy (TLE) is a common and heterogeneous focal epilepsy syndrome with a complex etiology, involving both environmental and genetic factors. Several familial forms of TLE have been described, including familial lateral TLE (FLTLE), familial mesial TLE (FMTLE) without hippocampal sclerosis, and FMTLE with hippocampal sclerosis. Mutations have been identified only in the leucine-rich, glioma-inactivated 1 (LGI1) gene on chromosome 10q22-q24 in FLTLE. Several loci have been mapped in families with FMTLE, but responsible genes have not been found. We report clinical evaluation in a large family with FMTLE and a new genetic locus. We conducted a genome-wide scan using 10cM-spaced microsatellite markers on a family with TLE. Seven individuals had TLE without antecedent FS; four other individuals had FS during childhood, but no subsequent epilepsy. Patients with TLE had infrequent simple partial, complex partial and secondarily generalized seizures that generally responded well to treatment. The proband had no hippocampal sclerosis. The mode of inheritance appeared to be autosomal dominant with incomplete penetrance. Linkage analysis was performed using the Genehunter software. Regions with LOD score>1 and those that were poorly informative in the first-pass scan were further genotyped. Linkage was identified on chromosome 3q25-q26 in a 13cM region flanked by markers D3S1584 and D3S3520, with a peak LOD score of 3.23. This interval does not correspond to any previously known locus for familial epilepsy or FS. KCNAB1, encoding a voltage-gated, shaker-related potassium channel, and NLGN1, encoding a member of a family of neuronal cell surface protein were excluded as disease causing mutations. We identified a novel locus for familial TLE with FS, providing additional evidence of the complexity and genetic heterogeneity of familial focal epilepsy.

Diagnosis and Management of Paraneoplastic Neurologic Disorders

Paraneoplastic neurologic disorders (PND) are a heterogeneous group of immune-mediated neurological disorders associated with systemic cancers. When a PND is diagnosed, prompt identification and treatment of the associated tumor is important as PND stabilization and in some cases improvement have been reported after tumor treatment. The cancer, however, may be small and difficult to detect or the onset of the PND may precede the development of the cancer by months or years. In the latter cases, patients often initially present to neurologists or internists who will need assistance from their oncology colleagues to uncover the cancer. It is therefore important to be aware of the associations of common cancers with specific PND syndromes and the significance in some PND of the presence in serum and/or cerebrospinal fluid (CSF) of specific antineuronal antibodies. Together, this information can focus the search for the tumor or support continued vigilance. Previously thought to be poorly responsive to therapies, it is now recognized that there is a subgroup of PND, mostly associated with antibodies to antigens on the neuronal cell surface that are highly treatment responsive. Treatments aimed at the PND are mostly immunosuppressive and include corticosteroids, plasma exchange and intravenous immunoglobulins (IVIg). Immunosuppressive chemotherapeutics and B-cell targeting drugs such as rituximab also may be useful. Although cancer patients tolerate these therapies, there is the risk of increased toxicity when combined with tumor-directed treatments and treatment plans should be coordinated between specialists.

Differentiation of human adipose stem cells into neural phenotype by neuroblastoma‐ or olfactory ensheathing cells‐conditioned medium

Olfactory ensheathing cells (OECs) are known to be capable of continuous neurogenesis throughout lifetime and are a source of multiple trophic factors important in central nervous system regeneration. B104 neuroblastoma cells are recognized to induce differentiation of neural stem cells into oligodendrocyte precursor cells. Therefore, the aim of this study was to verify if conditioned medium (CM) obtained from OECs or B104 cells was capable of inducing differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) to a neuronal phenotype. In order to this goal, immunocytochemical procedures and flow cytometry analysis were used and some neural markers, as nestin, protein gene product 9.5 (PGP 9.5), microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and neuron cell surface antigen (A2B5) were examined 24 h and 7 days after the treatment. The results showed that both OECs- or B104-CM treated AT-MSCs express markers of progenitor and mature neurons (nestin, PGP 9.5 and MAP2) in time-dependent manner, display morphological features resembling neuronal cells, and result negative for GFAP and A2B5, astrocyte and oligodendrocyte markers, respectively. This study demonstrated that AT-MSCs can be influenced by the environment, indicating that these cells can respond to environmental cues also versus a neuronal phenotype.

Post-transcriptional regulation of GABAB receptor and GIRK1 channels by Nogo receptor 1

BackgroundType B GABA receptors (GABA Rs) play a critical role in synaptic transmission. We carried out studies to determine whether neuronal cell surface expression of GABAB-Rs might be regulated by the Nogo receptor 1 (NgR1).ResultssiRNA knock-down of NgR1 resulted in a selective increase of GABAB R1 and GABAB R2 protein without altering the expression of GABAA receptor or GAD65. The increase in GABAB receptor subunits was unaccompanied by a change in mRNA, but inhibition of mTOR by rapamycin blocked the increase in GABAB protein. NgR1 siRNA also caused an increase in G protein coupled inwardly rectifying potassium channel (GIRK1). The increase in GABAB receptor and GIRK1 channel proteins was in the plasma membrane, determined by cell surface biotinylation. In NgR1 knockout mice, the amount of GABAB R2 and GIRK1 in hippocampus-derived synaptosomes was increased.ConclusionsTogether these findings suggest that NgR1 mediated modulation of synaptic transmission may be accomplished, at least in part, through modulation of G protein coupled receptors and channels.

Propofol and Thiopental Suppress Amyloid Fibril Formation and GM1 Ganglioside Expression through the γ-Aminobutyric Acid A Receptor

The incidence of Alzheimer disease may increase after surgical interventions. Amyloid β-protein (Aβ) fibrillogenesis, which is closely related to Alzheimer disease, is reportedly accelerated by exposure to anesthetics. However, the effects of GM1 ganglioside (GM1) on Αβ fibrillogenesis have not yet been reported. The current study was designed to examine whether the anesthetics propofol and thiopental are associated with Αβ assembly and GM1 expression on the neuronal cell surface. PC12N cells and cultured neuronal cells were treated with propofol or thiopental, and GM1 expression in treated and untreated cells was determined by the specific binding of horseradish peroxidase-conjugated cholera toxin subunit B (n = 5). The effects of an inhibitor of the γ-aminobutyric acid A receptor was also examined (n= 5). In addition, the effects of the anesthetics on GM1 liposome-induced Αβ assembly were investigated (n = 5). Finally, the neurotoxicity of the assembled Αβ fibrils was studied by the lactate dehydrogenase release assay (n = 6). Propofol (31.2 ± 4.7%) and thiopental (34.6 ± 10.5%) decreased GM1 expression on the cell surface through the γ-aminobutyric acid A receptor. The anesthetics inhibited Αβ fibril formation from soluble Αβ in cultured neurons. Moreover, propofol and thiopental suppressed GM1-induced fibril formation in a cell-free system (propofol, 75.8 ± 1.9%; thiopental, 83.6 ± 1.9%) and reduced the neurotoxicity of a mixture containing Aβ and GM1 liposomes (propofol, 35.3 ± 16.4%; thiopental, 21.3 ± 11.6%). Propofol and thiopental have direct and indirect inhibitory effects on Αβ fibrillogenesis.

The Neuroactive Steroid Pregnenolone Sulfate Stimulates Trafficking of FunctionalN-Methyl D-Aspartate Receptors to the Cell Surface via a Noncanonical, G Protein, and Ca2+-Dependent Mechanism

N-methyl D-aspartate (NMDA) receptors (NMDARs) mediate fast excitatory synaptic transmission and play a critical role in synaptic plasticity associated with learning and memory. NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, and clinical studies have revealed reduced negative symptoms of schizophrenia with a dose of pregnenolone that elevates serum levels of the neuroactive steroid pregnenolone sulfate (PregS). This report describes a novel process of delayed-onset potentiation whereby PregS approximately doubles the cell's response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS. The number of functional cell-surface NMDARs in cortical neurons increases 60-100% within 10 minutes of exposure to PregS, as shown by surface biotinylation and affinity purification. Delayed-onset potentiation is reversible and selective for expressed receptors containing the NMDAR subunit subtype 2A (NR2A) or NR2B, but not the NR2C or NR2D, subunits. Moreover, substitution of NR2B J/K helices and M4 domain with the corresponding region of NR2D ablates rapid allosteric potentiation of the NMDA response by PregS but not delayed-onset potentiation. This demonstrates that the initial phase of rapid positive allosteric modulation is not a first step in NMDAR upregulation. Delayed-onset potentiation by PregS occurs via a noncanonical, pertussis toxin-sensitive, G protein-coupled, and Ca(2+)-dependent mechanism that is independent of NMDAR ion channel activation. Further investigation into the sequelae for PregS-stimulated trafficking of NMDARs to the neuronal cell surface may uncover a new target for the pharmacological treatment of disorders in which NMDAR hypofunction has been implicated.

Prions disturb post-Golgi trafficking of membrane proteins

Conformational conversion of normal cellular prion protein PrP(C) into pathogenic PrP(Sc) is central to the pathogenesis of prion diseases. However, the pathogenic mechanism remains unknown. Here we show that post-Golgi vesicular trafficking is significantly delayed in prion-infected N2a cells. Accordingly, cell surface expression of membrane proteins examined, including PrP(C), insulin receptor involved in neuroprotection, and attractin, whose mutation causes prion disease-like spongiform neurodegeneration, is reduced. Instead, they accumulate in the Golgi apparatus. PrP(Sc) is detected throughout endosomal compartments, being particularly abundant in recycling endosome. We also show reduced surface expression of PrP(C) and insulin receptor in prion-infected mouse brains well before the onset of disease. These results suggest that prion infection might impair post-Golgi trafficking of membrane proteins to the cell surface in neurons via PrP(Sc) accumulated in recycling endosome, and eventually induce neuronal dysfunctions associated with prion diseases.

Meigo governs dendrite targeting specificity by modulating Ephrin level and N-glycosylation

Neural circuit assembly requires precise dendrite and axon targeting. We identified an evolutionarily conserved endoplasmic reticulum (ER) protein, Meigo, from a mosaic genetic screen in Drosophila melanogaster. Meigo was cell-autonomously required in olfactory receptor neurons and projection neurons to target their axons and dendrites to the lateral antennal lobe and to refine projection neuron dendrites into individual glomeruli. Loss of Meigo induced an unfolded protein response and reduced the amount of neuronal cell surface proteins, including Ephrin. Ephrin overexpression specifically suppressed the projection neuron dendrite refinement defect present in meigo mutant flies, and ephrin knockdown caused a similar projection neuron dendrite refinement defect. Meigo positively regulated the level of Ephrin N-glycosylation, which was required for its optimal function in vivo. Thus, Meigo, an ER-resident protein, governs neuronal targeting specificity by regulating ER folding capacity and protein N-glycosylation. Furthermore, Ephrin appears to be an important substrate that mediates Meigo's function in refinement of glomerular targeting.

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein, Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca2+- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer's disease.