Atomic-force microscopy imaging of plasma membranes purified from spinach leaves

Summary: Plasma membranes purified from spinach leaves by aqueous two-phase partitioning were examined by atomic-force microscopy (AFM) in phosphate buffer, and details on their structure were reported at nanometric scale. Examination of the fresh membrane preparation deposited on mica revealed a complex organization of the surface. It appeared composed of a first layer of material, about 8 nm in thickness, that practically covered all the mica surface and on which stand structures highly heterogeneous in shape and size. High-resolution imaging showed that the surface of the first layer appeared relatively smooth in some regions, whereas different characteristic features were observed in other regions. They consisted of globular-to-elliptical protruding particles of various sizes, from 4-5 nm x-y size for the smallest to 40-70 nm for the largest, and of channel-like structures 25-30 nm in diameter with a central hole. Macromolecular assemblies of protruding particles of various shapes were imaged. Addition of the proteolytic enzyme pronase led to a net roughness decrease in regions covered with particles, indicating their proteinaceous nature. The results open fascinating perspectives in the investigation of membrane surfaces in plant cells with the possibility to get structural information at the nanometric rang

Crystal structure, biochemical and biophysical characterisation of NHR1 domain of E3 Ubiquitin ligase neutralized

International audienceNotch signaling controls diverse developmental decisions of central importance to cell activity. One of the conserved positive regulators of No- tch signaling is Neuralized, the E3 Ubiquitin li-gase enzyme that regulates signaling activity by endocytosis. Neuralized has two novel repeats, NHR1 and NHR2, with a RING finger motif at the C-terminus. Both endocytosis of the Notch ligand, Delta, and inhibition of Notch signaling by Tom, a bearded family member, require the NHR1 domain. Here we describe the first crystal structure of NHR1 domain from Drosophila me- lanogaster, solved to 2.1 Å resolution by X-ray analysis. Using NMR and other biophysical tech- niques we define a minimal binding region of Tom, consisting of 12 residues, which interacts with NHR1 and show by interfacial analysis of protein monolayers that NHR1 binds PI4P. Taken together, the studies provide insight into mo-lecular interactions that are important for Notch signaling

Disruption of striatal glutamatergic transmission induced by mutant huntingtin involves remodelling of both postsynaptic density and NMDA receptor signalling

We study the striatal susceptibility to NMDA receptor (NMDAR)-mediated injury of two Huntington’s disease (HD) transgenic mice:R6/1 and R6/1:BDNF+/−. We found that R6/1:BDNF+/− mice – whichexpress reduced levels of BDNF – were more resistant than R6/1 miceto intrastriatal injection of quinolinate. This increased resistance isrelated to a differential reduction in expression of NMDAR scaffoldingproteins, MAGUKs (PSD-95, PSD-93, SAP-102 and SAP-97) but notto altered levels or synaptic location of NMDAR. A robust reorganizationof postsynaptic density (PSD) was detected in HD transgenicmice, shown by a switch of PSD-93 by PSD-95 in PSD. Furthermore,NMDAR signaling pathways were affected by different BDNF levels inHD mice; we found a reduction of synaptic αCaMKII (but not ofnNOS) in R6/1:BDNF+/− compared to R6/1 mice. The specific regulationof MAGUKs and αCaMKII in striatal neurons may reflect aprotective mechanism against expression of mutant huntingtin exon-1

Dissociation between CA3-CA1 synaptic plasticity and associative learning in TgNTRK3 transgenic mice

Neurotrophins and their cognate receptors might serve as feedback regulators for the efficacy of synaptic transmission.Weanalyzed miceoverexpressing TrkC (TgNTRK3) for synaptic plasticity and the expression of glutamate receptor subunits. Animals were conditionedusing a trace [conditioned stimulus (CS), tone; unconditioned stimulus (US), shock] paradigm. A single electrical pulse presented to theSchaffer collateral– commissural pathway during the CS–US interval evoked a monosynaptic field EPSP (fEPSP) at ipsilateral CA1pyramidal cells. In wild types, fEPSP slopes increased across conditioning sessions and decreased during extinction, being linearlyrelated to learning evolution. In contrast, fEPSPs in TgNTRK3 animals reached extremely high values, not accompanied with a proportionateincrease in their learning curves. Long-term potentiation evoked in conscious TgNTRK3 was also significantly longer lasting thanin wild-type mice. These functional alterations were accompanied by significant changes inNR1andNR2BNMDAreceptor subunits, withno modification of NR1Ser 896 or NR1Ser 897 phosphorylation. No changes of AMPA and kainate subunits were detected. Results indicatethat the NT-3/TrkC cascade could regulate synaptic transmission and plasticity through modulation of glutamatergic transmission at theCA3–CA1 synapse

Developmental alterations in Huntington's disease neural cells and pharmacological rescue in cells and mice

Neural cultures derived from Huntington's disease (HD) patient-derived induced pluripotent stem cells were used for 'omics' analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling, axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development, the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks, gene expression and unique peak profiles associated with dysregulated genes, suggesting a coordinated epigenetic program. Treatment with isoxazole-9, which targets key dysregulated pathways, led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time

Astrocytic BDNF and TrkB regulate severity and neuronal activity in mouse models of temporal lobe epilepsy

Astrocytes have emerged as crucial regulators of neuronal network activity, synapse formation, and underlying behavioral and cognitive processes. Despite some pathways have been identified, the communication between astrocytes and neurons remains to be completely elucidated. Unraveling this communication is crucial to design potential treatments for neurological disorders like temporal lobe epilepsy (TLE). The BDNF and TrkB molecules have emerged as very promising therapeutic targets. However, their modulation can be accompanied by several off-target effects such as excitotoxicity in case of uncontrolled upregulation or dementia, amnesia, and other memory disorders in case of downregulation. Here, we show that BDNF and TrkB from astrocytes modulate neuronal dysfunction in TLE models. First, conditional overexpression of BDNF from astrocytes worsened the phenotype in the lithium-pilocarpine mouse model. Our evidences pointed out to the astrocytic pro-BDNF isoform as a major player of this altered phenotype. Conversely, specific genetic deletion of BDNF in astrocytes prevented the increase in the number of firing neurons and the global firing rate in an in vitro model of TLE. Regarding to the TrkB, we generated mice with a genetic deletion of TrkB specifically in hippocampal neurons or astrocytes. Interestingly, both lines displayed neuroprotection in the lithium-pilocarpine model but only the mice with genetic deletion of TrkB in astrocytes showed significantly preserved spatial learning skills. These data identify the astrocytic BDNF and TrkB molecules as promising therapeutic targets for the treatment of TLE

Reduced fractalkine levels lead to striatal synaptic plasticity deficits in Huntington's disease

Huntington's disease (HD) is an inherited neurodegenerative disorder in which the striatum is the most affected brain region. Although a chronic inflammatory microglial reaction that amplifies disease progression has been described in HD patients, some murine models develop symptoms without inflammatory microglial activation. Thus, dysfunction of non-inflammatory microglial activity could also contribute to the early HD pathological process. Here, we show the involvement of microglia and particularly fractalkine signaling in the striatal synaptic dysfunction of R6/1 mice. We found reduced fractalkine gene expression and protein concentration in R6/1 striata from 8 to 20 weeks of age. Consistently, we also observed a down-regulation of fractalkine levels in the putamen of HD patients and in HD patient hiPSC-derived neurons. Automated cell morphology analysis showed a non-inflammatory ramified microglia in the striatum of R6/1 mice. However, we found increased PSD-95-positive puncta inside microglia, indicative of synaptic pruning, before HD motor symptoms start to manifest. Indeed, microglia appeared to be essential for striatal synaptic function, as the inhibition of microglial activity with minocycline impaired the induction of corticostriatal long-term depression (LTD) in wild-type mice. Notably, fractalkine administration restored impaired corticostriatal LTD in R6/1 mice. Our results unveil a role for fractalkine-dependent neuron-microglia interactions in the early striatal synaptic dysfunction characteristic of HD

The AMPA receptor positive allosteric modulator S 47445 rescues in vivo CA3-CA1 long-term potentiation and structural synaptic changes in old mice

Positive allosteric modulators of cc-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are small molecules that decrease deactivation of AMPARs via an allosteric site. These molecules keep the receptor in an active state. Interestingly, this type of modulator has been proposed for treating cognitive decline in ageing, dementias, and Alzheimer's disease (AD). S 47445 (8-cyclopropyl-3[2-(3-fluorophenyflethy1]-7,8-dihydro-3H-[1,3]oxazino[6,5-g][1,2,3]benzotriazine-4,9-dione) is a novel AMPAR positive allosteric modulator (AMPA-PAM). Here, the mechanisms by which S 47445 could improve synaptic strength and connectivity were studied and compared between young and old mice. A single oral administration of S 47445 at 10 mg/kg significantly increased long-term potentiation (LTP) in CA3-CA1 hippocampal synapses in alert young mice in comparison to control mice. Moreover, chronic treatment with S 47445 at 10 mg/kg in old alert animals significantly counteracted the deficit of LTP due to age. Accordingly, chronic treatment with S 47445 at 10 mg/kg seems to preserve synaptic cytoarchitecture in old mice as compared with young control mice. It was shown that the significant decreases in number and size of pre-synaptic buttons stained for VGlutl, and post-synaptic dendritic spines stained for spinophilin, observed in old mice were significantly prevented after chronic treatment with 10 mg/kg of S 47445. Altogether, by its different effects on LTP, VGlutl-positive particles, and spinophilin, S 47445 is able to modulate both the structure and function of hippocampal excitatory synapses known to be involved in learning and memory processes. These results open a new window for the treatment of specific age-dependent cognitive decline and dementias such as AD. (C) 2017 The Authors. Published by Elsevier Ltd

Lack of Helios during neural development induces adult schizophrenia-like behaviors associated with aberrant levels of the TRIF-recruiter protein WDFY1

The role of the WDFY1 protein has been studied as a TLR3/4 scaffold/recruiting protein in the immune system and in different oncogenic conditions. However, its function in brain remains poorly understood. We have found that in mice devoid of Helios (He−/− mice), a transcription factor specifically expressed during the development of the immune cells and the central nervous system, there is a permanent and sustained increase of Wdfy1 gene expression in the striatum and hippocampus. Interestingly, we observed that WDFY1 protein levels were also increased in the hippocampus and dorsolateral prefrontal cortex of schizophrenic patients, but not in the hippocampus of Alzheimer's disease patients with an associated psychotic disorder. Accordingly, young He−/− mice displayed several schizophrenic-like behaviors related to dysfunctions in the striatum and hippocampus. These changes were associated with an increase in spine density in medium spiny neurons (MSNs) and with a decrease in the number and size of PSD-95-positive clusters in the stratum radiatum of the CA1. Moreover, these alterations in structural synaptic plasticity were associated with a strong reduction of neuronal NF-κB in the pyramidal layer of the CA1 in He−/− mice. Altogether, our data indicate that alterations involving the molecular axis Helios-WDFY1 in neurons during the development of core brain regions could be relevant for the pathophysiology of neuropsychiatric disorders such as schizophrenia

RTP801 is involved in mutant huntingtin-induced cell death

RTP801 expression is induced by cellular stress and has a pro-apoptotic function in non-proliferating differentiated cells such as neurons. In several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease, elevated levels of RTP801 have been observed, which suggests a role for RTP801 in neuronal death. Neuronal death is also a pathological hallmark in Huntington's disease (HD), an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Currently, the exact mechanisms underlying mutant huntingtin (mhtt)-induced toxicity are still unclear. Here, we investigated whether RTP801 is involved in (mhtt)-induced cell death. Ectopic exon-1 mhtt elevated RTP801 mRNA and protein levels in nerve growth factor (NGF)-differentiated PC12 cells and in rat primary cortical neurons. In neuronal PC12 cells, mhtt also contributed to RTP801 protein elevation by reducing its proteasomal degradation rate, in addition to promoting RTP801 gene expression. Interestingly, silencing RTP801 expression with short hairpin RNAs (shRNAs) blocked mhtt-induced cell death in NGF-differentiated PC12 cells. However, RTP801 protein levels were not altered in the striatum of Hdh(Q7/Q111) and R6/1 mice, two HD models that display motor deficits but not neuronal death. Importantly, RTP801 protein levels were elevated in both neural telencephalic progenitors differentiated from HD patient-derived induced pluripotent stem cells and in the putamen and cerebellum of human HD postmortem brains. Taken together, our results suggest that RTP801 is a novel downstream effector of mhtt-induced toxicity and that it may be relevant to the human disease