Biology subjects

Triller, A.

Publications and source records attributed to Triller, A..

12 recordsLinked to original sources

bioRxiv2022

AUTS2 gene dosage affects synaptic AMPA receptors via a local dendritic spine AUTS2-TTC3-AKT-mTORC1 signaling dysfunction

The Human 1.2-Mb AUTS2 locus on chromosome 7q11.22 encodes a 1259-aa full-length protein, and a 711-aa C-terminal isoform. Functions of these AUTS2 proteins are only partly known. The major traits found in patients displaying AUTS2 locus mutations are Intellectual Disabilities, microcephaly attention deficit hyperactivity disorder (ADHD) (54%), and autistic traits. Furthermore, AUTS2 common variants were recently found associated to alcohol consumption and dyslexia using GWAS approaches. Auts2 localizes mainly in cell nuclei. We evidenced by super-resolution that Auts2 is present in dendritic spines. Auts2 interacts with Ttc3, the Akt2 E3 ligase, and negatively regulates Akt2 ubiquitination. Auts2 haploinsufficiency affects Akt/mTorc1 pathway with a decrease in AMPA and NMDA receptor subunits and in synaptic currents. Akt2 injection in postsynaptic neurons is sufficient to reverse changes in synaptic currents generated by Auts2 haploinsufficiency. Using chromosome engineering based on targeted meiotic recombination, we generated two mouse models with Auts2 locus deletion and duplication. Deleted Auts2 locus mice display stereotypies (rearing), perseveration and abnormal recognition memory. Duplicated Auts2 locus mice display similar perseveration and abnormal recognition memory but also a decrease in cued and contextual fear memory. Gene dosage induce changes in brain sub-region neuronal networks. In the thalamo-lateral amygdala pathway linked to cued fear memory, we found synaptic impairments linked to AMPA receptors, with a specific decrease in pAKT/total AKT ratio in duplicated Auts2 mice. Altogether, our study thereby provides a novel mechanistic and potentially therapeutic understanding of synaptic AKT/mTORC1 deregulated signaling and its related behavioral and cognitive phenotypes.

neuroscience↗

bioRxiv2022

Microglial TNFα orchestrates brain phosphorylation during the sleep period and controls homeostatic sleep.

The time we spend asleep is adjusted to previous time spent awake, and therefore believed to be under tight homeostatic control. Here, we establish microglia as a new cellular component of the sleep homeostat circuit. By using quantitative phosphoproteomics we demonstrate that microglia-derived TNF controls thousands of phosphorylation sites during the sleep period. Substrates of microglial TNF comprise sleep-promoting kinases and numerous synaptic proteins, including a subset whose phosphorylation status codes sleep need and determines sleep duration. As a result, lack of microglial TNF attenuates the build-up of sleep need, as measured by slow wave activity, and prevents immediate compensation for loss of sleep. Together, we propose that microglia control sleep homeostasis by releasing TNF that acts at the neuronal circuitry through dynamic control of phosphorylation.

neuroscience↗

bioRxiv2022

Microglial TNFα controls GABAAR plasticity, slow waves and memory consolidation during sleep

Microglia sense the changes in their environment. How microglia actively translate these changes into suitable cues to adapt brain physiology is unknown. We reveal an activity-dependent regulation of cortical inhibitory synapses by microglia, driven by purinergic signaling acting on P2RX7 and mediated by microglia-derived TNF. We demonstrate that sleep induces microglia-dependent synaptic enrichment of GABAARs in a manner dependent on microglial TNF and P2RX7. We further show that microglia-specific depletion of TNF alters slow waves during NREM sleep and blunt memory consolidation in sleep-dependent learning tasks. Together, our results reveal that microglia orchestrate sleep-intrinsic plasticity of synaptic GABAARs, sculpt sleep slow waves and support memory consolidation.

neuroscience↗

bioRxiv2021

Identification of a stereotypic molecular arrangement of glycine receptors at native spinal cord synapses

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy (SR-CLEM). We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs m-2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

molecular biology↗

bioRxiv2021

Ligands binding to the cellular prion protein induce its protective proteolytic release with therapeutic potential in neurodegenerative proteinopathies

The cellular prion protein (PrPC) is a central player in neurodegenerative diseases caused by protein misfolding, such as prion diseases or Alzheimers disease (AD). Expression levels of this GPI-anchored glycoprotein, especially at the neuronal cell surface, critically correlate with various pathomechanistic aspects underlying these diseases, such as templated misfolding (in prion diseases) and neurotoxicity and, hence, with disease progression and severity. In stark contrast to cell-associated PrPC, soluble extracellular forms or fragments of PrP are linked with neuroprotective effects, which is likely due to their ability to interfere with neurotoxic disease-associated protein conformers in the interstitial fluid. Fittingly, the endogenous proteolytic release of PrPC by the metalloprotease ADAM10 ( shedding) was characterized as a protective mechanism. Here, using a recently generated cleavage-site specific antibody, we shed new light on earlier studies by demonstrating that shed PrP (sPrP) negatively correlates with conformational conversion (in prion disease) and is markedly redistributed in murine brain in the presence of prion deposits or AD-associated amyloid plaques indicating a blocking and sequestrating activity. Importantly, we reveal that administration of certain PrP-directed antibodies and other ligands results in increased PrP shedding in cells and organotypic brain slice cultures. We also provide mechanistic and structural insight into this shedding-stimulating effect. In addition, we identified a striking exception to this, as one particular neuroprotective antibody, due to its special binding characteristics, did not cause increased shedding but rather strong surface clustering followed by fast endocytosis and degradation of PrPC. Both mechanisms may contribute to the beneficial action described for some PrP-directed antibodies/ligands and pave the way for new therapeutic strategies against devastating and currently incurable neurodegenerative diseases.

neuroscience↗

bioRxiv2021

NMDARs expressed in Granule Cells are involved in cerebellar plasticity and motor learning

Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic NMDA receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers nitric oxide signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-Purkinje cell (PF-PC) synapses. Consistent with this finding, the selective deletion of GCs NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning. Significance StatementLearning depends on synaptic plasticity. The signaling mechanisms that control induction of plasticity determine the learning rules at the specific synapse involved. Moreover, the relationship between the activity patterns of synaptic inputs and the type, direction, and level of plasticity induced may evolve during development. Here, we establish a key link between NMDA receptor activation presynaptic to cerebellar Purkinje cells, downstream signaling mechanisms, and the ability of adult animals to learn a cerebellar motor task.

neuroscience↗

bioRxiv2020

Reciprocal stabilisation of glycine receptors and gephyrin scaffold proteins at inhibitory synapses

Postsynaptic scaffold proteins immobilise neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses where they form receptor-scaffold complexes. While several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterise the GlyR and gephyrin dynamics at inhibitory synapses we performed fluorescence time-lapse imaging after photoconversion in order to directly visualise the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilisation of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the crosslinking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared to control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR crosslinking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilisation of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organisation. Statement of significanceThe efficiency of signal transmission between neurons depends strongly on the number of available neurotransmitter receptors in the postsynaptic membrane. Postsynaptic scaffold proteins provide binding sites for receptors, thus setting the gain of synaptic transmission. However, the importance of receptor-scaffold interactions for the stability of the postsynaptic scaffold itself has received relatively little attention. Using time-lapse imaging of glycine receptors and gephyrin scaffolds at inhibitory synapses in spinal cord neurons together with biophysical modelling, we show that receptor mobility controls the exchange but not the total number of gephyrin molecules at the synapse, and predict that glycine receptors and gephyrin scaffolds dynamically organise into different subpopulations with varying degrees of reciprocal stabilisation.

neuroscience↗

bioRxiv2020

Differential homeostatic regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses

Super-resolution imaging of synapses has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). At mixed inhibitory synapses in spinal cord neurons, both GlyRs and GABAARs reside at the same post-synaptic density (PSD). To examine how the different inhibitory receptors are organized and regulated, we carried out dual-color direct stochastic optical reconstruction microscopy (dSTORM). We found that endogenous GlyRs and GABAARs as well as their common scaffold protein gephyrin form SSDs that align with pre-synaptic RIM1/2, thus forming trans-synaptic nanocolumns. Strikingly, GlyRs and GABAARs occupy different sub-synaptic spaces, exhibiting only a partial overlap at mixed inhibitory synapses. When network activity was increased by pharmacological treatment using the K+ channel blocker 4-aminopyridine (4-AP), the GABAAR copy numbers of as well as the number of GABAAR SSDs were reduced, while GlyRs remained largely unchanged. This differential regulation is likely the result of changes in gephyrin phosphorylation that preferentially occurred outside of the SSDs. The total gephyrin content was not altered by 4-AP application. The activity-dependent regulation of GABAARs versus GlyRs suggests that different signaling pathways control their respective sub-synaptic organization. Whereas gephyrin serves as a scaffold protein that upholds GlyR numbers at SSDs, it may act as a switch regulating GABAARs via its phosphorylation state. Taken together, our data reinforce the notion that the precise sub-synaptic organization of GlyRs, GABAARs and gephyrin has functional consequences for the homeostatic regulation of mixed inhibitory synapses. HighlightsAlignment of sub-synaptic domains (SSDs) in trans-synaptic nanocolumns at inhibitory synapses Differential spatial organization of SSDs formed by GlyRs and GABAARs at mixed inhibitory synapses Activity-dependent regulation of GABAARs but not GlyRs at mixed inhibitory synapses Gephyrin phosphorylation is compartmentalized in SSDs within the synaptic scaffold

neuroscience↗

bioRxiv2020

Morphologically constrained modeling of spinous inhibition in the somato-sensory cortex

Pyramidal neurons are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light-electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 pyramidal neurons of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually-innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibition and that it can decouple voltage and calcium signaling, potentially impacting synaptic plasticity.

neuroscience↗

bioRxiv2019

SUSD4 Controls Activity-Dependent Degradation of AMPA Receptor GLUA2 and Synaptic Plasticity

Fine control of protein stoichiometry at synapses underlies brain function and plasticity. How proteostasis is controlled independently for each type of synaptic protein in a synapse-specific and activity-dependent manner remains unclear. Here we show that SUSD4, a complement-related transmembrane protein, binds the AMPA receptor subunit GLUA2 and controls its activity-dependent degradation. Several proteins with known roles in the regulation of AMPA receptor turnover, in particular ubiquitin ligases of the NEDD4 subfamily, are identified as SUSD4 binding partners. SUSD4 is expressed by many neuronal populations starting at the time of synapse formation. Loss-of-function of Susd4 in the mouse prevents long-term depression at cerebellar synapses, and leads to impairment in motor coordination adaptation and learning. Our findings reveal that activity-dependent synaptic plasticity relies on a transmembrane CCP domain-containing protein that regulates the degradation of specific substrates. This mechanism potentially accounts for the role of SUSD4 mutations in neurodevelopmental diseases.

neuroscience↗

bioRxiv2019

Feasibility analysis of semiconductor voltage nanosensors for neuronal membrane potential sensing

In the last decade, optical imaging methods have significantly improved our understanding of the information processing principles in the brain. Although many promising tools have been designed, sensors of membrane potential are lagging behind the rest. Semiconductor nanoparticles are an attractive alternative to classical voltage indicators, such as voltage-sensitive dyes and proteins. Such nanoparticles exhibit high sensitivity to external electric fields via the quantum-confined Stark effect. Here we report the development of lipid-coated semiconductor voltage-sensitive nanorods (vsNRs) that self-insert into the neuronal membrane. We describe a workflow to detect and process the photoluminescent signal of vsNRs after wide-field time-lapse recordings. We also present data indicating that vsNRs are feasible for sensing membrane potential in neurons at a single-particle level. This shows the potential of vsNRs for detection of neuronal activity with unprecedentedly high spatial and temporal resolution.

bioengineering↗

bioRxiv2017

Continuous rearrangement of the postsynaptic gephyrin scaffolding domain: a super-resolution quantified and energetic approach

Synaptic function and plasticity requires a delicate balance between overall structural stability and the continuous rearrangement of the components that make up the presynaptic active zone and the postsynaptic density (PSD). Photoactivated localization microscopy (PALM) has provided a detailed view of the nanoscopic structure and organization of some of these synaptic elements. Still lacking, are tools to address the morphing and stability of such complexes at super-resolution. We describe an approach to quantify morphological changes and energetic states of multimolecular assemblies over time. With this method, we studied the scaffold protein gephyrin, which forms postsynaptic clusters that play a key role in the stabilization of receptors at inhibitory synapses. Postsynaptic gephyrin clusters exhibit an internal microstructure composed of nanodomains. We found, that within the PSD, gephyrin molecules continuously undergo spatial reorganization. This dynamic behavior depends on neuronal activity and cytoskeleton integrity. The proposed approach also allowed access to the effective energy responsible for the tenacity of the PSD despite molecular instability.\n\nSignificant statementSuper-resolution microscopy has become an important tool for the study of biological systems, allowing detailed, nano-scale structural reconstruction, single molecule tracking, particle counting, and interaction studies. However, quantification tools that take full advantage of the information provided by this technology are still lacking. We describe a novel quantification method to obtain information related to the size, directionality, dynamics, and stability of clustered structures from super-resolution microscopy. With this method, we studied the stability of gephyrin clusters, the main inhibitory scaffold protein. We found that gephyrin molecules continuously undergo reorganization based on neuronal activity and changes in the cytoskeleton.

biophysics↗