Publications by authors named "Carlo Sala"

84 Publications

A literature overview on epilepsy and inflammasome activation.

Brain Res Bull 2021 Jul 5;172:229-235. Epub 2021 May 5.

CNR Neuroscience Institute, Milan, Italy.

Epilepsy is one of the most prevalent serious brain disorders worldwide. Accumulating evidence has suggested that inflammation participates in the progression and pathogenesis of epilepsy. During inflammation, a cytosolic multimolecular complex called the "inflammasome" is activated, driving the innate immune response. This inflammatory pathway by sensing various pathogens and molecules from damaged cells and then activation of caspase-1 enzyme initiates inflammatory responses. Activated caspase-1 leads to the proteolytic cleavage of the pro-inflammatory cytokines, interleukin-1β (IL-1β) and interleukin-18 (IL-18), and also induction of an inflammatory programmed cell death termed pyroptosis. NLR family pyrin domain-containing 1 (NLRP1) and NLRP3 are the two best-characterized inflammasome members, and both basic and clinical research has reported their activation during epilepsy. This overview is intended to summarize the current literature concerning NLRP1 and NLRP3 inflammasome activation and epilepsy.
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http://dx.doi.org/10.1016/j.brainresbull.2021.05.001DOI Listing
July 2021

Editorial: Dendritic Spines: From Biophysics to Neuropathology.

Front Synaptic Neurosci 2021 18;13:652117. Epub 2021 Feb 18.

Neurobiology Department, Weizmann Institute, Rehovot, Israel.

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http://dx.doi.org/10.3389/fnsyn.2021.652117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930062PMC
February 2021

Activation of the medial preoptic area (MPOA) ameliorates loss of maternal behavior in a Shank2 mouse model for autism.

EMBO J 2021 Mar 25;40(5):e104267. Epub 2021 Jan 25.

Institute for Anatomy and Cell Biology, Ulm University, Ulm, Germany.

Impairments in social relationships and awareness are features observed in autism spectrum disorders (ASDs). However, the underlying mechanisms remain poorly understood. Shank2 is a high-confidence ASD candidate gene and localizes primarily to postsynaptic densities (PSDs) of excitatory synapses in the central nervous system (CNS). We show here that loss of Shank2 in mice leads to a lack of social attachment and bonding behavior towards pubs independent of hormonal, cognitive, or sensitive deficits. Shank2 mice display functional changes in nuclei of the social attachment circuit that were most prominent in the medial preoptic area (MPOA) of the hypothalamus. Selective enhancement of MPOA activity by DREADD technology re-established social bonding behavior in Shank2 mice, providing evidence that the identified circuit might be crucial for explaining how social deficits in ASD can arise.
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http://dx.doi.org/10.15252/embj.2019104267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917557PMC
March 2021

N-methyl-d-aspartate receptor function in neuronal and synaptic development and signaling.

Curr Opin Pharmacol 2021 Feb 8;56:93-101. Epub 2021 Jan 8.

CNR Neuroscience Institute, Milano and NeuroMi Milan Center for Neuroscience, University of Milano-Bicocca, Milan, Italy. Electronic address:

The N-methyl-d-aspartate (NMDA) receptor, among the ionotropic glutamate receptors, are fundamental to integrating and transducing complex signaling in neurons. Glutamate activation of these receptors mediates intracellular signals essential to neuronal and synaptic formation and synaptic plasticity and also contribute to excitotoxic processes in several neurological disorders. The NMDA receptor signaling is mediated by the permeability to Ca2+ and by the large network of signaling and scaffolding proteins associated mostly with the large C-terminal domain of GluN2 subunits. Important studies showed that GluN2 C-terminal interactions differ in accordance with the GluN2 subtype, and this influences the type of signaling that NMDA receptor activity controls. Thus, it is not surprising that mutations in genes that codify for NMDA receptor subunits have been associated with severe neuronal diseases. We will review recent advances and explore outstanding problems in this active area of research.
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http://dx.doi.org/10.1016/j.coph.2020.12.006DOI Listing
February 2021

Developmental impaired Akt signaling in the Shank1 and Shank3 double knock-out mice.

Mol Psychiatry 2021 Jan 5. Epub 2021 Jan 5.

CNR Neuroscience Institute, Milan, Milano, Italy.

Human mutations and haploinsufficiency of the SHANK family genes are associated with autism spectrum disorders (ASD) and intellectual disability (ID). Complex phenotypes have been also described in all mouse models of Shank mutations and deletions, consistent with the heterogeneity of the human phenotypes. However, the specific role of Shank proteins in synapse and neuronal functions remain to be elucidated. Here, we generated a new mouse model to investigate how simultaneously deletion of Shank1 and Shank3 affects brain development and behavior in mice. Shank1-Shank3 DKO mice showed a low survival rate, a developmental strong reduction in the activation of intracellular signaling pathways involving Akt, S6, ERK1/2, and eEF2 during development and a severe behavioral impairments. Our study suggests that Shank1 and Shank3 proteins are essential to developmentally regulate the activation of Akt and correlated intracellular pathways crucial for mammalian postnatal brain development and synaptic plasticity. Therefore, Akt function might represent a new therapeutic target for enhancing cognitive abilities of syndromic ASD patients.
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http://dx.doi.org/10.1038/s41380-020-00979-xDOI Listing
January 2021

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter.

Nat Commun 2020 12 3;11(1):6194. Epub 2020 Dec 3.

National Enterprise for Nanoscience and Nanotechnology (NEST), Istituto Nanoscienze Consiglio Nazionale delle Ricerche (CNR) and Scuola Normale Superiore Pisa, 56127, Pisa, Italy.

Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.
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http://dx.doi.org/10.1038/s41467-020-19864-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713426PMC
December 2020

Human induced pluripotent stem cells technology in treatment resistant depression: novel strategies and opportunities to unravel ketamine's fast-acting antidepressant mechanisms.

Ther Adv Psychopharmacol 2020 2;10:2045125320968331. Epub 2020 Nov 2.

Psychiatric Department, San Gerardo Hospital, ASST Monza, Monza, Italy.

Approximately 30% of Major Depressive Disorder (MDD) patients develop treatment-resistant depression (TRD). Among the different causes that make TRD so challenging in both clinical and research contexts, major roles are played by the inadequate understanding of MDD pathophysiology and the limitations of current pharmacological treatments. Nevertheless, the field of psychiatry is facing exciting times. Combined with recent advances in genome editing techniques, human induced pluripotent stem cell (hiPSC) technology is offering novel and unique opportunities in both disease modelling and drug discovery. This technology has allowed innovative disease-relevant patient-specific models to be set up for many psychiatric disorders. Such models hold great potential in enhancing our understanding of MDD pathophysiology and overcoming many of the well-known practical limitations inherent to animal and post-mortem models. Moreover, the field is approaching the advent of (es)ketamine, a glutamate N-methyl-d-aspartate (NMDA) receptor antagonist, claimed as one of the first and exemplary agents with rapid (in hours) antidepressant effects, even in TRD patients. Although ketamine seems poised to transform the treatment of depression, its exact mechanisms of action are still unclear but greatly demanded, as the resulting knowledge may provide a model to understand the mechanisms behind rapid-acting antidepressants, which may lead to the discovery of novel compounds for the treatment of depression. After reviewing insights into ketamine's mechanisms of action (derived from preclinical animal studies) and depicting the current state of the art of hiPSC technology below, we will consider the implementation of an hiPSC technology-based TRD model for the study of ketamine's fast acting antidepressant mechanisms of action.
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http://dx.doi.org/10.1177/2045125320968331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649879PMC
November 2020

Eukaryotic Elongation Factor 2 Kinase a Pharmacological Target to Regulate Protein Translation Dysfunction in Neurological Diseases.

Neuroscience 2020 10 21;445:42-49. Epub 2020 Feb 21.

CNR Neuroscience Institute, Milano, Italy. Electronic address:

Two major processes tightly regulate protein synthesis, the initiation of mRNA translation and elongation phase that mediates the movement of ribosomes along the mRNA. The elongation phase is a high energy-consuming process, and is mainly regulated by the eukaryotic elongation factor 2 kinase (eEF2K) activity that phosphorylates and inhibits eEF2, the only known substrate of the kinase. eEF2K activity is closely regulated by several signaling pathways because the translation elongation phase strongly influences the cellular energy demand and can change the expression of specific proteins in different tissues. An increasing number of recent findings link eEF2k over activation to an array of human diseases, such as atherosclerosis, pulmonary arterial hypertension, progression of solid tumors, and some major neurological disorders. Several neurological studies suggest that eEF2K is a valuable target in treating epilepsy, depression and major neurodegenerative diseases. Despite eEF2k is an ubiquitous and conserved protein, it has been proved that its deletion does not affect development in animal models and in general cell viability. Therefore, it is possible to postulate that inhibiting its function may not cause serious side effects. In addition, eEF2K is a peculiar kinase molecularly different from most of other mammalian kinases and new compounds that inhibit eEF2K should not necessarily interfere with other important protein kinases. In this review we will critically summarize the evidence supporting the role of the altered eEF2K/eEF2 pathway in defined neurological diseases and its implications in curing these diseases in animal models, and possibly in humans, by targeting eEF2K activity.
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http://dx.doi.org/10.1016/j.neuroscience.2020.02.015DOI Listing
October 2020

High-Aspect-Ratio Semiconducting Polymer Pillars for 3D Cell Cultures.

ACS Appl Mater Interfaces 2019 Aug 29;11(31):28125-28137. Epub 2019 Jul 29.

Center for Nano Science and [email protected] , Istituto Italiano di Tecnologia , via Pascoli 70/3 , 20133 Milano , Italy.

Hybrid interfaces between living cells and nano/microstructured scaffolds have huge application potential in biotechnology, spanning from regenerative medicine and stem cell therapies to localized drug delivery and from biosensing and tissue engineering to neural computing. However, 3D architectures based on semiconducting polymers, endowed with responsivity to visible light, have never been considered. Here, we apply for the first time a push-coating technique to realize high aspect ratio polymeric pillars, based on polythiophene, showing optimal biocompatibility and allowing for the realization of soft, 3D cell cultures of both primary neurons and cell line models. HEK-293 cells cultured on top of polymer pillars display a remarkable change in the cell morphology and a sizable enhancement of the membrane capacitance due to the cell membrane thinning in correspondence to the pillars' top surface, without negatively affecting cell proliferation. Electrophysiology properties and synapse number of primary neurons are also very well preserved. In perspective, high aspect ratio semiconducting polymer pillars may find interesting applications as soft, photoactive elements for cell activity sensing and modulation.
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http://dx.doi.org/10.1021/acsami.9b08822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943816PMC
August 2019

SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse.

Neuron 2019 07 3;103(2):217-234.e4. Epub 2019 Jun 3.

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau, 08025 Barcelona, Spain; Universitat Autònoma de Barcelona, 08193 Bellaterra, Cerdanyola del Vallès, Spain.

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).
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http://dx.doi.org/10.1016/j.neuron.2019.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764089PMC
July 2019

IL-38 Ameliorates Skin Inflammation and Limits IL-17 Production from γδ T Cells.

Cell Rep 2019 04;27(3):835-846.e5

Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt, Germany. Electronic address:

Interleukin-38 (IL-38) is a cytokine of the IL-1 family with a role in chronic inflammation. However, its main cellular targets and receptors remain obscure. IL-38 is highly expressed in the skin and downregulated in psoriasis patients. We report an investigation in cellular targets of IL-38 during the progression of imiquimod-induced psoriasis. In this model, IL-38 knockout (IL-38 KO) mice show delayed disease resolution with exacerbated IL-17-mediated inflammation, which is reversed by the administration of mature IL-38 or γδ T cell-receptor-blocking antibodies. Mechanistically, X-linked IL-1 receptor accessory protein-like 1 (IL1RAPL1) is upregulated upon γδ T cell activation to feedforward-amplify IL-17 production and is required for IL-38 to suppress γδ T cell IL-17 production. Accordingly, psoriatic IL1RAPL1 KO mice show reduced inflammation and IL-17 production by γδ T cells. Our findings indicate a role for IL-38 in the regulation of γδ T cell activation through IL1RAPL1, with consequences for auto-inflammatory disease.
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http://dx.doi.org/10.1016/j.celrep.2019.03.082DOI Listing
April 2019

The Up and Down of the N-Methyl-D-Aspartate Receptor That Causes Autism.

Biol Psychiatry 2019 04;85(7):530-531

Consiglio Nazionale delle Ricerche Neuroscience Institute and Department of Biotechnology and Translational Medicine, University of Milan, Milan, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.biopsych.2019.01.020DOI Listing
April 2019

Different attentional dysfunctions in eEF2K , IL1RAPL1 and SHANK3Δ11 mice.

Genes Brain Behav 2019 06 27;18(5):e12563. Epub 2019 Mar 27.

Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy.

A common feature of several psychiatric disorders is the attentional impairment. eEF2K , IL1RAPL1 and SHANK3Δ11 mice were used as animal models consistently linked to changes in synaptic plasticity, learning and memory. All knockout (KO) mice and their corresponding littermates were submitted to the novel object recognition (NOR) and visual object recognition (VOR) tasks. In the NOR, eEF2K mice exhibited a normal performance in terms of mean discrimination index, while SHANK3Δ11 and IL1RAPL1 mice were impaired when a delay of 2 and 24 hours was introduced. Surprisingly, when submitted to VOR, where the two objects were replaced with two shapes delivered from two iPods, all the mutant mice performed worse than those in the NOR. In VOR, the application of motion to different shapes, to increase attention, improved performance in eEF2K and IL1RAPL1 but not in SHANK3Δ11 mice. In SHANK3Δ11 mice, attentional deficit was also present even if different motions were applied to the same shapes or when these mice were repeatedly exposed for 5 days to the context. Behavioral analysis showed that eEF2K and IL1RAPL1 mice had a good flexibility tested in the T-maze. eEF2K showed normal self-grooming. On the basis of previous literature data indicating that SHANK3Δ11 showed impaired flexibility and reduced sociability, we identified in this genotype the most exhaustive model showing all the core symptoms of autism spectrum disorder including a heavy visual attention deficit. These findings show the importance of VOR to identify mouse models of autism.
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http://dx.doi.org/10.1111/gbb.12563DOI Listing
June 2019

The Synaptic and Neuronal Functions of the X-Linked Intellectual Disability Protein Interleukin-1 Receptor Accessory Protein Like 1 (IL1RAPL1).

Dev Neurobiol 2019 01 21;79(1):85-95. Epub 2018 Dec 21.

CNR Neuroscience Institute, Milan, Italy.

Since the first observation that described a patient with a mutation in IL1RAPL1 gene associated with intellectual disability in 1999, the function of IL1RAPL1 has been extensively studied by a number of laboratories. In this review, we summarize all the major data describing the synaptic and neuronal functions of IL1RAPL1 and recapitulate most of the genetic deletion identified in humans and associated to intellectual disability (ID) and autism spectrum disorders (ASD). All the data clearly demonstrate that IL1RAPL1 is a synaptic adhesion molecule localized at the postsynaptic membrane. Mutations in IL1RAPL1 gene cause either the absence of the protein or the production of a dysfunctional protein. More recently it has been demonstrated that IL1RAPL1 regulated dendrite formation and mediates the activity of IL-1β on dendrite morphology. All these data will possibly contribute to identifying therapies for patients carrying mutations in IL1RAPL1 gene.
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http://dx.doi.org/10.1002/dneu.22657DOI Listing
January 2019

SOD1 stimulates lamellipodial protrusions in Neuro 2A cell lines.

Commun Integr Biol 2018 9;11(3):1-7. Epub 2018 Aug 9.

Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy.

We here investigated the effects of overexpressed superoxide dismutase (SOD)1 and amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants G93A and G147S in Neuro 2A (N2A) cell lines, and found a three-fold increase in lamellipodia either in cells cultured under differentiated or undifferentiated growth conditions. In undifferentiated N2A cells, SOD1 constructs promoted lamellipodial protrusions to similar extent as the overexpression of Rac1, and SOD1-mediated lamellipodia were prevented by coexpression of the N17 dominant-negative form of Rac1, or shRNA for a downstream effector of Rac1, the insulin receptor tyrosine kinase substrate p53 (IRSp53) or its binding partner LIN7. Moreover, no additive effect was measured by coexpression of the SOD1 constructs with Rac1, IRSp53 or LIN7. Collectively these data support a role for SOD1 in the regulation of Rac1-mediated lamellipodia pathway, a property fully retained by the two SOD1 mutants.
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http://dx.doi.org/10.1080/19420889.2018.1486652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6132423PMC
August 2018

SHANK genes in autism: Defining therapeutic targets.

Prog Neuropsychopharmacol Biol Psychiatry 2018 06 22;84(Pt B):416-423. Epub 2017 Nov 22.

CNR Neuroscience Institute, Department of Biotechnology and Translational Medicine, University of Milan, Milan, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.pnpbp.2017.11.019DOI Listing
June 2018

The X-Linked Intellectual Disability Protein IL1RAPL1 Regulates Dendrite Complexity.

J Neurosci 2017 07 2;37(28):6606-6627. Epub 2017 Jun 2.

National Research Council Neuroscience Institute, 20129 Milan, Italy,

Mutations and deletions of the () gene, located on the X chromosome, are associated with intellectual disability (ID) and autism spectrum disorder (ASD). IL1RAPL1 protein is located at the postsynaptic compartment of excitatory synapses and plays a role in synapse formation and stabilization. Here, using primary neuronal cultures and -KO mice, we characterized the role of IL1RAPL1 in regulating dendrite morphology. In -KO mice we identified an increased number of dendrite branching points in CA1 and CA2 hippocampal neurons associated to hippocampal cognitive impairment. Similarly, induced pluripotent stem cell-derived neurons from a patient carrying a null mutation of the gene had more dendrites. In hippocampal neurons, the overexpression of full-length IL1RAPL1 and mutants lacking part of C-terminal domains leads to simplified neuronal arborization. This effect is abolished when we overexpressed mutants lacking part of N-terminal domains, indicating that the IL1RAPL1 extracellular domain is required for regulating dendrite development. We also demonstrate that PTPδ interaction is not required for this activity, while IL1RAPL1 mediates the activity of IL-1β on dendrite morphology. Our data reveal a novel specific function for IL1RAPL1 in regulating dendrite morphology that can help clarify how changes in IL1RAPL1-regulated pathways can lead to cognitive disorders in humans. Abnormalities in the architecture of dendrites have been observed in a variety of neurodevelopmental, neurodegenerative, and neuropsychiatric disorders. Here we show that the X-linked intellectual disability protein interleukin-1 receptor accessory protein like 1 (IL1RAPL1) regulates dendrite morphology of mice hippocampal neurons and induced pluripotent stem cell-derived neurons from a patient carrying a null mutation of gene. We also found that the extracellular domain of IL1RAPL1 is required for this effect, independently of the interaction with PTPδ, but IL1RAPL1 mediates the activity of IL-1β on dendrite morphology. Our data reveal a novel specific function for IL1RAPL1 in regulating dendrite morphology that can help clarify how changes in IL1RAPL1-regulated pathways can lead to cognitive disorders in humans.
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http://dx.doi.org/10.1523/JNEUROSCI.3775-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596553PMC
July 2017

Modelling Autistic Neurons with Induced Pluripotent Stem Cells.

Adv Anat Embryol Cell Biol 2017 ;224:49-64

Centre for Stem Cells and Regenerative Medicine, King's College, London, UK.

Autism spectrum disorder (ASD) is a neurodevelopmental condition that affects more than 1% of children per current estimates. It has been characterised by the following two core behavioural phenotypes: (1) deficits in social interaction and communication and (2) repetitive behaviours, restricted interests and activities. Due to the complex nature of ASD, there are currently no effective treatments. The reason behind this is the clinical and genetic heterogeneity between affected individuals on the one hand and the lack of understanding of the underpinning pathophysiological mechanisms on the other hand. Induced pluripotent stem cells (iPSCs) are reprogrammed stem cells from adult cells. These have the capacity to self-renew and differentiate into any type of cells in the body. Therefore, human iPSCs provide a unique opportunity to study the human cellular and molecular phenotypes associated with ASD. Here, we systematically review various ASD variants and co-morbid diseases modelled using human iPSCs.
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http://dx.doi.org/10.1007/978-3-319-52498-6_3DOI Listing
June 2018

Epilepsy and intellectual disability linked protein Shrm4 interaction with GABARs shapes inhibitory neurotransmission.

Nat Commun 2017 03 6;8:14536. Epub 2017 Mar 6.

CNR, Institute of Neuroscience, Via Vanvitelli, 32, 20129 Milano, Italy.

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABA receptors (GABARs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABAR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABARs and extrasynaptic δ-subunit-containing GABARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABAR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.
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http://dx.doi.org/10.1038/ncomms14536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343488PMC
March 2017

Proteomic Analysis of Post-synaptic Density Fractions from Mutant Mice Reveals Brain Region Specific Changes Relevant to Autism Spectrum Disorder.

Front Mol Neurosci 2017 14;10:26. Epub 2017 Feb 14.

Institute for Anatomy and Cell Biology, Ulm UniversityUlm, Germany; Division of Neuroanatomy, Institute of Anatomy, Otto-von-Guericke UniversityMagdeburg, Germany; Leibniz Institute for NeurobiologyMagdeburg, Germany.

Disruption of the human gene can cause several neuropsychiatric disease entities including Phelan-McDermid syndrome, autism spectrum disorder (ASD), and intellectual disability. Although, a wide array of neurobiological studies strongly supports a major role for SHANK3 in organizing the post-synaptic protein scaffold, the molecular processes at synapses of individuals harboring mutations are still far from being understood. In this study, we biochemically isolated the post-synaptic density (PSD) fraction from striatum and hippocampus of adult mutant mice and performed ion-mobility enhanced data-independent label-free LC-MS/MS to obtain the corresponding PSD proteomes (Data are available via ProteomeXchange with identifier PXD005192). This unbiased approach to identify molecular disturbances at mutant PSDs revealed hitherto unknown brain region specific alterations including a striatal decrease of several molecules encoded by ASD susceptibility genes such as the serine/threonine kinase Cdkl5 and the potassium channel K1.1. Being the first comprehensive analysis of brain region specific PSD proteomes from a mutant line, our study provides crucial information on molecular alterations that could foster translational treatment studies for mutation-associated synaptopathies and possibly also ASD in general.
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http://dx.doi.org/10.3389/fnmol.2017.00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5306440PMC
February 2017

Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors.

Nat Nanotechnol 2017 05 28;12(4):322-328. Epub 2016 Nov 28.

Laboratoire Aimé Cotton, CNRS, Université Paris-Sud, ENS Cachan, Université Paris-Saclay, 91405 Orsay, France.

Brain diseases such as autism and Alzheimer's disease (each inflicting >1% of the world population) involve a large network of genes displaying subtle changes in their expression. Abnormalities in intraneuronal transport have been linked to genetic risk factors found in patients, suggesting the relevance of measuring this key biological process. However, current techniques are not sensitive enough to detect minor abnormalities. Here we report a sensitive method to measure the changes in intraneuronal transport induced by brain-disease-related genetic risk factors using fluorescent nanodiamonds (FNDs). We show that the high brightness, photostability and absence of cytotoxicity allow FNDs to be tracked inside the branches of dissociated neurons with a spatial resolution of 12 nm and a temporal resolution of 50 ms. As proof of principle, we applied the FND tracking assay on two transgenic mouse lines that mimic the slight changes in protein concentration (∼30%) found in the brains of patients. In both cases, we show that the FND assay is sufficiently sensitive to detect these changes.
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http://dx.doi.org/10.1038/nnano.2016.260DOI Listing
May 2017

The differential role of cortical protein synthesis in taste memory formation and persistence.

NPJ Sci Learn 2016 11;1:16001. Epub 2016 May 11.

Sagol Department of Neurobiology, Center for Gene Manipulation in the Brain, University of Haifa, Mt Carmel, Haifa, Israel.

The current dogma suggests that the formation of long-term memory (LTM) is dependent on protein synthesis but persistence of the memory trace is not. However, many of the studies examining the effect of protein synthesis inhibitors (PSIs) on LTM persistence were performed in the hippocampus, which is known to have a time-dependent role in memory storage, rather than the cortex, which is considered to be the main structure to store long-term memories. Here we studied the effect of PSIs on LTM formation and persistence in male Wistar Hola ( ≥ 5) rats by infusing the protein synthesis inhibitor, anisomycin (100 μg, 1 μl), into the gustatory cortex (GC) during LTM formation and persistence in conditioned taste aversion (CTA). We found that local anisomycin infusion to the GC before memory acquisition impaired LTM formation ( = 8.9E - 5), but had no effect on LTM persistence when infused 3 days post acquisition ( = 0.94). However, when we extended the time interval between treatment with anisomycin and testing from 3 days to 14 days, LTM persistence was enhanced ( = 0.01). The enhancement was on the background of stable and non-declining memory, and was not recapitulated by another amnesic agent, APV (10 μg, 1 μl), an -methyl-d-aspartate receptor antagonist ( = 0.54). In conclusion, CTA LTM remains sensitive to the action of PSIs in the GC even 3 days following memory acquisition. This sensitivity is differentially expressed between the formation and persistence of LTM, suggesting that increased cortical protein synthesis promotes LTM formation, whereas decreased protein synthesis promotes LTM persistence.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053367PMC
http://dx.doi.org/10.1038/npjscilearn.2016.1DOI Listing
May 2016

Glial degeneration with oxidative damage drives neuronal demise in MPSII disease.

Cell Death Dis 2016 08 11;7(8):e2331. Epub 2016 Aug 11.

Department of Biotechnology and Biosciences, University Milan Bicocca, Piazza della Scienza 2, Milano 20126, Italy.

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the iduronate 2-sulfatase (IDS) enzyme, causing progressive neurodegeneration in patients. Neural stem cells (NSCs) derived from the IDS-ko mouse can recapitulate MPSII pathogenesis in vitro. In differentiating IDS-ko NSCs and in the aging IDS-ko mouse brain, glial degeneration precedes neuronal degeneration. Here we show that pure IDS-ko NSC-derived astrocytes are selectively able to drive neuronal degeneration when cocultured with healthy neurons. This phenotype suggests concurrent oxidative damage with metabolic dysfunction. Similar patterns were observed in murine IDS-ko animals and in human MPSII brains. Most importantly, the mutant phenotype of IDS-ko astrocytes was reversed by low oxygen conditions and treatment with vitamin E, which also reversed the toxic effect on cocultured neurons. Moreover, at very early stages of disease we detected in vivo the development of a neuroinflammatory background that precedes astroglial degeneration, thus suggesting a novel model of MPSII pathogenesis, with neuroinflammation preceding glial degeneration, which is finally followed by neuronal death. This hypothesis is also consistent with the progression of white matter abnormalities in MPSII patients. Our study represents a novel breakthrough in the elucidation of MPSII brain pathogenesis and suggests the antioxidant molecules as potential therapeutic tools to delay MPSII onset and progression.
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http://dx.doi.org/10.1038/cddis.2016.231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108318PMC
August 2016

Homer1 Scaffold Proteins Govern Ca2+ Dynamics in Normal and Reactive Astrocytes.

Cereb Cortex 2017 03;27(3):2365-2384

Department of Fundamental Neurosciences, University of Lausanne, CH1005Lausanne, Switzerland.

In astrocytes, the intracellular calcium (Ca2+) signaling mediated by activation of metabotropic glutamate receptor 5 (mGlu5) is crucially involved in the modulation of many aspects of brain physiology, including gliotransmission. Here, we find that the mGlu5-mediated Ca2+ signaling leading to release of glutamate is governed by mGlu5 interaction with Homer1 scaffolding proteins. We show that the long splice variants Homer1b/c are expressed in astrocytic processes, where they cluster with mGlu5 at sites displaying intense local Ca2+ activity. We show that the structural and functional significance of the Homer1b/c-mGlu5 interaction is to relocate endoplasmic reticulum (ER) to the proximity of the plasma membrane and to optimize Ca2+ signaling and glutamate release. We also show that in reactive astrocytes the short dominant-negative splice variant Homer1a is upregulated. Homer1a, by precluding the mGlu5-ER interaction decreases the intensity of Ca2+ signaling thus limiting the intensity and the duration of glutamate release by astrocytes. Hindering upregulation of Homer1a with a local injection of short interfering RNA in vivo restores mGlu5-mediated Ca2+ signaling and glutamate release and sensitizes astrocytes to apoptosis. We propose that Homer1a may represent one of the cellular mechanisms by which inflammatory astrocytic reactions are beneficial for limiting brain injury.
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http://dx.doi.org/10.1093/cercor/bhw078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963825PMC
March 2017

eEF2K/eEF2 Pathway Controls the Excitation/Inhibition Balance and Susceptibility to Epileptic Seizures.

Cereb Cortex 2017 03;27(3):2226-2248

CNR Neuroscience Institute, Milan, Italy, Università degli Studi di Milano, Milan, Italy.

Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.
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http://dx.doi.org/10.1093/cercor/bhw075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963824PMC
March 2017

Shank synaptic scaffold proteins: keys to understanding the pathogenesis of autism and other synaptic disorders.

J Neurochem 2015 Dec 3;135(5):849-58. Epub 2015 Sep 3.

CNR Neuroscience Institute and Department of Biotechnology and Translational Medicine, University of Milan, Milan, Italy.

Shank/ProSAP proteins are essential to synaptic formation, development, and function. Mutations in the family of SHANK genes are strongly associated with autism spectrum disorders (ASD) and other neurodevelopmental and neuropsychiatric disorders, such as intellectual disability (ID), and schizophrenia. Thus, the term 'Shankopathies' identifies a number of neuronal diseases caused by alteration of Shank protein expression leading to abnormal synaptic development. With this review we want to summarize the major genetic, molecular, behavior and electrophysiological studies that provide new clues into the function of Shanks and pave the way for the discovery of new therapeutic drugs targeted to treat patients with SHANK mutations and also patients affected by other neurodevelopmental and neuropsychiatric disorders. Shank/ProSAP proteins are essential to synaptic formation, development, and function. Mutations in the family of SHANK genes are strongly associated with autism spectrum disorders (ASD) and other neurodevelopmental and neuropsychiatric disorders, such as intellectual disability (ID), and schizophrenia (SCZ). With this review we want to summarize the major genetic, molecular, behavior and electrophysiological studies that provide new clues into the function of Shanks and pave the way for the discovery of new therapeutic drugs targeted to treat patients with SHANK mutations.
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http://dx.doi.org/10.1111/jnc.13232DOI Listing
December 2015

Novel IL1RAPL1 mutations associated with intellectual disability impair synaptogenesis.

Hum Mol Genet 2015 Feb 9;24(4):1106-18. Epub 2014 Oct 9.

Institut Cochin, INSERM U1016, CNRS UMR8104, Université Paris Descartes, Paris 75014, France,

Mutations in interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene have been associated with non-syndromic intellectual disability (ID) and autism spectrum disorder. This protein interacts with synaptic partners like PSD-95 and PTPδ, regulating the formation and function of excitatory synapses. The aim of this work was to characterize the synaptic consequences of three IL1RAPL1 mutations, two novel causing the deletion of exon 6 (Δex6) and one point mutation (C31R), identified in patients with ID. Using immunofluorescence and electrophysiological recordings, we examined the effects of IL1RAPL1 mutant over-expression on synapse formation and function in cultured rodent hippocampal neurons. Δex6 but not C31R mutation leads to IL1RAPL1 protein instability and mislocalization within dendrites. Analysis of different markers of excitatory synapses and sEPSC recording revealed that both mutants fail to induce pre- and post-synaptic differentiation, contrary to WT IL1RAPL1 protein. Cell aggregation and immunoprecipitation assays in HEK293 cells showed a reduction of the interaction between IL1RAPL1 mutants and PTPδ that could explain the observed synaptogenic defect in neurons. However, these mutants do not affect all cellular signaling because their over-expression still activates JNK pathway. We conclude that both mutations described in this study lead to a partial loss of function of the IL1RAPL1 protein through different mechanisms. Our work highlights the important function of the trans-synaptic PTPδ/IL1RAPL1 interaction in synaptogenesis and as such in ID in the patients.
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http://dx.doi.org/10.1093/hmg/ddu523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867007PMC
February 2015

LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex.

Front Mol Neurosci 2014 27;7:49. Epub 2014 May 27.

Division of Neuroscience, San Raffaele Scientific Institute and Vita-Salute University Milan, Italy ; Department of Molecular and Cellular Pharmacology, National Research Council, Neuroscience Institute Milan, Italy.

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex.
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http://dx.doi.org/10.3389/fnmol.2014.00049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4034499PMC
June 2014

Leucine-rich repeat kinase 2 binds to neuronal vesicles through protein interactions mediated by its C-terminal WD40 domain.

Mol Cell Biol 2014 Jun 31;34(12):2147-61. Epub 2014 Mar 31.

Helmholtz Zentrum München, German Research Center for Environmental Health, Research Unit Protein Science, Neuherberg, Germany Eberhard Karls University Tübingen, Institute for Ophthalmic Research, Medical Proteome Center, Tübingen, Germany

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.
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http://dx.doi.org/10.1128/MCB.00914-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054300PMC
June 2014

Elongation factor-2 phosphorylation in dendrites and the regulation of dendritic mRNA translation in neurons.

Front Cell Neurosci 2014 10;8:35. Epub 2014 Feb 10.

CNR Institute of Neuroscience and Department of Biotechnology and Translational Medicine, University of Milan Milan, Italy ; Neuromuscular Diseases and Neuroimmunology, Foundation Carlo Besta Neurological Institute Milan, Italy.

Neuronal activity results in long lasting changes in synaptic structure and function by regulating mRNA translation in dendrites. These activity dependent events yield the synthesis of proteins known to be important for synaptic modifications and diverse forms of synaptic plasticity. Worthy of note, there is accumulating evidence that the eukaryotic Elongation Factor 2 Kinase (eEF2K)/eukaryotic Elongation Factor 2 (eEF2) pathway may be strongly involved in this process. Upon activation, eEF2K phosphorylates and thereby inhibits eEF2, resulting in a dramatic reduction of mRNA translation. eEF2K is activated by elevated levels of calcium and binding of Calmodulin (CaM), hence its alternative name calcium/CaM-dependent protein kinase III (CaMKIII). In dendrites, this process depends on glutamate signaling and N-methyl-D-aspartate receptor (NMDAR) activation. Interestingly, it has been shown that eEF2K can be activated in dendrites by metabotropic glutamate receptor (mGluR) 1/5 signaling, as well. Therefore, neuronal activity can induce local proteomic changes at the postsynapse by altering eEF2K activity. Well-established targets of eEF2K in dendrites include brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeletal-associated protein (Arc), the alpha subunit of calcium/CaM-dependent protein kinase II (αCaMKII), and microtubule-associated protein 1B (MAP1B), all of which have well-known functions in different forms of synaptic plasticity. In this review we will give an overview of the involvement of the eEF2K/eEF2 pathway at dendrites in regulating the translation of dendritic mRNA in the context of altered NMDAR- and neuronal activity, and diverse forms of synaptic plasticity, such as metabotropic glutamate receptor-dependent-long-term depression (mGluR-LTD). For this, we draw on studies carried out both in vitro and in vivo.
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http://dx.doi.org/10.3389/fncel.2014.00035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918593PMC
March 2014