Publications by authors named "Maria Passafaro"

56 Publications

The DNA repair protein ATM as a target in autism spectrum disorder.

JCI Insight 2021 Feb 8;6(3). Epub 2021 Feb 8.

Department of Medical Biotechnology and Translational Medicine (BIOMETRA), University of Milan, Milan, Italy.

Impairment of the GABAergic system has been reported in epilepsy, autism, attention deficit hyperactivity disorder, and schizophrenia. We recently demonstrated that ataxia telangiectasia mutated (ATM) directly shapes the development of the GABAergic system. Here, we show for the first time to our knowledge how the abnormal expression of ATM affects the pathological condition of autism. We exploited 2 different animal models of autism, the methyl CpG binding protein 2-null (Mecp2y/-) mouse model of Rett syndrome and mice prenatally exposed to valproic acid, and found increased ATM levels. Accordingly, treatment with the specific ATM kinase inhibitor KU55933 (KU) normalized molecular, functional, and behavioral defects in these mouse models, such as (a) delayed GABAergic development, (b) hippocampal hyperexcitability, (c) low cognitive performances, and (d) social impairments. Mechanistically, we demonstrate that KU administration to WT hippocampal neurons leads to (a) higher early growth response 4 activity on Kcc2b promoter, (b) increased expression of Mecp2, and (c) potentiated GABA transmission. These results provide evidence and molecular substrates for the pharmacological development of ATM inhibition in autism spectrum disorders.
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http://dx.doi.org/10.1172/jci.insight.133654DOI Listing
February 2021

Right Place at the Right Time: How Changes in Protocadherins Affect Synaptic Connections Contributing to the Etiology of Neurodevelopmental Disorders.

Cells 2020 Dec 18;9(12). Epub 2020 Dec 18.

Consiglio Nazionale delle Ricerche (CNR), Istituto di Neuroscienze, 20129 Milan, Italy.

During brain development, neurons need to form the correct connections with one another in order to give rise to a functional neuronal circuitry. Mistakes during this process, leading to the formation of improper neuronal connectivity, can result in a number of brain abnormalities and impairments collectively referred to as neurodevelopmental disorders. Cell adhesion molecules (CAMs), present on the cell surface, take part in the neurodevelopmental process regulating migration and recognition of specific cells to form functional neuronal assemblies. Among CAMs, the members of the protocadherin (PCDH) group stand out because they are involved in cell adhesion, neurite initiation and outgrowth, axon pathfinding and fasciculation, and synapse formation and stabilization. Given the critical role of these macromolecules in the major neurodevelopmental processes, it is not surprising that clinical and basic research in the past two decades has identified several genes as responsible for a large fraction of neurodevelopmental disorders. In the present article, we review these findings with a focus on the non-clustered PCDH sub-group, discussing the proteins implicated in the main neurodevelopmental disorders.
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http://dx.doi.org/10.3390/cells9122711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766791PMC
December 2020

Lateral habenula dysfunctions in Tm4sf2 mice model for neurodevelopmental disorder.

Neurobiol Dis 2021 Jan 20;148:105189. Epub 2020 Nov 20.

Institute of Neuroscience, CNR, Milan 20129, Italy; NeuroMI Milan Center for Neuroscience, Università Milano-Bicocca, Milan 20126, Italy. Electronic address:

Mutations in the TM4SF2 gene, which encodes TSPAN7, cause a severe form of intellectual disability (ID) often comorbid with autism spectrum disorder (ASD). Recently, we found that TM4SF2 loss in mice affects cognition. Here, we report that Tm4sf2 mice, beyond an ID-like phenotype, display altered sociability, increased repetitive behaviors, anhedonic- and depressive-like states. Cognition relies on the integration of information from several brain areas. In this context, the lateral habenula (LHb) is strategically positioned to coordinate the brain regions involved in higher cognitive functions. Furthermore, in Tm4sf2 mice we found that LHb neurons present hypoexcitability, aberrant neuronal firing pattern and altered sodium and potassium voltage-gated ion channels function. Interestingly, we also found a reduced expression of voltage-gated sodium channel and a hyperactivity of the PKC-ERK pathway, a well-known modulator of ion channels activity, which might explain the functional phenotype showed by Tm4sf2 mice LHb neurons. These findings support Tm4sf2 mice as useful in modeling some ASD-like symptoms. Additionally, we can speculate that LHb functional alteration in Tm4sf2 mice might play a role in the disease pathophysiology.
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http://dx.doi.org/10.1016/j.nbd.2020.105189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840593PMC
January 2021

Pharmacological antagonism of kainate receptor rescues dysfunction and loss of dopamine neurons in a mouse model of human parkin-induced toxicity.

Cell Death Dis 2020 11 10;11(11):963. Epub 2020 Nov 10.

Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132, Milan, Italy.

Mutations in the PARK2 gene encoding the protein parkin cause autosomal recessive juvenile Parkinsonism (ARJP), a neurodegenerative disease characterized by dysfunction and death of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Since a neuroprotective therapy for ARJP does not exist, research efforts aimed at discovering targets for neuroprotection are critically needed. A previous study demonstrated that loss of parkin function or expression of parkin mutants associated with ARJP causes an accumulation of glutamate kainate receptors (KARs) in human brain tissues and an increase of KAR-mediated currents in neurons in vitro. Based on the hypothesis that such KAR hyperactivation may contribute to the death of nigral DA neurons, we investigated the effect of KAR antagonism on the DA neuron dysfunction and death that occur in the parkinQ311X mouse, a model of human parkin-induced toxicity. We found that early accumulation of KARs occurs in the DA neurons of the parkinQ311X mouse, and that chronic administration of the KAR antagonist UBP310 prevents DA neuron loss. This neuroprotective effect is associated with the rescue of the abnormal firing rate of nigral DA neurons and downregulation of GluK2, the key KAR subunit. This study provides novel evidence of a causal role of glutamate KARs in the DA neuron dysfunction and loss occurring in a mouse model of human parkin-induced toxicity. Our results support KAR as a potential target in the development of neuroprotective therapy for ARJP.
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http://dx.doi.org/10.1038/s41419-020-03172-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7656261PMC
November 2020

The Epilepsy-Related Protein PCDH19 Regulates Tonic Inhibition, GABAR Kinetics, and the Intrinsic Excitability of Hippocampal Neurons.

Mol Neurobiol 2020 Dec 3;57(12):5336-5351. Epub 2020 Sep 3.

Institute of Neuroscience, CNR, 20129, Milan, Italy.

PCDH19 encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily preferentially expressed in the brain. PCDH19 mutations cause a neurodevelopmental syndrome named epileptic encephalopathy, early infantile, 9 (EIEE9) characterized by seizures associated with cognitive and behavioral deficits. We recently reported that PCDH19 binds the alpha subunits of GABA receptors (GABARs), modulating their surface availability and miniature inhibitory postsynaptic currents (mIPSCs). Here, we investigated whether PCDH19 regulatory function on GABARs extends to the extrasynaptic receptor pool that mediates tonic current. In fact, the latter shapes neuronal excitability and network properties at the base of information processing. By combining patch-clamp recordings in whole-cell and cell-attached configurations, we provided a functional characterization of primary hippocampal neurons from embryonic rats of either sex expressing a specific PCDH19 short hairpin (sh)RNA. We first demonstrated that PCDH19 downregulation reduces GABAR-mediated tonic current, evaluated by current shift and baseline noise analysis. Next, by single-channel recordings, we showed that PCDH19 regulates GABARs kinetics without altering their conductance. In particular, GABARs of shRNA-expressing neurons preferentially exhibit brief openings at the expense of long ones, thus displaying a flickering behavior. Finally, we showed that PCDH19 downregulation reduces the rheobase and increases the frequency of action potential firing, thus indicating neuronal hyperexcitability. These findings establish PCDH19 as a critical determinant of GABAR-mediated tonic transmission and GABARs gating, and provide the first mechanistic insights into PCDH19-related hyperexcitability and comorbidities.
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http://dx.doi.org/10.1007/s12035-020-02099-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541378PMC
December 2020

Nesfatin-1 decreases the motivational and rewarding value of food.

Neuropsychopharmacology 2020 09 30;45(10):1645-1655. Epub 2020 Apr 30.

Department of Internal Medicine I, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.

Homeostatic and hedonic pathways distinctly interact to control food intake. Dysregulations of circuitries controlling hedonic feeding may disrupt homeostatic mechanisms and lead to eating disorders. The anorexigenic peptides nucleobindin-2 (NUCB2)/nesfatin-1 may be involved in the interaction of these pathways. The endogenous levels of this peptide are regulated by the feeding state, with reduced levels following fasting and normalized by refeeding. The fasting state is associated with biochemical and behavioral adaptations ultimately leading to enhanced sensitization of reward circuitries towards food reward. Although NUCB2/nesfatin-1 is expressed in reward-related brain areas, its role in regulating motivation and preference for nutrients has not yet been investigated. We here report that both dopamine and GABA neurons express NUCB2/nesfatin-1 in the VTA. Ex vivo electrophysiological recordings show that nesfatin-1 hyperpolarizes dopamine, but not GABA, neurons of the VTA by inducing an outward potassium current. In vivo, central administration of nesfatin-1 reduces motivation for food reward in a high-effort condition, sucrose intake and preference. We next adopted a 2-bottle choice procedure, whereby the reward value of sucrose was compared with that of a reference stimulus (sucralose + optogenetic stimulation of VTA dopamine neurons) and found that nesfatin-1 fully abolishes the fasting-induced increase in the reward value of sucrose. These findings indicate that nesfatin-1 reduces energy intake by negatively modulating dopaminergic neuron activity and, in turn, hedonic aspects of food intake. Since nesfatin-1´s actions are preserved in conditions of leptin resistance, the present findings render the NUCB2/nesfatin-1 system an appealing target for the development of novel therapeutical treatments towards obesity.
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http://dx.doi.org/10.1038/s41386-020-0682-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419560PMC
September 2020

TSPAN5 Enriched Microdomains Provide a Platform for Dendritic Spine Maturation through Neuroligin-1 Clustering.

Cell Rep 2019 10;29(5):1130-1146.e8

CNR, Institute of Neuroscience, Milan 20129, Italy. Electronic address:

Tetraspanins are a class of evolutionarily conserved transmembrane proteins with 33 members identified in mammals that have the ability to organize specific membrane domains, named tetraspanin-enriched microdomains (TEMs). Despite the relative abundance of different tetraspanins in the CNS, few studies have explored their role at synapses. Here, we investigate the function of TSPAN5, a member of the tetraspanin superfamily for which mRNA transcripts are found at high levels in the mouse brain. We demonstrate that TSPAN5 is localized in dendritic spines of pyramidal excitatory neurons and that TSPAN5 knockdown induces a dramatic decrease in spine number because of defects in the spine maturation process. Moreover, we show that TSPAN5 interacts with the postsynaptic adhesion molecule neuroligin-1, promoting its correct surface clustering. We propose that membrane compartmentalization by tetraspanins represents an additional mechanism for regulating excitatory synapses.
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http://dx.doi.org/10.1016/j.celrep.2019.09.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899445PMC
October 2019

The Role of Protocadherin 19 (PCDH19) in Neurodevelopment and in the Pathophysiology of Early Infantile Epileptic Encephalopathy-9 (EIEE9).

Dev Neurobiol 2019 01 18;79(1):75-84. Epub 2019 Jan 18.

CNR Institute of Neuroscience, Milano, Italy.

PCDH19 is considered one of the most clinically relevant genes in epilepsy, second only to SCN1A. To date about 150 mutations have been identified as causative for PCDH19-female epilepsy (also known as early infantile epileptic encephalopathy-9, EIEE9), which is characterized by early onset epilepsy, intellectual disabilities, and behavioral disturbances. Although little is known about the physiological role of PCDH19 and the pathogenic mechanisms that lead to EIEE9, in this review, we will present latest researches focused on these aspects, underlining protein expression, its known functions and the mechanisms by which the protein acts, with particular interest in PCDH19 extracellular and intracellular roles in neurons.
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http://dx.doi.org/10.1002/dneu.22654DOI Listing
January 2019

Mitochondrial Dysregulation and Impaired Autophagy in iPSC-Derived Dopaminergic Neurons of Multiple System Atrophy.

Stem Cell Reports 2018 11 18;11(5):1185-1198. Epub 2018 Oct 18.

IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico, Dino Ferrari Center, Neuroscience Section, Department of Pathophysiology and Transplantation, University of Milan, Milan 20122, Italy. Electronic address:

Multiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets.
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http://dx.doi.org/10.1016/j.stemcr.2018.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234905PMC
November 2018

Recent Findings on AMPA Receptor Recycling.

Front Cell Neurosci 2018 3;12:286. Epub 2018 Sep 3.

Institute of Neuroscience, Consiglio Nazionale delle Ricerche (CNR), Milan, Italy.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) are tetrameric protein complexes that mediate most of the fast-excitatory transmission in response to the neurotransmitter glutamate in neurons. The abundance of AMPA-Rs at the surface of excitatory synapses establishes the strength of the response to glutamate. It is thus evident that neurons need to tightly regulate this feature, particularly in the context of all synaptic plasticity events, which are considered the biological correlates of higher cognitive functions such as learning and memory. AMPA-R levels at the synapse are regulated by insertion of newly synthesized receptors, lateral diffusion on the plasma membrane and endosomal cycling. The latter is likely the most important especially for synaptic plasticity. This process starts with the endocytosis of the receptor from the cell surface and is followed by either degradation, if the receptor is directed to the lysosomal compartment, or reinsertion at the cell surface through a specialized endosomal compartment called recycling endosomes. Although the basic steps of this process have been discovered, the details and participation of additional regulatory proteins are still being discovered. In this review article, we describe the most recent findings shedding light on this crucial mechanism of synaptic regulation.
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http://dx.doi.org/10.3389/fncel.2018.00286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129582PMC
September 2018

Rho GTPases in Intellectual Disability: From Genetics to Therapeutic Opportunities.

Int J Mol Sci 2018 Jun 20;19(6). Epub 2018 Jun 20.

Department Molecular Biotechnology and Health Science, University of Torino, Via Nizza 52, 10126 Turin, Italy.

Rho-class small GTPases are implicated in basic cellular processes at nearly all brain developmental steps, from neurogenesis and migration to axon guidance and synaptic plasticity. GTPases are key signal transducing enzymes that link extracellular cues to the neuronal responses required for the construction of neuronal networks, as well as for synaptic function and plasticity. Rho GTPases are highly regulated by a complex set of activating (GEFs) and inactivating (GAPs) partners, via protein:protein interactions (PPI). Misregulated RhoA, Rac1/Rac3 and cdc42 activity has been linked with intellectual disability (ID) and other neurodevelopmental conditions that comprise ID. All genetic evidences indicate that in these disorders the RhoA pathway is hyperactive while the Rac1 and cdc42 pathways are consistently hypoactive. Adopting cultured neurons for in vitro testing and specific animal models of ID for in vivo examination, the endophenotypes associated with these conditions are emerging and include altered neuronal networking, unbalanced excitation/inhibition and altered synaptic activity and plasticity. As we approach a clearer definition of these phenotype(s) and the role of hyper- and hypo-active GTPases in the construction of neuronal networks, there is an increasing possibility that selective inhibitors and activators might be designed via PPI, or identified by screening, that counteract the misregulation of small GTPases and result in alleviation of the cognitive condition. Here we review all knowledge in support of this possibility.
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http://dx.doi.org/10.3390/ijms19061821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032284PMC
June 2018

Trehalose reduces retinal degeneration, neuroinflammation and storage burden caused by a lysosomal hydrolase deficiency.

Autophagy 2018 23;14(8):1419-1434. Epub 2018 Jul 23.

a Department of Molecular and Human Genetics , Baylor College of Medicine, Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital , Houston , TX , USA.

The accumulation of undegraded molecular material leads to progressive neurodegeneration in a number of lysosomal storage disorders (LSDs) that are caused by functional deficiencies of lysosomal hydrolases. To determine whether inducing macroautophagy/autophagy via small-molecule therapy would be effective for neuropathic LSDs due to enzyme deficiency, we treated a mouse model of mucopolysaccharidosis IIIB (MPS IIIB), a storage disorder caused by deficiency of the enzyme NAGLU (alpha-N-acetylglucosaminidase [Sanfilippo disease IIIB]), with the autophagy-inducing compound trehalose. Treated naglu mice lived longer, displayed less hyperactivity and anxiety, retained their vision (and retinal photoreceptors), and showed reduced inflammation in the brain and retina. Treated mice also showed improved clearance of autophagic vacuoles in neuronal and glial cells, accompanied by activation of the TFEB transcriptional network that controls lysosomal biogenesis and autophagic flux. Therefore, small-molecule-induced autophagy enhancement can improve the neurological symptoms associated with a lysosomal enzyme deficiency and could provide a viable therapeutic approach to neuropathic LSDs.

Abbreviations: ANOVA: analysis of variance; Atg7: autophagy related 7; AV: autophagic vacuoles; CD68: cd68 antigen; ERG: electroretinogram; ERT: enzyme replacement therapy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GNAT2: guanine nucleotide binding protein, alpha transducing 2; HSCT: hematopoietic stem cell transplantation; INL: inner nuclear layer; LC3: microtubule-associated protein 1 light chain 3 alpha; MPS: mucopolysaccharidoses; NAGLU: alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); ONL: outer nuclear layer; PBS: phosphate-buffered saline; PRKCA/PKCα: protein kinase C, alpha; S1BF: somatosensory cortex; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; VMP/VPL: ventral posterior nuclei of the thalamus.
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http://dx.doi.org/10.1080/15548627.2018.1474313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6103706PMC
October 2019

Hyperactivity of Rac1-GTPase pathway impairs neuritogenesis of cortical neurons by altering actin dynamics.

Sci Rep 2018 05 8;8(1):7254. Epub 2018 May 8.

Department of Molecular Biotechnology and Health Sciences, University of Torino, Turin, Italy.

The small-GTPase Rac1 is a key molecular regulator linking extracellular signals to actin cytoskeleton dynamics. Loss-of-function mutations in RAC1 and other genes of the Rac signaling pathway have been implicated in the pathogenesis of Intellectual Disability (ID). The Rac1 activity is negatively controlled by GAP proteins, however the effect of Rac1 hyperactivity on neuronal networking in vivo has been poorly studied. ArhGAP15 is a Rac-specific negative regulator, expressed in the main subtypes of pyramidal cortical neurons. In the absence of ArhGAP15, cortical pyramidal neurons show defective neuritogenesis, delayed axonal elongation, reduced dendritic branching, both in vitro and in vivo. These phenotypes are associated with altered actin dynamics at the growth cone due to increased activity of the PAK-LIMK pathway and hyperphosphorylation of ADF/cofilin. These results can be explained by shootin1 hypo-phosphorylation and uncoupling with the adhesion system. Functionally, ArhGAP15 mice exhibit decreased synaptic density, altered electroencephalographic rhythms and cognitive deficits. These data suggest that both hypo- and hyperactivation of the Rac pathway due to mutations in Rac1 regulators can result in conditions of ID, and that a tight regulation of Rac1 activity is required to attain the full complexity of the cortical networks.
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http://dx.doi.org/10.1038/s41598-018-25354-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940682PMC
May 2018

Tetraspanins shape the synapse.

Mol Cell Neurosci 2018 09 6;91:76-81. Epub 2018 Apr 6.

CNR Institute of Neuroscience, Via L. Vanvitelli, 32-20129 Milano, Italy. Electronic address:

Tetraspanins are a family of proteins largely expressed in mammals. These proteins share very similar structures and are involved in several biological processes spanning from the immune system to cancer growth regulation. Moreover, tetraspanins are scaffold proteins that are able to interact with each other and with a subset of proteins involved in the regulation of the central nervous system, including synapse formation, function and plasticity. In this review, we will focus on the analysis of the literature on tetraspanins, highlighting their involvement in synapse formation and function through direct or indirect modulation of synaptic proteins.
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http://dx.doi.org/10.1016/j.mcn.2018.04.001DOI Listing
September 2018

The female epilepsy protein PCDH19 is a new GABAAR-binding partner that regulates GABAergic transmission as well as migration and morphological maturation of hippocampal neurons.

Hum Mol Genet 2018 03;27(6):1027-1038

CNR Institute of Neuroscience, Milan 20129, Italy.

The PCDH19 gene (Xp22.1) encodes the cell-adhesion protein protocadherin-19 (PCDH19) and is responsible for a neurodevelopmental pathology characterized by female-limited epilepsy, cognitive impairment and autistic features, the pathogenic mechanisms of which remain to be elucidated. Here, we identified a new interaction between PCDH19 and GABAA receptor (GABAAR) alpha subunits in the rat brain. PCDH19 shRNA-mediated downregulation reduces GABAAR surface expression and affects the frequency and kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in cultured hippocampal neurons. In vivo, PCDH19 downregulation impairs migration, orientation and dendritic arborization of CA1 hippocampal neurons and increases rat seizure susceptibility. In sum, these data indicate a role for PCDH19 in GABAergic transmission as well as migration and morphological maturation of neurons.
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http://dx.doi.org/10.1093/hmg/ddy019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886308PMC
March 2018

Pharmacological Modulation of AMPAR Rescues Intellectual Disability-Like Phenotype in Tm4sf2-/y Mice.

Cereb Cortex 2017 11;27(11):5369-5384

CNR Institute of Neuroscience, 20129 Milano, Italy.

Intellectual disability affects 2-3% of the world's population and typically begins during childhood, causing impairments in social skills and cognitive abilities. Mutations in the TM4SF2 gene, which encodes the TSPAN7 protein, cause a severe form of intellectual disability, and currently, no therapy is able to ameliorate this cognitive impairment. We previously reported that, in cultured neurons, shRNA-mediated down-regulation of TSPAN7 affects AMPAR trafficking by enhancing PICK1-GluA2 interaction, thereby increasing the intracellular retention of AMPAR. Here, we found that loss of TSPAN7 function in mice causes alterations in hippocampal excitatory synapse structure and functionality as well as cognitive impairment. These changes occurred along with alterations in AMPAR expression levels. We also found that interfering with PICK1-GluA2 binding restored synaptic function in Tm4sf2-/y mice. Moreover, potentiation of AMPAR activity via the administration of the ampakine CX516 reverted the neurological phenotype observed in Tm4sf2-/y mice, suggesting that pharmacological modulation of AMPAR may represent a new approach for treating patients affected by TM4SF2 mutations and intellectual disability.
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http://dx.doi.org/10.1093/cercor/bhx221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939231PMC
November 2017

Glutamatergic synapses in neurodevelopmental disorders.

Prog Neuropsychopharmacol Biol Psychiatry 2018 06 19;84(Pt B):328-342. Epub 2017 Sep 19.

CNR, Institute of Neuroscience, Via Vanvitelli 32, 20129 Milan, Italy. Electronic address:

Neurodevelopmental disorders (NDDs) are a group of diseases whose symptoms arise during childhood or adolescence and that impact several higher cognitive functions such as learning, sociability and mood. Accruing evidence suggests that a shared pathogenic mechanism underlying these diseases is the dysfunction of glutamatergic synapses. We summarize present knowledge on autism spectrum disorders (ASD), intellectual disability (ID), Down syndrome (DS), Rett syndrome (RS) and attention-deficit hyperactivity disorder (ADHD), highlighting the involvement of glutamatergic synapses and receptors in these disorders. The most commonly shared defects involve α-amino-3-hydroxy-5-methyl- 4-isoxazole propionic acid receptors (AMPARs), N-methyl-d-aspartate receptors (NMDARs) and metabotropic glutamate receptors (mGluRs), whose functions are strongly linked to synaptic plasticity, affecting both cell-autonomous features as well as circuit formation. Moreover, the major scaffolding proteins and, thus, the general structure of the synapse are often deregulated in neurodevelopmental disorders, which is not surprising considering their crucial role in the regulation of glutamate receptor positioning and functioning. This convergence of defects supports the definition of neurodevelopmental disorders as a continuum of pathological manifestations, suggesting that glutamatergic synapses could be a therapeutic target to ameliorate patient symptomatology.
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http://dx.doi.org/10.1016/j.pnpbp.2017.09.014DOI Listing
June 2018

Biosynthesis of Astrocytic Trehalose Regulates Neuronal Arborization in Hippocampal Neurons.

ACS Chem Neurosci 2017 09 13;8(9):1865-1872. Epub 2017 Jul 13.

Institute of Neuroscience, CNR , Via Luigi Vanvitelli, 32, I-20129 Milan, Italy.

Trehalose is a nonreducing disaccharide that has recently attracted much attention because of its ability to inhibit protein aggregation, induce autophagy, and protect against dissections and strokes. In vertebrates, the biosynthesis of trehalose was long considered absent due to the lack of annotated genes involved in this process. In contrast, trehalase (TreH), which is an enzyme required for the cleavage of trehalose, is known to be conserved and expressed. Here, we show that trehalose is present as an endogenous metabolite in the rodent hippocampus. We found that primary astrocytes were able to synthesize trehalose and release it into the extracellular space. Notably, the TreH enzyme was observed only in the soma of neurons, which are the exclusive users of this substrate. A statistical analysis of the metabolome during different stages of maturation indicated that this metabolite is implicated in neuronal maturation. A morphological analysis of primary neurons confirmed that trehalose is required for neuronal arborization.
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http://dx.doi.org/10.1021/acschemneuro.7b00177DOI Listing
September 2017

The synaptic function of parkin.

Brain 2017 Sep;140(9):2265-2272

IRCCS Istituto Auxologico Italiano, Department of Neurology and Laboratory of Neuroscience, Milan, Italy.

Loss of function mutations in the gene PARK2, which encodes the protein parkin, cause autosomal recessive juvenile parkinsonism, a neurodegenerative disease characterized by degeneration of the dopaminergic neurons localized in the substantia nigra pars compacta. No therapy is effective in slowing disease progression mostly because the pathogenesis of the disease is yet to be understood. From accruing evidence suggesting that the protein parkin directly regulates synapses it can be hypothesized that PARK2 gene mutations lead to early synaptic damage that results in dopaminergic neuron loss over time. We review evidence that supports the role of parkin in modulating excitatory and dopaminergic synapse functions. We also discuss how these findings underpin the concept that autosomal recessive juvenile parkinsonism can be primarily a synaptopathy. Investigation into the molecular interactions between parkin and synaptic proteins may yield novel targets for pharmacologic interventions.
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http://dx.doi.org/10.1093/brain/awx006DOI Listing
September 2017

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

Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate.

Sci Rep 2016 12 1;6:38167. Epub 2016 Dec 1.

Department of Medicine and Health Sciences, University of Molise, Campobasso, Italy.

Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies.
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http://dx.doi.org/10.1038/srep38167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131271PMC
December 2016

Oligophrenin-1 regulates number, morphology and synaptic properties of adult-born inhibitory interneurons in the olfactory bulb.

Hum Mol Genet 2016 12;25(23):5198-5211

Neuroscience Institute - CNR, Padova, Italy.

Among the X-linked genes associated with intellectual disability, Oligophrenin-1 (OPHN1) encodes for a Rho GTPase-activating protein, a key regulator of several developmental processes, such as dendrite and spine formation and synaptic activity. Inhibitory interneurons play a key role in the development and function of neuronal circuits. Whether a mutation of OPHN1 can affect morphology and synaptic properties of inhibitory interneurons remains poorly understood. To address these open questions, we studied in a well-established mouse model of X-linked intellectual disability, i.e. a line of mice carrying a null mutation of OPHN1, the development and function of adult generated inhibitory interneurons in the olfactory bulb. Combining quantitative morphological analysis and electrophysiological recordings we found that the adult generated inhibitory interneurons were dramatically reduced in number and exhibited a higher proportion of filopodia-like spines, with the consequences on their synaptic function, in OPHN1 ko mice. Furthermore, we found that olfactory behaviour was perturbed in OPHN1 ko mice. Chronic treatment with a Rho kinase inhibitor rescued most of the defects of the newly generated neurons. Altogether, our data indicated that OPHN1 plays a key role in regulating the number, morphology and function of adult-born inhibitory interneurons and contributed to identify potential therapeutic targets.
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http://dx.doi.org/10.1093/hmg/ddw340DOI Listing
December 2016

Disruption of ArhGAP15 results in hyperactive Rac1, affects the architecture and function of hippocampal inhibitory neurons and causes cognitive deficits.

Sci Rep 2016 10 7;6:34877. Epub 2016 Oct 7.

Department of Molecular Biotechnologies and Health Sciences, University of Torino, Italy.

During brain development, the small GTPases Rac1/Rac3 play key roles in neuronal migration, neuritogenesis, synaptic formation and plasticity, via control of actin cytoskeleton dynamic. Their activity is positively and negatively regulated by GEFs and GAPs molecules, respectively. However their in vivo roles are poorly known. The ArhGAP15 gene, coding for a Rac-specific GAP protein, is expressed in both excitatory and inhibitory neurons of the adult hippocampus, and its loss results in the hyperactivation of Rac1/Rac3. In the CA3 and dentate gyrus (DG) regions of the ArhGAP15 mutant hippocampus the CR+, PV+ and SST+ inhibitory neurons are reduced in number, due to reduced efficiency and directionality of their migration, while pyramidal neurons are unaffected. Loss of ArhGAP15 alters neuritogenesis and the balance between excitatory and inhibitory synapses, with a net functional result consisting in increased spike frequency and bursts, accompanied by poor synchronization. Thus, the loss of ArhGAP15 mainly impacts on interneuron-dependent inhibition. Adult ArhGAP15 mice showed defective hippocampus-dependent functions such as working and associative memories. These findings indicate that a normal architecture and function of hippocampal inhibitory neurons is essential for higher hippocampal functions, and is exquisitely sensitive to ArhGAP15-dependent modulation of Rac1/Rac3.
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http://dx.doi.org/10.1038/srep34877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054378PMC
October 2016

Biosynthesis of glycerol phosphate is associated with long-term potentiation in hippocampal neurons.

Metabolomics 2016;12:133. Epub 2016 Jul 23.

Institute of Neuroscience, CNR, Via L. Vanvitelli 32, 20129 Milan, Italy.

Introduction: Neurons have a very high energy requirement, and their metabolism is tightly regulated to ensure delivery of adequate substrate to sustain neuronal activity and neuroplastic changes. The mechanisms underlying the regulation of neuronal metabolism, however, are not completely clear.

Objective: The objective of this study was to investigate the central carbon metabolism in neurons, in order to identify the regulatory pathways governing neuronal anabolism and catabolism.

Methods: Here we first have applied MS-based endometabolomics to elucidate the metabolic dynamics in cultured hippocampal primary neurons. Using nanoLC-ESI-LTQ Orbitrap MS approach followed by statistical analysis, we measure the dynamics of uniformly labeled C-glucose entering neurons. We adapted the method by coupling offline patch-clamp setup with MS to confirm findings in vivo.

Results: According to non-parametric statistical analysis of metabolic dynamics, in cultured hippocampal neurons, the glycerol phosphate shuttle is active and correlates with the metabolic flux in the pentose phosphate pathway. In the hippocampus, glycerol-3-phosphate biosynthesis was activated in response to long-term potentiation together with the upregulation of glycolysis and the TCA cycle, but was inactive or silenced in basal conditions.

Conclusions: We identified the biosynthesis of glycerol-3-phosphate as a key regulator in mechanisms implicated in learning and memory. Notably, defects in enzymes linked with the glycerol phosphate shuttle have been implicated in neurological disorders and intellectual disability These results could improve our understanding of the general mechanisms of learning and memory and facilitate the development of novel therapies for metabolic disorders linked with intellectual disability.
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http://dx.doi.org/10.1007/s11306-016-1083-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958395PMC
July 2016

SNX27, a protein involved in down syndrome, regulates GPR17 trafficking and oligodendrocyte differentiation.

Glia 2016 08 6;64(8):1437-60. Epub 2016 Jun 6.

CNR - Institute of Neuroscience, Department of Medical Biotechnologies and Translational Medicine (BIOMETRA), Università Degli Studi Di Milano, Milan, Italy.

The G protein-coupled receptor 17 (GPR17) plays crucial roles in myelination. It is highly expressed during transition of oligodendrocyte progenitor cells to immature oligodendrocytes, but, after this stage, it must be down-regulated to allow generation of mature myelinating cells. After endocytosis, GPR17 is sorted into lysosomes for degradation or recycled to the plasma membrane. Balance between degradation and recycling is important for modulation of receptor levels at the cell surface and thus for the silencing/activation of GPR17-signaling pathways that, in turn, affect oligodendrocyte differentiation. The molecular mechanisms at the basis of these processes are still partially unknown and their characterization will allow a better understanding of myelination and provide cues to interpret the consequences of GPR17 dysfunction in diseases. Here, we demonstrate that the endocytic trafficking of GPR17 is mediated by the interaction of a type I PDZ-binding motif located at the C-terminus of the receptor and SNX27, a recently identified protein of the endosome-associated retromer complex and whose functions in oligodendrocytes have never been studied. SNX27 knock-down significantly reduces GPR17 plasma membrane recycling in differentiating oligodendrocytes while accelerating cells' terminal maturation. Interestingly, trisomy-linked down-regulation of SNX27 expression in the brain of Ts65Dn mice, a model of Down syndrome, correlates with a decrease in GPR17(+) cells and an increase in mature oligodendrocytes, which, however, fail in reaching full maturation, eventually leading to hypomyelination. Our data demonstrate that SNX27 modulates GPR17 plasma membrane recycling and stability, and that disruption of the SNX27/GPR17 interaction might contribute to pathological oligodendrocyte differentiation defects. GLIA 2016. GLIA 2016;64:1437-1460.
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http://dx.doi.org/10.1002/glia.23015DOI Listing
August 2016

New Role of ATM in Controlling GABAergic Tone During Development.

Cereb Cortex 2016 10 10;26(10):3879-88. Epub 2016 May 10.

Department of Biotechnology and Translational Medicine, University of Milan, 20129 Milan, Italy Institute of Neuroscience, C.N.R., 20129 Milan, Italy.

The capacity to guarantee the proper excitatory/inhibitory balance is one of the most critical steps during early development responsible for the correct brain organization, function, and plasticity. GABAergic neurons guide this process leading to the right structural organization, brain circuitry, and neuronal firing. Here, we identified the ataxia telangiectasia mutated (ATM), a serine/threonine protein kinase linked to DNA damage response, as crucial in regulating neurotransmission. We found that reduced levels of ATM in the hippocampal neuronal cultures produce an excitatory/inhibitory unbalance toward inhibition as indicated by the higher frequency of miniature inhibitory postsynaptic current events and an increased number of GABAergic synapses. In vivo, the increased inhibition still persists and, even if a higher excitation is also present, a reduced neuronal excitability is found as indicated by the lower action potential frequency generated in response to high-current intensity stimuli. Finally, we found an elevated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in heterozygous hippocampi associated with lower expression levels of the ERK1/2 phosphatase PP1. Given that the neurodegenerative condition associated with genetic mutations in the Atm gene, ataxia telangiectasia, presents a variable phenotype with impairment in cognition, our molecular findings provide a logical frame for a more clear comprehension of cognitive defects in the pathology, opening to novel therapeutic strategies.
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http://dx.doi.org/10.1093/cercor/bhw125DOI Listing
October 2016

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

LSD1 modulates stress-evoked transcription of immediate early genes and emotional behavior.

Proc Natl Acad Sci U S A 2016 Mar 14;113(13):3651-6. Epub 2016 Mar 14.

Laboratory of Neuroepigenetics, Dipartimento di Biotecnologie Mediche e Medicina Traslazionale and Centro di Eccellenza per le Malattie Neurodegenerative (CEND), Università degli Studi di Milano, 20133 Milan, Italy; National Research Council Institute of Neuroscience, 20129 Milan, Italy;

Behavioral changes in response to stressful stimuli can be controlled via adaptive epigenetic changes in neuronal gene expression. Here we indicate a role for the transcriptional corepressor Lysine-Specific Demethylase 1 (LSD1) and its dominant-negative splicing isoform neuroLSD1, in the modulation of emotional behavior. In mouse hippocampus, we show that LSD1 and neuroLSD1 can interact with transcription factor serum response factor (SRF) and set the chromatin state of SRF-targeted genes early growth response 1 (egr1) and c-fos Deletion or reduction of neuro LSD1 in mutant mice translates into decreased levels of activating histone marks at egr1 and c-fos promoters, dampening their psychosocial stress-induced transcription and resulting in low anxiety-like behavior. Administration of suberoylanilide hydroxamine to neuroLSD1(KO)mice reactivates egr1 and c-fos transcription and restores the behavioral phenotype. These findings indicate that LSD1 is a molecular transducer of stressful stimuli as well as a stress-response modifier. Indeed, LSD1 expression itself is increased acutely at both the transcriptional and splicing levels by psychosocial stress, suggesting that LSD1 is involved in the adaptive response to stress.
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http://dx.doi.org/10.1073/pnas.1511974113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822633PMC
March 2016

Bcl-2/adenovirus E1B 19-kDa interacting protein (BNip3) has a key role in the mitochondrial dysfunction induced by mutant huntingtin.

Hum Mol Genet 2015 Nov 10;24(22):6530-9. Epub 2015 Sep 10.

Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, 20149 Milan, Italy,

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the IT15 gene that encodes the protein huntingtin (htt). Evidence shows that mutant htt causes mitochondrial depolarization and fragmentation, but the underlying molecular mechanism has yet to be clarified. Bax/Bak and BNip3 are pro-apoptotic members of the Bcl-2 family protein whose activation triggers mitochondrial depolarization and fragmentation inducing cell death. Evidence suggests that Bax/Bak and BNip3 undergo activation upon mutant htt expression but whether these proteins are required for mitochondrial depolarization and fragmentation induced by mutant htt is unclear. Our results show that BNip3 knock-out cells are protected from mitochondrial damage and cell death induced by mutant htt whereas Bax/Bak knock-out cells are not. Moreover, deletion of BNip3 C-terminal transmembrane domain, required for mitochondrial targeting, suppresses mitochondrial depolarization and fragmentation in a cell culture model of HD. Hence, our results suggest that changes in mitochondrial morphology and transmembrane potential, induced by mutant htt protein, are dependent and linked to BNip3 and not to Bax/Bak activation. These results provide new compelling evidence that underlies the molecular mechanisms by which mutant htt causes mitochondrial dysfunction and cell death, suggesting BNip3 as a potential target for HD therapy.
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http://dx.doi.org/10.1093/hmg/ddv362DOI Listing
November 2015

The intellectual disability protein RAB39B selectively regulates GluA2 trafficking to determine synaptic AMPAR composition.

Nat Commun 2015 Mar 18;6:6504. Epub 2015 Mar 18.

Dulbecco Telethon Institute at IRCCS San Raffaele Scientific Institute, Division of Neuroscience, 20132 Milan, Italy.

RAB39B is a member of the RAB family of small GTPases that controls intracellular vesicular trafficking in a compartment-specific manner. Mutations in the RAB39B gene cause intellectual disability comorbid with autism spectrum disorder and epilepsy, but the impact of RAB39B loss of function on synaptic activity is largely unexplained. Here we show that protein interacting with C-kinase 1 (PICK1) is a downstream effector of GTP-bound RAB39B and that RAB39B-PICK1 controls trafficking from the endoplasmic reticulum to the Golgi and, hence, surface expression of GluA2, a subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). The role of AMPARs in synaptic transmission varies depending on the combination of subunits (GluA1, GluA2 and GluA3) they incorporate. RAB39B downregulation in mouse hippocampal neurons skews AMPAR composition towards non GluA2-containing Ca(2+)-permeable forms and thereby alters synaptic activity, specifically in hippocampal neurons. We posit that the resulting alteration in synaptic function underlies cognitive dysfunction in RAB39B-related disorders.
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http://dx.doi.org/10.1038/ncomms7504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383008PMC
March 2015