The Postsynaptic Density Proteins Homer and Shank Form a Polymeric Network Structure

The postsynaptic density (PSD) is crucial for synaptic functions, but the molecular architecture retaining its structure and components remains elusive. Homer and Shank are among the most abundant scaffolding proteins in the PSD, working synergistically for maturation of dendritic spines. Here, we demonstrate that Homer and Shank, together, form a mesh-like matrix structure. Crystallographic analysis of this region revealed a pair of parallel dimeric coiled coils intercalated in a tail-to-tail fashion to form a tetramer, giving rise to the unique configuration of a pair of N-terminal EVH1 domains at each end of the coiled coil. In neurons, the tetramerization is required for structural integrity of the dendritic spines and recruitment of proteins to synapses. We propose that the Homer-Shank complex serves as a structural framework and as an assembly platform for other PSD proteins.Massachusetts Institute of Technology. Biophysical Instrumentation FacilityRIKEN Brain Science InstituteNational Institutes of Health (U.S.) (Grant R01DA17310)Japan. Ministry of Education, Culture, Sports, Science and Technology (Grant-in-Aid for Scientific Research

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

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 (\u3b1CaMKII), 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

Definition and clinical variability of SHANK3-related Phelan-McDermid syndrome

Phelan-McDermid syndrome (PMS) is an infrequently described syndrome that presents with a disturbed development, neurological and psychiatric characteristics, and sometimes other comorbidities. As part of the development of European medical guidelines we studied the definition, phenotype, genotype-phenotype characteristics, and natural history of the syndrome. The number of confirmed diagnoses of PMS in different European countries was also assessed and it could be concluded that PMS is underdiagnosed. The incidence of PMS in European countries is estimated to be at least 1 in 30,000. Next generation sequencing, including analysis of copy number variations, as first tier in diagnostics of individuals with intellectual disability will likely yield a larger number of individuals with PMS than presently known. A definition of PMS by its phenotype is at the present not possible, and therefore PMS-SHANK3 related is defined by the presence of SHANK3 haploinsufficiency, either by a deletion involving region 22q13.2–33 or a pathogenic/likely pathogenic variant in SHANK3. In summarizing the phenotype, we subdivided it into that of individuals with a 22q13 deletion and that of those with a pathogenic/likely pathogenic SHANK3 variant. The phenotype of individuals with PMS is variable, depending in part on the deletion size or whether only a variant of SHANK3 is present. The core phenotype in the domains development, neurology, and senses are similar in those with deletions and SHANK3 variants, but individuals with a SHANK3 variant more often are reported to have behavioural disorders and less often urogenital malformations and lymphedema. The behavioural disorders may, however, be a less outstanding feature in individuals with deletions accompanied by more severe intellectual disability. Data available on the natural history are limited. Results of clinical trials using IGF-1, intranasal insulin, and oxytocin are available, other trials are in progress. The present guidelines for PMS aim at offering tools to caregivers and families to provide optimal care to individuals with PMS.</p

Alternative splicing of the histone demethylase LSD1/KDM1 contributes to the modulation of neurite morphogenesis in the mammalian nervous system

A variety of chromatin remodeling complexes are thought to orchestrate transcriptional programs that lead neuronal precursors from earliest commitment to terminal differentiation. Here we show that mammalian neurons have a specialized chromatin remodeling enzyme arising from a neurospecific splice variant of LSD1/KDM1, histone lysine specific demethylase 1, whose demethylase activity on Lys4 of histone H3 has been related to gene repression. We found that alternative splicing of LSD1 transcript generates four full-length isoforms from combinatorial retention of two identified exons: the 4 aa exon E8a is internal to the amine oxidase domain, and its inclusion is restricted to the nervous system. Remarkably, the expression of LSD1 splice variants is dynamically regulated throughout cortical development, particularly during perinatal stages, with a progressive increase of LSD1 neurospecific isoforms over the ubiquitous ones. Notably, the same LSD1 splice dynamics can be fairly recapitulated in cultured cortical neurons. Functionally, LSD1 isoforms display in vitro a comparable demethylase activity, yet the inclusion of the sole exon E8a reduces LSD1 repressor activity on a reporter gene. Additional distinction among isoforms is supported by the knockdown of neurospecific variants in cortical neurons resulting in the inhibition of neurite maturation, whereas overexpression of the same variants enhances it. Instead, perturbation of LSD1 isoforms that are devoid of the neurospecific exon elicits no morphogenic effect. Collectively, results demonstrate that the arousal of neuronal LSD1 isoforms pacemakes early neurite morphogenesis, conferring a neurospecific function to LSD1 epigenetic activity

The X-linked intellectual disability protein IL1RAPL1 regulates excitatory synapse formation by binding PTPδ and RhoGAP2

Mutations of the Interleukin-1-receptor accessory protein like 1 (IL1RAPL1) gene are associated with cognitive impairment ranging from non-syndromic X-linked mental retardation to autism. IL1RAPL1 belongs to a novel family of IL1/Toll receptors, which is localized at excitatory synapses and interacts with PSD-95. We previously showed that IL1RAPL1 regulates the synaptic localization of PSD-95 by controlling c-Jun N-terminal kinase activity and PSD-95 phosphorylation. Here, we show that the IgG-like extracellular domains of IL1RAPL1 induce excitatory pre-synapse formation by interacting with protein tyrosine phosphatase delta (PTPδ). We also found that IL1RAPL1 TIR domains interact with RhoGAP2, which is localized at the excitatory post-synaptic density. More interestingly, the IL1RAPL1/PTPδ complex recruits RhoGAP2 at excitatory synapses to induce dendritic spine formation. We also found that the IL1RAPL1 paralog, IL1RAPL2, interacts with PTPδ and induces excitatory synapse and dendritic spine formation. The interaction of the IL1RAPL1 family of proteins with PTPδ and RhoGAP2 reveals a pathophysiological mechanism of cognitive impairment associated with a novel type of trans-synaptic signaling that regulates excitatory synapse and dendritic spine formation

Recommendations, guidelines, and best practice for the use of human induced pluripotent stem cells for neuropharmacological studies of neuropsychiatric disorders

The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including genetics, imaging, clinical and animal models has advanced our knowledge regarding NPDs, we still lack basic knowledge regarding the underlying pathophysiological mechanisms. Moreover, there is an urgent need for highly effective therapeutics for NPDs. Human induced pluripotent stem cells (hiPSCs) generated from somatic cells enabled scientists to create brain cells in a patient-specific manner. However, there are challenges to the use of hiPSCs that need to be addressed. In the current paper, consideration of best practices for neuropharmacological and neuropsychiatric research using hiPSCs will be discussed. Specifically, we provide recommendations for best practice in patient recruitment, including collecting demographic, clinical, medical (before and after treatment and response), diagnostic (including scales) and genetic data from the donors. We highlight considerations regarding donor genetics and sex, in addition to discussing biological and technical replicates. Furthermore, we present our views on selecting control groups/lines, experimental designs, and considerations for conducting neuropharmacological studies using hiPSC-based models in the context of NPDs. In doing so, we explore key issues in the field concerning reproducibility, statistical analysis, and how to translate in vitro studies into clinically relevant observations. The aim of this article is to provide a key resource for hiPSC researchers to perform robust and reproducible neuropharmacological studies, with the ultimate aim of improving identification and clinical translation of novel therapeutic drugs for NPDs

Anti-Angiogenic Therapy Induces Integrin-Linked Kinase 1 Up-Regulation in a Mouse Model of Glioblastoma

BACKGROUND: In order to improve our understanding of the molecular pathways that mediate tumor proliferation and angiogenesis, and to evaluate the biological response to anti-angiogenic therapy, we analyzed the changes in the protein profile of glioblastoma in response to treatment with recombinant human Platelet Factor 4-DLR mutated protein (PF4-DLR), an inhibitor of angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: U87-derived experimental glioblastomas were grown in the brain of xenografted nude mice, treated with PF4-DLR, and processed for proteomic analysis. More than fifty proteins were differentially expressed in response to PF4-DLR treatment. Among them, integrin-linked kinase 1 (ILK1) signaling pathway was first down-regulated but then up-regulated after treatment for prolonged period. The activity of PF4-DLR can be increased by simultaneously treating mice orthotopically implanted with glioblastomas, with ILK1-specific siRNA. As ILK1 is related to malignant progression and a poor prognosis in various types of tumors, we measured ILK1 expression in human glioblastomas, astrocytomas and oligodendrogliomas, and found that it varied widely; however, a high level of ILK1 expression was correlated to a poor prognosis. CONCLUSIONS/SIGNIFICANCE: Our results suggest that identifying the molecular pathways induced by anti-angiogenic therapies may help the development of combinatorial treatment strategies that increase the therapeutic efficacy of angiogenesis inhibitors by association with specific agents that disrupt signaling in tumor cells

SHANK3 controls maturation of social reward circuits in the VTA.

Haploinsufficiency of SHANK3, encoding the synapse scaffolding protein SHANK3, leads to a highly penetrant form of autism spectrum disorder. How SHANK3 insufficiency affects specific neural circuits and how this is related to specific symptoms remains elusive. Here we used shRNA to model Shank3 insufficiency in the ventral tegmental area of mice. We identified dopamine (DA) and GABA cell-type-specific changes in excitatory synapse transmission that converge to reduce DA neuron activity and generate behavioral deficits, including impaired social preference. Administration of a positive allosteric modulator of the type 1 metabotropic glutamate receptors mGluR1 during the first postnatal week restored DA neuron excitatory synapse transmission and partially rescued the social preference defects, while optogenetic DA neuron stimulation was sufficient to enhance social preference. Collectively, these data reveal the contribution of impaired ventral tegmental area function to social behaviors and identify mGluR1 modulation during postnatal development as a potential treatment strategy

Combination of temozolomide with immunocytokine F16–IL2 for the treatment of glioblastoma

Glioblastoma patients are still not cured by the treatments available at the moment. We investigated the therapeutic properties of temozolomide in combination with F16-IL2, a clinical-stage immunocytokine consisting of human interleukin (IL)-2 fused to the human antibody F16, specific to the A1 domain of tenascin-C