Synaptic plasticity is a process that shapes neuronal connections during neurodevelopment and learning and memory. Autophagy is a mechanism that allows the cell to degrade its unnecessary or dysfunctional components. Autophagosomes appear at dendritic spines in response to plasticity-inducing stimuli. Autophagy defects contribute to altered dendritic spine development, autistic-like behavior in mice, and neurological disease. While several studies have explored the involvement of autophagy in synaptic plasticity, the initial steps of the emergence of autophagosomes at the postsynapse remain unknown. Here, we demonstrate a postsynaptic association of autophagy-related protein 9A (Atg9A), known to be involved in the early stages of autophagosome formation, with Rab11, a small GTPase that regulates endosomal trafficking. Rab11 activity was necessary to maintain Atg9A-positive structures at dendritic spines. Inhibition of mTOR increased Rab11 and Atg9A interaction and increased the emergence of LC3 positive vesicles, an autophagosome membrane-associated protein marker, in dendritic spines when coupled to NMDA receptor stimulation. Dendritic spines with newly formed LC3+ vesicles were more resistant to NMDA-induced morphologic change. Rab11 DN overexpression suppressed appearance of LC3+ vesicles. Collectively, these results suggest that initiation of autophagy in dendritic spines depends on neuronal activity and Rab11a-dependent Atg9A interaction that is regulated by mTOR activity.

We present a comprehensive multi-omic analysis of the EPISTOP prospective clinical trial of early intervention with vigabatrin for pre-symptomatic epilepsy treatment in Tuberous Sclerosis Complex (TSC), in which 93 infants with TSC were followed from birth to age 2 years, seeking biomarkers of epilepsy development. Vigabatrin had profound effects on many metabolites, increasing serum deoxycytidine monophosphate (dCMP) levels 52-fold. Most serum proteins and metabolites, and blood RNA species showed significant change with age. Thirty-nine proteins, metabolites, and genes showed significant differences between age-matched control and TSC infants. Six also showed a progressive difference in expression between control, TSC without epilepsy, and TSC with epilepsy groups. A multivariate approach using enrollment samples identified multiple 3-variable predictors of epilepsy, with the best having a positive predictive value of 0.987. This rich dataset will enable further discovery and analysis of developmental effects, and associations with seizure development in TSC.

Tuberous sclerosis complex (TSC) is a rare multi-system genetic disorder characterized by a high incidence of epilepsy and neuropsychiatric manifestations known as tuberous-sclerosis-associated neuropsychiatric disorders (TANDs), including autism spectrum disorder (ASD) and intellectual disability (ID). MicroRNAs (miRNAs) are small regulatory non-coding RNAs that regulate the expression of more than 60% of all protein-coding genes in humans and have been reported to be dysregulated in several diseases, including TSC. In the current study, RNA sequencing analysis was performed to define the miRNA and isoform (isomiR) expression patterns in serum. A Receiver Operating Characteristic (ROC) curve analysis was used to identify circulating molecular biomarkers, miRNAs, and isomiRs, able to discriminate the development of neuropsychiatric comorbidity, either ASD, ID, or ASD + ID, in patients with TSC. Part of our bioinformatics predictions was verified with RT-qPCR performed on RNA isolated from patients’ serum. Our results support the notion that circulating miRNAs and isomiRs have the potential to aid standard clinical testing in the early risk assessment of ASD and ID development in TSC patients.

Autism spectrum disorder (ASD) is highly prevalent in subjects with Tuberous Sclerosis Complex (TSC), but we are not still able to reliably predict which infants will develop ASD. This study aimed to identify the early clinical markers of ASD and/or developmental delay (DD) in infants with an early diagnosis of TSC. We prospectively evaluated 82 infants with TSC (6–24 months of age), using a detailed neuropsychological assessment (Bayley Scales of Infant Development—BSID, and Autism Diagnostic Observation Schedule—ADOS), in the context of the EPISTOP (Long-term, prospective study evaluating clinical and molecular biomarkers of EPIleptogenesiS in a genetic model of epilepsy—Tuberous SclerOsis ComPlex) project (NCT02098759). Normal cognitive developmental quotient at 12 months excluded subsequent ASD (negative predictive value 100%). The total score of ADOS at 12 months clearly differentiated children with a future diagnosis of ASD from children without (p = 0.012). Atypical socio-communication behaviors (p < 0.001) were more frequently observed than stereotyped/repetitive behaviors in children with ASD at 24 months. The combined use of BSID and ADOS can reliably identify infants with TSC with a higher risk for ASD at age 6–12 months, allowing for clinicians to target the earliest symptoms of abnormal neurodevelopment with tailored intervention strategies.

Several mosaic mutations of the mammalian/mechanistic target of rapamycin (mTOR) have recently been found in patients with cortical malformations, such as hemimegalencephaly (HME) and focal cortical dysplasia (FCD). Although all of them should activate mTOR signaling, comparisons of the impact of different mTOR mutations on brain development have been lacking. Also it remains unknown if any potential differences these mutations may have on cortical development are directly related to a degree of mTOR signaling increase. The present study assessed levels of mTORC1 pathway activity in cell lines and rat primary neurons overexpressing several mTOR mutants that were previously found in HME, FCD, cancer patients and in vitro mutagenesis screens. Next we introduced the mutants, enhancing mTORC1 signaling most potently, into developing mouse brains and assessed electroporated cell morphology and migratory phenotype using immunofluorescent staining. We observed the differential inhibition of neuronal progenitor cortical migration, which partly corresponded with a degree of mTORC1 signaling enhancement these mutants induced in cultured cells. The most potent quadruple mutant prevented most of the progenitors from entering the cortical plate. Cells that expressed less potent, single-point, mTOR mutants entered the cortical plate but failed to reach its upper layers and had enlarged soma. Our findings suggest a correlation between the potency of mTOR mutation to activate mTORC1 pathway and disruption of cortical migration.

Epileptogenesis is a process triggered by initial environmental or genetic factors that result in epilepsy and may continue during disease progression. Important parts of this process include changes in transcriptome and the pathological rewiring of neuronal circuits that involves changes in neuronal morphology. Mammalian/mechanistic target of rapamycin (mTOR) is upregulated by proconvulsive drugs, e.g., kainic acid, and is needed for progression of epileptogenesis, but molecular aspects of its contribution are not fully understood. Since mTOR can modulate transcription, we tested if rapamycin, an mTOR complex 1 inhibitor, affects kainic acid-evoked transcriptome changes. Using microarray technology, we showed that rapamycin inhibits the kainic acid-induced expression of multiple functionally heterogeneous genes. We further focused on engulfment and cell motility 1 (Elmo1), which is a modulator of actin dynamics and therefore could contribute to pathological rewiring of neuronal circuits during epileptogenesis. We showed that prolonged overexpression of Elmo1 in cultured hippocampal neurons increased axonal growth, decreased dendritic spine density, and affected their shape. In conclusion, data presented herein show that increased mTORC1 activity in response to kainic acid has no global effect on gene expression. Instead, our findings suggest that mTORC1 inhibition may affect development of epilepsy, by modulating expression of specific subset of genes, including Elmo1, and point to a potential role for Elmo1 in morphological changes that accompany epileptogenesis.

The behavioral changes that comprise operant learning are associated with plasticity in early sensory cortices as well as with modulation of gene expression, but the connection between the behavioral, electrophysiological, and molecular changes is only partially understood. We specifically manipulated c-Fos expression, a hallmark of learning-induced synaptic plasticity, in auditory cortex of adult mice using a novel approach based on RNA interference. Locally blocking c-Fos expression caused a specific behavioral deficit in a sound discrimination task, in parallel with decreased cortical experience-dependent plasticity, without affecting baseline excitability or basic auditory processing. Thus, c-Fos-dependent experience-dependent cortical plasticity is necessary for frequency discrimination in an operant behavioral task. Our results connect behavioral, molecular and physiological changes and demonstrate a role of c-Fos in experience-dependent plasticity and learning.

The selective and neuronal activity-dependent degradation of synaptic proteins appears to be crucial for long-term synaptic plasticity. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which regulates the synaptic content of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), excitatory synapse strength and dendritic spine morphology. The levels of Arc protein are tightly regulated, and its removal occurs via proteasome-mediated degradation that requires prior ubiquitination. Glycogen synthase kinases α and β (GSK3α, GSKβ; collectively named GSK3α/β) are serine-threonine kinases with abundant expression in the central nervous system. Both GSK3 isozymes are tonically active under basal conditions, but their activity is regulated by intra- and extracellular factors, intimately involved in neuronal activity. Similar to Arc, GSK3α and GSK3β contribute to synaptic plasticity and the structural plasticity of dendritic spines. The present study identified Arc as a GSK3α/β substrate and showed that GSKβ promotes Arc degradation under conditions that induce de novo Arc synthesis. We also found that GSK3α/β inhibition potentiated spine head thinning that was caused by the prolonged stimulation of N-methyl-D-aspartate receptors (NMDAR). Furthermore, overexpression of Arc mutants that were resistant to GSK3β-mediated phosphorylation or ubiquitination resulted in a stronger reduction of dendritic spine width than wildtype Arc overexpression. Thus, GSK3β terminates Arc expression and limits its effect on dendritic spine morphology. Taken together, the results identify GSK3α/β-catalyzed Arc phosphorylation and degradation as a novel mechanism for controlling the duration of Arc expression and function.

International Journal of Developmental NeuroscienceVolume 47, Issue Part_A p. 19-20 Article ISDN2014_0076: Modeling tuberous sclerosis with induced pluripotent stem cells Ewa Liszewska, Corresponding Author Ewa Liszewska n/[email protected] International Institute of Molecular and Cell Biology in Warsaw, PolandSearch for more papers by this authorKatarzyna Rydz, Katarzyna Rydz International Institute of Molecular and Cell Biology in Warsaw, PolandSearch for more papers by this authorMagdalena Blazejczyk, Magdalena Blazejczyk International Institute of Molecular and Cell Biology in Warsaw, PolandSearch for more papers by this authorKatarzyna Kotulska, Katarzyna Kotulska The Children's Memorial Health Institute, Warsaw, PolandSearch for more papers by this authorSergiusz Jozwiak, Sergiusz Jozwiak The Children's Memorial Health Institute, Warsaw, PolandSearch for more papers by this authorJacek Jaworski, Jacek Jaworski International Institute of Molecular and Cell Biology in Warsaw, PolandSearch for more papers by this author Ewa Liszewska, Corresponding Author Ewa Liszewska n/[email protected] International Institute of Molecular and Cell Biology in Warsaw, PolandSearch for more papers by this authorKatarzyna Rydz, Katarzyna Rydz International Institute of Molecular and Cell Biology in Warsaw, PolandSearch for more papers by this authorMagdalena Blazejczyk, Magdalena Blazejczyk International Institute of Molecular and Cell Biology in Warsaw, PolandSearch for more papers by this authorKatarzyna Kotulska, Katarzyna Kotulska The Children's Memorial Health Institute, Warsaw, PolandSearch for more papers by this authorSergiusz Jozwiak, Sergiusz Jozwiak The Children's Memorial Health Institute, Warsaw, PolandSearch for more papers by this authorJacek Jaworski, Jacek Jaworski International Institute of Molecular and Cell Biology in Warsaw, PolandSearch for more papers by this author First published: 05 November 2015 https://doi.org/10.1016/j.ijdevneu.2015.04.062Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume47, IssuePart_AISDN 2014 AbstractsDecember 2015Pages 19-20 RelatedInformation

The shape of the dendritic arbor is one of the criteria of neuron classification and reflects functional specialization of particular classes of neurons. The development of a proper dendritic branching pattern strongly relies on interactions between the extracellular environment and intracellular processes responsible for dendrite growth and stability. We previously showed that mammalian target of rapamycin (mTOR) kinase is crucial for this process. In this work, we performed a screen for modifiers of dendritic growth in hippocampal neurons, the expression of which is potentially regulated by mTOR. As a result, we identified Cyr61, an angiogenic factor with unknown neuronal function, as a novel regulator of dendritic growth, which controls dendritic growth in a beta 1-integrin-dependent manner.

Mammalian target of rapamycin (mTOR) is a protein kinase that senses nutrient availability, trophic factors support, cellular energy level, cellular stress, and neurotransmitters and adjusts cellular metabolism accordingly. Adequate mTOR activity is needed for development as well as proper physiology of mature neurons. Consequently, changes in mTOR activity are often observed in neuropathology. Recently, several groups reported that seizures increase mammalian target of rapamycin (mTOR) kinase activity, and such increased activity in genetic models can contribute to spontaneous seizures. However, the current knowledge about the spatiotemporal pattern of mTOR activation induced by proconvulsive agents is rather rudimentary. Also consequences of insufficient mTOR activity on a status epilepticus are poorly understood. Here, we systematically investigated these two issues. We showed that mTOR signaling was activated by kainic acid (KA)-induced status epilepticus through several brain areas, including the hippocampus and cortex as well as revealed two waves of mTOR activation: an early wave (2 h) that occurs in neurons and a late wave that predominantly occurs in astrocytes. Unexpectedly, we found that pretreatment with rapamycin, a potent mTOR inhibitor, gradually (i) sensitized animals to KA treatment and (ii) induced gross anatomical changes in the brain.

Dendritic arbors are compartments of neurons dedicated to receiving synaptic inputs. Their shape is an outcome of both the intrinsic genetic program and environmental signals. The microtubules and actin cytoskeleton are both crucial for proper dendritic morphology, but how they interact is unclear. The present study demonstrates that microtubule plus-end tracking protein CLIP-170 and actin-binding protein IQGAP1 regulate dendrite morphology of rat neurons by coordinating the interaction between microtubules and the actin cytoskeleton. Moreover, we show that mTOR kinase interacts with CLIP-170 and is needed for efficient formation of a protein complex containing CLIP-170 and IQGAP1. Dynamic microtubules, CLIP-170, and IQGAP1 are required for proper dendritic arbor morphology and PI3K-mTOR-induced increase in dendritic arbor complexity. Moreover, CLIP-170 and IQGAP1 knockdown modulates dendritic arbor growth via regulation of the actin cytoskeleton. We postulate that mTOR controls dendritic arbor morphology by enhancing cross talk between dynamic microtubules and actin through CLIP-170 and IQGAP1.

Calmyrin1 (CaMy1) is an EF-hand Ca2+-binding protein expressed in several cell types, including brain neurons. Using a yeast two-hybrid screen of a human fetal brain cDNA library, we identified SCG10 protein (stathmin2) as a CaMy1 partner. SCG10 is a microtubule-destabilizing factor involved in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels. In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca2+-dependent in vivo and requires the C-terminal domain of CaMy1 (residues 99–192) and the N-terminal domain of SCG10 (residues 1–35). CaMy1 did not interact with stathmin1, a protein that is homologous with SCG10 but lacks the N-terminal domain characteristic of SCG10. CaMy1 interfered with SCG10 inhibitory activity in a microtubule polymerization assay. Moreover, CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. This CaMy1 activity did not occur when an N-terminally truncated SCG10 mutant unable to interact with CaMy1 was expressed. Altogether, these data suggest that CaMy1 via SCG10 couples Ca2+ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Genome-wide association studies have revealed a link between essential tremor (ET) and the gene SLC1A2, which encodes excitatory amino acid transporter type 2 (EAAT2). We explored EAAT biology in ET by quantifying EAAT2 and EAAT1 levels in the cerebellar dentate nucleus, and expanded our prior analysis of EAAT2 levels in the cerebellar cortex.To quantify EAAT2 and EAAT1 levels in the cerebellar dentate nucleus and cerebellar cortex of ET cases vs. controls.We used immunohistochemistry to quantify EAAT2 and EAAT1 levels in the dentate nucleus of a discovery cohort of 16 ET cases and 16 controls. Furthermore, we quantified EAAT2 levels in the dentate nucleus in a replicate cohort (61 ET cases, 25 controls). Cortical EAAT2 levels in all 77 ET cases and 41 controls were quantified.In the discovery cohort, dentate EAAT2 levels were 1.5-fold higher in 16 ET cases vs. 16 controls (p = 0.007), but EAAT1 levels did not differ significantly (p = 0.279). Dentate EAAT2 levels were 1.3-fold higher in 61 ET cases vs. 25 controls in the replicate cohort (p = 0.022). Cerebellar cortical EAAT2 levels were 20% and 40% lower in ET cases vs. controls in the discovery and the replicate cohorts (respective p values = 0.045 and < 0.001).EAAT2 expression is enhanced in the ET dentate nucleus, in contrast to differentially reduced EAAT2 levels in the ET cerebellar cortex, which might reflect a compensatory mechanism to maintain excitation-inhibition balance in cerebellar nuclei.