Rab11 regulates autophagy at dendritic spines in an mTOR- and NMDA-dependent manner.

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.

Molecular EPISTOP, a comprehensive multi-omic analysis of blood from Tuberous Sclerosis Complex infants age birth to two years.

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.

miRNAs and isomiRs: Serum-Based Biomarkers for the Development of Intellectual Disability and Autism Spectrum Disorder in Tuberous Sclerosis Complex.

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.

Pathological mTOR mutations impact cortical development.

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.

GSK3α and GSK3ß Phosphorylate Arc and Regulate its Degradation.

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 ß (GSK3α, GSKß; collectively named GSK3α/ß) 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 GSK3ß contribute to synaptic plasticity and the structural plasticity of dendritic spines. The present study identified Arc as a GSK3α/ß substrate and showed that GSKß promotes Arc degradation under conditions that induce de novo Arc synthesis. We also found that GSK3α/ß 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 GSK3ß-mediated phosphorylation or ubiquitination resulted in a stronger reduction of dendritic spine width than wildtype Arc overexpression. Thus, GSK3ß terminates Arc expression and limits its effect on dendritic spine morphology. Taken together, the results identify GSK3α/ß-catalyzed Arc phosphorylation and degradation as a novel mechanism for controlling the duration of Arc expression and function.

Kainic Acid Induces mTORC1-Dependent Expression of Elmo1 in Hippocampal Neurons.

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.

Spatiotemporal characterization of mTOR kinase activity following kainic acid induced status epilepticus and analysis of rat brain response to chronic rapamycin treatment.

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.

Cyr61, a matricellular protein, is needed for dendritic arborization of hippocampal neurons.

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 ß1-integrin-dependent manner.

CLIP-170 and IQGAP1 cooperatively regulate dendrite morphology.

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 binds to SCG10 protein (stathmin2) to modulate neurite outgrowth.

Calmyrin1 (CaMy1) is an EF-hand Ca(2+)-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 Ca(2+)-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 Ca(2+) 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.

Biochemical characterization and expression analysis of a novel EF-hand Ca2+ binding protein calmyrin2 (Cib2) in brain indicates its function in NMDA receptor mediated Ca2+ signaling.

Calmyrin2 (CaMy2, Cib2) is a novel EF-hand calcium-binding protein found recently in skeletal muscles. CaMy2 mRNA was also detected in brain, but nothing is known about CaMy2 protein localization and properties in the brain. We report cloning and characterization of CaMy2 in rat brain: its expression pattern, intracellular localization and biochemical features. CaMy2 binds Ca2+ and exhibits Ca2+/conformational switch. Moreover, CaMy2 undergoes N-myristoylation without Ca2+/myristoyl switch, is membrane-associated and localizes in neurons together with Golgi apparatus and dendrite markers. CaMy2 transcript and protein are present mainly in the hippocampus and cortex. In cultured hippocampal neurons, CaMy2 is induced upon neuronal activation. Most prominent increase in CaMy2 protein (7-fold), and mRNA (2-fold) occurs upon stimulation of NMDA receptor (NMDAR). The induction is blocked by translation inhibitors, specific antagonists of NMDAR, the Ca2+-chelator BAPTA, and inhibitors of ERK1/2 and PKC, kinases transmitting NMDAR-linked Ca2+ signal. Our results show that CaMy2 level is controlled by NMDAR and Ca2+ and suggest CaMy2 role in Ca2+ signaling underlying NMDAR activation.

Expression of STIM1 in brain and puncta-like co-localization of STIM1 and ORAI1 upon depletion of Ca(2+) store in neurons.

Recent findings indicate that Store Operated Ca(2+) Entry (SOCE) in non-excitable cells is based on the interaction of ER calcium sensor STIM1 with the plasma membrane Ca(2+) channel protein ORAI1. However, despite physiological evidence for functional SOCE in neurons, its mechanism is not known. Using PCR, immunoblotting and immunohistochemical methods we show that STIM1 protein is present in the mouse brain. The protein and mRNA levels of STIM1 are similar in the thalamus, the hippocampus, the cortex and the amygdala and the higher level is observed in the cerebellum. Immunohistochemistry of the cortex and the hippocampus of brain sections shows that STIM1 is present in cell bodies and dendrites of pyramidal neurons. In the cerebellum STIM1 is present in Purkinje and granule cells. The same immunostaining pattern is observed in cultured hippocampal and cortical neurons. Localization of YFP-STIM1 and ORAI1 changes from a dispersed pattern in untreated cortical neurons to puncta-like pattern in cells with a Ca(2+) store depleted by thapsigargin treatment. The YFP-STIM1(D76A) dominant positive mutant, which is active regardless of the Ca(2+) level in ER, concentrates as puncta even without depletion of the neuronal Ca(2+) store. Also, this mutant forces ORAI1 redistribution to form puncta-like staining. We suggest that in neurons, just as in non-excitable cells, the STIM1 and ORAI1 proteins are involved in SOCE.

Molecular basis of dendritic arborization.

The pattern of dendritic branching along with the receptor and channel composition and density of synapses regulate the electrical properties of neurons. Abnormalities in dendritic tree development lead to serious dysfunction of neuronal circuits and, consequently, the whole nervous system. Not surprisingly, the complicated and multi-step process of dendritic arbor development is highly regulated and controlled at every stage by both extrinsic signals and intrinsic molecular mechanisms. In this review, we analyze the molecular mechanisms that contribute to cellular processes that are crucial for the proper formation and stability of dendritic arbors, in such distant organisms as insects (e.g. Drosophila melanogaster) amphibians (Xenopus laevis) and mammals.

Biochemical properties of endogenous presenilin 1 and presenilin 2 in cultured human B-lymphocytes.

BACKGROUND: Presenilin 1 (PS1) and presenilin 2 (PS2) are membranous proteins involved in the pathology of Alzheimer's disease. The development of specific therapies targeted at PS1 or PS2 requires the determination of biochemical properties of presenilins. Hence, in this study we analyzed the hydrophobic and ionic properties of endogenous presenilins. METHODS: Lysates of immortalized human B-lymphocytes were used as a source of endogenous presenilins. The presence of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) detergent in lysates favored preservation of PS1 and PS2 native protein complexes. We compared Kyte-Doolittle hydropathicity profiles and hydrophobic interactions of PS1 and PS2 with phenyl-agarose. We also compared the ionic properties of presenilins using anion-exchange chromatography. RESULTS: The hydropathicity profiles of PS1 and PS2 revealed similarly located hydrophobic regions and more hydrophobic region in the C-terminal fragment of PS2. However, both PS1 and PS2 under physiological conditions showed no interactions with phenyl-agarose. Despite similar predicted isoelectric points, PS1 and PS2 exhibited different ionic behavior during anion-exchange chromatography. CONCLUSIONS: The different than expected hydrophobic and ionic behavior of PS1 and PS2 may be caused by interactions with other proteins present in complexes formed by endogenous presenilins. The observed difference in ionic properties of PS1 and PS2 can be further explained assuming that PS1 and PS2 form complexes with different sets of proteins. The composition of such variegated PS1 and PS2 complexes can be explored using a proteomic approach. The difference in PS1 and PS2 ionic behavior can be used for purification of endogenous PS1 from PS2, which has not yet been achieved by any other means.

Ca2+-independent binding and cellular expression profiles question a significant role of calmyrin in transduction of Ca2+-signals to Alzheimer's disease-related presenilin 2 in forebrain.

The interaction between the EF-hand Ca(2+)-binding protein calmyrin and presenilin 2 (PS2) has been suggested to play a role in Alzheimer's disease (AD). We now report that calmyrin binds specifically endogenous PS2 and not PS1. However, binding appears to be Ca(2+)-independent and calmyrin does not exhibit a Ca(2+)-dependent translocation to intracellular membranes as demonstrated in a Ca(2+)-myristoyl switch assay. Moreover, calmyrin is only present at very low levels in brain areas associated with the onset of AD. In rat, forebrain calmyrin is localized only in a subset of principal neurons, similarly as in human forebrain. Finally, subcellular fractionation demonstrates only a limited overlap of calmyrin and PS2 at neuronal membranes. We therefore conclude that calmyrin will not contribute significantly as a Ca(2+)-sensor that transduces Ca(2+)-signaling events to PS2 in forebrain.

Coronary artery embryogenesis in cardiac defects induced by retinoic acid in mice.

BACKGROUND: Although normal coronary artery embryogenesis is well described in the literature, little is known about the development of coronary vessels in abnormal hearts. METHODS: We used an animal model of retinoic acid (RA)-evoked outflow tract malformations (e.g., double outlet right ventricle [DORV], transposition of the great arteries [TGA], and common truncus arteriosus [CTA]) to study the embryogenesis of coronary arteries using endothelial cell markers (anti-PECAM-1 antibodies and Griffonia simplicifolia I (GSI) lectin). These markers were applied to serial sections of staged mouse hearts to demonstrate the location of coronary artery primordia. RESULTS: In malformations with a dextropositioned aorta, the shape of the peritruncal plexus, from which the coronary arteries develop, differed from that of control hearts. This difference in the shape of the early capillary plexus in the control and RA-treated hearts depends on the position of the aorta relative to the pulmonary trunk. In both normal and RA-treated hearts, there are several capillary penetrations to each aortic sinus facing the pulmonary trunk, but eventually only 1 coronary artery establishes patency with 1 aortic sinus. CONCLUSIONS: The abnormal location of the vessel primordia induces defective courses of coronary arteries; creates fistulas, a single coronary artery, and dilated vessel lumens; and leaves certain areas of the heart devoid of coronary artery branches. RA-evoked heart malformations may be a useful model for elucidating abnormal patterns of coronary artery development and may shed some light on the angiogenesis of coronary artery formation.

Calcium-binding calmyrin forms stable covalent dimers in vitro, but in vivo is found in monomeric form.

The EF-hand Ca(2+)-binding protein calmyrin is expressed in many tissues and can interact with multiple effector proteins, probably as a sensor transferring Ca(2+) signals. As oligomerization may represent one of Ca(2+)-signal transduction mechanisms, we characterised recombinant calmyrin forms using non-reducing SDS/PAGE, analytical ultracentrifugation and gel filtration. We also aimed at identification of biologically active calmyrin forms. Non-reducing SDS/PAGE showed that in vitro apo- and Ca(2+)-bound calmyrin oligomerizes forming stable intermolecular disulfide bridges. Ultracentrifugation indicated that at a 220 microM initial protein concentration apo-calmyrin existed in an equilibrium of a 21.9 kDa monomer and a 43.8 kDa dimer (trimeric or tetrameric species were not detected). The dimerization constant was calculated as Ka = 1.78 x 103 M(-1) at 6 degrees C. Gel filtration of apo- and Ca(2+)-bound calmyrin at a 100 microM protein concentration confirmed an equilibrium of a monomer and a covalent dimer state. Importantly, both monomer and dimer underwent significant conformational changes in response to binding of Ca(2+). However, when calmyrin forms were analyzed under non-reducing conditions in cell extracts by Western blotting, only monomeric calmyrin was detected in human platelets and lymphocytes, and in rat brain. Moreover, in contrast to recombinant calmyrin, crosslinking did not preserve any dimeric species of calmyrin regardless of Ca(2+) concentrations. In summary, our data indicate that although calmyrin forms stable covalent dimers in vitro, it most probably functions as a monomer in vivo.