ASD-Associated De Novo Mutations in Five Actin Regulators Show Both Shared and Distinct Defects in Dendritic Spines and Inhibitory Synapses in Cultured Hippocampal Neurons

Many actin cytoskeleton-regulating proteins control dendritic spine morphology and density, which are cellular features often altered in autism spectrum disorder (ASD). Recent studies using animal models show that autism-related behavior can be rescued by either manipulating actin regulators or by reversing dendritic spine density or morphology. Based on these studies, the actin cytoskeleton is a potential target pathway for developing new ASD treatments. Thus, it is important to understand how different ASD-associated actin regulators contribute to the regulation of dendritic spines and how ASD-associated mutations modulate this regulation. For this study, we selected five genes encoding different actin-regulating proteins and induced ASD-associated de novo missense mutations in these proteins. We assessed the functionality of the wild-type and mutated proteins by analyzing their subcellular localization, and by analyzing the dendritic spine phenotypes induced by the expression of these proteins. As the imbalance between excitation and inhibition has been suggested to have a central role in ASD, we additionally evaluated the density, size and subcellular localization of inhibitory synapses. Common for all the proteins studied was the enrichment in dendritic spines. ASD-associated mutations induced changes in the localization of alpha-actinin-4, which localized less to dendritic spines, and for SWAP-70 and SrGAP3, which localized more to dendritic spines. Among the wild-type proteins studied, only alpha-actinin-4 expression caused a significant change in dendritic spine morphology by increasing the mushroom spine density and decreasing thin spine density. We hypothesized that mutations associated with ASD shift dendritic spine morphology from mushroom to thin spines. An M554V mutation in alpha-actinin-4 (ACTN4) resulted in the expected shift in dendritic spine morphology by increasing the density of thin spines. In addition, we observed a trend toward higher thin spine density withmutations inmyosin IXb and SWAP-70. Myosin IIb and myosin IXb expression increased the proportion of inhibitory synapses in spines. The expression of mutated myosin IIb (Y265C), SrGAP3 (E469K), and SWAP-70 (L544F) induced variable changes in inhibitory synapses.Peer reviewe

MIM-Deficient Mice Exhibit Anatomical Changes in Dendritic Spines, Cortex Volume and Brain Ventricles, and Functional Changes in Motor Coordination and Learning

In this study, we performed a comprehensive behavioral and anatomical analysis of the Missing in Metastasis (Mtss1/MIM) knockout (KO) mouse brain. We also analyzed the expression of MIM in different brain regions at different ages. MIM is an I-BAR containing membrane curving protein, shown to be involved in dendritic spine initiation and dendritic branching in Purkinje cells in the cerebellum. Behavioral analysis of MIM KO mice revealed defects in both learning and reverse-learning, alterations in anxiety levels and reduced dominant behavior, and confirmed the previously described deficiency in motor coordination and pre-pulse inhibition. Anatomically, we observed enlarged brain ventricles and decreased cortical volume. Although MIM expression was relatively low in hippocampus after early development, hippocampal pyramidal neurons exhibited reduced density of thin and stubby dendritic spines. Learning deficiencies can be connected to all detected anatomical changes. Both behavioral and anatomical findings are typical for schizophrenia mouse models.Peer reviewe

Dendritic Spine Initiation in Brain Development, Learning and Diseases and Impact of BAR-Domain Proteins

Dendritic spines are small, bulbous protrusions along neuronal dendrites where most of the excitatory synapses are located. Dendritic spine density in normal human brain increases rapidly before and after birth achieving the highest density around 2-8 years. Density decreases during adolescence, reaching a stable level in adulthood. The changes in dendritic spines are considered structural correlates for synaptic plasticity as well as the basis of experience-dependent remodeling of neuronal circuits. Alterations in spine density correspond to aberrant brain function observed in various neurodevelopmental and neuropsychiatric disorders. Dendritic spine initiation affects spine density. In this review, we discuss the importance of spine initiation in brain development, learning, and potential complications resulting from altered spine initiation in neurological diseases. Current literature shows that two Bin Amphiphysin Rvs (BAR) domain-containing proteins, MIM/Mtss1 and SrGAP3, are involved in spine initiation. We review existing literature and open databases to discuss whether other BAR-domain proteins could also take part in spine initiation. Finally, we discuss the potential molecular mechanisms on how BAR-domain proteins could regulate spine initiation.Peer reviewe

Molecular Regulation of the Dendritic Spine Initiation

This thesis elucidates the molecular regulation of the dendritic spine initiation. Dendritic spines are tiny actin-rich protrusions along the neuronal dendrites and the structural markers for neuronal connections in the brain. Formation of new dendritic spines represents the formation of new connections, which is the basis for all major brain functions. Various neurodevelopmental and neuropsychiatric disorders exhibit abnormal dendritic spine density and morphology. To understand how brain functions at cellular and molecular level and what goes wrong in different neurological diseases, it is important to study the molecular and signaling mechanisms regulating the dendritic spine formation. In this thesis, we examined how different BAR domain proteins are involved in dendritic spine initiation. We also studied 5 different ASD-associated actin regulating proteins and how the disease-associated mutation affects their functions in dendritic spines and inhibitory synapses. In study I, we investigated MIM/Mtss1, a previously identified dendritic spine initiation factor. We showed that MIM expression is developmentally regulated in different brain regions. Although MIM expression is relatively high in cortex, hippocampus, and cerebellum in developing (P7) mouse brain, the expression is dramatically reduced in cortex and hippocampus after P7, therefore limiting MIM expression only to the cerebellum in the adult brain. Results from this study suggest that there might be other potential spine initiation factors that are more broadly expressed in developing and adult brain. In study II, we reviewed the current literature on molecular regulation of the dendritic spine initiation and how it relates to normal brain functions and diseases. We described different mechanisms on how BAR-domain proteins could regulate dendritic spine initiation. Furthermore, we highlighted the gaps in knowledge in the field and proposed the potential candidates from BAR-domain protein family for future studies in spine initiation. In study III, we identified the growth-arrest-specific 7 (Gas7), a member of the BAR-domain protein family as a novel spine initiation factor as well as a molecular link between neuronal activity and formation of new dendritic spines. We showed that Gas7 initiates new dendritic spines in neuron-activity dependent manner. Our results revealed that neuronal activation leads to rapid translocation of cytoplasmic Gas7 to the dendritic plasma membrane forming bigger Gas7 clusters that act as the hotspots for the formation of new dendritic spines. PI3- kinase activity and intact F-BAR domain were required for Gas7 clustering and localization. As expected, actin regulation was also important for the Gas7- mediated spine initiation. We showed that Gas7 regulates the localization and clustering of N-WASP, a key actin regulator. Furthermore, Gas7 enhances actin clustering and Arp2/3 complex (an actin nucleator) activity is important for the Gas7-induced actin clustering. In study IV, we studied five different actin regulating proteins (-actinin-4, myosin IIb, myosin IXb, SWAP-70, and SrGAP3) that have been associated with the autism spectrum disorder (ASD). Numerous ASD susceptibility genes are involved in regulating the postsynaptic site of glutamatergic synapses, the development and maturation of synaptic contacts, or synaptic transmission. Studies of postmortem human ASD brains have revealed increased spine density. Recent studies using animal models show that autism-related behavior can be rescued by either manipulating actin regulators or by reversing dendritic spine density or morphology. Based on these studies, the actin cytoskeleton is a potential target pathway for developing new ASD treatments. Thus, it is important to understand how different ASD-associated actin regulators contribute to the regulation of dendritic spines and how ASD-associated mutations affect this regulation. Here, we introduced ASD-associated de novo missense mutations in five actin regulating proteins and studied the effect of these mutations in protein localization, dendritic spine density and morphology, as well as the inhibitory synapses. The M554V mutation in -actinin-4 decreased its localization to the dendritic spines whereas L544F in SWAP-70 and E469K in SrGAP3 enhanced their localization to the dendritic spines. Among the wild type proteins, only the expression of -actinin-4 resulted in a significant change in spine morphology. While the overexpression of wild type -actinin-4 significantly increased the mushroom spine density and decreased the thin spine density, expression of mutant -actinin-4 failed to induce such changes compared to the control. Moreover, we noted a trend towards increased thin spine density with mutant protein expression for myosin IXb and SWAP-70. In addition, expression of wild type proteins myosin IIb and myosin IXb significantly increased the ratio of inhibitory synapses on dendritic spines. Furthermore, the expression of myosin IIb (Y265C), SrGAP3 (E469K), and SWAP-70 (L544F) resulted in variable changes in different parameters of the inhibitory synapses. These results suggest that ASD-associated mutations in different actin regulators exhibit both shared and distinct defects in dendritic spines and inhibitory synapses. With these studies, we demonstrated the involvement of different regulatory mechanisms for different dendritic spine initiation factors (study I and study III). Identification of a novel molecular link between neuronal activity and formation of new dendritic spines provides new insights into the molecular level understanding of basic brain functions as well as the diseases associated with the disruption of those functions. Moreover, a single de novo mutation in actin regulating proteins could have notable effect on both dendritic spines and inhibitory synapses (study IV). Altogether, this thesis work extends the molecular level understanding of the dendritic spine formation and how it relates to normal brain functions and diseases.Hermosolut viestivät toisten hermosolujen kanssa synapseiksi kutsuttujen liitosten kautta. Hermosoluja aktivoivat synapsit sijaitsevat hermosolujen dendriiteillä pienen pienissä nystyröissä, joita kutsutaan dendriittien okasiksi. Kun opimme uusia taitoja, meille muodostuu uusia okasia. Samalla ”turhat” okaset surkastuvat pois. Okasia voidaankin pitää aivojen pieninä muistiyksiköinä. Niitä lisäämällä ja poistamalla, ja niiden tehoa kasvattamalla tai vähentämällä, aivot muokkaavat toimintaansa ja tallentavat uusia taitoja muokattuihin synapsipolkuihin. Dendriittien okasten säätely on häiriintynyt useissa neurologisissa sairauksissa. Ymmärtääksemme kuinka aivot toimivat solu- ja molekyylitasolla ja mikä menee pieleen erilaisissa neurologisissa sairauksissa, on tärkeää tutkia dendriittien okasten muodostumista sääteleviä molekyylimekanismeja. Tämä opinnäytetyö selvitti miten dendriittien okasten muodostumista säädellään molekyylitasolla. Tutkimme, kuinka erilaiset BAR-domeeniproteiinit osallistuvat dendriittien okasten muodostumisen aloittamiseen. BAR-domeeni on proteiinin osa, joka sitoutuu solukalvolle. Jotkut BAR-domeenit pystyvät vääntämään solukalvoa, mikä edesauttaa uusien ulokkeiden muodostumista. Tutkimme myös viittä erilaista autismiin liitettyä aktiinitukirankaa säätelevää proteiinia ja kuinka autismiin liitetty mutaatio vaikuttaa näiden proteiinien toimintoihin dendriittien okasissa ja inhiboivissa synapseissa. Hermosoluissa on kahta pääsynapsityyppiä, toiset lisäävät hermosolun aktiivisuutta, kun taas toiset vähentävät, inhiboivat aktiivisuutta. Aktivoivat synapsit ovat pääasiassa okasissa, kun taas inhiboivat synapsit ovat sekä okasissa, että dendriitin rungossa. Ensimmäisessä osatyössä tutkimme MIM/Mtss1-proteiinia. MIM/Mtss1-proteiinilla on I-BAR-domeeni, joka pystyy kääntämään solukalvoa ulospäin tehden näin pienen ulokkeen solun pinnalle tai hermosolussa dendriitin runkoon. Osoitimme, että MIM-proteiinin tuotto on kehityksellisesti säädeltyä aivojen eri alueilla. Vaikka MIM-proteiinia tuotetaan suhteellisen paljon aivokuoressa ja hippokampuksessa heti syntymän jälkeen, sen tuotto vähenee voimakkaasti seitsemän päivän jälkeen syntymästä. Pikkuaivoissa MIM-proteiinia sen sijaan tuotetaan runsaasti läpi koko elämän. Tämän tutkimuksen tulokset viittaavat siihen, että todennäköisesti aivoissa on myös muita proteiineja, jotka voivat aloittaa dendriittien okasia. Toisessa osatyössä tarkastelimme nykyistä kirjallisuutta dendriittien okasten aloittamisen molekyylisäätelystä ja sen suhteesta normaaleihin aivotoimintoihin ja sairauksiin. Kolmannessa osatyössä tunnistimme Gas7 proteiinin uudeksi okasten aloittaja-proteiiniksi. Gas7-proteiini omaa BAR-domeenin, joka ei näyttäisi kääntävän solukalvoa, vaan ohjaavan proteiinin solukalvolle. Tuloksemme osoittivat, että hermosolujen aktivaatio vie solulimassa vapaana kulkevan Gas7-proteiinin solukalvolle muodostaen Gas7-klustereita, jotka toimivat uusien dendriittien okasten aloituspaikkoina. Gas7 proteiini ohjasi myös aktiinitukirangan rakenteiden muodostumista solukalvon sisäpinnalle edesauttaen näin uusien ulokkeiden muodostumista. Aktiinitukiranka on kuin solujen lihakset ja luusto ja aktiinitukiranka säätelee solujen muodon muutoksia ja muodon ylläpitoa. Aktiinitukiranka on hyvin tärkeä dendriittien okasten muodostumisessa ja ylläpidossa. Neljännessä osatyössä tutkimme viittä erilaista aktiinitukirankaa säätelevää proteiinia (alfa-aktiniini-4, myosiini IIb, myosiini IXb, SWAP-70 ja SrGAP3), jotka on liitetty autismiin. Moni autismiin liitetty geenimutaatio löytyy proteiineista, jotka säätelevät aktiinitukirankaa tai dendriittien okasia ja autismikrirjon henkilöillä on usein neuronormaaleihin verrattuna poikkeava okasten tiheys. Yksittäisten mutaatioiden vaikutusta ei voi kuitenkaan suoraan ennustaa muuttuneesta proteiinin aminohappojärjestyksestä. Siten oli kiinnostavaa testata muuttaako autismiin liitetyt mutaatiot proteiinien toimintaa tai vaikuttavatko mutaatiot okasten tiheyteen tai muotoon. Lisäksi katsoimme vaikuttaako mutaatiot inhiboivien synapsien tiheyteen tai kokoon. Alfa-aktiniini-4:n mutaatio muutti selvästi proteiinin paikkaa solussa ja proteiinin vaikutusta okasiin. Muissa proteiineissa mutaation vaikutus oli pienempi. Yhteenvetona tämä työ tuotti uutta tietoa siitä, miten okasia aloitetaan ja miten okasten aloittajien aktiivisuutta tai tuottoa säädellään eri tavoin. Kun opimme uusia taitoja, tietyt hermosolut aktivoituvat ja yleensä nämä aktiiviset hermosolut muodostavat keskenään uusia yhteyksiä. Yhteyksien muodostumista edesauttaa uusien okasten synty. Näin ollen se, että pystyimme yhdistämään hermosolun aktiivisuuden Gas7-proteiinin sijainnin muutokseen, mikä edesauttoi uusien okasten muodostumista, auttaa ymmärtämään miten oppimisessa juuri aktiiviset hermosolut muodostavat uusia yhteyksiä. Lisäksi tämä työ tuotti uutta tietoa siitä, miten autismiin liitetyt geenimutaatiot muuttavat proteiinien toimintaa ja niiden vaikutusta okasiin

Efficacy of Biorational Compounds against Mustard Aphid (Lipaphis erysimi Kalt.) and English Grain Aphid (Sitobion avenae Fab.) under Laboratory Conditions in Nepal

Mustard aphid (Lipaphis erysimi) and English grain aphid (Sitobion avenae) are among the most important pests in mustard and wheat fields in Nepal. Biocide Manic (Metarhizium anisopliae a.i. = 1 × 109 spores/ml) at 3 ml/l water, Agri Sakti (Beauveria bassiana a.i. = 1 × 109 spores/ml) at 3.3 ml/l water, Varunastra (Verticillium lecanii spores 2% aqueous suspension, 2 × 108 CFU/ml) at 6 ml/l water, Mahastra (Bacillus thuringiensis var. kurstaki 0.5% wettable powder) at 6 g/l water, Neemraj Super (Azadirachitin 0.3% w/w) at 3.3 ml/l water, Tracer (Spinosad 90% spinosyns) at 0.33 ml/l water, and control treatment (pure water) were used to test their efficacy against L. erysimi and S. avenae, using leaf dip and spray methods under laboratory conditions in Rupandehi, Nepal, in the year 2018. Each treatment was replicated four times, and the experiment was carried out in a randomized complete block design. Mortality of aphids was recorded at 24, 48, 72, and 98 hours after treatment application. The result revealed highest mortality of mustard aphids with Agri Sakti at 24 hours after treatment (HAT); however, Neemraj Super was found to be the most effective at 48, 72, and 96 HAT with the leaf spray method. With the leaf dip method, Neemraj Super killed more mustard aphids than other treatments at all observed time points. Among tested biorational products, Agri Sakti was found to be most effective against English grain aphids in both leaf spray and leaf dip methods. In all the bioassays, the mortality caused by biorational compounds over control was highly significant. The present study suggests for further verification of the biorational products in the field and development of novel management strategies against different species of aphids

Gas7 Is a Novel Dendritic Spine Initiation Factor

Brain stores new information by modifying connections between neurons. When new information is learnt, a group of neurons gets activated and they are connected to each other via synapses. Dendritic spines are protrusions along neuronal dendrites where excitatory synapses are located. Dendritic spines are the first structures to protrude out from the dendrite to reach out to other neurons and establish a new connection. Thus, it is expected that neuronal activity enhances spine initiation. However, the molecular mechanisms linking neuronal activity to spine initiation are poorly known. Membrane binding BAR domain proteins are involved in spine initiation, but it is not known whether neuronal activity affects BAR domain proteins. Here, we used bicuculline treatment to activate excitatory neurons in organotypic hippocampal slices. With this experimental setup, we identified F-BAR domain containing growth arrest-specific protein (Gas7) as a novel spine initiation factor responding to neuron activity. Upon bicuculline addition, Gas7 clustered to create spine initiation hotspots, thus increasing the probability to form new spines in activated neurons. Gas7 clustering and localization was dependent on PI3-kinase (PI3K) activity and intact F-BAR domain. Gas7 overexpression enhanced N-WASP localization to clusters as well as it increased the clustering of actin. Arp2/3 complex was required for normal Gas7-induced actin clustering. Gas7 overexpression increased and knock-down decreased spine density in hippocampal pyramidal neurons. Taken together, we suggest that Gas7 creates platforms under the dendritic plasma membrane which facilitate spine initiation. These platforms grow on neuronal activation, increasing the probability of making new spines and new connections between active neurons. As such, we identified a novel molecular mechanism to link neuronal activity to the formation of new connections between neurons.Peer reviewe

Efficacy of entomo-pathogenic fungus and botanical pesticides against mustard aphid (Lipaphis erysimi Kalt.) at field condition Rupandehi Nepal

Mustard aphid is the most concerning pest of rapeseed in warm and humid areas of Nepal because of its widespread prevalence and increasing severity. There is increasing use of chemicals, as only resort, to manage this pest. The experiment was carried out to evaluate the effectiveness of different bio-friendly management techniques against Mustard aphid, at the Institute of Agriculture and Animal Science, Paklihawa campus. Treatments like “Jholmal” (250 ml/L), Beauveria bassiana (4gm/L) Abamectin @ 1 ml/L of water, Metarhizium anisopliae (2 gm/L), Verticillium lecanii 2% A.S (5 ml/L) and Neem oil (5 ml/L) were used at post-infestation condition. Results revealed that the overall performance of Abamectin was found to be remarkably effective as compared to others. However, the performance of “Jholmal” and Neem was also found similar for both adult and nymph management. Also, the yield and yield attributing characters in “Jholmal”, Neem, and Abamectin treated plots were similar. However, Abamectin was not found to be convincing considering its impact on natural enemies and thus “Jholmal” and Neem are suggested from the experimental results for the management of mustard aphids at the farmer's level

Insecticide residue analysis on vegetable crops through Rapid Bioassay of Pesticide Residue (RBPR) technique in Nepal

Insecticides applied on food crops and vegetables reduce the pest population and leave chemical residues that may result in serious health consequences. In Nepalese context, farmers use pesticides repeatedly to get rid of pests and also don’t consider the waiting period. The study was conducted to evaluate the amount of pesticide residue after application of different organophosphate and carbamate insecticides in vegetable crops. Five insecticides were used in seven different combinations (Dimethoate, Malathion, dichlorvos, Chlorpyriphos, Dimethoate + dichlorvos, Malathion + Chlorpyriphos and Carbofuran) and applied in mustard and broccoli. The residues were assessed using the “Rapid Bioassay of Pesticide Residue technique”. Results exhibited that Chlorpyriphos treated mustard leaves were edible in 3 DAA (Days after application) and in 6 DAA when sticker was applied with treatment. With or without sticker Dimethoate followed by Malathion applied mustard leaves were edible in 6 DAA. With or without sticker Dichlorvos took longest (12 DAA) to reach the safe limits for mustard leaves. In contrary the Dichlorvos treated broccoli was edible in 3 DAA but took 6 DAA when sticker was applied. Malathion treated broccoli, with and without sticker, was edible in 6 DAA. For both crops Carbofuran exhibited anomalous nature showing lower residue level in the beginning and higher later. When both vegetables were applied treatments with stickers, they showed significantly higher residue and longer time to reach edible limit. The applied insecticides took relatively longer to reach safe level in mustard leaf as compared to broccoli. The study suggests use of Chlorpyriphos for mustard leaves and Malathion for broccoli with at least 6 days of waiting period, with or without use of sticker