Ste20-like kinase is critical for inhibitory synapse maintenance and its deficiency confers a developmental dendritopathy

SummaryThe size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ 3rd, order dendrites. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, while excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and of inhibitory synapse maintenance.

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Multiple Alu exonization in 3’UTR of a primate specific isoform of CYP20A1 creates a potential miRNA sponge

Genome Biology and Evolution ◽

10.1093/gbe/evaa233 ◽

2020 ◽

Author(s):

Aniket Bhattacharya ◽

Vineet Jha ◽

Khushboo Singhal ◽

Mahar Fatima ◽

Dayanidhi Singh ◽

...

Keyword(s):

Heat Shock ◽

Cortical Neurons ◽

Regulatory Networks ◽

Large Scale ◽

Neuronal Development ◽

Random Sets ◽

Rna Seq ◽

Orphan Gene ◽

Mirna Sponge ◽

Human Neurons

Abstract Alu repeats contribute to phylogenetic novelties in conserved regulatory networks in primates. Our study highlights how exonized Alus could nucleate large-scale mRNA-miRNA interactions. Using a functional genomics approach, we characterize a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) that has exonization of 23 Alus in its 3’UTR. CYP20A1_Alu-LT, confirmed by 3’RACE, is an outlier in length (9 kb 3’UTR) and widely expressed. Using publically available datasets, we demonstrate its expression in higher primates and presence in single nucleus RNA-seq of 15928 human cortical neurons. miRanda predicts ∼4700 miRNA recognition elements (MREs) for ∼1000 miRNAs, primarily originated within these 3’UTR-Alus. CYP20A1_Alu-LT could be a potential multi-miRNA sponge as it harbors ≥10 MREs for 140 miRNAs and has cytosolic localization. We further tested whether expression of CYP20A1_Alu-LT correlates with mRNAs harboring similar MRE targets. RNA-seq with conjoint miRNA-seq analysis was done in primary human neurons where we observed CYP20A1_Alu-LT to be downregulated during heat shock response and upregulated in HIV1-Tat treatment. 380 genes were positively correlated with its expression (significantly downregulated in heat shock and upregulated in Tat) and they harbored MREs for nine expressed miRNAs which were also enriched in CYP20A1_Alu-LT. MREs were significantly enriched in these 380 genes compared to random sets of differentially expressed genes (p = 8.134e-12). Gene ontology suggested involvement of these genes in neuronal development and hemostasis pathways thus proposing a novel component of Alu-miRNA mediated transcriptional modulation that could govern specific physiological outcomes in higher primates.

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KBP interacts with SCG10, linking Goldberg–Shprintzen syndrome to microtubule dynamics and neuronal differentiation

Human Molecular Genetics ◽

10.1093/hmg/ddq280 ◽

2010 ◽

Vol 19 (18) ◽

pp. 3642-3651 ◽

Cited By ~ 30

Author(s):

Maria M. Alves ◽

Grzegorz Burzynski ◽

Jean-Marie Delalande ◽

Jan Osinga ◽

Annemieke van der Goot ◽

...

Keyword(s):

Nervous System ◽

Enteric Nervous System ◽

Neuronal Differentiation ◽

Cortical Neurons ◽

Neuronal Development ◽

Direct Interaction ◽

Clinical Disorder ◽

Neurite Development

Abstract Goldberg–Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show that KBP expression is up-regulated during neuronal development in mouse cortical neurons. Moreover, KBP-depleted PC12 cells were defective in nerve growth factor-induced differentiation and neurite outgrowth, suggesting that KBP is required for cell differentiation and neurite development. To identify KBP interacting proteins, we performed a yeast two-hybrid screen and found that KBP binds almost exclusively to microtubule associated or related proteins, specifically SCG10 and several kinesins. We confirmed these results by validating KBP interaction with one of these proteins: SCG10, a microtubule destabilizing protein. Zebrafish studies further demonstrated an epistatic interaction between KBP and SCG10 in vivo . To investigate the possibility of direct interaction between KBP and microtubules, we undertook co-localization and in vitro binding assays, but found no evidence of direct binding. Thus, our data indicate that KBP is involved in neuronal differentiation and that the central and enteric nervous system defects seen in GOSHS are likely caused by microtubule-related defects.

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Dyrk1A Influences Neuronal Morphogenesis Through Regulation of Cytoskeletal Dynamics in Mammalian Cortical Neurons

Cerebral Cortex ◽

10.1093/cercor/bhr362 ◽

2012 ◽

Vol 22 (12) ◽

pp. 2867-2877 ◽

Cited By ~ 44

Author(s):

M. Martinez de Lagran ◽

R. Benavides-Piccione ◽

I. Ballesteros-Yanez ◽

M. Calvo ◽

M. Morales ◽

...

Keyword(s):

Cortical Neurons ◽

Neuronal Morphogenesis ◽

Cytoskeletal Dynamics

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Correct Laminar Positioning in the Neocortex Influences Proper Dendritic and Synaptic Development

Cerebral Cortex ◽

10.1093/cercor/bhy113 ◽

2018 ◽

Vol 28 (8) ◽

pp. 2976-2990 ◽

Cited By ~ 8

Author(s):

Fanny Sandrine Martineau ◽

Surajit Sahu ◽

Vanessa Plantier ◽

Emmanuelle Buhler ◽

Fabienne Schaller ◽

...

Keyword(s):

Neuronal Migration ◽

Cortical Neurons ◽

Neuronal Development ◽

Synapse Formation ◽

Functional Organization ◽

Functional Integration ◽

Proper Function ◽

Cortical Circuits ◽

Gabaergic Synapses ◽

Dendrite Morphogenesis

Abstract The neocortex is a 6-layered laminated structure with a precise anatomical and functional organization ensuring proper function. Laminar positioning of cortical neurons, as determined by termination of neuronal migration, is a key determinant of their ability to assemble into functional circuits. However, the exact contribution of laminar placement to dendrite morphogenesis and synapse formation remains unclear. Here we manipulated the laminar position of cortical neurons by knocking down doublecortin (Dcx), a crucial effector of migration, and show that misplaced neurons fail to properly form dendrites, spines, and functional glutamatergic and GABAergic synapses. We further show that knocking down Dcx in properly positioned neurons induces similar but milder defects, suggesting that the laminar misplacement is the primary cause of altered neuronal development. Thus, the specific laminar environment of their fated layers is crucial for the maturation of cortical neurons, and influences their functional integration into developing cortical circuits.

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Valproic acid selectively suppresses the formation of inhibitory synapses in cultured cortical neurons

Neuroscience Letters ◽

10.1016/j.neulet.2014.03.066 ◽

2014 ◽

Vol 569 ◽

pp. 142-147 ◽

Cited By ~ 19

Author(s):

Emi Kumamaru ◽

Yoshihiro Egashira ◽

Rie Takenaka ◽

Shigeo Takamori

Keyword(s):

Valproic Acid ◽

Cortical Neurons ◽

Inhibitory Synapses ◽

Cultured Cortical Neurons

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Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death

Journal of Neurobiology ◽

10.1002/neu.10037 ◽

2002 ◽

Vol 51 (1) ◽

pp. 9-23 ◽

Cited By ~ 154

Author(s):

Christian Lesuisse ◽

Lee J. Martin

Keyword(s):

Cortical Neurons ◽

Neuronal Development ◽

Long Term Culture

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Interaction between autism-linked MDGAs and neuroligins suppresses inhibitory synapse development

The Journal of Cell Biology ◽

10.1083/jcb.201206028 ◽

2013 ◽

Vol 200 (3) ◽

pp. 321-336 ◽

Cited By ~ 82

Author(s):

Katherine L. Pettem ◽

Daisaku Yokomaku ◽

Hideto Takahashi ◽

Yuan Ge ◽

Ann Marie Craig

Keyword(s):

Neurodevelopmental Disorders ◽

Hippocampal Neurons ◽

Rare Variants ◽

Inhibitory Synapse ◽

Excitatory Synapse ◽

Inhibitory Synapses ◽

Excitatory Synapses ◽

Synapse Development ◽

Multiple Protein ◽

Synapse Density

Rare variants in MDGAs (MAM domain–containing glycosylphosphatidylinositol anchors), including multiple protein-truncating deletions, are linked to autism and schizophrenia, but the function of these genes is poorly understood. Here, we show that MDGA1 and MDGA2 bound to neuroligin-2 inhibitory synapse–organizing protein, also implicated in neurodevelopmental disorders. MDGA1 inhibited the synapse-promoting activity of neuroligin-2, without altering neuroligin-2 surface trafficking, by inhibiting interaction of neuroligin-2 with neurexin. MDGA binding and suppression of synaptogenic activity was selective for neuroligin-2 and not neuroligin-1 excitatory synapse organizer. Overexpression of MDGA1 in cultured rat hippocampal neurons reduced inhibitory synapse density without altering excitatory synapse density. Furthermore, RNAi-mediated knockdown of MDGA1 selectively increased inhibitory but not excitatory synapse density. These results identify MDGA1 as one of few identified negative regulators of synapse development with a unique selectivity for inhibitory synapses. These results also place MDGAs in the neurexin–neuroligin synaptic pathway implicated in neurodevelopmental disorders and support the idea that an imbalance between inhibitory and excitatory synapses may contribute to these disorders.

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Activity-dependent inhibitory synapse remodeling through gephyrin phosphorylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1411170112 ◽

2014 ◽

Vol 112 (1) ◽

pp. E65-E72 ◽

Cited By ~ 54

Author(s):

Carmen E. Flores ◽

Irina Nikonenko ◽

Pablo Mendez ◽

Jean-Marc Fritschy ◽

Shiva K. Tyagarajan ◽

...

Keyword(s):

Activity Patterns ◽

Phosphorylation Site ◽

Morphological Plasticity ◽

Long Term Potentiation ◽

Inhibitory Synapse ◽

Inhibitory Synapses ◽

Synapse Plasticity ◽

Inhibitory Plasticity ◽

And Function ◽

Activity Dependent

Maintaining a proper balance between excitation and inhibition is essential for the functioning of neuronal networks. However, little is known about the mechanisms through which excitatory activity can affect inhibitory synapse plasticity. Here we used tagged gephyrin, one of the main scaffolding proteins of the postsynaptic density at GABAergic synapses, to monitor the activity-dependent adaptation of perisomatic inhibitory synapses over prolonged periods of time in hippocampal slice cultures. We find that learning-related activity patterns known to induce N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation and transient optogenetic activation of single neurons induce within hours a robust increase in the formation and size of gephyrin-tagged clusters at inhibitory synapses identified by correlated confocal electron microscopy. This inhibitory morphological plasticity was associated with an increase in spontaneous inhibitory activity but did not require activation of GABAA receptors. Importantly, this activity-dependent inhibitory plasticity was prevented by pharmacological blockade of Ca2+/calmodulin-dependent protein kinase II (CaMKII), it was associated with an increased phosphorylation of gephyrin on a site targeted by CaMKII, and could be prevented or mimicked by gephyrin phospho-mutants for this site. These results reveal a homeostatic mechanism through which activity regulates the dynamics and function of perisomatic inhibitory synapses, and they identify a CaMKII-dependent phosphorylation site on gephyrin as critically important for this process.

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