The molecular mechanisms that coordinate postnatal brain enlargement, synaptic properties and cognition remain an enigma. This study demonstrates that neuronal complexity controlled by p21-activated kinases (PAKs) is a key determinant for postnatal brain enlargement and synaptic properties. Double knockout (DK) mice lacking both PAK1 and PAK3 were severely impaired in postnatal brain growth, resulting in a dramatic reduction in brain volume at maturity. Remarkably, the reduced brain was accompanied by minimal changes in total cell count, due to a significant increase in cell density. However, the DK neurons have smaller soma, markedly simplified dendritic arbors/axons and reduced synapse density. Surprisingly, the DK mice were elevated in basal synaptic responses due to enhanced individual synaptic potency, but severely impaired in bi-directional synaptic plasticity. The PAK1/3 action is likely mediated by cofilin-dependent actin regulation because the activity of cofilin and the properties of actin filaments were specifically altered in the DK mice.

The molecular mechanisms that coordinate postnatal brain enlargement, synaptic properties and cognition remain an enigma. This study demonstrates that neuronal complexity controlled by p21-activated kinases (PAKs) is a key determinant for postnatal brain enlargement and synaptic properties. Double knockout (DK) mice lacking both PAK1 and PAK3 were severely impaired in postnatal brain growth, resulting in a dramatic reduction in brain volume at maturity. Remarkably, the reduced brain was accompanied by minimal changes in total cell count, due to a significant increase in cell density. However, the DK neurons have smaller soma, markedly simplified dendritic arbors/axons and reduced synapse density. Surprisingly, the DK mice were elevated in basal synaptic responses due to enhanced individual synaptic potency, but severely impaired in bi-directional synaptic plasticity. The PAK1/3 action is likely mediated by cofilin-dependent actin regulation because the activity of cofilin and the properties of actin filaments were specifically altered in the DK mice.

<p>The proper modification and trafficking of plasma membrane proteins are essential for normal neuronal function, such as dendrite morphogenesis, spine formation and synaptic plasticity. The secretory organelles including endoplasmic reticulum and Golgi apparatus are critical for the trafficking of these molecules as shown in fibroblasts. Although these secretory organelles have been observed in neurons including dendritic branches, their spatial organization and function in protein trafficking, neuronal development and plasticity are not clear yet. Here, I used photobleaching and photoactivation approaches combined with electron microscopy to show that although rapidly diffusing within the continuous network of the somato-dendritic ER, membrane proteins such as nascent AMPA receptors are confined by ER spatial complexity. The spatial range of ER membrane protein mobility becomes progressively confined over neuronal development and is rapidly restricted by synaptic activity. Thus, constrained lateral mobility within the ER provides a novel mechanism for compartmentalized trafficking of nascent receptors throughout dendrites. I also identified an ER protein as a novel microtubule-associated protein regulating dendritic ER spatial complexity, neuronal dendrite elongation and spine formation. Together, these results describe the spatial organization of dendritic ER and its role in regulating membrane protein trafficking, neuronal morphogenesis and postsynaptic functions.</p> / Dissertation

Development of functional neural circuits involves a series of complicated steps, including neurogenesis and neuronal morphogenesis. To understand the molecular mechasnims of neurite complexity, especially neurite branching/arborization, the Drosophila brain, especially MBNs (mushroom body neurons) and PNs (projection neurons) in olfactory circuitry, was used in this dissertation work as the model system to study how two molecules, Dscam and Kr-h1 affect neurite complexity in the Drosophilabrain. For the Drosophila Dscam, through alternative splicing it could encode up to 152,064 distinct immunoglobulin/fibronectin type cell adhesion molecules. Each Dscam isoform is derived from one of the 19,008 ectodomain variants connected with one of the two alternative transmembrane segments and one of the four possible endodomain portions. Recent studies revealed that Dscam was widely required for neurite branching/arborizaiton. However, due to the technical difficulty, the functional roles of Dscam transmembrane variants and ectodomain variants remain unclear. In this thesis work, a microRNA based RNA interference was used to knock down distinct subsets of Dscam isoform. First, loss of Dscam[TM1] versus Dscam[TM2], two distinct Dscam transmembrane variants, disrupted the dendritic versus axonal morphogenesis, respectively. Furthermore, structural analysis suggested that the juxtamembrane portion of transmembrane segment was required for the Dscam protein targeting in dendrites/axons and this differential protein targeting might account for the functional distinction between Dscam[TM1] and Dscam[TM2]. Second, to further address the functional significance of having two Dscam transmembrane variants in axons versus dendrites, the possibility that there might be different usage of Dscam repertoire between axons and dendrites that lead to different levels of morphological complexity between axons and dendrites in the same neuron was examined. To this end, end-in targeting approaches were used to exchange Dscam populations between axons and dendrites. Though the genetic data suggested that Dscam populations were exchanged between axons and dendrites, the phenotypic analysis in various neuronal types revealed that depending on the neuronal types, exchange of Dscam populations between axons and dendrites might primarily affect either axonal or dendritic morphology, suggesting that different usage of Dscam population between axons and dendrites might regulate complex patterns of neurite morphology. Finally, the functions of Dscam exon 4 variants had been addressed in different model neurons in the Drosophilabrain. First, 12 Dscam exon 4 variants were divided into three groups based on their phylogenetic distance. Then, three miRNA constructs were engineered to knock down one group at a time. The genetic data suggested that different Dscam exon 4 variants are differentially required in different neurons to support their proper neuronal morphogenesis. In summary, this part of my thesis work identified and characterized previously unrecognized functions of all these distinct Dscam variants and provided novel insights into how diverse Dscam isoforms regulate the different aspects of neuronal morphogenesis. In the honey bee brain, Kr-h1 is upregulated during the behavioral shift from nursing to foraging when there is increased neurite branching in the brain. To directly examine the hypothesis that altered Kr-h1 expression might regulate morphological complexity of neurites, this research work involved the MARCM (mosaic analysis with a repressible cell marker) and TARGET (temporal and regional gene expression targeting) techniques to analyze the roles of Kr-h1 in Drosophila neuronal morphogenesis. Interestingly, increased expression of Kr-h1 blocked the axon branching and further disrupted the lobe formation in the mushroom body whereas the loss-of-Kr-h1 did not show any apparent neuronal morphogenetic defects. In addition, it was observed that Kr-h1 was expressed when MB (mushroom body) did not undergo active morphogenesis, suggesting its potential anti-morphogenetic activity. Indeed, loss of Kr-h1 (Kruppel homolog 1) enhanced the neuronal morphogenesis that was otherwise delayed due to the defective TGF-beta signaling. Furthermore, Kr-h1 expression was closely linked to ecdysone dependent signaling: Kr-h1 was first regulated by usp (ultraspiracle), which dimerized with various ecdysone receptors and then Kr-h1 expression was essential for proper ecdysone patterning in the larval CNS (central nervous system). Together, though Kr-h1could potentially regulate the neurite complexity, it seems primarily involved in the coordinating ecdysone signaling. In conclusion, the powerful genetic toolkit available in the Drosophila has allowed the investigation in the molecular mechanisms of neuronal morphogenesis and understanding of these mechanisms will enhance our understanding of how the complex nervous system is wired to perform the delicate behaviors.

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