Maya Shelly

New dentate granule cells (DGCs) are continuously generated, and integrate into the preexisting hippocampal network in the adult brain. How an adult-born neuron with initially simple spindle-like morphology develops into a DGC, consisting of a single apical dendrite with further branches, remains largely unknown. Here, using retroviruses to birth date and manipulate newborn neurons, we examined initial dendritic formation and possible underlying mechanisms. We found that GFP-expressing newborn cells began to establish a DGC-like morphology at ∼7 d after birth, with a primary dendrite pointing to the molecular layer, but at this stage, with several neurites in the neurogenic zone. Interestingly, the Golgi apparatus, an essential organelle for neurite growth and maintenance, was dynamically repositioning in the soma of newborn cells during this initial integration stage. Two weeks after birth, by which time most neurites in the neurogenic zone were eliminated, a compact Golgi apparatus was positioned exclusively at the base of the primary dendrite. We analyzed the presence of Golgi-associated genes using single-cell transcriptomes of newborn DGCs, and among Golgi-related genes, found the presence ofSTK25andSTRAD, regulators of embryonic neuronal development. When we knocked down either of these two proteins, we found Golgi mislocalization and extensive aberrant dendrite formation. Furthermore, overexpression of a mutated form of STRAD, underlying the disorder polyhydramnios, megalencephaly, and symptomatic epilepsy, characterized by abnormal brain development and intractable epilepsy, caused similar defects in Golgi localization and dendrite formation in adult-born neurons. Together, our findings reveal a role for Golgi repositioning in regulating the initial integration of adult-born DGCs.SIGNIFICANCE STATEMENTSince the discovery of the continuous generation of new neurons in the adult hippocampus, extensive effort was directed toward understanding the functional contribution of these newborn neurons to the existing hippocampal circuit and associated behaviors, while the molecular mechanisms controlling their early morphological integration are less well understood. Dentate granule cells (DGCs) have a single, complex, apical dendrite. The events leading adult-born DGCs' to transition from simple spindle-like morphology to mature dendrite morphology are largely unknown. We studied establishment of newborn DGCs dendritic pattern and found it was mediated by a signaling pathway regulating precise localization of the Golgi apparatus. Furthermore, this Golgi-associated mechanism for dendrite establishment might be impaired in a human genetic epilepsy syndrome, polyhydramnios, megalencephaly, and symptomatic epilepsy.

Protein micropatterning techniques, including microfluidic devices and protein micro-contact printing, enable the generation of highly controllable substrates for spatial manipulation of intracellular and extracellular signaling determinants to examine the development of cultured dissociated neurons in vitro. In particular, culture substrates coated with proteins of interest in defined stripes, including cell adhesion molecules and secreted proteins, have been successfully used to study neuronal polarization, a process in which the neuron establishes axon and dendrite identities, a critical architecture for the input/output functions of the neuron. We have recently used this methodology to pattern the extracellular protein Semaphorin 3A (Sema3A), a secreted factor known to control neuronal development in the mammalian embryonic cortex. We showed that stripe-patterned Sema3A regulates axon and dendrite formation during the early phase of neuronal polarization in cultured rat hippocampal neurons. Here, we describe microfabrication and substrate stripe micropatterning of Sema3A. We note that same methodologies can be applied to pattern other extracellular proteins that regulate neuronal development in the embryonic brain, as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and Netrin-1. We describe modifications of these methodologies for stripe micropatterning of membrane-permeable analog of the second messengers cyclic AMP (cAMP) and cyclic GMP (cGMP), intracellular regulators of neuronal polarization that might act downstream of Sema3A.

γ-Aminobutyric acid is the principal inhibitory neurotransmitter in adults, acting through ionotropic chloride-permeable GABA_A receptors (GABA_ARs), and metabotropic GABA_BRs coupled to calcium or potassium channels, and cyclic AMP signalling. During early development, γ-aminobutyric acid is the main neurotransmitter and is not hyperpolarizing, as GABA_AR activation is depolarizing while GABA_BRs lack coupling to potassium channels. Despite extensive knowledge on GABA_ARs as key factors in neuronal development, the role of GABA_BRs remains unclear. Here we address GABA_BR function during rat cortical development by in utero knockdown (short interfering RNA) of GABA_BR in pyramidal-neuron progenitors. GABA_BR short interfering RNA impairs neuronal migration and axon/dendrite morphological maturation by disrupting cyclic AMP signalling. Furthermore, GABA_BR activation reduces cyclic AMP-dependent phosphorylation of LKB1, a kinase involved in neuronal polarization, and rescues LKB1 overexpression-induced defects in cortical development. Thus, non-hyperpolarizing activation of GABA_BRs during development promotes neuronal migration and morphological maturation by cyclic AMP/LKB1 signalling.

The formation of axon/dendrite polarity is critical for the neuron to perform its signaling function in the brain. Recent advance in our understanding of cellular and molecular mechanisms underlying the development and maintenance of neuronal polarity has been greatly facilitated by the use of the culture system of dissociated hippocampal neurons. Among many polarization‐related proteins, we here focus on the mammalian LKB1, the counterpart of the C. elegans Par‐4, which is an upstream regulator among six Par (partitioning‐defective) genes that act as master regulators of cell polarity in different cell types across evolutionary distant species. Recent studies have identified LKB1 and its downstream targets SAD/MARK kinases (mammalian homologs of Par‐1) as key regulators of neuronal polarization and axon development in cultured neurons and in developing cortical neurons in vivo. We will review the properties of and interactions among proteins in this LKB1‐SAD/MARK pathway, drawing upon information obtained from both neuronal and non‐neuronal systems. Due to central role of the protein kinase A ‐ dependent phosphorylation of LKB1 in the activation of this pathway, we will review recent findings on how cAMP and cGMP signaling may serve as antagonistic second messengers for axon/dendrite development, and how these cyclic nucleotides may mediate the action of extracellular polarizing factors by modulating the activity of the LKB1‐SAD/MARK pathway. © 2011 Wiley Periodicals, Inc. Develop Neurobiol 71: 508–527, 2011