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 of and, 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. Since 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.

Studies in cultured neurons have established that axon specification instructs neuronal polarization and is necessary for dendrite development. However, dendrite formation in vivo occurs when axon formation is prevented. The mechanisms promoting dendrite development remain elusive. We find that apical dendrite development is directed by a localized cyclic guanosine monophosphate (cGMP)-synthesizing complex. We show that the scaffolding protein Scribble associates with cGMP-synthesizing enzymes soluble-guanylate-cyclase (sGC) and neuronal nitric oxide synthase (nNOS). The Scribble scaffold is preferentially localized to and mediates cGMP increase in dendrites. These events are regulated by kinesin KifC2. Knockdown of Scribble, sGC-b1, or KifC2 or disrupting their associations prevents cGMP increase in dendrites and causes severe defects in apical dendrite development.Local cGMP elevation or sGC expression rescues the effects of Scribble knockdown on dendrite development, indicating that Scribble is an upstream regulator of cGMP. During neuronal polarization, dendrite development is directed by the Scribble scaffold that might link extracellular cues to localized cGMP increase.

The metabolism of the amyloid precursor protein (APP) has been extensively investigated because its processing generates the amyloid-␤-peptide (A␤), which is a likely cause of Alzheimer disease. Much prior research has focused on APP processing using transgenic constructs and heterologous cell lines. Work to date in native neuronal cultures suggests that A␤ is produced in very large amounts. We sought to investigate APP metabolism and A␤ production simultaneously under more physiological conditions in vivo and in vitro using cultured rat cortical neurons and live pigs. We found in cultured neurons that both APP and A␤ are secreted rapidly and at extremely high rates into the extracellular space (2-4 molecules/neuron/s for A␤). Little APP is degraded outside of the pathway that leads to extracellular release. Two metabolic pools of APP are identified, one that is metabolized extremely rapidly (t1 ⁄ 2 ‫؍‬ 2.2 h), and another, surface pool, composed of both synaptic and extrasynaptic elements, that turns over very slowly. A␤ release and accumulation in the extracellular medium can be accounted for stoichiometrically by the extracellular release of ␤-cleaved forms of the APP ectodomain. Two ␣-cleavages of APP occur for every ␤-cleavage. Consistent with the results seen in cultured neurons, an extremely high rate of A␤ production and secretion from the brain was seen in juvenile pigs. In summary, our experiments show an enormous and rapid production and extracellular release of A␤ and the soluble APP ectodomain. A small, slowly metabolized, surface pool of full-length APP is also identified. Alzheimer disease (AD)2 is a progressive, neurodegenerative process characterized pathologically by accumulation of the ␤-amyloid peptide (A␤) in the form of extracellular plaques (1). On the basis of genetic, cellular, and animal studies the amyloid hypothesis postulates that increased deposition of A␤ in the brain is the primary influence in the pathogenesis of AD (2). A␤ is part of the amyloid precursor protein (APP) that is expressed ubiquitously by neuronal and non-neuronal cells (3-5). APP is a type 1 transmembrane glycoprotein that undergoes sequential site-specific proteolytic cleavages by either ␣-or ␤-secretase followed by ␥-secretase, which cleaves APP in the transmembrane domain to yield several secreted derivatives. Cleavage of APP by the ␤-secretase BACE1 releases a large fragment called sAPP␤ leaving behind a fragment that is acted upon by ␥-secretase at different sites to release several short peptides of which A␤40 and A␤42 are the major components (6). Alternatively, cleavage of APP by ␣-secretase occurs within the A␤ domain, precluding the formation of A␤ and resulting in the secretion of sAPP␣, a short peptide called p3, and a C-terminal domain, none of which are amyloidogenic.Both in vitro and in vivo evidence suggests that A␤ is secreted from cells under normal physiological conditions (3, 4, 7-9) but the absolute amount, an important consideration given its proposed roles and pharmacologic interest, has not...

During mammalian embryonic development, neurons polarize to create distinct cellular compartments of axon and dendrite that inherently differ in form and function, providing the foundation for directional signaling in the nervous system. Polarization results from spatiotemporal segregation of specific proteins’ activities to discrete regions of the neuron to dictate axonal vs. dendritic fate. We aim to manipulate axon formation by directed subcellular localization of crucial intracellular protein function. Here we report critical steps toward the development of a nanotechnology for localized subcellular introduction and retention of an intracellular kinase, LKB1, crucial regulator of axon formation. This nanotechnology will spatially manipulate LKB1-linked biomagnetic nanocomplexes (LKB1-NCs) in developing rodent neurons in culture and in vivo. We created a supramolecular assembly for LKB1 rapid neuronal uptake and prolonged cytoplasmic stability. LKB1-NCs retained kinase activity and phosphorylated downstream targets. NCs were successfully delivered to cultured embryonic hippocampal neurons, and were stable in the cytoplasm for 2 days, sufficient time for axon formation. Importantly, LKB1-NCs promoted axon formation in these neurons, representing unique proof of concept for the sufficiency of intracellular protein function in dictating a central developmental event. Lastly, we established NC delivery into cortical progenitors in live rat embryonic brain in utero. Our nanotechnology provides a viable platform for spatial manipulation of intracellular protein-activity, to dictate central events during neuronal development.

Up-regulation of Mirta22/Emc10 is a major transcriptional effect of the 22q11.2-associated microRNA dysregulation and underlies key cellular as well as behavioral deficits. EMC10 is a component of the ER membrane complex, which promotes membrane insertion of a subset of polytopic and tail-anchored membrane proteins. Here we show that EMC10 expression is elevated in hiPSC-derived neurons from 22q11.2 deletion carriers and that reduction of EMC10 levels restores defects in neurite outgrowth and calcium signaling. Moreover, using injection of Antisense Oligonucleotides, we demonstrate that normalization of Emc10 levels in adult mouse brain rescues social memory deficits. The observations that elevated EMC10 expression is deleterious in 22q11.2 deletion carriers and that sustained elevation of EMC10 throughout the adult life can interfere with neural processes point to manipulations of EMC10 levels and downstream targets as a specific venue to ameliorate or even prevent disease progression in 22q11.2 deletion syndrome.