Woo Yang Kim

GSK-3 is a master regulator of neural progenitor homeostasis

The development of the brain requires the exquisite coordination of progenitor proliferation and differentiation to achieve complex circuit assembly. It has been suggested that glycogen synthase kinase 3 (GSK-3) acts as an integrating molecule for multiple proliferation and differentiation signals because of its essential role in the RTK, Wnt and Shh signaling pathways. We created conditional mutations that deleted both the α and β forms of GSK-3 in mouse neural progenitors. GSK-3 deletion resulted in massive hyperproliferation of neural progenitors along the entire neuraxis. Generation of both intermediate neural progenitors and postmitotic neurons was markedly suppressed. These effects were associated with the dysregulation of β-catenin, Sonic Hedgehog, Notch and fibroblast growth factor signaling. Our results indicate that GSK-3 signaling is an essential mediator of homeostatic controls that regulate neural progenitors during mammalian brain development.

Essential Roles for GSK-3s and GSK-3-Primed Substrates in Neurotrophin-Induced and Hippocampal Axon Growth

Glycogen synthase kinase-3beta (GSK-3beta) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly, we found no gross morphological deficits in nervous system development in GSK-3beta null mice. We therefore designed an shRNA that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3alpha and beta markedly reduced axon growth in dissociated cultures and slice preparations. We then assessed the role of different GSK-3 substrates in regulating axon morphology. Elimination of activity toward primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase-dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. Our results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome.

The Adenomatous Polyposis Coli Protein Is an Essential Regulator of Radial Glial Polarity and Construction of the Cerebral Cortex

Radial glia are highly polarized cells that serve as neuronal progenitors and as scaffolds for neuronal migration during construction of the cerebral cortex. How radial glial cells establish and maintain their morphological polarity is unknown. Using conditional gene targeting in mice, we demonstrate that adenomatous polyposis coli (APC) serves an essential function in the maintenance of polarized radial glial scaffold during brain development. In the absence of APC, radial glial cells lose their polarity and responsiveness to the extracellular polarity maintenance cues, such as neuregulin-1. Elimination of APC further leads to marked instability of the radial glial microtubule cytoskeleton. The resultant changes in radial glial function and loss of APC in radial glial progeny lead to defective generation and migration of cortical neurons, severely disrupted cortical layer formation, and aberrant axonal tract development. Thus, APC is an essential regulator of radial glial polarity and is critical for the construction of cerebral cortex in mammals.

Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain

J. Neurochem. (2010) 115, 974–983.

Functions of GSK-3 signaling in development of the nervous system

Glycogen synthase kinase-3 (GSK-3) is central to multiple intracellular pathways including those activated by Wnt/β-catenin, Sonic Hedgehog, Notch, growth factor/RTK, and G protein-coupled receptor signals. All of these signals importantly contribute to neural development. Early attention on GSK-3 signaling in neural development centered on the regulation of neuronal polarity using in vitro paradigms. However, recent creation of appropriate genetic models has demonstrated the importance of GSK-3 to multiple aspects of neural development including neural progenitor self-renewal, neurogenesis, neuronal migration, neural differentiation, and synaptic development.

MACF1 regulates the migration of pyramidal neurons via microtubule dynamics and GSK-3 signaling

Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons.

Cdc42 and Gsk3 modulate the dynamics of radial glial growth, inter-radial glial interactions and polarity in the developing cerebral cortex

Polarized radial glia are crucial to the formation of the cerebral cortex. They serve as neural progenitors and as guides for neuronal placement in the developing cerebral cortex. The maintenance of polarized morphology is essential for radial glial functions, but the extent to which the polarized radial glial scaffold is static or dynamic during corticogenesis remains an open question. The developmental dynamics of radial glial morphology, inter-radial glial interactions during corticogenesis, and the role of the cell polarity complexes in these activities remain undefined. Here, using real-time imaging of cohorts of mouse radial glia cells, we show that the radial glial scaffold, upon which the cortex is constructed, is highly dynamic. Radial glial cells within the scaffold constantly interact with one another. These interactions are mediated by growth cone-like endfeet and filopodia-like protrusions. Polarized expression of the cell polarity regulator Cdc42 in radial glia regulates glial endfeet activities and inter-radial glial interactions. Furthermore, appropriate regulation of Gsk3 activity is required to maintain the overall polarity of the radial glia scaffold. These findings reveal dynamism and interactions among radial glia that appear to be crucial contributors to the formation of the cerebral cortex. Related cell polarity determinants (Cdc42, Gsk3) differentially influence radial glial activities within the evolving radial glia scaffold to coordinate the formation of cerebral cortex.

mTOR regulates brain morphogenesis by mediating GSK3 signaling

Balanced control of neural progenitor maintenance and neuron production is crucial in establishing functional neural circuits during brain development, and abnormalities in this process are implicated in many neurological diseases. However, the regulatory mechanisms of neural progenitor homeostasis remain poorly understood. Here, we show that mammalian target of rapamycin (mTOR) is required for maintaining neural progenitor pools and plays a key role in mediating glycogen synthase kinase 3 (GSK3) signaling during brain development. First, we generated and characterized conditional mutant mice exhibiting deletion of mTOR in neural progenitors and neurons in the developing brain using Nestin-cre and Nex-cre lines, respectively. The elimination of mTOR resulted in abnormal cell cycle progression of neural progenitors in the developing brain and thereby disruption of progenitor self-renewal. Accordingly, production of intermediate progenitors and postmitotic neurons were markedly suppressed. Next, we discovered that GSK3, a master regulator of neural progenitors, interacts with mTOR and controls its activity in cortical progenitors. Finally, we found that inactivation of mTOR activity suppresses the abnormal proliferation of neural progenitors induced by GSK3 deletion. Our findings reveal that the interaction between mTOR and GSK3 signaling plays an essential role in dynamic homeostasis of neural progenitors during brain development.

Statins decrease dendritic arborization in rat sympathetic neurons by blocking RhoA activation

Clinical and experimental evidence suggest that statins decrease sympathetic activity, but whether peripheral mechanisms involving direct actions on post‐ganglionic sympathetic neurons contribute to this effect is not known. Because tonic activity of these neurons is directly correlated with the size of their dendritic arbor, we tested the hypothesis that statins decrease dendritic arborization in sympathetic neurons. Oral administration of atorvastatin (20u2003mg/kg/day for 7u2003days) significantly reduced dendritic arborization in vivo in sympathetic ganglia of adult male rats. In cultured sympathetic neurons, statins caused dendrite retraction and reversibly blocked bone morphogenetic protein‐induced dendritic growth without altering cell survival or axonal growth. Supplementation with mevalonate or isoprenoids, but not cholesterol, attenuated the inhibitory effects of statins on dendritic growth, whereas specific inhibition of isoprenoid synthesis mimicked these statin effects. Statins blocked RhoA translocation to the membrane, an event that requires isoprenylation, and constitutively active RhoA reversed statin effects on dendrites. These observations that statins decrease dendritic arborization in sympathetic neurons by blocking RhoA activation suggest a novel mechanism by which statins decrease sympathetic activity and protect against cardiovascular and cerebrovascular disease.

Statins decrease expression of the proinflammatory neuropeptides calcitonin gene-related peptide and substance P in sensory neurons

Clinical and experimental observations suggest that statins may be useful for treating diseases presenting with predominant neurogenic inflammation, but the mechanism(s) mediating this potential therapeutic effect are poorly understood. In this study, we tested the hypothesis that statins act directly on sensory neurons to decrease expression of proinflammatory neuropeptides that trigger neurogenic inflammation, specifically calcitonin gene-related peptide (CGRP) and substance P. Reverse transcriptase-polymerase chain reaction, radioimmunoassay, and immunocytochemistry were used to quantify CGRP and substance P expression in dorsal root ganglia (DRG) harvested from adult male rats and in primary cultures of sensory neurons derived from embryonic rat DRG. Systemic administration of statins at pharmacologically relevant doses significantly reduced CGRP and substance P levels in DRG in vivo. In cultured sensory neurons, statins blocked bone morphogenetic protein (BMP)-induced CGRP and substance P expression and decreased expression of these neuropeptides in sensory neurons pretreated with BMPs. These effects were concentration-dependent and occurred independent of effects on cell survival or axon growth. Statin inhibition of neuropeptide expression was reversed by supplementation with mevalonate and cholesterol, but not isoprenoid precursors. BMPs signal via Smad activation, and cholesterol depletion by statins inhibited Smad1 phosphorylation and nuclear translocation. These findings identify a novel action of statins involving down-regulation of proinflammatory neuropeptide expression in sensory ganglia via cholesterol depletion and decreased Smad1 activation and suggest that statins may be effective in attenuating neurogenic inflammation.