β-PIX, a Rac1/Cdc42-specific guanine nucleotide exchange factor, is known to regulate actin cytoskeleton remodeling during cell migration. In this study, we investigated the effects of β-PIX-d, an isoform of β-PIX, on neocortical development and neuritogenesis. Overexpression of β-PIX-d in the embryonic neocortex induced increased cell clusters and enhanced neurite outgrowth in cortical neurons. Following in utero electroporation of β-PIX-d expression vectors into neuronal progenitor cells at embryonic day 13.5 (E13.5), histological analysis at postnatal day 0 (P0) revealed the presence of clustered neurons and neurites outside of the marginal zone (MZ). Immunofluorescence staining with the neuronal marker TuJ1 confirmed that the clustered structures were predominantly composed of neurons. Layer-specific marker analysis further demonstrated the misplacement of layer V-VI neurons into layer I and the subarachnoid space. In primary neocortical cultures, β-PIX-d overexpression promoted neuritogenesis and increased Rac1 activity, as detected by pull-down assays. These findings suggest that β-PIX-d and Rac1 interactions play a critical role in the formation of neocortical clustering and the regulation of neuritogenesis.

β-PIX, a Rac1/Cdc42-specific guanine nucleotide exchange factor, is known to regulate actin cytoskeleton remodeling during cell migration. In this study, we investigated the effects of β-PIX-d, an isoform of β-PIX, on neocortical development and neuritogenesis. Overexpression of β-PIX-d in the embryonic neocortex induced increased cell clusters and enhanced neurite outgrowth in cortical neurons. Following in utero electroporation of β-PIX-d expression vectors into neuronal progenitor cells at embryonic day 13.5 (E13.5), histological analysis at postnatal day 0 (P0) revealed the presence of clustered neurons and neurites outside of the marginal zone (MZ). Immunofluorescence staining with the neuronal marker TuJ1 confirmed that the clustered structures were predominantly composed of neurons. Layer-specific marker analysis further demonstrated the misplacement of layer V-VI neurons into layer I and the subarachnoid space. In primary neocortical cultures, β-PIX-d overexpression promoted neuritogenesis and increased Rac1 activity, as detected by pull-down assays. These findings suggest that β-PIX-d and Rac1 interactions play a critical role in the formation of neocortical clustering and the regulation of neuritogenesis.

β-PIX, a Rac1/Cdc42-specific guanine nucleotide exchange factor, is known to regulate actin cytoskeleton remodeling during cell migration. In this study, we investigated the effects of β-PIX-d, an isoform of β-PIX, on neocortical development and neuritogenesis. Overexpression of β-PIX-d in the embryonic neocortex induced increased cell clusters and enhanced neurite outgrowth in cortical neurons. Following in utero electroporation of β-PIX-d expression vectors into neuronal progenitor cells at embryonic day 13.5 (E13.5), histological analysis at postnatal day 0 (P0) revealed the presence of clustered neurons and neurites outside of the marginal zone (MZ). Immunofluorescence staining with the neuronal marker TuJ1 confirmed that the clustered structures were predominantly composed of neurons. Layer-specific marker analysis further demonstrated the misplacement of layer V-VI neurons into layer I and the subarachnoid space. In primary neocortical cultures, β-PIX-d overexpression promoted neuritogenesis and increased Rac1 activity, as detected by pull-down assays. These findings suggest that β-PIX-d and Rac1 interactions play a critical role in the formation of neocortical clustering and the regulation of neuritogenesis.

β-PIX, a Rac1/Cdc42-specific guanine nucleotide exchange factor, is known to regulate actin cytoskeleton remodeling during cell migration. In this study, we investigated the effects of β-PIX-d, an isoform of β-PIX, on neocortical development and neuritogenesis. Overexpression of β-PIX-d in the embryonic neocortex induced increased cell clusters and enhanced neurite outgrowth in cortical neurons. Following in utero electroporation of β-PIX-d expression vectors into neuronal progenitor cells at embryonic day 13.5 (E13.5), histological analysis at postnatal day 0 (P0) revealed the presence of clustered neurons and neurites outside of the marginal zone (MZ). Immunofluorescence staining with the neuronal marker TuJ1 confirmed that the clustered structures were predominantly composed of neurons. Layer-specific marker analysis further demonstrated the misplacement of layer V-VI neurons into layer I and the subarachnoid space. In primary neocortical cultures, β-PIX-d overexpression promoted neuritogenesis and increased Rac1 activity, as detected by pull-down assays. These findings suggest that β-PIX-d and Rac1 interactions play a critical role in the formation of neocortical clustering and the regulation of neuritogenesis.

Rheb (Ras homolog enriched in the brain) is a small GTPase protein that plays an important role in cell signaling for development of the neocortex through modulation of mTORC1 (mammalian-target-of-rapamycin-complex-1) activity. mTORC1 is known to control various biological processes including axonal growth in forming complexes at the lysosomal membrane compartment. As such, anchoring of Rheb on the lysosomal membrane via the farnesylation of Rheb at its cysteine residue (C180) is required for its promotion of mTOR activity. To test the significance of Rheb farnesylation, we overexpressed a farnesylation mutant form of Rheb, Rheb C180S, in primary rat hippocampal neurons and also in mouse embryonic neurons using in utero electroporation. Interestingly, we found that Rheb C180S maintained promotional effect of axonal elongation similar to the wild-type Rheb in both test systems. On the other hand, Rheb C180S failed to exhibit the multiple axon-promoting effect which is found in wild-type Rheb. The levels of phospho-4EBP1, a downstream target of mTORC1, were surprisingly increased in Rheb C180S transfected neurons, despite the levels of phosphorylated mTOR being significantly decreased compared to control vector transfectants. A specific mTORC1 inhibitor, rapamycin, also could not completely abolish axon elongation characteristics of Rheb C180S in transfected cells. Our data suggests that Rheb in a non-membrane compartment can promote the axonal elongation via phosphorylation of 4EBP1 and through an mTORC1-independent pathway.
Animals ; Axons ; Biological Processes ; Cysteine ; Electroporation ; GTP Phosphohydrolases ; Hand ; Membranes ; Mice ; Neocortex ; Neurons ; Phosphorylation ; Prenylation ; Protein Prenylation ; Rats ; Sirolimus ; TOR Serine-Threonine Kinases