The anaphase-promoting complex (APC) is highly expressed in postmitotic neurons, but its function in the nervous system was previously unknown. We report that the inhibition of Cdh1-APC in primary neurons specifically enhanced axonal growth. Cdh1 knockdown in cerebellar slice overlay assays and in the developing rat cerebellum in vivo revealed cell-autonomous abnormalities in layer-specific growth of granule neuron axons and parallel fiber patterning. Cdh1 RNA interference in neurons was also found to override the inhibitory influence of myelin on axonal growth. Thus, Cdh1-APC appears to play a role in regulating axonal growth and patterning in the developing brain that may also limit the growth of injured axons in the adult brain.
Neurons form distinctive axonal and dendritic compartments that are important for directional signaling, but the mechanisms that discriminate between axons and dendrites remain elusive. Previous studies have demonstrated that the kinesin-1 motor domain is capable of distinguishing the axon from dendrites. Here we found that the amino acid substitutions in the beta5-loop8 region transformed truncated kinesin-1 from a uni-destination (that is, the axon-specific destination) to a bi-destination (that is, axons and dendrites) state. Furthermore, tyrosinated tubulins that are abundant in somatodendrites prevent the wild-type kinesin-1 from binding to microtubules, whereas the bi-destination-type kinesin-1 does not have this inhibition. Consistently, inhibition of tubulin tyrosination in rat hippocampal neurons resulted in the distribution of truncated kinesin-1 in both axons and dendrites. Our study identifies a molecular mechanism that discriminates the axonal microtubules from somatodendritic microtubules, as well as a previously unknown linkage between tubulin modification and polarized trafficking in neurons.
A mechanism that triggers neuronal apoptosis has been characterized. We report that the cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and that Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. Our results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions.
The elaboration of dendrites is fundamental to the establishment of neuronal polarity and connectivity, but the mechanisms that underlie dendritic morphogenesis are poorly understood. We found that the genetic knockdown of the transcription factor NeuroD in primary granule neurons including in organotypic cerebellar slices profoundly impaired the generation and maintenance of dendrites while sparing the development of axons. We also found that NeuroD mediated neuronal activity-dependent dendritogenesis. The activity-induced protein kinase CaMKII catalyzed the phosphorylation of NeuroD at distinct sites, including endogenous NeuroD at Ser336 in primary neurons, and thereby stimulated dendritic growth. These findings uncover an essential function for NeuroD in granule neuron dendritic morphogenesis. Our study also defines the CaMKII-NeuroD signaling pathway as a novel mechanism underlying activity-regulated dendritic growth that may play important roles in the developing and mature brain.
Axonal growth is fundamental to the establishment of neuronal connectivity in the brain. However, the cell-intrinsic mechanisms that govern axonal morphogenesis remain to be elucidated. The ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC) suppresses the growth of axons in postmitotic neurons. Here, we report that Cdh1-APC operates in the nucleus to inhibit axonal growth. We also identify the transcriptional corepressor SnoN as a key target of neuronal Cdh1-APC that promotes axonal growth. Cdh1 forms a physical complex with SnoN and stimulates the ubiquitin-dependent proteasomal degradation of SnoN in neurons. Knockdown of SnoN in neurons significantly reduces axonal growth and suppresses Cdh1 RNAi enhancement of axonal growth. In addition, SnoN knockdown in vivo suggests an essential function for SnoN in the development of granule neuron parallel fibers in the cerebellar cortex. These findings define Cdh1-APC and SnoN as components of a cell-intrinsic pathway that orchestrates axonal morphogenesis in a transcription-dependent manner in the mammalian brain.
The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in the developing and mature organism. The JNK signaling pathway is thought to induce apoptosis via transcription-dependent and transcription-independent mechanisms that remain to be elucidated. In this study, we report a novel mechanism by which the JNK signaling pathway directly activates a component of the cell death machinery. We have found that JNK catalyzes the phosphorylation of the BH3-only protein BAD at the distinct site of serine 128 in vitro. Activation of the JNK signaling pathway induces the BAD serine 128 phosphorylation in vivo, including in primary granule neurons of the developing rat cerebellum. The JNK-induced BAD serine 128 phosphorylation promotes the apoptotic effect of BAD in primary neurons by antagonizing the ability of growth factors to inhibit BAD-mediated apoptosis. These findings indicate that BAD is a novel substrate of JNK that links the stress-activated signaling pathway to the cell death machinery.Regulation of cell death is critical to the normal development and homeostasis of multicellular organisms. In the developing nervous system, neurons are produced in excess, and naturally occurring neuronal cell death is thus critical in ensuring that neurons form the appropriate connections (1). In the mature nervous system, abnormally occurring neuronal cell death contributes to the pathogenesis of a variety of diseases including stroke, epilepsy, and neurodegenerative diseases (2-4). Not surprisingly, the mechanisms that underlie neuronal cell death have been the subject of intense interest.Studies of cell death in a variety of organisms have revealed that members of the Bcl-2 family of proteins act as gatekeepers of the cell death machinery (5-7). Under conditions in which cells are destined to undergo programmed cell death (apoptosis), the proapoptotic multidomain Bcl-2 proteins, including Bax, induce the release of cytochrome c from mitochondria leading to the activation of a caspase cascade that executes the cell death program (8, 9). The prosurvival multidomain Bcl-2 proteins, including Bcl-2 and Bcl-xl, interact with and inhibit the proapoptotic multidomain Bcl-2 proteins (10).The BH3-only subfamily of Bcl-2 proteins appears to play an important role in regulating the function of the multidomain Bcl-2 protein in response to signals from the cell surface or the cytoskeleton (11). The BH3-only protein BAD promotes cell death by interacting with and inhibiting the prosurvival multidomain Bcl-2 proteins (10). Survival factors suppress BADmediated apoptosis by inducing the phosphorylation of BAD at serine 136, serine 112, or both, culminating in the interaction and sequestration of phosphorylated BAD by members of the 14-3-3 family of proteins (12). The protein kinases Akt, p21-activated kinase 1, and p70S6 kinase appear to mediate survival factor-induced phosphorylation of BAD serine 136 (13-17). On the other hand, the protein kinases Rsk, protein kinase A, and p21-activated kinase 1 ar...
Activation of cyclin-dependent kinase 1 (Cdk1) has been linked to cell death of postmitotic neurons in brain development and disease. We found that Cdk1 phosphorylated the transcription factor FOXO1 at Ser249 in vitro and in vivo. The phosphorylation of FOXO1 at Ser249 disrupted FOXO1 binding with 14-3-3 proteins and thereby promoted the nuclear accumulation of FOXO1 and stimulated FOXO1-dependent transcription, leading to cell death in neurons. In proliferating cells, Cdk1 induced FOXO1 Ser249 phosphorylation at the G2/M phase of the cell cycle, resulting in FOXO1-dependent expression of the mitotic regulator Polo-like kinase (Plk). These findings define a conserved signaling link between Cdk1 and FOXO1 that may have a key role in diverse biological processes, including the degeneration of postmitotic neurons.
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