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Freda D Miller

Publications and source records attributed to Freda D Miller.

At least 19 recordsLinked to original sources
PubMed2006

An essential role for the integrin-linked kinase-glycogen synthase kinase-3 beta pathway during dendrite initiation and growth.

Multiple cues, including growth factors and circuit activity, signal to regulate the initiation and growth of mammalian dendrites. In this study, we have asked how these environmental cues regulate dendrite formation, and in particular, whether dendrite initiation and growth requires integrin-linked kinase (ILK) or its downstream effector, glycogen synthase kinase-3beta (GSK-3beta). In cultured sympathetic neurons, NGF and neuronal depolarization activated ILK and promoted dendrite initiation and growth, and inhibition of ILK (either pharmacologically, with a dominant-negative form of ILK, or by genetic knockdown) reduced depolarization-induced dendrite formation. In sympathetic neurons, ILK phosphorylated and inhibited GSK-3beta, and inhibition of GSK-3beta (either pharmacologically, with dominant-negative GSK-3beta, or by genetic knockdown) caused robust dendrite initiation. GSK-3beta inhibition also caused dendrite initiation in cultured cortical neurons and growth of hippocampal neurons in slice cultures. GSK-3beta functioned downstream of ILK to regulate dendrite formation, because inhibition of GSK-3beta promoted dendrite initiation even when ILK was simultaneously inhibited. Moreover, GSK-3beta promoted dendrite formation in sympathetic neurons by regulating the activity of a key dendrite formation effector, the MAP (microtubule-associated protein) kinase kinase (MEK)-extracellular signal-regulated protein kinase (ERK) pathway. Specifically, inhibition of GSK-3beta led to increased ERK phosphorylation, and inhibition of MEK completely blocked the effects of GSK-3beta inhibition on dendrite initiation and growth. Thus, the ILK-GSK-3beta pathway plays a key role in regulating dendrite formation in developing mammalian neurons.

Animals↗
PubMed2006

APPL1 associates with TrkA and GIPC1 and is required for nerve growth factor-mediated signal transduction.

The neurotrophin receptor TrkA plays critical roles in the nervous system by recruiting signaling molecules that activate pathways required for the growth and survival of neurons. Here, we report APPL1 as a TrkA-associated protein. APPL1 and TrkA co-immunoprecipitated in sympathetic neurons. We have identified two routes through which this association can occur. APPL1 was isolated as a binding partner for the TrkA-interacting protein GIPC1 from rat brain lysate by mass spectrometry. The PDZ domain of GIPC1 directly engaged the C-terminal sequence of APPL1. This interaction provides a means through which APPL1 may be recruited to TrkA. In addition, the APPL1 PTB domain bound to TrkA, indicating that APPL1 may associate with TrkA independently of GIPC1. Isolation of endosomal fractions by high-resolution centrifugation determined that APPL1, GIPC1, and phosphorylated TrkA are enriched in the same fractions. Reduction of APPL1 or GIPC1 protein levels suppressed nerve growth factor (NGF)-dependent MEK, extracellular signal-regulated kinase, and Akt activation and neurite outgrowth in PC12 cells. Together, these results indicate that GIPC1 and APPL1 play a role in TrkA function and suggest that a population of endosomes bearing a complex of APPL1, GIPC1, and activated TrkA may transmit NGF signals.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The localization, trafficking and retrograde transport of BDNF bound to p75NTR in sympathetic neurons.

BDNF, through p75NTR, promotes apoptosis and inhibits axonal growth of sympathetic neurons, antagonizing the pro-survival and axon growth-promoting actions of NGF through TrkA. While the trafficking of the TrkA:NGF complex is well characterized, little is known about p75NTR:BDNF trafficking in these neurons. Here we show that BDNF binds to and appears inside sympathetic neurons relatively slowly, although the temperature-sensitive internalization step itself is rapid. P75NTR internalization is partially sensitive to disruption of clathrin- or raft-mediated internalization, while that of TrkA is entirely clathrin-mediated. P75NTR, but not Trk, associates with neurotrophins in lipid rafts and coimmunoprecipitates with the truncated beta-caveolin-1 isoform. Finally, we directly visualize the retrograde transport of p75NTR ligands to cell bodies, which is insensitive to inhibitors of Trk retrograde transport, suggesting mechanistic differences. We postulate that beta-caveolin-1-containing lipid rafts and possibly intracellular endosomes might be compartments to which p75NTR:BDNF complexes are trafficked separately from Trk.

Animals↗
PubMed2006

Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system.

Although neural stem cells hold considerable promise for treatment of the injured or degenerating nervous system, their current human sources are embryonic stem cells and fetally derived neural tissue. Here, we asked whether rodent and human skin-derived precursors (SKPs), neural crest-related precursors found in neonatal dermis, represent a source of functional, myelinating Schwann cells. Specifically, cultured SKPs responded to neural crest cues such as neuregulins to generate Schwann cells, and these Schwann cells proliferated and induced myelin proteins when in contact with sensory neuron axons in culture. Similar results were obtained in vivo; 6 weeks after transplantation of naive SKPs or SKP-derived Schwann cells into the injured peripheral nerve of wild-type or shiverer mutant mice (which are genetically deficient in myelin basic protein), the majority of SKP-derived cells had associated with and myelinated axons. Naive rodent or human SKPs also generated Schwann cells that myelinated CNS axons when transplanted into the dysmyelinated brain of neonatal shiverer mice. Thus, neonatal SKPs generate functional neural progeny in response to appropriate neural crest cues and, in so doing, provide a highly accessible source of myelinating cells for treatment of nervous system injury, congenital leukodystrophies, and dysmyelinating disorders.

Animals↗
PubMed2006

Analysis of the neurogenic potential of multipotent skin-derived precursors.

Multipotent precursors similar to stem cells of the embryonic neural crest (NC) have been identified in several postnatal tissues, and are potentially useful for research and therapeutic purposes. However, their neurogenic potential, including their ability to produce electrophysiologically active neurons, is largely unexplored. We investigated this issue with regard to skin-derived precursors (SKPs), multipotent NC-related precursors isolated from the dermis of skin. SKP cultures follow an appropriate pattern and time-course of neuronal differentiation, with proliferating nestin-expressing SKPs generating post-mitotic neuronal cells that co-express pan-neuronal and peripheral autonomic lineage markers. These SKP-derived neuron-like cells survive and maintain their peripheral phenotype for at least 5 weeks when transplanted into the CNS environment of normal or kainate-injured hippocampal slices. Undifferentiated SKPs retain key neural precursor properties after multi-passage expansion, including growth factor dependence, nestin expression, neurogenic potential, and responsiveness to embryonic neural crest fate determinants. Despite undergoing an apparently appropriate neurogenic process, however, SKP-derived neuron-like cells possess an immature electrophysiological profile. These findings indicate that SKPs retain latent neurogenic properties after residing in a non-neural tissue, but that additional measures will be necessary to promote their differentiation into electrophysiologically active neurons.

Animals↗
PubMed2006

When a motor goes bad: a kinesin regulates neuronal survival.

In this issue of Cell, Midorikawa et al. (2006) demonstrate that the kinesin superfamily member KIF4, a microtubule-based molecular motor, regulates the survival of electrically active neurons in the developing brain by modulating the function of poly(ADP-ribose) polymerase-1 in an unexpected way.

Animals↗
PubMed2006

The p53 family in nervous system development and disease.

The p53 family, consisting of the tumor suppressors p53, p63 and p73, play a vital role as regulators of survival and apoptosis in the developing, adult and injured nervous system. These proteins function as key survival and apoptosis checkpoints in neurons, acting as either rheostats or sensors responsible for integrating multiple pro-apoptotic and survival cues. A dramatic example of this checkpoint function is observed in developing sympathetic neurons, where a pro-survival and truncated form of p73 antagonizes the apoptotic functions of p53 and p63. Thus the levels and activities of the different p53 family members may ultimately determine whether neurons either live or die during nervous system development and disease.

Animals↗
PubMed2005

p63 is an essential proapoptotic protein during neural development.

The p53 family member p63 is required for nonneural development, but has no known role in the nervous system. Here, we define an essential proapoptotic role for p63 during naturally occurring neuronal death. Sympathetic neurons express full-length TAp63 during the developmental death period, and TAp63 levels increase following NGF withdrawal. Overexpression of TAp63 causes neuronal apoptosis in the presence of NGF, while cultured p63-/- neurons are resistant to apoptosis following NGF withdrawal. TAp63 is also essential in vivo, since embryonic p63-/- mice display a deficit in naturally occurring sympathetic neuron death. While both TAp63 and p53 induce similar apoptotic signaling proteins and require BAX expression and function for their effects, TAp63 induces neuronal death in the absence of p53, but p53 requires coincident p63 expression for its proapoptotic actions. Thus, p63 is essential for developmental neuronal death, likely functioning both on its own, and as an obligate proapoptotic partner for p53.

Aging↗
PubMed2005

CCAAT/enhancer-binding protein phosphorylation biases cortical precursors to generate neurons rather than astrocytes in vivo.

The intracellular mechanisms that bias mammalian neural precursors to generate neurons versus glial cells are not well understood. We demonstrated previously that the growth factor-regulated mitogen-activated protein kinase kinase (MEK) and its downstream target, the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, are essential for neurogenesis in cultured cortical precursor cells (Ménard et al., 2002). Here, we examined a role for this pathway during cortical cell fate determination in vivo using in utero electroporation of the embryonic cortex. These studies demonstrate that inhibition of the activity of either MEK or the C/EBPs inhibits the genesis of neurons in vivo. Moreover, the MEK pathway mediates phosphorylation of C/EBPbeta in cortical precursors, and expression of a C/EBPbeta construct in which the MEK pathway phosphorylation sites are mutated inhibits neurogenesis. Conversely, expression of a C/EBPbeta construct, in which the same sites are mutated to glutamate and therefore are "constitutively" phosphorylated, enhances neurogenesis in the early embryonic cortex. A subpopulation of precursors in which C/EBP activity is inhibited are maintained as cycling precursors in the ventricular/subventricular zone of the cortex until early in postnatal life, when they have an enhanced propensity to generate astrocytes, presumably in response to gliogenic signals in the neonatal environment. Thus, activation of an MEK-C/EBP pathway in cortical precursors in vivo biases them to become neurons and against becoming astrocytes, thereby acting as a growth factor-regulated switch.

Animals↗
PubMed2005

Evidence that embryonic neurons regulate the onset of cortical gliogenesis via cardiotrophin-1.

Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition are unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrate that cortical neurons synthesize and secrete the neurotrophic cytokine cardiotrophin-1, which activates the gp130-JAK-STAT pathway and is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo. In utero electroporation of neurotrophic cytokines in the environment of embryonic cortical precursors causes premature gliogenesis, while acute perturbation of gp130 in cortical precursors delays the normal timed appearance of astrocytes. Moreover, the neonatal cardiotrophin-1-/- cortex contains fewer astrocytes. Together, these results describe a neural feedback mechanism; newly born neurons produce cardiotrophin-1, which instructs multipotent cortical precursors to generate astrocytes, thereby ensuring that gliogenesis does not occur until neurogenesis is largely complete.

Analysis of Variance↗
PubMed2005

TrkA induces apoptosis of neuroblastoma cells and does so via a p53-dependent mechanism.

Neuroblastoma (NB) is the most frequent solid extracranial tumor in children. Its clinical prognosis correlates with the expression of members of the Trk neurotrophin receptor family, which includes TrkA and TrkB. TrkA expression is associated with favorable prognosis, whereas TrkB expression is associated with poor prognosis. Here we show that TrkA expression induces the apoptosis of NB cells and does so by modulating the levels or activities of a number of proteins involved in regulating cell survival and apoptosis, including p53, Bcl-2, and caspase-3. TrkA increased the expression of p53 target proteins and failed to induce apoptosis in cells where p53 was inactivated by mutation or via expression of dominant inhibitory p53 or E1B55K, indicating that TrkA mediates apoptosis, at least in part, through p53. Treatment with a caspase inhibitor or overexpression of Bcl-X(L) also prevented TrkA from inducing apoptosis. In contrast, elevated expression of TrkA in non-transformed sympathetic neurons resulted in the suppression of p53 levels and enhanced survival. These results identify apoptosis as a novel biological response of TrkA in NB cells and imply that TrkA is a good prognosis marker for NB due in part to its ability to mediate apoptosis when expressed at sufficient levels.

Adenoviridae↗
PubMed2005

Activity regulates positive and negative neurotrophin-derived signals to determine axon competition.

Developmental axon competition plays a key role in sculpting neural circuitry. Here, we have asked how activity and neurotrophins could interact to select one axon over another. Using compartmented cultures of sympathetic neurons, we show that, in the presence of NGF, local depolarization confers a competitive growth advantage on the depolarized axon collaterals and at the same time disadvantages the growth of unstimulated axons from the same and competing neurons. Depolarization mediates the competitive advantage by activating a CaMKII-MEK pathway, which converges to enhance local NGF-mediated downstream growth signals. Patterned electrical stimulation also acts via this pathway to enhance NGF-promoted axonal growth. In contrast, the competitive disadvantage is due to BDNF secreted from and acting on the unstimulated, competing axons through p75NTR. Thus, activity regulates both positive and negative neurotrophin-derived signaling cascades to confer a competitive growth advantage on one axon versus another, thereby providing a cellular mechanism for developmental axon selection.

Action Potentials↗
PubMed2005

Role for Runx1 in the proliferation and neuronal differentiation of selected progenitor cells in the mammalian nervous system.

Neurogenesis requires factors that regulate the decision of dividing progenitors to leave the cell cycle and activate the neuronal differentiation program. It is shown here that the murine runt-related gene Runx1 is expressed in proliferating cells on the basal side of the olfactory epithelium. These include both Mash1+ olfactory receptor neuron (ORN) progenitors and NeuroD+ ORN precursors. Disruption of Runx1 function in vivo does not cause a change in Mash1 expression but leads to a decrease in the number of NeuroD+ neuronal precursors and an increase in differentiated ORNs. These effects result in premature and ectopic ORN differentiation. It is shown further that exogenous Runx1 expression in cultured olfactory neural progenitors causes an expansion of the mitotic cell population. In agreement with these findings, exogenous Runx1 expression also promotes cortical neural progenitor cell proliferation without inhibiting neuronal differentiation. These effects are phenocopied by a chimeric protein containing ETO, the eight twenty one transcriptional repressor, fused to the Runx1 DNA-binding domain, which suggests the involvement of transcription repression mechanisms. Consistent with this possibility, Runx1 represses transcription driven by the promoter of the cell cycle inhibitor p21Cip 1 in cortical progenitors. Together, these findings suggest a previously unrecognized role for Runx1 in coordinating the proliferation and neuronal differentiation of selected populations of neural progenitors.

Amino Acid Substitution↗
PubMed2005

A transcriptional role for C/EBP beta in the neuronal response to axonal injury.

The molecular mechanisms responsible for inducing gene expression following neuronal injury are not well understood. Here, we address this issue by focusing upon C/EBPbeta, a transcription factor implicated in cellular injury and regeneration. We show that C/EBPbeta mRNA is expressed in neurons throughout the mature brain and that levels of both C/EBPbeta mRNA and phosphoprotein are increased in facial motor neurons following axonal injury. To determine the importance of these increases, we examined the regeneration-associated Talpha1 alpha-tubulin gene which contains functional C/EBP binding sites in its promoter. In transgenic mice, expression of a minimal 176 nucleotide Talpha1 alpha-tubulin promoter:nlacZ reporter gene was upregulated in injured facial motor neurons. This injury-induced transcriptional increase was inhibited in C/EBPbeta -/- mice. A similar inhibition was observed in C/EBPbeta -/- mice that carried a larger 1.1-kb promoter Talpha1:nlacZ reporter construct. Moreover, in situ hybridization revealed that the injury-induced upregulation of the endogenous mouse alpha1 alpha-tubulin mRNA, and of a second regeneration-associated mRNA, GAP-43, was inhibited in C/EBPbeta -/- mice. Thus, C/EBPbeta is essential for the neuronal injury response, acting to transcriptionally activate regeneration-associated gene expression.

Animals↗
PubMed2004

The invulnerability of adult neurons: a critical role for p73.

Here, we investigated the intracellular mechanisms that underlie the relative invulnerability of adult versus developing dorsal root ganglion (DRG) sensory neurons. In culture, adult neurons were resistant to stimuli that caused apoptosis of their neonatal counterparts. In both adult and neonatal neurons, death stimuli induced the apoptotic c-Jun N-terminal protein kinase (JNK) pathway, but JNK activation only caused death of neonatal neurons, indicating that adult neurons have a downstream block to apoptosis. Expression of the dominant-inhibitory p53 family member, DeltaNp73, rescued JNK-induced apoptosis of neonatal neurons, suggesting that it might participate in the downstream apoptotic block in adult neurons. To test this possibility, we examined adult DRG neurons cultured from p73+/- mice. Adult p73+/- DRG neurons were more vulnerable to apoptotic stimuli than their p73+/+ counterparts, and invulnerability could be restored to the p73+/- neurons by increased expression of DeltaNp73. Moreover, although DRG neuron development was normal in p73+/- animals in vivo, axotomy caused death of adult p73+/- but not p73+/+ DRG neurons. Thus, one way adult neurons become invulnerable is to enhance endogenous survival pathways, and one critical component of these survival pathways is p73.

Aging↗
PubMed2004

Evidence that DeltaNp73 promotes neuronal survival by p53-dependent and p53-independent mechanisms.

The p53 family member, p73, is essential for the survival of sympathetic neurons during the developmental period of naturally occurring neuronal death. Here, we have asked whether DeltaNp73, which is the only p73 isoform expressed in sympathetic neurons, mediates this survival by p53-dependent and/or p53-independent mechanisms. Initially, we used a genetic approach and crossed p53+/- and p73+/- mice. Quantitation of neurons in the sympathetic superior cervical ganglion during the period of naturally occurring cell death revealed that the loss of p53 partially rescued the death of neurons seen in p73-/- animals. Moreover, exogenous expression of DeltaNp73 in cultured p53-/- sympathetic neurons rescued these neurons from apoptosis after NGF withdrawal. Biochemical studies asking how DeltaNp73 inhibited NGF withdrawal-induced apoptosis in wild-type neurons demonstrated that it prevented the upregulation of the direct p53 targets p21 and Apaf-1 as well as cleavage of caspase-3. It also inhibited events at the mitochondrial apoptotic checkpoint, suppressing the induction of BimEL and the release of mitochondrial cytochrome c. Interestingly, DeltaNp73 expression also inhibited one very early event in the apoptotic cascade, the activation of c-Jun N-terminal protein kinase (JNK), likely by binding directly to JNK. Finally, we show that neuronal cell size is decreased in p73-/- mice, and that this decrease is not rescued by the lack of p53, suggesting a role for p73 in regulating cell size that does not involve interactions with p53. Thus, DeltaNp73 promotes neuronal survival via p53-dependent and -independent mechanisms, and it does so at multiple points, including some of the most proximal events that occur after NGF withdrawal.

Animals↗
PubMed2004

Neuronal MEK is important for normal fear conditioning in mice.

The extracellular signal-regulated kinase (ERK) cascade has received much attention for its possible role in neuronal synaptic plasticity. Although ERK activation has been linked to learning behaviors and activity-dependent neuronal function, much of the acquired data has relied upon pharmacological agents that suppress ERK function in both neurons and nonneuronal cells. To determine the function of neuronal ERK activity in learning, a new line of transgenic mice was generated wherein dominant-negative MEK1, the upstream obligate activator of ERK1/2, was expressed by using a neuronal-specific and pan-neuronal Talpha1 alpha-tubulin promoter element. Mice expressing this construct exhibited decreased ERK1/2 activity in the hippocampus and thus were tested for learning impairments. In a battery of control tests, including open field, rotarod, and shock threshold, the transgenic mice displayed no deficits and performed as well as their wild-type littermate counterparts. However, the mice displayed a significant impairment in contextual fear conditioning compared with the wild-type littermates. These findings indicate that the MEK1/ERK1/2 cascade within neurons plays an important role in the processes of learning and memory.

Animals↗