During development of the nervous system, axonal projections and synaptic contacts are supported by target-derived neuroptrophic factors, such as the neurotrophins and the glial cell line-derived (GDNF) family ligands (GFLs). During this period of target innervation, neurons often make excessive projections into their targets that are later pruned back. Development of the neuromuscular junction (NMJ), for example, undergoes a process in which NMJs are initially innervated by several axons, known as polyneuronal innervation. Then, through a process known as synaptic elimination, weaker connections are eliminated resulting in a 1:1 pairing between the presynaptic motor neuron and the postsynaptic receptor clusters in the muscle. This competitive “pushing out” of weaker axonal connections occurs by the release of inhibitory factors by the successful axons. The delicate balance between growth and survival-promoting neurotrophic factors, and inhibitory competitive factors, is thought to ultimately sculpt the architecture of mature circuits. My laboratory is interested in understanding the ligand and receptor mechanisms responsible for growth and survival promotion, synaptic elimination and apoptosis, and regeneration of peripheral neurons. We utilize biochemical and cell biological methods for the analysis of transgenic mice in which these receptors and ligands are deleted at specific developmental times. The determination of these mechanisms of survival, cell death and circuit maintenance will enable a more rational approach for the development of therapeutic strategies for diseases and injuries of the nervous system.
Brian Pierchala received his B.S. in Biochemistry from Oakland University in Michigan and obtained a Ph.D. in Neuroscience from the Johns Hopkins University School of Medicine. In the laboratory of David D. Ginty, his doctoral research investigated the ability of Nerve Growth Factor, a potent survival factor for sensory and sympathetic neurons, to support neuronal function when only activating receptors on axon terminals. His work on “retrograde” NGF signaling influenced the most widely accepted view of the field, namely that stable ligand-receptor complexes are trafficked in neurons over long distances to regulate biochemical events in the cell body necessary for survival, growth, and differentiation. Dr. Pierchala conducted his postdoctoral training in the laboratory of Eugene M. Johnson, Jr. at Washington University School of Medicine in Saint Louis. There, he investigated a newly discovered family of neuronal growth factors, the Glial Cell Line-derived Neurotrophic Factor (GDNF) Family Ligands (GFLs) and made several contributions to the understanding of how the GDNF receptor complex signals survival and differentiation. He continued his investigation of GDNF receptor signal transduction as an Assistant Professor at the State University of New York at Buffalo prior to his arrival to the Department of Biologic and Materials Sciences. His laboratory investigates the mechanisms of action of neurotrophic factors and proapoptotic factors in the development, maintenance and regeneration of the peripheral nervous system.
Dr. Pierchala is a Principal Investigator on research projects funded by the NIH and CHDI. He has instructed undergraduate students, Ph.D. graduate students and dental students in neurobiology, signal transduction, and cell biology. Dr. Pierchala serves as a reviewer for multiple journals including The Journal of Neuroscience, Nature and Science, and has served as a reviewer on NIH study sections.
The most recent publications are reported below via PubMed search.
To see all PubMed results go to the complete listing of publications by Dr. Pierchala.
Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo.
J Neurosci. 2015 Sep 23;35(38):13233-43
Authors: Tsui CC, Gabreski NA, Hein SJ, Pierchala BA
UNLABELLED: Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF-Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1-TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1-TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1(TM/TM) mice, GFRα1-TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1(+/TM) mice are viable, Gfrα1(TM/TM) mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1(-/-) mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates.
SIGNIFICANCE STATEMENT: Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex into lipid rafts.
PMID: 26400951 [PubMed - in process]
Polarized expression of p75(NTR) specifies axons during development and adult neurogenesis.
Cell Rep. 2014 Apr 10;7(1):138-52
Authors: Zuccaro E, Bergami M, Vignoli B, Bony G, Pierchala BA, Santi S, Cancedda L, Canossa M
VIDEO ABSTRACT: Newly generated neurons initiate polarizing signals that specify a single axon and multiple dendrites, a process critical for patterning neuronal circuits in vivo. Here, we report that the pan-neurotrophin receptor p75(NTR) is a polarity regulator that localizes asymmetrically in differentiating neurons in response to neurotrophins and is required for specification of the future axon. In cultured hippocampal neurons, local exposure to neurotrophins causes early accumulation of p75(NTR) into one undifferentiated neurite to specify axon fate. Moreover, knockout or knockdown of p75(NTR) results in failure to initiate an axon in newborn neurons upon cell-cycle exit in vitro and in the developing cortex, as well as during adult hippocampal neurogenesis in vivo. Hence, p75(NTR) governs neuronal polarity, determining pattern and assembly of neuronal circuits in adult hippocampus and cortical development.
PMID: 24685135 [PubMed - indexed for MEDLINE]
CD2-associated protein (CD2AP) enhances casitas B lineage lymphoma-3/c (Cbl-3/c)-mediated Ret isoform-specific ubiquitination and degradation via its amino-terminal Src homology 3 domains.
J Biol Chem. 2014 Mar 14;289(11):7307-19
Authors: Calco GN, Stephens OR, Donahue LM, Tsui CC, Pierchala BA
Ret is the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family of neuronal growth factors. Upon activation by GDNF, Ret is rapidly polyubiquitinated and degraded. This degradation process is isoform-selective, with the longer Ret51 isoform exhibiting different degradation kinetics than the shorter isoform, Ret9. In sympathetic neurons, Ret degradation is induced, at least in part, by a complex consisting of the adaptor protein CD2AP and the E3-ligase Cbl-3/c. Knockdown of Cbl-3/c using siRNA reduced the GDNF-induced ubiquitination and degradation of Ret51 in neurons and podocytes, suggesting that Cbl-3/c was a predominant E3 ligase for Ret. Coexpression of CD2AP with Cbl-3/c augmented the ubiquitination of Ret51 as compared with the expression of Cbl-3/c alone. Ret51 ubiquitination by the CD2AP·Cbl-3/c complex required a functional ring finger and TKB domain in Cbl-3/c. The SH3 domains of CD2AP were sufficient to drive the Cbl-3/c-dependent ubiquitination of Ret51, whereas the carboxyl-terminal coiled-coil domain of CD2AP was dispensable. Interestingly, activated Ret induced the degradation of CD2AP, but not Cbl-3/c, suggesting a potential inhibitory feedback mechanism. There were only two major ubiquitination sites in Ret51, Lys(1060) and Lys(1107), and the combined mutation of these lysines almost completely eliminated both the ubiquitination and degradation of Ret51. Ret9 was not ubiquitinated by the CD2AP·Cbl-3/c complex, suggesting that Ret9 was down-regulated by a fundamentally different mechanism. Taken together, these results suggest that only the SH3 domains of CD2AP were necessary to enhance the E3 ligase activity of Cbl-3/c toward Ret51.
PMID: 24425877 [PubMed - indexed for MEDLINE]
Expression of axonal protein degradation machinery in sympathetic neurons is regulated by nerve growth factor.
J Neurosci Res. 2012 Aug;90(8):1533-46
Authors: Frampton JP, Guo C, Pierchala BA
Deficiencies in protein degradation and proteolytic function within neurons are linked to a number of neurodegenerative diseases and developmental disorders. Compartmentalized cultures of peripheral neurons were used to investigate the properties and relative abundance of the proteolytic machinery in the axons and cell bodies of sympathetic and sensory neurons. Immunoblotting of axonal proteins demonstrated that LAMP2, LC3, and PSMA2 were abundant in axons, suggesting that lysosomes, autophagosomes and proteasomes were located in axons. Interestingly, the expression of proteins associated with lysosomes and proteasomes were upregulated selectively in axons by NGF stimulation of the distal axons of sympathetic neurons, suggesting that axonal growth and maintenance requires local protein turnover. The regulation of the abundance of both proteasomes and lysosomes in axons by NGF provides a link between protein degradation and the trophic status of peripheral neurons. Inhibition of proteasomes located in axons resulted in an accumulation of ubiquitinated proteins in these axons. In contrast, lysosome inhibition in axons did not result in an accumulation of ubiquitinated proteins or the transferrin receptor, a transmembrane protein degraded by lysosomes. Interestingly, lysosomes were transported both retrogradely and anterogradely, so it is likely that ubiquitinated proteins that are normally destined for degradation by lysosomes in axons can be transported to the cell bodies for degradation. In summary, proteasomal degradation occurs locally, whereas proteins degraded by lysosomes can most likely either be degraded locally in axons or be transported to cell bodies for degradation.
PMID: 22411744 [PubMed - indexed for MEDLINE]
Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals.
Cell. 2012 Feb 3;148(3):568-82
Authors: Bonanomi D, Chivatakarn O, Bai G, Abdesselem H, Lettieri K, Marquardt T, Pierchala BA, Pfaff SL
Growing axons encounter multiple guidance cues, but it is unclear how separate signals are resolved and integrated into coherent instructions for growth cone navigation. We report that glycosylphosphatidylinositol (GPI)-anchored ephrin-As function as "reverse" signaling receptors for motor axons when contacted by transmembrane EphAs present in the dorsal limb. Ephrin-A receptors are thought to depend on transmembrane coreceptors for transmitting signals intracellularly. We show that the receptor tyrosine kinase Ret is required for motor axon attraction mediated by ephrin-A reverse signaling. Ret also mediates GPI-anchored GFRα1 signaling in response to GDNF, a diffusible chemoattractant in the limb, indicating that Ret is a multifunctional coreceptor for guidance molecules. Axons respond synergistically to coactivation by GDNF and EphA ligands, and these cooperative interactions are gated by GFRα1 levels. Our studies uncover a hierarchical GPI-receptor signaling network that is constructed from combinatorial components and integrated through Ret using ligand coincidence detection.
PMID: 22304922 [PubMed - indexed for MEDLINE]
Generation of mice with a conditional allele for the p75(NTR) neurotrophin receptor gene.
Genesis. 2011 Nov;49(11):862-9
Authors: Bogenmann E, Thomas PS, Li Q, Kim J, Yang LT, Pierchala B, Kaartinen V
The p75(NTR) neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75(NTR) , many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (Exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75(NTR) (p75(NTR-FX) ) allele made by flanking Exons 4-6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75(NTR) /Wnt1-Cre mutants and conventional p75(NTR) null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75(NTR) in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75(NTR) mutants. This novel conditional p75(NTR) allele will offer new opportunities to investigate the role of p75(NTR) in specific tissues and cells.
PMID: 21413144 [PubMed - indexed for MEDLINE]
Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function.
Kidney Int. 2010 Nov;78(9):868-82
Authors: Pierchala BA, Muñoz MR, Tsui CC
Podocytes are morphologically complex cells, the junctions of which form critical elements of the final filtration barrier. Disruption of their foot processes and slit diaphragms occur early in the development of many glomerular diseases. Here, we biochemically purified fractions enriched with slit diaphragm proteins and performed a proteomic analysis to identify new components of this important structure. Several known slit diaphragm proteins were found, such as podocin and nephrin, confirming the validity of the purification scheme. However, proteins on the apical membrane such as podocalyxin were neither enriched nor identified in our analysis. The chloride intracellular channel protein 5 (CLIC5), predominantly expressed in podocytes, was enriched in these fractions and localized in the foot process apical and basal membranes. CLIC5 colocalized and associated with the ezrin/radixin/moesin complex and with podocalyxin in podocytes in vivo. It is important to note that CLIC5(-/-) mice were found to have significantly decreased foot process length, widespread foot process abnormalities, and developed proteinuria. The ezrin/radixin/moesin complex and podocalyxin were significantly decreased in podocytes from CLIC5(-/-) mice. Thus, our study identifies CLIC5 as a new component that is enriched in and necessary for foot process integrity and podocyte function in vivo.
PMID: 20664558 [PubMed - indexed for MEDLINE]
The differential axonal degradation of Ret accounts for cell-type-specific function of glial cell line-derived neurotrophic factor as a retrograde survival factor.
J Neurosci. 2010 Apr 14;30(15):5149-58
Authors: Tsui CC, Pierchala BA
Glial cell line-derived neurotrophic factor (GDNF) is a neuronal growth factor critical for the development and maintenance of central and peripheral neurons. GDNF is expressed in targets of innervation and provides support to several populations of large, projection neurons. To determine whether GDNF promotes retrograde survival over long axonal distances to cell bodies, we used a compartmentalized culture system. GDNF supported only modest and transient survival of postnatal sympathetic neurons when applied to their distal axons, in contrast to dorsal root ganglion (DRG) sensory neurons in which GDNF promoted survival equally well from either distal axons or cell bodies. Ret, the receptor tyrosine kinase for GDNF, underwent rapid proteasomal degradation in the axons of sympathetic neurons. Interestingly, the level of activated Ret in DRG neurons was sustained in the axons and also appeared in the cell bodies, suggesting that Ret was not degraded in sensory axons and was retrogradely transported. Pharmacologic inhibition of proteasomes only in the distal axons of sympathetic neurons caused an accumulation of activated Ret in both the axons and cell bodies during GDNF stimulation. Furthermore, exposure of the distal axons of sympathetic neurons to both GDNF and proteasome inhibitors, but neither one alone, promoted robust survival, identical to GDNF applied directly to the cell bodies. This differential responsiveness of sympathetic and sensory neurons to target-derived GDNF was attributable to the differential expression and degradation of the Ret9 and Ret51 isoforms. Therefore, the local degradation of Ret in axons dictates whether GDNF family ligands act as retrograde survival factors.
PMID: 20392937 [PubMed - indexed for MEDLINE]
CD2AP and Cbl-3/Cbl-c constitute a critical checkpoint in the regulation of ret signal transduction.
J Neurosci. 2008 Aug 27;28(35):8789-800
Authors: Tsui CC, Pierchala BA
The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are critical for nervous system development and maintenance. GFLs promote survival and growth via activation of the receptor tyrosine kinase (RTK) Ret. In sympathetic neurons, the duration of Ret signaling is governed by how rapidly Ret is degraded after its activation. In an effort to elucidate mechanisms that control the half-life of Ret, we have identified two novel Ret interactors, CD2-associated protein (CD2AP) and Cbl-3. CD2AP, an adaptor molecule involved in the internalization of ubiquitinated RTKs, is associated with Ret under basal, unstimulated conditions in neurons. After Ret activation by GDNF, CD2AP dissociates. Similarly, the E3-ligase Cbl-3 interacts with unphosphorylated Ret and dissociates from Ret after Ret activation. In contrast to their dissociation from autophosphorylated Ret, an interaction between CD2AP and Cbl-3 is induced by GDNF stimulation of sympathetic neurons, suggesting that CD2AP and Cbl-3 dissociate from Ret as a complex. In neurons, the overexpression of CD2AP enhances the degradation of Ret and inhibits GDNF-dependent survival, and gene silencing of CD2AP blocks Ret degradation and promotes GDNF-mediated survival. Surprisingly, Cbl-3 overexpression dramatically stabilizes activated Ret and enhances neuronal survival, even though Cbl-family E3 ligases normally function to trigger RTK downregulation. In combination with CD2AP, however, Cbl-3 promotes Ret degradation rapidly and almost completely blocks survival promotion by GDNF, suggesting that Cbl-3 acts as a switch that is triggered by CD2AP and oscillates between inhibition and promotion of Ret degradation. Consistent with the hypothesis, Cbl-3 silencing in neurons only inhibited Ret degradation and enhanced neuronal survival in combination with CD2AP silencing. CD2AP and Cbl-3, therefore, constitute a checkpoint that controls the extent of Ret downregulation and, thereby, the sensitivity of neurons to GFLs.
PMID: 18753381 [PubMed - indexed for MEDLINE]
NGF augments the autophosphorylation of Ret via inhibition of ubiquitin-dependent degradation.
J Neurochem. 2007 Mar;100(5):1169-76
Authors: Pierchala BA, Tsui CC, Milbrandt J, Johnson EM
Nerve growth factor (NGF) is required for the trophic maintenance of postnatal sympathetic neurons. A significant portion of the growth-promoting activity of NGF is from NGF-dependent phosphorylation of the heterologous receptor tyrosine kinase, Ret. We found that NGF applied selectively to distal axons of sympathetic neurons maintained in compartmentalized cultures activated Ret located in these distal axons. Inhibition of either proteasomal or lysosomal degradation pathways mimicked the effect of NGF on Ret activation. Likewise, NGF inhibited the degradation of Ret induced by glial cell line-derived neurotrophic factor-dependent activation, a process that requires ubiquitination and proteasomal degradation. NGF induced the accumulation of autophosphorylated Ret predominantly in the plasma membrane, in contrast to GDNF, which promoted the internalization of activated Ret. An accretion of monoubiquitinated, but not polyubiquitinated, Ret occurred in NGF-treated neurons, in contrast to glial cell line-derived neurotrophic factor that promoted the robust polyubiquitination of Ret. Thus, NGF stimulates Ret activity in mature sympathetic neurons by inhibiting the ongoing ubiquitin-mediated degradation of Ret before its internalization and polyubiquitination.
PMID: 17241133 [PubMed - indexed for MEDLINE]
Glial cell line-derived neurotrophic factor and its receptor ret is a novel ligand-receptor complex critical for survival response during podocyte injury.
J Am Soc Nephrol. 2006 Jun;17(6):1543-52
Authors: Tsui CC, Shankland SJ, Pierchala BA
Glomerulosclerosis correlates with a reduction in podocyte number that occurs through mechanisms that include apoptosis. Whether glial cell line-derived neurotrophic factor (GDNF), a growth factor that is critical for neural and renal development, is a survival factor for injured podocytes was investigated. Ret, the GDNF receptor tyrosine kinase, was upregulated in podocytes in the passive Heymann nephritis and puromycin aminonucleoside (PA) nephrosis rat models of podocyte injury. In addition, Ret mRNA and protein were upregulated in mouse podocytes in vitro after injury that was induced by sublytic C5b-9 and PA. GDNF, which also was induced during podocyte injury, inhibited significantly the apoptosis of podocytes that was induced by ultraviolet C irradiation. Knockdown of Ret expression by small interference RNA in podocytes exacerbated apoptosis that was induced by both ultraviolet C and PA. Ret knockdown, upon injury, decreased AKT phosphorylation, suggesting that the phosphoinositol-3 kinase/AKT pathway mediated the survival effect of GDNF on podocytes. Consistent with this hypothesis, the selective phosphoinositol-3 kinase inhibitor LY294002 blocked the survival-promoting effects of GDNF. In conclusion, GDNF is a novel podocyte survival factor. Furthermore, Ret is highly upregulated during podocyte injury in vitro and in vivo, suggesting that Ret activation is a critical adaptive response for podocyte remodeling and repair.
PMID: 16672314 [PubMed - indexed for MEDLINE]
Glial cell line-derived neurotrophic factor-dependent recruitment of Ret into lipid rafts enhances signaling by partitioning Ret from proteasome-dependent degradation.
J Neurosci. 2006 Mar 8;26(10):2777-87
Authors: Pierchala BA, Milbrandt J, Johnson EM
The receptor tyrosine kinase (RTK) Ret is activated by the formation of a complex consisting of ligands such as glial cell line-derived neurotrophic factor (GDNF) and glycerophosphatidylinositol-anchored coreceptors termed GFRalphas. During activation, Ret translocates into lipid rafts, which is critical for functional responses to GDNF. We found that Ret was rapidly ubiquitinated and degraded in sympathetic neurons when activated with GDNF, but, unlike other RTKs that are trafficked to lysosomes for degradation, Ret was degraded predominantly by the proteasome. After GDNF stimulation, the majority of ubiquitinated Ret was located outside of lipid rafts and Ret was lost predominantly from nonraft membrane domains. Consistent with the predominance of Ret degradation outside of rafts, disruption of lipid rafts in neurons did not alter either the GDNF-dependent ubiquitination or degradation of Ret. GDNF-mediated survival of sympathetic neurons was inhibited by lipid raft depletion, and this inhibitory effect of raft disruption on GDNF-mediated survival was reversed if Ret degradation was blocked via proteasome inhibition. Therefore, lipid rafts sequester Ret away from the degradation machinery located in nonraft membrane domains, such as Cbl family E3 ligases, thereby sustaining Ret signaling.
PMID: 16525057 [PubMed - indexed for MEDLINE]
Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells.
Exp Neurol. 2005 Jan;191(1):65-76
Authors: Lee CS, Tee LY, Dusenbery S, Takata T, Golden JP, Pierchala BA, Gottlieb DI, Johnson EM, Choi DW, Snider BJ
Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.
PMID: 15589513 [PubMed - indexed for MEDLINE]
Nerve growth factor promotes the survival of sympathetic neurons through the cooperative function of the protein kinase C and phosphatidylinositol 3-kinase pathways.
J Biol Chem. 2004 Jul 2;279(27):27986-93
Authors: Pierchala BA, Ahrens RC, Paden AJ, Johnson EM
The signaling pathways activated by nerve growth factor (NGF) that account for its ability to promote the survival of neurons are not completely understood. Phosphatidylinositol 3-kinase (PI3K) is critical for the survival of several cell types, including neurons. To determine whether additional signaling pathways cooperate with PI3K to promote survival, we examined other pathways known to be activated by NGF. NGF activated protein kinases C (PKCs) in sympathetic neurons, and pharmacologic PKC activation rescued neurons from apoptosis induced by the withdrawal of NGF. Inhibition of PKCs did not inhibit the survival of NGF-maintained neurons. Similarly, inhibition of PI3K caused only a modest attrition of neurons in the presence of NGF. In contrast, the simultaneous inhibition of both PKCs and PI3K induced the apoptotic death of NGF-maintained sympathetic neurons. Inhibition of both PI3K and PKCs promoted the expression and phosphorylation of the proapoptotic transcription factor c-Jun, indicating that these pathways inhibit programmed cell death at the stage of proapoptotic gene expression. In culture conditions under which PI3K inhibition alone kills NGF-maintained neurons, PKC inhibition also led to a significant loss of viability, indicating that both pathways are required. Therefore, PKC and PI3K, regardless of the culture conditions, cooperate to promote the NGF-dependent survival of sympathetic neurons.
PMID: 15117960 [PubMed - indexed for MEDLINE]
Lipid rafts in neuronal signaling and function.
Trends Neurosci. 2002 Aug;25(8):412-7
Authors: Tsui-Pierchala BA, Encinas M, Milbrandt J, Johnson EM
Lipid rafts are plasma membrane microdomains rich in cholesterol and sphingolipids, which provide a particularly ordered lipid environment. Rafts are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, as well as proteins involved in signal transduction and intracellular trafficking. In neurons, lipid rafts act as platforms for the signal transduction initiated by several classes of neurotrophic factors, including neurotrophins and glial-derived neurotrophic factor (GDNF)-family ligands. Emerging evidence also indicates that such rafts are important for neuronal cell adhesion, axon guidance and synaptic transmission. Thus, lipid rafts are structurally unique components of plasma membranes, crucial for neural development and function.
PMID: 12127758 [PubMed - indexed for MEDLINE]
The long and short isoforms of Ret function as independent signaling complexes.
J Biol Chem. 2002 Sep 13;277(37):34618-25
Authors: Tsui-Pierchala BA, Ahrens RC, Crowder RJ, Milbrandt J, Johnson EM
Ret, the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family ligands (GFLs), is alternatively spliced to yield at least two isoforms, Ret9 and Ret51, which differ only in their C termini. To identify tyrosines in Ret that are autophosphorylation sites in neurons, we generated antibodies specific to phosphorylated Y905Ret, Y1015Ret, Y1062Ret, and Y1096Ret, all of which are autophosphorylated in cell lines. All four of these tyrosines in Ret became phosphorylated rapidly upon activation by GFLs in sympathetic neurons. These tyrosines remained phosphorylated in sympathetic neurons in the continued presence of GFLs, albeit at a lower level than immediately after GFL treatment. Comparison of GFL activation of Ret9 and Ret51 revealed that phosphorylation of Tyr(905) and Tyr(1062) was greater and more sustained in Ret9 as compared with Ret51. In contrast, Tyr(1015) was more highly phosphorylated over time in Ret51 than in Ret9. Surprisingly, Ret9 and Ret51 did not associate with each other in sympathetic neurons after glial cell line-derived neurotrophic factor stimulation, even though they share identical extracellular domains. Furthermore, the signaling complex associated with Ret9 was markedly different from the Ret51-associated signaling complex. Taken together, these data provide a biochemical basis for the dramatic functional differences between Ret9 and Ret 51 in vivo.
PMID: 12091387 [PubMed - indexed for MEDLINE]
NGF utilizes c-Ret via a novel GFL-independent, inter-RTK signaling mechanism to maintain the trophic status of mature sympathetic neurons.
Neuron. 2002 Jan 17;33(2):261-73
Authors: Tsui-Pierchala BA, Milbrandt J, Johnson EM
During postnatal development, sympathetic neurons lose their dependence upon NGF for survival but continue to require NGF for soma and process growth and for development of a mature neurotransmitter phenotype. Although c-Ret is expressed in sympathetic neurons during this period, its function in these transitional processes is unclear. The level of Ret phosphorylation markedly increased with postnatal age in SCG neurons in vitro and in vivo. Postnatal Ret phosphorylation did not require either GFLs or GFR(alpha) coreceptors. Instead, NGF promoted age-dependent Ret phosphorylation with delayed kinetics both in vitro and in vivo. Functionally, maximal NGF-dependent trophism of mature sympathetic neurons required Ret, but not GFR(alpha) coreceptors. Therefore, NGF promotes phosphorylation of the heterologous RTK Ret resulting in augmented growth, metabolism, and gene expression.
PMID: 11804573 [PubMed - indexed for MEDLINE]
Inhibition of the c-Jun N-terminal kinase signaling pathway by the mixed lineage kinase inhibitor CEP-1347 (KT7515) preserves metabolism and growth of trophic factor-deprived neurons.
J Neurosci. 2002 Jan 1;22(1):103-13
Authors: Harris CA, Deshmukh M, Tsui-Pierchala B, Maroney AC, Johnson EM
Nerve growth factor (NGF) deprivation triggers metabolic changes in sympathetic neurons that precede cell death. Here, we investigate the role of the c-Jun N-terminal kinase (JNK) pathway in downregulating neuronal metabolism. We show that, in the presence of CEP-1347 (KT7515), a small molecule known to block cell death upstream of JNK, cellular metabolism is preserved in neurons deprived of NGF. Biochemical data that are presented are consistent with the mechanism of action of CEP-1347 being the inhibition of the mixed lineage kinases (MLKs), known activators of JNK signaling. We demonstrate that CEP-1347-saved neurons continue to grow even in the absence of NGF, indicating that inhibition of the JNK pathway is permissive for neuronal growth in the absence of trophic support. These trophic effects are seen despite the fact that CEP-1347 does not stimulate several known survival kinase pathways. In addition to blocking Bax-dependent cytochrome c release, the inhibition of the JNK signaling pathway with CEP-1347 also blocks the development of competence-to-die in response to cytosolic cytochrome c. Therefore, inhibition of the JNK signaling pathway with the MLK inhibitor CEP-1347 inhibits both limbs of the apoptotic pathway. Finally, we demonstrate that neurons that have been NGF-deprived long-term but that have been kept alive by caspase inhibitors can be rescued metabolically by CEP-1347 as assessed by soma size, cytochrome c localization, and protein synthesis rates. Therefore, we conclude that, in addition to converting extracellular signals into decisions of life and death, the JNK pathway can modulate cellular metabolism directly and thereby maintain not only survival but the "quality of life" of neurons.
PMID: 11756493 [PubMed - indexed for MEDLINE]
c-Src is required for glial cell line-derived neurotrophic factor (GDNF) family ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway.
J Neurosci. 2001 Mar 1;21(5):1464-72
Authors: Encinas M, Tansey MG, Tsui-Pierchala BA, Comella JX, Milbrandt J, Johnson EM
The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), consisting of GDNF, neurturin, persephin, and artemin, signal via a multicomponent complex composed of Ret tyrosine kinase and the glycosyl-phosphatidylinositol (GPI)-anchored coreceptors GFRalpha1-alpha4. In previous work we have demonstrated that the localization of Ret to membrane microdomains known as lipid rafts is essential for GDNF-induced downstream signaling, differentiation, and neuronal survival. Moreover, we have found that Ret interacts with members of the Src family kinases (SFK) only when it is localized to these microdomains. In the present work we show by pharmacological and genetic approaches that Src activity was necessary to elicit optimal GDNF-mediated signaling, neurite outgrowth, and survival. In particular, p60Src, but not the other ubiquitous SFKs, Fyn and Yes, was responsible for the observed effects. Moreover, Src appeared to promote neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)-dependent pathway because the PI-3K inhibitor LY294002 prevented GFL-mediated neuronal survival and prevented activated Src-mediated neuronal survival. In contrast, the inhibition of Src activity had no effects on NGF-mediated survival, indicating that the requirement for Src was selective for GFL-mediated neuronal survival. These data confirm the importance of protein-protein interactions between Ret and raft-associated proteins in the signaling pathways elicited by GDNF, and the data implicate Src as one of the major signaling molecules involved in GDNF-mediated bioactivity.
PMID: 11222636 [PubMed - indexed for MEDLINE]
Phosphatidylinositol 3-kinase is required for the trophic, but not the survival-promoting, actions of NGF on sympathetic neurons.
J Neurosci. 2000 Oct 1;20(19):7228-37
Authors: Tsui-Pierchala BA, Putcha GV, Johnson EM
Nerve growth factor (NGF) supports target-dependent survival of sympathetic and other neurons during development; however, the NGF-regulated signaling pathways required for survival are not fully understood. Sympathetic neurons are able to abort acutely the cell death pathway initiated by NGF deprivation at early, as well as late, time points after readdition of NGF. We found that NGF-dependent phosphatidylinositol 3-kinase (PI-3-K) activity inhibited an early cell death event proximal to c-Jun phosphorylation. However, PI-3-K activity was not required for NGF to inhibit the translocation of Bax from the cytoplasm to the mitochondria, nor was it required for NGF to inhibit the subsequent release of mitochondrial cytochrome c, two events required for NGF deprivation-induced apoptosis. MEK/MAPK activity did not account for any of these NGF-dependent events. When subjected to long-term PI-3-K inhibition in the presence of NGF, the majority of sympathetic neurons did not die. Those that did die exhibited significant differences in the characteristics of death caused by PI-3-K inhibition as compared with NGF deprivation. Additionally, PI-3-K inhibition in the presence of NGF did not induce release of mitochondrial cytochrome c, indicating that these neurons were unable to complete the apoptotic program. In contrast to its modest effects on survival, inhibition of PI-3-K induced marked decreases in somal diameter and metabolic function, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, suggesting that PI-3-K is required for the trophic effects of NGF. Therefore, although PI-3-K is important for the trophic effects of NGF, it is not required for survival. Other, or at least additional, signaling pathways contribute to NGF-mediated survival of sympathetic neurons.
PMID: 11007879 [PubMed - indexed for MEDLINE]
Characterization of an NGF-P-TrkA retrograde-signaling complex and age-dependent regulation of TrkA phosphorylation in sympathetic neurons.
J Neurosci. 1999 Oct 1;19(19):8207-18
Authors: Tsui-Pierchala BA, Ginty DD
Nerve growth factor (NGF) is a target-derived trophic factor for developing sympathetic and cutaneous sensory neurons. NGF promotes growth and survival of neurons via activation of the receptor tyrosine kinase TrkA. We used compartmentalized cultures of sympathetic neurons to address the mechanism of NGF signaling from distal axons and terminals to proximal axons and cell bodies. Our results demonstrate that an NGF-phospho-TrkA (NGF-P-TrkA)-signaling complex forms in distal axons and is retrogradely transported as a complex to cell bodies of sympathetic neurons. Although a minor fraction of both NGF and TrkA is retrogradely transported, a large fraction of the NGF that is retrogradely transported is found complexed with retrogradely transported TrkA. Interestingly, the metabolism of the P-TrkA complex is dramatically different in young, NGF-dependent sympathetic neurons as compared to older, NGF-independent sympathetic neurons. After withdrawal of NGF from distal axons of young neurons, P-TrkA within distal axons, as well as within proximal axons and cell bodies, dephosphorylates rapidly. In contrast, after withdrawal of NGF from distal axons of older neurons, P-TrkA within distal axons dephosphorylates completely, although more slowly than that in young neurons, whereas dephosphorylation of P-TrkA within proximal axons and cell bodies occurs markedly more slowly, with at least one-half of the level of P-TrkA remaining 2 d after NGF withdrawal. Thus, P-TrkA within the cell bodies of young, NGF-dependent sympathetic neurons is derived from distal axons. A more stable P-TrkA complex within cell bodies of mature sympathetic neurons may contribute to the acquisition of NGF independence for survival of mature sympathetic neurons.
PMID: 10493722 [PubMed - indexed for MEDLINE]
An NGF-TrkA-mediated retrograde signal to transcription factor CREB in sympathetic neurons.
Science. 1997 Aug 22;277(5329):1097-100
Authors: Riccio A, Pierchala BA, Ciarallo CL, Ginty DD
Nerve growth factor (NGF) is a neurotrophic factor secreted by cells that are the targets of innervation of sympathetic and some sensory neurons. However, the mechanism by which the NGF signal is propagated from the axon terminal to the cell body, which can be more than 1 meter away, to influence biochemical events critical for growth and survival of neurons has remained unclear. An NGF-mediated signal transmitted from the terminals and distal axons of cultured rat sympathetic neurons to their nuclei regulated phosphorylation of the transcription factor CREB (cyclic adenosine monophosphate response element-binding protein). Internalization of NGF and its receptor tyrosine kinase TrkA, and their transport to the cell body, were required for transmission of this signal. The tyrosine kinase activity of TrkA was required to maintain it in an autophosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus, an NGF-TrkA complex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons.
PMID: 9262478 [PubMed - indexed for MEDLINE]
The rapamycin and FKBP12 target (RAFT) displays phosphatidylinositol 4-kinase activity.
J Biol Chem. 1995 Sep 8;270(36):20875-8
Authors: Sabatini DM, Pierchala BA, Barrow RK, Schell MJ, Snyder SH
The immunosuppressant rapamycin prevents cell cycle progression in several mammalian cell lines and the yeast Saccharomyces cerevisiae. In mammalian cells, rapamycin binds to the small FK506-binding protein, FKBP12, allowing the drug-receptor complex to interact with the 289-kDa RAFT1/FRAP proteins. These proteins, along with their yeast homologs, TOR1/DRR1 and TOR2/DRR2, contain a C-terminal domain with amino acid homology to several phosphatidylinositol (PI) 4- and 3-kinases. However, no direct demonstration of kinase activity for this family of proteins has been reported. We now show that RAFT1, immunoprecipitated from rat brain and MG63 and HEK293 cells, contains PI 4-kinase activity and that rapamycin-FKBP12 has no effect on this activity. Thus, it is likely that, in vivo, rapamycin does not directly inhibit the PI 4-kinase activity and affects the RAFT1/FRAP protein through another mechanism.
PMID: 7673106 [PubMed - indexed for MEDLINE]
A p75-Ret Signaling Complex Mediates Neuronal Survival and Death in the Developing Peripheral Nervous