Quantitative systems pharmacology model of the amyloid pathway in Alzheimer's disease: Insights into the therapeutic mechanisms of clinical candidates.

Despite considerable investment into potential therapeutic approaches for Alzheimer's disease (AD), currently approved treatment options are limited. Predictive modeling using quantitative systems pharmacology (QSP) can be used to guide the design of clinical trials in AD. This study developed a QSP model representing amyloid beta (Aß) pathophysiology in AD. The model included mechanisms of Aß monomer production and aggregation to form insoluble fibrils and plaques; the transport of soluble species between the compartments of brain, cerebrospinal fluid (CSF), and plasma; and the pharmacokinetics, transport, and binding of monoclonal antibodies to targets in the three compartments. Ordinary differential equations were used to describe these processes quantitatively. The model components were calibrated to data from the literature and internal studies, including quantitative data supporting the underlying AD biology and clinical data from clinical trials for anti-Aß monoclonal antibodies (mAbs) aducanumab, crenezumab, gantenerumab, and solanezumab. The model was developed for an apolipoprotein E (APOE) ɛ4 allele carrier and tested for an APOE ɛ4 noncarrier. Results indicate that the model is consistent with data on clinical Aß accumulation in untreated individuals and those treated with monoclonal antibodies, capturing increases in Aß load accurately. This model may be used to investigate additional AD mechanisms and their impact on biomarkers, as well as predict Aß load at different dose levels for mAbs with known targets and binding affinities. This model may facilitate the design of scientifically enriched and efficient clinical trials by enabling a priori prediction of biomarker dynamics in the brain and CSF.

Antibody semorinemab reduces tau pathology in a transgenic mouse model and engages tau in patients with Alzheimer's disease.

Tau has become an attractive alternative target for passive immunotherapy efforts for Alzheimer's disease (AD). The anatomical distribution and extent of tau pathology correlate with disease course and severity better than other disease markers to date. We describe here the generation, preclinical characterization, and phase 1 clinical characterization of semorinemab, a humanized anti-tau monoclonal antibody with an immunoglobulin G4 (igG4) isotype backbone. Semorinemab binds all six human tau isoforms and protects neurons against tau oligomer neurotoxicity in cocultures of neurons and microglia. In addition, when administered intraperitoneally once weekly for 13 weeks, murine versions of semorinemab reduced the accumulation of tau pathology in a transgenic mouse model of tauopathy, independent of antibody effector function status. Semorinemab also showed clear evidence of target engagement in vivo, with increases in systemic tau concentrations observed in tau transgenic mice, nonhuman primates, and humans. Higher concentrations of systemic tau were observed after dosing in AD participants compared to healthy control participants. No concerning safety signals were observed in the phase 1 clinical trial at single doses up to 16,800 mg and multiple doses totaling 33,600 mg in a month.

Mechanistic Modeling of Soluble Aß Dynamics and Target Engagement in the Brain by Anti-Aß mAbs in Alzheimer's Disease.

BACKGROUND: Anti-amyloid-ß (Aß) monoclonal antibodies (mAbs) are currently in development for treating Alzheimer's disease. OBJECTIVES: To address the complexity of Aß target engagement profiles, improve the understanding of crenezumab Pharmacokinetics (PK) and Aß Pharmacodynamics (PD) in the brain, and facilitate comparison of anti-Aß therapies with different binding characteristics. METHODS: A mechanistic mathematical model was developed describing the distribution, elimination, and binding kinetics of anti-Aß mAbs and Aß (monomeric and oligomeric forms of Aß1-40 and Aß1-42) in the brain, Cerebrospinal Fluid (CSF), and plasma. Physiologically meaningful values were assigned to the model parameters based on the previous data, with remaining parameters fitted to clinical measurements of Aß concentrations in CSF and plasma, and PK/PD data of patients undergoing anti-Aß therapy. Aß target engagement profiles were simulated using a Monte Carlo approach to explore the impact of biological uncertainty in the model parameters. RESULTS: Model-based estimates of in vivo affinity of the antibody to monomeric Aß were qualitatively consistent with the previous data. Simulations of Aß target engagement profiles captured observed mean and variance of clinical PK/PD data. CONCLUSION: This model is useful for comparing target engagement profiles of different anti-Aß therapies and demonstrates that 60 mg/kg crenezumab yields a significant increase in Aß engagement compared with lower doses of solanezumab, supporting the selection of 60 mg/kg crenezumab for phase 3 studies. The model also provides evidence that the delivery of sufficient quantities of mAb to brain interstitial fluid is a limiting step with respect to the magnitude of soluble Aß oligomer neutralization.

Characterization of the selective in vitro and in vivo binding properties of crenezumab to oligomeric Aß.

BACKGROUND: Accumulation of amyloid ß (Aß) in the brain is proposed as a cause of Alzheimer's disease (AD), with Aß oligomers hypothesized to be the primary mediators of neurotoxicity. Crenezumab is a humanized immunoglobulin G4 monoclonal antibody that has been shown to bind to synthetic monomeric and aggregated Aß in vitro; however, less is known about the binding characteristic in vivo. In this study, we evaluated the binding patterns of crenezumab to synthetic and native forms of Aß both in vitro and in vivo. METHODS: Crenezumab was used to immunoprecipitate Aß from synthetic Aß preparations or brain homogenates from a PS2APP mouse model of AD to determine the forms of Aß that crenezumab interacts with. Following systemic dosing in PS2APP or nontransgenic control mice, immunohistochemistry was used to localize crenezumab and assess its relative distribution in the brain, compared with amyloid plaques and markers of neuritic dystrophies (BACE1; LAMP1). Pharmacodynamic correlations were performed to investigate the relationship between peripheral and central target engagement. RESULTS: In vitro, crenezumab immunoprecipitated Aß oligomers from both synthetic Aß preparations and endogenous brain homogenates from PS2APP mice. In vivo studies in the PS2APP mouse showed that crenezumab localizes to regions surrounding the periphery of amyloid plaques in addition to the hippocampal mossy fibers. These regions around the plaques are reported to be enriched in oligomeric Aß, actively incorporate soluble Aß, and contribute to Aß-induced neurotoxicity and axonal dystrophy. In addition, crenezumab did not appear to bind to the dense core region of plaques or vascular amyloid. CONCLUSIONS: Crenezumab binds to multiple forms of amyloid ß (Aß), particularly oligomeric forms, and localizes to brain areas rich in Aß oligomers, including the halo around plaques and hippocampal mossy fibers, but not to vascular Aß. These insights highlight a unique mechanism of action for crenezumab of engaging Aß oligomers.

Widespread brain distribution and activity following i.c.v. infusion of anti-ß-secretase (BACE1) in nonhuman primates.

BACKGROUND AND PURPOSE: The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood-brain barrier. I.c.v. delivery is a promising route to the brain; however, it is unclear how efficiently antibodies delivered i.c.v. penetrate the cerebrospinal spinal fluid (CSF)-brain barrier and distribute throughout the brain parenchyma. EXPERIMENTAL APPROACH: We evaluated the pharmacokinetics and pharmacodynamics of an inhibitory monoclonal antibody against ß-secretase 1 (anti-BACE1) following continuous infusion into the left lateral ventricle of healthy adult cynomolgus monkeys. KEY RESULTS: Animals infused with anti-BACE1 i.c.v. showed a robust and sustained reduction (~70%) of CSF amyloid-ß (Aß) peptides. Antibody distribution was near uniform across the brain parenchyma, ranging from 20 to 40 nM, resulting in a ~50% reduction of Aß in the cortical parenchyma. In contrast, animals administered anti-BACE1 i.v. showed no significant change in CSF or cortical Aß levels and had a low (~0.6 nM) antibody concentration in the brain. CONCLUSION AND IMPLICATIONS: I.c.v. administration of anti-BACE1 resulted in enhanced BACE1 target engagement and inhibition, with a corresponding dramatic reduction in CNS Aß concentrations, due to enhanced brain exposure to antibody.

Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice.

The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.

Structure of Crenezumab Complex with Aß Shows Loss of ß-Hairpin.

Accumulation of amyloid-ß (Aß) peptides and amyloid plaque deposition in brain is postulated as a cause of Alzheimer's disease (AD). The precise pathological species of Aß remains elusive although evidence suggests soluble oligomers may be primarily responsible for neurotoxicity. Crenezumab is a humanized anti-Aß monoclonal IgG4 that binds multiple forms of Aß, with higher affinity for aggregated forms, and that blocks Aß aggregation, and promotes disaggregation. To understand the structural basis for this binding profile and activity, we determined the crystal structure of crenezumab in complex with Aß. The structure reveals a sequential epitope and conformational requirements for epitope recognition, which include a subtle but critical element that is likely the basis for crenezumab's versatile binding profile. We find interactions consistent with high affinity for multiple forms of Aß, particularly oligomers. Of note, crenezumab also sequesters the hydrophobic core of Aß and breaks an essential salt-bridge characteristic of the ß-hairpin conformation, eliminating features characteristic of the basic organization in Aß oligomers and fibrils, and explains crenezumab's inhibition of aggregation and promotion of disaggregation. These insights highlight crenezumab's unique mechanism of action, particularly regarding Aß oligomers, and provide a strong rationale for the evaluation of crenezumab as a potential AD therapy.

Characterization of a sensitive mouse Aß40 PD biomarker assay for Alzheimer's disease drug development in wild-type mice.

AIM: Transgenic mice that overexpress human amyloid precursor protein with Swedish or London (APPswe or APPlon) mutations have been widely used for preclinical Alzheimer's disease (AD) drug development. AD patients, however, rarely possess these mutations or overexpress APP. RESULTS: We developed a sensitive ELISA that specifically and accurately measures low levels of endogenous Aß40 in mouse plasma, brain and CSF. In wild-type mice treated with a bispecific anti-TfR/BACE1 antibody, significant Aß reductions were observed in the periphery and the brain. APPlon transgenic mice showed a slightly less reduction, whereas APPswe mice did not have any decrease. CONCLUSION: This sensitive and well-characterized mouse Aß40 assay enables the use of wild-type mice for preclinical PK/PD and efficacy studies of potential AD therapeutics.

Mathematical PKPD and safety model of bispecific TfR/BACE1 antibodies for the optimization of antibody uptake in brain.

Treatment of diseases of the central nervous system by monoclonal antibodies may be limited by the restricted uptake of antibodies across the blood-brain barrier (BBB). An antibody targeting transferrin receptor (TfR) has been shown to take advantage of the receptor-mediated transcytosis properties of TfR in order to cross the BBB in mice, with the uptake in the brain being dependent on the affinity to TfR. In the bispecific format with arms targeting both TfR and ß-secretase 1 (BACE1), altering the affinity to TfR has been shown to impact systemic exposure and safety profiles. In this work, a mathematical model incorporating pharmacokinetic/pharmacodynamic (PKPD) and safety profiles is developed for bispecific TfR/BACE1 antibodies with a range of affinities to TfR in order to guide candidate selection. The model captures the dependence of both systemic and brain exposure on TfR affinity and the subsequent impact on brain Aß40 lowering and circulating reticulocyte levels. Model simulations identify the optimal affinity for the TfR arm of the bispecific to maximize Aß reduction while maintaining reticulocyte levels. The model serves as a useful tool to prioritize and optimize preclinical studies and has been used to support the selection of additional candidates for further development.

Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates.

Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target ß-secretase (BACE1) can cross the BBB and reduce brain amyloid-ß (Aß) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain Aß in a TfR affinity-dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced Aß both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain.

A rare mutation in UNC5C predisposes to late-onset Alzheimer's disease and increases neuronal cell death.

We have identified a rare coding mutation, T835M (rs137875858), in the UNC5C netrin receptor gene that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer's disease and that was associated with disease across four large case-control cohorts (odds ratio = 2.15, Pmeta = 0.0095). T835M alters a conserved residue in the hinge region of UNC5C, and in vitro studies demonstrate that this mutation leads to increased cell death in human HEK293T cells and in rodent neurons. Furthermore, neurons expressing T835M UNC5C are more susceptible to cell death from multiple neurotoxic stimuli, including ß-amyloid (Aß), glutamate and staurosporine. On the basis of these data and the enriched hippocampal expression of UNC5C in the adult nervous system, we propose that one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer's disease is by increasing susceptibility to neuronal cell death, particularly in vulnerable regions of the Alzheimer's disease brain.

Molecular mechanisms of Alzheimer disease protection by the A673T allele of amyloid precursor protein.

Pathogenic mutations in the amyloid precursor protein (APP) gene have been described as causing early onset familial Alzheimer disease (AD). We recently identified a rare APP variant encoding an alanine-to-threonine substitution at residue 673 (A673T) that confers protection against development of AD (Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) Nature 488, 96-99). The Ala-673 residue lies within the ß-secretase recognition sequence and is part of the amyloid-ß (Aß) peptide cleavage product (position 2 of Aß). We previously demonstrated that the A673T substitution makes APP a less favorable substrate for cleavage by BACE1. In follow-up studies, we confirm that A673T APP shows reduced cleavage by BACE1 in transfected mouse primary neurons and in isogenic human induced pluripotent stem cell-derived neurons. Using a biochemical approach, we show that the A673T substitution modulates the catalytic turnover rate (V(max)) of APP by the BACE1 enzyme, without affecting the affinity (K(m)) of the APP substrate for BACE1. We also show a reduced level of Aß(1-42) aggregation with A2T Aß peptides, an observation not conserved in Aß(1-40) peptides. When combined in a ratio of 1:9 Aß(1-42)/Aß(1-40) to mimic physiologically relevant mixtures, A2T retains a trend toward slowed aggregation kinetics. Microglial uptake of the mutant Aß(1-42) peptides correlated with their aggregation level. Cytotoxicity of the mutant Aß peptides was not dramatically altered. Taken together, our findings demonstrate that A673T, a protective allele of APP, reproducibly reduces amyloidogenic processing of APP and also mildly decreases Aß aggregation. These effects could together have an additive or even synergistic impact on the risk of developing AD.

Mechanistic pharmacokinetic-pharmacodynamic modeling of BACE1 inhibition in monkeys: development of a predictive model for amyloid precursor protein processing.

This study was conducted to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of two novel inhibitors of ß-site amyloid precursor protein (APP)-cleaving enzyme (BACE1), GNE-629 [(4S,4a'S,10a'S)-2-amino-8'-(2-fluoropyridin-3-yl)-1-methyl-3',4',4a',10a'-tetrahydro-1'H-spiro[imidazole-4,10'-pyrano[4,3-b]chromen]-5(1H)-one] and GNE-892 [(R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one], and to develop a PK-PD model to predict in vivo effects based solely on in vitro activity and PK. GNE-629 and GNE-892 concentrations and PD biomarkers including amyloid ß (Aß) in the plasma and cerebrospinal fluid (CSF), and secreted APPß (sAPPß) and secreted APPα (sAPPα) in the CSF were measured after a single oral administration of GNE-629 (100 mg/kg) or GNE-892 (30 or 100 mg/kg) in cynomolgus monkeys. A mechanistic PK-PD model was developed to simultaneously characterize the plasma Aß and CSF Aß, sAPPα, and sAPPß using GNE-629 in vivo data. This model was used to predict the in vivo effects of GNE-892 after adjustments based on differences in in vitro cellular activity and PK. The PK-PD model estimated GNE-629 CSF and free plasma IC50 of 0.0033 µM and 0.065 µM, respectively. These differences in CSF and free plasma IC50 suggest that different mechanisms are involved in Aß formation in these two compartments. The predicted in vivo effects for GNE-892 using the PK-PD model were consistent with the observed data. In conclusion, a PK-PD model was developed to mechanistically describe the effects of BACE1 inhibition on Aß, sAPPß, and sAPPα in the CSF, and Aß in the plasma. This model can be used to prospectively predict in vivo effects of new BACE1 inhibitors using just their in vitro activity and PK data.

Spatially coordinated kinase signaling regulates local axon degeneration.

In addition to being a hallmark of neurodegenerative disease, axon degeneration is used during development of the nervous system to prune unwanted connections. In development, axon degeneration is tightly regulated both temporally and spatially. Here, we provide evidence that degeneration cues are transduced through various kinase pathways functioning in spatially distinct compartments to regulate axon degeneration. Intriguingly, glycogen synthase kinase-3 (GSK3) acts centrally, likely modulating gene expression in the cell body to regulate distally restricted axon degeneration. Through a combination of genetic and pharmacological manipulations, including the generation of an analog-sensitive kinase allele mutant mouse for GSK3ß, we show that the ß isoform of GSK3, not the α isoform, is essential for developmental axon pruning in vitro and in vivo. Additionally, we identify the dleu2/mir15a/16-1 cluster, previously characterized as a regulator of B-cell proliferation, and the transcription factor tbx6, as likely downstream effectors of GSK3ß in axon degeneration.

An effector-reduced anti-ß-amyloid (Aß) antibody with unique aß binding properties promotes neuroprotection and glial engulfment of Aß.

Passive immunization against ß-amyloid (Aß) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aß monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aß, protected against Aß1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aß oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aß, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aß antibody that takes advantage of a unique Aß binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

A mutation in APP protects against Alzheimer's disease and age-related cognitive decline.

The prevalence of dementia in the Western world in people over the age of 60 has been estimated to be greater than 5%, about two-thirds of which are due to Alzheimer's disease. The age-specific prevalence of Alzheimer's disease nearly doubles every 5 years after age 65, leading to a prevalence of greater than 25% in those over the age of 90 (ref. 3). Here, to search for low-frequency variants in the amyloid-ß precursor protein (APP) gene with a significant effect on the risk of Alzheimer's disease, we studied coding variants in APP in a set of whole-genome sequence data from 1,795 Icelanders. We found a coding mutation (A673T) in the APP gene that protects against Alzheimer's disease and cognitive decline in the elderly without Alzheimer's disease. This substitution is adjacent to the aspartyl protease ß-site in APP, and results in an approximately 40% reduction in the formation of amyloidogenic peptides in vitro. The strong protective effect of the A673T substitution against Alzheimer's disease provides proof of principle for the hypothesis that reducing the ß-cleavage of APP may protect against the disease. Furthermore, as the A673T allele also protects against cognitive decline in the elderly without Alzheimer's disease, the two may be mediated through the same or similar mechanisms.

A therapeutic antibody targeting BACE1 inhibits amyloid-ß production in vivo.

Reducing production of amyloid-ß (Aß) peptide by direct inhibition of the enzymes that process amyloid precursor protein (APP) is a central therapeutic strategy for treating Alzheimer's disease. However, small-molecule inhibitors of the ß-secretase (BACE1) and γ-secretase APP processing enzymes have shown a lack of target selectivity and poor penetrance of the blood-brain barrier (BBB). Here, we have developed a high-affinity, phage-derived human antibody that targets BACE1 (anti-BACE1) and is anti-amyloidogenic. Anti-BACE1 reduces endogenous BACE1 activity and Aß production in human cell lines expressing APP and in cultured primary neurons. Anti-BACE1 is highly selective and does not inhibit the related enzymes BACE2 or cathepsin D. Competitive binding assays and x-ray crystallography indicate that anti-BACE1 binds noncompetitively to an exosite on BACE1 and not to the catalytic site. Systemic dosing of mice and nonhuman primates with anti-BACE1 resulted in sustained reductions in peripheral Aß peptide concentrations. Anti-BACE1 also reduces central nervous system Aß concentrations in mouse and monkey, consistent with a measurable uptake of antibody across the BBB. Thus, BACE1 can be targeted in a highly selective manner through passive immunization with anti-BACE1, providing a potential approach for treating Alzheimer's disease. Nevertheless, therapeutic success with anti-BACE1 will depend on improving antibody uptake into the brain.

PirB is a functional receptor for myelin inhibitors of axonal regeneration.

A major barrier to regenerating axons after injury in the mammalian central nervous system is an unfavorable milieu. Three proteins found in myelin--Nogo, MAG, and OMgp--inhibit axon regeneration in vitro and bind to the glycosylphosphatidylinositol-anchored Nogo receptor (NgR). However, genetic deletion of NgR has only a modest disinhibitory effect, suggesting that other binding receptors for these molecules probably exist. With the use of expression cloning, we have found that paired immunoglobulin-like receptor B (PirB), which has been implicated in nervous system plasticity, is a high-affinity receptor for Nogo, MAG, and OMgp. Interfering with PirB activity, either with antibodies or genetically, partially rescues neurite inhibition by Nogo66, MAG, OMgp, and myelin in cultured neurons. Blocking both PirB and NgR activities leads to near-complete release from myelin inhibition. Our results implicate PirB in mediating regeneration block, identify PirB as a potential target for axon regeneration therapies, and provide an explanation for the similar enhancements of visual system plasticity in PirB and NgR knockout mice.

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.

Semaphorin 3F antagonizes neurotrophin-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase signaling: a mechanism for growth cone collapse.

Peripheral nerve growth is regulated by the coordinated action of numerous external stimuli, including positively acting neurotrophin-derived growth cues and restrictive semaphorin cues. Here, we show that Semaphorin 3F (Sema 3F) can antagonize nerve growth factor (NGF)-stimulated TrkA (tyrosine receptor kinase A) signaling in sympathetic neurons, thereby apparently contributing to growth cone collapse. Sema 3F suppressed NGF-induced activation of the phosphatidylinositol 3 (PI3)-kinase-Akt and MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase) pathways, both of which we show to be required to maintain growth cone structure. Sema 3F-induced growth cone collapse was partially reversed by sustained activation of the PI3-kinase and MEK pathways, which was achieved by overexpression of the Gab-1 (growth-associated binder 1) docking protein. These data indicate that a novel mechanism used by Sema 3F to collapse growth cones in sympathetic neurons is to dampen neurotrophin signaling, providing an intracellular mechanism for cross talk between positive and negative axon growth cues.