John W. Winslow

Neurotrophin-5: A novel neurotrophic factor that activates trk and trkB

In vertebrates, the formation and maintenance of neuronal connections are subject to regulation by multiple target-derived, diffusible (neurotrophic) factors. Here we describe the identification and characterization of a novel neurotrophic factor designated neurotrophin-5 (NT-5). NT-5 is structurally related to nerve growth factor and is expressed in embryonic as well as adult tissues. Recombinant NT-5 promotes the survival of peripheral sensory and sympathetic neurons and induces differentiation of the pheochromocytoma cell line PC12. NT-5 activates two trk-related tyrosine kinase receptors and shares these receptors with other neurotrophins. Activation of multiple receptors may permit a single neurotrophin to control target innervation by distinct neuronal populations. Receptor sharing could enable neurotrophic factors emanating from distinct targets to cooperate in regulating neurons with multiple connections.

BDNF mRNA is decreased in the hippocampus of individuals with Alzheimer's disease

In recent years, nerve growth factor (NGF) has gained attention as a potential therapeutic agent for Alzheimers disease (AD). To study the expression of NGF and its homologs, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3), postmortem samples of hippocampus from AD and control donors were examined by in situ hybridization. Hybridization signal for BDNF, but not NGF or NT-3, was decreased in samples of hippocampus from donors with AD. Decreased transcript abundance of BDNF mRNA in hippocampi of individuals with AD was verified by an RNAase protection assay. These results suggest the possibility that decreased expression of BDNF may contribute to the progression of cell death in AD.

Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes

Muscarinic acetylcholine receptors (mAChRs), like many other neurotransmitter and hormone receptors, transduce agonist signals by activating G proteins to regulate ion channel activity and the generation of second messengers via the phosphoinositide (PI) and adenylyl cyclase systems1,2. Human mAChRs are a family of at least four gene products which have distinct primary structures, ligand-binding properties and patterns of tissue-specific expression3. To examine the question of whether functional differences exist between multiple receptor subtypes, we have investigated the ability of each subtype to regulate PI hydrolysis and adenylyl cyclase when expressed individually in a cell lacking endogenous mAChRs. We show that the HM2 and HM3 mAChRs efficiently inhibit adenylyl cyclase activity but poorly activate PI hydrolysis. In contrast, the HM1 and HM4 mAChRs strongly activate PI hydrolysis, but do not inhibit adenylyl cyclase, and in fact can substantially elevate cAMP levels. Interestingly, the subtypes that we find to be functionally similar are also more similar in sequence. Our results indicate that the different receptor subtypes are functionally specialized.

Primary structure and biological activity of a novel human neurotrophic factor

During development, each tissue receives and maintains a number of specific neuronal projections that are adequate to sustain its function. The mechanism by which this intricate process occurs is not well understood, but it has been proposed that diffusible neurotrophic factors derived from the target tissue may be involved. Here we describe the identification of a novel human protein that is important for the growth, differentiation, and survival of primary sympathetic and placode-derived sensory neurons. This polypeptide, designated neuronotrophin-3, has a broad tissue distribution and is structurally related to both nerve growth factor and brain-derived neurotrophic factor. Its unique range of trophic and differentiation-inducing activities suggests that it is likely to play a wide role in defining the fate and function of nerve cells during development.

Evidence that brain-derived neurotrophic factor is a trophic factor for motor neurons in vivo

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) act upon populations of neurons that express specific receptors. The present study demonstrates that BDNF rescues motor neurons from degeneration and may also play a role in the normal physiology of these cells. BDNF is expressed in the local environment and in muscle targets of motor neurons; muscle expression is up-regulated by denervation. The alpha motor neurons express the gene encoding p145trkB, a receptor involved in BDNF signal transduction, whereas a subset of motor neurons express p75NGFR. BDNF is transported selectively to alpha motor neurons from skeletal muscles. Finally, BDNF prevents motor neuron death in the axotomized facial nucleus of the neonatal rat. The effects of BDNF on motor neurons raise the possibility that some neurotrophins may be useful in treating patients with motor neuropathies and amyotrophic lateral sclerosis.

Cloning of AL-1, a ligand for an Eph-related tyrosine kinase receptor involved in axon bundle formation

REK7 is an Eph-related tyrosine kinase receptor expressed exclusively in the nervous system, predominantly in hippocampus and cortex. A soluble REK7-IgG fusion protein, produced to analyze the biological role of REK7, prevents axon bundling in cocultures of cortical neurons with astrocytes, a model of late stage nervous system development and differentiation. Using REK7-IgG as an affinity reagent, we purified and cloned a novel REK7 ligand called AL-1, a GPI-linked protein homologous to other members of an emerging ligand family. Membrane attachment of AL-1 appears necessary for receptor activation, since REK7 on cortical neurons is efficiently activated by transfected cells expressing GPI-linked AL-1, but not by soluble AL-1. Consistent with this, soluble AL-1 blocks axon bundling. Our findings, together with the observation that both molecules are expressed in the brain, suggest a role in the formation of neuronal pathways, a crucial feature of nervous system development and regeneration.

Functionally distinct G proteins selectively couple different receptors to PI hydrolysis in the same cell

The number of G proteins identified by molecular cloning exceeds the number of known G protein functions. Here we show that a cell can possess multiple G proteins that carry out a similar function, the activation of phospholipase C, but couple selectively to different receptors, which are endogenous to the cell or introduced by DNA transfection. These G proteins (termed Gp) can be distinguished by their sensitivity to pertussis toxin. The assignment of a given Gp pathway to specific receptors is confirmed by the additivity relationships of the PI hydrolysis response mediated by the different receptors. Significantly different amounts of PI hydrolysis are activated through each Gp pathway, suggesting that Gp proteins also differ in their coupling to phospholipase C. These results indicate that distinct Gp pathways in a given cell exist to couple different receptors to PI hydrolysis selectively, and may specify the nature of the cellular response to different receptors by determining the magnitude of PI hydrolysis.

AL‐1‐induced Growth Cone Collapse of Rat Cortical Neurons is Correlated with REK7 Expression and Rearrangement of the Actin Cytoskeleton

Previous experiments identified AL‐1 as a glycosylphosphatidylinositol (GPI)‐linked ligand for the Eph‐related receptor, REK7, and showed that a REK7‐IgG fusion protein blocks axon bundling in co‐cultures of cortical neurons on astrocytes, suggesting a role for REK7 and AL‐1 in axon fasciculation. Subsequent identification of RAGS, the chick homologue of AL‐1, as a repellent axon guidance molecule in the developing chick visual system led to speculation that AL‐1, expressed on astrocytes, provides a repellent stimulus for cortical axons, inducing them to bundle as an avoidance mechanism. Using a growth cone collapse assay to test this hypothesis, we show that a soluble AL‐1‐IgG fusion protein is a potent collapsing factor for embryonic rat cortical neurons. The response is strongly correlated with REK7 expression, implicating REK7 as a receptor mediating AL‐1‐induced collapse. Morphological collapse is preceded by an AL‐1‐IgG‐induced reorganization of the actin cytoskeleton that resembles the effects of cytochalasin D. This suggests a pathway whereby REK7 activation by AL‐1 leads to perturbation of the actin cytoskeleton, possibly by an effect on actin polymerization, followed by growth cone collapse. We further show that AL‐1‐IgG causes collapse of rat hippocampal neurons and rat retinal ganglion cells. These data suggest a role for REK7 and AL‐1 in the patterning of axonal connections in the developing cortex, hippocampus and visual system.

K‐252b Selectively Potentiates Cellular Actions and trk Tyrosine Phosphorylation Mediated by Neurotrophin‐3

Abstract: K‐252b, a protein kinase inhibitor, has been shown earlier to inhibit nerve growth factor actions on cholinergic neurons of the basal forebrain. In the present study, K‐252b was found to prevent trophic actions of two other neurotrophins, brain‐derived neurotrophic factor, and neurotrophin‐3, on central cholinergic and dopaminergic neurons, peripheral sensory neurons, and PC 12 pheochromocytoma cells, when used at >2 μM concentration. Comparable actions of nonneurotrophin growth factors were not affected. Surprisingly, at 0.1‐100 nM, K‐252b selectively enhanced the trophic action of neurotrophin‐3 on central cholinergic neurons, peripheral sensory neurons, and PC 12 cells. In PC 12 cells, K‐252b potentiated the neurotrophin‐3‐induced tyrosine phosphorylation of trk, a protein kinase responsible for transmitting neurotrophin signals. Of the three structurally related nerve growth factor inhibitors, K‐252a, K‐252b, and staurosporine, only the first two also mediated neurotrophin‐3 potentiation. These findings indicate that K‐252b generally and selectively potentiates the neurotrophic action of neurotrophin‐3 and suggest that this action involves trk‐type neurotrophin receptors.

A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue.

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy.