Mahesh B. Lachyankar

Embryonic precursor cells that express Trk receptors: induction of different cell fates by NGF, BDNF, NT-3, and CNTF

Epidermal growth factor (EGF)-treated neurosphere cultures from embryonal striatum contain multipotential cells capable of neuronal, astrocytic, and oligodendroglial differentiation. In this study, we tested whether these neural precursor cells differentiate in the presence of neurotrophic factors. We first assayed neurosphere cells for expression of neurotrophin receptors. TrkA, TrkB, TrkC, and gp75 were detected by immunofluorescence microscopy in 60-80% of cells. In addition, the ciliary neurotrophic factor receptor alpha was expressed in 50-60% of cells. In the presence of the mitogen, EGF, treatment of stem cells with neurotrophic factors had no apparent effect. Removal of EGF from cells resulted in cessation of cell proliferation and pronounced astrocytic (glial fibrillary acidic protein+) differentiation. Neuronal (neurofilament+) and oligodendroglial (galactocerebroside+) cells appeared in cultures treated with neurotrophic factors. Nerve growth factor (NGF) resulted in bipolar neuronal cells, and brain-derived neurotrophic factor led to multipolar neuronal cells. Treatment with neurotrophin-3 or ciliary neurotrophic factor resulted in bipolar neuronal cells and oligodendrocytes. Neuronal differentiation in the presence of NGF was enhanced by extracellular matrix, and the resulting neuronal cells expressed choline acetyltransferase and, to a lesser degree, tyrosine hydroxylase. These studies demonstrate that neurotrophic factors influence the fates of these multipotential precursor cells. Indeed, the true utility of multipotential precursor cells is the production of different types of cells in different situations. Local cues, such as neurotrophic factors and extracellular matrix, may regulate production of different types of neural cells during development or in response to other stimuli, such as injury.

Novel functional interactions between Trk kinase and p75 neurotrophin receptor in neuroblastoma cells.

To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET‐R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR), and the TrkA transmembrane and intracellular domains. EGF activated the ET‐R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation but enhanced the EGF‐induced response, leading to differentiation of almost all cells. A mutated NGF, 3T‐NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF‐induced differentiation required at least nanomolar concentrations of NGF, consistent with the low‐affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhanced apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF seems to enhance survival through the phosphatidylinositol‐3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET‐R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase‐3. An anti‐p75 antibody blocked the survival effect of NGF. Brain‐derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.

Retrograde axonal transport and lesion-induced upregulation of the TrkA high-affinity NGF receptor

Long-term physiological responses of nerve growth factor (NGF) and other neurotrophins require gene regulation and likely depend on retrograde axonal transport of NGF or a signaling molecule activated by ligand-receptor interaction. The low-affinity neurotrophin receptor p75LANR is retrogradely transported, but this receptor is not sufficient for NGF-dependent cell survival or differentiation. In this study we examined the distribution and transport of the TrkA NGF receptor using two anti-peptide polyclonal antibodies and a monoclonal antibody, all of which are TrkA specific. We find that (1) in the adult rat brain TrkA-like immunoreactivity is similar with all antibodies in striatal and basal forebrain neurons, (2) TrkA is upregulated in neuronal and nonneuronal cells near the sites of injury, and (3) TrkA immunoreactivity builds up within the proximal and distal segments of transected fimbrial axons, which is consistent with its transport in the anterograde and retrograde directions. Thus, TrkA may itself be, or be a component of, the neurotrophic intraaxonal messenger by which NGF regulates gene expression in sensitive neurons.

PTEN: A Newly Identified Regulator of Neuronal Differentiation

Even though phosphorylation of phosphatidylinositols by phosphoinositide 3-kinase has an important and pervasive role in the nervous system, little is known about the phosphatases that reverse this reaction. Recently, such a phosphatase, PTEN, was cloned as a tumor suppressor for gliomas. We now know that PTEN is a tumor suppressor for many tumor types and is a phosphatidylinositol phosphatase specific for the 3-position of the inositol ring. PTEN is expressed in most, if not all, neurons and is localized in the nucleus and cytoplasm. PTEN is not evident in neural processes or synapses. PTEN is induced during neuronal differentiation and is required for survival of differentiating neuronal cells. In summary, PTEN is a regulatory molecule with multiple functions at multiple subcellular sites. Further studies are required to determine which downstream pathways are regulated by PTEN, by which mechanisms PTEN activity is regulated, which stimuli regulate PTEN activity, and why a molecule that inhibits several survival pathways is induced during neurogenesis.

Chapter 2 Axonal transport of the trkA high-affinity NGF receptor

Publisher Summary Nerve growth factor (NGF) is the best characterized neurotrophic factors (NTF). NGF is a 26,000-Da polypeptide that modulates differentiation of rat pheochromocytoma PC12 cells to sympathetic neuron-like cells and can act as a chemoattractant. It is a survival factor for sympathetic and sensory neurons. NGF microinjected into the cytoplasm or nucleus does not induce a biological response, and microinjection of anti-NGF antibodies into the nucleus does not block the response to exogenous NGF. The low-affinity NGF receptor (NGFR) also is transported to the cell body but does not appear to allow neuronal survival in the absence of trkA. The anti-trkA antibodies were used to test for axonal transport of trkA. The fimbria–fornix was bilaterally transected at the level at which it joins the rostral pole of the hippocampus. There seems no possibility that the trkA immunoreactivity results from glial cells. The build-up of trkA immunoreactivity indicates that trkA is transported in both the retrograde and anterograde directions.