Graham L. Barrett

The p75 neurotrophin receptor and neuronal apoptosis.

Although evidence continues to accumulate for the apoptosis-inducing role of the p75 neurotrophin receptor, several outstanding questions remain. One of these concerns the signal transduction pathway of p75, which continues to be elusive. The evidence for the roles of ceramide, c-jun kinase and NF-kappaB is discussed: none of these are able to account satisfactorily for p75 death signalling. Negative modulation of Trk signalling by p75 could account for part of the pro-apoptotic effect, but is unlikely to be a major component. Although recent evidence indicates that the juxtamembrane region is critical for causing cell death, p75 has a well-conserved death domain. This may be important for functions other than killing. In glial cells and some neurons that express p75 but not TrkA, p75 causes cell death in response to nerve growth factor (NGF) binding. In sensory neurons and PC12 cells, p75 appears to signal constitutively. In cholinergic forebrain neurons, p75 expression leads to atrophy and downregulation of cholinergic markers, rather than cell death. The major challenges in p75 research are to define its signalling pathways, and particularly the intracellular proteins with which it interacts. Another major challenge is to develop a model that reconciles the different facets of p75, such as its ability in some situations to assist TrkA to rescue NGF-dependent neurons, but to stimulate apoptosis in others.

Effect of I.C.V. injection of AT4 receptor ligands, NLE1-angiotensin IV and LVV-hemorphin 7, on spatial learning in rats

Central administration of angiotensin IV (Ang IV) or its analogues enhance performance of rats in passive avoidance and spatial memory paradigms. The purpose of this study was to examine the effect of a single bolus injection of two distinct AT4 ligands, Nle1-Ang IV or LVV-haemorphin-7, on spatial learning in the Barnes circular maze. Mean number of days for rats treated with either Nle1-Ang IV or LVV-haemorphin-7 to achieve learner criterion is significantly reduced compared with controls (P < 0.001 and P < 0.05 respectively). This is due to enhanced ability of the peptide-treated rats to adopt a spatial strategy for finding the escape hatch. In all three measures of learning performance, (1) the number of errors made, (2) the distance travelled and (3) the latency in finding the escape hatch, rats treated with either 100 pmol or 1 nmol of Nle1-Ang IV or 100 pmol LVV-haemorphin-7 performed significantly better than the control groups. As early as the first day of testing, the rats treated with the lower dose of Nle1-Ang IV or LVV-haemorphin-7 made fewer errors (P < 0.01 and P < 0.05 respectively) and travelled shorter distances (P < 0.05 for both groups) than the control animals. The enhanced spatial learning induced by Nle1-Ang IV (100 pmol) was attenuated by the co-administration of the AT4 receptor antagonist, divalinal-Ang IV (10 nmol). Thus, administration of AT4 ligands results in an immediate potentiation of learning, which may be associated with facilitation of synaptic transmission and/or enhancement of acetylcholine release.

Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice

The p75 low affinity neurotrophin receptor (p75) can induce apoptosis in various neuronal and glial cell types. Because p75 is expressed in the cholinergic neurons of the basal forebrain, p75 knockout mice may be expected to show an increased number of neurons in this region. Previous studies, however, have produced conflicting results, suggesting that genetic background and choice of control mice are critical. To try to clarify the conflicting results from previous reports, we undertook a further study of the basal forebrain in p75 knockout mice, paying particular attention to the use of genetically valid controls. The genetic backgrounds of p75 knockout and control mice used in this study were identical at 95% of loci. There was a small decrease in the number of cholinergic basal forebrain neurons in p75 knockout mice at four months of age compared with controls. This difference was no longer apparent at 15 months due to a reduction in numbers in control mice between the ages of 4 and 15 months. Cholinergic cell size in the basal forebrain was markedly increased in p75 knockout mice compared with controls. Spatial learning performance was consistently better in p75 knockout mice than in controls, and did not show any deterioration with age. The results indicate that p75 exerts a negative influence on the size of cholinergic forebrain neurons, but little effect on neuronal numbers. The markedly better spatial learning suggests that the function, as well as the size, of cholinergic neurons is negatively modulated by p75.

Reducing p75 nerve growth factor receptor levels using antisense oligonucleotides prevents the loss of axotomized sensory neurons in the dorsal root ganglia of newborn rats

The low‐affinity p75 receptor for nerve growth factor (p75NGFR) has been implicated in mediating neuronal cell death in vitro. A recent in vitro study from our laboratory showed that the death of sensory neurons can be prevented by reducing the levels of p75NGFR with antisense oligonucleotides. To determine if p75NGFR also functions as a death signal in vivo, we have attempted to reduce its expression in peripheral sensory neurons by applying antisense oligonucleotides to the proximal end of the transected sciatic or median and ulnar nerves. We report here that antisense oligonucleotides, when applied to the proximal stump of a transected peripheral nerve, are retrogradely transported and effectively reduce p75NGFR protein levels in sensory neurons located in the dorsal root ganglia. Furthermore, treatment of the proximal nerve stump with antisense p75NGFR oligonucleotides significantly reduced the loss of these axotomized sensory neurons. These findings further support the view that p75NGFR is a death signaling molecule and that it signals death in axotomized neurons in the neonatal sensory nervous system.

Nerve growth factor determines survival and death of PC12 cells by regulation of the bcl-x, bax, and caspase-3 genes.

Abstract: We investigated the effects of nerve growth factor (NGF) and NGF withdrawal on expression of members of the bcl‐2 family of genes and caspase‐3 in PC12 cells. NGF regulated several members of the bcl‐2 family and caspase‐3 in a manner consistent with its effect on apoptosis in PC12 cells. Levels of bcl‐xl, bcl‐xs, and caspase‐3 mRNAs were increased by NGF treatment. The increases in caspase‐3 and bcl‐xs levels should have disposed the cells toward apoptosis but were opposed by the simultaneous increase in bcl‐xl level. NGF withdrawal resulted in abrupt down‐regulation of bcl‐xl and up‐regulation of bax, favoring apoptosis. Forced expression of bcl‐xl after NGF withdrawal was sufficient to prevent cell death. Cell death was rapid when NGF was withdrawn after 5 days of treatment but relatively slow when NGF was withdrawn after only 1 or 2 days of treatment. This was consistent with the reduced accumulation of caspase‐3 mRNA with shorter NGF treatments. These results indicate that Bcl‐xl, Bcl‐xs, Bax, and caspase‐3 are important regulators of apoptosis in PC12 cells. Furthermore, regulation of their mRNA levels is implicated in the signal transduction of NGF.

Ceramide can induce cell death in sensory neurons, whereas ceramide analogues and sphingosine promote survival

Ceramide has been shown to induce apoptosis in leukaemic cells and some other cell types, but there are few data on its role in neuronal cells. We investigated the effect of ceramide and its analogues in cultured sensory neurons from neonatal mice. These cells undergo apoptosis in the absence of neurotrophins. Treatment with ceramide or its analogues increased survival, both in the presence and absence of NGF. Sphingosine treatment also increased survival. In the presence of the ceramidase inhibitor N‐oleoyl ethanolamine, which blocks conversion of ceramide to sphingosine, the addition of natural ceramide‐induced cell death, even in the presence of nerve growth factor (NGF). N‐oleoyl ethanolamine did not cause cell death by itself. N‐oleoyl ethanolamine did not alter the response to ceramide analogues, indicating that they were not ceramidase substrates. These results indicate that, in sensory neurons, exogenous ceramide is converted to sphingosine, which promotes cell survival. When conversion is blocked by ceramidase inhibition, exogenous ceramide causes cell death, presumably due to the high levels of ceramide itself. The ceramide analogues all mimicked the effect of sphingosine rather than ceramide, casting serious doubt on their validity as models of ceramide action. Ceramide analogues could prevent neuronal death even in the combined presence of N‐oleoyl ethanolamine and natural ceramide. Surprisingly, dihydro C2‐ceramide, which is frequently used as a control for C2‐ceramide, had the same effect as ceramide analogues. J. Neurosci. Res. 54:206–213, 1998.

The p75 neurotrophin receptor enhances TrkA signalling by binding to Shc and augmenting its phosphorylation

Nerve growth factor (NGF) is an important neuronal survival factor, especially during development. Optimal sensitivity of the survival response to NGF requires the presence of TrkA and the p75 neurotrophin receptor, p75NTR. Signalling pathways used by TrkA are well established, but the mechanisms by which p75NTR enhances NGF signalling remain far from clear. A prevalent view is that p75NTR and TrkA combine to form a high‐affinity receptor, but definitive evidence for this is still lacking. We therefore investigated the possibility that p75NTR and TrkA interact via their signal transduction pathways. Using antisense techniques to down‐regulate p75NTR and TrkA, we found that p75NTR specifically enhanced phosphorylation of the 46‐ and 52‐kDa isoforms of Shc during nerve growth factor‐induced TrkA activation. p75NTR did not enhance tyrosine phosphorylation of other TrkA substrates. Serine phosphorylation of Akt, downstream of Shc activation, was also p75NTR‐dependent. We consistently detected co‐immunoprecipitation of p75NTR and Shc. These data indicate that p75NTR interacts with Shc physically, via a binding interaction, and functionally, by assisting its phosphorylation. Whilst providing evidence that p75NTR augments TrkA signal transduction, these results do not preclude the presence of a p75NTR‐TrkA high‐affinity NGF receptor.

BDNF Exerts Contrasting Effects on Peripheral Myelination of NGF-Dependent and BDNF-Dependent DRG Neurons

Although brain-derived neurotrophic factor (BDNF) has been shown to promote peripheral myelination during development and remyelination after injury, the precise mechanisms mediating this effect remain unknown. Here, we determine that BDNF promotes myelination of nerve growth factor-dependent neurons, an effect dependent on neuronal expression of the p75 neurotrophin receptor, whereas BDNF inhibits myelination of BDNF-dependent neurons via the full-length TrkB receptor. Thus, BDNF exerts contrasting effects on Schwann cell myelination, depending on the complement of BDNF receptors that are expressed by different subpopulations of dorsal root ganglion neurons. These results demonstrate that BDNF exerts contrasting modulatory roles in peripheral nervous system myelination, and that its mechanism of action is acutely regulated and specifically targeted to neurons.

Effects of estrogen on basal forebrain cholinergic neurons and spatial learning.

Estrogen receptors are expressed in several areas of the brain associated with cognition, including the basal forebrain cholinergic nuclei, and numerous reports have described improvements in memory in response to estrogen supplementation. The relationship between estrogens effects on the basal cholinergic system and improvements in cognitive function, however, are obscure. We therefore undertook a study to determine the effects of estrogen on several parameters of the cholinergic system in ovariectomized rats and measured the concomitant effects on performance in the Barnes maze, a test of spatial memory. Six weeks of estradiol treatment caused an increase in choline acetyltransferase activity throughout the projection fields of the basal forebrain, including the hippocampal formation (14%), olfactory bulb (30%), and cerebral cortex (35%). Estrogen treatment also caused an increase in cell soma size of cholinergic neurons in the horizontal diagonal limb of the band of Broca and in the basal nucleus of Meynert. There was no change in the number of neurons positive for p75NTR, nor in the level of p75NTR expression per neuron. Barnes maze performance was markedly improved after estradiol treatment, reinforcing the view that estrogen has beneficial cognitive effects, particularly on spatial memory. The beneficial cognitive effect was likely mediated in part by stimulation of the basal forebrain cholinergic system, especially in its neocortical projection, but was not associated with changes in the level of p75NTR expression.

Treatment of experimental autoimmune encephalomyelitis with antisense oligonucleotides against the low affinity neurotrophin receptor

Upregulated expression of the low‐affinity neurotrophin receptor (p75) in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE) has recently been demonstrated. To investigate whether p75 plays a role in disease pathogenesis, we adopted a gene therapy approach, utilizing antisense oligonucleotides to downregulate p75 expression during EAE. Phosphorothioate antisense oligonucleotides (AS), nonsense oligonucleotides (NS) or phosphate buffered saline (PBS) were injected daily for 18 days after immunization of SJL/J (H‐2s)‐mice with myelin proteolipid protein (PLP) peptide 139‐151. In the AS group, there was a statistically significant reduction in both the mean maximal disease score (1.85 in the AS, 2.94 in the NS and 2.75 in the PBS‐groups, respectively, P < 0.025) and in the cumulative disease incidence (≈60% in the AS group and ≈90% in the control groups). Histological and immunohistochemical analysis showed reduced inflammation and demyelination, as well as reduced p75 expression at the blood‐brain barrier (BBB) in the AS‐treated mice in comparison with both control groups. There was no difference, however, in p75 expression on neural cells within the CNS between the three groups of mice. We conclude that p75 could play a proactive role in the pathogenesis of EAE and may exert its effect at the level of the BBB. J. Neurosci. Res. 59:712–721, 2000