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During the course of normal aging, certain populations of nerve growth factor (NGF)-responsive neurons become selectively vulnerable to cell death. Studies using dissociated neurons isolated from neonates have shown that c-Jun N-terminal kinases (JNKs) are important in regulating the survival and neurite outgrowth of NGF-responsive sympathetic neurons. Unlike neonatal neurons, adult sympathetic neurons are not dependent on NGF for their survival. Moreover, the NGF precursor, proNGF, is neurotoxic for aging but not young adult NGF-responsive neurons. Because of these age-related differences, the effects of JNK inhibition on the survival and growth of sympathetic neurons isolated from aged mice were studied. Aged neurons, as well as glia, were found to be dependent on JNK for their growth but not their survival. Conversely, proNGF neurotoxicity was JNK-dependent and mediated by the p75-interacting protein NRAGE, whereas neurite outgrowth was independent of NRAGE. These results have implications for the potential use of JNK inhibitors as therapies for ameliorating age-related neurodegenerative disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We have examined the hypothesis that differences in nerve growth factor (NGF) uptake and transport determine vulnerability to age-related neurodegeneration. Neurons projecting to cerebral blood vessels (CV) in aged rats are more vulnerable to age-related degeneration than those projecting to the iris. Uptake of NGF was therefore examined in sympathetic neurons projecting from the superior cervical ganglion (SCG) to CV and iris in young and old rats by treating the peripheral processes of these neurons with different doses of I125-NGF. Total uptake of I125-NGF was reduced in old CV-projecting, but not iris-projecting, neurons. Numbers of radiolabelled neurons projecting to each target were counted in sectioned ganglia. The data showed age-related reductions in numbers of labelled neurons projecting to CV, but no change in numbers of neurons projecting to the iris. Calculation of uptake of I125-NGF per neuron unexpectedly showed no major age-related differences in either of the two neuron populations. However, uptake per neuron was considerably lower for young and old CV-projecting, compared to iris-projecting, SCG neurons. We hypothesized that variations in NGF uptake might affect neuronal survival in old age. Counts of SCG neurons using a physical disector following retrograde tracing with Fluorogold confirmed the selective vulnerability of CV-projecting neurons by showing a significant 37% loss of these neurons in the period between 15 and 24 months. In contrast, there was no significant loss of iris-projecting neurons. We conclude that vulnerability to, or protection from, age-related neurodegeneration and neuronal cell death are associated with life-long low, or high, levels of NGF uptake, respectively.

We have developed and tested the biological activity and specificity of a novel fluorescent dextran-Texas Red-nerve growth factor (DTR-NGF) conjugate. DTR-NGF was found to promote survival and neurite outgrowth in cultured dissociated sympathetic neurons similarly to native NGF. The conjugate was taken up and transported retrogradely by terminal sympathetic nerves innervating the iris to neurons in the ipsilateral superior cervical ganglion (SCG) of young adult rats. Uptake and transport was assessed by counting numbers of labelled neurons and by measuring intensity of neuronal labelling using confocal microscopy and image analysis. DTR-NGF labelling in SCG neurons was shown to be dose-dependent with an EC(50) of 75 ng. Similar concentrations of unconjugated DTR resulted in no neuronal labelling. DTR-NGF uptake was competed off using a 50-fold excess of native NGF, resulting in a 73% reduction in numbers of labelled neurons. Pretreatment of nerve terminals with function-blocking antibodies against the low (p75) and high (TrkA) affinity NGF receptors resulted in a large (85-93%) reduction in numbers of DTR-NGF labelled neurons. Anti-p75 and anti-TrkA antibodies had comparable effects which were concentration-dependent. These findings indicate that both receptors are required for uptake of NGF in adult rat sympathetic neurons. In particular, the results provide strong evidence that the p75 receptor plays a more active role in transducing the NGF signal than has been proposed.

The factors that determine the ability of some, but not all neurons, to sustain their axonal projections during aging remain largely unknown. Because sympathetic neurons remain responsive to nerve growth factor (NGF) in old age, it has been proposed that the selective decrease observed in the sympathetic innervation to some targets in aged rats may be the result of a deficit in target-derived NGF. In this study we utilized two different techniques to demonstrate decreased target innervation by sympathetic fibers in the aged rat pineal gland, which is an appropriate and relevant model for examining mechanisms of neuron-target interactions in aging. Tyrosine hydroxylase immunoreactive profiles were quantified in pineal glands of young and aged male Sprague-Dawley rats. The density of tyrosine hydroxylase-immunoreactive fibers was 30% lower in aged pineals, although the remaining fibers contained 20% more tyrosine hydroxylase-immunoreactivity. Othograde tracing of the pineal sympathetic innervation using biotinylated dextran revealed that average axon length, varicosity numbers, branch point numbers, and numbers of terminations were all decreased by approximately 50% in aged tissues, indicating possible functional deficits. These findings suggest that whole branches, along with their associated varicosities were lost in old age. A sensitive quantitative ribonuclease protection assay and a two-site ELISA assay were used to examine whether reduced NGF availability might correlate with sympathetic nerve atrophy. No significant differences were detected in either NGF mRNA or NGF protein levels when comparing young and aged pineal glands, suggesting that atrophy in aged sympathetic neurons is not causally related to reduced availability of NGF at the target. Our results indicate that mechanisms other than NGF expression need to be explored in order to explain the age-related axonal regression observed in this target.

Sympathetic nerves to the enlarged fetus-containing region of the uterus undergo degenerative changes during late pregnancy and show slow regrowth after parturition. It is not known whether this unusual response of sympathetic nerves to smooth muscle hypertrophy is due to the sensitivity of short adrenergic neurons to hormonal changes, or whether the nerves respond to changes in the neurotrophic capacity of the target. We have investigated this question using in oculo transplantation. Small pieces of myometrium from the uterine horn of virgin guinea pigs, or from the region previously occupied by the placenta and fetus in postpartum guinea pigs, were transplanted into the anterior eye chamber. After 3 wk in oculo, the pattern of reinnervation of the transplants was assessed on whole mount stretch preparations stained for tyrosine hydroxylase. The histology of the transplants was examined in toluidine blue-stained semithin sections. Myometrial transplants from virgin donors and uterine artery transplants from both virgin and postpartum donors became organotypically reinnervated by sympathetic fibres from the host iris. In contrast, sympathetic nerves did not reinnervate myometrial transplants from postpartum donors, although they approached the transplants and became distributed in the surrounding connective tissue. All transplanted tissues showed a normal histological appearance. Both the myometrium and uterine artery from postpartum donors retained a hypertrophic appearance after 3 wk in oculo. We interpret these results to indicate that the degeneration of sympathetic nerves in late pregnancy, as well as their slow regrowth to the uterus after delivery, may be due to changes in uterine smooth muscle rather than a particular sensitivity of short adrenergic neurons to hormonal changes.

Contact with sweat gland acini causes sympathetic neurons to switch from a catecholaminergic to a cholinergic phenotype during development and following experimental manipulations. Substantial reductions of cholinergic innervation have been shown in the sweat glands of ageing rats and humans. Using in oculo transplantation, we have now studied whether sweat gland target tissues retain the capacity to regulate changes in the phenotype of sympathetic neurons observed in maturity and old age, including a switch from catecholaminergic to cholinergic characters. Markers have been used which indicate changes in nerve fibre morphology (the pan-neuronal marker, PGP9.5) as well as neurotransmitter expression (acetylcholinesterase (AChE), vasocative intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). Sweat glands from young and old donor rats became reinnervated by an organotypic pattern of cholinergic host nerves. Surgical sympathectomy demonstrated that these cholinergic nerve fibres originate from sympathetic neurons of the host superior cervical ganglion (SCG). Retrograde tracing combined with staining for VIP (a marker associated with cholinergic phenotype in neurons supplying sweat glands) showed that SCG neurons projecting to irises with sweat gland implants may be induced to express VIP. We hypothesise that these neurons have been switched from their normal catecholaminergic phenotype to a cholinergic one by contact with the sweat gland implants. Transplants from old donors attracted a density of reinnervation by young host nerves which was appropriate to the age of the donor, thus old sweat glands received a significantly reduced density of innervation compared to young glands. Despite the reduced density of innervation, there was no obvious difference in the ability of young and old implants to induce the switch to a cholinergic phenotype, suggesting that different mechanisms regulate nerve growth and neurotransmitter phenotype.

Previous studies in our laboratory using a transplantation model have shown that target tissues of some autonomic neurons, including cerebral blood vessels, exert a controlling influence on nerve fibre loss in old age. The present study was undertaken in order to discover whether the influence of targets extends to controlling age changes in specific populations of nerves. In old rats, we have demonstrated a significant decrease of approximately 50% in the sympathetic innervation of middle cerebral arteries, using tyrosine hydroxylase-like immunoreactivity. Following transplantation, tyrosine hydroxylase-like immunoreactive nerve density on both young and old implanted middle cerebral arteries mirrored the nerve densities seen in normal, non-transplanted vessels. Furthermore, implanted tissue from old donors became reinnervated with a nerve density approximately 50% less than that of young implanted vessels. Treatment of transplants with nerve growth factor, however, was able to reverse these age changes and restore the sympathetic innervation of aged middle cerebral arteries to levels above those seen in young middle cerebral arteries. These results suggest that the pattern and density of sympathetic innervation that the middle cerebral artery receives is determined by the target rather than by the neurons supplying the tissue. The ability of nerve growth factor to induce regrowth in sympathetic neurons innervating ageing target tissues implies that age-related neuronal atrophy may be due to reduced synthesis or availability of target-derived neurotrophic factors.