Eugene M. Johnson

Expression of Neurturin, GDNF, and GDNF Family-Receptor mRNA in the Developing and Mature Mouse

The GDNF family of neurotrophic factors currently has four members: neurturin (NRTN), glial cell line-derived neurotrophic factor (GDNF), persephin, and artemin. These proteins are potent survival factors for several populations of central and peripheral neurons. The receptors for these factors are complexes that include the Ret tyrosine kinase receptor and a GPI-linked, ligand-binding component called GDNF family receptor alpha 1-4 (GFRalpha1-4). We have used in situ hybridization to study the mRNA expression of NRTN, GDNF, Ret, GFRalpha1, and GFRalpha2 during embryonic development and in the adult mouse. GDNF receptors were prominently expressed during embryonic development in the nervous system, the urogenital system, the digestive system, the respiratory system, and in developing skin, bone, muscle, and endocrine glands. In some regions, incomplete receptor complexes were expressed suggesting that other, as yet unidentified, receptor components exist or that receptor complexes are formed in trans. NRTN and GDNF were expressed in many trigeminal targets during embryonic development including the nasal epithelium, the teeth, and the whisker follicles. NRTN and GDNF were also expressed in the developing limbs and urogenital system. In the embryo, GDNF factors and receptors were expressed at several sites of mesenchyme/epithelial induction, including the kidney, tooth, and submandibular gland. This expression pattern is consistent with the possibility that the GDNF factors function in inductive processes during embryonic development and with the recently discovered role of NRTN as a necessary trophic factor for the development of some parasympathetic neurons. In the mature animal, receptor expression was more limited than in the embryo. In the adult mouse, NRTN was most prominently expressed in the gut, prostate testicle, and oviduct; GDNF was most prominently expressed in the ovary.

Effects of long-term nerve growth factor deprivation on the nervous system of the adult rat: An experimental autoimmune approach

Adult rats immunized with 2.5S mouse nerve growth factor (NGF) produced antibodies which cross-reacted with rat NGF. By the criterion of ammonium sulfate precipitation followed by Sephadex G-200 chromatography, all serum anti-NGF activity was retained in the IgG fraction. Animals which developed and maintained chronic (5-6 months) serum titers of anti-NGF demonstrated a pronounced biochemical and morphological atrophy of the superior cervical ganglion which was accompanied by a 35-40% reduction in neuronal number. Norepinephrine was reduced by approximately 90% in heart and brown fat. The extent of biochemical atrophy correlated well with serum titers of anti-NGF. No effects were observed on the short adrenergic neurons of the vas deferens, adrenal medullary chromaffin cells, central adrenergic neurons, of peripheral sensory neurons. These results strongly suggest that mature peripheral sympathetic neurons remain dependent on NGF for survival as well as for maintenance.

Characterization of the retrograde transport of nerve growth factor (NGF) using high specific activity [125I]NGF

The process of the retrograde transport of nerve growth factor (NGF) has been recharacterized using a high specific activity preparation of[125I]NGF. Most of the general conclusions reached in the previous studies of Hendry, Thoenen and co-workers have been confirmed. However, significant quantitative differences were noted. Intraocular (anterior eye chamber) administration of[125I]NGF (less than 10 ng) resulted in accumulation in the superior cervical ganglia beginning at about 4 h. The ratio of radioactivity in the ipsilateral contralateral ganglia was 15--30:1. Maximal accumulation was seen at about 12h in the hamster and 16 h in rats. This pattern was quite different from that seen in other tissues. The uptake system from the eye of the rat was saturable (half-maximal at 15 ng) with maximal accumulation of 35--40 pg/ganglion. Systemic administration of[125I]NGF (200 ng) to adult rats resulted in no accumulation in SGG or celiac ganglion prior to 3 h, with subsequent rapid accumulation by 6 h and a rapid fall in radioactivity after 12 h. A similar time course was seen in 5-day-old rats, although the time curve was shifted slightly toward shorter time. The radioactivity in ganglia co-migrated with native NGF by SDS gell electrophoresis. Cytochrome c of comparable specific activity was not transported, and NGF did not stimulate the uptake and transport of cytochrome c. The retrograde transport of[125I]NGF was inhibited by the co-administration of biologically active, but not inactive, oxidized derivatives of NGF. By any route of administration, a significant percentage of the transported[125I]NGF was found in a purified nuclear fraction of the ganglia. Coupled with previous observations of specific nuclear NGF receptors in embryonic chick and sympathetic ganglia, this suggests that, after internalization and retrograde transport, NGF may directly act on the nucleus to produce at least some of its effects on the responsive cell.

Guanethidine-Induced Destruction of Sympathetic Neurons

Publisher Summary This chapter describes several aspects of the phenomenon of guanethidine-induced destruction of sympathetic neurons and emphasizes the discovery of guanethidine as a means of producing sympathectomy, structure–activity relationships of guanethidine and its analogs for neuronal destruction, the mechanism by which guanethidine destroys the sympathetic nervous system in the rat, and the utility of guanethidine as a means of producing animals with permanent peripheral sympathectomy. Guanethidine can be considered the prototypical adrenergic neuron blocking agent and act to dissociate the action potential from subsequent release of norepinephrine in sympathetic neurons. Results show that guanethidine, particularly when administered to neonates, produces a permanent, complete, and highly specific sympathectomy; unfortunately, guanethidine only produces sympathectomy in the rat, limiting its utility as an experimental tool. Guanethidine-induced destruction of sympathetic neurons offers many advantages as an experimental model of autoimmune-mediated destruction of the nervous system and/or drug-induced autoimmunity. Eventual elucidation of the antigen being attacked and the mechanism by which nerve growth factor prevents the destruction will provide insights into other autoimmune disorders.

Destruction of the sympathetic nervous system in neonatal rats and hamsters by vinblastine: prevention by concomitant administration of nerve growth factor

Abstract If the primary mechanism by which nerve growth factor (NGF) gains access to the cell body is a specific uptake and retrograde transport from the periphery, then an inhibitor of axoplasmic transport would be expected to produce cell death. Such an inhibitor was administered to neonatal and adult rats and hamsters. A single injection of vinblastine (0.25–0.5 mg/kg s.c.) to 2-day-old rats produced massive cell death in the superior cervical ganglia (SCG). The levels of tyrosine hydroxylase in the SCG were reduced 80% at doses of 0.4 mg/kg, a dose which was uniformly fatal. Doses of 0.25 mg/kg at two days of age resulted in a 50% mortality rate and the survivors showed a permanent, partial sympathectomy. The adrenal medulla and sensory neurons did not appear to be affected. Administration of a single injection of vinblastine at maximally tolerated doses did not destroy sympathetic neurons in animals 14 days of age or older. Chronic administration of vinblastine (0.5 mg/kg every other day for two weeks) to adult rats did not destroy sympathetic neurons. Similar results were obtained in hamsters. Concomitant administration of NGF appeared to completely prevent the cytotoxic effects of vinblastine assessed by both morphological and enzymatic criteria. Several explanations are discussed which might explain these results. The similarities in the effects of vinblastine, other drugs, and axonal crush on sympathetic neurons are noted. It is proposed that all drugs which destroy sympathetic neurons (guanethidine, 6-hydroxydopamine, vinblastine) as well as anti-NGF and axonal ligation destroy the neurons by depriving the cell body of NGF from the periphery.

Properly scaled and targeted AAV2-NRTN (neurturin) to the substantia nigra is safe, effective and causes no weight loss: support for nigral targeting in Parkinson's disease.

Recent analyses of autopsied brains from subjects previously administered AAV2-neurturin (NRTN) gene transfer argues that optimizing the effects of neurotrophic factors in Parkinsons disease (PD) likely requires delivery to both the degenerating cell bodies (in substantia nigra) and their terminals (in striatum). Prior to implementing this novel dosing paradigm in humans, we conducted eight nonclinical experiments with three general objectives: (1) evaluate the feasibility, safety and effectiveness of targeting the substantia nigra (SN) with AAV2-NRTN, (2) better understand and appraise recent warnings of serious weight loss that might occur with targeting the SN with neurotrophic factors, and (3) define an appropriate dose of AAV2-NRTN that should safely and effectively cover the SN in PD patients. Toward these ends, we first determined SN volume for rats, monkeys and humans, and employed these values to calculate comparable dose equivalents for each species by scaling each dose, based on relative SN volume. Using this information, we next injected AAV2-GFP to monkey SN to quantify AAV2-vector distribution and confirm reasonable SN coverage. We then selected and administered a ~200-fold range of AAV2-NRTN doses (and a single AAV2-GDNF dose) to rat SN, producing a wide range of protein expression. In contrast to recent warnings regarding nigra targeting, no dose produced any serious side effects or toxicity, though we replicated the modest reduction in weight gain reported by others with the highest AAV2-NRTN and the AAV2-GDNF dose. A dose-related increase in NRTN expression was seen, with the lower doses limiting NRTN to the peri-SN and the highest dose producing mistargeted NRTN well outside the SN. We then demonstrated that the reduction in weight gain following excessive-doses can be dissociated from NRTN in the targeted SN, and is linked to mistargeted NRTN in the diencephalon. We also showed that prior destruction of the dopaminergic SN neurons via 6-OHDA had no impact on the weight loss phenomenon, further dissociating neurotrophic exposure to the SN as the culprit for weight changes. Finally, low AAV2-NRTN doses provided significant neuroprotection against 6-OHDA toxicity, establishing a wide therapeutic index for nigral targeting. These data support targeting the SN with AAV2-NRTN in PD patients, demonstrating that properly targeted and scaled AAV2-NRTN provides safe and effective NRTN expression. They also provided the means to define an appropriate human-equivalent dose for proceeding into an ongoing clinical trial, using empirically-based scaling to account for marked differences in SN volume between species.

Effects of exposure to nerve growth factor antibodies on the developing nervous system of the rat: An experimental autoimmune approach

An autoimmune method of NGF deprivation was used to characterize the effects of exposure to anti-NGF prenatally in utero and postnatally in milk. Offspring of NGF-immunized female rats were cross-fostered at birth with offspring of control female rats to separate the effects of in utero vs in milk exposure to maternal anti-NGF. In order to determine whether the effects of early exposure to maternal anti-NGF were permanent, rats were evaluated at maturity. Exposure to anti-NGF in utero or in milk resulted in significant decreases in protein and in both pre- and postsynaptic markers in the superior cervical ganglia (choline acetyltransferase and tyrosine hydroxylase, respectively). Significant decreases in norepinephrine levels were measured in peripheral tissues receiving sympathetic innervation (heart, uterus, and brown fat). In rats exposed to anti-NGF in utero , but not in milk, there was an approximately 30% decrease in total protein content in the dorsal root ganglia which was present at birth and in adulthood. The persistence of a decrease in protein content in mature sensory neurons reported here correlated with the previously reported loss of ability of sensory neurons exposed to anti-NGF in utero to retrogradely transport 125 I-NGF. Short adrenergic neurons, central adrenergic neurons, and adrenal medullary cells did not appear to be affected by either prenatal or postnatal exposure to maternal anti-NGF. The potential of the autoimmune approach in the study of the physiological role of NGF and other growth factors is discussed.

Effects of autoimmune NGF deprivation in the adult rabbit and offspring

An experimental autoimmune approach to the production of nerve growth factor deprivation, which we have previously described in the rat and guinea pig, has been applied to the rabbit. This species was chosen for study because of several potential advantages. The rabbit produces large litters and has a relatively short gestation period. More importantly, rabbits generate high titers of antibody against mouse NGF and large amounts of maternal antibody are passively transferred to the developing rabbit fetus compared to most other species, particularly the rat. The sympathetic nervous system of adult rabbit immunized against mouse NGF underwent degeneration with up to an 85% decrease in neuronal numbers in the superior cervical ganglion after 10 months of immunization, thus providing further evidence that NGF is required for the survival of mature sympathetic neurons. Despite the fact that newborn rabbits born to anti-NGF producing mothers had much higher titers of anti-NGF than did rats, the effects on the developing sympathetic and sensory nervous systems were not found to be any greater than in rats. Reductions in norepinephrine levels in the heart and spleen of adult rabbits born to anti-NGF producing mothers were greater than in small intestine. Prenatal exposure to maternal anti-NGF caused reductions (up to 70%) in the number of neurons in the dorsal root ganglia. Substance-P immunoreactivity was reduced in the substantia gelatinosa of the spinal cord of rabbit exposed to maternal anti-NGF. These changes, however, were not greater than seen in the rat. We conclude that although the rabbits offers some advantage in the study of the effects of NGF deprivation in the adult animal, it appears less well suited than the rat or guinea pig to the study of the effects of NGF deprivation on development.

Destruction of sympathetic and sensory neurons in the developing rat by a monoclonal antibody against the nerve growth factor (NGF) receptor.

The ability of the monoclonal antibody, 192-IgG, directed against the rat nerve growth factor (NGF) receptor to mimic or inhibit the actions of NGF was examined in vitro and in vivo. 192-IgG had no effect on morphology, survival, or protein synthesis rates of sympathetic neuronal cultures. When injected into newborn rats, destruction of sympathetic, but not sensory, neurons was produced. Injection prenatally produced more dramatic destruction of sympathetic neurons and, in addition, destruction of neural crest-derived sensory neurons. Therefore, although 192-IgG had no discernible effects in vitro, it produced a pattern of neuronal destruction in vivo qualitatively similar to that produced by antibodies to NGF itself.

Clinical tests of neurotrophic factors for human neurodegenerative diseases, part 1: Where have we been and what have we learned?

Over the past 25years, about 3 dozen clinical reports have been published regarding the safety and possible efficacy of neurotrophic factors in patients with various neurodegenerative diseases. This effort involved a half dozen different neurotrophic factors, using at least 5 different general delivery approaches for ALS (amyolateral sclerosis), peripheral neuropathies, PD (Parkinsons disease) and AD (Alzheimers disease). While none of these efforts have yet produced efficacy data sufficiently robust or reliable to establish neurotrophic factors as treatments for any human disease, the obstacles encountered and novel information reported, when viewed collectively, provide important insight to help future efforts. Three consistent themes emerge from these publications: (1) unexpected and undesirable side effects, at times serious, have plagued many efforts to deliver neurotrophic factors to humans; (2) the magnitude and consistency of clinical benefit has been disappointing; (3) by far that most consistently proposed reason for the side effects and poor efficacy has been inadequate dosing and delivery. This paper reviews and attempts to synthesize the available data derived from clinical tests of neurotrophic factors for neurodegenerative diseases. The obstacles encountered, the solutions attempted, and the lessons learned are discussed. The vast majority of solutions have involved changes in dosing paradigms and dose levels, which has primarily led to improved safety outcomes. However, lack of adequate efficacy remains a significant issue. While current efforts continue to focus exclusively on still-further changes in dosing parameters, a review of available data argues that it may now be the time to ask whether other, non-dose-related variables should be given more serious consideration as being responsible for the great divide that exists between the robust effects seen in animal models and the relatively weak effects seen in human neurodegenerative patients. Foremost among these appears to be the severe degeneration seen in the majority of patients enrolled in past and current trials testing neurotrophic factors in humans. A companion paper (Bartus and Johnson, 2016), reviews the contemporary data and concludes that compelling empirical evidence already exists for enrolling earlier-stage subjects as likely essential to achieving more robust and reliable benefit.