Systemic administration of ciliary neurotrophic factor induces cachexia in rodents.

Ciliary neurotrophic factor (CNTF) has previously been shown to promote the survival of several classes of neurons and glial. We report here that in addition to its effects on the nervous system, CNTF can induce potent effects in extra-neural tissues. Implantation of C6 glioma cells engineered to secrete CNTF either subcutaneously or into the peritoneal cavity of adult mice, or systemic injections of purified rat or human recombinant CNTF, resulted in a rapid syndrome of weight loss resulting in death over a period of 7-10 d. This weight loss could not be explained by a reduction in food intake and involved losses of both fat and skeletal muscle. CNTF also induced the synthesis of acute phase proteins such as haptoglobin. Implantation of C6 lines expressing a nonsecreted form of CNTF, or the parental C6 line itself, did not result in wasting effects. Analysis of this CNTF-induced wasting indicates similarities with the previously described cachectins, tumor necrosis factor, interleukin 6, and leukemia inhibitory factor, but does not involve the induction of these cytokines.

Localization of CNTF immunoreactivity to neurons and astroglia in the CNS.

Species specific antibodies were raised to a peptide of rat ciliary neurotrophic factor (CNTF-amino acids number 131-147). Following affinity purification, these antibodies were used to determine the pattern of CNTF immunoreactivity in adult rat and mouse brain, spinal cord, and sciatic nerve. Alternate sections stained using neurofilament and the affinity purified anti-CNTF antibody (HARC-1) demonstrate that CNTF immunoreactive neurons are present within the facial nucleus, dentate gyrus, olfactory bulb, basal forebrain, locus coeruleus, cortex and substantia nigra. In addition, neurons throughout the hippocampus, and Purkinje cells within the cerebellum also exhibit CNTF immunoreactivity. CNTF immunopositive neurons demonstrate a preponderance of nuclear staining, with some staining present in the cytoplasm. Alternate sections incubated with glial fibrillary acidic protein (GFAP) antibody also demonstrate glia which are positive for CNTF. In the peripheral nervous system, Schwann cells of the sciatic nerve exhibit strong immunoreactivity for CNTF, however staining is confined to the cytoplasm and is absent from the cell nucleus. These data demonstrate that CNTF immunoreactivity is broadly distributed throughout neurons and glia of the adult rodent nervous system.

Increased CNTF gene expression in process-bearing astrocytes following injury is augmented by R(-)-deprenyl.

R(-)-deprenyl has been shown to rescue axotomized immature facial motoneurons with an efficacy comparable to that of the neurotrophic factors CNTF and BDNF (Salo and Tatton, J Neurosci Res 31:394-400, 1992; Ansari et al., J Neurosci 13:4042-4053, 1993). Recent work has suggested that some of the actions of (-)-deprenyl may be mediated through reactive astrocytes (Biagini et al., NeuroReport 4:955-958, 1993). To test this proposal we have developed an in vitro model of reactive gliosis consisting of a mixed astrocyte population of flat and process-bearing (PB) astroglia taken from postnatal day (PD) 2 or PD5 rat cerebral cortex. After mechanical wounding, PB astrocytes preferentially migrate into the wound zone while flat astrocytes maintain their position at the wound edge. CNTF mRNA was localized to PB astrocytes, but not flat astrocytes, as determined by in situ hybridization using biotin-labelled riboprobes. Following "wounding," there was an increase in CNTF mRNA in PB astrocytes only, which could be further enhanced by a single pulse of (-)-deprenyl (10(-8)-10(-11) M) 48 hr after injury. (-)-Deprenyl also increased the total process length of PB astrocytes after wounding by an average of 50%. The stereoisomer (+)-deprenyl (10(-9) M) had no effect on either astrocyte process length or CNTF mRNA content. This is the first report to our knowledge of an agent which can upregulate CNTF gene expression in astroglial cell culture as well as influence glial cell process length.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between MPTP-induced and age-related neuronal death in a murine model of Parkinson's disease.

Abiotrophy is hypothesized to explain the onset and time course of deficits in Parkinson's disease (PD) Abiotrophy includes: 1) exposure to agent(s) causing the death of dopaminergic nigrostriatal neurons (DNSns), 2) gradual death of DNSns with age, 3) summation of 1) and 2) until DNSn numbers fall below a threshold for detectable neurological deficits. Murine DNSn death following methyl-phenyl-tetrahydropyridine (MPTP) exposure occurs according to an exponential relationship while age-related death of DNSns occurs according to a second exponential relationship. Summing the two exponential losses overestimates experimental DNSn death showing a simple abiotrophic model is not sufficient. Aged murine DNSns greatly increase their dopamine synthesis and the density of their striatal axon terminals which may explain the above threshold. Murine DNSns die gradually after MPTP exposure and L-deprenyl treatment rescues MPTP-damaged DNSns by a previously undiscovered action, altering the abiotrophic interactions and possibly explaining the slowed progression of PD found with deprenyl treatment.

Transmitter synthesis increases in substantia nigra neurons of the aged mouse.

Striatal dopamine concentrations are relatively well maintained with age despite extensive death of the nigrostriatal neurons whose terminals contain the dopamine. Counts of nigrostriatal dopaminergic neurons in C57BL mice identified using immunocytochemistry, Fluoro-Gold retrograde axonal transport and Nissl staining were combined with measures of striatal dopamine and DOPA after saline, pargyline or NSD-1015 treatment. On average, 68% of the dopaminergic nigrostriatal neurons died between ages 8 and 104 weeks and there was a 3-fold increase in dopamine synthesis per average neuron in the aged mice. Increased transmitter synthesis by surviving neurons may serve to compensate brain function in old age.

Increased dopamine synthesis in aging substantia nigra neurons.

Striatal dopamine (DA) and metabolite (DOPAC) levels in 8-, 21-, 52- and 104-week-old C57BL mice were compared with those in 11-week-old mice, 20 days after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. DA and DOPAC concentrations expressed relative to striatal wet weight did not change with age. In contrast, DA and DOPAC levels increased almost linearly when values were expressed relative to the proportion of remaining tyrosine hydroxylase-positive (TH+) SNc neurons, reaching a 5-7-fold increase per average remaining TH+ neuron by 104 weeks of age (corresponding to neuronal loss of 70%) relative to that found per average neuron in 8-week-old mice. DA and DOPAC levels per average remaining TH+ SNc neuron following MPTP increased for low doses (neuronal losses less than 42%) but decreased for higher doses (55 and 70% losses) but the DOPAC/DA ratio per SNc neuron increased and was 9-fold higher in the 300 mg/kg MPTP-treated animals in comparison to saline controls. Cytoplasmic TH protein (estimated by somal TH immunodensity) was increased by 45% in SNc somata from mice treated with 150 mg/kg MPTP in comparison to saline controls, and by 63% in 104-week-old mice in comparison to 8-week-old animals. This study provides evidence that an average surviving TH+ SNc neuron compensates for the age-related loss of other SNc neurons by increasing dopamine synthesis similar to younger SNc neurons surviving low levels of toxically induced damage and that the compensation may be in part mediated by increased synthesis of TH.

First expression of protamine message in trout testis.

In situ hybridizations were performed using a biotinylated riboprobe complementary to protamine messenger RNA in order to directly examine the various cell types in the trout testis for the presence of protamine message. Computer-aided optical density measurements were used to provide estimates of transcript abundance for cells identified by their DAPI-labeled nuclei. Optically detectable protamine hybridization occurred only in spermatid cells. These findings are in accord with results obtained in other species which report protamine mRNA only in the post-meiotic spermatid cell; but they are in conflict with a previous study employing solution hybridization which noted that protamine message first appears in the spermatocytes of rainbow trout.

MPTP produces reversible disappearance of tyrosine hydroxylase-containing retinal amacrine cells.

To determine whether 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) alters the tyrosine hydroxylase (TH) immunoreactivity of murine dopaminergic retinal amacrine cells, 8-10-week-old C57BL/6J mice were treated with i.p. with saline or cumulative doses of MPTP ranging from 10 to 300 mg/kg. Paraformaldehyde-fixed retinal whole mounts and cross sections were examined using immunochemistry with a tyrosine hydroxylase (TH) or a choline acetyltransferase (ChAT) polyclonal antibody and an avidin-biotin peroxidase reaction. Both TH+ amacrines and ChAT+ retinal neurons showed somal and process morphology and distributions that were commensurate with previous studies of the same or several related species. At 20 days following the MPTP treatment, there was a loss of TH+ amacrines according to a logarithmic relationship relative to MPTP dosage. The loss ranged from 18 to 87% for the dosage range without any decrease in the numbers of ChAT+ neurons. The TH+ amacrines were deleted randomly from the retinas without any peripheral-central predilection. By 273 days after MPTP treatment, the number of TH+ amacrines had returned to values found for age-matched controls demonstrating that the loss of TH immunoreactivity was reversible and occurred without destruction of TH+ amacrines. Computer densitometry revealed that the MPTP-treated TH+ amacrines were divided into two distinct populations: one with normal TH immunodensity levels and a second with TH immunodensity levels below our detection capability. Increasing the MPTP dosage increased the proportion of TH amacrines in the second population. The transient and completely reversible disappearance in the number of TH+ amacrines: (1) appears to form the basis for the decreased concentrations of dopamine and the loss of catecholamine fluorescent neurons previously described for MPTP-treated mouse retinae; (2) may underlie the defects in the electroretinograms of MPTP-treated monkeys, and (3) may result as a response to neurite damage similarly to the alterations in protein synthesis in other central neurons following axonal damage.

Dose-dependent destruction of the coeruleus-cortical and nigral-striatal projections by MPTP.

In order to determine whether 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces neuronal death or the loss of tyrosine hydroxylase (TH) immunoreactivity, 4 catecholaminergic nuclei in the mouse: substantia nigra compacta (SNc), locus coeruleus (LC), ventral tegmental area (VTA) and the A13 nucleus in the hypothalamus were quantitatively examined. Serial sections were taken through the rostrocaudal extent of each nucleus: alternate sections were incubated with TH antiserum and reacted with an immunoperoxidase technique while the alternate set was Nissl stained. Counts and 3 dimensional reconstructions of TH reactive somata were made for each nucleus for saline-treated controls and mice treated with different doses of MPTP (37.5, 75, 150 and 300 mg/kg). TH-positive neurons were counted along with their counterparts on the Nissl-stained alternative sections to both identify the catecholaminergic neurons and to measure their destruction. Concentrations of striatal dopamine and cortical norepinephrine were measured for all dosages of MPTP in order to determine the relationship between dosage, target tissue neurotransmitter concentration and neuronal destruction. By 20 days after MPTP injection there was a dose-dependent random loss of TH-immunoreactive neurons that was almost identical in all 4 nuclei examined. Analysis of the Nissl versus TH cell counts revealed that MPTP resulted in neuronal destruction in the SNc and the LC rather than just a loss of TH immunoreactivity. There was no difference in sensitivity to MPTP between the SNc and the LC. Decreases in cortical norepinephrine concentrations were about one third of the decreases of LC neuronal counts for all MPTP doses; while decreases in striatal dopamine and SNc cell loss was similar to the LC for the two lower doses of MPTP but for the higher doses, the relationship approached or exceeded a one to one ratio. Hence estimates of neuronal death based upon target tissue transmitter concentrations could not be made using the same relationship for SNc and the LC catecholaminergic neurons and use of the same relationship for higher MPTP dosages results in an underestimate of LC neuronal destruction relative to that in the SNc.