Suppression of terminal axonal sprouting at the neuromuscular junction by monoclonal antibodies against a muscle-derived antigen of 56,000 daltons.

After the partial denervation or paralysis of a muscle, the remaining motor axon terminals may sprout fine, neuritic processes (terminal sprouts) which escape the endplate region of the neuromuscular junction. We previously identified a muscle-derived, protein antigen of 56,000 daltons (56 kD) which plays a necessary role in terminal sprouting. A panel of monoclonal antibodies have been produced against the 56-kD antigen, some of which also partially suppress motor axon terminal sprouting. These monoclonal antibodies define at least two different epitopes upon the surface of the antigen, one of which is necessary for it to effect its biological role in vivo.

Correction.

In the report "Motor neuron degeneration in mice that express a human Cu, Zn super-oxide dismutase mutation" by M. E. Gurney et al. (17 June 1994, p. 1772)(1), a systematic, 10-fold error was made in calculating the dilutions of brain extract used for determinations of total brain superoxide dismutase (SOD) activity shown in column 6 of table 1 (p. 1774). Each value reported should have been reduced by that factor, for example, the total SOD activity reported for the G1 line should have been 4.26 +/- 0.2 SOD (U)/total protein (microg), not 42.6 +/- 2.1 U/microg, and so forth.

Hormone-induced sexual differentiation of brain and behavior in zebra finches.

The male zebra finch sings, whereas the female does not. This behavioral dimorphism is correlated with the presence of morphological sex differences within the neural substrate that mediates this behavior, the song system. When a female chick is exposed to 17beta-estradiol her song system is subsequently masculinized. Either testosterone or 5alpha-dihydrotestosterone may then induce such a female to sing when an adult.

Functional interaction and partial homology between human immunodeficiency virus and neuroleukin.

Dementia is common in patients with AIDS, but the mechanism by which the human immunodeficiency virus type 1 (HIV-1) causes the neurological impairment is unknown. In this study the possibility that an antigen of HIV-1 suppresses neuronal responses to neurotrophic factors was examined. Both HIV-1 and a related retrovirus, simian immunodeficiency virus (SIV), inhibited the growth of sensory neurons from chick dorsal root ganglia in medium containing neuroleukin (NLK) but not in medium containing nerve growth factor. An unrelated type D retrovirus, simian acquired immunodeficiency syndrome virus, did not affect the growth of neurons in the presence of either neurotrophic factor. The inhibition by HIV-1 of neuron growth in the presence of NLK was found to be due to the gp120 envelope glycoprotein. Regions of sequence homology between gp120 and NLK may account for this inhibitory property of gp120 and functional interactions between gp120 and NLK may be important in the pathogenesis of the AIDS dementia complex.

Second conserved domain of gp120 is important for HIV infectivity and antibody neutralization.

Rabbit antisera were raised against three overlapping synthetic peptides with sequence homology to the second conserved domain of the external envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV). All of the antisera immunoprecipitated the envelope glycoprotein. In particular, an antiserum directed against amino acids 254 to 274 of env was efficient in neutralizing three different isolates of HIV in vitro, without affecting the binding of the virus to CD4-positive cells. Therefore, this conserved region of gp120 appears to be critical in a postbinding event during virus penetration and may represent a target for antibody neutralization of HIV. These findings may be applicable in the design of a vaccine for the acquired immunodeficiency syndrome.

Molecular cloning and expression of neuroleukin, a neurotrophic factor for spinal and sensory neurons.

A novel 56,000-dalton growth factor found in mouse salivary gland was purified, molecularly cloned, and expressed in monkey COS cells. The protein is a neurotrophic factor and also, surprisingly, a lymphokine product of lectin-stimulated T cells. The factor was therefore named neuroleukin. Neuroleukin promotes the survival in culture of a subpopulation of embryonic spinal neurons that probably includes skeletal motor neurons. Neuroleukin also supports the survival of cultured sensory neurons that are insensitive to nerve growth factor, but has no effect on sympathetic or parasympathetic neurons. The amino acid sequence of neuroleukin is partly homologous to a highly conserved region of the external envelope protein of HTLV-III/LAV, the retrovirus that causes acquired immune deficiency syndrome.

Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.

Mutations of human Cu,Zn superoxide dismutase (SOD) are found in about 20 percent of patients with familial amyotrophic lateral sclerosis (ALS). Expression of high levels of human SOD containing a substitution of glycine to alanine at position 93--a change that has little effect on enzyme activity--caused motor neuron disease in transgenic mice. The mice became paralyzed in one or more limbs as a result of motor neuron loss from the spinal cord and died by 5 to 6 months of age. The results show that dominant, gain-of-function mutations in SOD contribute to the pathogenesis of familial ALS.

Neuroleukin: a lymphokine product of lectin-stimulated T cells.

Neuroleukin is a lymphokine product of lectin-stimulated T cells that induces immunoglobulin secretion by cultured human peripheral blood mononuclear cells. Neuroleukin acts early in the in vitro response that leads to formation of antibody-secreting cells, but continued production of immunoglobulin by differentiated antibody-secreting cells is neuroleukin-independent. Although the factor is not directly mitogenic, cellular proliferation is a late component of the response to neuroleukin. Neuroleukin does not have B-cell growth factor (BCGF) or B-cell differentiation factor (BCDF) activity in defined assays. Neuroleukin-evoked induction of immunoglobulin secretion is both monocyte- and T-cell-dependent.

Intracellular calcium parallels motoneuron degeneration in SOD-1 mutant mice.

Transgenic mice with Cu,Zn superoxide dismutase (SOD-1) mutations provide a unique model to examine altered Ca homeostasis in selectively vulnerable and resistant motoneurons. In degenerating spinal motoneurons of G93 A SOD-1 mice, developing vacuoles were filled with calcium, while calcium was gradually depleted from the cytoplasm and intact mitochondria. In oculomotor neurons, no degenerative changes, vacuolization, or increased calcium were noted. Motor axon terminals of interosseus muscle gradually degenerated and intracellular calcium was depleted. Oculomotor terminals of mutant SOD-1 mice were smaller and exhibited no degenerative changes, but did exhibit unique membrane-enclosed organelles containing calcium. Spinal motoneurons of SOD-1 mice were shown to have fewer calcium binding proteins, such as parvalbumin, compared with oculomotor neurons. These data suggest that the SOD-1 mutation is associated with impaired calcium homeostasis in motoneurons in vivo, with increased likelihood of degeneration associated with higher levels of intracellular calcium and lower to absent levels of calbindin-D28K and/or parvalbumin, and decreased likelihood of degeneration associated with minimally changed calcium and ample calbindin-D28K and/or parvalbumin.

Riluzole preserves motor function in a transgenic model of familial amyotrophic lateral sclerosis.

Riluzole was tested in a dose-ranging study for preservation of motor function in a transgenic mouse model of familial ALS. The model is based on expression of mutant human Cu,Zn superoxide dismutase in mouse brain and spinal cord. In contrast with the human ALS trials, in the mouse model, riluzole significantly preserved motor function as assessed by nightly running in a wheel. The effect of riluzole on motor performance was greater earlier in disease than later, suggesting that riluzole may have benefit for "quality-of-life" measures in ALS. Treatment with riluzole was initiated earlier in the transgenic model than in the human ALS trials, which may account for the significantly better outcome.

Quantitative immunoassay of recombinant murine neuroleukin.

A eukaryotic, transient expression system was used to produce recombinant neuroleukin, a growth factor for neurons. Neuroleukin in media conditioned by transfected monkey COS-1 cells was purified to homogeneity in one step by high-performance cation-exchange chromatography. Purified neuroleukin was used to establish a quantitative two-site enzyme-linked immunosorbent assay and the accuracy of the assay was confirmed by analytical high-performance liquid chromatography. The amount of recombinant neuroleukin secreted by the transfected COS-1 cells and the content of endogenous neuroleukin in various murine cell lines was determined. Neuroleukin levels were nearly undetectable in Balb/3T3 embryo cells, intermediate in several leukocytic cell lines and highest in mouse LBRM-33 T lymphoma cells. Maximal survival of sensory neurons was obtained with approximately 10(-9) M recombinant neuroleukin although tissue derived neuroleukin appeared to be significantly more active. Dialysis of the transfected COS-1 cell conditioned medium resulted in increased neuroleukin bioactivity, while binding to the cation-exchange column reduced bioactivity. The expression and purification of the recombinant protein and the detection of natural sources expressing high levels of neuroleukin will greatly facilitate studies of its biological effects.

Lack of involvement of neuronal nitric oxide synthase in the pathogenesis of a transgenic mouse model of familial amyotrophic lateral sclerosis.

A subset of familial cases of amyotrophic lateral sclerosis are linked to missense mutations in copper/zinc superoxide dismutase type 1. Patients with missense mutations in copper/zinc superoxide dismutase type 1 develop a paralytic disease indistinguishable from sporadic amyotrophic lateral sclerosis through an unknown toxic gain of function. Nitric oxide reacts with the superoxide anion to form the strong oxidant, peroxynitrite, which participates in neuronal injury in a variety of model systems. Peroxynitrite is an alternate substrate for copper/zinc superoxide dismutase type 1, causing catalytic nitration of tyrosine residues in other proteins. Mutations in copper/zinc superoxide dismutase type 1 may disrupt the active site of the enzyme and permit greater access of peroxynitrite to copper, leading to increased nitration by peroxynitrite of critical cellular targets. To investigate whether neuronal-derived nitric oxide plays a role in the pathogenesis of familial amyotrophic lateral sclerosis, we examined the effects of three different nitric oxide synthase inhibitors: a non-selective nitric oxide synthase inhibitor, nitro-L-arginine methyl ester; a relatively selective inhibitor of neuronal nitric oxide synthase, 7-nitroindazole; and a novel highly selective neuronal nitric oxide synthase inhibitor, AR-R 17,477, in transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 (Gly-->Ala at position 93; G93A) containing a high transgene copy number and a low transgene copy number. AR-R 17,477, but not nitro-L-arginine methyl ester or 7-nitroindazole, significantly prolonged survival in both the high and low transgene transgenic mice. To determine whether neuronal nitric oxide synthase is involved in the pathogenesis resulting from the familial amyotrophic lateral sclerosis copper/zinc superoxide dismutase type 1 mutation, we produced mice with the copper/zinc superoxide dismutase type 1 mutation which lack the neuronal nitric oxide synthase gene. The transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background do not live significantly longer than transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1. Western blot analysis indicates the presence of two neuronal nitric oxide synthase-like immunoreactive bands in spinal cord homogenates of the neuronal nitric oxide synthase null mice, and residual neuronal nitric oxide synthase catalytic activity ( > 7%) is detected in the spinal cord of the transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background. This amount of residual activity probably does not account for lack of protection afforded by the disrupted neuronal nitric oxide synthase gene in the familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 mice. Immunological nitric oxide synthase is not detected in the copper/zinc superoxide dismutase type 1 mutant mice at several different ages, thus excluding immunological nitric oxide synthase as a contributor to the pathogenesis of familial amyotrophic lateral sclerosis. Levels of neuronal nitric oxide synthase as well as Ca2+-dependent nitric oxide synthase catalytic activity in the copper/zinc superoxide dismutase type 1 mutant mice do not differ from wild type mice. Endothelial nitric oxide synthase levels may be decreased in the copper/zinc superoxide dismutase type 1 mutant mice. Together, these results do not support a significant role for neuronal-derived nitric oxide in the pathogenesis of familial amyotrophic lateral sclerosis transgenic mice.

Single basic amino acid substitutions at position 302 or 320 in the V3 domain of HIV type 1 are not sufficient to alter the antiviral activity of dextran sulfate and heparin.

The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 contains a substantial number of positively charged amino acid residues. We previously demonstrated that mutation of basic amino acid residues at position 303, 306, 309, 313, and 325 in the V3 domain of HIV-1 strain NL4-3 resulted in a dramatic elimination of both virus infectivity and syncytium-inducing ability. Mutations of arginine at position 302 to serine (R302S) or lysine at position 320 to glutamine (K320Q) had variable effects on infectivity for a panel of T cell lines tested. These mutations are located on opposite sides of the Gly-Pro-Gly-Arg-Ala sequence in the center of the V3 domain. The R302S and K320Q mutations allowed us to determine if these basic residues are important for virus neutralization by polyanionic compounds. Dextran sulfate and heparin inhibited the cytopathogenicities of both mutants for MT-4 cells, although their 50% antiviral effective doses were slightly higher than those required to achieve complete protection against wild-type HIV-1NL4-3 replication. This result emphasizes that the basic amino acids of Arg302 and Lys320 are not essential for the inhibitory effect of dextran sulfate and heparin on HIV-1 infection.

HIV type 1 infection of CD4+ T cells depends critically on basic amino acid residues in the V3 domain of envelope glycoprotein 120.

The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a major determinant of phenotypic variability. The V3 domain of HIV-1 has many basic amino acid residues. Lymphocytotropic HIV-1 tends to have a V3 domain with a higher density of positive charge than does monocytotropic HIV-1. The importance of basic residues in the V3 domain for the HIV-1 infectivity, however, has not been well investigated. Here we show that mutation of basic amino acid residues at positions 303, 306, 309, 313, and 325 in the V3 domain of the lymphocytotropic isolate NL4-3 results in a dramatic elimination of both virus infectivity and syncytium-inducing ability. Three basic amino acid substitutions (at position 306, 309, and 313) induced a decrease in the binding ability of two kinds of neutralizing antibodies (NEA9284 and 0.5 beta) that recognize a different site in the V3 domain. This suggests that the basic residues play an important role in maintaining the tertiary structure of the V3 domain. Monocytotropism was not simply dependent on either decreased positive charge in the V3 domain of NL4-3 or on mutation of lysine to glutamate at position 320, which is a characteristic amino acid of monocytotropic HIV-1. These findings contribute to our understanding of the significance of basic residues on the function of envelope glycoprotein.

Systemic injections of ciliary neurotrophic factor induce sprouting by adult motor neurons.

The ability of ciliary neurotrophic factor (CNTF) to induce sprouting by undamaged adult motor neurons was studied in gluteal muscles of adult ICR mice. Low doses of CNTF (0.02 mg kg-1 day-1) only induced sprouting in gluteus muscles that were beneath the site of injection, whereas high doses of CNTF (0.4-1.2 mg kg-1 day-1) acted systemically to induce motor neuron sprouting. We found little difference between the type or quality of sprouting induced by CNTF compared with muscle paralysis. Both stimuli induced sprouts of the same length, although muscle paralysis tended to induce more sprouts per end-plate. Paralysis also induced more nodal sprouting than did CNTF, but both were weaker stimuli for nodal sprouting than was partial denervation. In addition to its effects on motor neuron sprouting, high doses of CNTF induced loss of up to 36% of the body weight of treated mice. The substantial wasting caused by CNTF indicates that the factor has potent cachectic activity.

Reactive astrocytes express nitric oxide synthase in the spinal cord of transgenic mice expressing a human Cu/Zn SOD mutation.

The distribution of the neuronal isoform of nitric oxide synthase (nNOS) in the spinal cord of transgenic mice expressing a mutated human copper/zinc superoxide dismutase gene was enhanced when investigated by immunocytochemistry. Immunocytochemistry showed intensely stained NOS-immunoreactive (IR) glial cells with the appearance of astrocytes in the spinal cord and brain stem of transgenic mice, but none were observed at these sites in control mice. Using antisera directed against GFAP, the specific marker for astrocyte, the glial cells were confirmed by immunocytochemistry to be astrocytes. This immunocytochemical evidence suggests that nitric oxide may mediate glutamate neurotoxicity, and this study provides the first in vivo evidence that nitric oxide may be implicated in the pathologic process of human familial amyotrophic lateral sclerosis.

Reactive astrocytes express p53 in the spinal cord of transgenic mice expressing a human Cu/Zn SOD mutation.

In a previous study, we reported increased NOS expression in the astrocytes in the spinal cord of SOD mutant transgenic mice that are used as ALS animal model. Recently, Messmer and Brune suggested that nitric oxide-induced apoptosis is intimately related with p53-dependent signaling pathway, and de la Monte et al. reported increased p53-immunoreactivity in the spinal cord of ALS patients. In the present study, we performed immunocytochemical studies to investigate the changes of p53-immunoreactivity in the brains of the mutant transgenic mice expressing a human Cu/Zn SOD mutation. Immunocytochemistry showed intensely stained p53-IR glial cells with the appearance of astrocytes in all levels of the spinal cord of the mutant transgenic mice, but no p53-IR glial cells were observed in the spinal cord of the control mice. P53-IR astrocytes were also detected in the brain stem of the mutant transgenic mice. In the medulla, they were observed in the medullary reticular formation, hypoglossal nucleus, vestibular nucleus, dorsal motor nucleus of the vagus and nucleus ambiguus. In the pons, their presences were noted in the pontine reticular formation, and trigeminal and facial nuclei. In the midbrain, astrocytes were detected in the mesencephalic reticular formation, red nucleus and periaqueductal gray matter. In the cerebellum, intensely stained p53-IR astrocytes were detected in the intracerebellar nuclei. In contrast to the mutant transgenic mice, no p53-IR astrocytes were detected in the brain stem and spinal cord of the control mice. Further multidisciplinary investigations involving p53-mediated cellular damage and pathogenesis of ALS are needed to clarify the importance of these results.

Behavioral correlates of sexual differentiation in the zebra finch song system.

The capacity for song is masculinized in female zebra finches by exposure to 17 beta-estradiol at hatching, and requires continual exposure to androgen for its behavioral expression in the adult. The stereotypy of the song developed by estrogenized females can be as good as that of male song. The sexual dimorphism in behavioral capacity for song parallels morphological sex differences in the efferent motor pathway which participates in the production of song. Estradiol induces the cytoarchitectonic masculinization of the telencephalic nuclei within the motor system for song, and renders them able to respond morphologically when song is induced in estrogenized females by exposure to androgen as adults.