TGFbeta trophic factors differentially modulate motor axon outgrowth and protection from excitotoxicity.

Transforming growth factor (TGF) beta-like trophic factors have been shown to be protective in acute neuronal injury paradigms. In the current study, we analyzed and compared members of this growing family, including glial cell line-derived neurotrophic factor (GDNF), neurturin, nodal, persephin, and TGFbeta1, for protection against chronic glutamate toxicity. In parallel, we developed a organotypic spinal cord culture system to study the ability of these factors to promote motor axon outgrowth across white matter. Using these systems, we were able to differentiate the neuroprotective effect of the TGFbeta-like factors from their motor axon outgrowth-promoting activity. GDNF, neurturin, persephin, nodal, and TGFbeta1 all protected against excitotoxic motor neuron degeneration. Low amounts of GDNF (1 ng/ml) and high concentrations of neurturin induced vigorous motor axon outgrowth. In contrast, nodal, persephin, and TGFbeta1 did not induce motor axon outgrowth. Both GDNF and neurturin bind to Ret receptor complexes and were capable of activating the MAP kinase pathway. A specific inhibitor of MAP kinase kinase, PD98059, inhibited the motor axon outgrowth-promoting activity of the GDNF but not the neuroprotective activity. Similarly, the specific PI3K inhibitors, LY294002 and wortmannin, were able to inhibit the promotion of motor axon outgrowth by GDNF, but did not affect neuroprotective activity. Our results suggest that the neurite outgrowth-promoting effect of GDNF is mediated through the PI3K and MAP kinase pathways. The neuroprotective effect of GDNF appears to be through a separate pathway.

Use of antisense oligodeoxynucleotides to inhibit expression of glutamate transporter subtypes.

Antisense ODN are highly effective tools to selectively inhibit synthesis of glutamate transporter subtypes. They have been used to evaluate the biology of individual transporter subtypes in vivo and in vitro. Appropriate use of antisense ODN, however, requires a number of important controls to validate the specificity of their effects. Ultimately, the efficacy at inhibiting the synthesis of transporters proteins reflects several variables: the actual efficacy of the antisense to directly inhibit synthesis, the rate of degradation of the ODN, the rate of intracellular penetration of the ODN, the rate of new protein synthesis for the transporter subtype, and the actual penetration of the ODN into CNS tissue. The methods described in this chapter will help one to optimize each of these parameters.

Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterized by selective upper and lower motor neuron degeneration, the pathogenesis of which is unknown. About 60%-70% of sporadic ALS patients have a 30%-95% loss of the astroglial glutamate transporter EAAT2 (excitatory amino acid transporter 2) protein in motor cortex and spinal cord. Loss of EAAT2 leads to increased extracellular glutamate and excitotoxic neuronal degeneration. Multiple abnormal EAAT2 mRNAs, including intron-retention and exon-skipping, have now been identified from the affected areas of ALS patients. The aberrant mRNAs were highly abundant and were found only in neuropathologically affected areas of ALS patients but not in other brain regions. They were found in 65% of sporadic ALS patients but were not found in nonneurologic disease or other disease controls. They were also detectable in the cerebrospinal fluid (CSF) of living ALS patients, early in the disease. In vitro expression studies suggest that proteins translated from these aberrant mRNAs may undergo rapid degradation and/ or produce a dominant negative effect on normal EAAT2 resulting in loss of protein and activity. These findings suggest that the loss of EAAT2 in ALS is due to aberrant mRNA and that these aberrant mRNAs could result from RNA processing errors. Aberrant RNA processing could be important in the pathophysiology of neurodegenerative disease and in excitotoxicity. The presence of these mRNA species in ALS CSF may have diagnostic utility.

Glutamate transporter gene expression in amyotrophic lateral sclerosis motor cortex.

Glutamate transport is critical for synaptic inactivation of glutamate and prevention of excitotoxicity. The following four glutamate transporters have been identified in human brain: EAAT1, EAAT2, EAAT3, and EAAT4. Deficient glutamate transport has been identified in the motor cortex and the spinal cord of tissue from amyotrophic lateral sclerosis (ALS) patients. The defect appears to be due to a selective loss of the astroglial specific glutamate transporter protein EAAT2. In these studies we sought to extend our understanding of glutamate transporters in ALS by examining the mRNA for each transporter subtype in ALS motor cortex. All tissue was matched for age and postmortem delay. There was no quantitative change in mRNA for EAAT1, EAAT2, or EAAT3 in ALS motor cortex, even in patients with a large loss of EAAT2 protein (95% decrease compared with control) and decreased tissue glutamate transport (73% decrease compared with control). These studies suggest that the dramatic abnormalities in EAAT2 may be due to translational or post-translational processes.

Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.

Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

Inhibition of CD26 enzyme activity with pro-boropro stimulates rat granulocyte/macrophage colony formation and thymocyte proliferation in vitro.

CD26 dipeptidyl peptidase (DPPIV) is involved in the regulation of proliferation of some hematopoietic and T-lineage cells. Here, we show that Pro-boropro a potent inhibitor of DPP activity has a costimulating effect in hematopoietic colony assays for macrophage and, to a lesser extent, for granulocyte colonies and has a stimulating effect in organ cultures of immature thymocytes. Based on these and other evidences, we propose that the mechanism by which CD26 regulates proliferation is associated with its DPP activity.

Chronic inhibition of superoxide dismutase produces apoptotic death of spinal neurons.

Mutations in the gene for Cu/Zn superoxide dismutase (SOD1) have been detected in some families with an autosomal dominant form of amyotrophic lateral sclerosis; these mutations appear to reduce the activity of this enzyme. To determine whether decreased SOD activity could contribute to motor neuron loss, SOD1 was inhibited chronically with either antisense oligodeoxynucleotides or diethyldithiocarbamate in spinal cord organotypic cultures. Chronic inhibition of SOD resulted in the apoptotic degeneration of spinal neurons, including motor neurons, over several weeks. Motor neuron loss was markedly potentiated by the inhibition of glutamate transport. In this paradigm, motor neuron toxicity could be entirely prevented by the antioxidant N-acetylcysteine and, to a lesser extent, by the non-N-methyl-D-aspartate glutamate receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride. These data support the hypothesis that the loss of motor neurons in familial amyotrophic lateral sclerosis could be due to a reduction in SOD1 activity, possibly potentiated by inefficient glutamate transport.

Upregulation of T cell receptor gamma chain transcription by interleukin-2.

Signals involved in TcR gene rearrangement and expression are poorly understood. The ability of interleukin 2 to control TcR gamma gene expression was examined. Precursor thymocytes grown in the presence of IL-1 and Con A develop to CD3+ T cells that express either the TcR alpha/beta or gamma/delta complex on the cell surface (1). We show here that a major subpopulation of these cells are CD4-/CD8-, gamma/delta cells expressing only low levels of TcR gamma transcripts, compared to TcR delta mRnA or to TcR gamma mRNA of gamma/delta cells grown from precursor thymocytes with IL-2 and Con A. The cells cultured with IL-1 and lectin then strongly reacted to IL-2 by upregulating the steady-state level of TcR gamma mRNA. Our findings indicate that IL-2 upregulates TcR gamma transcription by transcriptional regulatory effects in this in vitro system.

Differential regulation of group A streptococcal peptidoglycan-polysaccharide (PG-APS)-stimulated macrophage production of IL-1 by rat strains susceptible and resistant to PG-APS-induced arthritis.

The magnitude of the response of rat macrophages to group A streptococcal cell wall peptidoglycan-polysaccharide (PG-APS) stimulation is strain-dependent. A comparison of IL-1 expression between macrophages from a PG-APS-resistant (BUF) and PG-APS-sensitive (LEW) rat strain revealed that while mRNA levels for IL-1 alpha and IL-1 beta were equivalent for the two strains, supernatants from BUF macrophages were less potent than LEW supernatants at stimulating thymocyte proliferation and contained less immunoreactive IL-1 alpha and IL-1 beta protein. BUF and LEW macrophages expressed nearly equivalent levels of IL-1 receptor antagonist (IL-1ra) mRNA and secreted IL-1ra protein in supernatants. The ratio of IL-1/IL-1ra produced by macrophages may influence susceptibility versus resistance to PG-APS and is strongly suggestive of a regulatory role for macrophages in the pathogenesis of PG-APS-induced arthritis.

Mammary epithelial cells of lactating rats express prolactin messenger ribonucleic acid.

The presence of prolactin (PRL) mRNA in the mammary gland, placenta, and pituitary gland of lactating and pregnant rats was investigated by polymerase chain reaction (PCR). Polyadenylated RNA was prepared from total RNA samples by oligo(dT)-cellulose chromatography, and complementary cDNAs were synthesized. A standardized amount of cDNA from each sample was used as the template in a Taq PCR under high-stringency conditions. PCR amplified a signal with the predicted size of approximately 375 bp in mammary and pituitary glands of lactating and pregnant rats, and in placentae of pregnant rats. This band specifically hybridized with a probe overlapping the entire sequence of the mature rat (r) PRL mRNA in Southern blot analysis. When the rPRL-specific primers were used, PCR revealed no signal in the liver or in lactating mammary gland explants cultured in vitro for 48 h, while the same cDNA preparations gave strong signals for beta-actin. The viability of the mammary gland explants was also suggested by their ability to secrete immunoreactive casein in vitro. PRL mRNA was localized in the epithelium of alveoli and ducts of the lactating mammary gland by in situ hybridization. These data provide evidence that the PRL gene is expressed in the mammary gland of pregnant and lactating rats, and suggest that the mammary gland might contribute to PRL in milk by de novo synthesis. Thus, while the placenta is an exogenous source of PRL-like activities for the fetus in utero, the mammary gland might take over this function after birth.

Induction of ornithine decarboxylase by N-methyl-D-aspartate receptor activation is unrelated to potentiation of glutamate excitotoxicity by polyamines in cerebellar granule neurons.

Polyamines positively modulate the activity of the N-methyl-D-aspartate (NMDA)-sensitive glutamate receptors. The concentration of polyamines in the brain increases in certain pathological conditions, such as ischemia and brain trauma, and these compounds have been postulated to play a role in excitotoxic neuronal death. In primary cultures of rat cerebellar granule neurons, exogenous application of the polyamines spermidine and spermine (but not putrescine) potentiated the delayed neurotoxicity elicited by NMDA receptor stimulation with glutamate. Furthermore, both toxic and nontoxic concentrations of glutamate stimulated the activity of ornithine decarboxylase (ODC)--the key regulatory enzyme in polyamine synthesis--and increased the concentration of ODC mRNA in cerebellar granule neurons but not in glial cells. Glutamate-induced ODC activation but not neurotoxicity was blocked by the ODC inhibitor difluoromethylornithine. Thus, high extracellular polyamine concentrations potentiate glutamate-triggered neuronal death, but the glutamate-induced increase in neuronal ODC activity may not play a determinant role in the cascade of intracellular events responsible for delayed excitotoxicity.

Thymocyte costimulating antigen is CD26 (dipeptidyl-peptidase IV). Costimulation of granulocyte, macrophage, and T lineage cell proliferation via CD26.

The rat thymocyte costimulating protein appears to be involved in the regulation of CD4-/CD8- thymocyte proliferation in vitro. We show that thymocyte costimulating protein is dipeptidyl peptidase IV (E.C.N.3.4.14.5) also known as CD26. Some bone marrow cells as well as CD4-/CD8- and, and to a lesser extent, more differentiated T cells respond by proliferating to a CD26 specific mAb-mediated costimulus that does not influence dipeptidyl dipeptidase IV enzyme activity. This suggests the existence of a CD26-linked regulatory mechanism of proliferation that is operational on granulocyte- and macrophage-lineage cells and throughout T cell development.

Characterization of a novel rat thymocyte costimulating antigen by the monoclonal antibody 1.3.

To define membrane-associated molecules that impart signals for the activation and expansion of double negative (DN) cells, mAb were raised against in vitro-cultured rat DN cells. One such mAb, 1.3, stimulated proliferation of DN cells along with submitogenic concentrations of PMA and IL-2 without affecting the mobilization of Ca2+. The 1.3 mAb precipitated a heterodimeric protein from DN cells and kidney (130/110 kDa). Although the tissue distribution and biochemical characteristics of the 1.3 determinant resemble the neutral aminopeptidase (AP-N) first described as the thymocyte activating molecule in the mouse, other data are contradictory; AP-N message was not detected in mRNA from 1.3 positive cells and the AP-N gene was absent in the genomic DNA from rat DN hybridomas expressing high levels of 1.3 Ag. In addition, the 1.3 mAb did not affect AP-N enzyme activity suggesting that 1.3 mAb does not function through this enzyme to transduce signals for proliferation. Thus, the 1.3 mAb defines a new and important thymocyte costimulating Ag.

IL-1 induces ornithine decarboxylase in normal T lymphocytes.

IL-1 alpha regulation of ornithine decarboxylase (ODC) was examined in T cells because IL-1 is a costimulus for T cell proliferation and ODC is a critical enzyme in the metabolic events associated with cellular proliferation. In the present study, we demonstrate that IL-1 alpha induces ODC mRNA and ODC enzyme activity in T cells. Unlike many IL-1 actions on T cells, this did not require a costimulus from the TCR, IL-1 alone being sufficient to induce ODC. The mechanism of IL-1 induction of ODC probably operates at several levels, including transcription, mRNA stability, and translation. Previous studies have shown that IL-1 prepares T cells for replication by increasing the production of c-jun, c-fos, c-myc, growth factors, growth factor receptors, and the response to growth factors. From the present study, ODC induction can be added to the list of IL-1-induced replicative machinery in T cells.

IL-1 alpha induces expression of active transforming growth factor-beta in nonproliferating T cells via a post-transcriptional mechanism.

IL-1 is a co-stimulus for T cell proliferation along with TCR cross-linking agents or lectins. Here we demonstrate that IL-1 alpha, in the absence of a TCR stimulus, induces active TGF-beta production by non-proliferating T cells. Thus, in addition to being an incomplete signal, IL-1 alpha alone induced the production of a negative modulator for T cell proliferation. Interestingly, when Con A and IL-1 were combined, active TGF-beta was not detected in supernatants. Production of active TGF-beta protein by T cells in response to IL-1 alpha was established by 1) growth inhibition of the TGF-beta-responsive CCL 64 target cell, 2) neutralization of the activity in supernatants with an anti-TGF-beta mAb, and 3) competition of supernatants with [125I]TGF-beta binding to CCL 64 membranes. The mechanism of IL-1 alpha induction of TGF-beta was examined: IL-1 alpha did not increase TGF beta-1 message over constitutive levels, nor did it induce transcription of TGF-beta 2 message. A latent acid-activatable form of the protein was detected in nonproliferating murine T cells. We therefore suggest that IL-1 alpha regulates TGF-beta expression in T cells through translational and/or post-translational mechanisms. IL-1 regulation of active TGF-beta production by T cells may be a mechanism for controlling the proliferative response of T cells to a co-stimulus, and may be relevant in the pathophysiology of certain chronic inflammatory disease states.

Cutaneous immunofluorescence studies in adult rheumatoid arthritis in sun-exposed and non-sun-exposed areas.

The incidence and significance of positive cutaneous immunofluorescence findings were assessed in biopsy specimens of both sun-exposed and non-sun-exposed skin of 34 adult patients with rheumatoid arthritis (RA) who were not receiving systemic corticosteroids. The incidence of lupus erythematosus (LE)-band was low (8.6%) in both groups. Twenty-eight percent of the patients had perivascular IgM and/or C3 deposits, and 74% had cytoid bodies in the papillary dermis. These studies indicate that the incidence of LE band is low in RA and that the detection of such a band in normal skin warrants close follow-up of RA patients for possible development of LE.