Molecular mimicry: anti-DNA antibodies bind microbial and nonnucleic acid self-antigens.

Although cells of the innate immune response have a variety of pattern recognition receptors that are triggered by blood classes of markers, a critical feature of the adaptive immune response is antigenic specificity. Yet it is becoming increasingly clear that the specificity of lymphocyte receptors admits of some laxity. Cross-reactivity may, in fact, be necessary for lymphocyte survival as antigen receptor signaling maintains cellular viability in the absence of antigen activation. Studies of molecular mimicry have revealed many instances in which antibodies to microbial antigens bind also to self-antigens; in some cases, this cross-reactivity has pathogenic potential. In this chapter, we describe cross-reactivity between two self-antigens, DNA and NMDA receptors, and how antibodies with specificity for DNA in patients with splenic lupus may cause central nervous system damage by virtue of binding also to neuronal receptors. This example serves as a reminder that cross-reactivity may exist among self-antigens as well as between foreign and self-antigens.

Cell-type specific signal transduction and gene regulation via mitogen-activated protein kinase pathway in catecholaminergic neurons by restraint stress.

It has been demonstrated that tyrosine hydroxylase (TH) gene is easily regulated in the CNS as well as peripheral nervous systems by stressful conditions. The stimuli, such as stress or reserpine administration, significantly increased the TH gene in noradrenergic neurons in the locus ceruleus (LC), but not in dopaminergic neurons in the substantia nigra (SN). To explore the molecular mechanisms governing differential TH gene regulation in catecholaminergic cells, the present study investigated the regulation of immediate early gene (c-Fos), transcription factors (pCREB, CREB binding protein [CBP]), mitogen-activated protein (MAP) kinases (phospho-extra-cellular regulated kinase [pERK]1/2, phospho-p38 MAP kinase [p-p38 MAPK], phospho-c-Jun N-terminal kinase [pJNK]) in the LC and SN in control conditions and in response to 2 h restraint stress (RS). Significant induction of c-Fos expression was observed in the LC, but not in the SN. In addition, pERK1/2 significantly increased following 2 h RS specifically in the LC, but not in the SN. No significant change was observed in p-p38 MAPK and pJNK. The expression of c-Fos and pERK1/2 preceded the upregulation of TH in the LC. Furthermore, pCREB and CBP also increased in the LC in response to 2 h RS. The induction of c-Fos prior to TH, in conjunction with the upregulation of pCREB and CBP in the LC, suggests that activator protein 1 and CRE transcription sites in the TH gene may be involved in the cell-type specific activation in the stress response, at least, by pERK1/2.

Temporal and sequential analysis of microglia in the substantia nigra following medial forebrain bundle axotomy in rat.

Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis after transection of the medial forebrain bundle. We have assessed the temporal and sequential activities of microglia in these events by examining the complement-3 (OX-42), major histocompatibility complex class II antigen presentation (OX-6) and phagocytic activity (ED1), and correlating these indicators with dopaminergic neuronal loss. Microglia in the ipsilateral substantia nigra pars reticulata evinced activation morphology at 12 h postaxotomy. Phagocytic microglia apposed dying dopaminergic neurons in the pars compacta starting at 3 days postlesion; their number increased through 14 days and slowly decreased. Nuclear chromatin condensation and significant loss of tyrosine hydroxylase-positive dopaminergic neurons occurred around 7 days postlesion. In contrast to microglial expression of interleukin-1beta and inducible nitric oxide synthase at the axotomy site, nigral microglia were interleukin-1beta and inducible nitric oxide synthase-negative. Consistently, RNase protection assays showed that interleukin-1beta and inducible nitric oxide synthase transcripts in nigra were equivocal. The present data support the idea that phagocytosis of axotomized neurons by activated microglia is not limited to dead neurons but includes dying neurons probably without cytotoxic effects of inflammatory substances, such as interleukin-1beta or nitric oxide.

A subset of lupus anti-DNA antibodies cross-reacts with the NR2 glutamate receptor in systemic lupus erythematosus.

In systemic lupus erythematosus, antibodies against double-stranded DNA are a major contributor to renal disease. We have previously demonstrated that the pentapeptide Asp/Glu-Trp-Asp/Glu-Tyr-Ser/Gly is a molecular mimic of double-stranded DNA. This sequence is also present in the extracellular domain of murine and human NMDA (N-methyl-D-aspartate) receptor subunits NR2a and NR2b. Here we show that the NR2 receptor is recognized by both murine and human anti-DNA antibodies. Moreover, anti-DNA antibodies with this cross-reactivity mediate apoptotic death of neurons in vivo and in vitro. Finally, we show that the cerebrospinal fluid of a patient with systemic lupus erythematosus contains these antibodies and also mediates neuronal death via an apoptotic pathway. These observations indicate that lupus antibodies cross-react with DNA and NMDA receptors, gain access to cerebrospinal fluid and may mediate non-thrombotic and non-vasculitic abnormalities of the central nervous system.

Dopaminergic cell death induced by MPP(+), oxidant and specific neurotoxicants shares the common molecular mechanism.

Recent etiological study in twins (Tanner et al. 1999) strongly suggests that environmental factors play an important role in typical, non-familial Parkinson's disease (PD), beginning after age 50. Epidemiological risk factor analyses of typical PD cases have identified several neurotoxicants, including MPP(+) (the active metabolite of MPTP), paraquat, dieldrin, manganese and salsolinol. Here, we tested the hypothesis that these neurotoxic agents might induce cell death in our nigral dopaminergic cell line, SN4741 (Son et al. 1999) through a common molecular mechanism. Our initial experiments revealed that treatment with both MPP(+) and the other PD-related neurotoxicants induced apoptotic cell death in SN4741 cells, following initial increases of H(2)O(2)-related ROS activity and subsequent activation of JNK1/2 MAP kinases. Moreover, we have demonstrated that during dopaminergic cell death cascades, MPP(+), the neurotoxicants and an oxidant, H(2)O(2) equally induce the ROS-dependent events. Remarkably, the oxidant treatment alone induced similar sequential molecular events: ROS increase, activation of JNK MAP kinases, activation of the PITSLRE kinase, p110, by both Caspase-1 and Caspase-3-like activities and apoptotic cell death. Pharmacological intervention using the combination of the antioxidant Trolox and a pan-caspase inhibitor Boc-(Asp)-fmk (BAF) exerted significant neuroprotection against ROS-induced dopaminergic cell death. Finally, the high throughput cDNA microarray screening using the current model identified downstream response genes, such as heme oxygenase-1, a constituent of Lewy bodies, that can be the useful biomarkers to monitor the pathological conditions of dopaminergic neurons under neurotoxic insult.

Neurotoxic APP C-terminal and beta-amyloid domains colocalize in the nuclei of substantia nigra pars reticulata neurons undergoing delayed degeneration.

Increased amyloid precursor protein (APP) expression and intracellular accumulation of its toxic fragments have been associated with acute neuronal death processes. However, the role of APP fragments in delayed neurodegeneration remains poorly understood. We have characterized the appearance of APP domains in rat substantia nigra pars reticulata (SNpR) neurons targeted for delayed degeneration following neurotoxic striatal lesion. From 4 to 8 days postlesion (dpl) SNpR neurons ipsilateral to the lesion showed marked cytosolic accumulation of full length APP. Moreover, the nuclei of affected neurons also showed intense immunoreactivity (IR) for APP C-terminal and beta-amyloid domains but not for an N-terminal sequence. These data suggested the presence of APP C-terminal fragment. The absence of nuclear IR for a beta1-40 specific antibody supports this conclusion. Ultrastructural analysis of nigral sections from 6 dpl rats using a beta-amyloid domain antibody showed pronounced accumulation of immunogold-silver reaction product in the nuclei of affected SNpR neurons that was absent in control, contralateral SNpR neurons. These findings suggest that intranuclear APP C-terminal fragment may play a role in genomic events contributing to delayed neuron degeneration in the SNpR.

Dizocilpine maleate, MK-801, but not 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline, NBQX, prevents transneuronal degeneration of nigral neurons after neurotoxic striatal-pallidal lesion.

Unilateral neurotoxin lesion of rat caudate-putamen and globus pallidus resulted in delayed, transneuronal degeneration of GABAergic substantia nigra pars reticulata neurons. To explore whether the disinhibition of endogenous glutamate excitatory input played a role in the degeneration of substantia nigra pars reticulata neurons, animals with unilateral striatal-pallidal lesions received three daily intraperitoneal injections of either dizocilpine maleate (MK-801, 1 or 10 mg/kg), an N-methyl-D-aspartate glutamate receptor blocker, or 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX, 30 mg/kg), an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor blocker, that began 24 h after the striatal-pallidal neurotoxin lesion. Drug treatment affected neither the volume of the initial lesion nor the volume of striatal-pallidal glial fibrillary acidic protein immunoreactivity. Neuron number in the substantia nigra pars reticulata ipsilateral to the lesioned striatopallidum was reduced on average by 37% in untreated control rats, in low dose MK-801, and NBQX-treated rats (P<0.0001). However, in animals treated with high doses of MK-801 there was no difference in the number of neurons in the substantia nigra pars reticulata ipsilateral or contralateral to the neurotoxin lesion. These data demonstrate that dose-related treatment with N-methyl-D-aspartate glutamate receptor blockers protects substantia nigra pars reticulata neurons, and suggests that glutamatergic mechanisms play a role in delayed transneuronal degeneration.

Histological and temporal characteristics of nigral transneuronal degeneration after striatal injury.

Neurotoxic injury of the caudate-putamen and lateral globus pallidus unilaterally initiated transneuronal degeneration of neurons in the ipsilateral substantia nigra reticulata (SNR). Quantification of SNR neurons using unbiased stereology demonstrated that neuron loss began 4 days after the initial striatal lesion, followed by significant loss (50%) at 6 days and a plateau at 8 days. Analysis at the light and ultrastructural levels revealed morphological changes consistent with a type of programmed cell death. These temporal and histological results refine an in vivo model in which to explore mechanisms of delayed neuronal degeneration.

Implantation of cultured human leptomeningeal cells into rat brain.

Since previous studies have shown that cells cultured from human leptomeninges can express neuronal and glial antigens under appropriate culture conditions [DeGiorgio L. A. et al. (1994) J. Neurol. Sci. 124, 141 148; Bernstein J. J. et al. (1996) Int. J. Derl Neurosci. 14(5), 681 687], we have studied the developmental characteristics of these cells further by grafting them into young adult rat brains. Cells were labeled in culture with Fast Blue and were identified unequivocally by hybridization with nick-translated human DNA. Intensely Fast Blue positive human leptomeningeal cells were concentrated in the implant pocket and adjacent rat leptomeninges al one and two weeks postimplant. Human and rat leptomeningeal cells were similar morphologically and were equally immunopositive for vimentin and fibronectin. Implanted human cells did not express the neuronal and glial proteins they had in vitro. Cells which hybridized with human DNA corresponded to the intensely Fast Blue positive cells. Small groups of human DNA hybridizing cells were also observed in the choroid plexus. Less intensely Fast Blue positive neurons and glia were found in the brain but these hybridized with rat DNA. A minority of human leptomeningeal cells implanted into rat brain are subsequently found in host leptomeninges where they demonstrate properties characteristic of leptomeningeal fibroblasts. Small numbers of implanted cells can survive for two weeks.

Human A beta-amyloid and amyloid precursor protein accumulates in rat brain cells after cultured human leptomeningeal fibroblast implants.

Cultured human leptomeningeal fibroblasts grafted into rat frontal cortex were localized to the implant pocket and to adjacent host leptomeninges. Immunohistochemical studies using a panel of human-specific and domain-specific APP antibodies revealed that all grafted cells expressed both APP and A beta in situ. Remarkably, these antibodies also labeled rat pial and ependymal cells as well as reactive astrocytes adjacent to vessels. In addition, apical projections and cell bodies of many cortical pyramidal neurons contained human-specific APP immunoreactive material. Groups of subcortical neurons, particularly those of the amygdala, hippocampal formation and suprachiasmatic nuclei, were similarly labeled. The presence of human APP in host brains was confirmed by immunoblotting. Birefringent Congo Red staining was observed in the cortical neuropil and in leptomeningeal vessels. These data indicate that grafted leptomeningeal fibroblasts hyperexpress APP and A beta which can diffuse into parenchyma and be taken up by specific rat cells.

Human leptomeningeal-derived cells express GFAP and HLADR when grafted into rat spinal cord.

The following series of experiments explores the post-xenografting differentiation of a naturally occurring, non-neuronal cell cultured from the leptomeninges of an 84-year-old woman. In culture, flat process-bearing human cells from the leptomeninges were positive for GFAP and 200 kDa neurofilament protein (negative for 68, 160 kDa neurofilament protein). The C3 spinal cord was exposed in 30 adult athymic rats. The hindlimb dorsal columns were transected at C3 and the nerve fibers aspirated to form a pocket, into which 10(6) fast blue-labeled, human leptomeningeal-derived cells were placed. The C3 spinal cord was studied immunohistochemically over 60 days. Three days later the dorsal horn contained fast blue-GFAP-positive astrocyte-like cells that were negative for neurofilament protein. By 7 days, large, process-bearing, fast blue-GFAP-positive (neurofilament protein-negative), astrocyte-like cells joined the native astrocytes of the pia-glia membrane and were in the gray matter of the spinal cord. Some of these astrocyte-like cells were also positive for the human specific histocompatibility complex, HLADR. These data extend the age, species and tissue of origin for pluripotential cells for CNS transplantation.

Culture from human leptomeninges of cells containing neurofilament protein and neuron-specific enolase.

Actively dividing cells cultured from human leptomeninges obtained at autopsy and from human pia obtained at neurosurgery expressed two antigens characteristic of a mature neuronal phenotype: the 160 kDa neurofilament protein (NF-M) and neuron-specific enolase (NSE). The cells lacked typical glial, epithelial and endothelial markers but did contain vimentin, a protein normally associated with but not limited to mesenchymally derived cell types. Immunocytochemical results using redundant antibodies were consistent over serial passage and were confirmed by Western blot analysis. Morphologically the cells were pleiomorphic with frequent long, NF-M + processes. The antigenic characteristics of the cultured leptomeningeal and pial cells were similar to those exhibited by human autopsy and biopsy brain-derived cultures described previously, suggesting that the latter may have originated from pial cells adherent to the autopsy and biopsy brain tissue specimens.

Expression of neuronal proteins in cells from normal adult rat brain propagated in serial culture.

Cells have been cultured from the brains of 60-day-old rats and propagated through 12 passages. The cells contain the high and middle, but not low, molecular weight neurofilament subunits and neuron-specific enolase, demonstrated by immunoblotting and immunocytochemistry with redundant antibodies. The cells did not have the morphology of neurons when cultured in medium containing fetal calf serum and growth factors. In low serum medium containing the same growth factors with the addition of dibutyryl cyclic AMP, the cells became smaller and developed long processes. Three clonal lines derived from these cultures had the same properties. These observations are in agreement with recent observations using mouse and human brain tissue and demonstrate that proteins normally associated with neurons can be found in dividing cells cultured from the brains of young adult rats.