We found a new target for anti-P autoantibody, termed NSPA, that is expressed at the surface of brain neurons and can potentially mediate anti-P–dependent NP symptoms. Sequence data and biochemical characterization showed that NSPA corresponds to a new integral membrane protein of high molecular mass (331 kD). A polyclonal antibody suitable for immunohistochemistry demonstrated that NSPA is expressed by neurons from specific regions of the brain, including regions involved in emotional responses, memory, and other higher brain functions that become affected in neuropshychiatric lupus. In addition, we disclosed for the first time that anti-P autoantibodies have neurotoxic potential, as shown on neurons in culture and in situ. Thus, our results contribute to mechanistic support for the role of anti-P autoantibodies in NP-SLE that has been studied for more than 20 years.
More than 100 autoantibodies have been described in SLE, but very few can be associated with clinical outcomes and even fewer revealed their pathogenic mechanism (1
). Particularly elusive has been the role of autoantibodies in the pathogenesis of NP-SLE (2
). The subset of anti–double-stranded DNA reported to cross react with NMDAR, resulting in apoptotic neuronal loss and cognitive impairment in mice, so far constitute the best documented factor with neuropathogenic potential to mediate CNS dysfunctions (19
). However, the clinical evidence still seems limited (57
). Two series coincidently reported their association with depression but differed in their association with cognitive decline in lupus patients (14
). In contrast, anti-P antibodies had remained for a long time as the most promising candidates for a neuropathogenic role in SLE, supported by numerous clinical data that reported their association with lupus psychosis and/or depression (25
). However, contradictory studies (5
), combined with the lack of any suitable neuronal target or evidence of deleterious actions on neuronal function, have consistently raised doubts about the pathogenic role of anti-P antibodies (25
The following evidence indicates that NSPA is a neuronal target of anti-P antibodies able to mediate neurotoxic effects. First, affinity-purified anti-P antibodies used in biotinylation and immunocapture assays detected NSPA as the only cell-surface target, both in N2a cells and in cortex neurons in primary culture. Experiments of alkaline extraction of synaptosomal membranes also showed NSPA as the only protein recognized by anti-P antibodies. Second, peptide competition blocked the interaction of anti-P antibodies with the cell surface, indicating that NSPA exposes a P epitope at the cell surface, very likely conformed by two separate regions encompassing residues 644DDLG647 and 2881GLFE2884. Third, affinity-purified anti-P antibodies from two SLE patients with psychosis and rabbit anti-NSPA antibodies both triggered calcium influx into neurons in primary culture showing specific target dependency, as demonstrated by the corresponding blocking peptide. Neither anti-P nor anti-NSPA antibodies elicited calcium influx in astrocytes that do not express NSPA. Fourth, anti-P antibodies elicited caspase-3 activation and neuronal death both in vitro and in the rat brain in vivo. Finally, experiments with rabbit anti-P antibodies raised against the 11 ribosomal C-terminal residues containing the P epitopes mimicked the reactivity and the effects of both α-hP11 and α-NSPA antibodies, including the recognition of cell surface and synaptosomal NSPA, and the induction of calcium influx and apoptosis in neurons (unpublished data). Collectively, these results indicate that anti-P antibodies interact with NSPA through a P epitope exposed at the cell surface, and as a consequence of such interaction, neurons experience enhanced calcium entry and undergo apoptosis.
Our anti-NSPA antibodies allowed us to define the distribution of a cell-surface protein bearing a P epitope in the brain. In immunohistochemistry, we found NSPA being expressed by neurons at specific brain regions, including neocortical layers II, V, and VI, and other zones of relevance for the pathogenesis of NP-SLE, such as the amygdala, which is involved in arousal and emotional responses (58
), the cortex and hippocampus, which are involved in memory and higher brain functions (59
), and the ventral tegmental area, which is associated with reward processing and drug addiction (60
). A recent study reported that anti-P antibodies selectively stained certain limbic regions of the mice brain (36
), but it did not clarify whether the immunostaining involved neuronal or glial cell bodies, or axons, and did not identify the antigen recognized by anti-P antibodies. Anti-P antibodies might decorate ribosomes in cells that could become inadvertently permeabilized during the immunohistochemistry. Instead, our α-NSPA antibody only showed immunohistochemical staining in neurons (neural bodies), and in some places we could distinguish immunostaining in axon terminals (synaptic buttons), including the calyx-like axon terminal in the central nucleus of the amygdala. Controls indicating that the immunohistochemical staining corresponds specifically to NSPA included competition with the immunogenic NSPA peptide and correlation with immunoblot detection in synaptosomes obtained from either the cortex or striatum, which displayed or lacked α-NSPA staining, respectively. Thus, our present work not only identifies a new P antigen that is expressed at the neuronal cell surface but also discloses in great detail its distribution in the brain. At least through interaction with NSPA, anti-P antibodies could provoke serious dysfunctions in the CNS and contribute to developing NP disorders.
Because not all SLE patients that produce anti-P antibodies develop an NP disease, an important yet unsolved question is whether anti-P antibodies from SLE patients that do not manifest psychiatric or other diffuse CNS symptoms are functionally equivalent to those produced by SLE patients with psychiatric compromise. We showed that anti-P antibodies from SLE patients without NP disease not only recognize NSPA but also induce calcium influx and apoptosis in neurons, indicating that they possess an equivalent pathogenic potential as psychiatrically associated anti-P antibodies. Therefore, besides the production of anti-P antibodies, additional risk factors are certainly required for eliciting psychiatric manifestations.
Both accessibility into the brain and the presence of other autoantibodies very likely cooperate in developing NP-SLE. Experiments in mice that produce or have been injected with antibodies against NMDAR recently highlighted the requirement of disrupting the blood–brain barrier (BBB) for autoantibody-mediated brain symptoms, which varied according to the region of the BBB breach (21
). Evidence of antibody production in situ in the brain and passage across the BBB have been described in SLE patients (22
). There are also several conditions that promote BBB breach, including infection, stress, hypertension, and nicotine exposure (21
), which can vary among patients. In addition, the variety of NP symptoms in SLE patients very likely involves coexpression of autoantibodies with direct or indirect neuropathogenic potential, such as anti-NMDAR antibodies (19
). In this paper, we show that anti-NSPA antibodies share with α-hp11
antibodies the capacity to trigger neuronal calcium influx. Future studies should determine whether SLE patients produce autoantibodies against NSPA, which might be distinct from anti-P antibodies. All of these factors could contribute to the NP outcome of SLE.
The identification of new neuronal surface molecules as autoimmune targets in the CNS also entails general interest. Disorders affecting the neuromuscular junction involve autoantibodies against nicotinic acetylcholine receptors in myasthenia gravis, calcium channels in the Lambert-Eaton syndrome, and potassium channels in Isaac's syndrome (18
). Whether autoantibodies underlie certain CNS disorders remains less apparent, as they would require a mechanism to access CNS targets (22
). Autoantibody targets with pathogenic potential include the glutamate receptor 3 in Rasmussen's encephalitis (64
), metabotropic glutamate receptor 1 in paraneoplastic cerebellar ataxia (65
), the NMDAR in SLE (19
), and neuropeptides in eating disorders (66
). The protein that we described in this study, NSPA, is a candidate for alterations caused either by autoantibodies or mutations that may eventually promote psychiatric disease, not only in SLE. Although the function of NSPA is unknown, we can speculate, by analogy with the mentioned disorders, that it may be related to cell-surface receptors and/or ion channels.
In summary, we have contributed a mechanistic link to the vast body of clinical evidence supporting a role of anti-P antibodies in psychiatric lupus that can explain why they might be pathogenic. Deleterious calcium influxes can be triggered by anti-P antibodies in brain neuronal cells through interaction with NSPA at specific brain zones.