In utero exposure to maternal anti-aquaporin-4 antibodies alters brain vasculature and neural dynamics in male mouse offspring.

The fetal brain is constantly exposed to maternal IgG before the formation of an effective blood-brain barrier (BBB). Here, we studied the consequences of fetal brain exposure to an antibody to the astrocytic protein aquaporin-4 (AQP4-IgG) in mice. AQP4-IgG was cloned from a patient with neuromyelitis optica spectrum disorder (NMOSD), an autoimmune disease that can affect women of childbearing age. We found that embryonic radial glia cells in neocortex express AQP4. These cells are critical for blood vessel and BBB formation through modulation of the WNT signaling pathway. Male fetuses exposed to AQP4-IgG had abnormal cortical vasculature and lower expression of WNT signaling molecules Wnt5a and Wnt7a. Positron emission tomography of adult male mice exposed in utero to AQP4-IgG revealed increased blood flow and BBB leakiness in the entorhinal cortex. Adult male mice exposed in utero to AQP4-IgG had abnormal cortical vessels, fewer dendritic spines in pyramidal and stellate neurons, and more S100ß+ astrocytes in the entorhinal cortex. Behaviorally, they showed impairments in the object-place memory task. Neural recordings indicated that their grid cell system, within the medial entorhinal cortex, did not map the local environment appropriately. Collectively, these data implicate in utero binding of AQP4-IgG to radial glia cells as a mechanism for alterations of the developing male brain and adds NMOSD to the conditions in which maternal IgG may cause persistent brain dysfunction in offspring.

In utero exposure to endogenous maternal polyclonal anti-Caspr2 antibody leads to behavioral abnormalities resembling autism spectrum disorder in male mice.

The concept that exposure in utero to maternal anti-brain antibodies contributes to the development of autism spectrum disorders (ASD) has been entertained for over a decade. We determined that antibodies targeting Caspr2 are present at high frequency in mothers with brain-reactive serology and a child with ASD, and further demonstrated that exposure in utero to a monoclonal anti-Caspr2 antibody, derived from a mother of an ASD child, led to an-ASD like phenotype in male offspring. Now we propose a new model to study the effects of in utero exposure to anti-Caspr2 antibody. Dams immunized with the extracellular portion of Caspr2 express anti-Caspr2 antibodies throughout gestation to better mimic the human condition. Male but not female mice born to dams harboring polyclonal anti-Caspr2 antibodies showed abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as repetitive behaviors and impairments in novelty interest in the social preference test as adults. These data supporting the pathogenicity of anti-Caspr2 antibodies are consistent with the concept that anti-brain antibodies present in women during gestation can alter fetal brain development, and confirm that males are peculiarly susceptible.

Selective Impairment of Spatial Cognition Caused by Autoantibodies to the N-Methyl-D-Aspartate Receptor.

Patients with systemic lupus erythematosus (SLE) experience cognitive abnormalities in multiple domains including processing speed, executive function, and memory. Here we show that SLE patients carrying antibodies that bind DNA and the GluN2A and GluN2B subunits of the N-methyl-d-aspartate receptor (NMDAR), termed DNRAbs, displayed a selective impairment in spatial recall. Neural recordings in a mouse model of SLE, in which circulating DNRAbs penetrate the hippocampus, revealed that CA1 place cells exhibited a significant expansion in place field size. Structural analysis showed that hippocampal pyramidal cells had substantial reductions in their dendritic processes and spines. Strikingly, these abnormalities became evident at a time when DNRAbs were no longer detectable in the hippocampus. These results suggest that antibody-mediated neurocognitive impairments may be highly specific, and that spatial cognition may be particularly vulnerable to DNRAb-mediated structural and functional injury to hippocampal cells that evolves after the triggering insult is no longer present.

Neurotoxic lupus autoantibodies alter brain function through two distinct mechanisms.

Damaging interactions between antibodies and brain antigenic targets may be responsible for an expanding range of neurological disorders. In the case of systemic lupus erythematosus (SLE), patients generate autoantibodies (AAbs) that frequently bind dsDNA. Although some symptoms of SLE may arise from direct reactivity to dsDNA, much of the AAb-mediated damage originates from cross-reactivity with other antigens. We have studied lupus AAbs that bind dsDNA and cross-react with the NR2A and NR2B subunits of the NMDA receptor (NMDAR). In adult mouse models, when the blood-brain barrier is compromised, these NMDAR-reactive AAbs access the brain and elicit neuronal death with ensuing cognitive dysfunction and emotional disturbance. The cellular mechanisms that underlie these deleterious effects remain incompletely understood. Here, we show that, at low concentration, the NMDAR-reactive AAbs are positive modulators of receptor function that increase the size of NMDAR-mediated excitatory postsynaptic potentials, whereas at high concentration, the AAbs promote excitotoxicity through enhanced mitochondrial permeability transition. Other synaptic receptors are completely unaffected by the AAbs. NMDAR activation is required for producing both the synaptic and the mitochondrial effects. Our study thus reveals the mechanisms by which NMDAR-reactive AAbs trigger graded cellular alterations, which are likely to be responsible for the transient and permanent neuropsychiatric symptoms observed in patients with SLE. Our study also provides a model in which local AAb concentration determines the exact nature of the cellular response.

Differential regulation of dopaminergic gene expression by Er81.

A recent study proposed that differentiation of dopaminergic neurons requires a conserved "dopamine motif" (DA-motif) that functions as a binding site for ETS DNA binding domain transcription factors. In the mammalian olfactory bulb (OB), the expression of a set of five genes [including tyrosine hydroxylase (Th)] that are necessary for differentiation of dopaminergic neurons was suggested to be regulated by the ETS-domain transcription factor ER81 via the DA-motif. To investigate this putative regulatory role of ER81, expression levels of these five genes were compared in both olfactory bulbs of adult wild-type mice subjected to unilateral naris closure and the olfactory bulbs of neonatal Er81 wild-type and mutant mice. These studies found that ER81 was necessary only for Th expression and not the other cassette genes. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) experiments showed that ER81 bound directly to a consensus binding site/DA-motif in the rodent Th proximal promoter. However, the ER81 binding site/DA-motif in the Th proximal promoter is poorly conserved in other mammals. Both ChIP assays with canine OB tissue and EMSA experiments with the human Th proximal promoter did not detect ER81 binding to the Th DA-motif from these species. These results suggest that regulation of Th expression by the direct binding of ER81 to the Th promoter is a species-specific mechanism. These findings indicate that ER81 is not necessary for expression of the OB dopaminergic gene cassette and that the DA-motif is not involved in differentiation of the mammalian OB dopaminergic phenotype.

Polyreactive autoantibodies in systemic lupus erythematosus have pathogenic potential.

The present study was undertaken to determine whether germline encoded and polyreactive antibodies might be pathogenic and whether the breach of early tolerance checkpoints in systemic lupus erythematosus (SLE) might lead to a population of B cells expressing germline encoded antibodies that become pathogenic merely by class switching to IgG in a pro-inflammatory milieu. We demonstrate here that IgM, DNA-reactive antibodies obtained from lupus patients that are unmutated and display polyreactivity can bind to isolated glomeruli and exhibit neurotoxic potential. Thus, the IgM polyreactive repertoire in SLE includes antibodies that may acquire pathogenic function merely by undergoing class-switch recombination to become IgG antibodies.

ER81 and CaMKIV identify anatomically and phenotypically defined subsets of mouse olfactory bulb interneurons.

The mechanisms underlying dopamine (DA) phenotypic differentiation in the olfactory bulb (OB) have not yet been fully elucidated and are the subject of some controversy. OB DA interneurons destined for the glomerular layer were shown to originate in the subventricular zone (SVZ) and in the rostral migratory stream (RMS). The current study investigated whether calcium/calmodulin-dependent protein kinase IV (CaMKIV) either alone or together with the Ets transcription factor ER81 was necessary for phenotypic determination during migration of progenitors. In most brain areas, including the OB, CaMKIV and ER81 displayed a reciprocal distribution. In the SVZ, only ER81 could be demonstrated. In the RMS, a subpopulation of progenitors contained ER81, but few, if any, contained CaMKIV. In OB, CaMKIV expression, restricted to deep granule cells, showed limited overlap with ER81. ER81 expression was weak in deep granule cells. Strong labeling occurred in the mitral and glomerular layers, where ER81 colabeled dopaminergic periglomerular cells that expressed either tyrosine hydroxylase (TH) or green fluorescent protein, the latter reporter gene under control of 9-kb of 5' TH promoter. Odor deprivation resulted in a significant 5.2-fold decline in TH immunoreactivity, but ER81 exhibited a relatively small 1.7-fold decline in immunoreactivity. TH expression as well as brain and bulb size were unchanged in CaMKIV knockout mice. These data suggest that ER81 may be required but is not sufficient for DA neuron differentiation and that CaMKIV is not directly involved in TH gene regulation.

Differentiation of the dopaminergic phenotype in the olfactory system of neonatal and adult mice.

Olfactory bulb (OB) interneurons are derived primarily postnatally from progenitors in the anterior subventricular zone (SVZa) and migrate to the OB in the rostral migratory stream (RMS). Progenitors differentiate into phenotypically diverse granule and periglomerular cells by as yet undefined mechanisms. To visualize spatiotemporal aspects of periglomerular dopamine (DA) neuron differentiation, two independently derived transgenic mouse lines were analyzed with a 9-kb tyrosine hydroxylase (TH) promoter to drive either a LacZ or an enhanced green fluorescent protein (EGFP) reporter gene. Both reporters showed similar neonatal expression that varied from low levels in RMS, to moderate in the superficial granule cell layer, to strong in relatively large cells, possibly external tufted cells, in the periglomerular region. TH mRNA and protein were not detected in the RMS but were colocalized with the transgenes in neonatal superficial granule and periglomerular cells. By comparison, TH protein in adults was further limited to periglomerular cells. To demonstrate that transcriptional regulation was the same for EGFP and TH, expression was shown to decline similarly in the OB ipsilateral to odor deprivation produced by adult unilateral naris closure. Of two genes previously hypothesized to regulate OB DA expression, only regulated expression of the orphan receptor Nurr1, but not the homeobox-containing genes Dlx-1 and -2, was consistent with a role in regulation of the DA phenotype. These data demonstrate for the first time that DA phenotypic differentiation in neonates begins with low-level transcription in migrating progenitors in the RMS and culminates with activity-dependent protein expression in periglomerular cells innervated by olfactory receptor cells.

Transient expression of tyrosine hydroxylase promoter/reporter gene constructs in the olfactory epithelium of transgenic mice.

Maturation and survival of olfactory receptor neurons (ORNs) are hypothesized to depend on trophic support from the olfactory bulb during both development and regeneration of the olfactory epithelium (OE). The current study characterized transgene expression in two independently derived transgenic mouse lines in which 9 kb of tyrosine hydroxylase (TH) promoter was utilized to drive either enhanced green fluorescent protein (TH/eGFP) or LacZ (TH/beta-gal) reporters. Transgene expression, found primarily on dorsal aspects of the OE, the dorsal septum and endoturbinate II, resembled the Zone one distribution of olfactory receptor genes. Labeled cells were ovoid to fusiform with dendrites that projected to the epithelial surface but only rarely exhibited discernable cilia. Axons were short and did not extend beyond the basal lamina. As only a subpopulation of the cells contained olfactory marker protein, indicative of ORN maturation, the transgene expressing cells were likely immature neuronal precursors. Demonstration of transgene expression without either TH mRNA or protein was consistent with low basal level transcriptional activity of endogenous TH that may reflect differences between TH and reporter protein stability. Molecules identifying specific olfactory-derived cell populations, PDE2 and LHRH, also did not co-localize with either reporter. A higher than predicted proportion of apoptotic neonatal transgene-expressing cells accounted for their apparent paucity in adult mice. These studies support the concept that transgene expressing cells exhibiting morphological and biochemical characteristics of presumptive ORNs are unable to mature and undergo apoptotic cell death possibly because they lack trophic support.

Dlx-1 and Dlx-2 expression in the adult mouse brain: relationship to dopaminergic phenotypic regulation.

Expression of the homeodomain-containing transcription factors Dlx-1 and Dlx-2 in the lateral (LGE) and medial (MGE) ganglionic eminences, subpallial embryonic structures, is required for generation of telencephalic interneurons. LGE- and MGE-derived progenitors migrate and populate a number of forebrain structures, including the cortex, hippocampus, and olfactory bulb (OB). Previous reports focusing on embryogenesis of telencephalic neurons in Dlx-1 and Dlx-2 null mice suggested a specific role for these genes in expression of the OB dopamine (DA) phenotype. We have investigated whether these genes also are expressed in adult brain, especially in those pallial derivatives, such as the OB, hippocampus, and possibly cortex, where neurogenesis continues in adults. With a highly sensitive, nonradioactive in situ hybridization technique and both DLX-2 and pan DLX antisera, widespread expression of both genes was found in adult mouse fore- but not mid- or hindbrain. The adult unilateral naris closure paradigm was employed to establish a causative role for Dlx in regulating tyrosine hydroxylase (TH) expression; TH is the first enzyme in DA biosynthesis. TH mRNA, but not Dlx expression, was significantly down-regulated in the OB ipsilateral to closure. These findings suggest that Dlx-1 and -2 do not play a direct role in DA phenotypic differentiation and TH gene regulation in adult OB. The widespread expression of Dlx mRNA and protein in the adult brain suggests that these genes may have additional roles in mature animals.