Jeffrey L. Bennett

Detection of autoantibodies to neural cells of the cerebellum in the plasma of subjects with autism spectrum disorders

Autism spectrum disorders (ASD) are a group of heterogeneous, behaviorally defined disorders characterized by disturbances in social interaction and communication, often with repetitive and stereotyped behavior. Previous studies have described the presence of antibodies to various neural proteins in autistic individuals as well as post-mortem evidence of neuropathology in the cerebellum. We examined plasma from children with ASD, as well as age-matched typically developing controls, for antibodies directed against human cerebellar protein extracts using Western blot analysis. In addition, the presence of cerebellar specific antibodies was assessed by immunohistochemical staining of sections from Macaca fascicularis monkey cerebellum. Western blot analysis revealed that 13/63 (21%) of subjects with ASD possessed antibodies that demonstrated specific reactivity to a cerebellar protein with an apparent molecular weight of approximately 52 kDa compared with only 1/63 (2%) of the typically developing controls (p=0.0010). Intense immunoreactivity, to what was determined morphologically to be the Golgi cell of the cerebellum, was noted for 7/34 (21%) of subjects with ASD, compared with 0/23 of the typically developing controls. Furthermore, there was a strong association between the presence of antibodies reactive to the 52 kDa protein by Western blot with positive immunohistochemical staining of cerebellar Golgi cells in the ASD group (r=0.76; p=0.001) but not controls. These studies suggest that when compared with age-matched typically developing controls, children with ASD exhibit a differential antibody response to specific cells located in the cerebellum and this response may be associated with a protein of approximately 52 kDa.

Autoantibodies in Autism Spectrum Disorders (ASD)

Abstract:  Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders defined behaviorally by abnormalities in social, verbal, and nonverbal communication. The etiologies of ASD are unknown, likely to be the result of a variety of numerous genetic, neurological, environmental, and immunological interactions that lead to a general behavioral phenotype defined as ASD. This review will focus on the various immune system anomalies, in particular, autoantibodies, which have been reported in subjects with ASD. In addition, we will discuss recent studies performed by our group concerning the presence of autoantibodies directed against neural antigens, which are observed in patients with ASD.

A comprehensive transcriptional map of primate brain development

The transcriptional underpinnings of brain development remain poorly understood, particularly in humans and closely related non-human primates. We describe a high-resolution transcriptional atlas of rhesus monkey (Macaca mulatta) brain development that combines dense temporal sampling of prenatal and postnatal periods with fine anatomical division of cortical and subcortical regions associated with human neuropsychiatric disease. Gene expression changes more rapidly before birth, both in progenitor cells and maturing neurons. Cortical layers and areas acquire adult-like molecular profiles surprisingly late in postnatal development. Disparate cell populations exhibit distinct developmental timing of gene expression, but also unexpected synchrony of processes underlying neural circuit construction including cell projection and adhesion. Candidate risk genes for neurodevelopmental disorders including primary microcephaly, autism spectrum disorder, intellectual disability, and schizophrenia show disease-specific spatiotemporal enrichment within developing neocortex. Human developmental expression trajectories are more similar to monkey than rodent, although approximately 9% of genes show human-specific regulation with evidence for prolonged maturation or neoteny compared to monkey.

Further characterization of autoantibodies to GABAergic neurons in the central nervous system produced by a subset of children with autism

BackgroundAutism is a neurodevelopmental disorder characterized by impairments in social interaction and deficits in verbal and nonverbal communication, together with the presence of repetitive behaviors or a limited repertoire of activities and interests. The causes of autism are currently unclear. In a previous study, we determined that 21% of children with autism have plasma autoantibodies that are immunoreactive with a population of neurons in the cerebellum that appear to be Golgi cells, which are GABAergic interneurons.MethodsWe have extended this analysis by examining plasma immunoreactivity in the remainder of the brain. To determine cell specificity, double-labeling studies that included one of the calcium-binding proteins that are commonly colocalized in GABAergic neurons (calbindin, parvalbumin or calretinin) were also carried out to determine which GABAergic neurons are immunoreactive. Coronal sections through the rostrocaudal extent of the macaque monkey brain were reacted with plasma from each of seven individuals with autism who had previously demonstrated positive Golgi cell staining, as well as six negative controls. In addition, brain sections from adult male mice were similarly examined.ResultsIn each case, specific staining was observed for neurons that had the morphological appearance of interneurons. By double-labeling sections with plasma and with antibodies directed against γ-aminobutyric acid (GABA), we determined that all autoantibody-positive neurons were GABAergic. However, not all GABAergic neurons were autoantibody-positive. Calbindin was colabeled in several of the autoantibody-labeled cells, while parvalbumin colabeling was less frequently observed. Autoantibody-positive cells rarely expressed calretinin. Sections from the mouse brain processed similarly to the primate sections also demonstrated immunoreactivity to interneurons distributed throughout the neocortex and many subcortical regions. Some cell populations stained in the primate (such as the Golgi neurons in the cerebellum) were not as robustly immunoreactive in the mouse brain.ConclusionsThese results suggest that the earlier report of autoantibody immunoreactivity to specific cells in the cerebellum extend to other regions of the brain. Further, these findings confirm the autoantibody-targeted cells to be a subpopulation of GABAergic interneurons. The potential impact of these autoantibodies on GABAergic disruption with respect to the etiology of autism is discussed herein.

The Rhesus Monkey Connectome Predicts Disrupted Functional Networks Resulting from Pharmacogenetic Inactivation of the Amygdala

Contemporary research suggests that the mammalian brain is a complex system, implying that damage to even a single functional area could have widespread consequences across the system. To test this hypothesis, we pharmacogenetically inactivated the rhesus monkey amygdala, a subcortical region with distributed and well-defined cortical connectivity. We then examined the impact of that perturbation on global network organization using resting-state functional connectivity MRI. Amygdala inactivation disrupted amygdalocortical communication and distributed corticocortical coupling across multiple functional brain systems. Altered coupling was explained using a graph-based analysis of experimentally established structural connectivity to simulate disconnection of the amygdala. Communication capacity via monosynaptic and polysynaptic pathways, in aggregate, largely accounted for the correlational structure of endogenous brain activity and many of the non-local changes that resulted from amygdala inactivation. These results highlight the structural basis of distributed neural activity and suggest a strategy for linking focal neuropathology to remote neurophysiological changes.

Conserved molecular signatures of neurogenesis in the hippocampal subgranular zone of rodents and primates

The neurogenic potential of the subgranular zone (SGZ) of the hippocampal dentate gyrus is likely to be regulated by molecular cues arising from its complex heterogeneous cellular environment. Through transcriptome analysis using laser microdissection coupled with DNA microarrays, in combination with analysis of genome-wide in situ hybridization data, we identified 363 genes selectively enriched in adult mouse SGZ. These genes reflect expression in the different constituent cell types, including progenitor and dividing cells, immature granule cells, astrocytes, oligodendrocytes and GABAergic interneurons. Similar transcriptional profiling in the rhesus monkey dentate gyrus across postnatal development identified a highly overlapping set of SGZ-enriched genes, which can be divided based on temporal profiles to reflect maturation of glia versus granule neurons. Furthermore, we identified a neurogenesis-related gene network with decreasing postnatal expression that is highly correlated with the declining number of proliferating cells in dentate gyrus over postnatal development. Many of the genes in this network showed similar postnatal downregulation in mouse, suggesting a conservation of molecular mechanisms underlying developmental and adult neurogenesis in rodents and primates. Conditional deletion of Sox4 and Sox11, encoding two neurogenesis-related transcription factors central in this network, produces a mouse with no hippocampus, confirming the crucial role for these genes in regulating hippocampal neurogenesis.

Spatiotemporal dynamics of the postnatal developing primate brain transcriptome

Developmental changes in the temporal and spatial regulation of gene expression drive the emergence of normal mature brain function, while disruptions in these processes underlie many neurodevelopmental abnormalities. To solidify our foundational knowledge of such changes in a primate brain with an extended period of postnatal maturation like in human, we investigated the whole-genome transcriptional profiles of rhesus monkey brains from birth to adulthood. We found that gene expression dynamics are largest from birth through infancy, after which gene expression profiles transition to a relatively stable state by young adulthood. Biological pathway enrichment analysis revealed that genes more highly expressed at birth are associated with cell adhesion and neuron differentiation, while genes more highly expressed in juveniles and adults are associated with cell death. Neocortex showed significantly greater differential expression over time than subcortical structures, and this trend likely reflects the protracted postnatal development of the cortex. Using network analysis, we identified 27 co-expression modules containing genes with highly correlated expression patterns that are associated with specific brain regions, ages or both. In particular, one module with high expression in neonatal cortex and striatum that decreases during infancy and juvenile development was significantly enriched for autism spectrum disorder (ASD)-related genes. This network was enriched for genes associated with axon guidance and interneuron differentiation, consistent with a disruption in the formation of functional cortical circuitry in ASD.

Neural Reorganization Due to Neonatal Amygdala Lesions in the Rhesus Monkey: Changes in Morphology and Network Structure

It is generally believed that neural damage that occurs early in development is associated with greater adaptive capacity relative to similar damage in an older individual. However, few studies have surveyed whole brain changes following early focal damage. In this report, we employed multimodal magnetic resonance imaging analyses of adult rhesus macaque monkeys who had previously undergone bilateral, neurotoxic lesions of the amygdala at about 2 weeks of age. A deformation-based morphometric approach demonstrated reduction of the volumes of the anterior temporal lobe, anterior commissure, basal ganglia, and pulvinar in animals with early amygdala lesions compared to controls. In contrast, animals with early amygdala lesions had an enlarged cingulate cortex, medial superior frontal gyrus, and medial parietal cortex. Diffusion-weighted imaging tractography and network analysis were also used to compare connectivity patterns and higher-level measures of communication across the brain. Using the communicability metric, which integrates direct and indirect paths between regions, lesioned animals showed extensive degradation of network integrity in the temporal and orbitofrontal cortices. This work demonstrates both degenerative as well as progressive large-scale neural changes following long-term recovery from neonatal focal brain damage.

Cytoarchitectonically‐driven MRI atlas of nonhuman primate hippocampus: Preservation of subfield volumes in aging

Identification of primate hippocampal subfields in vivo using structural MRI imaging relies on variable anatomical guidelines, signal intensity differences, and heuristics to differentiate between regions (Yushkevich et al., 2015a). Thus, a clear anatomically‐driven basis for subfield demarcation is lacking. Recent work, however, has begun to develop methods to use ex vivo histology or ex vivo MRI (Adler et al., 2014; Iglesias et al., 2015) that have the potential to inform subfield demarcations of in vivo images. For optimal results, however, ex vivo and in vivo images should ideally be matched within the same healthy brains, with the goal to develop a neuroanatomically‐driven basis for in vivo structural MRI images. Here, we address this issue in young and aging rhesus macaques (young n = 5 and old n = 5) using ex vivo Nissl‐stained sections in which we identified the dentate gyrus, CA3, CA2, CA1, subiculum, presubiculum, and parasubiculum guided by morphological cell properties (30 μm thick sections spaced at 240 μm intervals and imaged at 161 nm/pixel). The histologically identified boundaries were merged with in vivo structural MRIs (0.625 × 0.625 × 1 mm) from the same subjects via iterative rigid and diffeomorphic registration resulting in probabilistic atlases of young and old rhesus macaques. Our results indicate stability in hippocampal subfield volumes over an age range of 13 to 32 years, consistent with previous results showing preserved whole hippocampal volume in aged macaques (Shamy et al., 2006). Together, our methods provide a novel approach for identifying hippocampal subfields in non‐human primates and a potential ‘ground truth’ for more accurate identification of hippocampal subfield boundaries on in vivo MRIs. This could, in turn, have applications in humans where accurately identifying hippocampal subfields in vivo is a critical research goal.

T35. DIFFUSION MEASURES OF EXTRACELLULAR FREE WATER IN A NON-HUMAN PRIMATE MODEL OF MATERNAL IMMUNE ACTIVATION: EXPLORING NEUROIMMUNE MECHANISMS OF PSYCHIATRIC DISORDERS

Abstract Background Evidence has been accumulating for an immune-based component of psychiatric disorder etiology, particularly schizophrenia. Early epidemiological studies found an increased incidence of schizophrenia in offspring of mothers who had an infection during pregnancy. Recent work has identified genetic links to the MHC complex, pro-inflammatory cytokine elevations in cerebrospinal fluid and plasma. We have developed a non-human primate (NHP) model of maternal immune activation (MIA) using a modified form of the viral mimic polyIC (polyICLC) examine the relationship between altered neuorimmune function may contribute to psychosis risk through effects on the developing brain and behavior of NHP offspring. In a previous cohort of MIA-exposed offspring, our group observed evidence for increased pre-synaptic dopamine levels in the striatum using 6-[18F]fluoro-L-m-tyrosine (FMT) positron emission tomography, in addition to pubertal-onset behavioral abnormalities, which may model part of the neurodevelopmental pathway towards psychosis. This study builds on this model and examines the effect of maternal immune activation on in vivo--extracellular free water--a diffusion magnetic resonance imaging measure obtained with a multi-shell acquisition. We sought to test the hypothesis that offspring of pregnant monkeys who received polyICLC injections at the end of the first trimester would show increased extracellular free water compared to control offspring. Methods Fourteen pregnant rhesus monkeys (Macaca mulatta) receiving polyICLC at the end of the first trimester were compared to 14 controls. The offspring from both groups underwent a multi-shell diffusion MRI scan at 3 Tesla. Diffusion data was collected when the offspring were one month, 6 months, and 12 months of age. Six month preliminary findings are currently presented. Diffusion images were aligned to individual subject MPRAGE scans. Individual subject structural scans were then nonlinearly aligned to generate a common group average template and the group average template was subsequently nonlinearly aligned to a neurodevelopmental rhesus atlas. For this preliminary analysis, the frontal cortex was selected as an a priori region of interest in addition to the more global whole-brain gray and white matter masks. Results Six month old MIA-exposed rhesus offspring showed a trend for increased whole-brain white matter extracellular free water (p=.09) with no significant difference in whole-brain gray matter free water (p=.27) compared to control offspring. However, analysis of the frontal ROI revealed significantly increased gray matter free water in the left hemisphere (p=.013) with a trend towards increased gray matter free water in the right hemisphere (p=.081). There were no significant differences between MIA-exposed and control offspring in basic motor and reflex development or growth trajectories. Discussion These data suggest that despite the lack of behavioral abnormalities at this early age, extracellular free water values are increased in MIA-exposed offspring, particularly in frontal gray matter. More global whole-brain free water group differences did not reach statistical significance, which may indicate some regional specificity to these changes early in development. The NHP MIA model complements the human schizophrenia literature in which extracellular free water increases have been repeatedly identified. Ultimately, these data provide validation of the clinical relevance of the NHP MIA model and improve our understanding of neuroimmune mechanisms in the development of psychiatric disorders, particularly schizophrenia.