Ulrike Weber-Stadlbauer

Translational evaluation of translocator protein as a marker of neuroinflammation in schizophrenia

Positron emission tomography (PET) imaging with radiotracers that target translocator protein 18 kDa (TSPO) has become a popular approach to assess putative neuroinflammatory processes and associated microglia activation in psychotic illnesses. It remains unclear, however, whether TSPO imaging can accurately capture low-grade inflammatory processes such as those present in schizophrenia and related disorders. Therefore, we evaluated the validity of TSPO as a disease-relevant marker of inflammation using a translational approach, which combined neurodevelopmental and neurodegenerative mouse models with PET imaging in patients with recent-onset schizophrenia and matched controls. Using an infection-mediated neurodevelopmental mouse model, we show that schizophrenia-relevant behavioral abnormalities and increased inflammatory cytokine expression are associated with reduced prefrontal TSPO levels. On the other hand, TSPO was markedly upregulated in a mouse model of acute neurodegeneration and reactive gliosis, which was induced by intrahippocampal injection of kainic acid. In both models, the changes in TSPO levels were not restricted to microglia but emerged in various cell types, including microglia, astrocytes and vascular endothelial cells. Human PET imaging using the second-generation TSPO radiotracer [11C]DPA-713 revealed a strong trend towards reduced TSPO binding in the middle frontal gyrus of patients with recent-onset schizophrenia, who were previously shown to display increased levels of inflammatory cytokines in peripheral and central tissues. Together, our findings challenge the common assumption that central low-grade inflammation in schizophrenia is mirrored by increased TSPO expression or ligand binding. Our study further underscores the need to interpret altered TSPO binding in schizophrenia with caution, especially when measures of TSPO are not complemented with other markers of inflammation. Unless more selective microglial markers are available for PET imaging, quantification of cytokines and other inflammatory biomarkers, along with their molecular signaling pathways, may be more accurate in attempts to characterize inflammatory profiles in schizophrenia and other mental disorders that lack robust reactive gliosis.

Prenatal immune activation causes hippocampal synaptic deficits in the absence of overt microglia anomalies.

Prenatal exposure to infectious or inflammatory insults can increase the risk of developing neuropsychiatric disorder in later life, including schizophrenia, bipolar disorder, and autism. These brain disorders are also characterized by pre- and postsynaptic deficits. Using a well-established mouse model of maternal exposure to the viral mimetic polyriboinosinic-polyribocytidilic acid [poly(I:C)], we examined whether prenatal immune activation might cause synaptic deficits in the hippocampal formation of pubescent and adult offspring. Based on the widely appreciated role of microglia in synaptic pruning, we further explored possible associations between synaptic deficits and microglia anomalies in offspring of poly(I:C)-exposed and control mothers. We found that prenatal immune activation induced an adult onset of presynaptic hippocampal deficits (as evaluated by synaptophysin and bassoon density). The early-life insult further caused postsynaptic hippocampal deficits in pubescence (as evaluated by PSD95 and SynGAP density), some of which persisted into adulthood. In contrast, prenatal immune activation did not change microglia (or astrocyte) density, nor did it alter their activation phenotypes. The prenatal manipulation did also not cause signs of persistent systemic inflammation. Despite the absence of overt glial anomalies or systemic inflammation, adult offspring exposed to prenatal immune activation displayed increased hippocampal IL-1β levels. Taken together, our findings demonstrate that age-dependent synaptic deficits and abnormal pro-inflammatory cytokine expression can occur during postnatal brain maturation in the absence of microglial anomalies or systemic inflammation.

Genome-wide DNA Methylation Changes in a Mouse Model of Infection-Mediated Neurodevelopmental Disorders

BACKGROUND Prenatal exposure to infectious or inflammatory insults increases the risk of neurodevelopmental disorders. Using a well-established mouse model of prenatal viral-like immune activation, we examined whether this pathological association involves genome-wide DNA methylation differences at single nucleotide resolution. METHODS Prenatal immune activation was induced by maternal treatment with the viral mimetic polyriboinosinic-polyribocytidylic acid in middle or late gestation. Following behavioral and cognitive characterization of the adult offspring (n = 12 per group), unbiased capture array bisulfite sequencing was combined with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantitative real-time polymerase chain reaction analyses to quantify DNA methylation changes and transcriptional abnormalities in the medial prefrontal cortex of immune-challenged and control offspring. Gene ontology term enrichment analysis was used to explore shared functional pathways of genes with differential DNA methylation. RESULTS Adult offspring of immune-challenged mothers displayed hyper- and hypomethylated CpGs at numerous loci and at distinct genomic regions, including genes relevant for gamma-aminobutyric acidergic differentiation and signaling (e.g., Dlx1, Lhx5, Lhx8), Wnt signaling (Wnt3, Wnt8a, Wnt7b), and neural development (e.g., Efnb3, Mid1, Nlgn1, Nrxn2). Altered DNA methylation was associated with transcriptional changes of the corresponding genes. The epigenetic and transcriptional effects were dependent on the offsprings age and were markedly influenced by the precise timing of prenatal immune activation. CONCLUSIONS Prenatal viral-like immune activation is capable of inducing stable DNA methylation changes in the medial prefrontal cortex. These long-term epigenetic modifications are a plausible mechanism underlying the disruption of prefrontal gene transcription and behavioral functions in subjects with prenatal infectious histories.

Hypervulnerability of the adolescent prefrontal cortex to nutritional stress via reelin deficiency

Overconsumption of high-fat diets (HFDs) can critically affect synaptic and cognitive functions within telencephalic structures such as the medial prefrontal cortex (mPFC). The underlying mechanisms, however, remain largely unknown. Here we show that adolescence is a sensitive period for the emergence of prefrontal cognitive deficits in response to HFD. We establish that the synaptic modulator reelin (RELN) is a critical mediator of this vulnerability because (1) periadolescent HFD (pHFD) selectively downregulates prefrontal RELN+ cells and (2) augmenting mPFC RELN levels using transgenesis or prefrontal pharmacology prevents the pHFD-induced prefrontal cognitive deficits. We further identify N-methyl-d-aspartate-dependent long-term depression (NMDA-LTD) at prefrontal excitatory synapses as a synaptic signature of this association because pHFD abolishes NMDA-LTD, a function that is restored by RELN overexpression. We believe this study provides the first mechanistic insight into the vulnerability of the adolescent mPFC towards nutritional stress, such as HFDs. Our findings have primary relevance to obese individuals who are at an increased risk of developing neurological cognitive comorbidities, and may extend to multiple neuropsychiatric and neurological disorders in which RELN deficiency is a common feature.

Epigenetic and transgenerational mechanisms in infection-mediated neurodevelopmental disorders

Prenatal infection is an environmental risk factor for various brain disorders with neurodevelopmental components, including autism spectrum disorder and schizophrenia. Modeling this association in animals shows that maternal immune activation negatively affects fetal brain development and leads to the emergence of behavioral disturbances later in life. Recent discoveries in these preclinical models suggest that epigenetic modifications may be a critical molecular mechanism by which prenatal immune activation can mediate changes in brain development and functions, even across generations. This review discusses the potential epigenetic mechanisms underlying the effects of prenatal infections, thereby highlighting how infection-mediated epigenetic reprogramming may contribute to the transgenerational transmission of pathological traits. The identification of epigenetic and transgenerational mechanisms in infection-mediated neurodevelopmental disorders appears relevant to brain disorders independently of existing diagnostic classifications and may help identifying complex patterns of transgenerational disease transmission beyond genetic inheritance. The consideration of ancestral infectious histories may be of great clinical interest and may be pivotal for developing new preventive treatment strategies against infection-mediated neurodevelopmental disorders.

Cognitive effects of subdiaphragmatic vagal deafferentation in rats

&NA; Vagal afferents are a crucial neuronal component of the gut‐brain axis and mediate the information flow from the viscera to the central nervous system. Based on the findings provided by experiments involving vagus nerve stimulation, it has been suggested that vagal afferent signaling may influence various cognitive functions such as recognition memory and cognitive flexibility. Here, we examined this hypothesis using a rat model of subdiaphragmatic vagal deafferentation (SDA), the most complete and selective abdominal vagal deafferentation method existing to date. We found that SDA did not affect working memory in a nonspatial alternation task, nor did it influence short‐, intermediate‐, and long‐term object recognition memory. SDA did also not affect the acquisition of positively reinforced left‐right discrimination learning, but it facilitated the subsequent reversal left‐right discrimination learning. The SDA‐induced effects on reversal learning emerged in the absence of concomitant changes in motivation towards the positive reinforcer, indicating selective effects on cognitive flexibility. Taken together, these findings suggest that the relative contribution of vagal afferent signaling to cognitive functions is limited. At the same time, our study demonstrates that cognitive flexibility, at least in the domains of positively reinforced learning, is subjected to visceral modulation through abdominal vagal afferents. HighlightsAbdominal vagal deafferentation facilitates positively motivated reversal learning.Abdominal vagal deafferentation does not affect object recognition memory.Abdominal vagal deafferentation does not affect working memory.

Mouse models of maternal immune activation: Mind your caging system!

Rodent models of maternal immune activation (MIA) are increasingly used as experimental tools to study neuronal and behavioral dysfunctions in relation to infection-mediated neurodevelopmental disorders. One of the most widely used MIA models is based on gestational administration of poly(I:C) (= polyriboinosinic-polyribocytdilic acid), a synthetic analog of double-stranded RNA that induces a cytokine-associated viral-like acute phase response. The effects of poly(I:C)-induced MIA on phenotypic changes in the offspring are known to be influenced by various factors, including the precise prenatal timing, genetic background, and immune stimulus intensity. Thus far, however, it has been largely ignored whether differences in the basic type of laboratory housing can similarly affect the outcomes of MIA models. Here, we examined this possibility by comparing the poly(I:C)-based MIA model in two housing systems that are commonly used in preclinical mouse research, namely the open cage (OC) and individually ventilated cage (IVC) systems. Pregnant C57BL6/N mice were kept in OCs or IVCs and treated with a low (1 mg/kg, i.v.) or high (5 mg/kg, i.v.) dose of poly(I:C), or with control vehicle solution. MIA or control treatment was induced on gestation day (GD) 9 or 12, and the resulting offspring were raised and maintained in OCs or IVCs until adulthood for behavioral testing. An additional cohort of dams was used to assess the influence of the different caging systems on poly(I:C)-induced cytokine and stress responses in the maternal plasma. Maternal poly(I:C) administration on GD9 caused a dose-dependent increase in spontaneous abortion in IVCs but not in OCs, whereas MIA in IVC systems during a later gestational time-point (GD12) did not affect pregnancy outcomes. Moreover, the precise type of caging system markedly affected maternal cytokines and chemokines at basal states and in response to poly(I:C) and further influenced the maternal levels of the stress hormone, corticosterone. The efficacy of MIA to induce deficits in working memory, social interaction, and sensorimotor gating in the adult offspring was influenced by the different housing conditions, the dosing of poly(I:C), and the precise prenatal timing. Taken together, the present study identifies the basic type of caging system as a novel factor that can confound the outcomes of MIA in mice. Our findings thus urge the need to consider and report the kind of laboratory housing systems used to implement MIA models. Providing this information seems pivotal to yield reproducible results in these models.

Prenatal exposure to TiO 2 nanoparticles in mice causes behavioral deficits with relevance to autism spectrum disorder and beyond

Environmental factors are involved in the etiology of autism spectrum disorder (ASD) and may contribute to the raise in its incidence rate. It is currently unknown whether the increasing use of nanoparticles such as titanium dioxide (TiO2 NPs) in consumer products and biomedical applications may play a role in these associations. While nano-sized TiO2 is generally regarded as safe and non-toxic, excessive exposure to TiO2 NPs may be associated with negative health consequences especially when occurring during sensitive developmental periods. To test if prenatal exposure to TiO2 NPs alters fetal development and behavioral functions relevant to ASD, C57Bl6/N dams were subjected to a single intravenous injection of a low (100 µg) or high (1000 µg) dose of TiO2 NPs or vehicle solution on gestation day 9. ASD-related behavioral functions were assessed in the offspring using paradigms that index murine versions of ASD symptoms. Maternal exposure to TiO2 NPs led to subtle and dose-dependent impairments in neonatal vocal communication and juvenile sociability, as well as a dose-dependent increase in prepulse inhibition of the acoustic startle reflex of both sexes. These behavioral alterations emerged in the absence of pregnancy complications. Prenatal exposure to TiO2 NPs did not cause overt fetal malformations or changes in pregnancy outcomes, nor did it affect postnatal growth of the offspring. Taken together, our study provides a first set of preliminary data suggesting that prenatal exposure to nano-sized TiO2 can induce behavioral deficits relevant to ASD and related neurodevelopmental disorders without inducing major changes in physiological development. If extended further, our preclinical findings may provide an incentive for epidemiological studies examining the role of prenatal TiO2 NPs exposure in the etiology of ASD and other neurodevelopmental disorders.

Abdominal Vagal Afferents Modulate the Brain Transcriptome and Behaviors Relevant to Schizophrenia

Reduced activity of vagal efferents has long been implicated in schizophrenia and appears to be responsible for diminished parasympathetic activity and associated peripheral symptoms such as low heart rate variability and cardiovascular complications in affected individuals. In contrast, only little attention has been paid to the possibility that impaired afferent vagal signaling may be relevant for the disorders pathophysiology as well. The present study explored this hypothesis using a model of subdiaphragmatic vagal deafferentation (SDA) in male rats. SDA represents the most complete and selective vagal deafferentation method existing to date as it leads to complete disconnection of all abdominal vagal afferents while sparing half of the abdominal vagal efferents. Using next-generation mRNA sequencing, we show that SDA leads to brain transcriptional changes in functional networks annotating with schizophrenia. We further demonstrate that SDA induces a hyperdopaminergic state, which manifests itself as increased sensitivity to acute amphetamine treatment and elevated accumbal levels of dopamine and its major metabolite, 3,4-dihydroxyphenylacetic acid. Our study also shows that SDA impairs sensorimotor gating and the attentional control of associative learning, which were assessed using the paradigms of prepulse inhibition and latent inhibition, respectively. These data provide converging evidence suggesting that the brain transcriptome, dopamine neurochemistry, and behavioral functions implicated in schizophrenia are subject to visceral modulation through abdominal vagal afferents. Our findings may encourage the further establishment and use of therapies for schizophrenia that are based on vagal interventions. SIGNIFICANCE STATEMENT The present work provides a better understanding of how disrupted vagal afferent signaling can contribute to schizophrenia-related brain and behavioral abnormalities. More specifically, it shows that subdiaphragmatic vagal deafferentation (SDA) in rats leads to (1) brain transcriptional changes in functional networks related to schizophrenia, (2) increased sensitivity to dopamine-stimulating drugs and elevated dopamine levels in the nucleus accumbens, and (3) impairments in sensorimotor gating and the attentional control of associative learning. These findings may encourage the further establishment of novel therapies for schizophrenia that are based on vagal interventions.

40.2 MATERNAL IMMUNE ACTIVATION LEADS TO INCREASED LEVELS OF INFLAMMATORY CYTOKINES IN THE ABSENCE OF OVERT MICROGLIA ANOMALIES IN THE MIDBRAIN

Abstract Background Inflammatory theories in schizophrenia have gained increasing recognition and acceptance in recent years. The evidence supporting a role of altered inflammatory processes in the etiology and pathophysiology of schizophrenia involves early-life exposure to infectious pathogens or inflammatory stimuli, increased expression of cytokines and other mediators of inflammation in the adult central nervous system (CNS) and periphery, as well as signs of glial anomalies. Given the role of dopaminergic deregulation in the pathophysiology of schizophrenia, inflammatory processes in the midbrain may contribute to dopamine abnormalities in the midbrain and its subcortical and cortical output regions. Here, we tested this hypothesis using an established neurodevelopmental mouse model with relevance to schizophrenia, namely the maternal immune activation (MIA) model. Methods Pregnant C57BL6/N mice on gestation day 17 were treated with the viral mimetic polyriboinosinic-polyribocytidilic acid (poly(I:C)) or vehicle control solution. We then quantified the gene transcripts of an array of pro-inflammatory cytokines, acute phase proteins, and dopaminergic markers in the midbrain of MIA offspring (N=32) and control offspring (N= 32) at adult age. We also assessed the cell density of microglial cells expressing Iba1 and CD68 by immunohistochemistry to ascertain whether putative inflammatory changes are accompanied by microglia anomalies. Given the large sample sizes, we performed two-step recursive cluster analyses in order to identify possible subgroups of offspring that are characterized by “high” and “low” inflammatory profiles. Results When considering the entire treatment group, MIA-exposed offspring displayed significantly increased expression of several inflammatory cytokines in the ventral midbrain, including IL-1b (p < 0.01), TNF-a (p < 0.01) and SERPINA3 (p < 0.01). These inflammatory changes occurred in the absence of overt microglia anomalies but were paralleled by changes in dopaminergic markers. The two-step cluster analyses further identified subgroups of MIA-exposed offspring that are characterized by a “high” (41 %, N = 13) and “low” (59 %, N = 19) inflammatory profiles. The “high” inflammatory subgroup of MIA-exposed offspring was defined by marked elevations of SERPINA3, IL-1β, IL-6, and TNFα mRNA levels (all p’s < 0.01). Discussion Maternal immune activation during pregnancy causes persistent signs of inflammation in the offspring’s midbrain. In agreement with post-mortem studies in schizophrenia, these inflammatory abnormalities are clearly noticeable in a subgroup of MIA-exposed offspring only. Hence, prenatal immune activation may be one of the factors inducing lasting inflammatory changes relevant to (some cases of) schizophrenia and may contribute to dopaminergic dysfunctions in this disorder.