Irene Knuesel

The time of prenatal immune challenge determines the specificity of inflammation-mediated brain and behavioral pathology.

Disturbance to early brain development is implicated in several neuropsychiatric disorders including autism, schizophrenia, and mental retardation. Epidemiological studies have indicated that the risk of developing these disorders is enhanced by prenatal maternal infection, presumably as a result of neurodevelopmental defects triggered by cytokine-related inflammatory events. Here, we demonstrate that the effects of maternal immune challenge between middle and late gestation periods in mice are dissociable in terms of fetal brain cytokine responses to maternal inflammation and the pathological consequences in brain and behavior. Specifically, the relative expression of pro- and anti-inflammatory cytokines in the fetal brains in response to maternal immune challenge may be an important determinant among other developmental factors for the precise pathological profile emerging in later life. Thus, the middle and late gestation periods correspond to two windows with differing vulnerability to adult behavioral dysfunction, brain neuropathology in early adolescence, and of the acute cytokine responses in the fetal brain.

Adult brain and behavioral pathological markers of prenatal immune challenge during early/middle and late fetal development in mice.

Maternal infection during pregnancy increases the risk for neurodevelopmental disorders such as schizophrenia and autism in the offspring. This association appears to be critically dependent on the precise prenatal timing. However, the extent to which distinct adult psychopathological and neuropathological traits may be sensitive to the precise times of prenatal immune activation remains to be further characterized. Here, we evaluated in a mouse model of prenatal immune challenge by the viral mimic, polyriboinosinic-polyribocytidilic acid (PolyIC), whether prenatal immune activation in early/middle and late gestation may influence the susceptibility to some of the critical cognitive, pharmacological, and neuroanatomical dysfunctions implicated in schizophrenia and autism. We revealed that PolyIC-induced prenatal immune challenge on gestation day (GD) 9 but not GD17 significantly impaired sensorimotor gating and reduced prefrontal dopamine D1 receptors in adulthood, whereas prenatal immune activation specifically in late gestation impaired working memory, potentiated the locomotor reaction to the NMDA-receptor antagonist dizocilpine, and reduced hippocampal NMDA-receptor subunit 1 expression. On the other hand, potentiation of the locomotor reaction to the dopamine-receptor agonist amphetamine and reduction in Reelin- and Parvalbumin-expressing prefrontal neurons emerged independently of the precise times of prenatal immune challenge. Our findings thus highlight that prenatal immune challenge during early/middle and late fetal development in mice leads to distinct brain and behavioral pathological symptom clusters in adulthood. Further examination and evaluation of in utero immune challenge at different times of gestation may provide important new insight into the neuroimmunological and neuropathological mechanisms underlying the segregation of different symptom clusters in heterogeneous neuropsychiatric disorders such as schizophrenia and autism.

Altered synaptic clustering of GABAA receptors in mice lacking dystrophin (mdx mice)

Dystrophin is selectively localized in the postsynaptic density of neurons in cerebral cortex, hippocampus and cerebellum. Here, we show by double‐immunofluorescence staining that dystrophin is extensively colocalized with GABAA receptor subunit clusters in these brain regions. To determine the relevance of this observation, we investigated in mdx mice, which provide a model of Duchenne muscular dystrophy, whether the absence of dystrophin affects the synaptic clustering of GABAA receptors. A marked reduction in the number of clusters immunoreactive for the α1 and α2 subunits was observed in, respectively, cerebellum and hippocampus of mdx mice, but not in striatum, which is normally devoid of dystrophin. Furthermore, these alterations were not accompanied by a change in gephyrin staining, although gephyrin is colocalized with the majority of GABAA receptor clusters in these regions. These results indicate that dystrophin may play an important role in the clustering or stabilization of GABAA receptors in a subset of central inhibitory synapses. These deficits may underlie the cognitive impairment seen in Duchenne patients.

Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice

BackgroundAlzheimer’s disease (AD) is the most prevalent form of age-related dementia, and its effect on society increases exponentially as the population ages. Accumulating evidence suggests that neuroinflammation, mediated by the brain’s innate immune system, contributes to AD neuropathology and exacerbates the course of the disease. However, there is no experimental evidence for a causal link between systemic inflammation or neuroinflammation and the onset of the disease.MethodsThe viral mimic, polyriboinosinic-polyribocytidilic acid (PolyI:C) was used to stimulate the immune system of experimental animals. Wild-type (WT) and transgenic mice were exposed to this cytokine inducer prenatally (gestation day (GD)17) and/or in adulthood. Behavioral, immunological, immunohistochemical, and biochemical analyses of AD-associated neuropathologic changes were performed during aging.ResultsWe found that a systemic immune challenge during late gestation predisposes WT mice to develop AD-like neuropathology during the course of aging. They display chronic elevation of inflammatory cytokines, an increase in the levels of hippocampal amyloid precursor protein (APP) and its proteolytic fragments, altered Tau phosphorylation, and mis-sorting to somatodendritic compartments, and significant impairments in working memory in old age. If this prenatal infection is followed by a second immune challenge in adulthood, the phenotype is strongly exacerbated, and mimics AD-like neuropathologic changes. These include deposition of APP and its proteolytic fragments, along with Tau aggregation, microglia activation and reactive gliosis. Whereas Aβ peptides were not significantly enriched in extracellular deposits of double immune-challenged WT mice at 15 months, they dramatically increased in age-matched immune-challenged transgenic AD mice, precisely around the inflammation-induced accumulations of APP and its proteolytic fragments, in striking similarity to the post-mortem findings in human patients with AD.ConclusionChronic inflammatory conditions induce age-associated development of an AD-like phenotype in WT mice, including the induction of APP accumulations, which represent a seed for deposition of aggregation-prone peptides. The PolyI:C mouse model therefore provides a unique tool to investigate the molecular mechanisms underlying the earliest pathophysiological changes preceding fibrillary Aβ plaque deposition and neurofibrillary tangle formations in a physiological context of aging. Based on the similarity between the changes in immune-challenged mice and the development of AD in humans, we suggest that systemic infections represent a major risk factor for the development of AD.

Relative Prenatal and Postnatal Maternal Contributions to Schizophrenia-Related Neurochemical Dysfunction after In Utero Immune Challenge

Prenatal exposure to infections represents a risk factor for the emergence of neuropsychiatric disorders in later life, including schizophrenia and autism. However, it remains essentially unknown whether this association is primarily attributable to prenatal and/or postnatal maternal effects on the offspring. Here, we addressed this issue by dissecting the relative contributions of prenatal inflammatory events and postnatal maternal factors in an animal model of prenatal viral-like infection. Pregnant mice were exposed to the inflammatory agent polyriboinosinic-polyribocytidilic acid (PolyI:C; 5 mg/kg, i.v.) or vehicle treatment on gestation day 9, and offspring born to PolyI:C- and vehicle-treated dams were cross fostered to surrogate rearing mothers that had either experienced inflammatory or sham treatment during pregnancy. We demonstrate that a variety of dopamine- and glutamate-related pharmacological and neuroanatomical disturbances emerge after prenatal immune challenge regardless of whether neonates were raised by vehicle- or PolyI:C-exposed surrogate mothers. However, the adoption of prenatal control animals to immune-challenged surrogate mothers was also sufficient to induce specific pharmacological and neuroanatomical abnormalities in the fostered offspring. Multiple schizophrenia-related dysfunctions emerging after prenatal immune challenge are thus mediated by prenatal but not postnatal maternal effects on the offspring, but immunological stress during pregnancy may affect postpartum maternal factors in such a way that being reared by an immune-challenged surrogate mother can confer risk for distinct forms of psychopathology in adult life.

SynGAP Regulates Spine Formation

SynGAP is a brain-specific ras GTPase-activating protein that is an abundant component of the signaling complex associated with the NMDA-type glutamate receptor. We generated mutant mice lacking synGAP to study its physiological role. Homozygous mutant mice die in the first few days after birth; however, neurons from mutant embryos can be maintained in culture. Here, we report that spine and synapse formation are accelerated in cultured mutant neurons, and the spines of mature mutant neurons are significantly larger than those of wild type. Clusters of PSD-95 and subunits of AMPA-type and NMDA-type glutamate receptors accumulate in spines of mutant neurons by day 10 in vitro, whereas in wild-type neurons they are still mostly located in dendritic shafts. The frequency and amplitude of miniature EPSCs are larger in mutant neurons at day 10 in vitro, confirming that they have more functional synapses. At day 21 in vitro, the spines of mutant neurons remain significantly larger than those of wild type. The mutant phenotype at day 10 in vitro can be rescued by introduction of recombinant wild-type synGAP on day 9. In contrast, introduction of mutant synGAP with a mutated GAP domain or lacking the terminal domain that binds to PSD-95 does not rescue the mutant phenotype, indicating that both domains play a role in control of spine formation. Thus, the GAP activity of synGAP and its association with PSD-95 are important for normal regulation of spine and synapse formation in hippocampal neurons.

Disruption of glycine transporter 1 restricted to forebrain neurons is associated with a procognitive and antipsychotic phenotypic profile.

The NMDA receptor is thought to play a central role in some forms of neuronal plasticity, including the induction of long-term potentiation. NMDA receptor hypofunction can result in mnemonic impairment and has been implicated in the cognitive symptoms of schizophrenia. The activity of NMDA receptors is controlled by its endogenous coagonist glycine, and a local elevation of glycine levels is expected to enhance NMDA receptor function. Here, we achieved this by the generation of a novel mouse line (CamKIIαCre;Glyt1tm1.2fl/fl) with a neuron and forebrain selective disruption of glycine transporter 1 (GlyT1). The mutation led to a significant reduction of GlyT1 and a corresponding reduction of glycine reuptake in forebrain samples, without affecting NMDA receptor expression. NMDA (but not AMPA) receptor-evoked EPSCs recorded in hippocampal slices of mutant mice were 2.5 times of those recorded in littermate controls, suggesting that neuronal GlyT1 normally assumes a specific role in the regulation of NMDA receptor responses. Concomitantly, the mutants were less responsive to phencyclidine than controls. The mutation enhanced aversive Pavlovian conditioning without affecting spontaneous anxiety-like behavior in the elevated plus maze and augmented a form of attentional learning called latent inhibition in three different experimental paradigms: conditioned freezing, conditioned active avoidance, conditioned taste aversion. The CamKIIαCre;Glyt1tm1.2fl/fl mouse model thus suggests that augmentation of forebrain neuronal glycine transmission is promnesic and may also offer an effective therapeutic intervention against the cognitive and attentional impairments characteristic of schizophrenia.

Maternal immune activation during pregnancy increases limbic GABAA receptor immunoreactivity in the adult offspring: Implications for schizophrenia

Prenatal exposures to a variety of infections have been associated with an increased incidence of schizophrenia. We have reported that a single injection of the synthetic cytokine releaser PolyI:C to pregnant mice produced offspring that exhibited multiple schizophrenia-related behavioral deficits in adulthood. Here, we characterized the effect of maternal inflammation during fetal brain development on adult limbic morphology and expression of GABAA-receptors. The PolyI:C treatment did not induce morphological abnormalities but resulted in a significant increase in GABAA receptor subunit alpha2 immunoreactivity (IR) in the ventral dentate gyrus and basolateral amygdala in adult treated compared to control subjects. Correlative analyses between the a2 subunit IR in the ventral dentate gyrus and the performance in the prepulse inhibition paradigm revealed a significant correlation in controls that was however absent in the pathological condition. These results suggest that prenatal immune activation-induced disturbances of early brain development result in profound alterations in the limbic expression of GABAA receptors that may underlie the schizophrenia-related behavioral deficits in the adult mice.

Reduced Reelin Expression Accelerates Amyloid-β Plaque Formation and Tau Pathology in Transgenic Alzheimer's Disease Mice

In addition to the fundamental role of the extracellular glycoprotein Reelin in neuronal development and adult synaptic plasticity, alterations in Reelin-mediated signaling have been suggested to contribute to neuronal dysfunction associated with Alzheimers disease (AD). In vitro data revealed a biochemical link between Reelin-mediated signaling, Tau phosphorylation, and amyloid precursor protein (APP) processing. To directly address the role of Reelin in amyloid-β plaque and Tau pathology in vivo, we crossed heterozygous Reelin knock-out mice (reeler) with transgenic AD mice to investigate the temporal and spatial AD-like neuropathology. We demonstrate that a reduction in Reelin expression results in enhanced amyloidogenic APP processing, as indicated by the precocious production of amyloid-β peptides, the significant increase in number and size of amyloid-β plaques, as well as age-related aggravation of plaque pathology in double mutant compared with single AD mutant mice of both sexes. Numerous amyloid-β plaques accumulate in the hippocampal formation and neocortex of double mutants, precisely in layers with strongest Reelin expression and highest accumulation of Reelin plaques in aged wild-type mice. Moreover, concentric accumulations of phosphorylated Tau-positive neurons around amyloid-β plaques were evident in 15-month-old double versus single mutant mice. Silver stainings indicated the presence of neurofibrillary tangles, selectively associated with amyloid-β plaques and dystrophic neurites in the entorhinal cortex and hippocampus. Our findings suggest that age-related Reelin aggregation and concomitant reduction in Reelin-mediated signaling play a proximal role in synaptic dysfunction associated with amyloid-β deposition, sufficient to enhance Tau phosphorylation and tangle formation in the hippocampal formation in aged Reelin-deficient transgenic AD mice.

Long-term effects of early life deprivation on brain glia in fischer rats

Both clinical and experimental studies have indicated that depression and depression-like animal conditions are associated with disruption of the intrinsic plasticity of the brain, resulting in neuronal atrophy. However, little is known about the brain glia in these conditions. Early life stress in the form of infant abuse or neglect constitutes a risk factor in the aetiology of major depressive disorder in later life. It is possible to model this relation between early life stress and depression in the rat through maternal deprivation; in adulthood, this postnatal manipulation is known to lead to depression-like behaviour. In the stress-hyperresponsive Fischer strain, P1-14 pups were isolated for 4 h/day (early deprivation, ED, n=6) or were nonhandled (NH, n=6); they were left undisturbed until adulthood. Postmortem quantitative analysis of regional astroglial distribution and morphology based on glial fibrillary acidic protein (GFAP) immunohistochemistry indicated a significant effect of ED on the density of GFAP-reactive astrocytes in brain areas implicated in stress-related behaviour. A moderate (10-22%) but consistent reduction in GFAP-reactive astrocyte density was seen in dorsal dentate gyrus, prefrontal cortex, ventral hippocampal CA1, cingulate cortex, dorsal hippocampal CA1 and basolateral amygdala. The ED-related reduction in GFAP-immunoreactive astrocyte density was more marked than the reduction in total cell density, which suggests that GFAP immunoreactivity, rather than the number of astrocytes, was reduced. This study provides evidence that early life stress leads to long-term changes in the density of astroglia in the brain regions involved in stress responses in the rat.