Pleiotropic effects of trisomy and pharmacologic modulation on structural, functional, molecular, and genetic systems in a Down syndrome mouse model.

Down syndrome (DS) is characterized by skeletal and brain structural malformations, cognitive impairment, altered hippocampal metabolite concentration and gene expression imbalance. These alterations were usually investigated separately, and the potential rescuing effects of green tea extracts enriched in epigallocatechin-3-gallate (GTE-EGCG) provided disparate results due to different experimental conditions. We overcame these limitations by conducting the first longitudinal controlled experiment evaluating genotype and GTE-EGCG prenatal chronic treatment effects before and after treatment discontinuation. Our findings revealed that the Ts65Dn mouse model reflected the pleiotropic nature of DS, exhibiting brachycephalic skull, ventriculomegaly, neurodevelopmental delay, hyperactivity, and impaired memory robustness with altered hippocampal metabolite concentration and gene expression. GTE-EGCG treatment modulated most systems simultaneously but did not rescue DS phenotypes. On the contrary, the treatment exacerbated trisomic phenotypes including body weight, tibia microarchitecture, neurodevelopment, adult cognition, and metabolite concentration, not supporting the therapeutic use of GTE-EGCG as a prenatal chronic treatment. Our results highlight the importance of longitudinal experiments assessing the co-modulation of multiple systems throughout development when characterizing preclinical models in complex disorders and evaluating the pleiotropic effects and general safety of pharmacological treatments.

An immunological puzzle: The adaptive immune system fuels Alzheimer's disease pathology.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by a concerning rise in prevalence. It is projected that the number of affected individuals will reach a staggering 150 million by 2050. While recent advancements in monoclonal antibodies targeting Aß have shown some clinical effects, there is an urgent need for improved therapies to effectively address the impeding surge of AD patients worldwide. To achieve this, a deeper understanding of the intricate mechanisms underlying the disease is crucial. In recent years, mounting evidence has underscored the vital role of the innate immune system in AD pathology. However, limited findings persist regarding the involvement of the adaptive immune system. Here, we report on the impact of the adaptive immune system on various aspects of AD by using AppNL-G-F mice crossed into a Rag2-/- background lacking mature adaptive immune cells. In addition, to simulate the continuous exposure to various challenges such as infections that is commonly observed in humans, the innate immune system was activated through the repetitive induction of peripheral inflammation. We observed a remarkably improved performance on complex cognitive tasks when a mature adaptive immune system is absent. Notably, this observation is pathologically associated with lower Aß plaque accumulation, reduced glial activation, and better-preserved neuronal networks in the mice lacking a mature adaptive immune system. Collectively, these findings highlight the detrimental role of the adaptive immune system in AD and underscore the need for effective strategies to modulate it for therapeutic purposes.

Neurobeachin haploinsufficient mice display sex-independent alterations in cued and contextual fear conditioning.

Neurobeachin ( NBEA ) is a cytoplasmic protein that regulates receptor trafficking, neurotransmitter and hormone secretion, as well as synaptic connectivity. Recently, hippocampus-dependent contextual extinction, the gradual decrease of a conditioned fear response to a context, was suggested to be specifically impaired in male mice with Nbea deficiency ( Nbea+/- ). The current study examines the role of sex in this effect and whether Nbea also influences cued fear conditioning. We included both female and male mice and used a phased contextual and cued fear acquisition protocol that consists of different phases allowing us to assess fear acquisition, cued and contextual fear memory and within-phase extinction. Performance of Nbea+/- mice during assessment of both contextual and cued fear memory was significantly altered compared to controls, independent of sex. Follow-up analyses revealed that this altered performance could be indicative of impaired within-phase extinction. Altered within-phase extinction was not exclusively attributable to hippocampus, and independent of sex. Our results rather suggest that Nbea influences complex learning more broadly across different brain structures.

Molecular and cognitive signatures of ageing partially restored through synthetic delivery of IL2 to the brain.

Cognitive decline is a common pathological outcome during aging, with an ill-defined molecular and cellular basis. In recent years, the concept of inflammaging, defined as a low-grade inflammation increasing with age, has emerged. Infiltrating T cells accumulate in the brain with age and may contribute to the amplification of inflammatory cascades and disruptions to the neurogenic niche observed with age. Recently, a small resident population of regulatory T cells has been identified in the brain, and the capacity of IL2-mediated expansion of this population to counter neuroinflammatory disease has been demonstrated. Here, we test a brain-specific IL2 delivery system for the prevention of neurological decline in aging mice. We identify the molecular hallmarks of aging in the brain glial compartments and identify partial restoration of this signature through IL2 treatment. At a behavioral level, brain IL2 delivery prevented the age-induced defect in spatial learning, without improving the general decline in motor skill or arousal. These results identify immune modulation as a potential path to preserving cognitive function for healthy aging.

Green Tea Catechins Modulate Skeletal Development with Effects Dependent on Dose, Time, and Structure in a down Syndrome Mouse Model.

Altered skeletal development in Down syndrome (DS) results in a brachycephalic skull, flattened face, shorter mandibular ramus, shorter limbs, and reduced bone mineral density (BMD). Our previous study showed that low doses of green tea extract enriched in epigallocatechin-3-gallate (GTE-EGCG), administered continuously from embryonic day 9 to postnatal day 29, reduced facial dysmorphologies in the Ts65Dn (TS) mouse model of DS, but high doses could exacerbate them. Here, we extended the analyses to other skeletal structures and systematically evaluated the effects of high and low doses of GTE-EGCG treatment over postnatal development in wild-type (WT) and TS mice using in vivo µCT and geometric morphometrics. TS mice developed shorter and wider faces, skulls, and mandibles, together with shorter and narrower humerus and scapula, and reduced BMD dynamically over time. Besides facial morphology, GTE-EGCG did not rescue any other skeletal phenotype in TS treated mice. In WT mice, GTE-EGCG significantly altered the shape of the skull and mandible, reduced the length and width of the long bones, and lowered the BMD. The disparate effects of GTE-EGCG depended on the dose, developmental timepoint, and anatomical structure analyzed, emphasizing the complex nature of DS and the need to further investigate the simultaneous effects of GTE-EGCG supplementation.

Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer's disease.

Dysfunctions of network activity and functional connectivity (FC) represent early events in Alzheimer's disease (AD), but the underlying mechanisms remain unclear. Astrocytes regulate local neuronal activity in the healthy brain, but their involvement in early network hyperactivity in AD is unknown. We show increased FC in the human cingulate cortex several years before amyloid deposition. We find the same early cingulate FC disruption and neuronal hyperactivity in AppNL-F mice. Crucially, these network disruptions are accompanied by decreased astrocyte calcium signaling. Recovery of astrocytic calcium activity normalizes neuronal hyperactivity and FC, as well as seizure susceptibility and day/night behavioral disruptions. In conclusion, we show that astrocytes mediate initial features of AD and drive clinically relevant phenotypes.

Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation.

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood-brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model.

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

Prenatal Radiation Exposure Leads to Higher-Order Telencephalic Dysfunctions in Adult Mice That Coincide with Reduced Synaptic Plasticity and Cerebral Hypersynchrony.

Higher-order telencephalic circuitry has been suggested to be especially vulnerable to irradiation or other developmentally toxic impact. This report details the adult effects of prenatal irradiation at a sensitive time point on clinically relevant brain functions controlled by telencephalic regions, hippocampus (HPC), and prefrontal cortex (PFC). Pregnant C57Bl6/J mice were whole-body irradiated at embryonic day 11 (start of neurogenesis) with X-ray intensities of 0.0, 0.5, or 1.0 Gy. Female offspring completed a broad test battery of HPC-/PFC-controlled tasks that included cognitive performance, fear extinction, exploratory, and depression-like behaviors. We examined neural functions that are mechanistically related to these behavioral and cognitive changes, such as hippocampal field potentials and long-term potentiation, functional brain connectivity (by resting-state functional magnetic resonance imaging), and expression of HPC vesicular neurotransmitter transporters (by immunohistochemical quantification). Prenatally exposed mice displayed several higher-order dysfunctions, such as decreased nychthemeral activity, working memory defects, delayed extinction of threat-evoked response suppression as well as indications of perseverative behavior. Electrophysiological examination indicated impaired hippocampal synaptic plasticity. Prenatal irradiation also induced cerebral hypersynchrony and increased the number of glutamatergic HPC terminals. These changes in brain connectivity and plasticity could mechanistically underlie the irradiation-induced defects in higher telencephalic functions.

MEK inhibition ameliorates social behavior phenotypes in a Spred1 knockout mouse model for RASopathy disorders.

BACKGROUND: RASopathies are a group of disorders that result from mutations in genes coding for proteins involved in regulating the Ras-MAPK signaling pathway, and have an increased incidence of autism spectrum disorder (ASD). Legius syndrome is a rare RASopathy caused by loss-of-function mutations in the SPRED1 gene. The patient phenotype is similar to, but milder than, Neurofibromatosis type 1-another RASopathy caused by loss-of-function mutations in the NF1 gene. RASopathies exhibit increased activation of Ras-MAPK signaling and commonly manifest with cognitive impairments and ASD. Here, we investigated if a Spred1-/- mouse model for Legius syndrome recapitulates ASD-like symptoms, and whether targeting the Ras-MAPK pathway has therapeutic potential in this RASopathy mouse model. METHODS: We investigated social and communicative behaviors in Spred1-/- mice and probed therapeutic mechanisms underlying the observed behavioral phenotypes by pharmacological targeting of the Ras-MAPK pathway with the MEK inhibitor PD325901. RESULTS: Spred1-/- mice have robust increases in social dominance in the automated tube test and reduced adult ultrasonic vocalizations during social communication. Neonatal ultrasonic vocalization was also altered, with significant differences in spectral properties. Spred1-/- mice also exhibit impaired nesting behavior. Acute MEK inhibitor treatment in adulthood with PD325901 reversed the enhanced social dominance in Spred1-/- mice to normal levels, and improved nesting behavior in adult Spred1-/- mice. LIMITATIONS: This study used an acute treatment protocol to administer the drug. It is not known what the effects of longer-term treatment would be on behavior. Further studies titrating the lowest dose of this drug that is required to alter Spred1-/- social behavior are still required. Finally, our findings are in a homozygous mouse model, whereas patients carry heterozygous mutations. These factors should be considered before any translational conclusions are drawn. CONCLUSIONS: These results demonstrate for the first time that social behavior phenotypes in a mouse model for RASopathies (Spred1-/-) can be acutely reversed. This highlights a key role for Ras-MAPK dysregulation in mediating social behavior phenotypes in mouse models for ASD, suggesting that proper regulation of Ras-MAPK signaling is important for social behavior.

Restoring miR-132 expression rescues adult hippocampal neurogenesis and memory deficits in Alzheimer's disease.

Neural stem cells residing in the hippocampal neurogenic niche sustain lifelong neurogenesis in the adult brain. Adult hippocampal neurogenesis (AHN) is functionally linked to mnemonic and cognitive plasticity in humans and rodents. In Alzheimer's disease (AD), the process of generating new neurons at the hippocampal neurogenic niche is impeded, yet the mechanisms involved are unknown. Here we identify miR-132, one of the most consistently downregulated microRNAs in AD, as a potent regulator of AHN, exerting cell-autonomous proneurogenic effects in adult neural stem cells and their progeny. Using distinct AD mouse models, cultured human primary and established neural stem cells, and human patient material, we demonstrate that AHN is directly affected by AD pathology. miR-132 replacement in adult mouse AD hippocampus restores AHN and relevant memory deficits. Our findings corroborate the significance of AHN in mouse models of AD and reveal the possible therapeutic potential of targeting miR-132 in neurodegeneration.

Effects of orbitofrontal cortex and ventral hippocampus disconnection on spatial reversal learning.

Behavioural flexibility is a cognition-related function that enables subjects to adapt to a changing environment. Orbitofrontal cortex (OFC) and hippocampus (HC) have been involved in cognitive flexibility, but the interaction between these structures might be of particular functional significance. We applied a disconnection model in C57BL/6JRj mice to investigate the importance of OFC and ventral HC (vHC) interaction. Spatial acquisition and reversal performance in the Morris water maze (MWM) was compared between animals with small contralateral excitotoxic lesions to OFC and vHC, ipsilateral lesions (i.e., OFC-vHC lesions in the same hemisphere), as well as small bilateral OFC or vHC lesions. Spatial learning and memory performance was mostly unimpaired or only slightly impaired in our brain-lesioned animals compared to sham-lesioned control mice. However, contralaterally lesioned mice were significantly impaired during the early phase of reversal learning, whereas the other lesion groups performed similar to controls. These mice might also have experienced some difficulties using cognitively advanced search strategies. Additional non-mnemonic tests indicated that none of the defects could be reduced to motor, motivational or anxiety-related changes. Our findings support the particular role of PFC-HC interaction in advanced cognitive processes and flexibility.

Lowering Synaptogyrin-3 expression rescues Tau-induced memory defects and synaptic loss in the presence of microglial activation.

Tau is a major driver of neurodegeneration and is implicated in over 20 diseases. Tauopathies are characterized by synaptic loss and neuroinflammation, but it is unclear if these pathological events are causally linked. Tau binds to Synaptogyrin-3 on synaptic vesicles. Here, we interfered with this function to determine the role of pathogenic Tau at pre-synaptic terminals. We show that heterozygous knockout of synaptogyrin-3 is benign in mice but strongly rescues mutant Tau-induced defects in long-term synaptic plasticity and working memory. It also significantly rescues the pre- and post-synaptic loss caused by mutant Tau. However, Tau-induced neuroinflammation remains clearly upregulated when we remove the expression of one allele of synaptogyrin-3. Hence neuroinflammation is not sufficient to cause synaptic loss, and these processes are separately induced in response to mutant Tau. In addition, the pre-synaptic defects caused by mutant Tau are enough to drive defects in cognitive tasks.

Comparison between touchscreen operant chambers and water maze to detect early prefrontal dysfunction in mice.

The relative lack of sensitive and clinically valid tests of rodent behavior might be one of the reasons for the limited success of the clinical translation of preclinical Alzheimer's disease (AD) research findings. There is a general interest in innovative behavioral methodology, and protocols have been proposed for touchscreen operant chambers that might be superior to existing cognitive assessment methods. We assessed and analyzed touchscreen performance in several novel ways to examine the possible occurrence of early signs of prefrontal (PFC) functional decline in the APP/PS1 mouse model of AD. Touchscreen learning performance was compared between APP/PS1-21 mice and wildtype littermates on a C57BL/6J background at 3, 6 and 12 months of age in parallel to the assessment of spatial learning, memory and cognitive flexibility in the Morris water maze (MWM). We found that older mice generally needed more training sessions to complete the touchscreen protocol than younger ones. Older mice also displayed defects in MWM working memory performance, but touchscreen protocols detected functional changes beginning at 3 months of age. Histological changes in PFC of APP/PS1 mice indeed occurred as early as 3 months. Our results suggest that touchscreen operant protocols are more sensitive to PFC dysfunction, which is of relevance to the use of these tasks and devices in preclinical AD research and experimental pharmacology.

Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.

Folic Acid Fortification Prevents Morphological and Behavioral Consequences of X-Ray Exposure During Neurulation.

Previous studies suggested a causal link between pre-natal exposure to ionizing radiation and birth defects such as microphthalmos and exencephaly. In mice, these defects arise primarily after high-dose X-irradiation during early neurulation. However, the impact of sublethal (low) X-ray doses during this early developmental time window on adult behavior and morphology of central nervous system structures is not known. In addition, the efficacy of folic acid (FA) in preventing radiation-induced birth defects and persistent radiation-induced anomalies has remained unexplored. To assess the efficacy of FA in preventing radiation-induced defects, pregnant C57BL6/J mice were X-irradiated at embryonic day (E)7.5 and were fed FA-fortified food. FA partially prevented radiation-induced (1.0 Gy) anophthalmos, exencephaly and gastroschisis at E18, and reduced the number of pre-natal deaths, fetal weight loss and defects in the cervical vertebrae resulting from irradiation. Furthermore, FA food fortification counteracted radiation-induced impairments in vision and olfaction, which were evidenced after exposure to doses ≥0.1 Gy. These findings coincided with the observation of a reduction in thickness of the retinal ganglion cell and nerve fiber layer, and a decreased axial length of the eye following exposure to 0.5 Gy. Finally, MRI studies revealed a volumetric decrease of the hippocampus, striatum, thalamus, midbrain and pons following 0.5 Gy irradiation, which could be partially ameliorated after FA food fortification. Altogether, our study is the first to offer detailed insights into the long-term consequences of X-ray exposure during neurulation, and supports the use of FA as a radioprotectant and antiteratogen to counter the detrimental effects of X-ray exposure during this crucial period of gestation.

Cognitive dysfunction in mice lacking proper glucocorticoid receptor dimerization.

Stress is a major risk factor for depression and anxiety. One of the effects of stress is the (over-) activation of the hypothalamic-pituitary-adrenal (HPA) axis and the release of stress hormones such as glucocorticoids (GCs). Chronically increased stress hormone levels have been shown to have detrimental effects on neuronal networks by inhibiting neurotrophic processes particularly in the hippocampus proper. Centrally, GCs modulate metabolic as well as behavioural processes by activating two classes of corticoid receptors, high-affinity mineralocorticoid receptors (MR) and low-affinity glucocorticoid receptors (GR). Upon activation, GR can modulate gene transcription either as a monomeric protein, or as a dimer interacting directly with DNA. GR can also modulate cellular processes via non-genomic mechanisms, for example via a GPCR-protein interaction. We evaluated the behavioral phenotype in mice with a targeted mutation in the GR in a FVB/NJ background. In GRdim/dim mice, GR proteins form poor homodimers, while the GR monomer remains intact. We evaluated the effect of poor GR dimerization on hippocampus-dependent cognition as well as on exploration and emotional behavior under baseline and chronically increased stress hormone levels. We found that GRdim/dim mice did not behave differently from GRwt/wt littermates under baseline conditions. However, after chronic elevation of stress hormone levels, GRdim/dim mice displayed a significant impairment in hippocampus-dependent memory compared to GRwt/wt mice, which correlated with differential expression of hippocampal Bdnf/TrkB and Fkbp5.

EphA4 loss improves social memory performance and alters dendritic spine morphology without changes in amyloid pathology in a mouse model of Alzheimer's disease.

BACKGROUND: EphA4 is a receptor of the ephrin system regulating spine morphology and plasticity in the brain. These processes are pivotal in the pathophysiology of Alzheimer's disease (AD), characterized by synapse dysfunction and loss, and the progressive loss of memory and other cognitive functions. Reduced EphA4 signaling has been shown to rescue beta-amyloid-induced dendritic spine loss and long-term potentiation (LTP) deficits in cultured hippocampal slices and primary hippocampal cultures. In this study, we investigated whether EphA4 ablation might preserve synapse function and ameliorate cognitive performance in the APPPS1 transgenic mouse model of AD. METHODS: A postnatal genetic ablation of EphA4 in the forebrain was established in the APPPS1 mouse model of AD, followed by a battery of cognitive tests at 9 months of age to investigate cognitive function upon EphA4 loss. A Golgi-Cox staining was used to explore alterations in dendritic spine density and morphology in the CA1 region of the hippocampus. RESULTS: Upon EphA4 loss in APPPS1 mice, we observed improved social memory in the preference for social novelty test without affecting other cognitive functions. Dendritic spine analysis revealed altered synapse morphology as characterized by increased dendritic spine length and head width. These modifications were independent of hippocampal plaque load and beta-amyloid peptide levels since these were similar in mice with normal versus reduced levels of EphA4. CONCLUSION: Loss of EphA4 improved social memory in a mouse model of Alzheimer's disease in association with alterations in spine morphology.

The autism- and schizophrenia-associated protein CYFIP1 regulates bilateral brain connectivity and behaviour.

Copy-number variants of the CYFIP1 gene in humans have been linked to autism spectrum disorders (ASD) and schizophrenia (SCZ), two neuropsychiatric disorders characterized by defects in brain connectivity. Here, we show that CYFIP1 plays an important role in brain functional connectivity and callosal functions. We find that Cyfip1-heterozygous mice have reduced functional connectivity and defects in white matter architecture, similar to phenotypes found in patients with ASD, SCZ and other neuropsychiatric disorders. Cyfip1-deficient mice also present decreased myelination in the callosal axons, altered presynaptic function, and impaired bilateral connectivity. Finally, Cyfip1 deficiency leads to abnormalities in motor coordination, sensorimotor gating and sensory perception, which are also known neuropsychiatric disorder-related symptoms. These results show that Cyfip1 haploinsufficiency compromises brain connectivity and function, which might explain its genetic association to neuropsychiatric disorders.

Differential effects of post-training scopolamine on spatial and non-spatial learning tasks in mice.

Muscarinic antagonist scopolamine has been extensively used to model amnesia in lab rodents, but most studies have focused on the effects of pre-training scopolamine administration. Here, we examined post-training scopolamine administration in C57BL/6JRj mice. Learning was assessed in three different procedures: odour discrimination in a digging paradigm, visual discrimination in a touchscreen-based setup, and spatial learning in the Morris water maze. Scopolamine administration affected performance in the odour discrimination task. More specifically, scopolamine decreased perseverance, which facilitated reversal learning. Similar results were obtained in the visual discrimination task, but scopolamine did not affect performance in the spatial learning task. It is unlikely that these results can be explained by non-memory-related cognitive effects (e.g., attention), non-cognitive behaviours (e.g., locomotor activity) or peripheral side-effects (e.g., mydriasis). They likely relate to the various neuropharmacological actions of scopolamine.