In and out: Benchmarking in vitro, in vivo, ex vivo, and xenografting approaches for an integrative brain disease modeling pipeline.

Human cellular models and their neuronal derivatives have afforded unprecedented advances in elucidating pathogenic mechanisms of neuropsychiatric diseases. Notwithstanding their indispensable contribution, animal models remain the benchmark in neurobiological research. In an attempt to harness the best of both worlds, researchers have increasingly relied on human/animal chimeras by xenografting human cells into the animal brain. Despite the unparalleled potential of xenografting approaches in the study of the human brain, literature resources that systematically examine their significance and advantages are surprisingly lacking. We fill this gap by providing a comprehensive account of brain diseases that were thus far subjected to all three modeling approaches (transgenic rodents, in vitro human lineages, human-animal xenografting) and provide a critical appraisal of the impact of xenografting approaches for advancing our understanding of those diseases and brain development. Next, we give our perspective on integrating xenografting modeling pipeline with recent cutting-edge technological advancements.

YY1 mutations disrupt corticogenesis through a cell-type specific rewiring of cell-autonomous and non-cell-autonomous transcriptional programs.

Germline mutations of YY1 cause Gabriele-de Vries syndrome (GADEVS), a neurodevelopmental disorder featuring intellectual disability and a wide range of systemic manifestations. To dissect the cellular and molecular mechanisms underlying GADEVS, we combined large-scale imaging, single-cell multiomics and gene regulatory network reconstruction in 2D and 3D patient-derived physiopathologically relevant cell lineages. YY1 haploinsufficiency causes a pervasive alteration of cell type specific transcriptional networks, disrupting corticogenesis at the level of neural progenitors and terminally differentiated neurons, including cytoarchitectural defects reminiscent of GADEVS clinical features. Transcriptional alterations in neurons propagated to neighboring astrocytes through a major non-cell autonomous pro-inflammatory effect that grounds the rationale for modulatory interventions. Together, neurodevelopmental trajectories, synaptic formation and neuronal-astrocyte cross talk emerged as salient domains of YY1 dosage-dependent vulnerability. Mechanistically, cell-type resolved reconstruction of gene regulatory networks uncovered the regulatory interplay between YY1, NEUROG2 and ETV5 and its aberrant rewiring in GADEVS. Our findings underscore the reach of advanced in vitro models in capturing developmental antecedents of clinical features and exposing their underlying mechanisms to guide the search for targeted interventions.

GTF2I dosage regulates neuronal differentiation and social behavior in 7q11.23 neurodevelopmental disorders.

Copy number variations at 7q11.23 cause neurodevelopmental disorders with shared and opposite manifestations. Deletion causes Williams-Beuren syndrome featuring hypersociability, while duplication causes 7q11.23 microduplication syndrome (7Dup), frequently exhibiting autism spectrum disorder (ASD). Converging evidence indicates GTF2I as key mediator of the cognitive-behavioral phenotypes, yet its role in cortical development and behavioral hallmarks remains largely unknown. We integrated proteomic and transcriptomic profiling of patient-derived cortical organoids, including longitudinally at single-cell resolution, to dissect 7q11.23 dosage-dependent and GTF2I-specific disease mechanisms. We observed dosage-dependent impaired dynamics of neural progenitor proliferation, transcriptional imbalances, and highly specific alterations in neuronal output, leading to precocious excitatory neuron production in 7Dup, which was rescued by restoring physiological GTF2I levels. Transgenic mice with Gtf2i duplication recapitulated progenitor proliferation and neuronal differentiation defects alongside ASD-like behaviors. Consistently, inhibition of lysine demethylase 1 (LSD1), a GTF2I effector, was sufficient to rescue ASD-like phenotypes in transgenic mice, establishing GTF2I-LSD1 axis as a molecular pathway amenable to therapeutic intervention in ASD.

High-throughput screening identifies histone deacetylase inhibitors that modulate GTF2I expression in 7q11.23 microduplication autism spectrum disorder patient-derived cortical neurons.

BACKGROUND: Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental condition affecting almost 1% of children, and represents a major unmet medical need with no effective drug treatment available. Duplication at 7q11.23 (7Dup), encompassing 26-28 genes, is one of the best characterized ASD-causing copy number variations and offers unique translational opportunities, because the hemideletion of the same interval causes Williams-Beuren syndrome (WBS), a condition defined by hypersociability and language strengths, thereby providing a unique reference to validate treatments for the ASD symptoms. In the above-indicated interval at 7q11.23, defined as WBS critical region, several genes, such as GTF2I, BAZ1B, CLIP2 and EIF4H, emerged as critical for their role in the pathogenesis of WBS and 7Dup both from mouse models and human studies. METHODS: We performed a high-throughput screening of 1478 compounds, including central nervous system agents, epigenetic modulators and experimental substances, on patient-derived cortical glutamatergic neurons differentiated from our cohort of induced pluripotent stem cell lines (iPSCs), monitoring the transcriptional modulation of WBS interval genes, with a special focus on GTF2I, in light of its overriding pathogenic role. The hits identified were validated by measuring gene expression by qRT-PCR and the results were confirmed by western blotting. RESULTS: We identified and selected three histone deacetylase inhibitors (HDACi) that decreased the abnormal expression level of GTF2I in 7Dup cortical glutamatergic neurons differentiated from four genetically different iPSC lines. We confirmed this effect also at the protein level. LIMITATIONS: In this study, we did not address the molecular mechanisms whereby HDAC inhibitors act on GTF2I. The lead compounds identified will now need to be advanced to further testing in additional models, including patient-derived brain organoids and mouse models recapitulating the gene imbalances of the 7q11.23 microduplication, in order to validate their efficacy in rescuing phenotypes across multiple functional layers within a translational pipeline towards clinical use. CONCLUSIONS: These results represent a unique opportunity for the development of a specific class of compounds for treating 7Dup and other forms of intellectual disability and autism.

Cortical Spreading Depression Causes Unique Dysregulation of Inflammatory Pathways in a Transgenic Mouse Model of Migraine.

Familial hemiplegic migraine type 1 (FHM1) is a rare monogenic subtype of migraine with aura caused by mutations in CACNA1A that encodes the α1A subunit of voltage-gated CaV2.1 calcium channels. Transgenic knock-in mice that carry the human FHM1 R192Q missense mutation ('FHM1 R192Q mice') exhibit an increased susceptibility to cortical spreading depression (CSD), the mechanism underlying migraine aura. Here, we analysed gene expression profiles from isolated cortical tissue of FHM1 R192Q mice 24 h after experimentally induced CSD in order to identify molecular pathways affected by CSD. Gene expression profiles were generated using deep serial analysis of gene expression sequencing. Our data reveal a signature of inflammatory signalling upon CSD in the cortex of both mutant and wild-type mice. However, only in the brains of FHM1 R192Q mice specific genes are up-regulated in response to CSD that are implicated in interferon-related inflammatory signalling. Our findings show that CSD modulates inflammatory processes in both wild-type and mutant brains, but that an additional unique inflammatory signature becomes expressed after CSD in a relevant mouse model of migraine.

Mass spectrometry imaging of amino neurotransmitters: a comparison of derivatization methods and application in mouse brain tissue.

The detection of small polar compounds such as amino neurotransmitters by MALDI mass spectrometry imaging has been hindered by low-detection sensitivity and background interferences. Recently, several of on-tissue chemical derivatization strategies have been independently reported that enable their detection. Here, we present a comparison between these methods, and demonstrate the visualization of the distributions of up to 23 amino metabolites in tissue. We applied this methodology to detect alterations of these compounds after inducing an experimental cortical spreading depression in mouse brain, which causes profound transient alterations in key neurotransmitters in one hemisphere and is relevant for migraine and various other neurological disorders.

Plasma metabolic profiling after cortical spreading depression in a transgenic mouse model of hemiplegic migraine by capillary electrophoresis--mass spectrometry.

Migraine is a common brain disorder characterized by recurrent attacks of severe headaches and other neurological symptoms. In one-third of patients headaches are accompanied by auras, which consist of transient visual and sensory disturbances, believed to be caused by cortical spreading depression (CSD). CSD is characterized by a wave of neuronal and glial depolarization with concomitant changes in metabolite concentrations in the brain and cerebrospinal fluid. It remains unknown whether CSD-induced brain metabolic changes can be captured outside the central nervous system, i.e., in peripheral fluids. This study investigated plasma metabolic changes in transgenic mice that harbor a gene mutation in voltage-gated CaV2.1 Ca(2+) channels previously identified in patients with familial hemiplegic migraine, a subtype of migraine with auras. The use of a mouse model allows investigation of molecular changes occurring shortly after CSD, which is notoriously difficult in patients. Capillary electrophoresis - mass spectrometry was used for the analysis of plasma samples to obtain, for the first time, a comprehensive view of molecular changes immediately after experimentally induced CSD. Multivariate data analysis showed a clear distinction between profiles of transgenic and wild-type animals after CSD. Two metabolites considered important for this discrimination were tentatively identified as being lysine and its by-product pipecolic acid with additional evidence provided by hydrophilic interaction chromatography combined with tandem mass spectrometry. The changed metabolites suggest a compensatory increase in GABAergic neurotransmission upon enhanced excitatory neurotransmission. These results show that CSD induces metabolic remodeling in transgenic migraine mice that can be captured and measured in plasma.

Large-scale mass spectrometry imaging investigation of consequences of cortical spreading depression in a transgenic mouse model of migraine.

Cortical spreading depression (CSD) is the electrophysiological correlate of migraine aura. Transgenic mice carrying the R192Q missense mutation in the Cacna1a gene, which in patients causes familial hemiplegic migraine type 1 (FHM1), exhibit increased propensity to CSD. Herein, mass spectrometry imaging (MSI) was applied for the first time to an animal cohort of transgenic and wild type mice to study the biomolecular changes following CSD in the brain. Ninety-six coronal brain sections from 32 mice were analyzed by MALDI-MSI. All MSI datasets were registered to the Allen Brain Atlas reference atlas of the mouse brain so that the molecular signatures of distinct brain regions could be compared. A number of metabolites and peptides showed substantial changes in the brain associated with CSD. Among those, different mass spectral features showed significant (t-test, P < 0.05) changes in the cortex, 146 and 377 Da, and in the thalamus, 1820 and 1834 Da, of the CSD-affected hemisphere of FHM1 R192Q mice. Our findings reveal CSD- and genotype-specific molecular changes in the brain of FHM1 transgenic mice that may further our understanding about the role of CSD in migraine pathophysiology. The results also demonstrate the utility of aligning MSI datasets to a common reference atlas for large-scale MSI investigations.

Stress hormone corticosterone enhances susceptibility to cortical spreading depression in familial hemiplegic migraine type 1 mutant mice.

Stress is a putative migraine trigger, but the pathogenic mechanisms involved are unknown. Stress and stress hormones increase neuronal excitability by enhancing glutamatergic neurotransmission, but inhibitory effects have also been reported. We hypothesise that an acute rise in stress hormones, such as corticosteroids which are released after stress, increase neuronal excitability and thereby may increase susceptibility to cortical spreading depression (CSD), the mechanism underlying the migraine aura. Here we investigated effects of acute restraint stress and of the stress hormone corticosterone on CSD susceptibility as surrogate migraine marker, in a transgenic mouse model of familial hemiplegic migraine type 1 (FHM1), which displays increased glutamatergic cortical neurotransmission and increased propensity for CSD. We found that 20-min and 3-h restraint stress did not influence CSD susceptibility in mutant or wild-type mice, despite elevated levels of plasma corticosterone. By contrast, subcutaneous administration of 20mg/kg corticosterone increased CSD frequency exclusively in mutant mice, while corticosterone plasma levels were similarly elevated in mutants and wild types. The effect of corticosterone on CSD frequency was normalised by pre-administration of the glucocorticoid receptor (GR) antagonist mifepristone. These findings suggest that corticosteroid-induced GR activation can enhance susceptibility to CSD in genetically susceptible individuals, and may predispose to attacks of migraine. Although corticosterone levels rise also during acute stress, the latter likely triggers a spatiotemporally more complex biological response with multiple positive and negative modulators which may not be adequately modeled by exogenous administration of corticosterone alone.

Automatic registration of mass spectrometry imaging data sets to the Allen brain atlas.

Mass spectrometry imaging holds great potential for understanding the molecular basis of neurological disease. Several key studies have demonstrated its ability to uncover disease-related biomolecular changes in rodent models of disease, even if highly localized or invisible to established histological methods. The high analytical reproducibility necessary for the biomedical application of mass spectrometry imaging means it is widely developed in mass spectrometry laboratories. However, many lack the expertise to correctly annotate the complex anatomy of brain tissue, or have the capacity to analyze the number of animals required in preclinical studies, especially considering the significant variability in sizes of brain regions. To address this issue, we have developed a pipeline to automatically map mass spectrometry imaging data sets of mouse brains to the Allen Brain Reference Atlas, which contains publically available data combining gene expression with brain anatomical locations. Our pipeline enables facile and rapid interanimal comparisons by first testing if each animal's tissue section was sampled at a similar location and enabling the extraction of the biomolecular signatures from specific brain regions.

Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS.

The need for sensitive analytical technologies applicable to metabolic profiling of volume-restricted biological samples is high. Here, we demonstrate feasibility of capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (MS) with sheathless nano-electrospray interface for non-targeted profiling of ionogenic metabolites in body fluids of experimental animals. A representative mixture of the metabolites and body fluids of mice such as cerebrospinal fluid (CSF), urine and plasma were used as examples of low-volume biological samples for method evaluation. An injection volume of only 9 nL resulted in limits of detection between 0.7 and 12 nM for the metabolite mixture. The method allowed the detection of ~350 molecular features in mouse CSF (an injection volume of ca. 45 nL), while ~400 features were observed in mouse plasma and ~3,500 features in mouse urine (an injection volume of ca. 9 nL). The low-volume body fluid samples were analyzed directly after only 1:1 dilution with water, thereby fully retaining sample integrity, which is of crucial importance for non-targeted metabolic profiling. As little is known about the metabolic composition of mouse CSF, we identified a fraction of the molecular features in mouse CSF using accurate mass information, migration times, MS/MS data, and comparison with authentic standards. We conclude that sheathless CE-MS can be used for sensitive metabolic profiling of volume-restricted biological samples.

Imaging mass spectrometry to visualize biomolecule distributions in mouse brain tissue following hemispheric cortical spreading depression.

MALDI mass spectrometry can simultaneously measure hundreds of biomolecules directly from tissue. Using essentially the same technique but different sample preparation strategies, metabolites, lipids, peptides and proteins can be analyzed. Spatially correlated analysis, imaging MS, enables the distributions of these biomolecular ions to be simultaneously measured in tissues. A key advantage of imaging MS is that it can annotate tissues based on their MS profiles and thereby distinguish biomolecularly distinct regions even if they were unexpected or are not distinct using established histological and histochemical methods e.g. neuropeptide and metabolite changes following transient electrophysiological events such as cortical spreading depression (CSD), which are spreading events of massive neuronal and glial depolarisations that occur in one hemisphere of the brain and do not pass to the other hemisphere , enabling the contralateral hemisphere to act as an internal control. A proof-of-principle imaging MS study, including 2D and 3D datasets, revealed substantial metabolite and neuropeptide changes immediately following CSD events which were absent in the protein imaging datasets. The large high dimensionality 3D datasets make even rudimentary contralateral comparisons difficult to visualize. Instead non-negative matrix factorization (NNMF), a multivariate factorization tool that is adept at highlighting latent features, such as MS signatures associated with CSD events, was applied to the 3D datasets. NNMF confirmed that the protein dataset did not contain substantial contralateral differences, while these were present in the neuropeptide dataset.

Purkinje cell-specific ablation of Cav2.1 channels is sufficient to cause cerebellar ataxia in mice.

The Cacna1a gene encodes the α(1A) subunit of voltage-gated Ca(V)2.1 Ca(2+) channels that are involved in neurotransmission at central synapses. Ca(V)2.1-α(1)-knockout (α1KO) mice, which lack Ca(V)2.1 channels in all neurons, have a very severe phenotype of cerebellar ataxia and dystonia, and usually die around postnatal day 20. This early lethality, combined with the wide expression of Ca(V)2.1 channels throughout the cerebellar cortex and nuclei, prohibited determination of the contribution of particular cerebellar cell types to the development of the severe neurobiological phenotype in Cacna1a mutant mice. Here, we crossed conditional Cacna1a mice with transgenic mice expressing Cre recombinase, driven by the Purkinje cell-specific Pcp2 promoter, to specifically ablate the Ca(V)2.1-α(1A) subunit and thereby Ca(V)2.1 channels in Purkinje cells. Purkinje cell Ca(V)2.1-α(1A)-knockout (PCα1KO) mice aged without difficulties, rescuing the lethal phenotype seen in α1KO mice. PCα1KO mice exhibited cerebellar ataxia starting around P12, much earlier than the first signs of progressive Purkinje cell loss, which appears in these mice between P30 and P45. Secondary cell loss was observed in the granular and molecular layers of the cerebellum and the volume of all individual cerebellar nuclei was reduced. In this mouse model with a cell type-specific ablation of Ca(V)2.1 channels, we show that ablation of Ca(V)2.1 channels restricted to Purkinje cells is sufficient to cause cerebellar ataxia. We demonstrate that spatial ablation of Ca(V)2.1 channels may help in unraveling mechanisms of human disease.

Migraine genes and the relation to gender.

Migraine is an episodic brain disorder that is characterized by recurrent attacks of severe unilateral headache that are accompanied by various neurological symptoms. In addition, many patients have what is called an aura with visual and sensory disturbances. The majority of patients are female, suggesting that female hormones play an important role in the pathophysiology of the disorder. The molecular mechanisms, however, underlying this female preponderance are not well understood. It can be expected that the field of genetics that aims at identifying genetic factors that cause migraine by lowering the threshold for attacks will unravel some of these mechanisms. The 3 best known migraine genes encode ion transporters and were identified in families with familial hemiplegic migraine (FHM), a rare subtype of migraine with aura. FHM gene mutations cause alterations in mechanisms that control and modulate the neurotransmitter balance in the brain. Transgenic mice knock-in with human pathogenic mutations that were shown to exhibit some migraine-relevant features were very helpful in dissecting molecular mechanisms of migraine and pointed to a central role for cortical glutamate. In addition, transgenic mice that overexpress human RAMP1 exist and exhibit an increased sensitivity to calcitonin gene-related peptide. Findings from genetic and animal experiments on gender differences in migraine are discussed. Recently, a role for glutamate also came forward from a genome-wide association study in common migraine. By deciphering genetic and pathogenic migraine pathways, it can be expected that in the near future we will better understand mechanisms behind the female preponderance in migraine.