A Cellular Ground Truth to Develop MRI Signatures in Glioma Models by Correlative Light Sheet Microscopy and Atlas-Based Coregistration.

Glioblastoma is the most common malignant primary brain tumor with poor overall survival. Magnetic resonance imaging (MRI) is the main imaging modality for glioblastoma but has inherent shortcomings. The molecular and cellular basis of MR signals is incompletely understood. We established a ground truth-based image analysis platform to coregister MRI and light sheet microscopy (LSM) data to each other and to an anatomic reference atlas for quantification of 20 predefined anatomic subregions. Our pipeline also includes a segmentation and quantification approach for single myeloid cells in entire LSM datasets. This method was applied to three preclinical glioma models in male and female mice (GL261, U87MG, and S24), which exhibit different key features of the human glioma. Multiparametric MR data including T2-weighted sequences, diffusion tensor imaging, T2 and T2* relaxometry were acquired. Following tissue clearing, LSM focused on the analysis of tumor cell density, microvasculature, and innate immune cell infiltration. Correlated analysis revealed differences in quantitative MRI metrics between the tumor-bearing and the contralateral hemisphere. LSM identified tumor subregions that differed in their MRI characteristics, indicating tumor heterogeneity. Interestingly, MRI signatures, defined as unique combinations of different MRI parameters, differed greatly between the models. The direct correlation of MRI and LSM allows an in-depth characterization of preclinical glioma and can be used to decipher the structural, cellular, and, likely, molecular basis of tumoral MRI biomarkers. Our approach may be applied in other preclinical brain tumor or neurologic disease models, and the derived MRI signatures could ultimately inform image interpretation in a clinical setting.SIGNIFICANCE STATEMENT We established a histologic ground truth-based approach for MR image analyses and tested this method in three preclinical glioma models exhibiting different features of glioblastoma. Coregistration of light sheet microscopy to MRI allowed for an evaluation of quantitative MRI data in histologically distinct tumor subregions. Coregistration to a mouse brain atlas enabled a regional comparison of MRI parameters with a histologically informed interpretation of the results. Our approach is transferable to other preclinical models of brain tumors and further neurologic disorders. The method can be used to decipher the structural, cellular, and molecular basis of MRI signal characteristics. Ultimately, information derived from such analyses could strengthen the neuroradiological evaluation of glioblastoma as they enhance the interpretation of MRI data.

Sphingosine-1-Phosphate Recruits Macrophages and Microglia and Induces a Pro-Tumorigenic Phenotype That Favors Glioma Progression.

Glioblastoma is the most aggressive brain tumor in adults. Treatment failure is predominantly caused by its high invasiveness and its ability to induce a supportive microenvironment. As part of this, a major role for tumor-associated macrophages/microglia (TAMs) in glioblastoma development was recognized. Phospholipids are important players in various fundamental biological processes, including tumor-stroma crosstalk, and the bioactive lipid sphingosine-1-phosphate (S1P) has been linked to glioblastoma cell proliferation, invasion, and survival. Despite the urgent need for better therapeutic approaches, novel strategies targeting sphingolipids in glioblastoma are still poorly explored. Here, we showed that higher amounts of S1P secreted by glioma cells are responsible for an active recruitment of TAMs, mediated by S1P receptor (S1PR) signaling through the modulation of Rac1/RhoA. This resulted in increased infiltration of TAMs in the tumor, which, in turn, triggered their pro-tumorigenic phenotype through the inhibition of NFkB-mediated inflammation. Gene set enrichment analyses showed that such an anti-inflammatory microenvironment correlated with shorter survival of glioblastoma patients. Inhibition of S1P restored a pro-inflammatory phenotype in TAMs and resulted in increased survival of tumor-bearing mice. Taken together, our results establish a crucial role for S1P in fine-tuning the crosstalk between glioma and infiltrating TAMs, thus pointing to the S1P-S1PR axis as an attractive target for glioma treatment.

Brain tumor classification of virtual NMR voxels based on realistic blood vessel-induced spin dephasing using support vector machines.

Remodeling of tissue microvasculature commonly promotes neoplastic growth; however, there is no imaging modality in oncology yet that noninvasively quantifies microvascular changes in clinical routine. Although blood capillaries cannot be resolved in typical magnetic resonance imaging (MRI) measurements, their geometry and distribution influence the integral nuclear magnetic resonance (NMR) signal from each macroscopic MRI voxel. We have numerically simulated the expected transverse relaxation in NMR voxels with different dimensions based on the realistic microvasculature in healthy and tumor-bearing mouse brains (U87 and GL261 glioblastoma). The 3D capillary structure in entire, undissected brains was acquired using light sheet fluorescence microscopy to produce large datasets of the highly resolved cerebrovasculature. Using this data, we trained support vector machines to classify virtual NMR voxels with different dimensions based on the simulated spin dephasing accountable to field inhomogeneities caused by the underlying vasculature. In prediction tests with previously blinded virtual voxels from healthy brain tissue and GL261 tumors, stable classification accuracies above 95% were reached. Our results indicate that high classification accuracies can be stably attained with achievable training set sizes and that larger MRI voxels facilitated increasingly successful classifications, even with small training datasets. We were able to prove that, theoretically, the transverse relaxation process can be harnessed to learn endogenous contrasts for single voxel tissue type classifications on tailored MRI acquisitions. If translatable to experimental MRI, this may augment diagnostic imaging in oncology with automated voxel-by-voxel signal interpretation to detect vascular pathologies.

Large-scale characterization of the microvascular geometry in development and disease by tissue clearing and quantitative ultramicroscopy.

Three-dimensional assessment of optically cleared, entire organs and organisms has recently become possible by tissue clearing and selective plane illumination microscopy ("ultramicroscopy"). Resulting datasets can be highly complex, encompass over a thousand images with millions of objects and data of several gigabytes per acquisition. This constitutes a major challenge for quantitative analysis. We have developed post-processing tools to quantify millions of microvessels and their distribution in three-dimensional datasets from ultramicroscopy and demonstrate the capabilities of our pipeline within entire mouse brains and embryos. Using our developed acquisition, segmentation, and analysis platform, we quantify physiological vascular networks in development and the healthy brain. We compare various geometric vessel parameters (e.g. vessel density, radius, tortuosity) in the embryonic spinal cord and brain as well as in different brain regions (basal ganglia, corpus callosum, cortex). White matter tract structures (corpus callosum, spinal cord) showed lower microvascular branch densities and longer vessel branch length compared to grey matter (cortex, basal ganglia). Furthermore, we assess tumor neoangiogenesis in a mouse glioma model to compare tumor core and tumor border. The developed methodology allows rapid quantification of three-dimensional datasets by semi-automated segmentation of fluorescently labeled objects with conventional computer hardware. Our approach can aid preclinical investigations and paves the way towards "quantitative ultramicroscopy".

A multi-omics analysis reveals the unfolded protein response regulon and stress-induced resistance to folate-based antimetabolites.

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.

Gibbs point field model quantifies disorder in microvasculature of U87-glioblastoma.

Microvascular proliferation in glioblastoma multiforme is a biological key mechanism to facilitate tumor growth and infiltration and a main target for treatment interventions. The vascular architecture can be obtained by Single Plane Illumination Microscopy (SPIM) to evaluate vascular heterogeneity in tumorous tissue. We make use of the Gibbs point field model to quantify the order of regularity in capillary distributions found in the U87 glioblastoma model in a murine model and to compare tumorous and healthy brain tissue. A single model parameter Γ was assigned that is linked to tissue-specific vascular topology through Monte-Carlo simulations. Distributions of the model parameter Γ differ significantly between glioblastoma tissue with mean 〈ΓG〉=2.1±0.4, as compared to healthy brain tissue with mean 〈ΓH〉=4.9±0.4, suggesting that the average Γ-value allows for tissue differentiation. These results may be used for diagnostic magnetic resonance imaging, where it has been shown recently that Γ is linked to tissue-inherent relaxation parameters.

PERK-mediated expression of peptidylglycine α-amidating monooxygenase supports angiogenesis in glioblastoma.

PKR-like kinase (PERK) plays a significant role in inducing angiogenesis in various cancer types including glioblastoma. By proteomics analysis of the conditioned medium from a glioblastoma cell line treated with a PERK inhibitor, we showed that peptidylglycine α-amidating monooxygenase (PAM) expression is regulated by PERK under hypoxic conditions. Moreover, PERK activation via CCT020312 (a PERK selective activator) increased the cleavage and thus the generation of PAM cleaved cytosolic domain (PAM sfCD) that acts as a signaling molecule from the cytoplasm to the nuclei. PERK was also found to interact with PAM, suggesting a possible involvement in the generation of PAM sfCD. Knockdown of PERK or PAM reduced the formation of tubes by HUVECs in vitro. Furthermore, in vivo data highlighted the importance of PAM in the growth of glioblastoma with reduction of PAM expression in engrafted tumor significantly increasing the survival in mice. In summary, our data revealed PAM as a potential target for antiangiogenic therapy in glioblastoma.

Glioblastoma multiforme restructures the topological connectivity of cerebrovascular networks.

Glioblastoma multiforme alters healthy tissue vasculature by inducing angiogenesis and vascular remodeling. To fully comprehend the structural and functional properties of the resulting vascular network, it needs to be studied collectively by considering both geometric and topological properties. Utilizing Single Plane Illumination Microscopy (SPIM), the detailed capillary structure in entire healthy and tumor-bearing mouse brains could be resolved in three dimensions. At the scale of the smallest capillaries, the entire vascular systems of bulk U87- and GL261-glioblastoma xenografts, their respective cores, and healthy brain hemispheres were modeled as complex networks and quantified with fundamental topological measures. All individual vessel segments were further quantified geometrically and modular clusters were uncovered and characterized as meta-networks, facilitating an analysis of large-scale connectivity. An inclusive comparison of large tissue sections revealed that geometric properties of individual vessels were altered in glioblastoma in a relatively subtle way, with high intra- and inter-tumor heterogeneity, compared to the impact on the vessel connectivity. A network topology analysis revealed a clear decomposition of large modular structures and hierarchical network organization, while preserving most fundamental topological classifications, in both tumor models with distinct growth patterns. These results augment our understanding of cerebrovascular networks and offer a topological assessment of glioma-induced vascular remodeling. The findings may help understand the emergence of hypoxia and necrosis, and prove valuable for therapeutic interventions such as radiation or antiangiogenic therapy.

A sensitive and simple targeted proteomics approach to quantify transcription factor and membrane proteins of the unfolded protein response pathway in glioblastoma cells.

Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines.

RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas.

Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.

A PRDX1-p38α heterodimer amplifies MET-driven invasion of IDH-wildtype and IDH-mutant gliomas.

The Peroxiredoxin 1 (PRDX1) gene maps to chromosome arm 1p and is hemizygously deleted and epigenetically silenced in isocitrate dehydrogenase 1 or 2 (IDH)-mutant and 1p/19q-codeleted oligodendroglial tumors. In contrast, IDH-wildtype astrocytic gliomas including glioblastomas mostly lack epigenetic silencing and express PRDX1 protein. In our study, we investigated how PRDX1 contributes to the infiltrative growth of IDH-wildtype gliomas. Focusing on p38α-dependent pathways, we analyzed clinical data from 133 patients of the NOA-04 trial cohort to look for differences in the gene expression profiles of gliomas with wildtype or mutant IDH. Biochemical interaction studies as well as in vitro and ex vivo migration studies were used to establish a biological role of PRDX1 in maintaining pathway activity. Whole-brain high-resolution ultramicroscopy and survival analyses of pre-clinical mouse models for IDH-wildtype gliomas were then used for in vivo confirmation. Based on clinical data, we found that the absence of PRDX1 is associated with changes in the expression of MET/HGF signaling components. PRDX1 forms a heterodimer with p38α mitogen-activated protein kinase 14 (MAPK14), stabilizing phospho-p38α in glioma cells. This process amplifies hepatocyte growth factor (HGF)-mediated signaling and stimulates actin cytoskeleton dynamics that promote glioma cell migration. Whole-brain high-resolution ultramicroscopy confirms these findings, indicating that PRDX1 promotes glioma brain invasion in vivo. Finally, reduced expression of PRDX1 increased survival in mouse glioma models. Thus, our preclinical findings suggest that PRDX1 expression levels may serve as a molecular marker for patients who could benefit from targeted inhibition of MET/HGF signaling.

Induction of ER and mitochondrial stress by the alkylphosphocholine erufosine in oral squamous cell carcinoma cells.

Endoplasmic reticulum (ER) plays an essential role in cell function and survival. Accumulation of unfolded or misfolded proteins in the lumen of the ER activates the unfolded protein response (UPR), resulting in ER stress and subsequent apoptosis. The alkylphosphocholine erufosine is a known Akt-mTOR inhibitor in oral squamous cell carcinoma (OSCC). In the present study, we evaluate erufosine's role to induce ER and mitochondrial stress leading to autophagy, apoptosis, and ROS induction. The cellular toxicity of erufosine was determined in two OSCC cell lines and gene expression and enrichment analyses were performed. A positive enrichment of ER stress upon erufosine exposure was observed, which was verified at protein levels for the ER stress sensors and their downstream mediators. Knockdown and pharmacological inhibition of the ER stress sensors PERK and XBP1 revealed their involvement into erufosine's cellular effects, including proliferation, apoptosis, and autophagy induction. Autophagy was confirmed by increased acidic vacuoles and LC3-B levels. Upon erufosine exposure, calcium influx into the cytoplasm of the two OSCC cell lines was seen. Apoptosis was confirmed by nuclear staining, Annexin-V, and immunoblotting of caspases. The induction of mitochondrial stress upon erufosine exposure was predicted by gene set enrichment analysis (GSEA) and shown by erufosine's effect on mitochondrial membrane potential, ATP, and ROS production in OSCC cells. These data show that ER and mitochondrial targeting by erufosine represents a new facet of its mechanism of action as well as a promising new framework in the treatment of head and neck cancers.

Correlated MRI and Ultramicroscopy (MR-UM) of Brain Tumors Reveals Vast Heterogeneity of Tumor Infiltration and Neoangiogenesis in Preclinical Models and Human Disease.

Diffuse tumor infiltration into the adjacent parenchyma is an effective dissemination mechanism of brain tumors. We have previously developed correlated high field magnetic resonance imaging and ultramicroscopy (MR-UM) to study neonangiogenesis in a glioma model. In the present study we used MR-UM to investigate tumor infiltration and neoangiogenesis in a translational approach. We compare infiltration and neoangiogenesis patterns in four brain tumor models and the human disease: whereas the U87MG glioma model resembles brain metastases with an encapsulated growth and extensive neoangiogenesis, S24 experimental gliomas mimic IDH1 wildtype glioblastomas, exhibiting infiltration into the adjacent parenchyma and along white matter tracts to the contralateral hemisphere. MR-UM resolves tumor infiltration and neoangiogenesis longitudinally based on the expression of fluorescent proteins, intravital dyes or endogenous contrasts. Our study demonstrates the huge morphological diversity of brain tumor models regarding their infiltrative and neoangiogenic capacities and further establishes MR-UM as a platform for translational neuroimaging.

A Comprehensive High-Resolution Targeted Workflow for the Deep Profiling of Sphingolipids.

Sphingolipids make up a highly diverse group of biomolecules that not only are membrane components but also are involved in various cellular functions such as signaling and protein sorting. To obtain a quantitative view of the sphingolipidome, sensitive, accurate, and comprehensive methods are needed. Here, we present a targeted reversed-phase liquid chromatography-high-resolution mass spectrometry-based workflow that significantly increases the accuracy of measured sphingolipids by resolving nearly isobaric and isobaric species; this is accomplished by a use of (i) an optimized extraction procedure, (ii) a segmented gradient, and (iii) parallel reaction monitoring of a sphingolipid specific fragmentation pattern. The workflow was benchmarked against an accepted sphingolipid model system, the RAW 264.7 cell line, and 61 sphingolipids were quantified over a dynamic range of 7 orders of magnitude, with detection limits in the low femtomole per milligram of protein level, making this workflow an extremely versatile tool for high-throughput sphingolipidomics.

In vivo nanoparticle imaging of innate immune cells can serve as a marker of disease severity in a model of multiple sclerosis.

Innate immune cells play a key role in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Current clinical imaging is restricted to visualizing secondary effects of inflammation, such as gliosis and blood-brain barrier disruption. Advanced molecular imaging, such as iron oxide nanoparticle imaging, can allow direct imaging of cellular and molecular activity, but the exact cell types that phagocytose nanoparticles in vivo and how phagocytic activity relates to disease severity is not well understood. In this study we used MRI to map inflammatory infiltrates using high-field MRI and fluorescently labeled cross-linked iron oxide nanoparticles for cell tracking. We confirmed nanoparticle uptake and MR detectability ex vivo. Using in vivo MRI, we identified extensive nanoparticle signal in the cerebellar white matter and circumscribed cortical gray matter lesions that developed during the disease course (4.6-fold increase of nanoparticle accumulation in EAE compared with healthy controls, P < 0.001). Nanoparticles showed good cellular specificity for innate immune cells in vivo, labeling activated microglia, infiltrating macrophages, and neutrophils, whereas there was only sparse uptake by adaptive immune cells. Importantly, nanoparticle signal correlated better with clinical disease than conventional gadolinium (Gd) imaging (r, 0.83 for nanoparticles vs. 0.71 for Gd-imaging, P < 0.001). We validated our approach using the Food and Drug Administration-approved iron oxide nanoparticle ferumoxytol. Our results show that noninvasive molecular imaging of innate immune responses can serve as an imaging biomarker of disease activity in autoimmune-mediated neuroinflammation with potential clinical applications in a wide range of inflammatory diseases.

Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain.

Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer's disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain.

Correlated magnetic resonance imaging and ultramicroscopy (MR-UM) is a tool kit to assess the dynamics of glioma angiogenesis.

Neoangiogenesis is a pivotal therapeutic target in glioblastoma. Tumor monitoring requires imaging methods to assess treatment effects and disease progression. Until now mapping of the tumor vasculature has been difficult. We have developed a combined magnetic resonance and optical toolkit to study neoangiogenesis in glioma models. We use in vivo magnetic resonance imaging (MRI) and correlative ultramicroscopy (UM) of ex vivo cleared whole brains to track neovascularization. T2* imaging allows the identification of single vessels in glioma development and the quantification of neovessels over time. Pharmacological VEGF inhibition leads to partial vascular normalization with decreased vessel caliber, density, and permeability. To further resolve the tumor microvasculature, we performed correlated UM of fluorescently labeled microvessels in cleared brains. UM resolved typical features of neoangiogenesis and tumor cell invasion with a spatial resolution of ~5 µm. MR-UM can be used as a platform for three-dimensional mapping and high-resolution quantification of tumor angiogenesis.

Tetraspanin-3 is an organizer of the multi-subunit Nogo-A signaling complex.

To ensure precision and specificity of ligand-receptor-induced signaling, co-receptors and modulatory factors play important roles. The membrane-bound ligand Nogo-A (an isoform encoded by RTN4) induces inhibition of neurite outgrowth, cell spreading, adhesion and migration through multi-subunit receptor complexes. Here, we identified the four-transmembrane-spanning protein tetraspanin-3 (TSPAN3) as a new modulatory co-receptor for the Nogo-A inhibitory domain Nogo-A-Δ20. Single-molecule tracking showed that TSPAN3 molecules in the cell membrane reacted to binding of Nogo-A with elevated mobility, which was followed by association with the signal-transducing Nogo-A receptor sphingosine-1-phosphate receptor 2 (S1PR2). Subsequently, TSPAN3 was co-internalized as part of the Nogo-A-ligand-receptor complex into early endosomes, where it subsequently separated from Nogo-A and S1PR2 to be recycled to the cell surface. The functional importance of the Nogo-A-TSPAN3 interaction is shown by the fact that knockdown of TSPAN3 strongly reduced the Nogo-A-induced S1PR2 clustering, RhoA activation, cell spreading and neurite outgrowth inhibition. In addition to the modulatory functions of TSPAN3 on Nogo-A-S1PR2 signaling, these results illustrate the very dynamic spatiotemporal reorganizations of membrane proteins during ligand-induced receptor complex organization.

Nogo-A-deficient Transgenic Rats Show Deficits in Higher Cognitive Functions, Decreased Anxiety, and Altered Circadian Activity Patterns.

Decreased levels of Nogo-A-dependent signaling have been shown to affect behavior and cognitive functions. In Nogo-A knockout and knockdown laboratory rodents, behavioral alterations were observed, possibly corresponding with human neuropsychiatric diseases of neurodevelopmental origin, particularly schizophrenia. This study offers further insight into behavioral manifestations of Nogo-A knockdown in laboratory rats, focusing on spatial and non-spatial cognition, anxiety levels, circadian rhythmicity, and activity patterns. Demonstrated is an impairment of cognitive functions and behavioral flexibility in a spatial active avoidance task, while non-spatial memory in a step-through avoidance task was spared. No signs of anhedonia, typical for schizophrenic patients, were observed in the animals. Some measures indicated lower anxiety levels in the Nogo-A-deficient group. Circadian rhythmicity in locomotor activity was preserved in the Nogo-A knockout rats and their circadian period (tau) did not differ from controls. However, daily activity patterns were slightly altered in the knockdown animals. We conclude that a reduction of Nogo-A levels induces changes in CNS development, manifested as subtle alterations in cognitive functions, emotionality, and activity patterns.