Biology subjects

Blaess, S.

Publications and source records attributed to Blaess, S..

8 recordsLinked to original sources

bioRxiv2026

A new conditional mouse model for Cre-mediated restoration of Hprt1 expression during brain development

A central challenge in genetic neurodevelopmental disorders is determining the developmental windows, brain regions, and cell lineages that are affected by gene dysfunction, and whether the resulting pathology remains reversible. The defined genotype-phenotype relationship in Lesch-Nyhan disease (LND), a monogenic disorder caused by pathogenic variants in HPRT1 and subsequent deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt), provides a well-characterized disease context for such mechanistic investigations. We present a new conditional Hprt1 mouse model carrying a loxP-flanked mCherry and transcriptional stop cassette, permitting Cre-driver-dependent analysis of spatially and temporally controlled restoration of endogenous Hprt1 expression during brain development. Hprt1 reactivation was confirmed at the genomic, transcriptional, and biochemical levels. Using the ubiquitous tamoxifen-inducible CAG-CreER driver, a single maternal tamoxifen dose at embryonic day (E)8.5 restored whole-brain Hprt1 mRNA expression and HGprt enzyme activity in E14.5 embryos to 72.4% and 13.4% of wild-type levels, respectively. Postnatal exposure through lactation during maternal treatment from postnatal day (P)0 to P4 yielded whole-brain HGprt activity equivalent to 28.3% of the WT level at P17. However, ligand-independent CAG-CreER activity and treatment-associated reproductive and developmental complications precluded extended longitudinal rescue studies with this Cre-driver. Developmentally regulated Wnt1-Cre2 activity, from E8 onwards, provided an alternative approach that permitted analysis through P60. Although the Wnt1 lineage constitutes only a fraction of the developing brain, Cre-dependent reactivation produced detectable HGprt activity in the forebrain, midbrain, and hindbrain. RNA fluorescent in situ hybridization (FISH) detected spatially distributed Hprt1 reactivation in the midbrain and prefrontal cortex at E14.5 and across multiple midbrain, cortical, and subcortical regions at P60. These findings establish the conditional Hprt1tm1.1(LoxP-mCherry-Stop-LoxP)Vsr mouse as a model for investigating when and where Hprt1 restoration could rescue abnormal brain development, while demonstrating that Cre-driver properties determine experimental specificity and longitudinal feasibility. Beyond LND, this conditional reactivation strategy provides an approach for defining developmental windows of reversibility and lineage-specific requirements for gene restoration in monogenic neurodevelopmental disorders.

neuroscience↗

bioRxiv2026

Basolateral amygdala dopamine signals behavioural salience across exploration and learning

Animals must balance exploration with threat avoidance, yet how neuromodulatory signals shape information-seeking states remains unclear. Using fibre photometry recordings in the basolateral amygdala in freely moving mice, we show that dopamine release is selectively elevated during self-initiated exploratory actions, adapts with repeated exposure, and tracks the behavioural relevance of predictive cues in associative learning and extinction. Our results advance current models of amygdala dopamine function by suggesting that it conveys a state-dependent salience signal that highlights behaviourally relevant moments arising from both environmental cues and self-generated exploration, thereby priming amygdala circuits for plasticity.

neuroscience↗

bioRxiv2026

A human developmental and adult brain atlas benchmarks dopaminergic stem cell models and cell therapy candidates

Parkinsons disease (PD) is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons1. Stem cell-derived mDA neurons hold promise for disease modelling2,3 and are currently in clinical trials for cell replacement therapy4-6. However, systematic benchmarking has been limited by the lack of a unified high-resolution reference and methods that quantify incomplete or mixed lineage specification in vitro7. We establish a single-cell and spatial atlas of the human developing diencephalon-midbrain-hindbrain axis resolving 93 cell subtypes, including 39 lacking prior single-cell characterization and 4 entirely novel populations. Using this atlas as a reference, we integrate 19 hPSC-derived mDA datasets, both published2,8-25 and unpublished, to build the Human Dopaminergic Neural Atlas (HDNA) spanning 2D, 3D, and graft models, including those used in clinical trials. To classify cells and quantify lineage fidelity, we develop CapybaraBrain, a marker-driven non-negative decomposition framework that assigns each cell continuous identity scores across all 93 developmental programs, enabling systematic discrimination of discrete, transitioning, and cross-lineage hybrid states26. We uncover a pervasive landscape of off-target populations reflecting relaxed transcriptional boundaries in vitro, including a previously unrecognized TH-PITX2 midbrain neuronal population, and we validate atlas-predicted latent lineage plasticity through inducible genetic fate mapping in mouse models. We further define maturation-associated transcriptional programs by harmonizing adult mDA subtype atlases, revealing that dopaminergic identity and maturation are partially decoupled across protocols. Finally, projecting PD patient-derived tri-cultures onto the HDNA uncovers genotype- and cell-type-specific transcriptional dysregulation. Together, these integrated atlases and computational framework establish a unified standard for benchmarking differentiation fidelity, exposing off-target states, and guiding next-generation PD models and cell therapies.

Developmental Biology↗

bioRxiv2024

Making the effect visible - OX40 targeting nanobodies for in vivo imaging of activated T cells

PurposeHuman OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. MethodsNbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. ResultsOur selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. ConclusionConsidering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40+ T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.

immunology↗

bioRxiv2023

A missense mutation (C667F) in beta-dystroglycan results in reduced dystroglycan protein levels leading to myopathy and destabilization of the blood-brain and blood-retinal barrier protein network

Dystroglycan (DG) is a glycoprotein and extracellular matrix receptor consisting of an -DG and a {beta}-DG subunit encoded by the gene DAG1. A homozygous missense mutation (c.2006G>T), resulting in an amino acid substitution (p.Cys669Phe) in the extracellular domain of {beta}-DG, causes severe Muscle-Eye-Brain disease with multicystic leukodystrophy. To investigate the mechanisms underlying the severe human pathology, we generated a mouse model of this primary dystroglycanopathy. We find that homozygous mutant mice show no obvious abnormalities during development and reach mature adulthood. However, - and {beta}-DG protein levels are significantly downregulated in muscle and brain of homozygous mutant mice. The mutant mice show a form of myopathy with late-onset and not fully penetrant histopathological changes in skeletal muscle and are impaired in their performance on an activity wheel. The brain and eyes of the homozygous mutant mice appear to be structurally normal, but the localization of mutant {beta}-DG is altered in the glial perivascular endfeet (PVE) at the blood-brain- and blood-retina barrier resulting in a perturbed protein composition in the PVE. In conclusion, the mouse model of the C669F {beta}-DG mutation does not seem to recapitulate the severe developmental phenotypes observed in human patients but represents a novel and highly valuable tool to study the impact of {beta}-DG functional changes at the molecular level and to gain insight into the pathogenesis of primary dystroglycanopathies.

pathology↗

bioRxiv2023

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Signal-regulatory protein (SIRP) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRP and the "dont eat me" ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRP (hSIRP)-specific nanobodies (Nbs). We identified three high-affinity Nbs targeting the hSIRP/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis (ADCP). For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography (PET) in novel hSIRP/hCD47 knock-in (KI) mice, we demonstrated the applicability of 64Cu-hSIRP-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRP-Nbs presented in this study have potential as versatile probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.

immunology↗

bioRxiv2020

The transcription factor BCL11A defines a distinctive subset of dopamine neurons in the developing and adult midbrain

Midbrain dopaminergic (mDA) neurons are diverse in their projection targets, impact on behavior and susceptibility to neurodegeneration. Little is known about the molecular mechanisms that establish this diversity in mDA neurons during development. We find that the transcription factor Bcl11a defines a subset of mDA neurons in the developing and adult murine brain. By combining intersectional labeling and viral-mediated tracing we show that Bcl11a-expressing mDA neurons form a highly specific subcircuit within the dopaminergic system. We demonstrate that Bcl11a-expressing mDA neurons in the substantia nigra (SN) are particularly vulnerable to neurodegeneration in an -synuclein overexpression model of Parkinsons disease. Inactivation of Bcl11a in developing mDA neurons results in anatomical changes, deficits in motor learning and a dramatic increase in the susceptibility to -synuclein-induced degeneration in SN-mDA neurons. In summary, we identify an mDA subpopulation with highly distinctive characteristics defined by the expression of the transcription factor Bcl11a already during development.

developmental biology↗

bioRxiv2018

Formation of the substantia nigra requires Reelin-mediated fast, laterally-directed migration of dopaminergic neurons

Midbrain dopaminergic (mDA) neurons migrate to form the laterally-located substantia nigra pars compacta (SN) and medially-located ventral tegmental area (VTA), but little is known about the underlying cellular and molecular processes. Reelin signaling regulates tangential migration of SN-mDA neurons, but whether Reelin acts directly on SN-mDA neurons and how it affects their cellular morphology and migratory behavior has not been explored. Here we visualize the dynamic cell morphologies of tangentially migrating SN-mDA neurons with 3D-time-lapse imaging and identify two distinct migration modes. Slow migration is the default mode in SN-mDA neurons, while fast, laterally-directed migration occurs infrequently and is strongly associated with bipolar cell morphology. By speci1cally inactivating Reelin signaling in mDA neurons we demonstrate its direct role in SN-mDA tangential migration. We show that Reelin signaling promotes laterally-biased movements in mDA neurons during their slow migration mode, stabilizes leading process morphology and increases the probability of fast, laterally-directed migration.

developmental biology↗