Sebastian Jessberger

Astrocyte loss occurs in various neurological conditions and can disrupt local tissue homeostasis. While astrocytes surrounding border-forming lesions adopt reactive states without restoring astrocyte networks, how astrocytes respond to spatially confined astrocyte loss remains poorly understood. Here we used longitudinal in vivo two-photon microscopy, combined with spatiotemporal transcriptional profiling, to examine astrocyte responses following focal aquaporin-4 antibody-mediated ablation in the somatosensory cortex of adult mouse brain, a model of astrocytopathy relevant to neuromyelitis optica spectrum disorder. Here we show that perilesional astrocytes undergo pronounced structural remodeling during lesion repopulation, characterized by cell proliferation, prolonged multinucleated astrocyte states, polarized process extension into the depleted area and gradual displacement of nuclei into previously unoccupied astrocyte territories. Spatial transcriptomics reveal an injury-associated molecular response that resolves as the astrocyte network is restored. Together, our findings delineate the spatiotemporal dynamics of astrocyte regeneration after astrocyte loss, extending current understanding of astroglial plasticity in the adult brain.

Neuronal activation drives activity-dependent gene expression that underlies experience-associated synaptic modifications, learning and memory. Here we show that the histone variant CENP-A, best known for specifying centromere identity, is dynamically regulated by synaptic activity at both the RNA and protein levels in postmitotic neurons. Neuronal activation increases a non-centromeric nuclear pool of CENP-A while leaving centromeric CENP-A levels unchanged. Downregulation of CENP-A selectively reduces the activity-associated non-centromeric pool, impairs activity-dependent induction of immediate-early genes such as FOS and ARC, and disrupts hippocampus-dependent learning and memory. Furthermore, we find that activity-dependent neuronal responses in human embryonic stem cell-derived forebrain organoids similarly require CENP-A. Our results reveal a mitosis-independent, conserved role of CENP-A for driving plasticity in mammalian neurons.

Adult neural stem cells (aNSCs) of the lateral ventricular sub-ventricular zone (V-SVZ) are set aside during embryogenesis from a pool of slowly dividing neural stem/progenitor cells (NSPCs) residing in the lateral ganglionic eminence (LGE). The time of aNSCs specification coincides with the peak of embryonic neurogenesis, raising the question of which mechanisms allow only a subpopulation of NSPCs to retain stem cell identity while others proceed to divide and differentiate into neurons. To address this, we isolated rapidly and slowly dividing NSPCs from the mouse LGE at mid-neurogenesis, when aNSCs are specified, and profiled the transcriptome at the single cell level. We find that slowly dividing NSPCs constitute a heterogeneous population encompassing both radial glia cells (RGCs) and intermediate progenitors (IPs) and characterized by different gene signatures. Focusing on RGCs, we show that slowly and rapidly dividing RGCs differentially express genes involved in the formation – such as Lamb2 – or degradation – such as Mmp15 – of the extracellular matrix (ECM), pointing to opposite niche-remodelling strategies as a potential mechanism of fate determination. In vivo perturbation of Lamb2 and Mmp15 affects the seeding and maintenance of NSCs in an opposing manner, with knockdown of Mmp15 increasing the NSC pool as also shown by single-cell RNA-sequencing. These findings suggest that opposite ECM remodelling by slowly and rapidly dividing RGCs constitutes a novel cross-regulatory niche-level mechanism regulating aNSCs emergence, opening new avenues for regulating their numbers and thereby long-term maintenance of the brain’s regenerative potential.

The epigenetic landscape of living cells is dynamic and heterogeneous. To track these changes with high temporal resolution, fluorescent biosensors can be powerful tools. We aimed to use the split-Venus system to monitor the dynamic changes in DNA methylation levels at the major satellite repeat regions. While the individual components of the biosensor showed specificity for their targets, key control experiments demonstrated that the combined biosensor constructs failed to respond to global DNA methylation changes. Specifically, the high intrinsic affinity of the split-Venus halves appeared to drive strong false-positive signal. The observed non-specific complementation highlights a limitation of split fluorophore systems, particularly regarding their need for careful construct testing and optimization.

Multiphoton imaging allows for the visualization of structural and functional plasticity within the central nervous system. However, gaining optical access to deep brain structures, such as the hippocampal dentate gyrus (DG), requires invasive approaches, causing brain damage. Here we optimize three-photon (3P) microscopy to perform longitudinal imaging of the DG in the intact brain at unprecedented depth of up to 1800 µm. We apply this approach to follow the dynamics of neural stem cells (NSCs) in the adult and developing DG, allowing for novel insights into structural plasticity deep within the intact mouse brain.

Astrocyte heterogeneity has been well explored, but our understanding of white matter (WM) astrocytes and their distinctions from gray matter (GM) astrocytes remains limited. Here, we compared astrocytes from cortical GM and WM/corpus callosum (WM/CC) using single-cell RNA sequencing and spatial transcriptomics of the murine forebrain. The comparison revealed similarities but also significant differences between WM and GM astrocytes, including cytoskeletal and metabolic hallmarks specific to WM astrocytes with molecular properties also shared with human WM astrocytes. When we compared murine astrocytes from two different WM regions, the cortex and cerebellum, we found that they exhibited distinct, region-specific molecular properties, with the cerebellum lacking, for example, a specific cluster of WM astrocytes expressing progenitor and proliferation genes. Functional experiments confirmed astrocyte proliferation in the WM/CC, but not in the cerebellar WM, suggesting that the WM/CC may be a source of continued astrogenesis.

During normal cellular homeostasis, unfolded and mislocalized proteins are recognized and removed, preventing the build-up of toxic byproducts¹. When protein homeostasis is perturbed during ageing, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites^2,3. Such modifications have been proposed to trigger the selective removal of chemically marked proteins^3–6; however, identifying modifications that are sufficient to induce protein degradation has remained challenging. Here, using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified FBXO31 as a reader of C-terminal amides. FBXO31 is a substrate receptor for the SKP1–CUL1–F-box protein (SCF) ubiquitin ligase SCF–FBXO31, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind to almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed after oxidative stress. A dominant human mutation found in neurodevelopmental disorders reverses CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins.

The mammalian dentate gyrus (DG) is involved in certain forms of learning and memory, and DG dysfunction has been implicated in age-related diseases. Although neurogenic potential is maintained throughout life in the DG as neural stem cells (NSCs) continue to generate new neurons, neurogenesis decreases with advancing age, with implications for age-related cognitive decline and disease. In this study, we used single-cell RNA sequencing to characterize transcriptomic signatures of neurogenic cells and their surrounding DG niche, identifying molecular changes associated with neurogenic aging from the activation of quiescent NSCs to the maturation of fate-committed progeny. By integrating spatial transcriptomics data, we identified the regional invasion of inflammatory cells into the hippocampus with age and show here that early-onset neuroinflammation decreases neurogenic activity. Our data reveal the lifelong molecular dynamics of NSCs and their surrounding neurogenic DG niche with age and provide a powerful resource to understand age-related molecular alterations in the aging hippocampus.

During human forebrain development, neural progenitor cells (NPCs) in the ventricular zone (VZ) undergo asymmetric cell divisions to produce a self-renewed progenitor cell, maintaining the potential to go through additional rounds of cell divisions, and differentiating daughter cells, populating the developing cortex. Previous work in the embryonic rodent brain suggested that the preferential inheritance of the pre-existing (older) centrosome to the self-renewed progenitor cell is required to maintain stem cell properties, ensuring proper neurogenesis. If asymmetric segregation of centrosomes occurs in NPCs of the developing human brain, which depends on unique molecular regulators and species-specific cellular composition, remains unknown. Using a novel, recombination-induced tag exchange-based genetic tool to birthdate and track the segregation of centrosomes over multiple cell divisions in human embryonic stem cell-derived regionalised forebrain organoids, we show the preferential inheritance of the older mother centrosome towards self-renewed NPCs. Aberration of asymmetric segregation of centrosomes by genetic manipulation of the centrosomal, microtubule-associated protein Ninein alters fate decisions of NPCs and their maintenance in the VZ of human cortical organoids. Thus, the data described here use a novel genetic approach to birthdate centrosomes in human cells and identify asymmetric inheritance of centrosomes as a mechanism to maintain self-renewal properties and to ensure proper neurogenesis in human NPCs.