Publications by authors named "Konstantin Khodosevich"

44 Publications

Identification of Vulnerable Interneuron Subtypes in 15q13.3 Microdeletion Syndrome Using Single-Cell Transcriptomics.

Biol Psychiatry 2021 Sep 24. Epub 2021 Sep 24.

Biotech Research and Innovation Center (BRIC), Copenhagen Biocenter, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Background: A number of rare copy number variants (CNVs) have been linked to neurodevelopmental disorders. However, because CNVs encompass many genes, it is often difficult to identify the mechanisms that lead to developmental perturbations.

Methods: We used 15q13.3 microdeletion to propose and validate a novel strategy to predict the impact of CNV genes on brain development that could further guide functional studies. We analyzed single-cell transcriptomics datasets containing cortical interneurons to identify their developmental vulnerability to 15q13.3 microdeletion, which was validated in mouse models.

Results: We found that Klf13-but not other 15q13.3 genes-is expressed by precursors and neuroblasts in the medial and caudal ganglionic eminences during development, with a peak of expression at embryonic day (E)13.5 and E18.5, respectively. In contrast, in the adult mouse brain, Klf13 expression is negligible. Using Df(h15q13.3)/+ and Klf13 embryos, we observed a precursor subtype-specific impairment in proliferation in the medial ganglionic eminence and caudal ganglionic eminence at E13.5 and E17.5, respectively, corresponding to vulnerability predicted by Klf13 expression patterns. Finally, Klf13 mice showed a layer-specific decrease in parvalbumin and somatostatin cortical interneurons accompanied by changes in locomotor and anxiety-related behavior.

Conclusions: We show that the impact of 15q13.3 microdeletion on precursor proliferation is grounded in a reduction in Klf13 expression. The lack of Klf13 in Df(h15q13.3)/+ cortex might be the major reason for perturbed density of cortical interneurons. Thus, the behavioral defects seen in 15q13.3 microdeletion could stem from a developmental perturbation owing to selective vulnerability of cortical interneurons during sensitive stages of their development.
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http://dx.doi.org/10.1016/j.biopsych.2021.09.012DOI Listing
September 2021

Coordinated maintenance of H3K36/K27 methylation by histone demethylases preserves germ cell identity and immortality.

Cell Rep 2021 Nov;37(8):110050

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes vej 5, Copenhagen DK-2200, Denmark. Electronic address:

Germ cells have evolved unique mechanisms to ensure the transmission of genetically and nongenetically encoded information, whose alteration compromises germ cell immortality. Chromatin factors play fundamental roles in these mechanisms. H3K36 and H3K27 methyltransferases shape and propagate a pattern of histone methylation essential for C. elegans germ cell maintenance, but the role of respective histone demethylases remains unexplored. Here, we show that jmjd-5 regulates H3K36me2 and H3K27me3 levels, preserves germline immortality, and protects germ cell identity by controlling gene expression. The transcriptional and biological effects of jmjd-5 loss can be hindered by the removal of H3K27demethylases, indicating that H3K36/K27 demethylases act in a transcriptional framework and promote the balance between H3K36 and H3K27 methylation required for germ cell immortality. Furthermore, we find that in wild-type, but not in jmjd-5 mutants, alterations of H3K36 methylation and transcription occur at high temperature, suggesting a role for jmjd-5 in adaptation to environmental changes.
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http://dx.doi.org/10.1016/j.celrep.2021.110050DOI Listing
November 2021

Cell segmentation in imaging-based spatial transcriptomics.

Nat Biotechnol 2021 Oct 14. Epub 2021 Oct 14.

Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA.

Single-molecule spatial transcriptomics protocols based on in situ sequencing or multiplexed RNA fluorescent hybridization can reveal detailed tissue organization. However, distinguishing the boundaries of individual cells in such data is challenging and can hamper downstream analysis. Current methods generally approximate cells positions using nuclei stains. We describe a segmentation method, Baysor, that optimizes two-dimensional (2D) or three-dimensional (3D) cell boundaries considering joint likelihood of transcriptional composition and cell morphology. While Baysor can take into account segmentation based on co-stains, it can also perform segmentation based on the detected transcripts alone. To evaluate performance, we extend multiplexed error-robust fluorescence in situ hybridization (MERFISH) to incorporate immunostaining of cell boundaries. Using this and other benchmarks, we show that Baysor segmentation can, in some cases, nearly double the number of cells compared to existing tools while reducing segmentation artifacts. We demonstrate that Baysor performs well on data acquired using five different protocols, making it a useful general tool for analysis of imaging-based spatial transcriptomics.
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http://dx.doi.org/10.1038/s41587-021-01044-wDOI Listing
October 2021

PIAS2-mediated blockade of IFN-β signaling: a basis for sporadic Parkinson disease dementia.

Mol Psychiatry 2021 Jul 8. Epub 2021 Jul 8.

Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark.

Familial Parkinson disease (PD) is associated with rare genetic mutations, but the etiology in most patients with sporadic (s)PD is largely unknown, and the basis for its progression to dementia (sPDD) is poorly characterized. We have identified that loss of IFNβ or IFNAR1, the receptor for IFNα/β, causes pathological and behavioral changes resembling PDD, prompting us to hypothesize that dysregulated genes in IFNβ-IFNAR signaling pathway predispose one to sPD. By transcriptomic analysis, we found defective neuronal IFNβ-IFNAR signaling, including particularly elevated PIAS2 associated with sPDD. With meta-analysis of GWASs, we identified sequence variants in IFNβ-IFNAR-related genes in sPD patients. Furthermore, sPDD patients expressed higher levels of PIAS2 mRNA and protein in neurons. To determine its function in brain, we overexpressed PIAS2 under a neuronal promoter, alone or with human α-synuclein, in the brains of mice, which caused motor and cognitive impairments and correlated with intraneuronal phosphorylated (p)α-synuclein accumulation and dopaminergic neuron loss. Ectopic expression of neuronal PIAS2 blocked mitophagy, increased the accumulation of senescent mitochondrial and oxidative stress, as evidenced by excessive oxDJ1 and 8OHdG, by inactivating ERK1/2-P53 signaling. Conversely, PIAS2 knockdown rescued the clinicopathological manifestations of PDD in Ifnb mice on restoring mitochondrial homeostasis, oxidative stress, and pERK1/2-pP53 signaling. The regulation of JAK-STAT2-PIAS2 signaling was crucial for neurite outgrowth and neuronal survival and excitability and thus might prevent cognitive impairments. Our findings provide insights into the progression of sPD and dementia and have implications for new therapeutic approaches.
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http://dx.doi.org/10.1038/s41380-021-01207-wDOI Listing
July 2021

Development of the Entorhinal Cortex Occurs via Parallel Lamination During Neurogenesis.

Front Neuroanat 2021 5;15:663667. Epub 2021 May 5.

Group of Brain Development and Disease, Section Pathobiological Sciences, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

The entorhinal cortex (EC) is the spatial processing center of the brain and structurally is an interface between the three layered paleocortex and six layered neocortex, known as the periarchicortex. Limited studies indicate peculiarities in the formation of the EC such as early emergence of cells in layers (L) II and late deposition of LIII, as well as divergence in the timing of maturation of cell types in the superficial layers. In this study, we examine developmental events in the entorhinal cortex using an understudied model in neuroanatomy and development, the pig and supplement the research with BrdU labeling in the developing mouse EC. We determine the pig serves as an excellent anatomical model for studying human neurogenesis, given its long gestational length, presence of a moderate sized outer subventricular zone and early cessation of neurogenesis during gestation. Immunohistochemistry identified prominent clusters of OLIG2 oligoprogenitor-like cells in the superficial layers of the lateral EC (LEC) that are sparser in the medial EC (MEC). These are first detected in the subplate during the early second trimester. MRI analyses reveal an acceleration of EC growth at the end of the second trimester. BrdU labeling of the developing MEC, shows the deeper layers form first and prior to the superficial layers, but the LV/VI emerges in parallel and the LII/III emerges later, but also in parallel. We coin this lamination pattern parallel lamination. The early born Reln stellate cells in the superficial layers express the classic LV marker, Bcl11b (Ctip2) and arise from a common progenitor that forms the late deep layer LV neurons. In summary, we characterize the developing EC in a novel animal model and outline in detail the formation of the EC. We further provide insight into how the periarchicortex forms in the brain, which differs remarkably to the inside-out lamination of the neocortex.
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http://dx.doi.org/10.3389/fnana.2021.663667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139189PMC
May 2021

The impact of (ab)normal maternal environment on cortical development.

Prog Neurobiol 2021 07 24;202:102054. Epub 2021 Apr 24.

Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200, Copenhagen, Denmark. Electronic address:

The cortex in the mammalian brain is the most complex brain region that integrates sensory information and coordinates motor and cognitive processes. To perform such functions, the cortex contains multiple subtypes of neurons that are generated during embryogenesis. Newly born neurons migrate to their proper location in the cortex, grow axons and dendrites, and form neuronal circuits. These developmental processes in the fetal brain are regulated to a large extent by a great variety of factors derived from the mother - starting from simple nutrients as building blocks and ending with hormones. Thus, when the normal maternal environment is disturbed due to maternal infection, stress, malnutrition, or toxic substances, it might have a profound impact on cortical development and the offspring can develop a variety of neurodevelopmental disorders. Here we first describe the major developmental processes which generate neuronal diversity in the cortex. We then review our knowledge of how most common maternal insults affect cortical development, perturb neuronal circuits, and lead to neurodevelopmental disorders. We further present a concept of selective vulnerability of cortical neuronal subtypes to maternal-derived insults, where the vulnerability of cortical neurons and their progenitors to an insult depends on the time (developmental period), place (location in the developing brain), and type (unique features of a cell type and an insult). Finally, we provide evidence for the existence of selective vulnerability during cortical development and identify the most vulnerable neuronal types, stages of differentiation, and developmental time for major maternal-derived insults.
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http://dx.doi.org/10.1016/j.pneurobio.2021.102054DOI Listing
July 2021

Author Correction: Identification of epilepsy-associated neuronal subtypes and gene expression underlying epileptogenesis.

Nat Commun 2020 Nov 19;11(1):5988. Epub 2020 Nov 19.

Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200, Copenhagen, Denmark.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19869-5.
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http://dx.doi.org/10.1038/s41467-020-19869-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678822PMC
November 2020

Identification of epilepsy-associated neuronal subtypes and gene expression underlying epileptogenesis.

Nat Commun 2020 10 7;11(1):5038. Epub 2020 Oct 7.

Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200, Copenhagen, Denmark.

Epilepsy is one of the most common neurological disorders, yet its pathophysiology is poorly understood due to the high complexity of affected neuronal circuits. To identify dysfunctional neuronal subtypes underlying seizure activity in the human brain, we have performed single-nucleus transcriptomics analysis of >110,000 neuronal transcriptomes derived from temporal cortex samples of multiple temporal lobe epilepsy and non-epileptic subjects. We found that the largest transcriptomic changes occur in distinct neuronal subtypes from several families of principal neurons (L5-6_Fezf2 and L2-3_Cux2) and GABAergic interneurons (Sst and Pvalb), whereas other subtypes in the same families were less affected. Furthermore, the subtypes with the largest epilepsy-related transcriptomic changes may belong to the same circuit, since we observed coordinated transcriptomic shifts across these subtypes. Glutamate signaling exhibited one of the strongest dysregulations in epilepsy, highlighted by layer-wise transcriptional changes in multiple glutamate receptor genes and strong upregulation of genes coding for AMPA receptor auxiliary subunits. Overall, our data reveal a neuronal subtype-specific molecular phenotype of epilepsy.
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http://dx.doi.org/10.1038/s41467-020-18752-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541486PMC
October 2020

A community-based transcriptomics classification and nomenclature of neocortical cell types.

Nat Neurosci 2020 12;23(12):1456-1468

Vanderbilt University, Nashville, TN, USA.

To understand the function of cortical circuits, it is necessary to catalog their cellular diversity. Past attempts to do so using anatomical, physiological or molecular features of cortical cells have not resulted in a unified taxonomy of neuronal or glial cell types, partly due to limited data. Single-cell transcriptomics is enabling, for the first time, systematic high-throughput measurements of cortical cells and generation of datasets that hold the promise of being complete, accurate and permanent. Statistical analyses of these data reveal clusters that often correspond to cell types previously defined by morphological or physiological criteria and that appear conserved across cortical areas and species. To capitalize on these new methods, we propose the adoption of a transcriptome-based taxonomy of cell types for mammalian neocortex. This classification should be hierarchical and use a standardized nomenclature. It should be based on a probabilistic definition of a cell type and incorporate data from different approaches, developmental stages and species. A community-based classification and data aggregation model, such as a knowledge graph, could provide a common foundation for the study of cortical circuits. This community-based classification, nomenclature and data aggregation could serve as an example for cell type atlases in other parts of the body.
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http://dx.doi.org/10.1038/s41593-020-0685-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683348PMC
December 2020

Complex IV subunit isoform COX6A2 protects fast-spiking interneurons from oxidative stress and supports their function.

EMBO J 2020 09 3;39(18):e105759. Epub 2020 Aug 3.

Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Parvalbumin-positive (PV ) fast-spiking interneurons are essential to control the firing activity of principal neuron ensembles, thereby regulating cognitive processes. The high firing frequency activity of PV interneurons imposes high-energy demands on their metabolism that must be supplied by distinctive machinery for energy generation. Exploring single-cell transcriptomic data for the mouse cortex, we identified a metabolism-associated gene with highly restricted expression to PV interneurons: Cox6a2, which codes for an isoform of a cytochrome c oxidase subunit. Cox6a2 deletion in mice disrupts perineuronal nets and enhances oxidative stress in PV interneurons, which in turn impairs the maturation of their morphological and functional properties. Such dramatic effects were likely due to an essential role of COX6A2 in energy balance of PV interneurons, underscored by a decrease in the ATP-to-ADP ratio in Cox6a2 PV interneurons. Energy disbalance and aberrant maturation likely hinder the integration of PV interneurons into cortical neuronal circuits, leading to behavioral alterations in mice. Additionally, in a human patient bearing mutations in COX6A2, we found a potential association of the mutations with mental/neurological abnormalities.
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http://dx.doi.org/10.15252/embj.2020105759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507454PMC
September 2020

Genetic modification increases the survival and the neuroregenerative properties of transplanted neural stem cells.

JCI Insight 2020 02 27;5(4). Epub 2020 Feb 27.

Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen, Denmark.

Cell therapy raises hopes high for better treatment of brain disorders. However, the majority of transplanted cells often die soon after transplantation, and those that survive initially continue to die in the subacute phase, diminishing the impact of transplantations. In this study, we genetically modified transplanted human neural stem cells (hNSCs), from 2 distant embryonic stem cell lines (H9 and RC17), to express 1 of 4 prosurvival factors - Hif1a, Akt1, Bcl-2, or Bcl-xl - and studied how these modifications improve short- and long-term survival of transplanted hNSCs. All genetic modifications dramatically increased survival of the transplanted hNSCs. Importantly, 3 out of 4 modifications also enhanced the exit of hNSCs from the cell cycle, thus avoiding aberrant growth of the transplants. Bcl-xl expression provided the strongest protection of transplanted cells, reducing both immediate and delayed cell death, and stimulated hNSC differentiation toward neuronal and oligodendroglial lineages. By designing hNSCs with drug-controlled expression of Bcl-xl, we demonstrated that short-term expression of a prosurvival factor can ensure the long-term survival of transplanted cells. Importantly, transplantation of Bcl-xl-expressing hNSCs into mice suffering from stroke improved behavioral outcome and recovery of motor activity in mice.
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http://dx.doi.org/10.1172/jci.insight.126268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101138PMC
February 2020

Dynamic Changes in Ultrastructure of the Primary Cilium in Migrating Neuroblasts in the Postnatal Brain.

J Neurosci 2019 12 4;39(50):9967-9988. Epub 2019 Nov 4.

Department of Developmental and Regenerative Neurobiology, Institute of Brain Science, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan,

New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain. Immature neurons (neuroblasts) generated in the postnatal brain have a mitotic potential and migrate in chain-like cell aggregates toward the olfactory bulb. Here we report that migrating neuroblasts possess a tiny cellular protrusion called a primary cilium. Immunohistochemical studies with zebrafish, mouse, and monkey brains suggest that the presence of the primary cilium in migrating neuroblasts is evolutionarily conserved. Ciliogenesis in migrating neuroblasts in the rostral migratory stream is suppressed during mitosis and promoted after cell cycle exit. Moreover, live imaging and 3D electron microscopy revealed that ciliary localization and orientation change during saltatory movement of neuroblasts. Our results reveal highly organized dynamics in maturation and positioning of the primary cilium during neuroblast migration that underlie saltatory movement of postnatal-born neuroblasts.
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http://dx.doi.org/10.1523/JNEUROSCI.1503-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978947PMC
December 2019

Maternal inflammation has a profound effect on cortical interneuron development in a stage and subtype-specific manner.

Mol Psychiatry 2020 10 8;25(10):2313-2329. Epub 2019 Oct 8.

Biotech Research and Innovation Centre (BRIC), Faculty of Health, University of Copenhagen, Copenhagen N, Denmark.

Severe infections during pregnancy are one of the major risk factors for cognitive impairment in the offspring. It has been suggested that maternal inflammation leads to dysfunction of cortical GABAergic interneurons that in turn underlies cognitive impairment of the affected offspring. However, the evidence comes largely from studies of adult or mature brains and how the impairment of inhibitory circuits arises upon maternal inflammation is unknown. Here we show that maternal inflammation affects multiple steps of cortical GABAergic interneuron development, i.e., proliferation of precursor cells, migration and positioning of neuroblasts, as well as neuronal maturation. Importantly, the development of distinct subtypes of cortical GABAergic interneurons was discretely impaired as a result of maternal inflammation. This translated into a reduction in cell numbers, redistribution across cortical regions and layers, and changes in morphology and cellular properties. Furthermore, selective vulnerability of GABAergic interneuron subtypes was associated with the stage of brain development. Thus, we propose that maternally derived insults have developmental stage-dependent effects, which contribute to the complex etiology of cognitive impairment in the affected offspring.
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http://dx.doi.org/10.1038/s41380-019-0539-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515848PMC
October 2020

GluA4-Targeted AAV Vectors Deliver Genes Selectively to Interneurons while Relying on the AAV Receptor for Entry.

Mol Ther Methods Clin Dev 2019 Sep 23;14:252-260. Epub 2019 Jul 23.

Division of Medical Biotechnology, Paul-Ehrlich-Institut, 63225 Langen, Germany.

Selective gene delivery into subtypes of interneurons remains an important challenge in vector development. Adeno-associated virus (AAV) vector particles are especially promising for intracerebral injections. For cell entry, AAV2 particles are supposed to attach to heparan-sulfate proteoglycans (HSPGs) followed by endocytosis via the AAV receptor (AAVR). Here, we assessed engineered AAV particles deficient in HSPG attachment but competent in recognizing the glutamate receptor 4 (GluA4, also known as GluRD or GRIA4) through a displayed GluA4-specific DARPin (designed ankyrin repeat protein). When injected into the mouse brain, histological evaluation revealed that in various regions, more than 90% of the transduced cells were interneurons, mainly of the parvalbumin-positive subtype. Although part of the selectivity was mediated by the DARPin, the chosen spleen focus-forming virus (SFFV) promoter had contributed as well. Further analysis revealed that the DARPin mediated selective attachment to GluA4-positive cells, whereas gene delivery required expression of AAVR. Our data suggest that cell selectivity of AAV particles can be modified rationally and efficiently through DARPins, but expression of the AAV entry receptor remains essential.
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http://dx.doi.org/10.1016/j.omtm.2019.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706527PMC
September 2019

Joint analysis of heterogeneous single-cell RNA-seq dataset collections.

Nat Methods 2019 08 15;16(8):695-698. Epub 2019 Jul 15.

Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA.

Single-cell RNA sequencing is often applied in study designs that include multiple individuals, conditions or tissues. To identify recurrent cell subpopulations in such heterogeneous collections, we developed Conos, an approach that relies on multiple plausible inter-sample mappings to construct a global graph connecting all measured cells. The graph enables identification of recurrent cell clusters and propagation of information between datasets in multi-sample or atlas-scale collections.
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http://dx.doi.org/10.1038/s41592-019-0466-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684315PMC
August 2019

Tracing the origin of adult intestinal stem cells.

Nature 2019 06 15;570(7759):107-111. Epub 2019 May 15.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.

Adult intestinal stem cells are located at the bottom of crypts of Lieberkühn, where they express markers such as LGR5 and fuel the constant replenishment of the intestinal epithelium. Although fetal LGR5-expressing cells can give rise to adult intestinal stem cells, it remains unclear whether this population in the patterned epithelium represents unique intestinal stem-cell precursors. Here we show, using unbiased quantitative lineage-tracing approaches, biophysical modelling and intestinal transplantation, that all cells of the mouse intestinal epithelium-irrespective of their location and pattern of LGR5 expression in the fetal gut tube-contribute actively to the adult intestinal stem cell pool. Using 3D imaging, we find that during fetal development the villus undergoes gross remodelling and fission. This brings epithelial cells from the non-proliferative villus into the proliferative intervillus region, which enables them to contribute to the adult stem-cell niche. Our results demonstrate that large-scale remodelling of the intestinal wall and cell-fate specification are closely linked. Moreover, these findings provide a direct link between the observed plasticity and cellular reprogramming of differentiating cells in adult tissues following damage, revealing that stem-cell identity is an induced rather than a hardwired property.
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http://dx.doi.org/10.1038/s41586-019-1212-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986928PMC
June 2019

Neuroserpin expression during human brain development and in adult brain revealed by immunohistochemistry and single cell RNA sequencing.

J Anat 2019 09 15;235(3):543-554. Epub 2019 Jan 15.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

Neuroserpin is a serine-protease inhibitor mainly expressed in the CNS and involved in the inhibition of the proteolytic cascade. Animal models confirmed its neuroprotective role in perinatal hypoxia-ischaemia and adult stroke. Although neuroserpin may be a potential therapeutic target in the treatment of the aforementioned conditions, there is still no information in the literature on its distribution during human brain development. The present study provides a detailed description of the changing spatiotemporal patterns of neuroserpin focusing on physiological human brain development. Five stages were distinguished within our examined age range which spanned from the 7th gestational week until adulthood. In particular, subplate and deep cortical plate neurons were identified as the main sources of neuroserpin production between the 25th gestational week and the first postnatal month. Our immunohistochemical findings were substantiated by single cell RNA sequencing data showing specific neuronal and glial cell types expressing neuroserpin. The characterization of neuroserpin expression during physiological human brain development is essential for forthcoming studies which will explore its involvement in pathological conditions, such as perinatal hypoxia-ischaemia and adult stroke in human.
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http://dx.doi.org/10.1111/joa.12931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704272PMC
September 2019

An advanced enrichment method for rare somatic retroelement insertions sequencing.

Mob DNA 2018 31;9:31. Epub 2018 Oct 31.

1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya str. 16/10, Moscow, 117997 Russia.

Background: There is increasing evidence that the transpositional activity of retroelements (REs) is not limited to germ line cells, but often occurs in tumor and normal somatic cells. Somatic transpositions were found in several human tissues and are especially typical for the brain. Several computational and experimental approaches for detection of somatic retroelement insertions was developed in the past few years. These approaches were successfully applied to detect somatic insertions in clonally expanded tumor cells. At the same time, identification of somatic insertions presented in small proportion of cells, such as neurons, remains a considerable challenge.

Results: In this study, we developed a normalization procedure for library enrichment by DNA sequences corresponding to rare somatic RE insertions. Two rounds of normalization increased the number of fragments adjacent to somatic REs in the sequenced sample by more than 26-fold, and the number of identified somatic REs was increased by 8-fold.

Conclusions: The developed technique can be used in combination with vast majority of modern RE identification approaches and can dramatically increase their capacity to detect rare somatic RE insertions in different types of cells.
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http://dx.doi.org/10.1186/s13100-018-0136-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208084PMC
October 2018

Maternal inhalation of carbon black nanoparticles induces neurodevelopmental changes in mouse offspring.

Part Fibre Toxicol 2018 09 10;15(1):36. Epub 2018 Sep 10.

National Research Centre for the Working Environment, Lersø Parkallé 105, DK-2100, Copenhagen Ø, Denmark.

Background: Engineered nanoparticles are smaller than 100 nm and designed to improve or creating even new physico-chemical properties. Consequently, toxicological properties of materials may change as size reaches the nm size-range. We examined outcomes related to the central nervous system in the offspring following maternal inhalation exposure to nanosized carbon black particles (Printex 90).

Methods: Time-mated mice (NMRI) were exposed by inhalation, for 45 min/day to 0, 4.6 or 37 mg/m aerosolized carbon black on gestation days 4-18, i.e. for a total of 15 days. Outcomes included maternal lung inflammation (differential cell count in bronchoalveolar lavage fluid and Saa3 mRNA expression in lung tissue), offspring neurohistopathology and behaviour in the open field test.

Results: Carbon black exposure did not cause lung inflammation in the exposed females, measured 11 or 28-29 days post-exposure. Glial fibrillary acidic protein (GFAP) expression levels were dose-dependently increased in astrocytes around blood vessels in the cerebral cortex and hippocampus in six weeks old offspring, indicative of reactive astrogliosis. Also enlarged lysosomal granules were observed in brain perivascular macrophages (PVMs) in the prenatally exposed offspring. The number of parvalbumin-positive interneurons and the expression levels of parvalbumin were decreased in the motor and prefrontal cortices at weaning and 120 days of age in the prenatally exposed offspring. In the open field test, behaviour was dose-dependently altered following maternal exposure to Printex 90, at 90 days of age. Prenatally exposed female offspring moved a longer total distance, and especially males spent significantly longer time in the central zone of the maze. In the offspring, the described effects were long-lasting as they were present at all time points investigated.

Conclusion: The present study reports for the first time that maternal inhalation exposure to Printex 90 carbon black induced dose-dependent denaturation of PVM and reactive astrocytes, similarly to the findings observed following maternal exposure to Printex 90 by airway instillation. Of note, some of the observed effects have striking similarities with those observed in mouse models of neurodevelopmental disorders.
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http://dx.doi.org/10.1186/s12989-018-0272-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131790PMC
September 2018

MANF Promotes Differentiation and Migration of Neural Progenitor Cells with Potential Neural Regenerative Effects in Stroke.

Mol Ther 2018 01 21;26(1):238-255. Epub 2017 Sep 21.

Institute of Biotechnology, HiLIFE Unit, University of Helsinki, Helsinki, Finland. Electronic address:

Cerebral ischemia activates endogenous reparative processes, such as increased proliferation of neural stem cells (NSCs) in the subventricular zone (SVZ) and migration of neural progenitor cells (NPCs) toward the ischemic area. However, this reparative process is limited because most of the NPCs die shortly after injury or are unable to arrive at the infarct boundary. In this study, we demonstrate for the first time that endogenous mesencephalic astrocyte-derived neurotrophic factor (MANF) protects NSCs against oxygen-glucose-deprivation-induced injury and has a crucial role in regulating NPC migration. In NSC cultures, MANF protein administration did not affect growth of cells but triggered neuronal and glial differentiation, followed by activation of STAT3. In SVZ explants, MANF overexpression facilitated cell migration and activated the STAT3 and ERK1/2 pathway. Using a rat model of cortical stroke, intracerebroventricular injections of MANF did not affect cell proliferation in the SVZ, but promoted migration of doublecortin (DCX) cells toward the corpus callosum and infarct boundary on day 14 post-stroke. Long-term infusion of MANF into the peri-infarct zone increased the recruitment of DCX cells in the infarct area. In conclusion, our data demonstrate a neuroregenerative activity of MANF that facilitates differentiation and migration of NPCs, thereby increasing recruitment of neuroblasts in stroke cortex.
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http://dx.doi.org/10.1016/j.ymthe.2017.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763030PMC
January 2018

Serotonergic Projections Govern Postnatal Neuroblast Migration.

Neuron 2017 May;94(3):534-549.e9

Department of Clinical Neurobiology, Medical Faculty of Heidelberg University and German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany. Electronic address:

In many vertebrates, postnatally generated neurons often migrate long distances to reach their final destination, where they help shape local circuit activity. Concerted action of extrinsic stimuli is required to regulate long-distance migration. Some migratory principles are evolutionarily conserved, whereas others are species and cell type specific. Here we identified a serotonergic mechanism that governs migration of postnatally generated neurons in the mouse brain. Serotonergic axons originating from the raphe nuclei exhibit a conspicuous alignment with subventricular zone-derived neuroblasts. Optogenetic axonal activation provides functional evidence for serotonergic modulation of neuroblast migration. Furthermore, we show that the underlying mechanism involves serotonin receptor 3A (5HT3A)-mediated calcium influx. Thus, 5HT3A receptor deletion in neuroblasts impaired speed and directionality of migration and abolished calcium spikes. We speculate that serotonergic modulation of postnatally generated neuroblast migration is evolutionarily conserved as indicated by the presence of serotonergic axons in migratory paths in other vertebrates.
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http://dx.doi.org/10.1016/j.neuron.2017.04.013DOI Listing
May 2017

Neuronal survival in the brain: neuron type-specific mechanisms.

Cell Death Dis 2017 03 2;8(3):e2643. Epub 2017 Mar 2.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.

Neurogenic regions of mammalian brain produce many more neurons that will eventually survive and reach a mature stage. Developmental cell death affects both embryonically produced immature neurons and those immature neurons that are generated in regions of adult neurogenesis. Removal of substantial numbers of neurons that are not yet completely integrated into the local circuits helps to ensure that maturation and homeostatic function of neuronal networks in the brain proceed correctly. External signals from brain microenvironment together with intrinsic signaling pathways determine whether a particular neuron will die. To accommodate this signaling, immature neurons in the brain express a number of transmembrane factors as well as intracellular signaling molecules that will regulate the cell survival/death decision, and many of these factors cease being expressed upon neuronal maturation. Furthermore, pro-survival factors and intracellular responses depend on the type of neuron and region of the brain. Thus, in addition to some common neuronal pro-survival signaling, different types of neurons possess a variety of 'neuron type-specific' pro-survival constituents that might help them to adapt for survival in a certain brain region. This review focuses on how immature neurons survive during normal and impaired brain development, both in the embryonic/neonatal brain and in brain regions associated with adult neurogenesis, and emphasizes neuron type-specific mechanisms that help to survive for various types of immature neurons. Importantly, we mainly focus on in vivo data to describe neuronal survival specifically in the brain, without extrapolating data obtained in the PNS or spinal cord, and thus emphasize the influence of the complex brain environment on neuronal survival during development.
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http://dx.doi.org/10.1038/cddis.2017.64DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386560PMC
March 2017

Auxiliary subunits of the CKAMP family differentially modulate AMPA receptor properties.

Elife 2015 Dec 1;4:e09693. Epub 2015 Dec 1.

Synaptic Signalling and Neurodegeneration, German Cancer Research Center, Heidelberg, Germany.

AMPA receptor (AMPAR) function is modulated by auxiliary subunits. Here, we report on three AMPAR interacting proteins-namely CKAMP39, CKAMP52 and CKAMP59-that, together with the previously characterized CKAMP44, constitute a novel family of auxiliary subunits distinct from other families of AMPAR interacting proteins. The new members of the CKAMP family display distinct regional and developmental expression profiles in the mouse brain. Notably, despite their structural similarities they exert diverse modulation on AMPAR gating by influencing deactivation, desensitization and recovery from desensitization, as well as glutamate and cyclothiazide potency to AMPARs. This study indicates that AMPAR function is very precisely controlled by the cell-type specific expression of the CKAMP family members.
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http://dx.doi.org/10.7554/eLife.09693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733035PMC
December 2015

The evidence for increased L1 activity in the site of human adult brain neurogenesis.

PLoS One 2015 17;10(2):e0117854. Epub 2015 Feb 17.

Laboratory of Comparative and Functional Genomics, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russ. Acad. of Sci., Moscow, Russia.

Retroelement activity is a common source of polymorphisms in human genome. The mechanism whereby retroelements contribute to the intraindividual genetic heterogeneity by inserting into the DNA of somatic cells is gaining increasing attention. Brain tissues are suspected to accumulate genetic heterogeneity as a result of the retroelements somatic activity. This study aims to expand our understanding of the role retroelements play in generating somatic mosaicism of neural tissues. Whole-genome Alu and L1 profiling of genomic DNA extracted from the cerebellum, frontal cortex, subventricular zone, dentate gyrus, and the myocardium revealed hundreds of somatic insertions in each of the analyzed tissues. Interestingly, the highest concentration of such insertions was detected in the dentate gyrus-the hotspot of adult neurogenesis. Insertions of retroelements and their activity could produce genetically diverse neuronal subsets, which can be involved in hippocampal-dependent learning and memory.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117854PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331437PMC
January 2016

Coexpressed auxiliary subunits exhibit distinct modulatory profiles on AMPA receptor function.

Neuron 2014 Aug 24;83(3):601-15. Epub 2014 Jul 24.

Synaptic Signalling and Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE), 53175 Bonn, Germany; Synaptic Signalling and Neurodegeneration, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany. Electronic address:

Gating properties and surface trafficking of AMPA receptors (AMPARs) are modulated by auxiliary subunits. Here we studied the function of coexpressed auxiliary subunits belonging to two different classes. We focused on TARP γ-8 and CKAMP44 in dentate gyrus (DG) granule cells, since both subunits are highly expressed in this cell type. TARP γ-8 and CKAMP44 decrease the rate of deactivation but have an opposing influence on receptor desensitization, which accounts for their differential modulation of synaptic short-term plasticity. Furthermore, long-term plasticity (LTP) requires TARP γ-8 but not CKAMP44. The coexpression of both auxiliary subunits is necessary for the efficient targeting of AMPARs to the cell surface of DG granule cells. Finally, electrophysiological and biochemical evidence support the notion that CKAMP44 and TARP γ-8 can be contained in the same AMPAR complex.
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http://dx.doi.org/10.1016/j.neuron.2014.07.004DOI Listing
August 2014

Dendrite development regulated by the schizophrenia-associated gene FEZ1 involves the ubiquitin proteasome system.

Cell Rep 2014 Apr 13;7(2):552-564. Epub 2014 Apr 13.

Department of Clinical Neurobiology at the German Cancer Research Center (DKFZ) and the Medical Faculty of Heidelberg University, Heidelberg 69120, Germany. Electronic address:

Downregulation of the schizophrenia-associated gene DISC1 and its interacting protein FEZ1 positively regulates dendrite growth in young neurons. However, little is known about the mechanism that controls these molecules during neuronal development. Here, we identify several components of the ubiquitin proteasome system and the cell-cycle machinery that act upstream of FEZ1. We demonstrate that the ubiquitin ligase cell division cycle 20/anaphase-promoting complex (Cdc20/APC) controls dendrite growth by regulating the degradation of FEZ1. Furthermore, dendrite growth is modulated by BubR1, whose known function so far has been restricted to control Cdc20/APC activity during the cell cycle. The modulatory function of BubR1 is dependent on its acetylation status. We show that BubR1 is deacetylated by Hdac11, thereby disinhibiting the Cdc20/APC complex. Because dendrite growth is affected both in hippocampal dentate granule cells and olfactory bulb neurons upon modifying expression of these genes, we conclude that the proposed mechanism governs neuronal development in a general fashion.
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http://dx.doi.org/10.1016/j.celrep.2014.03.022DOI Listing
April 2014

Connective tissue growth factor regulates interneuron survival and information processing in the olfactory bulb.

Neuron 2013 Sep 29;79(6):1136-51. Epub 2013 Aug 29.

Department of Clinical Neurobiology, Heidelberg University Medical Center, 69120 Heidelberg, Germany; Department of Clinical Neurobiology/A230, German Center for Cancer Research (DKFZ), 69120 Heidelberg, Germany.

Neurogenesis underlies plastic changes in defined neuronal circuits in the postnatal and adult brain. Here we identify connective tissue growth factor (CTGF) as a critical factor in the mouse olfactory bulb (OB) in determining the efficiency of incorporation of postnatally born inhibitory neurons, thus gating the output of glomeruli, the first relay station of olfactory processing in the brain. In the OB, CTGF expression was restricted to prenatally born external tufted cells. CTGF enhanced the proapoptotic activity of glial-derived TGF-β2, decreasing the survival of periglomerular inhibitory neurons. Changes in CTGF expression levels in the OB led to modifications in local neuronal circuitry and olfactory behaviors. We show that the odorant-specific recruitment of distinct glomeruli resulted in enhanced local CTGF expression levels in the activated glomeruli. Collectively our data reveal a molecular mechanism controlling the survival of defined postnatally born neurons, thus adapting neuronal integration to the sensory experiences.
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http://dx.doi.org/10.1016/j.neuron.2013.07.011DOI Listing
September 2013
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