Biology subjects

Krsnik, Z.

Publications and source records attributed to Krsnik, Z..

10 recordsLinked to original sources

bioRxiv2026

Diffusion MRI of cortical organoids reveals protocol-associated spatial reproducibility and increasing diffusivity with diffusion time

Early human cortical development involves rapid changes in cytoarchitecture that remain difficult to interrogate non-invasively. Biophysical diffusion MRI models provide a potential window onto this microstructure, including water exchange across cell membranes, but their assumptions are difficult to validate directly in developing human tissue. Human cortical organoids provide a tractable biological model in which diffusion measurements can be related to tissue architecture within the same specimen. We scanned six fixed cortical organoids at 9.4~T, three grown under a directed and three under an undirected differentiation protocol, at three diffusion times and b-values up to 7000~s/mm$^2$. Center-to-periphery profiles of DKI-derived diffusion metrics were reproducible across the directed organoids but more variable across the undirected organoids, while nuclear staining revealed greater variability in the internal architecture of the undirected organoids. Unexpectedly, mean diffusivity increased with diffusion time in more than 80% of tissue voxels in both batches, opposite to previous organoid measurements. Across 12,348 simulated two-compartment substrates, this increase was reproduced only with permeable membranes and an extracellular diffusivity substantially exceeding the apparent intracellular diffusivity, a regime that challenges assumptions commonly used in current gray-matter exchange models. These results support cortical organoids as biologically realistic platforms for probing early cortical microstructure and testing diffusion models under controlled conditions, while remaining amenable to post-MRI histological validation.

neuroscience↗

bioRxiv2026

Abnormal neuronal and synaptic morphology in Down syndrome brains reproduces in 2D and 3D human cellular models

Down syndrome (DS) is the most common autosomal aneuploidy compatible with postnatal survival and is caused by full or partial trisomy of chromosome 21. In this study, human isogenic induced pluripotent stem cells (iPSCs) were differentiated into 2D neurons and cortico-striatal assembloids (hCSAs). Abnormal neuronal morphology was observed: trisomic (T21) neurons formed aggregates of cell bodies interconnected with thick neurite bundles radiating outwards, connected and overlapped with other neurons in between the bundles while disomic (D21) neurons were evenly distributed across the surface and formed strong neuronal networks. Detailed analysis revealed significantly shorter neurites with larger diameter, fewer branches, and fewer terminal points in T21 neurons compared to D21 neurons in both 2D cultures and hCSAs. We observed similar phenotypes in foetal and postnatal human brain tissue. Furthermore, we observed abnormal mitochondrial morphology with an excess of some mitochondrial proteins (AIF, TOMM20), abnormal synaptic morphology and significantly lower expression of both presynaptic and postsynaptic markers (SYN-1, PSD95 and GEPH) throughout in vitro differentiation of T21 neurons. Finally, our data showed that T21 spheroids were significantly smaller throughout in vitro differentiation compared to D21 spheroids. T21 spheroids also exhibited significantly higher expression of neural stem cells (SOX2), significantly lower expression of proliferating cells (Ki67) and significantly higher expression of apoptotic cells (CCaspase-3). Overall, our study demonstrates that T21 leads to abnormal neuronal morphology in both 2D neurons and hCSAs, consistent with observations in the human brain.

neuroscience↗

bioRxiv2025

Single-cell atlas of the developing Down syndrome brain cortex

Down syndrome (DS), caused by trisomy of chromosome 21, is the leading genetic cause of intellectual disability, yet the mechanisms disrupting fetal brain development remain unclear. We performed single-cell transcriptomic and chromatin accessibility profiling of approximately 250,000 cells from 15 DS and 15 control human fetal cortices (10-20 weeks post-conception). Our analysis revealed a subtype-specific reduction in RORB/FOXP1-expressing excitatory neurons and widespread disruption of neurodevelopmental transcriptional programs. Chromosome 21 transcription factors BACH1, PKNOX1, and GABPA emerged as dosage-sensitive hubs regulating genes linked to intellectual disability. Antisense oligonucleotide-mediated normalization of these factors in human neural progenitors in vitro partially rescued target gene expression. Benchmarking a humanized in vivo model captured additional molecular and cellular signatures of DS, complementing the in vitro model. Together, this resource defines the gene-regulatory landscape underlying cortical development in DS and highlights candidate molecular targets and preclinical models for future intervention studies. HighlightsO_LISingle-cell atlas of Down syndrome fetal cortex links transcriptional dysregulation to reduction of layer 4 neurons C_LIO_LIChromosome 21 transcription factors PKNOX1, BACH1, and GABPA drive intellectual disability gene dysregulation C_LIO_LITransplanted human cells model late-stage DS phenotypes, bypassing scarcity of fetal tissue C_LIO_LIASO targeting chromosome 21 transcription factors restores DS-associated molecular signatures C_LI

neuroscience↗

bioRxiv2025

Insights into Temporal and Spatial Dynamics of Short Association Fiber Formation in the Human Fetal Brain

Short association fibers (SAFs) form the local scaffold of cortical connectivity, supporting early functional specialization and marking sites of neurodevelopmental vulnerability. However, their development before birth remains largely unknown. Leveraging advanced fetal diffusion MRI and a histology-validated framework, we present the first in-utero reconstruction of SAF pathways in the human brain. We tracked their volumetric and microstructural developmental trajectories in 243 fetuses spanning a critical period when the brains connectome is rapidly forming. We found that SAFs emerge before sulcal folding, initially as flat, loosely arranged pathways along the subplate-white matter interface, and later reorganize into coherent U-shaped bundles. Their maturation followed a sensorimotor-to-association gradient, paralleling cortical development. Nonlinear, bundle-specific trajectories captured multiphasic maturation, with subplate dynamics preceding increases in axonal coherence and early myelination. By filling a missing link in lifespan brain connectivity, this study establishes a prenatal reference for cortical wiring and provides tools for investigating the origins of neurodevelopmental disorders.

neuroscience↗

bioRxiv2024

Unraveling Microglial Spatial Organization in the Developing Human Brain with DeepCellMap, a Deep Learning Approach Coupled to Spatial Statistics

Mapping cellular organization in the developing brain presents significant challenges due to the multidimensional nature of the data, characterized by complex spatial patterns that are difficult to interpret without high-throughput tools. We developed DeepCellMap, a deep-learning-assisted tool that integrates multi-scale image processing with advanced spatial and clustering statistics. This pipeline was designed to map microglial organization during normal and pathological brain development but can be adapted to any cell type. Using DeepCellMap, we capture the morphological diversity of microglia, identify strong coupling between proliferative and phagocytic phenotypes, and show that distinct spatial clusters rarely overlap as human brain development progresses. Additionally, we uncover a novel association between microglia and blood vessels in fetal brains exposed to maternal SARS-CoV-2. These findings offer insights into whether various microglial phenotypes form networks in the developing brain to occupy space, and in conditions involving haemorrhages, whether microglia respond to, or influence changes in blood vessel integrity. DeepCellMap is available as open-source software and is a powerful tool for extracting spatial statistics and analyzing cellular organization in large tissue sections, accommodating various imaging modalities. This platform could open new avenues for studying brain development and related pathologies.

developmental biology↗

bioRxiv2023

Deep learning microstructure estimation of developing brains from diffusion MRI: a newborn and fetal study

Diffusion-weighted magnetic resonance imaging (dMRI) is widely used to assess the brain white matter. Fiber orientation distribution functions (FODs) are a common way of representing the orientation and density of white matter fibers. However, with standard FOD computation methods, accurate estimation of FODs requires a large number of measurements that usually cannot be acquired for newborns and fetuses. We propose to overcome this limitation by using a deep learning method to map as few as six diffusion-weighted measurements to the target FOD. To train the model, we use the FODs computed using multi-shell high angular resolution measurements as target. Extensive quantitative evaluations show that the new deep learning method, using significantly fewer measurements, achieves comparable or superior results to standard methods such as Constrained Spherical Deconvolution. We demonstrate the generalizability of the new deep learning method across scanners, acquisition protocols, and anatomy on two clinical datasets of newborns and fetuses. Additionally, we compute agreement metrics within the HARDI newborn dataset, and validate fetal FODs with post-mortem histological data. The results of this study show the advantage of deep learning in inferring the microstructure of the developing brain from in-vivo dMRI measurements that are often very limited due to subject motion and limited acquisition times, but also highlight the intrinsic limitations of dMRI in the analysis of the developing brain microstructure. These findings, therefore, advocate for the need for improved methods that are tailored to studying the early development of human brain.

bioinformatics↗

bioRxiv2022

Neurodevelopmental Patterns of Early Postnatal White Matter Maturation Represent Distinct Underlying Microstructure and Histology

During the early postnatal period, cerebral white matter undergoes rapid maturation through a complex series of interrelated cellular and histogenetic processes. Accurately quantifying these processes is important for improving understanding of early brain development, developmental abnormalities related to prematurity, and neurodevelopmental diseases. Past efforts have used magnetic resonance imaging (MRI) to track these developmental processes in vivo. However, most previous studies have relied on single imaging modality data and have often been limited by small samples and analytics that do not evaluate complex multivariate imaging patterns. Here, we applied an advanced unsupervised multivariate pattern analysis technique, non-negative matrix factorization (NMF), to T2w/T1w signal ratio maps from a large cohort of newborns (Developing Human Connectome Project [dHCP], n=342), revealing patterns of synchronous white matter maturation. These patterns showed divergent age-related maturational trajectories and differential susceptibility to premature birth, which were replicated in an independent large sample of newborns (Early Life Adversity, Biological Embedding, and Risk for Developmental Precursors of Mental Disorders [eLABE], n=239). Furthermore, we showed that T2w/T1w signal variations in white matter maturational patterns are explained by differential contributions of white matter microstructure indices (i.e., free water content and neurite density index) derived from neurite orientation dispersion and density imaging (NODDI) modeling of diffusion-weighted MRI. Finally, we demonstrated how white matter maturation patterns relate to distinct histological features by comparing our findings with postmortem late fetal/early postnatal brain tissue staining. Together, these results delineate a novel MRI representation of white matter microstructural and histological reorganization during the early postnatal development.

neuroscience↗

bioRxiv2021

Spatiotemporal dynamics of human microglia are linked with brain developmental processes across the lifespan

Microglia, the brains resident macrophages, shape neural development and wiring, and are key neuroimmune hubs in the pathological signature of neurodevelopmental disorders. In the human brain, microglial development has not been carefully examined yet, and most of our knowledge derives from rodents. We established an unprecedented collection of 97 postmortem tissues enabling quantitative, sex-matched, detailed analysis of microglial across the human lifespan. We identify the dynamics of these cells in the human telencephalon, describing novel waves in microglial density across gestation and infancy, controlled by a balance of proliferation and apoptosis, which track key neurodevelopmental milestones. These profound changes in microglia are also observed in bulk RNAseq and single-cell RNAseq datasets. This study provides unparalleled insight and detail into the spatiotemporal dynamics of microglia across the human lifespan. Our findings serve as a solid foundation for elucidating how microglia contribute to shaping neurodevelopment in humans.

neuroscience↗

bioRxiv2020

Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain

A population of >6 million people worldwide at high risk of Alzheimers disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of {beta}-amyloid-(A{beta})-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar A{beta} deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome-21-gene BACE2, but prevented by combined chemical {beta} and {gamma}-secretase inhibition. We found that T21-organoids secrete increased proportions of A{beta}-preventing (A{beta}1-19) and A{beta}-degradation products (A{beta}1-20 and A{beta}1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1-inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in [~]30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

neuroscience↗

bioRxiv2017

Multiple roles of PIWIL1 in mouse neocorticogenesis

PIWI-interacting RNAs (piRNAs) and their associated PIWI proteins play an important role in repressing transposable elements in animal germlines. However, little is known about the function of PIWI proteins and piRNAs in the developing brain. Here, we investigated the role of an important PIWI family member, Piwi-like protein 1 (Piwil1; also known as Miwi in mouse) in the developing mouse neocortex. Using a Piwil1 knock-out (Piwil1 KO) mouse strain, we found that Piwil1 is essential for several steps of neocorticogenesis, including neocortical cell cycle, neuron migration and dendritogenesis. Piwil1 deletion resulted in increased cell cycle re-entry at embryonic day 17 (E17) when predominantly intracortically projecting neurons are being produced. Prenatal Piwil1 deletion increased the number of Pax6+ radial glia at postnatal day 0 (P0). Furthermore, Piwil1 deletion disrupted migration of Satb2+ neurons within deep layers at E17, P0 and P7. Satb2+ neurons showed increased co-localization with Bcl11b (also known as Ctip2), marker of subcortically projecting neurons. Piwil1 knockouts had disrupted neocortical circuitry represented by thinning of the corpus callosum and altered dendritogenesis. We further investigated if Piwil1 deletion disrupted expression levels of neocortical piRNAs by small RNA-sequencing in neocortex. We did not find differential expression of piRNAs in the neocortices of Piwil1 KO, while differences were observed in other Piwil1 KO tissues. This result suggests that Piwil1 may act independently of piRNAs and have novel roles in higher cognitive centers, such as neocortex. In addition, we report a screen of piRNAs derived from tRNA fragments in developing neocortices. Our result is the first report of selective subsets of piRNAs and tRNA fragments in developing prenatal neocortices and helps clarify some outstanding questions about the role of the piRNA pathway in the brain.

neuroscience↗