Biology subjects

Pietilainen, O.

Publications and source records attributed to Pietilainen, O..

11 recordsLinked to original sources

bioRxiv2025

Astrocytic-supplied cholesterol drives synaptic gene expression programs in developing neurons and downstream astrocytic transcriptional programs

Astrocytes participate in neuronal synaptic programs that are enriched for genetic associations in schizophrenia and autism spectrum disorders (ASD). To better understand how these co-regulated cellular programs are induced during early neuronal development, we studied astrocytes and iPSC-derived neurons in co-cultures and mono-cultures at 16 time points spanning 0.5 hours to 8 days. We found that upregulation in astrocytes of genes involved in cholesterol biosynthesis preceded the activation of synaptic gene programs in neurons and upregulation of the astrocytic Nrxn1. Neuronal knockdown of key cholesterol receptors led to downregulation of neuronal synaptic genes and induced a robust transcriptional response in the astrocytes, including further upregulation of Nrxn1. This suggests that astrocyte-supplied cholesterol drives these neuronal changes and that bi-directional signalling is occuring. The genes upregulated in neurons were enriched for deleterious variants in schizophrenia and neurodevelopmental disorders, suggesting that their pathogenic effect may be, in part, mediated by reduced buffering capacity for changes in the astrocyte cholesterol supply to neurons. These findings highlight the critical role of astrocyte-neuron interactions in psychiatric and neurodevelopmental disorders, particularly in relation to lipid metabolism and synaptic plasticity.

molecular biology↗

bioRxiv2024

Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol

Sac1 is a conserved phosphoinositide phosphatase, whose loss-of-function compromises cell and organism viability. Here, we employed acute auxin-inducible Sac1 degradation to identify its immediate downstream effectors in human cells. Most of Sac1 was degraded in [~]1 h, paralleled by increased PI(4)P and decreased cholesterol in the trans-Golgi network (TGN) during the following hour, and superseded by Golgi fragmentation, impaired glycosylation, and selective degradation of TGN proteins by [~]4 h. The TGN disintegration resulted from its acute deacidification caused by disassembly of the Golgi V-ATPase. Mechanistically, Sac1 mediated TGN membrane composition maintained an assembly promoting conformation of the V0a2 subunit. Key phenotypes of acute Sac1 degradation were recapitulated in human differentiated trophoblasts, causing processing defects of chorionic gonadotropin, in line with loss-of-function intolerance of the human SACML1 gene. Collectively, our findings reveal that the assembly of the Golgi V-ATPase is controlled by the TGN membrane via Sac1 fuelled lipid exchange.

cell biology↗

bioRxiv2023

Astrocytes regulate neuronal network burst frequency through NMDA receptors species- anddonor-specifically

BackgroundDevelopment of synaptic activity is a neuronal key characteristic that relies largely on interactions between neurons and astrocytes. Although astrocytes have known roles in regulating synaptic function and malfunction, the use of human or donor-specific astrocytes in disease models is still rare. Rodent astrocytes are routinely used to enhance neuronal activity in cell cultures, but less is known how human astrocytes influence neuronal activity. MethodsWe established human induced pluripotent stem cell (hiPSC)-derived neuron-astrocyte co-cultures and studied their functional development on microelectrode array (MEA). We used cell lines from 5 neurotypical control individuals and 3 pairs of monozygotic twins discordant for schizophrenia. A method combining Ngn2 overexpression and dual SMAD inhibition was used for neuronal differentiation. The neurons were co-cultured with hiPSC-derived astrocytes differentiated from 6-month-old astrospheres or rat astrocytes. ResultsWe found that the hiPSC-derived co-cultures develop complex network bursting activity similarly to neuronal co-cultures with rat astrocytes. However, the effect of NMDA receptors on neuronal network burst frequency (NBF) differed between co-cultures containing human or rat astrocytes. By using co-cultures derived from patients with schizophrenia and unaffected individuals, we found lowered NBF in the affected cells. We continued to demonstrate how astrocytes from an unaffected individual rescue the lowered NBF in the affected neurons by increasing NMDA receptor activity. ConclusionsOur results indicate that astrocytes participate in the regulation of neuronal NBF through a mechanism involving NMDA receptors. These findings shed light on the importance of using human and donor-specific astrocytes in disease modeling.

neuroscience↗

bioRxiv2023

X-chromosome inactivation in human iPSCs provides insight into escape-regulated gene expression.

Epigenetic variation in the X chromosome inactivation (XCI) of human induced pluripotent stem cells (hiPSCs) can impact their ability to accurately model biological sex biases. However, the gene-wise landscape of escape from XCI remains unresolved in female hiPSCs. To characterize the patterns of escape from inactivation, we performed a systematic survey of allele specific expression (ASE) in 165 female hiPSC lines. The analysis revealed that the escape from XCI was non-random and affected primarily genes that escape also in human tissues. However, individual genes and cell lines varied in the frequency and degree of escape. The escape increased gradually after modest decrease of XIST in cultures, whose loss is commonly used to mark lines with eroded XCI. We identified three clusters of female lines at different stages of XCI. The increased degree of escape from XCI amplified female-biased expression and reduced male-female differences in genes with male-biased expression in the X chromosome. In autosomes, the increased escape modified genuine sex differences in a dose-dependent way suggesting that escape from XCI directly regulated autosomal gene expression. The variation in escape was sufficient to compensate for a dominant loss of function effect in several disease genes. The study presents a comprehensive view of escape from XCI in hiPSCs and emphasizes the need to monitor the XCI status at gene level for disease modeling. It further suggests that the uncommon and variable escape in hiPSCs can provide insight into X chromosomes role in regulating gene expression and sex differences in humans.

genomics↗

bioRxiv2022

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells

Astrocytes play essential roles in normal brain function, with dysfunction implicated in diverse developmental and degenerative disease processes. Emerging evidence of profound species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy, incompletely characterized, or require intermediate purification steps, limiting their utility for multi-cell line, adequately powered functional studies. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs) based upon transient Neurogenin 2 (NGN2) induction of neural progenitor-like cells followed by maturation in astrocyte media, which demonstrate remarkable homogeneity within the population and across 11 independent cell lines in the absence of additional purification steps. These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal maturation in vitro. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, most closely resembling human fetal astrocytes, and highly similar to hPSC-derived astrocytes generated using more complex approaches. Finally, the hiAs capture key molecular hallmarks in a trisomy 21 disease model. Thus, hiAs provide a valuable and practical resource well-suited for study of basic human astrocyte function and dysfunction in disease.

neuroscience↗

bioRxiv2021

The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia

To study how the 22q11.2 deletion predisposes to psychiatric disease, we generated induced pluripotent stem cells from deletion carriers and controls, as well as utilized CRISPR/Cas9 to introduce the heterozygous deletion into a control cell line. Upon differentiation into neural progenitor cells, we found the deletion acted in trans to alter the abundance of transcripts associated with risk for neurodevelopmental disorders including Autism Spectrum Disorder. In more differentiated excitatory neurons, altered transcripts encoded presynaptic factors and were associated with genetic risk for schizophrenia, including common (per-SNP heritability p ({tau}c)= 4.2 x 10-6) and rare, loss of function variants (p = 1.29x10-12). These findings suggest a potential relationship between cellular states, developmental windows and susceptibility to psychiatric conditions with different ages of onset. To understand how the deletion contributed to these observed changes in gene expression, we developed and applied PPItools, which identifies the minimal protein-protein interaction network that best explains an observed set of gene expression alterations. We found that many of the genes in the 22q11.2 interval interact in presynaptic, proteasome, and JUN/FOS transcriptional pathways that underlie the broader alterations in psychiatric risk gene expression we identified. Our findings suggest that the 22q11.2 deletion impacts genes and pathways that may converge with risk loci implicated by psychiatric genetic studies to influence disease manifestation in each deletion carrier.

neuroscience↗

bioRxiv2021

Astrocytic cell adhesion genes linked to schizophrenia promote synaptic programs in neurons

The maturation of neurons and the development of synapses - while emblematic of neurons - also rely on interactions with astrocytes and other glia. To study the role of glia-neuron interactions, we analyzed the transcriptomes of human pluripotent stem cell (hPSC)-derived neurons, from a total of 80 human donors, that were cultured with or without contact with glial cells. We found that the presence of astrocytes enhanced synaptic gene-expression programs in neurons. These changes in neuronal synaptic gene expression correlated with increased expression in the co-cultured glia of genes that encode synaptic cell adhesion molecules, and they were greatly enhanced in the glia in coculture. Both the neuronal and astrocyte gene-expression programs were enriched for genes that are linked to schizophrenia risk. Physical contact between the two cell types was required for the induction of synaptic programs in neurons. Our results suggest that astrocyte-expressed genes with synaptic functions are associated with stronger expression of synaptic genetic programs in neurons and suggest a potential role for astrocyte-neuron interactions in schizophrenia.

molecular biology↗

bioRxiv2021

Single-nucleus sequencing reveals enriched expression of genetic risk factors sensitises Motor Neurons to degeneration in ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterised by a progressive loss of motor function. The eponymous spinal sclerosis observed at autopsy is the result of the degeneration of extratelencephalic neurons, Betz cells (ETNs, Cortico-Spinal Motor Neuron). It remains unclear why this neuronal subtype is selectively affected. To understand the unique molecular properties that sensitise these cells to ALS, we performed RNA sequencing of 79,169 single nuclei from cortices of patients and controls. In unaffected individuals, we found that expression of ALS risk genes was significantly enriched in THY1+-ETNs and not in other cell types. In patients, these genetic risk factors, as well as genes involved in protein homeostasis and stress responses, were significantly induced in a wide collection of ETNs, but not in neurons with more superficial identities. Examination of oligodendroglial and microglial nuclei revealed patient-specific changes that were at least in part a response to alterations in neurons: downregulation of myelinating genes in oligodendrocytes and upregulation of a reactive state connected to endo-lysosomal pathways in microglia. Our findings suggest that the selective vulnerability of extratelencephalic neurons is partly connected to their intrinsic molecular properties sensitising them to genetics and mechanisms of degeneration. Graphical abstract and working model O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/452054v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@11237fdorg.highwire.dtl.DTLVardef@119f3ceorg.highwire.dtl.DTLVardef@d56c69org.highwire.dtl.DTLVardef@1c087e1_HPS_FORMAT_FIGEXP M_FIG Our study highlights cell type specific changes in premotor/motor cortex of sporadic ALS patients. Specifically, we identify upregulation of synaptic molecules in excitatory neurons of upper cortical layers, interestingly correlating to hyperexcitability phenotypes seen in patients. Moreover, excitatory neurons of the deeper layers of the cortex, that project to the spinal cord and are most affected by the disease, show higher levels of cellular stresses than other neuronal types. Correspondently, oligodendrocytes transition from a highly myelinating state to a more neuronally engaged state, probably to counteract stressed phenotypes seen in excitatory neurons. At the same time, microglia show a reactive state with specific upregulation of endo-lysosomal pathways. C_FIG

neuroscience↗

bioRxiv2018

Genome-wide screens in accelerated human stem cell-derived neural progenitor cells identify Zika virus host factors and drivers of proliferation

Neural progenitor cells (NPCs) are essential to brain development and their dysfunction is linked to several disorders, including autism, Zika Virus Congenital Syndrome, and cancer. Understanding of these conditions has been improved by advancements with stem cell-derived NPC models. However, current differentiation methods require many days or weeks to generate NPCs and show variability in efficacy among cell lines. Here, we describe human Stem cell-derived NGN2-accelerated Progenitor cells (SNaPs), which are produced in only 48 hours. SNaPs express canonical forebrain NPC protein markers, are proliferative, multipotent, and like other human NPCs, are susceptible to Zika-mediated death. We further demonstrate SNaPs are valuable for large-scale investigations of genetic and environmental influencers of neurodevelopment by deploying them for genome-wide CRISPR-Cas9 screens. Our studies expand knowledge of NPCs by identifying known and novel Zika host factors, as well as new regulators of NPC proliferation validated by re-identification of the autism spectrum gene PTEN.

neuroscience↗

bioRxiv2018

Contribution of rare and common variants to intellectual disability in a high-risk population sub-isolate of Northern Finland

The contribution of de novo and ultra-rare genetic variants in severe and moderate intellectual disability (ID) has been extensively studied whereas the genetic architecture of mild ID has been less well characterized. To elucidate the genetic background of milder ID we studied a regional cohort of 442 ID patients enriched for mild ID (>50%) from a population isolate of Finland. We analyzed rare variants using exome sequencing and CNV genotyping and common variants using common variant polygenic risk scores. As controls we used a Finnish collection of exome sequenced (n=11311) and GWAS chip genotyped (n=11699) individuals.\n\nWe show that rare damaging variants in genes known to be associated with cognitive defects are observed more often in severe (27%) than in mild ID (13%) patients (p-value: 7.0e-4). We further observed a significant enrichment of protein truncating variants in loss-of-function intolerant genes, as well as damaging missense variants in genes not yet associated with cognitive defects (OR: 2.1, p-value: 3e-8). For the first time to our knowledge, we show that a common variant polygenic load significantly contributes to all severity forms of ID. The heritability explained was the highest for educational attainment (EDU) in mild ID explaining 2.2% of the heritability on liability scale. For more severe ID it was lower at 0.6%. Finally, we identified a homozygote variant in the CRADD gene to be a cause of a specific syndrome with ID and pachygyria. The frequency of this variant is 50x higher in the Finnish population than in non-Finnish Europeans, demonstrating the benefits of utilizing population isolates in rare variant analysis of diseases under negative selection.

genetics↗

bioRxiv2017

Quantifying the impact of rare and ultra-rare coding variation across the phenotypic spectrum

There is a limited understanding about the impact of rare protein truncating variants across multiple phenotypes. We explore the impact of this class of variants on 13 quantitative traits and 10 diseases using whole-exome sequencing data from 100,296 individuals. Protein truncating variants in genes intolerant to this class of mutations increased risk of autism, schizophrenia, bipolar disorder, intellectual disability, ADHD. In individuals without these disorders, there was an association with shorter height, lower education, increased hospitalization and reduced age. Gene sets implicated from GWAS did not show a significant protein truncating variants-burden beyond what captured by established Mendelian genes. In conclusion, we provide the most thorough investigation to date of the impact of rare deleterious coding variants on complex traits, suggesting widespread pleiotropic risk.\n\nMain abbreviations

genetics↗