Biology subjects

Pietiläinen, O.

Publications and source records attributed to Pietiläinen, O..

3 recordsLinked to original sources

bioRxiv2026

INTS6 loss of function disrupts transcriptional regulation in mild intellectual disability

Pathogenic variants in genes involved in transcriptional regulation and RNA processing have emerged as points of functional convergence in neurodevelopmental disorders (NDDs), but their specific disease mechanisms remain unknown. By screening 1,562 Finnish extended families from the Northern Finland Intellectual Disability cohort affected by cognitive impairment, we discovered a family with six affected members carrying a heterozygous loss- of-function variant in INTS6. INTS6 is a conserved member of the phosphatase module of the Integrator complex, which regulates RNA polymerase II activity, with a reported role in the pathogenesis of NDDs. To determine the variants transcriptomic effects, we performed RNA-sequencing of induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells from cases and controls, revealing transcriptome-wide splicing defects, with increased intron retention observed in genes involved in translation, cell cycle and RNA processing in variant carriers. CRISPR-Cas9 knock-in iPSCs confirmed that the variant was associated with downregulation of transcription factors and developmental processes in early neuron differentiation. In addition, downregulated genes in variant carrier neurons were enriched for synaptic genes, suggesting effects on neuronal development. These findings highlight the critical role of INTS6 in transcriptional regulation of human neurodevelopment and reinforce its association with NDDs.

genetics↗

bioRxiv2022

SARS-CoV-2 infection of human neurons requires endosomal cell entry and can be blocked by inhibitors of host phosphoinositol-5 kinase

COVID-19 is a disease caused by coronavirus SARS-CoV-2. In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms that can last from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to infection of brain cells. We found that a small fraction of cortical neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), which was expressed at very low levels. Although only a limited number of neurons was infectable, the infection was productive, as demonstrated by the presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout the neuronal cell, and an increase in viral RNA in the culture medium within the first 48 h of infection (viral release). The productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface protein spike, which can occur at the plasma membrane or from endo/lysosomes. Using specific combinations of small-molecule inhibitors, we found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases found on the cell surface, including TMPRSS2. In contrast, the infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K) that regulates endosomal maturation. ImportanceCOVID-19 is a disease caused by coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures and encephalopathy, as well as anatomical abnormalities such as changes in brain morphology. Whether these symptoms are linked to brain infection is not clear. The mechanism of the virus entry into neurons has also not been characterized. Here we investigated SARS-CoV-2 infection using a human iPSC-derived neural cell model and found that a small fraction of cortical neurons was naturally susceptible to infection. The infection depended on the ACE2 receptor and was productive. We also found that the virus used the late endosomal/lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of the cellular phosphatidyl-inositol 5 kinase.

neuroscience↗

bioRxiv2022

Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using anHb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

neuroscience↗