Pharmacological modulation of developmental and synaptic phenotypes in human SHANK3 deficient stem cell-derived neuronal models.

Phelan-McDermid syndrome (PMDS) arises from mutations in the terminal region of chromosome 22q13, impacting the SHANK3 gene. The resulting deficiency of the postsynaptic density scaffolding protein SHANK3 is associated with autism spectrum disorder (ASD). We examined 12 different PMDS patient and CRISPR-engineered stem cell-derived neuronal models and controls and found that reduced expression of SHANK3 leads to neuronal hyperdifferentiation, increased synapse formation, and decreased neuronal activity. We performed automated imaging-based screening of 7,120 target-annotated small molecules and identified three compounds that rescued SHANK3-dependent neuronal hyperdifferentiation. One compound, Benproperine, rescued the decreased colocalization of Actin Related Protein 2/3 Complex Subunit 2 (ARPC2) with ß-actin and rescued increased synapse formation in SHANK3 deficient neurons when administered early during differentiation. Neuronal activity was only mildly affected, highlighting Benproperine's effects as a neurodevelopmental modulator. This study demonstrates that small molecular compounds that reverse developmental phenotypes can be identified in human neuronal PMDS models.

Machine learning identifies phenotypic profile alterations of human dopaminergic neurons exposed to bisphenols and perfluoroalkyls.

Parkinson's disease (PD) is the second most common neurodegenerative disease and is characterized by the loss of midbrain dopaminergic neurons. Endocrine disrupting chemicals (EDCs) are active substances that interfere with hormonal signaling. Among EDCs, bisphenols (BPs) and perfluoroalkyls (PFs) are chemicals leached from plastics and other household products, and humans are unavoidably exposed to these xenobiotics. Data from animal studies suggest that EDCs exposure may play a role in PD, but data about the effect of BPs and PFs on human models of the nervous system are lacking. Previous studies demonstrated that machine learning (ML) applied to microscopy data can classify different cell phenotypes based on image features. In this study, the effect of BPs and PFs at different concentrations within the real-life exposure range (0.01, 0.1, 1, and 2 µM) on the phenotypic profile of human stem cell-derived midbrain dopaminergic neurons (mDANs) was analyzed. Cells exposed for 72 h to the xenobiotics were stained with neuronal markers and evaluated using high content microscopy yielding 126 different phenotypic features. Three different ML models (LDA, XGBoost and LightGBM) were trained to classify EDC-treated versus control mDANs. EDC treated mDANs were identified with high accuracies (0.88-0.96). Assessment of the phenotypic feature contribution to the classification showed that EDCs induced a significant increase of alpha-synuclein (αSyn) and tyrosine hydroxylase (TH) staining intensity within the neurons. Moreover, microtubule-associated protein 2 (MAP2) neurite length and branching were significantly diminished in treated neurons. Our study shows that human mDANs are adversely impacted by exposure to EDCs, causing their phenotype to shift and exhibit more characteristics of PD. Importantly, ML-supported high-content imaging can identify concrete but subtle subcellular phenotypic changes that can be easily overlooked by visual inspection alone and that define EDCs effects in mDANs, thus enabling further pathological characterization in the future.

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons.

Parkinson's disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro PD cellular models lack complexity and do not take multiple phenotypes into account. Phenotypic profiling in human induced pluripotent stem cell (iPSC)-derived mDA neurons can address these shortcomings by simultaneously measuring a range of neuronal phenotypes in a PD-relevant cell type in parallel. Here, we describe a protocol to obtain and analyze phenotypic profiles from commercially available human mDA neurons. A neuron-specific fluorescent staining panel is used to visualize the nuclear, α-synuclein, Tyrosine hydroxylase (TH), and Microtubule-associated protein 2 (MAP2) related phenotypes. The described phenotypic profiling protocol is scalable as it uses 384-well plates, automatic liquid handling and high-throughput microscopy. The utility of the protocol is exemplified using healthy donor mDA neurons and mDA neurons carrying the PD-linked G2019S mutation in the Leucine-rich repeat kinase 2 (LRRK2) gene. Both cell lines were treated with the LRRK2 kinase inhibitor PFE-360 and phenotypic changes were measured. Additionally, we demonstrate how multidimensional phenotypic profiles can be analyzed using clustering or machine learning-driven supervised classification methods. The described protocol will particularly interest researchers working on neuronal disease modeling or studying chemical compound effects in human neurons.

High-content phenotyping of Parkinson's disease patient stem cell-derived midbrain dopaminergic neurons using machine learning classification.

Combining multiple Parkinson's disease (PD) relevant cellular phenotypes might increase the accuracy of midbrain dopaminergic neuron (mDAN) in vitro models. We differentiated patient-derived induced pluripotent stem cells (iPSCs) with a LRRK2 G2019S mutation, isogenic control, and genetically unrelated iPSCs into mDANs. Using automated fluorescence microscopy in 384-well-plate format, we identified elevated levels of α-synuclein (αSyn) and serine 129 phosphorylation, reduced dendritic complexity, and mitochondrial dysfunction. Next, we measured additional image-based phenotypes and used machine learning (ML) to accurately classify mDANs according to their genotype. Additionally, we show that chemical compound treatments, targeting LRRK2 kinase activity or αSyn levels, are detectable when using ML classification based on multiple image-based phenotypes. We validated our approach using a second isogenic patient-derived SNCA gene triplication mDAN model which overexpresses αSyn. This phenotyping and classification strategy improves the practical exploitability of mDANs for disease modeling and the identification of novel LRRK2-associated drug targets.

Time-resolved FRET screening identifies small molecular modifiers of mutant Huntingtin conformational inflexibility in patient-derived cells.

Huntington's disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1 encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTT's conformational flexibility in human patient fibroblasts. We have applied a well-established antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay, which measures endogenous HTT flexibility using two validated HTT-specific antibodies, to a high-throughput screening platform. By performing a small-scale compound screen, we identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. Thus, we demonstrated that the HTT TR-FRET immunoassay can be miniaturized and applied to a compound screening workflow in patient cells. This automated assay can now be used in large screening campaigns to identify previously unknown HD drugs and drug targets.

Author Correction: CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.

Single-Molecule Imaging of mRNA Localization and Regulation during the Integrated Stress Response.

Biological phase transitions form membrane-less organelles that generate distinct cellular environments. How molecules are partitioned between these compartments and the surrounding cellular space and the functional consequence of this localization is not well understood. Here, we report the localization of mRNA to stress granules (SGs) and processing bodies (PBs) and its effect on translation and degradation during the integrated stress response. Using single mRNA imaging in living human cells, we find that the interactions of mRNAs with SGs and PBs have different dynamics, very few mRNAs directly move between SGs and PBs, and that specific RNA-binding proteins can anchor mRNAs within these compartments. During recovery from stress, we show that mRNAs that were within SGs and PBs are translated and degraded at similar rates as their cytosolic counterparts. Our work provides a framework for using single-molecule measurements to directly investigate the molecular mechanisms of phase-separated compartments within their cellular environment.

Exchange between Escherichia coli polymerases II and III on a processivity clamp.

Escherichia coli has three DNA polymerases implicated in the bypass of DNA damage, a process called translesion synthesis (TLS) that alleviates replication stalling. Although these polymerases are specialized for different DNA lesions, it is unclear if they interact differently with the replication machinery. Of the three, DNA polymerase (Pol) II remains the most enigmatic. Here we report a stable ternary complex of Pol II, the replicative polymerase Pol III core complex and the dimeric processivity clamp, ß. Single-molecule experiments reveal that the interactions of Pol II and Pol III with ß allow for rapid exchange during DNA synthesis. As with another TLS polymerase, Pol IV, increasing concentrations of Pol II displace the Pol III core during DNA synthesis in a minimal reconstitution of primer extension. However, in contrast to Pol IV, Pol II is inefficient at disrupting rolling-circle synthesis by the fully reconstituted Pol III replisome. Together, these data suggest a ß-mediated mechanism of exchange between Pol II and Pol III that occurs outside the replication fork.

Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals.

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells.

The acyl-CoA binding protein is required for normal epidermal barrier function in mice.

The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ∼50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.

Serine protease inhibitor A3 in atherosclerosis and aneurysm disease.

Remodeling of extracellular matrix (ECM) plays an important role in both atherosclerosis and aneurysm disease. Serine protease inhibitor A3 (serpinA3) is an inhibitor of several proteases such as elastase, cathepsin G and chymase derived from mast cells and neutrophils. In this study, we investigated the putative role of serpinA3 in atherosclerosis and aneurysm formation. SerpinA3 was expressed in endothelial cells and medial smooth muscle cells in human atherosclerotic lesions and a 14-fold increased expression of serpinA3n mRNA was found in lesions from Apoe-/- mice compared to lesion-free vessels. In contrast, decreased mRNA expression (-80%) of serpinA3 was found in biopsies of human abdominal aortic aneurysm (AAA) compared to non-dilated aortas. Overexpression of serpinA3n in transgenic mice did not influence the development of atherosclerosis or CaCl2-induced aneurysm formation. In situ zymography analysis showed that the transgenic mice had lower cathepsin G and elastase activity, and more elastin in the aortas compared to wild-type mice, which could indicate a more stable aortic phenotype. Differential vascular expression of serpinA3 is clearly associated with human atherosclerosis and AAA but serpinA3 had no major effect on experimentally induced atherosclerosis or AAA development in mouse. However, serpinA3 may be involved in a phenotypic stabilization of the aorta.

Disruption of the acyl-CoA-binding protein gene delays hepatic adaptation to metabolic changes at weaning.

The acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor is an intracellular protein that binds C(14)-C(22) acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however, little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP(-/-)). These mice are viable and fertile and develop normally. However, around weaning, the ACBP(-/-) mice go through a crisis with overall weakness and a slightly decreased growth rate. Using microarray analysis, we show that the liver of ACBP(-/-) mice displays a significantly delayed adaptation to weaning with late induction of target genes of the sterol regulatory element-binding protein (SREBP) family. As a result, hepatic de novo cholesterogenesis is decreased at weaning. The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors, leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads to delayed induction of the lipogenic gene program in the liver.

Heparan sulfation-dependent fibroblast growth factor signaling maintains embryonic stem cells primed for differentiation in a heterogeneous state.

Embryonic stem (ES) cells continuously decide whether to maintain pluripotency or differentiate. While exogenous leukemia inhibitory factor and BMP4 perpetuate a pluripotent state, less is known about the factors initiating differentiation. We show that heparan sulfate (HS) proteoglycans are critical coreceptors for signals inducing ES cell differentiation. Genetic targeting of NDST1 and NDST2, two enzymes required for N-sulfation of proteoglycans, blocked differentiation. This phenotype was rescued by HS presented in trans or by soluble heparin. NaClO(3) (-), which reduces sulfation of proteoglycans, potently blocked differentiation of wild-type cells. Mechanistically, N-sulfation was identified to be critical for functional autocrine fibroblast growth factor 4 (FGF4) signaling. Microarray analysis identified the pluripotency maintaining transcription factors Nanog, KLF2/4/8, Tbx3, and Tcf3 to be negatively regulated, whereas markers of differentiation such as Gbx2, Dnmt3b, FGF5, and Brachyury were induced by sulfation-dependent FGF receptor (FGFR) signaling. We show that several of these genes are heterogeneously expressed in ES cells, and that targeting of heparan sulfation or FGFR-signaling facilitated a homogenous Nanog/KLF4/Tbx3 positive ES cell state. This finding suggests that the recently discovered heterogeneous state of ES cells is regulated by HS-dependent FGFR signaling. Similarly, culturing blastocysts with NaClO(3) (-) eliminated GATA6-positive primitive endoderm progenitors generating a homogenous Nanog-positive inner cell mass. Functionally, reduction of sulfation robustly improved de novo ES cell derivation efficiency. We conclude that N-sulfated HS is required for FGF4 signaling to maintain ES cells primed for differentiation in a heterogeneous state. Inhibiting this pathway facilitates a more naïve ground state.

Transgenic overexpression of the alpha-synuclein interacting protein synphilin-1 leads to behavioral and neuropathological alterations in mice.

Synphilin-1 has been identified as an interacting protein of alpha-synuclein, Parkin, and LRRK2, proteins which are mutated in familial forms of Parkinson disease (PD). Subsequently, synphilin-1 has also been shown to be an intrinsic component of Lewy bodies in sporadic PD. In order to elucidate the role of synphilin-1 in the pathogenesis of PD, we generated transgenic mice overexpressing wild-type and mutant (R621C) synphilin-1 driven by a mouse prion protein promoter. Transgenic expression of both wild-type and the R621C variant synphilin-1 resulted in increased dopamine levels of the nigrostriatal system in 3-month-old mice. Furthermore, we found pathological ubiquitin-positive inclusions in cerebellar sections and dark-cell degeneration of Purkinje cells. Both transgenic mouse lines showed significant reduction of motor skill learning and motor performance. These findings suggest a pathological role of overexpressed synphilin-1 in vivo and will help to further elucidate the mechanisms of protein aggregation and neuronal cell death.

Noninvasive in vivo imaging of pancreatic islet cell biology.

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.

Age-associated mosaic respiratory chain deficiency causes trans-neuronal degeneration.

Heteroplasmic mitochondrial DNA (mtDNA) mutations (mutations present only in a subset of cellular mtDNA copies) arise de novo during the normal ageing process or may be maternally inherited in pedigrees with mitochondrial disease syndromes. A pathogenic mtDNA mutation causes respiratory chain deficiency only if the fraction of mutated mtDNA exceeds a certain threshold level. These mutations often undergo apparently random mitotic segregation and the levels of normal and mutated mtDNA can vary considerably between cells of the same tissue. In human ageing, segregation of somatic mtDNA mutations leads to mosaic respiratory chain deficiency in a variety of tissues, such as brain, heart and skeletal muscle. A similar pattern of mutation segregation with mosaic respiratory chain deficiency is seen in patients with mitochondrial disease syndromes caused by inherited pathogenic mtDNA mutations. We have experimentally addressed the role of mosaic respiratory chain deficiency in ageing and mitochondrial disease by creating mouse chimeras with a mixture of normal and respiratory chain-deficient neurons in cerebral cortex. We report here that a low proportion (>20%) of respiratory chain-deficient neurons in the forebrain are sufficient to cause symptoms, whereas premature death of the animal occurs only if the proportion is high (>60-80%). The presence of neurons with normal respiratory chain function does not only prevent mortality but also delays the age at which onset of disease symptoms occur. Unexpectedly, respiratory chain-deficient neurons have adverse effect on normal adjacent neurons and induce trans-neuronal degeneration. In summary, our study defines the minimal threshold level of respiratory chain-deficient neurons needed to cause symptoms and also demonstrate that neurons with normal respiratory chain function ameliorate disease progression. Finally, we show that respiratory chain-deficient neurons induce death of normal neurons by a trans-neuronal degeneration mechanism. These findings provide novel insights into the pathogenesis of mosaic respiratory chain deficiency in ageing and mitochondrial disease.

Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation.

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the 'DNA damage' S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine gamma-aminobutyric acid (GABA) signalling by means of GABA(A) receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABA(A) receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABA(A) receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.

Nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3: in vivo evidence.

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat in the MJD1 gene resulting in an expanded polyglutamine repeat in the ataxin-3 protein. To study the course of the disease, we generated transgenic mice for SCA3 using full-length ataxin-3 constructs containing 15, 70, or 148 CAG repeats, respectively. Control mice (15 CAGs) were phenotypically normal and had no neuropathological findings. However, mice transgenic for ataxin-3 with expanded polyglutamine repeats were severely affected by a strong neurological phenotype with tremor, behavioral deficits, strongly reduced motor and exploratory activity, a hunchback, and premature death at 3 to 6 months of age. Neuropathological examination by immunohistochemical staining revealed ubiquitin- and ataxin-3-positive intranuclear inclusion bodies in a multitude of neurons. Directing ataxin-3 with 148 CAGs to the nucleus revealed an even more pronounced phenotype with more inclusions and earlier death, whereas mice transgenic with the same construct but attached to a nuclear export signal developed a milder phenotype with less inclusions. These studies indicate that nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3 in vivo.