Abstract Neurons derived from induced pluripotent stem cells (h-iPSC-Ns) provide an invaluable model for studying the physiological aspects of neuronal development and diseases. However, multiple studies have also demonstrated that h-iPSC-Ns exhibit a high degree of functional and epigenetic diversity. Due to the imprecise characterization and significant variation among the currently available maturation protocols, it is essential to establish a set of criteria to standardize models and accurately characterize and define the developmental properties of neurons derived from iPSCs. In this study, we conducted a comprehensive analysis of the h-iPSC-Ns via electrophysiological and microscopic techniques to follow their functional development at the cellular and network levels. This enabled us to provide a thorough description of the maturation process of h-iPSC-Ns over a 10-week period in vitro . Specifically, we have used conventional whole-cell patch-clamp and dynamic clamp techniques, alongside morphometry, to assess the characteristics of maturing h-iPSC-Ns. Additionally, we utilized calcium imaging to monitor the progression of synaptic activity and network communication. At the single cell level, human neurons exhibited gradually decreasing membrane resistance in parallel with improved excitability by 5 weeks of maturation. Their firing profiles were consistent with those of mature regular firing type of neurons. At the network level we observed the development of abundant fast glutamatergic and depolarizing GABAergic synaptic connections together with synchronized network activity. The identified sequence of differentiation events are consistent and offers a robust framework for developing targeted experiments at varying stages of neuronal maturation. This framework allows for the use of different, age-related methodologies or a singular set of experiments for a culture’s maturation.
Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging.
J. Neurochem. (2010) 115 , 314–324. Abstract Trypsinogen 4 is specifically expressed in the human brain, mainly by astroglial cells. Although its exact role in the nervous tissue is yet unclear, trypsin 4‐mediated pathological processes were suggested in Alzheimer’s disease, multiple sclerosis and ischemic injury. In the present study, we analyzed the intracellular distribution of fluorescently tagged human trypsinogen 4 isoforms during normal and anoxic conditions in transfected mouse primary astrocytes. Our results show that initiation of anoxic milieu by the combined action of KCN treatment and glucose deprivation rapidly leads to the association of leader peptide containing trypsinogen 4 constructs to the plasma membrane. Using rhodamine 110 bis‐(CBZ‐L‐isoleucyl‐L‐prolyl‐L‐arginine amide), a synthetic chromogen peptide substrate of trypsin, we show that anoxia can promote extracellular activation of trypsinogen 4 indicating that extracellular activation of human trypsinogen 4 can be an important component in neuropathological changes of the injured human brain.
Bacterial single-stranded (ss)DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL), and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the Escherichia coli SSB protein forms liquid-liquid phase-separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA, and SSB-interacting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage.
ABSTRACT Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) plays central roles in the repair of DNA breaks and oxidized DNA lesions. Here we show that hSSB1 undergoes liquid-liquid phase separation (LLPS) that is redox-dependent and requires the presence of single-stranded DNA or RNA, features that are distinct from those of LLPS by bacterial SSB. hSSB1 nucleoprotein droplets form under physiological ionic conditions, in response to treatment resulting in cellular oxidative stress. hSSB1’s intrinsically disordered region (IDR) is indispensable for LLPS, whereas all three cysteine residues of the oligonucleotide/oligosaccharide-binding (OB) fold are necessary to maintain redox-sensitive droplet formation. Proteins interacting with hSSB1 show selective enrichment inside hSSB1 droplets, suggesting tight content control and recruitment functions for the condensates. While these features appear instrumental for genome repair, we also detected hSSB1 condensates in the cytoplasm in response to oxidative stress in various cell lines. hSSB1 condensates colocalize with stress granules, implying unexplored extranuclear roles in cellular stress response. Our results suggest novel, condensation-linked roles for hSSB1, linking genome repair and cytoplasmic defense.
Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.
Az NR2B alegyseg tultermeltetese rendellenes kisagy-fejlődeshez vezet: a kisagyi szemcsesejtek es a parallel rost szinapszisok szama lecsokken, a Purkinje sejtek dendritikus arborizacioja kevesbe fejlett. In vivo BrdU jelolesekkel igazoltuk, hogy az NR2C-2B kisagyi szemcsesejtek vandorlasa felgyorsul. NR2C ko. allatok felhasznalasaval bizonyitottuk, hogy a megfigyelt valtozasok nem az NR2C alegyseg hianyanak, hanem az NR2B alegyseg tultermeltetesenek a kovetkezmenyei. A kisagyi szemcsesejt-tenyeszeteket ugy keszitettuk, hogy a vad tipusu es NR2C-2B szemcsesejtekben kifejeződő NMDA receptorok osszetetelukben es funkcionalisan kulonbozzenek. Az NR2C-2B alegysegcsere a nyulvanyok mozgasara nincs hatassal, ezzel szemben a sejttestek vandorlasi atlagsebesseget megnoveli. NR2B alegyseg-specifikus gatloszerekkel vegzett kiserletek alapjan a transzgen sejtekben nagyobb az NR1/NR2B diheteromer receptorok aranya. Adataink alapjan a diheteromer NMDA receptorok aranyanak es/vagy műkodesenek novekedese allhat a transzgen sejtekre jellemző, megnovekedett migracios sebesseg mogott. Nem vart eredmenyunk, hogy a kulonboző eletkoru allatokbol szarmazo szemcsesejtek mozgasi aktivitasa azonos tenyesztesi feltetelek mellett is jelentősen kulonbozik. Habar a tenyeszetek morfologiaja, valamint szamos molekularis jellemzője az izolalasi eletkortol fuggetlenul rendkivul hasonlo, a fiatalabb allatokbol szarmazo idegsejtek mozgasa gyorsabb, mint az oregebb allatokbol szarmazo idegsejteke. | Long-term cerebellar NR2B overexpression led to aberrant Purkinje cell morphology, a decrease in granule cell number and parallel fiber input. BrdU labeling showed that in vivo migratory rate of NR2C-2B granule cells was increased. As similar histological changes were not observed in NR2C ko. mice, developmental abnormalities reported in NR2C-2B mice were due to the overexpression of NR2B and not to the lack of NR2C subunit expression. Culture conditions were developed to obtain differences in NMDA receptor subunit composition and functioning between wild-type and NR2C-2B granule cells in vitro. Computer-controlled videomicroscopy showed that NR2C-2B subunit exchange did not affect neurite motility while significantly increased average migratory speed of granule cell bodies. NR2B subunit-specific NMDA receptor antagonists altered cell motility differently in wild-type and mutant cultures, indicating that NR1/NR2B diheteromer subunits were more characteristic to NR2C-2B cultures and that increase in the amount and/or in the activity of NR1/NR2B NMDA receptors might be responsible for an increase in migratory speed. Unexpected findings were that despite similarities in culture morphology, development or gene expression patterns, granule cells isolated from younger postnatal ages showed increased migratory speed and activity compared to granule cells isolated from older animals.
