Generation and characterization of human induced pluripotent stem cell line METUi002-A from a patient with primary familial brain calcification (PFBC) carrying a heterozygous mutation (c.687dupT (p.Val230CysfsTer28)) in the SLC20A2 gene.

Primary familial brain calcification (PFBC) is a rare neurological condition characterized by abnormal calcification commonly in basal ganglia and multiple other brain regions, leading to neuropsychiatric, cognitive, and motor symptoms. SLC20A2, one of the known causative genes for PFBC, contains the highest number of variants directly associated with the disease. Here, we established an iPSC line (METUi002-A) from the peripheral blood mononuclear cells of a clinically diagnosed PFBC patient carrying a SLC20A2 mutation (c.687dupT) using the integration-free Sendai reprogramming. METUi002-A can serve as a valuable tool to generate cellular models to investigate the mechanistic effects of this mutation in PFBC.

TrkA-cholinergic signaling modulates fear encoding and extinction learning in PTSD-like behavior.

Recent studies have suggested that the use of cognitive enhancers as adjuncts to exposure-based therapy in individuals suffering from post-traumatic stress disorder (PTSD) may be beneficial. Brain cholinergic signaling through basal forebrain projections to the hippocampus is an established pathway mediating fear response and cognitive flexibility. Here we employed a genetic strategy to enhance cholinergic activity through increased signaling of the NGF receptor TrkA. This strategy leads to increased levels of the marker of cholinergic activation, acetylcholine synthesizing enzyme choline acetyltransferase, in forebrain cholinergic regions and their projection areas such as the hippocampus. Mice with increased cholinergic activity do not display any neurobehavioral abnormalities except a selective attenuation of fear response and lower fear expression in extinction trials. Reduction in fear response is rescued by the GABA antagonist picrotoxin in mutant mice, and, in wild-type mice, is mimicked by the GABA agonist midazolam suggesting that GABA can modulate cholinergic functions on fear circuitries. Importantly, mutant mice also show a reduction in fear processing under stress conditions in a single prolonged stress (SPS) model of PTSD-like behavior, and augmentation of cholinergic signaling by the drug donepezil in wild-type mice promotes extinction learning in a similar SPS model of PTSD-like behavior. Donepezil is already in clinical use for the treatment of dementia suggesting a new translational application of this drug for improving exposure-based psychotherapy in PTSD patients.

Establishment of Human Induced Pluripotent Stem Cells from Multiple Sclerosis Patients.

The patient-derived iPSC lines provide valuable resources as these cells can be utilized to generate human cell types relevant to the disease of interest. In this context, human iPSC-based model systems are particularly useful for neurological diseases as the neuron and glial cell types affected by such diseases are difficult to obtain. Multiple sclerosis is a demyelinating central nervous system disease characterized by inflammation and eventually axonal damage. iPS cells generated from MS patients may allow for unique approaches for studying the disease in a species-specific manner, with a potentially limitless supply of patients' own glial and neuronal cells differentiated from the iPSCs. Here we describe the detailed protocol for establishing iPSCs from peripheral blood mononuclear cells that we have utilized to model multiple sclerosis. We particularly focused on optimized and cost-effective procedures using the integration-free Sendai virus-based reprogramming method for the generation and characterization of MS iPSCs.

Human-Induced Pluripotent Stem Cell-Based Models for Studying Sex-Specific Differences in Neurodegenerative Diseases.

The prevalence of neurodegenerative diseases is steadily increasing worldwide, and epidemiological studies strongly suggest that many of the diseases are sex-biased. It has long been suggested that biological sex differences are crucial for neurodegenerative diseases; however, how biological sex affects disease initiation, progression, and severity is not well-understood. Sex is a critical biological variable that should be taken into account in basic research, and this review aims to highlight the utility of human-induced pluripotent stem cells (iPSC)-derived models for studying sex-specific differences in neurodegenerative diseases, with advantages and limitations. In vitro systems utilizing species-specific, renewable, and physiologically relevant cell sources can provide powerful platforms for mechanistic studies, toxicity testings, and drug discovery. Matched healthy, patient-derived, and gene-corrected human iPSCs, from both sexes, can be utilized to generate neuronal and glial cell types affected by specific neurodegenerative diseases to study sex-specific differences in two-dimensional (2D) and three-dimensional (3D) human culture systems. Such relatively simple and well-controlled systems can significantly contribute to the elucidation of molecular mechanisms underlying sex-specific differences, which can yield effective, and potentially sex-based strategies, against neurodegenerative diseases.

Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity.

Botulinum neurotoxins (BoNTs) are known as the most potent bacterial toxins, which can cause potentially deadly disease botulism. BoNT Serotype A (BoNT/A) is the most studied serotype as it is responsible for most human botulism cases, and its formulations are extensively utilized in clinics for therapeutic and cosmetic applications. BoNT/A has the longest-lasting effect in neurons compared to other serotypes, and there has been high interest in understanding how BoNT/A manages to escape protein degradation machinery in neurons for months. Recent work demonstrated that an E3 ligase, HECTD2, leads to efficient ubiquitination of the BoNT/A Light Chain (A/LC); however, the dominant activity of a deubiquitinase (DUB), VCIP135, inhibits the degradation of the enzymatic component. Another DUB, USP9X, was also identified as a potential indirect contributor to A/LC degradation. In this study, we screened a focused ubiquitin-proteasome pathway inhibitor library, including VCIP135 and USP9X inhibitors, and identified ten potential lead compounds affecting BoNT/A mediated SNAP-25 cleavage in neurons in pre-intoxication conditions. We then tested the dose-dependent effects of the compounds and their potential toxic effects in cells. A subset of the lead compounds demonstrated efficacy on the stability and ubiquitination of A/LC in cells. Three of the compounds, WP1130 (degrasyn), PR-619, and Celastrol, further demonstrated efficacy against BoNT/A holotoxin in an in vitro post-intoxication model. Excitingly, PR-619 and WP1130 are known inhibitors of VCIP135 and USP9X, respectively. Modulation of BoNT turnover in cells by small molecules can potentially lead to the development of effective countermeasures against botulism.

Generation and characterization of human induced pluripotent stem cell line METUi001-A from a 25-year-old male patient with relapsing-remitting multiple sclerosis.

Multiple sclerosis is a chronic disease characterized by inflammation, demyelination, and axonal damage in the central nervous system. Here, we established an induced pluripotent stem cell (iPSC) line METUi001-A from the peripheral blood mononuclear cells of a 25-year-old male individual with clinically diagnosed Relapsing-Remitting Multiple Sclerosis (RRMS) using the integration-free Sendai reprogramming method. We demonstrated that the iPSCs are free of exogenous Sendai reprogramming vectors, have a normal male karyotype, express pluripotency markers, and differentiate into the three germ layers. The iPSC line can serve as a valuable resource to generate cellular model systems to investigate molecular mechanisms underlying RRMS.

Correction to: A study of the radiological baseline conditions around the planned Sinop (Turkey) nuclear power plant using the mapping method.

The original version of this article unfortunately contained a mistake.

A study of the radiological baseline conditions around the planned Sinop (Turkey) nuclear power plant using the mapping method.

This study makes a first attempt at a detailed estimation of the background radioactivity level and its distribution at the Sinop nuclear power plant site. The activity concentration levels of 226Ra, 232Th, 40K and 137Cs radionuclides in soil samples collected from 88 locations around Sinop Province, Turkey, in November 2016, were measured using gamma spectrometry. The distributions of radionuclide levels obtained from the results were evaluated using a geostatistical method, and the estimated radiation levels were determined using the ordinary kriging (OK) method, which is the best linear unbiased estimator (BLUE) for unmeasured points. Estimates of distribution results were evaluated using cross-validation diagrams, and it was shown that the OK method could predict radiological distributions for appropriate criteria. Finally, using the kriging parameters, distributions of radiation levels for the entire work area were mapped at a spatial resolution of 100 × 100 m2. These maps show that the natural radionuclides (226Ra, 232Th and 40K) are distributed at higher levels to the southeast of Sinop than in the other regions, and the activity of an artificial radionuclide (137Cs) is high in the interior and northern sections.

Determination of radiological hazard parameters in sea snails (Rapana venosa) in the East Black Sea Coast of Turkey.

The aim of this study was to determine the activity concentrations and radiological hazard parameters of 137Cs and 40K in soft tissue samples of sea snails (Rapana venosa) collected from 12 different stations in the East Black Sea coastal area of Turkey. The values in soft tissues of sea snail samples with three different sizes were between 0.31 and 1.79 Bq kg-1 for 137Cs and 163.9 and 269.4 Bq kg-1 for 40K. The mean activity concentrations of 137Cs and 40K in tissues of small-sized sea snails were determined to be higher than those of 137Cs and 40K in soft tissues of the other sizes. The determined activity concentration values were lower than the concentrations reported in similar studies. Radiological hazard parameters such as the daily intake (Dint) of 137Cs and 40K, annual committed effective dose (Eeff), and lifetime cancer risk (LCR) were calculated and compared with the international recommended values.

New Steroidal 4-Aminoquinolines Antagonize Botulinum Neurotoxin Serotype A in Mouse Embryonic Stem Cell Derived Motor Neurons in Postintoxication Model.

The synthesis and inhibitory potencies against botulinum neurotoxin serotype A light chain (BoNT/A LC) using in vitro HPLC based enzymatic assay for various steroidal, benzothiophene, thiophene, and adamantane 4-aminoquinoline derivatives are described. In addition, the compounds were evaluated for the activity against BoNT/A holotoxin in mouse embryonic stem cell derived motor neurons. Steroidal derivative 16 showed remarkable protection (up to 89% of uncleaved SNAP-25) even when administered 30 min postintoxication. This appears to be the first example of LC inhibitors antagonizing BoNT intoxication in mouse embryonic stem cell derived motor neurons (mES-MNs) in a postexposure model. Oral administration of 16 was well tolerated in the mouse up to 600 mg/kg, q.d. Although adequate unbound drug levels were not achieved at this dose, the favorable in vitro ADMET results strongly support further work in this series.

Deubiquitinating enzyme VCIP135 dictates the duration of botulinum neurotoxin type A intoxication.

Botulism is characterized by flaccid paralysis, which can be caused by intoxication with any of the seven known serotypes of botulinum neurotoxin (BoNT), all of which disrupt synaptic transmission by endoproteolytic cleavage of SNARE proteins. BoNT serotype A (BoNT/A) has the most prolonged or persistent effects, which can last several months, and exerts its effects by specifically cleaving and inactivating SNAP25. A major factor contributing to the persistence of intoxication is the long half-life of the catalytic light chain, which remains enzymatically active months after entry into cells. Here we report that BoNT/A catalytic light chain binds to, and is a substrate for, the ubiquitin ligase HECTD2. However, the light chain evades proteasomal degradation by the dominant effect of a deubiquitinating enzyme, VCIP135/VCPIP1. This deubiquitinating enzyme binds BoNT/A light chain directly, with the two associating in cells through the C-terminal 77 amino acids of the light chain protease. The development of specific DUB inhibitors, together with inhibitors of BoNT/A proteolytic activity, may be useful for reducing the morbidity and public health costs associated with BoNT/A intoxication and could have potential biodefense implications.

Determination of radioactivity levels and heavy metal concentrations in seawater, sediment and anchovy (Engraulis encrasicolus) from the Black Sea in Rize, Turkey.

Seawater, sediment and fish (anchovy) samples consumed in the Rize province of the Eastern Black Sea region of Turkey were collected from five different stations. The radioactivity levels (226Ra, 232Th, 40K and 137Cs) were determined in all the samples using a high-purity germanium detector. While 226Ra, 232Th and 40K radionuclides were detected in all samples, the radionuclide concentration of 137Cs, except for the sediment samples (mean activity is 9±1.4Bqkg-1), was not detected for the seawater and fish samples. The total annual effective dose rates from the ingestion of these radionuclides for fish were calculated using the measured activity concentrations in radionuclides and their ingested dose conversion factor. Also, the concentrations of some heavy metals in all the samples were determined. The activity and heavy metal concentration values that were determined for the seawater, sediment and fish samples were compared among the locations themselves and with literature values.

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

Phosphatase Inhibitors Function as Novel, Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays.

There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.

SRC family kinase inhibitors antagonize the toxicity of multiple serotypes of botulinum neurotoxin in human embryonic stem cell-derived motor neurons.

Botulinum neurotoxins (BoNTs), the causative agents of botulism, are potent inhibitors of neurotransmitter release from motor neurons. There are currently no drugs to treat BoNT intoxication after the onset of the disease symptoms. In this study, we explored how modulation of key host pathways affects the process of BoNT intoxication in human motor neurons, focusing on Src family kinase (SFK) signaling. Motor neurons derived from human embryonic stem (hES) cells were treated with a panel of SFK inhibitors and intoxicated with BoNT serotypes A, B, or E (which are responsible for >95 % of human botulism cases). Subsequently, it was found that bosutinib, dasatinib, KX2-391, PP1, PP2, Src inhibitor-1, and SU6656 significantly antagonized all three of the serotypes. Furthermore, the data indicated that the treatment of hES-derived motor neurons with multiple SFK inhibitors increased the antagonistic effect synergistically. Mechanistically, the small molecules appear to inhibit BoNTs by targeting host pathways necessary for intoxication and not by directly inhibiting the toxins' proteolytic activity. Importantly, the identified inhibitors are all well-studied with some in clinical trials while others are FDA-approved drugs. Overall, this study emphasizes the importance of targeting host neuronal pathways, rather than the toxin's enzymatic components, to antagonize multiple BoNT serotypes in motor neurons.

A high content imaging assay for identification of Botulinum neurotoxin inhibitors.

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Recent advances in botulinum neurotoxin inhibitor development.

Botulinum neurotoxins (BoNTs) are endopeptidases that target motor neurons and block acetylcholine neurotransmitter release. This action results in the muscle paralysis that defines the disease botulism. To date, there are no FDA-approved therapeutics to treat BoNT-mediated paralysis after intoxication of the motor neuron. Importantly, the rationale for pursuing treatments to counter these toxins is driven by their potential misuse. Current drug discovery efforts have mainly focused on small molecules, peptides, and peptidomimetics that can directly and competitively inhibit BoNT light chain proteolytic activity. Although this is a rational approach, direct inhibition of the Zn(2+) metalloprotease activity has been elusive as demonstrated by the dearth of candidates undergoing clinical evaluation. Therefore, broadening the scope of viable targets beyond that of active site protease inhibitors represents an additional strategy that could move the field closer to the clinic. Here we review the rationale, and discuss the outcomes of earlier approaches and highlight potential new targets for BoNT inhibition. These include BoNT uptake and processing inhibitors, enzymatic inhibitors, and modulators of neuronal processes associated with toxin clearance, neurotransmitter potentiation, and other pathways geared towards neuronal recovery and repair.

TrkA in vivo function is negatively regulated by ubiquitination.

TrkA is a tyrosine kinase receptor required for development and survival of the peripheral nervous system. In the adult, TrkA and its ligand NGF are peripheral pain mediators, particularly in inflammatory pain states. However, how TrkA regulates the function of nociceptive neurons and whether its activity levels may lead to sensory abnormalities is still unclear. Here we report the characterization of a 3 aa (KFG) domain that negatively regulates TrkA level and function in response to NGF. Deletion of this domain in mouse causes a reduction of TrkA ubiquitination leading to an increase in TrkA protein levels and activity. The number of dorsal root ganglia neurons is not affected by the mutation. However, mutant mice have enhanced thermal sensitivity and inflammatory pain. Together, these data suggest that ubiquitination is a mechanism used in nociceptive neurons to regulate TrkA level and function. Our results may enhance our understanding of how ubiquitination affects TrkA activation following noxious thermal stimulation and inflammatory pain.

Recent developments in cell-based assays and stem cell technologies for botulinum neurotoxin research and drug discovery.

Botulinum neurotoxins (BoNTs) are exceptionally potent inhibitors of neurotransmission, causing muscle paralysis and respiratory failure associated with the disease botulism. Currently, no drugs are available to counter intracellular BoNT poisoning. To develop effective medical treatments, cell-based assays provide a valuable system to identify novel inhibitors in a time- and cost-efficient manner. Consequently, cell-based systems including immortalized cells, primary neurons and stem cell-derived neurons have been established. Stem cell-derived neurons are highly sensitive to BoNT intoxication and represent an ideal model to study the biological effects of BoNTs. Robust immunoassays are used to quantify BoNT activity and play a central role during inhibitor screening. In this review, we examine recent progress in physiologically relevant cell-based assays and high-throughput screening approaches for the identification of both direct and indirect BoNT inhibitors.

Embryonic stem cell-derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery.

Botulinum neurotoxins (BoNTs) inhibit cholinergic synaptic transmission by specifically cleaving proteins that are crucial for neurotransmitter exocytosis. Due to the lethality of these toxins, there are elevated concerns regarding their possible use as bioterrorism agents. Moreover, their widespread use for cosmetic purposes, and as medical treatments, has increased the potential risk of accidental overdosing and environmental exposure. Hence, there is an urgent need to develop novel modalities to counter BoNT intoxication. Mammalian motoneurons are the main target of BoNTs; however, due to the difficulty and poor efficiency of the procedures required to isolate the cells, they are not suitable for high-throughput drug screening assays. Here, we explored the suitability of embryonic stem (ES) cell-derived motoneurons as a renewable, reproducible, and physiologically relevant system for BoNT studies. We found that the sensitivity of ES-derived motoneurons to BoNT/A intoxication is comparable to that of primary mouse spinal motoneurons. Additionally, we demonstrated that several BoNT/A inhibitors protected SNAP-25, the BoNT/A substrate, in the ES-derived motoneuron system. Furthermore, this system is compatible with immunofluorescence-based high-throughput studies. These data suggest that ES-derived motoneurons provide a highly sensitive system that is amenable to large-scale screenings to rapidly identify and evaluate the biological efficacies of novel therapeutics.