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Heavy metal concentrations of Mn, Cr, Fe, Ni, Cu, Zn, Cd and Pb in sediment samples collected from 12 stations in the Black Sea coast, Buyukdere and Sabuncular streams around Cayeli copper mine are...

Natural and artificial radionuclide concentrations were determined and evaluated in 12 different medicinal plants used in Rize Province of Turkey. The levels of 226Ra, 232Th, 40K and 137Cs were spe...

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.

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.

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.

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.

Zinc oxide thin films were grown on a glass substrate by a sol-gel process using a spin-coating technique. The obtained thin films were annealed between 350 °C and 550 °C in 50 °C steps and were then characterized using X-ray diffraction, scanning electron microscopy, and X-ray fluorescence techniques. The samples were stimulated by 59.5 keV gamma rays emitted from an Americium-241 annular radioisotope source. K X-rays emitted by samples were counted using an ultra-low energy germanium detector with a resolution of 150 eV at 5.96 keV. It was found that there was generally a decrease in both the Kβ/Kα X-ray intensity ratios and the K X-ray fluorescence cross sections for zinc oxide between 350 °C and 500 °C, but not at 550 °C. In addition, the X-ray diffraction patterns of the films showed that the transition phase from an amorphous to a polycrystalline hexagonal wurtzite structure was complete at an annealing temperature of 500 °C. The results show that variations in these parameters can be explai...

Buçalışma Doğu Karadeniz Bölgesinde on dört farklı istasyondan toplanan karayemiş(Prunus laurocerasus L.)‘in meyve kısmında ve bu türün köklerindeki toprakörneklerinde 226Ra, 232Th, 137Cs ve 40Kradyonüklid konsantrasyon sonuçlarını göstermektedir. Karayemişin meyvekısmında 226Ra, 232Th, 137Cs ve 40K’ınortalama aktivite konsantrasyonları sırasıyla 1.75, 1.03, 2.07 ve 215.38 Bq kg-1(kuruağırlık)’dır. Toprak örneklerinde 226Ra, 232Th ve 40K’ınortalama aktivite konsantransyonları Dünya ortalaması değerlerinden daha düşükolarak belirlenmiştir. Karayemiş meyvesi ve toprak örneklerinde en yüksek 137Csradyonüklid konsantrasyonu Rize Merkez istasyonunda gözlemlenmiştir. Bunlara ekolarak, karayemişin tüketimine bağlı olarak 226Ra, 232Th,137Cs ve 40K’ın günlük alımı, yıllık alınan etkin doz vekanserojen risk değerleri hesaplanmış ve uluslararası değerlerle karşılaştırılmıştır.Ayrıca toprak örneklerinde radyum eşdeğer aktivitesi, soğurulmuş doz oranı, dıştehlike indeksi ve yıllık etkin doz eşdeğeri hesaplanmıştır. Bu hesaplamalarailaveten topraktan karayemişin meyve kısmına geçen 226Ra, 232Th,137Cs ve 40K radyonüklidleri için transfer faktörleribelirlenmiştir. Bu çalışmada elde edilen sonuçlar kanserojen risk ve radyolojiketki parametre değerlerinin herhangi bir risk taşımadığını göstermektedir., This study presentsthe results of 226Ra, 232Th, 137Cs and 40Kradionuclide concentrations in the fruit part of cherry laurel (Prunuslaurocerasus L.) and soil samples in root of this species were collected fromfourteen different stations in the Eastern Black Sea Region. The mean activityconcentrations of 226Ra, 232Th, 137Cs and 40Kin the fruit part of cherry laurel were 1.75, 1.03, 2.07 and 215.38 Bq kg-1,respectively. The mean activity concentrations of 226Ra, 232Thand 40K in soil samples were determined to be lower than the worldaverage value. The highest 137Cs radionuclide concentration in fruitof cherry laurel and soil samples was monitored in the Rize center location. Inaddition, radiological impact parameters such as daily intake of 226Ra,232Th, 137Cs and 40K, annual committedeffective dose and carcinogenic risk due to the consumption of cherry laurelwere calculated and compared with the international data. The radium equivalentactivity (Raeq), the absorbed dose rate (D), the external hazardindex (Hex) and the annual effective dose equivalent (AEDE) for soilsamples were also evaluated. Moreover, transfer factors of 226Ra, 232Th,137Cs and 40K from soil to fruit part of cherry laurelwere determined. The results indicate that the lifetime cancer risk and theradiological impact parameters values in the samples from the area studied inthis present work is not significant.

This study presents the results of (137)Cs and (40)K radionuclide concentrations in mussel (Mytilus galloprovincialis) samples collected during the period of February-November 2014 from twelve different stations within the border of the eastern Black Sea region of Turkey. Also, these radionuclide concentrations were determined in sea water and sediment samples. The activity concentrations in seawater, sediment and mussel tissue samples were between 1.12-1.69mBqL(-1), 3.26-30.74 and 1.61-3.16Bqkg(-1) for (137)Cs and 231.41-399.49mBqL(-1), 215.71-450.07 and 286.84-382.16Bqkg(-1) for (40)K, respectively. These values are also in accordance with the concentrations reported for similar regions. Additionally, radiological impact parameters such as daily intake of (137)Cs and (40)K, annual committed effective dose and carcinogenic risk due to the consumption of mussel were calculated and compared with the international data. Lifetime cancer risk values are lower than the limit of 10(-3).

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. The peer-reviewed version of the article: Konstantinović, J. M., Kiris, E., Kota, K. P., Kugelman-Tonos, J., Videnović, M., Cazares, L. H., Terzić-Jovanović, N., Verbić, T., Anđelković, B. D., Duplantier, A. J., Bavari, S.,& Šolaja, B. (2018). New Steroidal 4-Aminoquinolines Antagonize Botulinum Neurotoxin Serotype A in Mouse Embryonic Stem Cell Derived Motor Neurons in Postintoxication Model. Journal of Medicinal Chemistry, American Chemical Society (ACS)., 61(4), 1595-1608.[https://doi.org/10.1021/acs.jmedchem.7b01710] Published version: [http://cer.ihtm.bg.ac.rs/handle/123456789/2325] Supporting information I: [https://cer.ihtm.bg.ac.rs/handle/123456789/4515] Supporting information II: [https://cer.ihtm.bg.ac.rs/handle/123456789/4516] Supporting information III: [https://cer.ihtm.bg.ac.rs/handle/123456789/4517]

The aim of this study was to determine the activity concentrations and radiological hazard parameters of Cs-137 and K-40 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 Cs-137 and 163.9 and 269.4 Bq kg(-1) for K-40. The mean activity concentrations of Cs-137 and K-40 in tissues of small-sized sea snails were determined to be higher than those of Cs-137 and K-40 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 Cs-137 and K-40, annual committed effective dose (E-eff), and lifetime cancer risk (LCR) were calculated and compared with the international recommended values.

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.

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.

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.

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.

// Sandrine Puverel 1, * , Erkan Kiris 1, * , Satyendra Singh 1 , Kimberly D. Klarmann 1, 2 , Vincenzo Coppola 3 , Jonathan R. Keller 1, 2 , Lino Tessarollo 1 1 Mouse Cancer Genetics Program, Center for Cancer Research, NCI, Frederick, MD 21702, USA 2 Basic Science Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, NCI, Frederick, MD 21702, USA 3 The Ohio State University, Department of Cancer, Biology and Genetics, Wexner Medical Center and James Comprehensive Cancer Center, Columbus, OH 43210, USA * These authors have contributed equally to this work Correspondence to: Lino Tessarollo, email: tessarol@mail.nih.gov Keywords: RanBP9, c-Kit signaling, hematopoietic system, spermatogenesis, stem cells Received: April 19, 2016 Accepted: October 26, 2016 Published: November 08, 2016 ABSTRACT 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.

y Germanium (HPGe) detector was used to measure natural (Ra-226, Th-232 K-40) radioactivity levels in sediment and seawater samples, and soft tissues of Mediterranean mussels (Mytilus galloprovincialis) of three different sizes, collected from twelve different stations along the coast of Giresun, Trabzon, Rize and Artvin provinces in Eastern Black Sea Region. The average activity concentrations of the sediment and seawater samples collected were 12.65 Bq kg(-1) and 3.57 mBq L-1 for Ra-226, 14.25 Bq kg(-1) and 4.01 mBq L-1 for Th-232 and 294.8 Bq kg(-1) and 317.8 mBq L-1 for K-40, respectively. The activity concentrations in soft tissues of three different sized mussel samples altered from 2.45 to 9.59 Bq kg(-1) for Ra-226 2.22-8.59 Bq kg(-1) for Th-232 and 251.9-382.1 Bq kg(-1) for K-40. Moreover, sediment distribution coefficients(K-d) and bioaccumulation factors (BAF(w) and BAF(s)) in the mussel samples were calculated. While the bioaccumulation of Ra-226 and Th-232 from seawater to mussel was observed, the bioaccumulation K-40 was observed from sediment to mussel. The ratio of the average concentration of radionuclides in large-sized mussels to that in water and sediment is greater as compared with smaller mussels. Although there were statistically differences (p < 0.05) among mussel sizes according to Th-232 levels for activity concentrations, BAF, and BAF, values in soft tissues of the mussel samples, it wasn't observed difference according to Ra-226 and K-40 levels (p > 0.05).

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.

Background Microtubules (MTs) are protein nanotubes comprised of straight protofilaments (PFs), head to tail assemblies of αβ-tubulin heterodimers. Previously, it was shown that Tau, a microtubule-associated protein (MAP) localized to neuronal axons, regulates the average number of PFs in microtubules with increasing inner radius observed for increasing Tau/tubulin-dimer molar ratio ΦTau at paclitaxel/tubulin-dimer molar ratio ΛPtxl = 1/1. Methods We report a synchrotron SAXS and TEM study of the phase behavior of microtubules as a function of varying concentrations of paclitaxel (1/32 ≤ ΛPtxl ≤ 1/4) and Tau (human isoform 3RS, 0 ≤ Φ3RS ≤ 1/2) at room temperature. Results Tau and paclitaxel have opposing regulatory effects on microtubule bundling architectures and microtubule diameter. Surprisingly and in contrast to previous results at ΛPtxl = 1/1 where microtubule bundles are absent, in the lower paclitaxel concentration regime (ΛPtxl ≤ 1/4), we observe both microtubule doublets and triplets with increasing Tau. Furthermore, increasing paclitaxel concentration (up to ΛPtxl = 1/1) slightly decreased the average microtubule diameter (by ~ 1 PF) while increasing Tau concentration (up to Φ3RS = 1/2) significantly increased the diameter (by ~ 2–3 PFs). Conclusions The suppression of Tau-mediated microtubule bundling with increasing paclitaxel is consistent with paclitaxel seeding more, but shorter, microtubules by rapidly exhausting tubulin available for polymerization. Microtubule bundles require the aggregate Tau-Tau attractions along the microtubule length to overcome individual microtubule thermal energies disrupting bundles. General significance Investigating MAP-mediated interactions between microtubules (as it relates to in vivo behavior) requires the elimination or minimization of paclitaxel.

Shielding of gamma-rays and neutrons by 12 concrete samples with and without mineral additives has been studied. The total mass attenuation and linear attenuation coefficients, half-value thicknesses, effective atomic numbers, effective electron densities and atomic cross-sections at photons energies of 59.5 and 661 keV have been measured and calculated. The measured and calculated values were compared and a reasonable agreement has been observed. Also the recorded values showed a change with energy and composition of the concrete samples. In addition, neutron shielding has been treated in terms of macroscopic removal cross-section (Σ R , cm −1 ) concept. The WinXCom and NXcom programs were employed to calculate the attenuation coefficients of gamma-rays and neutrons, respectively.

Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr231, Ser262, Ser396, and Ser404) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.

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.

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.

Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr(231), Ser(262), Ser(396), and Ser(404)) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.

The regulation of microtubule growing and shortening dynamics is essential for proper cell function and viability, and microtubule-associated proteins (MAPs) such as the neural protein tau are critical regulators of these dynamic processes. Further, we and our colleagues have proposed that misregulation of microtubule dynamics may contribute to tau-mediated neuronal cell death and dementia in Alzheimer's and related diseases. In the first part of this chapter, we present a general background on microtubule dynamics and then focus in on tau. We review the literature on the roles of tau in normal neuronal cell biology, the tau structure-function relationship, regulatory mechanisms influencing tau action, and pathological tau action, including normal and aberrant regulation of microtubule dynamics. In the second part of this chapter, we present detailed protocols for various in vitro procedures often used in studying tau-mediated regulation of microtubule dynamics, including purification and characterization of necessary reagents, microtubule assembly assays, and microtubule dynamics assays. Importantly, these assays are readily adaptable to examine other regulators of microtubule dynamics besides tau. In the final analysis, in vitro analyses of MAP-mediated regulation of microtubule dynamics will provide extremely valuable insights into our understanding of normal and pathological cell biology.

The regulation of microtubule growing and shortening dynamics is essential for proper cell function and viability, and microtubule-associated proteins (MAPs) such as the neural protein tau are critical regulators of these dynamic processes. Further, we and our colleagues have proposed that misregulation of microtubule dynamics may contribute to tau-mediated neuronal cell death and dementia in Alzheimer's and related diseases. In the first part of this chapter, we present a general background on microtubule dynamics and then focus in on tau. We review the literature on the roles of tau in normal neuronal cell biology, the tau structure-function relationship, regulatory mechanisms influencing tau action, and pathological tau action, including normal and aberrant regulation of microtubule dynamics. In the second part of this chapter, we present detailed protocols for various in vitro procedures often used in studying tau-mediated regulation of microtubule dynamics, including purification and characterization of necessary reagents, microtubule assembly assays, and microtubule dynamics assays. Importantly, these assays are readily adaptable to examine other regulators of microtubule dynamics besides tau. In the final analysis, in vitro analyses of MAP-mediated regulation of microtubule dynamics will provide extremely valuable insights into our understanding of normal and pathological cell biology.

Microtubule (MT) dynamics are traditionally analyzed from time lapse images by manual techniques that are laborious, approximate and often limited. Recently, computer vision techniques have been applied to the problem of automated tracking of MTs in live cell images. Aside of very low signal to noise ratios, live cell images of MTs exhibit severe clutter for accurate tracing of MT body. Moreover, intersecting and overlapping MT regions appear brighter due to additive fluorescence. In this paper, we present a MT body tracing algorithm that addresses the clutter without imposing directional constraints. We show that MT dynamics can be quantified with enhanced precision, and novel measurements that are beyond manual feasibility, can be obtained accurately. We demonstrate our results on actual images of MTs obtained by live cell fluorescence microscopy.

Neuronal microtubules (MTs) are 25 nm protein nanotubes used as tracks for intracellular trafficking of biomolecules, for example, those involved in transmitting signals between neurons. Distinct members of MAP tau isoforms regulate microtubule assembly and stabilization. Altered tau-MT interactions lead to MT depolymerization and tau tangles, which are implicated in a large number of neurodegenerative diseases. We describe our recent findings about the effect of human wild type MAP tau on interprotofilament and intermicrotubule interactions, by using synchrotron small angle x-ray scattering. Supported by DOE DE-FG02-06ER46314, NSF DMR-0503347, NIH GM-59288, NIHI RO1-NS35010 and NS13560.