Elevated parkinsonism pathological markers in dopaminergic neurons with developmental exposure to atrazine.

Atrazine (ATZ) is one of the most used herbicides in the US and a known endocrine disruptor. ATZ is frequently detected in drinking water, especially in Midwestern regions of the United States, exceeding the EPA regulation of maximum contamination level (MCL) of 3 ppb. Epidemiology studies have suggested an association between ATZ exposure and neurodegeneration. Less, however, is known about the neurotoxic mechanism of ATZ, particularly for exposures at a developmental stage. Here, we exposed floor plate progenitors (FPPs) derived from human induced pluripotent stem cells (hiPSCs) to low concentrations of ATZ at 0.3 and 3 ppb for two days followed by differentiation into dopaminergic (DA) neurons in ATZ-free medium. We then examined the morphology, activity, pathological protein aggregation, and transcriptomic changes of differentiated DA neurons. We observed significant decrease in the complexity of neurite network, increase of neuronal activity, and elevated tau- and α-synuclein (aSyn) pathologies after ATZ exposure. The ATZ-induced neuronal changes observed here align with pathological characteristics in Parkinson's disease (PD). Transcriptomic analysis further corroborates our findings; and collectively provides a strong evidence base that low-concentration ATZ exposure during development can elicit increased risk of neurodegeneration.

Effects of Neighboring Phosphorylation Events on the Affinities of pT181-Tau Antibodies.

A tau variant phosphorylated on threonine 181 (pT181-tau) has been widely investigated as a potential Alzheimer's disease (AD) biomarker in cerebrospinal fluid (CSF) and blood. pT181-tau is present in neurofibrillary tangles (NFTs) of AD brains, and CSF levels of pT181-tau correlate with the overall NFT burden. Various immunobased analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids. The reliability of these methods is dependent on the affinity and binding specificity of the antibodies used to measure pT181-tau levels. Although both of these properties could, in principle, be affected by phosphorylation within or near the antibody's cognate antigen, such effects have not been extensively studied. Here, we developed a biolayer interferometry assay to determine the degree to which the affinity of pT181-tau antibodies is altered by the phosphorylation of serine or threonine residues near the target epitope. Our results revealed that phosphorylation near T181 negatively affected the binding of pT181-tau antibodies to their cognate antigen to varying degrees. In particular, two of three antibodies tested showed a complete loss of affinity for the pT181 target when S184 or S185 was phosphorylated. These findings highlight the importance of selecting antibodies that have been thoroughly characterized in terms of affinity and binding specificity, addressing the potential disruptive effects of post-translational modifications in the epitope region to ensure accurate biomarker quantitation.

Evaluation of N- and O-Linked Indole Triazines for a Dual Effect on α-Synuclein and Tau Aggregation.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder underlying dementia in the geriatric population. AD manifests by two pathological hallmarks: extracellular amyloid-ß (Aß) peptide-containing senile plaques and intraneuronal neurofibrillary tangles comprised of aggregated hyperphosphorylated tau protein (p-tau). However, more than half of AD cases also display the presence of aggregated α-synuclein (α-syn)-containing Lewy bodies. Conversely, Lewy bodies disorders have been reported to have concomitant Aß plaques and neurofibrillary tangles. Our drug discovery program focuses on the synthesis of multitarget-directed ligands to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to slow the progression of AD and related dementias. To this end, we synthesized 11 compounds with a triazine-linker and evaluated their effectiveness in reducing α-syn, tau isoform 2N4R, and p-tau isoform 1N4R aggregation. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), and M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. We also performed disaggregation assays with isolated Aß-plaques from human AD brains. Our results demonstrated that compound 10 was effective in reducing both oligomerization and fibril formation of α-syn and tau isoform 2N4R in a dose-dependent manner via ThT and PICUP assays. Compound 10 was also effective at reducing the formation of recombinant α-syn, tau 2N4R, and p-tau 1N4R fibrils by TEM. Compound 10 reduced the development of α-syn inclusions in M17D neuroblastoma cells and stopped the seeding of tau P301S using biosensor cells. Disaggregation experiments showed smaller Aß-plaques and less paired helical filaments with compound 10. Compound 10 may provide molecular scaffolds for further optimization and preclinical studies for neurodegenerative proteinopathies.

Developmental Pb exposure increases AD risk via altered intracellular Ca2+ homeostasis in hiPSC-derived cortical neurons.

Exposure to environmental chemicals such as lead (Pb) during vulnerable developmental periods can result in adverse health outcomes later in life. Human cohort studies have demonstrated associations between developmental Pb exposure and Alzheimer's disease (AD) onset in later life which were further corroborated by findings from animal studies. The molecular pathway linking developmental Pb exposure and increased AD risk, however, remains elusive. In this work, we used human iPSC-derived cortical neurons as a model system to study the effects of Pb exposure on AD-like pathogenesis in human cortical neurons. We exposed neural progenitor cells derived from human iPSC to 0, 15, and 50 ppb Pb for 48 h, removed Pb-containing medium, and further differentiated them into cortical neurons. Immunofluorescence, Western blotting, RNA-sequencing, ELISA, and FRET reporter cell lines were used to determine changes in AD-like pathogenesis in differentiated cortical neurons. Exposing neural progenitor cells to low-dose Pb, mimicking a developmental exposure, can result in altered neurite morphology. Differentiated neurons exhibit altered calcium homeostasis, synaptic plasticity, and epigenetic landscape along with elevated AD-like pathogenesis markers, including phosphorylated tau, tau aggregates, and Aß42/40. Collectively, our findings provide an evidence base for Ca dysregulation caused by developmental Pb exposure as a plausible molecular mechanism accounting for increased AD risk in populations with developmental Pb exposure.

Discovery of 4-aminoindole carboxamide derivatives to curtail alpha-synuclein and tau isoform 2N4R oligomer formation.

Alzheimer's disease (AD) is a multifactorial, chronic neurodegenerative disease characterized by the presence of extracellular ß-amyloid (Aß) plaques, intraneuronal neurofibrillary tangles (NFTs), activated microglial cells, and an inflammatory state (involving reactive oxygen species production) in the brain. NFTs are comprised of misfolded and hyperphosphorylated forms of the microtubule-binding protein tau. Interestingly, the trimeric form of the 2N4R splice isoform of tau has been found to be more toxic than the trimeric 1N4R isoform in neuron precursor cells. Few drug discovery programs have focused on specific tau isoforms. The present drug discovery project is centered on the anti-aggregation effect of a series of seventeen 4- or 5-aminoindole carboxamides on the 2N4R isoform of tau. The selection of the best compounds was performed using α-synuclein (α-syn). The anti-oligomer and -fibril activities of newly synthesized aminoindole carboxamide derivatives were evaluated with biophysical methods, such as thioflavin T fluorescence assays, photo-induced cross-linking of unmodified proteins, and transmission electron microscopy. To evaluate the reduction of inclusions and cytoprotective effects, M17D neuroblastoma cells expressing inclusion-forming α-syn were treated with the best amide representatives. The 4-aminoindole carboxamide derivatives exhibited a better anti-fibrillar activity compared to their 5-aminoindole counterparts. The amide derivatives 2, 8, and 17 exerted anti-oligomer and anti-fibril activities on α-syn and the 2N4R isoform of tau. At a concentration of 40 µM, compound 8 reduced inclusion formation in M17D neuroblastoma cells expressing inclusion-prone αSynuclein3K::YFP. Our results demonstrate the potential of 4-aminoindole carboxamide derivatives with regard to inhibiting the oligomer formation of α-syn and tau (2N4R isoform) for further optimization prior to pre-clinical studies.

5-Nitro-1,2-benzothiazol-3-amine and N-Ethyl-1-[(ethylcarbamoyl)(5-nitro-1,2-benzothiazol-3-yl)amino]formamide Modulate α-Synuclein and Tau Aggregation.

Protein misfolding results in a plethora of known diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, transthyretin-related amyloidosis, type 2 diabetes, Lewy body dementia, and spongiform encephalopathy. To provide a diverse portfolio of therapeutic small molecules with the ability to reduce protein misfolding, we evaluated a set of 13 compounds: 4-(benzo[d]thiazol-2-yl)aniline (BTA) and its derivatives containing urea (1), thiourea (2), sulfonamide (3), triazole (4), and triazine (5) linker. In addition, we explored small modifications on a very potent antioligomer 5-nitro-1,2-benzothiazol-3-amine (5-NBA) (compounds 6-13). This study aims to define the activity of BTA and its derivatives on a variety of prone-to-aggregate proteins such as transthyretin (TTR81-127, TTR101-125), α-synuclein (α-syn), and tau isoform 2N4R (tau 2N4R) through various biophysical methods. Thioflavin T (ThT) fluorescence assay was used to monitor fibril formation of the previously mentioned proteins after treatment with BTA and its derivatives. Antifibrillary activity was confirmed using transmission electron microscopy (TEM). Photoreactive cross-linking assay (PICUP) was utilized to detect antioligomer activity and lead to the identification of 5-NBA (at low micromolar concentration) and compound 13 (at high concentration) as the most promising in reducing oligomerization. 5-NBA and not BTA inhibited the inclusion formation based on the cell-based assay using M17D neuroblastoma cells that express inclusion-prone αS-3K::YFP. 5-NBA abrogated the fibril, oligomer, and inclusion formation in a dose-dependent manner. 5-NBA derivatives could be the key to mitigate protein aggregation. In the future, the results made from this study will provide an initial platform to generate more potent inhibitors of α-syn and tau 2N4R oligomer and fibril formation.

Bassoon contributes to tau-seed propagation and neurotoxicity.

Tau aggregation is a defining histopathological feature of Alzheimer's disease and other tauopathies. However, the cellular mechanisms involved in tau propagation remain unclear. Here, we performed an unbiased quantitative proteomic study to identify proteins that specifically interact with this tau seed. We identified Bassoon (BSN), a presynaptic scaffolding protein, as an interactor of the tau seed isolated from a mouse model of tauopathy, and from Alzheimer's disease and progressive supranuclear palsy postmortem samples. We show that BSN exacerbates tau seeding and toxicity in both mouse and Drosophila models for tauopathy, and that BSN downregulation decreases tau spreading and overall disease pathology, rescuing synaptic and behavioral impairments and reducing brain atrophy. Our findings improve the understanding of how tau seeds can be stabilized by interactors such as BSN. Inhibiting tau-seed interactions is a potential new therapeutic approach for neurodegenerative tauopathies.

Identification of Novel Kinases of Tau Using Fluorescence Complementation Mass Spectrometry (FCMS).

Hyperphosphorylation of the microtubule-associated protein Tau is a major hallmark of Alzheimer's disease and other tauopathies. Understanding the protein kinases that phosphorylate Tau is critical for the development of new drugs that target Tau phosphorylation. At present, the repertoire of the Tau kinases remains incomplete, and methods to uncover novel upstream protein kinases are still limited. Here, we apply our newly developed proteomic strategy, fluorescence complementation mass spectrometry, to identify novel kinase candidates of Tau. By constructing Tau- and kinase-fluorescent fragment library, we detected 59 Tau-associated kinases, including 23 known kinases of Tau and 36 novel candidate kinases. In the validation phase using in vitro phosphorylation, among 15 candidate kinases we attempted to purify and test, four candidate kinases, OXSR1 (oxidative-stress responsive gene 1), DAPK2 (death-associated protein kinase 2), CSK (C-terminal SRC kinase), and ZAP70 (zeta chain of T-cell receptor-associated protein kinase 70), displayed the ability to phosphorylate Tau in time-course experiments. Furthermore, coexpression of these four kinases along with Tau increased the phosphorylation of Tau in human neuroglioma H4 cells. We demonstrate that fluorescence complementation mass spectrometry is a powerful proteomic strategy to systematically identify potential kinases that can phosphorylate Tau in cells. Our discovery of new candidate kinases of Tau can present new opportunities for developing Alzheimer's disease therapeutic strategies.

Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor.

Luciferase-based reporters provide a key measurement approach in a broad range of applications, from in vitro high-throughput screening to whole animal imaging. For example, luminescence intensity is widely used to measure promoter activity, protein expression levels, and cell growth. However, luminescence intensity measurements are subject to quantitative irregularities caused by luminescence decay and variation in reporter expression level. In contrast, bioluminescence resonance energy transfer (BRET) sensors provide the advantages of luciferase-based reporters but overcome the aforementioned irregularities because of the inherently ratiometric readout. Here, we generated a new ratiometric BRET sensor of ATP (ARSeNL-ATP detection with a Ratiometric mScarlet-NanoLuc sensor), and we demonstrated that it provides a stable and robust readout across protein, cell, and whole animal tissue contexts. The ARSeNL sensor was engineered by screening a color palette of sensors utilizing variants of the high photon flux NanoLuc luciferase as donors and a panel of red fluorescent proteins as acceptors. We found that the novel combination of NanoLuc and mScarlet exhibited the largest dynamic range, with a 5-fold change in the BRET ratio upon saturation with ATP. Importantly, the NanoLuc-mScarlet BRET pair provided a large spectral separation between luminescence emission channels that is compatible with green and red filter sets extensively used in typical biological microscopes and animal imaging systems. Using this new sensor, we showed that the BRET ratio was independent of luminescence intensity decay and sensor expression level, and the BRET ratio faithfully reported differences in live-cell energy metabolism whether in culture or within mouse tissue. In particular, BRET analyte sensors have not been used broadly in tissue contexts, and thus, in principle, our sensor could provide a new tool for in vivo imaging of metabolic status.

Differential zinc permeation and blockade of L-type Ca2+ channel isoforms Cav1.2 and Cav1.3.

Certain voltage-activated Ca2+ channels have been reported to act as potential zinc entry routes. However, it remains to be determined whether zinc can permeate individual Ca2+ channel isoforms. We expressed recombinant Ca2+ channel isoforms in Xenopus oocytes and attempted to record zinc currents from them using a two-electrode voltage clamp method. We found that, in an extracellular zinc solution, inward currents arising from zinc permeation could be recorded from Xenopus oocytes expressing L-type Cav1.2 or Cav1.3 isoforms, but not from oocytes expressing Cav2.2, Cav2.3, Cav3.1, or Cav3.2. Zinc currents through Cav1.2 and Cav1.3 were blocked by nimodipine, but enhanced by (±)Bay K8644, supporting the finding that zinc can permeate both L-type Cav1.2 and Cav1.3 channel isoforms. We also examined the blocking effects of low concentrations of zinc on Ca2+ currents through the L-type channel isoforms. Low micro-molar zinc potently blocked Ca2+ currents through Cav1.2 and Cav1.3 with different sensitivities (IC50 for Cav1.2 and Cav1.3=18.4 and 34.1 µM) and de-accelerated the activation and inactivation kinetics in a concentration-dependent manner. Notably, mild acidifications of the external zinc solution increased zinc currents through Cav1.2 and Cav1.3, with the increment level for Cav1.3 being greater than that for Cav1.2. In overall, we provide evidence that Cav1.2 and Cav1.3 isoforms are capable of potentially functioning as zinc permeation routes, through which zinc entry can be differentially augmented by mild acidifications.

Asn-Linked Glycosylation Contributes to Surface Expression and Voltage-Dependent Gating of Cav1.2 Ca²âº Channel.

The Cav1.2 Ca2+ channel is essential for cardiac and smooth muscle contractility and many physiological functions. We mutated single, double, and quadruple sites of the four potential Asn (N)-glycosylation sites in the rabbit Cav1.2 into Gln (Q) to explore the effects of Nglycosylation. When a single mutant (N124Q, N299Q, N1359Q, or N1410Q) or Cav1.2/WT was expressed in Xenopus oocytes, the biophysical properties of single mutants were not significantly different from Cav1.2/WT. In comparison, the double mutant N124,299Q showed a positive shift in voltage-dependent gating. Furthermore, the quadruple mutant (QM; N124,299,1359,1410Q) showed a positive shift in voltage-dependent gating as well as a reduction of current. We tagged EGFP to the QM, double mutants, and Cav1.2/WT to chase the mechanisms underlying the reduced currents of QM. The surface fluorescence intensity of QM was weaker than that of Cav1.2/WT, suggesting that the reduced current of QM arises from its lower surface expression than Cav1.2/WT. Tunicamycin treatment of oocytes expressing Cav1.2/WT mimicked the effects of the quadruple mutations. These findings suggest that Nglycosylation contributes to the surface expression and voltage-dependent gating of Cav1.2.