Modifications of the tau protein and their varied effects on aggregation and function

Tau is a microtubule-associated protein that is typically found in the axons of neurons. Six isoforms of the protein can be generated through alternative mRNA splicing and all are found in the adult brain. The protein is closely associated with a group of neurodegenerative diseases, including Alzheimer's disease, collectively known as tauopathies. In these diseases tau dissociates from microtubules and begins to polymerize into aggregates that are typically fibrillary in nature. The deposition of these aggregates is closely linked to the death and dysfunction of neurons and eventually leads to atrophy of specific regions of the brain. Tauopathies display a wide variety of pathologies that distinguish them from each other, including aggregate morphology and isoform inclusion. Differential conformational changes in pathological forms of the protein may affect its propensity for aggregation and function. Hyperphosphorylation of tau or inherited mutations, known as FTDP-17 mutations, may induce these conformational changes and alter aggregation and function. The studies described here used in vitro assays to determine how hyperphosphorylation affects each of the tau isoforms and how various FTDP-17 mutations can alter aggregation and function. This information helps to describe how intrinsic differences due to modifications of tau can manifest themselves in the varying pathologies of tauopathies. This can be applied to C. elegans models of tauopathies to determine the effects in living neurons. The results demonstrate that similar phosphorylation patterns in tau can result in very different effects on the protein's aggregation and ability to stabilize microtubule polymerization depending on the isoform background. This suggests that phosphorylation pattern is sufficient to induce differential aggregation and may affect the morphology and isoform contents of aggregates. Similarly, FTDP-17 mutations also induced very different effects on tau aggregation and function. This indicates that these mutations may be affecting disease pathologies in very different manners. Differences between tau isoforms and FTDP-17 mutations are likely affecting the phenotypes seen in animal models of disease and should not be ignored. This is being tested by developing several unique C. elegans models of tauopathies

FTDP-17 tau mutations induce distinct effects on aggregation and microtubule interactions

FTDP-17 mutations in the tau gene lead to early-onset frontotemporal dementias characterized by the pathological aggregation of the microtubule-associated protein tau. Tau aggregation is closely correlated with the progression and severity of localized atrophy of certain regions in the brain. These mutations are primarily located in or near the microtubule-binding repeat regions of tau and can have vastly different effects on the protein. Some mutations have been linked to effects such as increased aggregation, hyperphosphorylation, defects in mRNA splicing, and decreased interaction with microtubules. Given the differential effects of the mutations it may not be surprising that the pathology associated with FTDP-17 can vary widely as well. Despite this variety, several of the mutations are commonly used interchangeably as aggregation inducers for in vitro and in vivo models of tauopathies. We generated recombinant forms of 12 FTDP-17 mutations chosen for their predicted effects on the charge, hydrophobicity, and secondary structure of the protein. We then examined the effects that the mutations had on the properties of in vitro aggregation of the protein and its ability to stabilize microtubule assembly. The group of mutations induced very different effects on the total amount of aggregation, the kinetics of aggregation, and filament morphology. Several of the mutations inhibited the microtubule-stabilization ability of tau while others had very little effect compared to wild-type tau. These results indicate that the mechanisms of disease progression may differ among FTDP-17 mutations and that the effects of the varying mutations may not be equal in all model systems

Feasibility of Long Range Wide Area Network (LoRaWAN) Technology for LEO Applications

Long Range (LoRa) is a widely used technology for terrestrial communications and is increasingly utilized for space related applications. On the ground, the LoRaWAN protocol allows gateways connected to the internet to serve as transceivers for any eligible end device in range of LoRa modulated signal. Certified gateways are produced by a variety of manufacturers with a range of capabilities, and publicly available networks contain thousands of gateways distributed around the world. Now, we propose to utilize the worldwide network of LoRaWAN gateways as a distributed ground station network capable of significantly reducing delays in link availability for a satellite in LEO. Using a COTS transceiver based on the LoRaWAN protocol, a module will be developed that functions as an end device in network architecture. As a half-duplex communication subsystem it will be capable of sending small packages of selective telemetry and receiving specific commands for essential functions, and will have worldwide compatibility in the range of 868 MHz to 915 MHz. Currently the module is planned to function as the secondary communication subsystem on the Cosmic X-Ray Background NanoSat-3 (CXBN-3) satellite, a 2U CubeSat mission under construction by students at Morehead State University to explore the diffuse emission of hard X-rays in the Cosmic X-Ray Background (CXB). This paper will describe the feasibility of LoRaWAN technology for LEO applications through the evaluation of a LoRaWAN space-based end device, its development based on a COTS transceiver, and validation testing to simulate LEO range by using LoRaWAN commercial gateways

Hsp70 alters tau function and aggregation in an isoform specific manner

Tauopathies are characterized by abnormal aggregation of the microtubule associated protein tau. This aggregation is thought to occur when tau undergoes shifts from its native conformation to one that exposes hydrophobic areas on separate monomers, allowing contact and subsequent association into oligomers and filaments. Molecular chaperones normally function by binding to exposed hydrophobic stretches on proteins and assisting in their refolding. Chaperones of the heat shock protein 70 (Hsp70) family have been implicated in the prevention of abnormal tau aggregation in adult neurons. Tau exists as six alternatively spliced isoforms, and all six isoforms appear capable of forming the pathological aggregates seen in Alzheimer's disease. Because tau isoforms differ in primary sequence, we sought to determine whether Hsp70 would differentially affect the aggregation and microtubule assembly characteristics of the various tau isoforms. We found that Hsp70 inhibits tau aggregation directly, and not through inducer mediated effects. We also determined that Hsp70 inhibits the aggregation of each individual tau isoform and was more effective at inhibiting the three repeat isoforms. . Finally, all tau isoforms robustly induced microtubule formation while in the presence of Hsp70. The results presented herein indicate that Hsp70 affects tau isoform dysfunction while having very little impact on the normal function of tau to mediate microtubule assembly. This indicates that targeting Hsp70 to tau may provide a therapeutic approach for the treatment of tauopathies that avoids disruption of normal tau function

Analysis of isoform-specific tau aggregates suggests a common toxic mechanism involving similar pathological conformations and axonal transport inhibition

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Neurobiology of Aging 47 (2016): 113–126, doi:10.1016/j.neurobiolaging.2016.07.015.Misfolded tau proteins are characteristic of tauopathies, but the isoform composition of tau inclusions varies by tauopathy. Using aggregates of the longest tau isoform (containing 4 microtubule-binding repeats and 4-repeat tau), we recently described a direct mechanism of toxicity that involves exposure of the N-terminal phosphatase-activating domain (PAD) in tau, which triggers a signaling pathway that disrupts axonal transport. However, the impact of aggregation on PAD exposure for other tau isoforms was unexplored. Here, results from immunochemical assays indicate that aggregation-induced increases in PAD exposure and oligomerization are common features among all tau isoforms. The extent of PAD exposure and oligomerization was larger for tau aggregates composed of 4-repeat isoforms compared with those made of 3-repeat isoforms. Most important, aggregates of all isoforms exhibited enough PAD exposure to significantly impair axonal transport in the squid axoplasm. We also show that PAD exposure and oligomerization represent common pathological characteristics in multiple tauopathies. Collectively, these results suggest a mechanism of toxicity common to each tau isoform that likely contributes to degeneration in different tauopathies.This work was supported by NIH grants R01 AG044372 (Nicholas M. Kanaan), R01 NS082730 (Nicholas M. Kanaan and Scott T. Brady), BrightFocus Foundation (A2013364S, Nicholas M. Kanaan), the Jean P. Schultz Biomedical Research Endowment (Nicholas M. Kanaan), the Secchia Family Foundation (Nicholas M. Kanaan) and NS066942A (Gerardo Morfini)

Intense high-quality medical proton beams via laser fields

During the past decade, the interaction of high-intensity lasers with solid targets has attracted much interest, regarding its potential in accelerating charged particles. In spite of tremendous progress in laser-plasma based acceleration, it is still not clear which particle beam quality will be accessible within the upcoming multi petawatt (1 PW = 10$^{15}$ W) laser generation. Here, we show with simulations based on the coupled relativistic equations of motion that protons stemming from laser-plasma processes can be efficiently post-accelerated using crossed laser beams focused to spot radii of a few laser wavelengths. We demonstrate that the crossed beams produce monoenergetic accelerated protons with kinetic energies $> 200$ MeV, small energy spreads ($\approx$ 1$$) and high densities as required for hadron cancer therapy. To our knowledge, this is the first scheme allowing for this important application based on an all-optical set-up.Comment: 14 pages, 3 figures, 1 tabl

Tau: a signaling hub protein

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Mueller, R. L., Combs, B., Alhadidy, M. M., Brady, S. T., Morfini, G. A., & Kanaan, N. M. Tau: a signaling hub protein. Frontiers in Molecular Neuroscience, 14, (2021): 647054, https://doi.org/10.3389/fnmol.2021.647054.Over four decades ago, in vitro experiments showed that tau protein interacts with and stabilizes microtubules in a phosphorylation-dependent manner. This observation fueled the widespread hypotheses that these properties extend to living neurons and that reduced stability of microtubules represents a major disease-driving event induced by pathological forms of tau in Alzheimer’s disease and other tauopathies. Accordingly, most research efforts to date have addressed this protein as a substrate, focusing on evaluating how specific mutations, phosphorylation, and other post-translational modifications impact its microtubule-binding and stabilizing properties. In contrast, fewer efforts were made to illuminate potential mechanisms linking physiological and disease-related forms of tau to the normal and pathological regulation of kinases and phosphatases. Here, we discuss published work indicating that, through interactions with various kinases and phosphatases, tau may normally act as a scaffolding protein to regulate phosphorylation-based signaling pathways. Expanding on this concept, we also review experimental evidence linking disease-related tau species to the misregulation of these pathways. Collectively, the available evidence supports the participation of tau in multiple cellular processes sustaining neuronal and glial function through various mechanisms involving the scaffolding and regulation of selected kinases and phosphatases at discrete subcellular compartments. The notion that the repertoire of tau functions includes a role as a signaling hub should widen our interpretation of experimental results and increase our understanding of tau biology in normal and disease conditions.This work was supported by NIH grants (R01AG067762 and R01AG044372 to NK, R01NS082730 to NK and SB, R01NS118177 and R21NS120126 to GM, R01NS023868 and R01NS041170 to SB), a gift from Neurodegenerative Research Inc. (GM), a Zenith Award from the Alzheimer’s Association (SB), a grant from the Secchia Family Foundation (NK), NIH/National Institute on Aging (NIA) funded Michigan Alzheimer’s Disease Research Center 5P30AG053760 (BC), the Office of the Assistant Secretary of Defense for Health Affairs through the Peer-Reviewed Alzheimer’s Research Program (Award No. W81XWH-20-1-0174 to BC), and an Alzheimer’s Association Research Grant 20-682085 (BC)

Pseudophosphorylation of tau at S422 enhances SDS-stable dimer formation and impairs both anterograde and retrograde fast axonal transport

AbstractIn Alzheimer's disease (AD), tau undergoes numerous modifications, including increased phosphorylation at serine-422 (pS422). In the human brain, pS422 tau protein is found in prodromal AD, correlates well with cognitive decline and neuropil thread pathology, and appears associated with increased oligomer formation and exposure of the N-terminal phosphatase-activating domain (PAD). However, whether S422 phosphorylation contributes to toxic mechanisms associated with disease-related forms of tau remains unknown. Here, we report that S422-pseudophosphorylated tau (S422E) lengthens the nucleation phase of aggregation without altering the extent of aggregation or the types of aggregates formed. When compared to unmodified tau aggregates, the S422E modification significantly increased the amount of SDS-stable tau dimers, despite similar levels of immunoreactivity with an oligomer-selective antibody (TOC1) and another antibody that reports PAD exposure (TNT1). Vesicle motility assays in isolated squid axoplasm further revealed that S422E tau monomers inhibited anterograde, kinesin-1 dependent fast axonal transport (FAT). Unexpectedly, and unlike unmodified tau aggregates, which selectively inhibit anterograde FAT, aggregates composed of S422E tau were found to inhibit both anterograde and retrograde FAT. Highlighting the relevance of these findings to human disease, pS422 tau was found to colocalize with tau oligomers and with a fraction of tau showing increased PAD exposure in the human AD brain. This study identifies novel effects of pS422 on tau biochemical properties, including prolonged nucleation and enhanced dimer formation, which correlate with a distinct inhibitory effect on FAT. Taken together, these findings identify a novel mechanistic basis by which pS422 confers upon tau a toxic effect that may directly contribute to axonal dysfunction in AD and other tauopathies

Secondary nucleating sequences affect kinetics and thermodynamics of tau aggregation

Tau protein was scanned for highly amyloidogenic sequences in amphiphilic motifs (X)nZ, Z(X)nZ (n≥2) or (XZ)n (n≥2), where X is a hydrophobic residue and Z is a charged or polar residue. N-acetyl peptides homologous to these sequences were used to study aggregation. Transmission electron microscopy (TEM) showed 7 peptides, in addition to well known primary nucleating sequences c275VQIINK (AcPHF6*) and Ac306VQIVYK (AcPHF6), formed fibers, tubes, ribbons or rolled sheets. Of the peptides shown by TEM to form amyloid, Ac10VME, AcPHF6*, Ac375KLTFR, and Ac393VYK were found to enhance the fraction of β-structure of AcPHF6 formed at equilibrium, and Ac375KLTFR was found to inhibit AcPHF6 and AcPHF6* aggregation kinetics in a dose-dependent manner, consistent with its participation in a hybrid steric zipper model. Single site mutants were generated which transformed predicted amyloidogenic sequences in tau into non-amyloidogenic ones. A M11K mutant had fewer filaments and showed a decrease in aggregation kinetics and an increased lag time compared to wild type tau, while a F378K mutant showed significantly more filaments. Our results infer that sequences throughout tau, in addition to PHF6 and PHF6*, can seed amyloid formation or affect aggregation kinetics or thermodynamics