Self-assembly of the microtubule-associated protein tau into neurotoxic oligomers, fibrils, and paired helical filaments, and cell-to-cell spreading of these pathological tau species are critical processes underlying the pathogenesis of Alzheimer’s disease and other tauopathies. Modulating the self-assembly process and inhibiting formation and spreading of such toxic species are promising strategies for therapy development. A challenge in investigating tau self-assembly in vitro is that, unlike most amyloidogenic proteins, tau does not aggregate in the absence of posttranslational modifications (PTM), aggregation inducers, or preformed seeds. The most common induction method is addition of polyanions, such as heparin; yet, this artificial system may not represent adequately tau self-assembly in vivo, which is driven by aberrant phosphorylation and other PTMs, potentially leading to in vitro data that do not reflect the behavior of tau and its interaction with modulators in vivo. To tackle these challenges, methods for in vitro phosphorylation of tau to produce aggregation-competent forms recently have been introduced (28784767Proc. Natl. Acad. Sci. U.S.A.201711490809085). However, the oligomerization, seeding, and interaction with assembly modulators of the different forms of tau have not been studied to date. To address these knowledge gaps, we compared here side-by-side the self-assembly and seeding activity of heparin-induced tau with two forms of in vitro phosphorylated tau and tested how the molecular tweezer CLR01, a negatively charged compound, affected these processes. Tau was phosphorylated by incubation either with activated extracellular signal-regulated kinase 2 or with a whole rat brain extract. Seeding activity was measured using a fluorescence-resonance energy transfer-based biosensor-cell method. We also used solution-state NMR to investigate the binding sites of CLR01 on tau and how they were impacted by phosphorylation. Our systematic structure–activity relationship study demonstrates that heparin-induced tau behaves differently from in vitro phosphorylated tau. The aggregation rates of the different forms are distinct as is the intracellular localization of the induced aggregates, which resemble brain-derived tau strains suggesting that heparin-induced tau and in vitro phosphorylated tau have different conformations, properties, and activities. CLR01 inhibits aggregation and seeding of both heparin-induced and in vitro phosphorylated tau dose-dependently, although heparin induction interferes with the interaction between CLR01 and tau.

Parkinson’s disease (PD) affects millions of patients worldwide and is characterized by alpha-synuclein aggregation in dopamine neurons. Molecular tweezers have shown high potential as anti-aggregation agents targeting positively charged residues of proteins undergoing amyloidogenic processes. Here we report that the molecular tweezer CLR01 decreased aggregation and toxicity in induced pluripotent stem cell-derived dopaminergic cultures treated with PD brain protein extracts. In microfluidic devices CLR01 reduced alpha-synuclein aggregation in cell somas when axonal terminals were exposed to alpha-synuclein oligomers. We then tested CLR01 in vivo in a humanized alpha-synuclein overexpressing mouse model; mice treated at 12 months of age when motor defects are mild exhibited an improvement in motor defects and a decreased oligomeric alpha-synuclein burden. Finally, CLR01 reduced alpha-synuclein-associated pathology in mice injected with alpha-synuclein aggregates into the striatum or substantia nigra. Taken together, these results highlight CLR01 as a disease-modifying therapy for PD and support further clinical investigation.

During 2017, the Vancouver Fraser Port Authority's Enhancing Cetacean Habitat and Observation program carried out a two-month voluntary vessel slowdown trial to determine whether slowing to 11 knots was an effective method for reducing underwater radiated vessel noise. The trial was carried out in Haro Strait, British Columbia, in critical habitat of endangered southern resident killer whales. During the trial, vessel noise measurements were collected next to shipping lanes on two hydrophones inside the Haro Strait slowdown zone, while a third hydrophone in Strait of Georgia measured vessels noise outside the slowdown zone. Vessel movements were tracked using the automated identification system (AIS), and vessel pilots logged slowdown participation information for each transit. An automated data processing system analyzed acoustical and AIS data from the three hydrophone stations to calculate radiated noise levels and monopole source levels (SLs) of passing vessels. Comparing measurements of vessels participating in the trial with measurements from control periods before and after the trial showed that slowing down was an effective method for reducing mean broadband SLs for five categories of piloted commercial vessels: containerships (11.5 dB), cruise vessels (10.5 dB), vehicle carriers (9.3 dB), tankers (6.1 dB), and bulkers (5.9 dB).

A voluntary commercial vessel slowdown trial was conducted through 16 nm of shipping lanes overlapping critical habitat of at-risk southern resident killer whales (SRKW) in the Salish Sea. From August 7 to October 6, 2017, the trial requested piloted vessels to slow to 11 knots speed-through-water. Analysis of AIS vessel tracking data showed that 350 of 951 (37%) piloted transits achieved this target speed, 421 of 951 (44%) transits achieved speeds within one knot of this target (i.e., ≤12 knots), and 55% achieved speeds ≤ 13 knots. Slowdown results were compared to 'Baseline' noise of the same region, matched across lunar months. A local hydrophone listening station in Lime Kiln State Park, 2.3 km from the shipping lane, recorded 1.2 dB reductions in median broadband noise (10-100,000 Hz, rms) compared to the Baseline period, despite longer transit. The median reduction was 2.5 dB when filtering only for periods when commercial vessels were within 6 km radius of Lime Kiln. The reductions were highest in the 1st decade band (−3.1 dB, 10-100 Hz) and lowest in the 4th decade band (−0.3 dB reduction, 10-100 kHz). A regional vessel noise model predicted noise for a range of traffic volume and vessel speed scenarios for a 1133 km 2 'Slowdown region' containing the 16 nm of shipping lanes. A temporally and spatially explicit simulation model evaluated the changes in traffic volume and speed on SRKW in their foraging habitat within this Slowdown region. The model tracked the number and magnitude of noise-exposure events that impacted each of 78 (simulated) SRKW across different traffic scenarios. These disturbance metrics were simplified to a cumulative effect termed 'potential lost foraging time' that corresponded to the sum of disturbance events described by assumptions of time that whales could not forage due to noise disturbance. The model predicted that the voluntary Slowdown trial achieved 22% reduction in 'potential lost foraging time' for SRKW, with 40% reductions under 100% 11-knot participation. Slower vessel speeds reduced underwater noise in the Slowdown area despite longer passage times and therefore suggest this is an effective way to benefit SRKW habitat function in the vicinity of shipping lanes.

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