Pathologic tau conformer ensembles induce dynamic, liquid-liquid phase separation events at the nuclear envelope.

BACKGROUND: The microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes. RESULTS: As starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 µm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions. CONCLUSIONS: Tau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

Evidence that Memantine Reduces Chronic Tinnitus Caused by Acoustic Trauma in Rats.

Subjective tinnitus is a chronic neurological disorder in which phantom sounds are perceived. Increasing evidence suggests that tinnitus is caused by neuronal hyperactivity in auditory brain regions, either due to a decrease in synaptic inhibition or an increase in synaptic excitation. One drug investigated for the treatment of tinnitus has been the uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, memantine, although the evidence relating to it has been unconvincing to date. We re-investigated the effects of memantine on the behavioral manifestations of tinnitus induced by acoustic trauma (a 16-kHz, 110-dB pure tone presented unilaterally for 1 h) in rats. We used a conditioned lick suppression model in which lick suppression was associated with the perception of high frequency sound resembling tinnitus and a suppression ratio (SR) was calculated by comparing the number of licks in the 15-s period preceding the stimulus presentation (A) and the 15-s period during the stimulus presentation (B), i.e., SR = B/(A + B). Acoustic trauma resulted in a significant increase in the auditory brainstem-evoked response (ABR) threshold in the affected ear (P ≤ 0.0001) and a decrease in the SR compared to sham controls in response to 32 kHz tones in five out of eight acoustic trauma-exposed animals. A 5-mg/kg dose of memantine significantly reduced the proportion of these animals which exhibited tinnitus-like behavior (2/5 compared to 5/5; P ≤ 0.006), suggesting that the drug reduced tinnitus. These results suggest that memantine may reduce tinnitus caused by acoustic trauma.

A dose-response analysis of the effects of L-baclofen on chronic tinnitus caused by acoustic trauma in rats.

Subjective tinnitus is a chronic neurological disorder in which phantom sounds are perceived. Drugs that increase GABAergic neurotransmission in the CNS are sometimes used as a treatment. One such drug is the GABA(B) receptor agonist L-baclofen. The aim of this study was to investigate the effects of L-baclofen on the psychophysical attributes of tinnitus in rats.The effects of 1, 3 or 5 mg/kg L-baclofen (s.c.) on the psychophysical attributes of tinnitus were investigated using a conditioned lick suppression model, following acoustic trauma (a 16 kHz, 110 dB pure tone presented unilaterally for 1 h) in rats. In pre-drug testing, acoustic trauma resulted in a significant increase in the auditory brainstem-evoked response (ABR) threshold in the affected ear (P < 0.008) and a significant decrease in the suppression ratio (SR) compared to sham controls in response to the 20 kHz tones, but not the broadband noise or the 10 kHz tones (P < 0.002). The 3 and 5 mg/kg doses of L-baclofen significantly reversed the frequency-specific decrease in the SR in the acoustic trauma group, indicating that the drug reduced tinnitus. Following washout from the 3 mg/kg dose, but not the 5 mg/kg dose, the significant decrease in the SR for the acoustic trauma group returned, suggesting a return of the tinnitus. These results suggest that L-baclofen should be reconsidered as a drug treatment for tinnitus. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.