Scanning ultrasound-mediated memory and functional improvements do not require amyloid-ß reduction.

A prevalent view in treating age-dependent disorders including Alzheimer's disease (AD) is that the underlying amyloid plaque pathology must be targeted for cognitive improvements. In contrast, we report here that repeated scanning ultrasound (SUS) treatment at 1 MHz frequency can ameliorate memory deficits in the APP23 mouse model of AD without reducing amyloid-ß (Aß) burden. Different from previous studies that had shown Aß clearance as a consequence of blood-brain barrier (BBB) opening, here, the BBB was not opened as no microbubbles were used. Quantitative SWATH proteomics and functional magnetic resonance imaging revealed that ultrasound induced long-lasting functional changes that correlate with the improvement in memory. Intriguingly, the treatment was more effective at a higher frequency (1 MHz) than at a frequency within the range currently explored in clinical trials in AD patients (286 kHz). Together, our data suggest frequency-dependent bio-effects of ultrasound and a dissociation of cognitive improvement and Aß clearance, with important implications for the design of trials for AD therapies.

Fyn nanoclustering requires switching to an open conformation and is enhanced by FTLD-Tau biomolecular condensates.

Fyn is a Src kinase that controls critical signalling cascades and has been implicated in learning and memory. Postsynaptic enrichment of Fyn underpins synaptotoxicity in dementias such as Alzheimer's disease and frontotemporal lobar degeneration with Tau pathology (FTLD-Tau). The FLTD P301L mutant Tau is associated with a higher propensity to undergo liquid-liquid phase separation (LLPS) and form biomolecular condensates. Expression of P301L mutant Tau promotes aberrant trapping of Fyn in nanoclusters within hippocampal dendrites by an unknown mechanism. Here, we used single-particle tracking photoactivated localisation microscopy to demonstrate that the opening of Fyn into its primed conformation promotes its nanoclustering in dendrites leading to increased Fyn/ERK/S6 downstream signalling. Preventing the auto-inhibitory closed conformation of Fyn through phospho-inhibition or through perturbation of its SH3 domain increased Fyn's nanoscale trapping, whereas inhibition of the catalytic domain had no impact. By combining pharmacological and genetic approaches, we demonstrate that P301L Tau enhanced both Fyn nanoclustering and Fyn/ERK/S6 signalling via its ability to form biomolecular condensates. Together, our findings demonstrate that Fyn alternates between a closed and an open conformation, the latter being enzymatically active and clustered. Furthermore, pathogenic immobilisation of Fyn relies on the ability of P301L Tau to form biomolecular condensates, thus highlighting the critical importance of LLPS in controlling nanoclustering and downstream intracellular signalling events.

Claudin-5 binder enhances focused ultrasound-mediated opening in an in vitro blood-brain barrier model.

Rationale: The blood-brain barrier (BBB) while functioning as a gatekeeper of the brain, impedes cerebral drug delivery. An emerging technology to overcome this limitation is focused ultrasound (FUS). When FUS interacts with intravenously injected microbubbles (FUS+MB), the BBB opens, transiently allowing the access of therapeutic agents into the brain. However, the ultrasound parameters need to be tightly tuned: when the acoustic pressure is too low there is no opening, and when it is too high, tissue damage can occur. We therefore asked whether barrier permeability can be increased by combining FUS+MB with a second modality such that in a clinical setting lower acoustic pressures could be used. Methods: Given that FUS+MB achieves BBB opening in part by disruption of tight junction (TJ) proteins such as claudin-5 of brain endothelial cells, we generated a stable MDCK (Madin-Darby Canine Kidney) II cell line (eGFP-hCldn5-MDCK II) that expresses fluorescently tagged human claudin-5. Two claudin-5 binders, the peptide mC5C2 and cCPEm (truncated form of an enterotoxin), reported previously to weaken the barrier, were synthesized and assessed for their abilities to enhance the permeability of cellular monolayers. We then performed a comparative analysis of single and combination treatments, measuring transendothelial electrical resistance (TEER) and cargo leakage, combined with confocal image analysis. Results: We successfully generated a novel cell line that formed functional monolayers as validated by an increased TEER reading and a low (< 0.2%) permeability to sodium fluorescein (376 Da). We found that the binders exerted a time- and concentration-dependent effect on barrier opening when incubated over an extended period, whereas FUS+MB caused a rapid opening followed by recovery after 12 hours within the tested pressure range. Importantly, preincubation with cCPEm prior to FUS+MB treatment resulted in greater barrier opening compared to either FUS+MB or cCPEm alone as measured by reduced TEER values and an increased permeability to fluorescently labelled 40 kDa dextran (FD40). Conclusion: The data suggest that pre incubation with clinically suitable binders to TJ proteins may be a general strategy to facilitate safer and more effective ultrasound-mediated BBB opening in cellular and animal systems and potentially also for the treatment of human diseases of the brain.

Low-intensity ultrasound restores long-term potentiation and memory in senescent mice through pleiotropic mechanisms including NMDAR signaling.

Advanced physiological aging is associated with impaired cognitive performance and the inability to induce long-term potentiation (LTP), an electrophysiological correlate of memory. Here, we demonstrate in the physiologically aged, senescent mouse brain that scanning ultrasound combined with microbubbles (SUS+MB), by transiently opening the blood-brain barrier, fully restores LTP induction in the dentate gyrus of the hippocampus. Intriguingly, SUS treatment without microbubbles (SUSonly), i.e., without the uptake of blood-borne factors, proved even more effective, not only restoring LTP, but also ameliorating the spatial learning deficits of the aged mice. This functional improvement is accompanied by an altered milieu of the aged hippocampus, including a lower density of perineuronal nets, increased neurogenesis, and synaptic signaling, which collectively results in improved spatial learning. We therefore conclude that therapeutic ultrasound is a non-invasive, pleiotropic modality that may enhance cognition in elderly humans.

Role for caveolin-mediated transcytosis in facilitating transport of large cargoes into the brain via ultrasound.

The blood-brain barrier (BBB) is a dynamic diffusional barrier regulating the molecular and chemical flux between the blood and brain, thereby preserving cerebral homeostasis. Endothelial cells form the core anatomical component of the BBB based on properties such as specialized junctional complexes between cells, which restricts paracellular transport, and extremely low levels of vesicular transport, restricting transcytosis. In performing its protective function, the BBB also constrains the entry of therapeutics into the brain, hampering the treatment of various neurological disorders. Focused ultrasound is a novel therapeutic modality that has shown efficacy in transiently and non-invasively opening the BBB for the targeted delivery of therapeutics to the brain. Although the ability of ultrasound to disrupt the junctional assembly of endothelial cells has been partially investigated, its effect on the transcellular mode of transport has been largely neglected. In this study, we found that ultrasound induces a pronounced increase in the levels of the vesicle-forming protein caveolin-1. In order to investigate the role of vesicle-mediated transcytoplasmic transport, we compared the leakage of various cargo sizes between a mouse model that lacks caveolin-1 and wild-type mice following sonication of the hippocampus. The absence of caveolin-1 did not lead to overt abnormalities in the cerebral vasculature in the mice. We found that caveolin-1 has a critical role specifically in the transport of large (500 kDa), but not smaller (3 and 70 kDa) cargoes. Our findings indicate differential effects of therapeutic ultrasound on cellular transport mechanisms, with implications for therapeutic interventions.