Improving Cognition Without Clearing Amyloid: Effects of Tau and Ultrasound Neuromodulation.

Alzheimer's disease is characterized by progressive impairment of neuronal functions culminating in neuronal loss and dementia. A universal feature of dementia is protein aggregation, a process by which a monomer forms intermediate oligomeric assembly states and filaments that develop into end-stage hallmark lesions. In Alzheimer's disease, this is exemplified by extracellular amyloid-ß (Aß) plaques which have been placed upstream of tau, found in intracellular neurofibrillary tangles and dystrophic neurites. This implies causality that can be modeled as a linear activation cascade. When Aß load is reduced, for example, in response to an anti-Aß immunotherapy, cognitive functions improve in plaque-forming mice. They also deteriorate less in clinical trial cohorts although real-world clinical benefits remain to be demonstrated. Given the existence of aged humans with unimpaired cognition despite a high plaque load, the central role of Aß has been challenged. A counter argument has been that clinical symptoms would eventually develop if these aged individuals were to live long enough. Alternatively, intrinsic mechanisms that protect the brain in the presence of pathology may exist. In fact, Aß toxicity can be abolished by either reducing or manipulating tau (through which Aß signals), at least in preclinical models. In addition to manipulating steps in this linear pathocascade model, mechanisms of restoring brain reserve can also counteract Aß toxicity. Low-intensity ultrasound is a neuromodulatory modality that can improve cognitive functions in Aß-depositing mice without the need for removing Aß. Together, this highlights a dissociation of Aß and cognition, with important implications for therapeutic interventions.

Multimodal evaluation of blood-brain barrier opening in mice in response to low-intensity ultrasound and a claudin-5 binder.

Background: The blood-brain barrier (BBB) is a major bottleneck in delivering therapeutics to the brain. Treatment strategies to transiently open this barrier include focused ultrasound combined with intravenously injected microbubbles (FUS+MB) and targeting of molecules that regulate BBB permeability. Methods: Here, we investigated BBB opening mediated by the claudin-5 binder cCPEm (a microorganismal toxin in a truncated form) and FUS+MB at a centre frequency of 1 MHz, assessing dextran uptake, broadband emission, and endogenous immunoglobulin G (IgG) extravasation. Results: FUS+MB-induced BBB opening was detectable at a pressure ≥0.35 MPa when assessed for leakage of 10 and 70 kDa dextran, and at ≥0.2 MPa for uptake of endogenous IgG. Treating mice with 20 mg/kg cCPEm failed to open the BBB, and pre-treatment with cCPEm followed by FUS+MB at 0.2 and 0.3 MPa did not overtly increase BBB opening compared to FUS+MB alone. Using passive cavitation detection (PCD), we found that broadband emission correlated with the peak negative pressure (PNP) and dextran leakage, indicating the possibility of using broadband emission for developing a feedback controller to monitor BBB opening. Conclusions: Together, our study highlights the challenges in developing combinatorial approaches to open the BBB and presents an additional IgG-based histological detection method for BBB opening.

Proteostasis as a fundamental principle of Tau immunotherapy.

The microtubule-associated protein Tau is a driver of neuronal dysfunction in Alzheimer's disease and other tauopathies. In this process, Tau initially undergoes subtle changes to its abundance, subcellular localisation and a vast array of post-translational modifications including phosphorylation, that progressively result in the protein's somatodendritic accumulation and dysregulation of multiple Tau-dependent cellular processes. Given the various loss- and gain-of-functions of Tau in disease and the brain-wide changes in the proteome that characterise tauopathies, we asked whether targeting Tau would restore the alterations in proteostasis observed in disease. Therefore, by phage display, we generated a novel pan-Tau antibody, RNJ1, that preferentially binds human Tau and neutralises proteopathic seeding activity in multiple cell lines, and benchmarked it against a clinically tested pan-Tau antibody, HJ8.5 (murine version of tilavonemab). We then evaluated both antibodies, alone and in combination, in the K3 tauopathy mouse model, showing reduced Tau pathology and improvements in neuronal function following 14 weekly treatments, without obtaining synergy for the combination. These effects were more pronounced in female mice. To investigate the molecular mechanisms contributing to improvements in neuronal function, we employed quantitative proteomics, phosphoproteomics and kinase prediction analysis to first establish alterations in K3 mice relative to WT controls at the proteome level. In female K3 mice, we found 342 differentially abundant proteins, which are predominantly involved in metabolic and microtubule-associated processes, strengthening previously reported findings of defects in several functional domains in multiple tauopathy models. We next asked whether antibody-mediated Tau target engagement indirectly affects levels of deregulated proteins in the K3 model. Importantly, both immunotherapies, in particular RNJ1, induced abundance shifts towards a restoration to wild-type levels (proteostasis). A total of 257 of 342 (∼75%) proteins altered in K3 were closer in abundance to WT levels after RNJ1 treatment, and 73% after HJ8.5 treatment. However, the magnitude of these changes was less pronounced than that observed with RNJ1, as reflected by a far smaller number of differentially abundant proteins. Furthermore, analysis of the phosphoproteome showed an even stronger restoration effect with RNJ1, with ∼82% of altered phosphopeptides in K3 showing a shift to WT levels, and 75% with HJ8.5. Gene set over-representation analysis (ORA) further confirmed that proteins undergoing restoration are involved in biological pathways affected in K3 mice. Together, our study suggests that a Tau immunotherapy-induced restoration of proteostasis links target engagement and treatment efficacy.

Single-molecule imaging of Tau reveals how phosphorylation affects its movement and confinement in living cells.

Tau is a microtubule-associated protein that is regulated by post-translational modifications. The most studied of these modifications is phosphorylation, which affects Tau's aggregation and loss- and gain-of-functions, including the interaction with microtubules, in Alzheimer's disease and primary tauopathies. However, little is known about how Tau's phosphorylation state affects its dynamics and organisation at the single-molecule level. Here, using quantitative single-molecule localisation microscopy, we examined how mimicking or abrogating phosphorylation at 14 disease-associated serine and threonine residues through mutagenesis influences the behaviour of Tau in live Neuro-2a cells. We observed that both pseudohyperphosphorylated Tau (TauE14) and phosphorylation-deficient Tau (TauA14) exhibit a heterogeneous mobility pattern near the plasma membrane. Notably, we found that the mobility of TauE14 molecules was higher than wild-type Tau molecules, while TauA14 molecules displayed lower mobility. Moreover, TauA14 was organised in a filament-like structure resembling cytoskeletal filaments, within which TauA14 exhibited spatial and kinetic heterogeneity. Our study provides a direct visualisation of how the phosphorylation state of Tau affects its spatial and temporal organisation, presumably reflecting the phosphorylation-dependent changes in the interactions between Tau and its partners. We suggest that alterations in Tau dynamics resulting from aberrant changes in phosphorylation could be a critical step in its pathological dysregulation.

Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology.

Aging is a major risk factor for neurodegenerative diseases, and coronavirus disease 2019 (COVID-19) is linked to severe neurological manifestations. Senescent cells contribute to brain aging, but the impact of virus-induced senescence on neuropathologies is unknown. Here we show that senescent cells accumulate in aged human brain organoids and that senolytics reduce age-related inflammation and rejuvenate transcriptomic aging clocks. In postmortem brains of patients with severe COVID-19 we observed increased senescent cell accumulation compared with age-matched controls. Exposure of human brain organoids to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced cellular senescence, and transcriptomic analysis revealed a unique SARS-CoV-2 inflammatory signature. Senolytic treatment of infected brain organoids blocked viral replication and prevented senescence in distinct neuronal populations. In human-ACE2-overexpressing mice, senolytics improved COVID-19 clinical outcomes, promoted dopaminergic neuron survival and alleviated viral and proinflammatory gene expression. Collectively our results demonstrate an important role for cellular senescence in driving brain aging and SARS-CoV-2-induced neuropathology, and a therapeutic benefit of senolytic treatments.

Modelling how increased Cathepsin B/L and decreased TMPRSS2 usage for cell entry by the SARS-CoV-2 Omicron variant may affect the efficacy and synergy of TMPRSS2 and Cathepsin B/L inhibitors.

The SARS-CoV-2 Omicron variant harbours many mutations in its spike protein compared to the original SARS-CoV-2 strain, which may alter its ability to enter cells, cell tropism, and response to interventions blocking virus entry. To elucidate these effects, we developed a mathematical model of SARS-CoV-2 entry into target cells and applied it to analyse recent in vitro data. SARS-CoV-2 can enter cells via two pathways, one using the host proteases Cathepsin B/L and the other using the host protease TMPRSS2. We found enhanced entry efficiency of the Omicron variant in cells where the original strain preferentially used Cathepsin B/L and reduced efficiency where it used TMPRSS2. The Omicron variant thus appears to have evolved to use the Cathepsin B/L pathway better but at the expense of its ability to use the TMPRSS2 pathway compared to the original strain. We estimated >4-fold enhanced efficiency of the Omicron variant in entry via the Cathepsin B/L pathway and >3-fold reduced efficiency via the TMPRSS2 pathway compared to the original or other strains in a cell type-dependent manner. Our model predicted that Cathepsin B/L inhibitors would be more efficacious and TMPRSS2 inhibitors less efficacious in blocking Omicron variant entry into cells than the original strain. Furthermore, model predictions suggested that drugs simultaneously targeting the two pathways would exhibit synergy. The maximum synergy and drug concentrations yielding it would differ for the Omicron variant compared to the original strain. Our findings provide insights into the cell entry mechanisms of the Omicron variant and have implications for intervention targeting these mechanisms.

Clinical relevance of animal models in aging-related dementia research.

Alzheimer's disease (AD) and other, less prevalent dementias are complex age-related disorders that exhibit multiple etiologies. Over the past decades, animal models have provided pathomechanistic insight and evaluated countless therapeutics; however, their value is increasingly being questioned due to the long history of drug failures. In this Perspective, we dispute this criticism. First, the utility of the models is limited by their design, as neither the etiology of AD nor whether interventions should occur at a cellular or network level is fully understood. Second, we highlight unmet challenges shared between animals and humans, including impeded drug transport across the blood-brain barrier, limiting effective treatment development. Third, alternative human-derived models also suffer from the limitations mentioned above and can only act as complementary resources. Finally, age being the strongest AD risk factor should be better incorporated into the experimental design, with computational modeling expected to enhance the value of animal models.

Single-molecule imaging reveals Tau trapping at nanometer-sized dynamic hot spots near the plasma membrane that persists after microtubule perturbation and cholesterol depletion.

Accumulation of aggregates of the microtubule-binding protein Tau is a pathological hallmark of Alzheimer's disease. While Tau is thought to primarily associate with microtubules, it also interacts with and localizes to the plasma membrane. However, little is known about how Tau behaves and organizes at the plasma membrane of live cells. Using quantitative, single-molecule imaging, we show that Tau exhibits spatial and kinetic heterogeneity near the plasma membrane of live cells, resulting in the formation of nanometer-sized hot spots. The hot spots lasted tens of seconds, much longer than the short dwell time (∼ 40 ms) of Tau on microtubules. Pharmacological and biochemical disruption of Tau/microtubule interactions did not prevent hot spot formation, suggesting that these are different from the reported Tau condensation on microtubules. Although cholesterol removal has been shown to reduce Tau pathology, its acute depletion did not affect Tau hot spot dynamics. Our study identifies an intrinsic dynamic property of Tau near the plasma membrane that may facilitate the formation of assembly sites for Tau to assume its physiological and pathological functions.

Opportunities and challenges in delivering biologics for Alzheimer's disease by low-intensity ultrasound.

Low-intensity ultrasound combined with intravenously injected microbubbles (US+MB) is a novel treatment modality for brain disorders, including Alzheimer's disease (AD), safely and transiently allowing therapeutic agents to overcome the blood-brain barrier (BBB) that constitutes a major barrier for therapeutic agents. Here, we first provide an update on immunotherapies in AD and how US+MB has been applied to AD mouse models and in clinical trials, considering the ultrasound and microbubble parameter space. In the second half of the review, we compare different in vitro BBB models and discuss strategies for combining US+MB with BBB modulators (targeting molecules such as claudin-5), and highlight the insight provided by super-resolution microscopy. Finally, we conclude with a short discussion on how in vitro findings can inform the design of animal studies, and how the insight gained may aid treatment optimization in the clinical ultrasound space.

Ultrasound-Mediated Bioeffects in Senescent Mice and Alzheimer's Mouse Models.

Ultrasound is routinely used for a wide range of diagnostic imaging applications. However, given that ultrasound can operate over a wide range of parameters that can all be modulated, its applicability extends far beyond the bioimaging field. In fact, the modality has emerged as a hybrid technology that effectively assists drug delivery by transiently opening the blood-brain barrier (BBB) when combined with intravenously injected microbubbles, and facilitates neuromodulation. Studies in aged mice contributed to an insight into how low-intensity ultrasound brings about its neuromodulatory effects, including increased synaptic plasticity and improved cognitive functions, with a potential role for neurogenesis and the modulation of NMDA receptor-mediated neuronal signalling. This work is complemented by studies in mouse models of Alzheimer's disease (AD), a form of pathological ageing. Here, ultrasound was mainly employed as a BBB-opening tool that clears protein aggregates via microglial activation and neuronal autophagy, thereby restoring cognition. We discuss the currently available ultrasound approaches and how studies in senescent mice are relevant for AD and can accelerate the application of low-intensity ultrasound in the clinic.

Mechanistic Models of COVID-19: Insights into Disease Progression, Vaccines, and Therapeutics.

The COVID-19 pandemic has severely impacted health systems and economies worldwide. Significant global efforts are therefore ongoing to improve vaccine efficacies, optimize vaccine deployment, and develop new antiviral therapies to combat the pandemic. Mechanistic viral dynamics and quantitative systems pharmacology models of SARS-CoV-2 infection, vaccines, immunomodulatory agents, and antiviral therapeutics have played a key role in advancing our understanding of SARS-CoV-2 pathogenesis and transmission, the interplay between innate and adaptive immunity to influence the outcomes of infection, effectiveness of treatments, mechanisms and performance of COVID-19 vaccines, and the impact of emerging SARS-CoV-2 variants. Here, we review some of the critical insights provided by these models and discuss the challenges ahead.

Modeling how antibody responses may determine the efficacy of COVID-19 vaccines.

Predicting the efficacy of COVID-19 vaccines would aid vaccine development and usage strategies, which is of importance given their limited supplies. Here we develop a multiscale mathematical model that proposes mechanistic links between COVID-19 vaccine efficacies and the neutralizing antibody (NAb) responses they elicit. We hypothesized that the collection of all NAbs would constitute a shape space and that responses of individuals are random samples from this space. We constructed the shape space by analyzing reported in vitro dose-response curves of ~80 NAbs. Sampling NAb subsets from the space, we recapitulated the responses of convalescent patients. We assumed that vaccination would elicit similar NAb responses. We developed a model of within-host SARS-CoV-2 dynamics, applied it to virtual patient populations and, invoking the NAb responses above, predicted vaccine efficacies. Our predictions quantitatively captured the efficacies from clinical trials. Our study thus suggests plausible mechanistic underpinnings of COVID-19 vaccines and generates testable hypotheses for establishing them.

Increased B Cell Selection Stringency In Germinal Centers Can Explain Improved COVID-19 Vaccine Efficacies With Low Dose Prime or Delayed Boost.

The efficacy of COVID-19 vaccines appears to depend in complex ways on the vaccine dosage and the interval between the prime and boost doses. Unexpectedly, lower dose prime and longer prime-boost intervals have yielded higher efficacies in clinical trials. To elucidate the origins of these effects, we developed a stochastic simulation model of the germinal center (GC) reaction and predicted the antibody responses elicited by different vaccination protocols. The simulations predicted that a lower dose prime could increase the selection stringency in GCs due to reduced antigen availability, resulting in the selection of GC B cells with higher affinities for the target antigen. The boost could relax this selection stringency and allow the expansion of the higher affinity GC B cells selected, improving the overall response. With a longer dosing interval, the decay in the antigen with time following the prime could further increase the selection stringency, amplifying this effect. The effect remained in our simulations even when new GCs following the boost had to be seeded by memory B cells formed following the prime. These predictions offer a plausible explanation of the observed paradoxical effects of dosage and dosing interval on vaccine efficacy. Tuning the selection stringency in the GCs using prime-boost dosages and dosing intervals as handles may help improve vaccine efficacies.

Super-resolution microscopy: a closer look at synaptic dysfunction in Alzheimer disease.

The synapse has emerged as a critical neuronal structure in the degenerative process of Alzheimer disease (AD), in which the pathogenic signals of two key players - amyloid-ß (Aß) and tau - converge, thereby causing synaptic dysfunction and cognitive deficits. The synapse presents a dynamic, confined microenvironment in which to explore how key molecules travel, localize, interact and assume different levels of organizational complexity, thereby affecting neuronal function. However, owing to their small size and the diffraction-limited resolution of conventional light microscopic approaches, investigating synaptic structure and dynamics has been challenging. Super-resolution microscopy (SRM) techniques have overcome the resolution barrier and are revolutionizing our quantitative understanding of biological systems in unprecedented spatio-temporal detail. Here we review critical new insights provided by SRM into the molecular architecture and dynamic organization of the synapse and, in particular, the interactions between Aß and tau in this compartment. We further highlight how SRM can transform our understanding of the molecular pathological mechanisms that underlie AD. The application of SRM for understanding the roles of synapses in AD pathology will provide a stepping stone towards a broader understanding of dysfunction in other subcellular compartments and at cellular and circuit levels in this disease.

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection.

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through 'repurposing' drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.

Modular transient nanoclustering of activated ß2-adrenergic receptors revealed by single-molecule tracking of conformation-specific nanobodies.

None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous ß2-adrenoreceptor (ß2-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated ß2-AR (Nb80) is highly immobile and organized in nanoclusters. The Gαs-GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated ß2-AR (Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.

Radial contractility of actomyosin rings facilitates axonal trafficking and structural stability.

Most mammalian neurons have a narrow axon, which constrains the passage of large cargoes such as autophagosomes that can be larger than the axon diameter. Radial axonal expansion must therefore occur to ensure efficient axonal trafficking. In this study, we reveal that the speed of various large cargoes undergoing axonal transport is significantly slower than that of small ones and that the transit of diverse-sized cargoes causes an acute, albeit transient, axonal radial expansion, which is immediately restored by constitutive axonal contractility. Using live super-resolution microscopy, we demonstrate that actomyosin-II controls axonal radial contractility and local expansion, and that NM-II filaments associate with periodic F-actin rings via their head domains. Pharmacological inhibition of NM-II activity significantly increases axon diameter by detaching the NM-II from F-actin and impacts the trafficking speed, directionality, and overall efficiency of long-range retrograde trafficking. Consequently, prolonged NM-II inactivation leads to disruption of periodic actin rings and formation of focal axonal swellings, a hallmark of axonal degeneration.

Need for speed: Super-resolving the dynamic nanoclustering of syntaxin-1 at exocytic fusion sites.

Communication between cells relies on regulated exocytosis, a multi-step process that involves the docking, priming and fusion of vesicles with the plasma membrane, culminating in the release of neurotransmitters and hormones. Key proteins and lipids involved in exocytosis are subjected to Brownian movement and constantly switch between distinct motion states which are governed by short-lived molecular interactions. Critical biochemical reactions between exocytic proteins that occur in the confinement of nanodomains underpin the precise sequence of priming steps which leads to the fusion of vesicles. The advent of super-resolution microscopy techniques has provided the means to visualize individual molecules on the plasma membrane with high spatiotemporal resolution in live cells. These techniques are revealing a highly dynamic nature of the nanoscale organization of the exocytic machinery. In this review, we focus on soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) syntaxin-1, which mediates vesicular fusion. Syntaxin-1 is highly mobile at the plasma membrane, and its inherent speed allows fast assembly and disassembly of syntaxin-1 nanoclusters which are associated with exocytosis. We reflect on recent studies which have revealed the mechanisms regulating syntaxin-1 nanoclustering on the plasma membrane and draw inferences on the effect of synaptic activity, phosphoinositides, N-ethylmaleimide-sensitive factor (NSF), α-soluble NSF attachment protein (α-SNAP) and SNARE complex assembly on the dynamic nanoscale organization of syntaxin-1. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.