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.

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.

Break and accelerator-The mechanics of Tau (and amyloid) toxicity.

Aggregates of the microtubule-associated protein Tau define more than a dozen primary tauopathies, and together with amyloid-ß, the secondary tauopathy Alzheimer's disease (AD). Historically, Tau has been viewed as executor of amyloid-ß toxicity, with the two molecules working together as "trigger and bullet." Given the two protein's opposing roles in protein translation, we wish to introduce another metaphor, borrowing from the mechanics of a car, with amyloid-ß boosting Tau translation, whereas Tau puts a break on global translation. The underlying studies entail an alternative hypothesis regarding Tau's subcellular accumulation in AD, namely its de novo synthesis in the somatodendritic domain rather than the relocalization from the axon upon dissociation from microtubules. We contest that it may be worth (given Tau's 50th birthday) to revisit some entrenched dogmas about Tau's pathophysiology.

Vascular senescence and leak are features of the early breakdown of the blood-brain barrier in Alzheimer's disease models.

Alzheimer's disease (AD) is an age-related disease, with loss of integrity of the blood-brain barrier (BBB) being an early feature. Cellular senescence is one of the reported nine hallmarks of aging. Here, we show for the first time the presence of senescent cells in the vasculature in AD patients and mouse models of AD. Senescent endothelial cells and pericytes are present in APP/PS1 transgenic mice but not in wild-type littermates at the time of amyloid deposition. In vitro, senescent endothelial cells display altered VE-cadherin expression and loss of cell junction formation and increased permeability. Consistent with this, senescent endothelial cells in APP/PS1 mice are present at areas of vascular leak that have decreased claudin-5 and VE-cadherin expression confirming BBB breakdown. Furthermore, single cell sequencing of endothelial cells from APP/PS1 transgenic mice confirms that adhesion molecule pathways are among the most highly altered pathways in these cells. At the pre-plaque stage, the vasculature shows significant signs of breakdown, with a general loss of VE-cadherin, leakage within the microcirculation, and obvious pericyte perturbation. Although senescent vascular cells were not directly observed at sites of vascular leak, senescent cells were close to the leak area. Thus, we would suggest in AD that there is a progressive induction of senescence in constituents of the neurovascular unit contributing to an increasing loss of vascular integrity. Targeting the vasculature early in AD, either with senolytics or with drugs that improve the integrity of the BBB may be valid therapeutic strategies.

Understanding the biases to sepsis surveillance and quality assurance caused by inaccurate coding in administrative health data.

PURPOSE: Timely and accurate data on the epidemiology of sepsis are essential to inform policy decisions and research priorities. We aimed to investigate the validity of inpatient administrative health data (IAHD) for surveillance and quality assurance of sepsis care. METHODS: We conducted a retrospective validation study in a disproportional stratified random sample of 10,334 inpatient cases of age ≥ 15 years treated in 2015-2017 in ten German hospitals. The accuracy of coding of sepsis and risk factors for mortality in IAHD was assessed compared to reference standard diagnoses obtained by a chart review. Hospital-level risk-adjusted mortality of sepsis as calculated from IAHD information was compared to mortality calculated from chart review information. RESULTS: ICD-coding of sepsis in IAHD showed high positive predictive value (76.9-85.7% depending on sepsis definition), but low sensitivity (26.8-38%), which led to an underestimation of sepsis incidence (1.4% vs. 3.3% for severe sepsis-1). Not naming sepsis in the chart was strongly associated with under-coding of sepsis. The frequency of correctly naming sepsis and ICD-coding of sepsis varied strongly between hospitals (range of sensitivity of naming: 29-71.7%, of ICD-diagnosis: 10.7-58.5%). Risk-adjusted mortality of sepsis per hospital calculated from coding in IAHD showed no substantial correlation to reference standard risk-adjusted mortality (r = 0.09). CONCLUSION: Due to the under-coding of sepsis in IAHD, previous epidemiological studies underestimated the burden of sepsis in Germany. There is a large variability between hospitals in accuracy of diagnosing and coding of sepsis. Therefore, IAHD alone is not suited to assess quality of sepsis care.

Three methods for examining the de novo proteome of microglia using BONCAT bioorthogonal labeling and FUNCAT click chemistry.

Bioorthogonal labeling and click chemistry techniques allow the detailed examination of cellular physiology through tagging and visualizing newly synthesized proteins. Here, we describe three methods applying bioorthogonal non-canonical amino acid tagging and fluorescent non-canonical amino acid tagging to quantify protein synthesis in microglia. We describe steps for cell seeding and labeling. We then detail microscopy, flow cytometry, and Western blotting techniques. These methods can be easily adapted for other cell types to explore cellular physiology in health and disease. For complete details on the use and execution of this protocol, please refer to Evans et al. (2021).1.

ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.

Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3ß decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.

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.

Special issue on "Cytoskeletal proteins in health and neurodegenerative disease: Concepts and methods".

Since 2016, when we compiled a very well-received special issue on "Cytoskeletal Proteins in Health and Neurodegenerative Disease" for Brain Research Bulletin, the field has rapidly evolved, to a large part thanks to the development and maturation of new methods including super-resolution microscopy. Being asked to create a sequel, we therefore decided to keep the main topic, but focus on emerging concepts and novel methods. As before, we compiled nine articles on the role of the neuronal cytoskeleton in both physiological and pathological conditions. Seven of the contributions present current concepts and discuss how cytoskeletal components develop and are maintained throughout a neuron's long lifespan, and also, how they may contribute to physiology and neurodegenerative diseases. Two contributions focus on novel methodological developments and how these techniques can be used to analyze the structure and function of the neuronal cytoskeleton in new ways. The compilation of the articles makes it clear that future approaches must consider the functional relationships between the individual filament systems and the influence different signal transduction mechanisms have on the cytoskeleton and vice versa, in order to adequately explore the causes and consequences of the role of cytoskeletal proteins in health and disease. We hope that this compilation will help in the design of appropriate experiments, aided by new methods, to test critical hypotheses in the field.

Ultrasound as a versatile tool for short- and long-term improvement and monitoring of brain function.

Treating the brain with focused ultrasound (FUS) at low intensities elicits diverse responses in neurons, astroglia, and the extracellular matrix. In combination with intravenously injected microbubbles, FUS also opens the blood-brain barrier (BBB) and facilitates focal drug delivery. However, an incompletely understood cellular specificity and a wide parameter space currently limit the optimal application of FUS in preclinical and human studies. In this perspective, we discuss how different FUS modalities can be utilized to achieve short- and long-term improvements, thereby potentially treating brain disorders. We review the ongoing efforts to determine which parameters induce neuronal inhibition versus activation and how mechanoreceptors and signaling cascades are activated to induce long-term changes, including memory improvements. We suggest that optimal FUS treatments may require different FUS modalities and devices, depending on the targeted brain area or local pathology, and will be greatly enhanced by new techniques for monitoring FUS efficacy.

Transcriptional signature in microglia isolated from an Alzheimer's disease mouse model treated with scanning ultrasound.

Transcranial scanning ultrasound combined with intravenously injected microbubbles (SUS+MB) has been shown to transiently open the blood-brain barrier and reduce the amyloid-ß (Aß) pathology in the APP23 mouse model of Alzheimer's disease (AD). This has been accomplished through the activation of microglial cells; however, their response to the SUS treatment is incompletely understood. Here, wild-type (WT) and APP23 mice were subjected to SUS+MB, using nonsonicated mice as sham controls. After 48 h, the APP23 mice were injected with methoxy-XO4 to label Aß aggregates, followed by microglial isolation into XO4+ and XO4- populations using flow cytometry. Both XO4+ and XO4- cells were subjected to RNA sequencing and transcriptome profiling. The analysis of the microglial cells revealed a clear segregation depending on genotype (AD model vs. WT mice) and Aß internalization (XO4+ vs. XO4- microglia), but interestingly, no differences were found between SUS+MB and sham in WT mice. Differential gene expression analysis in APP23 mice detected 278 genes that were significantly changed by SUS+MB in the XO4+ cells (248 up/30 down) and 242 in XO- cells (225 up/17 down). Pathway analysis highlighted differential expression of genes related to the phagosome pathway and marked upregulation of cell cycle-related transcripts in XO4+ and XO4- microglia isolated from SUS+MB-treated APP23 mice. Together, this highlights the complexity of the microglial response to transcranial ultrasound, with potential applications for the treatment of AD.

Tyrosine phosphatase STEP61 in human dementia and in animal models with amyloid and tau pathology.

Synaptic degeneration is a precursor of synaptic and neuronal loss in neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal dementia with tau pathology (FTD-tau), a group of primary tauopathies. A critical role in this degenerative process is assumed by enzymes such as the kinase Fyn and its counterpart, the phosphatase striatal-enriched tyrosine phosphatase 61 (STEP61). Whereas the role of Fyn has been widely explored, less is known about STEP61 that localises to the postsynaptic density (PSD) of glutamatergic neurons. In dementias, synaptic loss is associated with an increased burden of pathological aggregates. Tau pathology is a hallmark of both AD (together with amyloid-ß deposition) and FTD-tau. Here, we examined STEP61 and its activity in human and animal brain tissue and observed a correlation between STEP61 and disease progression. In early-stage human AD, an initial increase in the level and activity of STEP61 was observed, which decreased with the loss of the synaptic marker PSD-95; in FTD-tau, there was a reduction in STEP61 and PSD-95 which correlated with clinical diagnosis. In APP23 mice with an amyloid-ß pathology, the level and activity of STEP61 were increased in the synaptic fraction compared to wild-type littermates. Similarly, in the K3 mouse model of FTD-tau, which we assessed at two ages compared to wild-type, expression and activity of STEP61 were increased with ageing. Together, these findings suggest that STEP contributes differently to the pathogenic process in AD and FTD-tau, and that its activation may be an early response to a degenerative process.

CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau.

The aggregation of the microtubule-associated protein tau is a defining feature of Alzheimer's disease and other tauopathies. Tau pathology is believed to be driven by free tau aggregates and tau carried within exosome-like extracellular vesicles, both of which propagate trans-synaptically and induce tau pathology in recipient neurons by a corrupting process of seeding. Here, we performed a genome-wide CRISPRi screen in tau biosensor cells and identified cellular regulators shared by both mechanisms of tau seeding. We identified ANKLE2, BANF1, NUSAP1, EIF1AD, and VPS18 as the top validated regulators that restrict tau aggregation initiated by both exosomal and vesicle-free tau seeds. None of our validated hits affected the uptake of either form of tau seeds, supporting the notion that they operate through a cell-autonomous mechanism downstream of the seed uptake. Lastly, validation studies with human brain tissue also revealed that several of the identified protein hits are down-regulated in the brains of Alzheimer's patients, suggesting that their decreased activity may be required for the emergence or progression of tau pathology in the human brain.

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.

A sporadic Alzheimer's blood-brain barrier model for developing ultrasound-mediated delivery of Aducanumab and anti-Tau antibodies.

Rationale: The blood-brain barrier (BBB) is a major impediment to therapeutic intracranial drug delivery for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD). Focused ultrasound applied together with microbubbles (FUS+MB) is a novel technique to transiently open the BBB and increase drug delivery. Evidence suggests that FUS+MB is safe, however, the effects of FUS+MB on human BBB cells, especially in the context of AD, remain sparsely investigated. In addition, there currently are no cell platforms to test for FUS+MB-mediated drug delivery. Methods: Here we generated BBB cells (induced brain endothelial-like cells (iBECs) and astrocytes (iAstrocytes)) from apolipoprotein E gene allele E4 (APOE4, high sporadic AD risk) and allele E3 (APOE3, lower AD risk) carrying patient-derived induced pluripotent stem cells (iPSCs). We established mono- and co-culture models of human sporadic AD and control BBB cells to investigate the effects of FUS+MB on BBB cell phenotype and to screen for the delivery of two potentially therapeutic AD antibodies, an Aducanumab-analogue (AduhelmTM; anti-amyloid-ß) and a novel anti-Tau antibody, RNF5. We then developed a novel hydrogel-based 2.5D BBB model as a step towards a more physiologically relevant FUS+MB drug delivery platform. Results: When compared to untreated cells, the delivery of Aducanumab-analogue and RNF5 was significantly increased (up to 1.73 fold), across the Transwell-based BBB models following FUS+MB treatment. Our results also demonstrated the safety of FUS+MB indicated by minimal changes in iBEC transcriptome as well as little or no changes in iBEC or iAstrocyte viability and inflammatory responses within the first 24 h post FUS+MB. Furthermore, we demonstrated successful iBEC barrier formation in our novel 2.5D hydrogel-based BBB model with significantly increased delivery (1.4 fold) of Aducanumab-analogue following FUS+MB. Conclusion: Our results demonstrate a robust and reproducible approach to utilize patient cells for FUS+MB-mediated drug delivery screening in vitro. With such a cell platform for FUS+MB research previously not reported, it has the potential to identify novel FUS+MB-deliverable drugs as well as screen for cell- and patient-specific effects of FUS+MB, accelerating the use of FUS+MB as a therapeutic modality in AD.

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 delivery of novel tau-specific monoclonal antibody enhances brain uptake but not therapeutic efficacy.

Tau-specific immunotherapy is an attractive strategy for the treatment of Alzheimer's disease and other tauopathies. However, effectively targeting tau in the brain remains a considerable challenge due to the restrictive nature of the blood-brain barrier (BBB), which excludes an estimated >99% of peripherally administered antibodies. However, their transport across the BBB can be facilitated by a novel modality, low-intensity scanning ultrasound used in combination with intravenously injected microbubbles (SUS+MB). We have previously shown that SUS+MB-mediated delivery of a tau-specific antibody in a single-chain (scFv) format to tau transgenic mice enhanced brain and neuronal uptake and subsequently, reduced tau pathology and improved behavioural outcomes to a larger extent than either scFv or SUS+MB on its own. Here we generated a novel tau-specific monoclonal antibody, RNF5, and validated it in its IgG format in the presence or absence of SUS+MB by treating K369I tau transgenic K3 mice once weekly for 12 weeks. We found that both RNF5 and SUS+MB treatments on their own significantly reduced tau pathology. In the combination group (RNF5 + SUS+MB), however, despite increased antibody localization in the brain, there were no further reductions in tau pathology when compared to RNF5 treatment alone. Furthermore, following SUS+MB, RNF5 accumulated heavily within cells across the pyramidal cell layer of the hippocampus, that were negative for MAP2 and p-tau, suggesting that SUS+MB may not facilitate enhanced RNF5 engagement of intraneuronal tau. Overall, our new findings reveal the complexities of combining tau immunotherapy with SUS+MB and challenge the view that this is a straight-forward approach.

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.