Focused ultrasound mitigates pathology and improves spatial memory in Alzheimer's mice and patients

Rationale: Bilateral sonication with focused ultrasound (FUS) in conjunction with microbubbles has been shown to separately reduce amyloid plaques and hyperphosphorylated tau protein in the hippocampal formation and the entorhinal cortex in different mouse models of Alzheimer's disease (AD) without any therapeutic agents. However, the two pathologies are expressed concurrently in human disease. Therefore, the objective of this study is to investigate the effects of repeated bilateral sonications in the presence of both pathologies. Methods: Herein, we investigate its functional and morphological outcomes on brains bearing both pathologies simultaneously. Eleven transgenic mice of the 3xTg-AD line (14 months old) expressing human amyloid beta and human tau and eleven age-matched wild-type littermates received four weekly bilateral sonications covering the hippocampus followed by working memory testing. Afterwards, immunohistochemistry and immunoassays (western blot and ELISA) were employed to assess any changes in amyloid beta and human tau. Furthermore, we present preliminary data from our clinical trial using a neuronavigation-guided FUS system for sonications in AD patients (NCT04118764). Results: Interestingly, both wild-type and transgenic animals that received FUS experienced improved working memory and spent significantly more time in the escape platform-quadrant, with wild-type animals spending 43.2% (sham: 37.7%) and transgenic animals spending 35.3% (sham: 31.0%) of the trial in the target quadrant. Furthermore, this behavioral amelioration in the transgenic animals correlated with a 58.3% decrease in the neuronal length affected by tau and a 27.2% reduction in total tau levels. Amyloid plaque population, volume and overall load were also reduced overall. Consistently, preliminary data from a clinical trial involving AD patients showed a 1.8% decrease of amyloid PET signal 3-weeks after treatment in the treated hemisphere compared to baseline. Conclusion: For the first time, it is shown that bilateral FUS-induced BBB opening significantly and simultaneously ameliorates both coexistent pathologies, which translated to improvements in spatial memory of transgenic animals with complex AD, the human mimicking phenotype. The level of cognitive improvement was significantly correlated with the volume of BBB opening. Non-transgenic animals were also shown to exhibit similar memory amelioration for the first time, indicating that BBB opening results into benefits in the neuronal function regardless of the existence of AD pathology. A potential mechanism of action for the reduction of the both pathologies investigated was the cholesterol metabolism, specifically the LRP1b receptor, which exhibited increased expression levels in transgenic mice following FUS-induced BBB opening. Initial clinical evidence supported that the beta amyloid reduction shown in rodents could be translatable to humans with significant amyloid reduction shown in the treated hemisphere

Quantitative analysis of the focused ultrasound-induced blood-brain barrier opening with applications in neurodegenerative disorders

The blood-brain barrier poses a formidable impediment to the treatment of adult-onset neurodegenerative disorders, by prevention of most drugs from gaining access to the brain parenchyma. Focused ultrasound (FUS), in conjunction with systemically administered microbubbles, has been shown to open the blood-brain barrier (BBB) locally, reversibly and non invasively both in rodents and in non-human-primates. Initially, we demonstrate a monotonic increase of the BBB opening volume with close to normal incidence angle, detectable by diffusion tensor imaging; the employed contrast-free magnetic resonance protocol that revealed the anisotropic nature of the diffusion gradient. Implementation of this optimized BBB opening technique in Parkinsonian mice, coupled with the administration of trophic growth factors, induced restorative effects in the dopaminergic neurons, the main cellular target of the pathological process in Parkinson’s disease. The immune response initiated by the FUS-induced BBB disruption has been proven pivotal in reducing proteinaceous aggregates from the brain through the activation of a gliosis cascade. Therefore, we investigated this immunomodulatory effect in Alzheimer’s disease. The neuropathological hallmarks of Alzheimer’s disease include aggregation of amyloid beta into plaques and accumulation of tau protein into neurofibrillary tangles. Tau pathology correlates well with impaired neuronal activity and dementia and was found to be attenuated after the application of ultrasound that correlated with increased microglia activity. Given the beneficial effect of this methodology on the Alzheimer’s pathologies when studied separately, we explored the application of FUS in brains subjected concurrently to amyloidosis and tau phosphorylation. Our findings indicate the reduction of tau protein and decrease in the amyloid load from brains treated with ultrasound, accompanied by spatial memory improvement. Overall, in this dissertation, we established an optimized targeting and detection protocol, pre-clinical implementation of which confirmed its ameliorative effects as a drug-delivery adjuvant or an immune response stimulant. These preclinical findings support the immense potential of such a methodology that significantly contributes to the treatment of different neurodegenerative disorders curbing their progression

Cutting-edge advances in modeling the blood–brain barrier and tools for its reversible permeabilization for enhanced drug delivery into the brain

The bloodâ brain barrier (BBB) is a sophisticated structure whose full functionality is required for maintaining the executive functions of the central nervous system (CNS). Tight control of transport across the barrier means that most drugs, particularly large size, which includes powerful biologicals, cannot reach their targets in the brain. Notwithstanding the remarkable advances in characterizing the cellular nature of the BBB and consequences of BBB dysfunction in pathology (brain metastasis, neurological diseases), it remains challenging to deliver drugs to the CNS. Herein, we outline the basic architecture and key molecular constituents of the BBB. In addition, we review the current status of approaches that are being explored to temporarily open the BBB in order to allow accumulation of therapeutics in the CNS. Undoubtedly, the major concern in field is whether it is possible to open the BBB in a meaningful way without causing negative consequences. In this context, we have also listed few other important key considerations that can improve our understanding about the dynamics of the BBB.The authors, DCF, RLR and JMO, would like to thank the funds under the project 2IQBIONEURO (reference: 0624_2IQBIONEURO_6_E) co-funded by INTERREG (Atlantic (Atlantic program or 622 V-A Spain-Portugal) and European fund for Regional Development (FEDER).Open Access funding enabled and organized by Projekt DEAL

Extensive frontal focused ultrasound mediated blood-brain barrier opening for the treatment of Alzheimer's disease: a proof-of-concept study

Background: Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening has shown efficacy in removal of amyloid plaque and improvement of cognitive functions in preclinical studies, but this is rarely reported in clinical studies. This study was conducted to evaluate the safety, feasibility and potential benefits of repeated extensive BBB opening. Methods: In this open-label, prospective study, six patients with Alzheimer's disease (AD) were enrolled at Severance Hospital in Korea between August 2020 and September 2020. Five of them completed the study. FUS-mediated BBB opening, targeting the bilateral frontal lobe regions over 20 cm3, was performed twice at three-month intervals. Magnetic resonance imaging, 18F-Florbetaben (FBB) positron emission tomography, Caregiver-Administered Neuropsychiatric Inventory (CGA-NPI) and comprehensive neuropsychological tests were performed before and after the procedures. Results: FUS targeted a mean volume of 21.1 ± 2.7 cm3 and BBB opening was confirmed at 95.7% ± 9.4% of the targeted volume. The frontal-to-other cortical region FBB standardized uptake value ratio at 3 months after the procedure showed a slight decrease, which was statistically significant, compared to the pre-procedure value (- 1.6%, 0.986 vs1.002, P = 0.043). The CGA-NPI score at 2 weeks after the second procedure significantly decreased compared to baseline (2.2 ± 3.0 vs 8.6 ± 6.0, P = 0.042), but recovered after 3 months (5.2 ± 5.8 vs 8.6 ± 6.0, P = 0.89). No adverse effects were observed. Conclusions: The repeated and extensive BBB opening in the frontal lobe is safe and feasible for patients with AD. In addition, the BBB opening is potentially beneficial for amyloid removal in AD patients.ope

Theranostic Strategy of Focused Ultrasound Induced Blood-Brain Barrier Opening for CNS Disease Treatment

Focused Ultrasound (FUS) in combination with gaseous microbubbles has emerged as a potential new means of effective drug delivery to the brain. Recent research has shown that, under burst-type energy exposure with the presence of microbubbles, this modality can transiently permeate the blood-brain barrier (BBB). The bioavailability of therapeutic agents is site-specifically augmented only in the zone where the FUS energy is targeted. The non-invasiveness of this approach makes FUS-induced BBB opening a novel and attractive means to perform localized CNS therapeutic agent delivery. Over the past decade, FUS-BBB opening has been preclinically confirmed to successfully enhance CNS penetration of therapeutic agents including chemotherapeutic agents, therapeutic peptides, monoclonal antibodies, and nanoparticles. Recently, a number of clinical human trials have begun to explore clinical utility. This review article, explores this technology through its physical mechanisms, summarizes the existing preclinical findings (including current medical device designs and technical approaches), and summarizes current ongoing clinical trials

Long-lasting restoration of memory function and hippocampal synaptic plasticity by focused ultrasound in Alzheimer's disease

Background: Focused ultrasound (FUS) is a medical technology that non-invasively stimulates the brain and has been applied in thermal ablation, blood–brain barrier (BBB) opening, and neuromodulation. In recent years, numerous experiences and indications for the use of FUS in clinical and preclinical studies have rapidly expanded. Focused ultrasound-mediated BBB opening induces cognitive enhancement and neurogenesis; however, the underlying mechanisms have not been elucidated. Methods: Here, we investigate the effects of FUS-mediated BBB opening on hippocampal long-term potentiation (LTP) and cognitive function in a 5xFAD mouse model of Alzheimer's disease (AD). We applied FUS with microbubble to the hippocampus and LTP was measured 6 weeks after BBB opening using FUS. Field recordings were made with a concentric bipolar electrode positioned in the CA1 region using an extracellular glass pipette filled with artificial cerebrospinal fluid. Morris water maze and Y-maze was performed to test cognitive function. Results: Our results demonstrated that FUS-mediated BBB opening has a significant impact on increasing LTP at Schaffer collateral - CA1 synapses and rescues cognitive dysfunction and working memory. These effects persisted for up to 7 weeks post-treatment. Also, FUS-mediated BBB opening in the hippocampus increased PKA phosphorylation. Conclusion: Therefore, it could be a promising treatment for neurodegenerative diseases as it remarkably increases LTP, thereby improving working memory. © 2023 The Author(s)ope

Synthesis, in vitro and in vivo evaluation of nanoparticles and metal complexes for the treatment of brain diseases

An intact blood-brain barrier is essential in order to maintain brain homeostasis and preserve neuronal function. However, its presence prevents over 98% of conventional small molecule drugs from entering the brain, making the treatment of brain diseases such as Alzheimer’s disease extremely challenging. Due to this, there has been great interest in designing methods to overcome the blood-brain barrier, but many of these are either highly invasive, non-targeted or show poor efficiency. Over the past twenty years, the use of focused ultrasound in combination with circulating microbubbles has emerged as a promising strategy, allowing the blood-brain barrier to be disrupted in a completely non-invasive, targeted and transient manner. In this thesis, we have developed two systems to take advantage of this technology: DNA-coated nanoparticles as potential delivery platforms for combination therapies and a series of metal complexes as potential inhibitors of amyloid beta aggregation for the treatment of Alzheimer’s disease. In Chapter 2, we began by synthesising a series of fluorescently-labelled DNA-coated nanoparticles and showed that they can be successfully delivered to a target location across the blood-brain barrier in mice using ultrasound. Ongoing work is seeking to evaluate drug-loaded derivatives of these nanoparticles in vitro and in vivo. In Chapters 3 and 4, a series of metal salphens and salnaphs were investigated for their ability to inhibit the aggregation of the amyloid beta peptide, which is one of the key hallmarks of Alzheimer’s disease. Through a series of in vitro assays, we showed that the synthesised metal complexes are able to inhibit amyloid beta aggregation and are non-cytotoxic. Using ultrasound-mediated blood-brain barrier opening methods, these complexes were successfully delivered into the brains of mice, allowing their therapeutic efficacy to be assessed in vivo. Preliminary findings obtained with our complexes in an Alzheimer’s disease mouse model are presented.Open Acces

Structure and properties of drug-loaded polymeric nanoparticles targeting β-amyloid

Polymere Nanopartikel sind ein vielversprechender Ansatz für die Diagnose und Therapie von Krankheiten. Sie ermöglichen den Einsatz von schwerlöslichen oder instabilen Wirkstoffen. Ein weiterer Vorteil ist die Möglichkeit das Targetings, durch gezielte Modifikationen des Nanopartikels wird der Wirkstoff zum Zielort transportiert und kann dort in der gewünschten Form freigesetzt werden; dadurch könnten bei erhöhter Wirksamkeit die Nebenwirkungen von Medikamenten reduziert werden. Ziel dieser Arbeit war die Untersuchung von physikalischen und biochemischen Eigenschaften von Nanopartikeln bestehend aus einem abbaustabilen Polystyren- Kern und einer biologisch abbaubaren Schale aus Polybutylcyanoacrylat. Es werden Methoden beschrieben, um die Größe, Struktur und den Abbau dieser Wirkstoffträger zu untersuchen. Die untersuchten Nanopartikel zeigen RAYLEIGH-Streuung, sowohl Größe als auch Abbau können durch Messung des Absorptionsspektrums bestimmt werden. Weiterhin konnten diese Eigenschaften mit Hilfe von dynamischer und statischer Lichtstreuung sowie Neutronenkleinwinkelstreuung untersucht werden. Bei letzterer Methode konnte gezeigt werden, dass die Schale größtenteils abgebaut werden kann, während der Kern intakt bleibt. In einem weiteren Teil der Arbeit wurde die Überwindung der Blut-Hirn-Schranke durch polymere Nanopartikel untersucht. Dabei wurde der fluoreszierende Thioflavine als Modellwirkstoffe eingesetzt. Das Durchdringen der Blut-Hirn-Schranke konnte nur mit Nanopartikeln erreicht werden, an deren Oberfläche ein Apolipoprotein E-Peptid gekoppelt war. Es konnte gezeigt werden, das die Nanopartikelschale im Gehirn abgebaut wird, der Wirkstoff freigesetzt wird und an Amyloid β, einem Marker der Alzheimer-Krankheit, bindet

Amyloid Beta Transport and Effects on Permeability in a Human Brain Endothelial Cell Line

The clearance of neurotoxic amyloid beta (Aβ) from the brain represents a novel therapeutic target for Alzheimer's disease (AD). The ability of two blood-brain barrier (BBB) drug transporters, P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP), to transport Aβ was investigated using a human brain endothelial cell (BEC) line, hCMEC/D3. P-gp expression by hCMEC/D3 cells was stable over a high passage number, polarised on the apical membrane, consistent with the blood side in vivo, and comparable, albeit slightly reduced, to primary isolated human BECs. The P-gp inhibitors tariquidar and vinblastine prevented the efflux of rhodamine 123 from hCMEC/D3 cells, indicative of functional P-gp expression. hCMEC/D3 cells therefore constituted a suitable model to investigate P-gp substrate interactions in vitro. P-gp, and to a lesser extent BCRP, inhibition, increased the net influx and decreased the efflux of 0.1 nM 125I Aβ 1-40 in hCMEC/D3 cells. Both P-gp and BCRP inhibition increased the apical-to-basolateral but not the basolateral-to-apical permeability of hCMEC/D3 cells to 125I Aβ 1-40. This data is consistent with P-gp and BCRP, acting in vivo to prevent blood-borne Aβ peptides entering the brain but not to clear Aβ load from the brain. Vascular dysfunction is emerging as a key pathological hallmark in AD, including increased BBB permeability. The effect of Aβ on the permeability of hCMEC/D3 cells was therefore investigated. Aβ 1-40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kDa FITC-dextran. Increased permeability was associated with a specific decrease in the tight junction protein occludin, but not claudin-5 or ZO-1, both at the protein and mRNA levels. JNK and p38MAPK inhibition prevented Aβ 1-40-mediated occludin down-regulation and increased paracellular permeability of hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing vascular dysfunction in AD

The Role of Perivascular FIbrosis in Post-Stroke Glymphatic Impairment and Cerebral Amyloid Angiopathy

In healthy brain tissue, toxic amyloid-β (Aβ) proteins are transported by the pulsatile flow of cerebrospinal fluid (CSF) along perivascular drainage pathways. Ischemic stroke may disrupt this process, leading to a perivascular build-up of Aβ, termed cerebral amyloid angiopathy (CAA). I hypothesize that an abnormal pattern of extracellular matrix deposition within the vascular basement membrane, termed fibrosis, impairs Aβ drainage from the aged brain after stroke. I further hypothesize that inhibition of astrocytic transforming growth factor-β (TGF-β) signaling can reverse these phenotypes. Finally, I also hypothesize that serum biomarkers of perivascular fibrosis can be used to diagnose CAA following intracerebral hemorrhage (ICH). To test these hypotheses, I first performed experimental stroke in young and aged wild-type mice, then measured basement membrane fibrosis and CSF flow using a variety of biochemical and physiological techniques. I also evaluated the contribution of astrocytes to these phenotypes using a primary cell culture model. Then, I treated aged mice with a TGF-β antagonist, and measured the impact on fibrosis and CSF flow. Finally, I explored the relevance of these findings to humans by measuring serum biomarkers of fibrosis after ICH, and correlating them to CAA etiology, injury severity and functional outcomes. Overall, my findings support a role for fibrosis in impairing perivascular Aβ drainage after stroke, which could lead to CAA and progressive cognitive decline in stroke survivors