"Focused Ultrasound-mediated Drug Delivery in Humans - a Path Towards Translation in Neurodegenerative Diseases".

The blood-brain barrier (BBB) has a major protective function in preventing the entry of harmful molecules into the brain, but is simultaneously limiting the delivery of drugs, restricting their potential clinical application in neurodegenerative diseases. Recent preclinical evidence demonstrates that following application of focused ultrasound with microbubbles (FUS+MB), the BBB becomes reversibly accessible to compounds that normally are brain-impermeable, suggesting FUS+MB as a promising new platform for delivery of therapeutic agents into the central nervous system. As a step towards translation, small cohort clinical studies were performed demonstrating safe BBB opening in Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS) patients following FUS+MB, however improved drug delivery has not yet been achieved in human. Simultaneously, rapid progress in the human induced pluripotent stem cell (hiPSC) modeling technology allowed for development of novel Alzheimer's disease patient-derived BBB in vitro model that reacts to FUS+MB with BBB opening and can be used to answer fundamental questions of human BBB responses to FUS+MB in health and disease. This review summarizes key features of the BBB that contribute to limited drug delivery, recapitulates recent advances in the FUS+MB mediated human BBB opening in vivo and in vitro in the context of neurodegenerative disorders, and highlights potential strategies for fast-track translation of the FUS+MB to improve bioavailability of drugs to the human brain. With safe and effective application, this innovative FUS+MB technology may open new avenues for therapeutic interventions in neurodegenerative diseases leading to improved clinical outcomes for patients.

Novel Anti-Neuroinflammatory Properties of a Thiosemicarbazone-Pyridylhydrazone Copper(II) Complex.

Neuroinflammation has a major role in several brain disorders including Alzheimer's disease (AD), yet at present there are no effective anti-neuroinflammatory therapeutics available. Copper(II) complexes of bis(thiosemicarbazones) (CuII(gtsm) and CuII(atsm)) have broad therapeutic actions in preclinical models of neurodegeneration, with CuII(atsm) demonstrating beneficial outcomes on neuroinflammatory markers in vitro and in vivo. These findings suggest that copper(II) complexes could be harnessed as a new approach to modulate immune function in neurodegenerative diseases. In this study, we examined the anti-neuroinflammatory action of several low-molecular-weight, charge-neutral and lipophilic copper(II) complexes. Our analysis revealed that one compound, a thiosemicarbazone-pyridylhydrazone copper(II) complex (CuL5), delivered copper into cells in vitro and increased the concentration of copper in the brain in vivo. In a primary murine microglia culture, CuL5 was shown to decrease secretion of pro-inflammatory cytokine macrophage chemoattractant protein 1 (MCP-1) and expression of tumor necrosis factor alpha (Tnf), increase expression of metallothionein (Mt1), and modulate expression of Alzheimer's disease-associated risk genes, Trem2 and Cd33. CuL5 also improved the phagocytic function of microglia in vitro. In 5xFAD model AD mice, treatment with CuL5 led to an improved performance in a spatial working memory test, while, interestingly, increased accumulation of amyloid plaques in treated mice. These findings demonstrate that CuL5 can induce anti-neuroinflammatory effects in vitro and provide selective benefit in vivo. The outcomes provide further support for the development of copper-based compounds to modulate neuroinflammation in brain diseases.

Alzheimer's disease brain endothelial-like cells reveal differential drug transporter expression and modulation by potentially therapeutic focused ultrasound.

The blood-brain barrier (BBB) has a key function in maintaining homeostasis in the brain, partly modulated by transporters, which are highly expressed in brain endothelial cells (BECs). Transporters mediate the uptake or efflux of compounds to and from the brain and they can also challenge the delivery of drugs for the treatment of Alzheimer's disease (AD). Currently there is a limited understanding of changes in BBB transporters in AD. To investigate this, we generated brain endothelial-like cells (iBECs) from induced pluripotent stem cells (iPSCs) with familial AD (FAD) Presenilin 1 (PSEN1) mutation and identified AD-specific differences in transporter expression compared to control (ctrl) iBECs. We first characterized the expression levels of 12 BBB transporters in AD-, Ctrl-, and isogenic (PSEN1 corrected) iBECs to identify any AD specific differences. We then exposed the cells to focused ultrasound (FUS) in the absence (FUSonly) or presence of microbubbles (MB) (FUS+MB), which is a novel therapeutic method that can be used to transiently open the BBB to increase drug delivery into the brain, however its effects on BBB transporter expression are largely unknown. Following FUSonly and FUS+MB, we investigated whether the expression or activity of key transporters could be modulated. Our findings demonstrate that PSEN1 mutant FAD (PSEN1AD) possess phenotypical differences compared to control iBECs in BBB transporter expression and function. Additionally, we show that FUSonly and FUS+MB can modulate BBB transporter expression and functional activity in iBECs, having potential implications on drug penetration and amyloid clearance. These findings highlight the differential responses of patient cells to FUS treatment, with patient-derived models likely providing an important tool for modelling therapeutic effects of FUS.

A patient-derived amyotrophic lateral sclerosis blood-brain barrier model for focused ultrasound-mediated anti-TDP-43 antibody delivery.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients' BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored. METHODS: Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro. RESULTS: Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model. CONCLUSIONS: Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.

Patient-Derived Blood-Brain Barrier Model for Screening Copper Bis(thiosemicarbazone) Complexes as Potential Therapeutics in Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent cause of dementia characterized by a progressive cognitive decline. Addressing neuroinflammation represents a promising therapeutic avenue to treat AD; however, the development of effective antineuroinflammatory compounds is often hindered by their limited blood-brain barrier (BBB) permeability. Consequently, there is an urgent need for accurate, preclinical AD patient-specific BBB models to facilitate the early identification of immunomodulatory drugs capable of efficiently crossing the human AD BBB. This study presents a unique approach to BBB drug permeability screening as it utilizes the familial AD patient-derived induced brain endothelial-like cell (iBEC)-based model, which exhibits increased disease relevance and serves as an improved BBB drug permeability assessment tool when compared to traditionally employed in vitro models. To demonstrate its utility as a small molecule drug candidate screening platform, we investigated the effects of diacetylbis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(atsm)) and a library of metal bis(thiosemicarbazone) complexes─a class of compounds exhibiting antineuroinflammatory therapeutic potential in neurodegenerative disorders. By evaluating the toxicity, cellular accumulation, and permeability of those compounds in the AD patient-derived iBEC, we have identified 3,4-hexanedione bis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(dtsm)) as a candidate with good transport across the AD BBB. Furthermore, we have developed a multiplex approach where AD patient-derived iBEC were combined with immune modulators TNFα and IFNγ to establish an in vitro model representing the characteristic neuroinflammatory phenotype at the patient's BBB. Here, we observed that treatment with CuII(dtsm) not only reduced the expression of proinflammatory cytokine genes but also reversed the detrimental effects of TNFα and IFNγ on the integrity and function of the AD iBEC monolayer. This suggests a novel pathway through which copper bis(thiosemicarbazone) complexes may exert neurotherapeutic effects on AD by mitigating BBB neuroinflammation and related BBB integrity impairment. Together, the presented model provides an effective and easily scalable in vitro BBB platform for screening AD drug candidates. Its improved translational potential makes it a valuable tool for advancing the development of metal-based compounds aimed at modulating neuroinflammation in AD.

Differential Cytokine Responses of APOE3 and APOE4 Blood-brain Barrier Cell Types to SARS-CoV-2 Spike Proteins.

SARS-CoV-2 spike proteins have been shown to cross the blood-brain barrier (BBB) in mice and affect the integrity of human BBB cell models. However, the effects of SARS-CoV-2 spike proteins in relation to sporadic, late onset, Alzheimer's disease (AD) risk have not been extensively investigated. Here we characterized the individual and combined effects of SARS-CoV-2 spike protein subunits S1 RBD, S1 and S2 on BBB cell types (induced brain endothelial-like cells (iBECs) and astrocytes (iAstrocytes)) generated from induced pluripotent stem cells (iPSCs) harboring low (APOE3 carrier) or high (APOE4 carrier) relative Alzheimer's risk. We found that treatment with spike proteins did not alter iBEC integrity, although they induced the expression of several inflammatory cytokines. iAstrocytes exhibited a robust inflammatory response to SARS-CoV-2 spike protein treatment, with differences found in the levels of cytokine secretion between spike protein-treated APOE3 and APOE4 iAstrocytes. Finally, we tested the effects of potentially anti-inflammatory drugs during SARS-CoV-2 spike protein exposure in iAstrocytes, and discovered different responses between spike protein treated APOE4 iAstrocytes and APOE3 iAstrocytes, specifically in relation to IL-6, IL-8 and CCL2 secretion. Overall, our results indicate that APOE3 and APOE4 iAstrocytes respond differently to anti-inflammatory drug treatment during SARS-CoV-2 spike protein exposure with potential implications to therapeutic responses.

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.

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.

Selenium mediates exercise-induced adult neurogenesis and reverses learning deficits induced by hippocampal injury and aging.

Although the neurogenesis-enhancing effects of exercise have been extensively studied, the molecular mechanisms underlying this response remain unclear. Here, we propose that this is mediated by the exercise-induced systemic release of the antioxidant selenium transport protein, selenoprotein P (SEPP1). Using knockout mouse models, we confirmed that SEPP1 and its receptor low-density lipoprotein receptor-related protein 8 (LRP8) are required for the exercise-induced increase in adult hippocampal neurogenesis. In vivo selenium infusion increased hippocampal neural precursor cell (NPC) proliferation and adult neurogenesis. Mimicking the effect of exercise through dietary selenium supplementation restored neurogenesis and reversed the cognitive decline associated with aging and hippocampal injury, suggesting potential therapeutic relevance. These results provide a molecular mechanism linking exercise-induced changes in the systemic environment to the activation of quiescent hippocampal NPCs and their subsequent recruitment into the neurogenic trajectory.

Author Correction: Mast cells increase adult neural precursor proliferation and differentiation but this potential is not realized in vivo under physiological conditions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Increasing neurogenesis refines hippocampal activity rejuvenating navigational learning strategies and contextual memory throughout life.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Increasing neurogenesis refines hippocampal activity rejuvenating navigational learning strategies and contextual memory throughout life.

Functional plasticity of the brain decreases during ageing causing marked deficits in contextual learning, allocentric navigation and episodic memory. Adult neurogenesis is a prime example of hippocampal plasticity promoting the contextualisation of information and dramatically decreases during ageing. We found that a genetically-driven expansion of neural stem cells by overexpression of the cell cycle regulators Cdk4/cyclinD1 compensated the age-related decline in neurogenesis. This triggered an overall inhibitory effect on the trisynaptic hippocampal circuit resulting in a changed profile of CA1 sharp-wave ripples known to underlie memory consolidation. Most importantly, increased neurogenesis rescued the age-related switch from hippocampal to striatal learning strategies by rescuing allocentric navigation and contextual memory. Our study demonstrates that critical aspects of hippocampal function can be reversed in old age, or compensated throughout life, by exploiting the brain's endogenous reserve of neural stem cells.

Mast cells increase adult neural precursor proliferation and differentiation but this potential is not realized in vivo under physiological conditions.

There is growing evidence that both peripheral and resident immune cells play an important part in regulating adult neural stem cell proliferation and neurogenesis, although the contribution of the various immune cell types is still unclear. Mast cells, a population of immune cells known for their role in the allergic response, have been implicated in the regulation of adult hippocampal neurogenesis. Mast cell-deficient c-kitW-sh/W-sh mice have previously been shown to exhibit significantly decreased adult hippocampal neurogenesis and associated learning and memory deficits. However, given that numerous other cell types also express high levels of c-kit, the utility of these mice as a reliable model of mast cell-specific depletion is questionable. We show here, using a different model of mast cell deficiency (Mcpt5CreR26DTA/DTA), that precursor proliferation and adult neurogenesis are not influenced by mast cells in vivo. Interestingly, when applied at supraphysiological doses, mast cells can activate latent hippocampal precursor cells and increase subventricular zone precursor proliferation in vitro, an effect that can be blocked with specific histamine-receptor antagonists. Thus, we conclude that while both mast cells and their major chemical mediator histamine have the potential to affect neural precursor proliferation and neurogenesis, this is unlikely to occur under physiological conditions.