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 agerelated 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.

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 (Aduhelm TM ; anti-amyloid-β) 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.

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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.

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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.

The blood-brain barrier is a semipermeable barrier structure that lines the walls of brain microvessels. Although the blood-brain barrier plays a key role in protecting the brain from unwanted molecules, it simultaneously challenges the delivery of drugs into the brain. In addition, the blood-brain barrier has been shown to be dysfunctional in Alzheimer' s disease, the most common cause of dementia for which there is no cure. Mouse models of Alzheimer' s disease have played a central role in investigating disease-specific changes in the blood-brain barrier, but the translation of findings from mouse models into the human system is hindered by interspecies differences. In an effort to develop new drug delivery techniques and/or understand changes in the human blood-brain barrier in Alzheimer' s disease, several human blood-brain barrier in vitro models have been developed. These comprise primary and immortalized human endothelial cell-based models as well as human induced pluripotent stem cell-derived brain microvascular endothelial cell models. Both two-and three-dimensional

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.