Ultrasound delivery of a TrkA agonist confers neuroprotection to Alzheimer-associated pathologies.

Early degeneration of basal forebrain cholinergic neurons contributes substantially to cognitive decline in Alzheimer's disease. Evidence from preclinical models of neuronal injury and aging support a pivotal role for nerve growth factor (NGF) in neuroprotection, resilience, and cognitive function. However, whether NGF can provide therapeutic benefit in the presence of Alzheimer's disease-related pathologies still unresolved. Perturbations in the NGF signalling system in Alzheimer's disease may render neurons unable to benefit from NGF administration. Additionally, challenges related to brain delivery remain for clinical translation of NGF-based therapies in Alzheimer's disease. To be safe and efficient, NGF-related agents should stimulate the NGF receptor, tropomyosin receptor kinase A (TrkA), avoid activation through the p75 neurotrophin receptor (p75NTR), and be delivered non-invasively to targeted brain areas using real-time monitoring. We addressed these limitations using MRI-guided focused ultrasound (MRIgFUS) to increase blood-brain barrier permeability locally and transiently, allowing an intravenously administered TrkA agonist that does not activate p75NTR, termed D3, to enter targeted brain areas. Here, we report the therapeutic potential of selective TrkA activation in a transgenic mouse model that recapitulates numerous Alzheimer's disease-associated pathologies. Repeated MRIgFUS-mediated delivery of D3 (D3/FUS) improved cognitive function in the TgCRND8 model of Alzheimer's disease. Mechanistically, D3/FUS treatment effectively attenuated cholinergic degeneration and promoted functional recovery. D3/FUS treatment also resulted in widespread reduction of brain amyloid pathology and dystrophic neurites surrounding amyloid plaques. Furthermore, D3/FUS markedly enhanced hippocampal neurogenesis in TgCRND8 mice, implicating TrkA agonism as a novel therapeutic target to promote neurogenesis in the context of Alzheimer's disease-related pathology. Thus, this study provides evidence that selective TrkA agonism confers neuroprotection to effectively counteract Alzheimer's disease-related vulnerability. Recent clinical trials demonstrate that non-invasive blood-brain barrier modulation using MRIgFUS is safe, feasible and reversible in Alzheimer's disease patients. TrkA receptor agonists coupled with MRIgFUS delivery constitute a promising disease-modifying strategy to foster brain health and counteract cognitive decline in Alzheimer's disease.

Vasculotide restores the blood-brain barrier after focused ultrasound-induced permeability in a mouse model of Alzheimer's disease.

Focused ultrasound (FUS) is used to locally and transiently induce blood-brain barrier (BBB) permeability, allowing targeted drug delivery to the brain. The purpose of the current study is to evaluate the potential of Vasculotide to accelerate the recovery of the BBB following FUS disruption in the TgCRND8 mouse model of amyloidosis, characteristic of Alzheimer's disease (AD). Accelerating the restoration of the BBB post-FUS would represent an additional safety procedure, which could be beneficial for clinical applications. Methods: TgCRND8 mice and their non-transgenic littermates were treated with Vasculotide (250 ng, intraperitoneal) every 48 hours for 3 months. BBB permeability was induced using FUS, in presence of intravenously injected microbubbles, in TgCRND8 and non-transgenic mice, and confirmed at time 0 by MRI enhancement using the contrast agent gadolinium. BBB closure was assessed at 6, 12 and 20 hours by MRI. In a separate cohort of animals, BBB closure was assessed at 24-hours post-FUS using Evans blue injected intravenously and followed by histological evaluation. Results: Chronic Vasculotide administration significantly reduces the ultra-harmonic threshold required for FUS-induced BBB permeability in the TgCRND8 mice. In addition, Vasculotide treatment led to a faster restoration of the BBB following FUS in TgCRND8 mice. BBB closure after FUS is not significantly different between TgCRND8 and non-transgenic mice. BBB permeability was assessed by gadolinium up to 20-hours post-FUS, demonstrating 87% closure in Vasculotide treated TgCRND8 mice, as opposed to 52% in PBS treated TgCRND8 mice, 58% in PBS treated non-transgenic mice, and 74% in Vasculotide treated non-transgenic mice. In both TgCRND8 mice and non-transgenic littermates the BBB was impermeable to Evans blue dye at 24-hours post-FUS. Conclusion: Vasculotide reduces the pressure required for microbubble ultra-harmonic onset for FUS-induced BBB permeability and it accelerates BBB restoration in a mouse model of amyloidosis, suggesting its potential clinical utility to promote vascular health, plasticity and repair in AD.

Focused ultrasound as a novel strategy for noninvasive gene delivery to retinal Müller glia.

Müller glia are specialized retinal cells with stem cell properties in fish and frogs but not in mammals. Current efforts to develop gene therapies to activate mammalian Müller glia for retinal repair will require safe and effective delivery strategies for recombinant adeno-associated viruses (AAVs), vectors of choice for clinical translation. Intravitreal and subretinal injections are currently used for AAV gene delivery in the eye, but less invasive methods efficiently targeting Müller glia have yet to be developed. Methods: As gene delivery strategies have been more extensively studied in the brain, to validate our vectors, we initially compared the glial tropism of AAV-PHP.eB, an AAV9 that crosses the blood-brain and blood-retinal barriers, for its ability to drive fluorescent protein expression in glial cells in both the brain and retina. We then tested the glial transduction of AAV2/8-GFAP-mCherry, a virus that does not cross blood-brain and blood-retinal barriers, for its effectiveness in transducing Müller glia in murine retinal explants ex vivo. For in vivo assays we used larger rat eyes, performing invasive intravitreal injections, and non-invasive intravenous delivery using focused ultrasound (FUS) (pressure amplitude: 0.360 - 0.84 MPa) and microbubbles (Definity, 0.2 ml/kg). Results: We showed that AAV-PHP.eB carrying a ubiquitous promoter (CAG) and green fluorescent protein (GFP) reporter, readily crossed the blood-brain and blood-retinal barriers after intravenous delivery in mice. However, murine Müller glia did not express GFP, suggesting that they were not transduced by AAV-PHP.eB. We thus tested an AAV2/8 variant, which was selected based on its safety record in multiple clinical trials, adding a glial fibrillary acidic protein (GFAP) promoter and mCherry (red fluorescent protein) reporter. We confirmed the glial specificity of AAV2/8-GFAP-mCherry, showing effective expression of mCherry in astrocytes after intracranial injection in the mouse brain, and of Müller glia in murine retinal explants. For in vivo experiments we switched to rats because of their larger size, injecting AAV2/8-GFAP-mCherry intravitreally, an invasive procedure, demonstrating passage across the inner limiting membrane, leading to Müller glia transduction. We then tested an alternative non-invasive delivery approach targeting a different barrier - the inner blood-retinal-barrier, applying focused ultrasound (FUS) to the retina after intravenous injection of AAV2/8 and microbubbles in rats, using magnetic resonance imaging (MRI) for FUS targeting. FUS permeabilized the rat blood-retinal-barrier and allowed the passage of macromolecules to the retina (Evans blue, IgG, IgM), with minimal extravasation of platelets and red blood cells. Intravenous injection of microbubbles and AAV2/8-GFAP-mCherry followed by FUS resulted in mCherry expression in rat Müller glia. However, systemic delivery of AAV2/8 also had off-target effects, transducing several murine peripheral organs, particularly the liver. Conclusions: Retinal permeabilisation via FUS in the presence of microbubbles is effective for delivering AAV2/8 across the inner blood-retinal-barrier, targeting Müller glia, which is less invasive than intravitreal injections that bypass the inner limiting membrane. However, implementing FUS in the clinic will require a comprehensive consideration of any off-target tropism of the AAV in peripheral organs, combined ideally, with the development of Müller glia-specific promoters.

Investigating the efficacy of a combination Aß-targeted treatment in a mouse model of Alzheimer's disease.

Amyloid-beta peptide (Aß) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Here, we explored the use of a combination treatment to reduce amyloid load through microglial phagocytosis in a mouse model of AD. We hypothesized that using an initial treatment of magnetic resonance image guided focused ultrasound (MRIgFUS) to transiently increase the blood-brain barrier (BBB) permeability and enhance the delivery of an Aß-antibody (BAM-10), followed by scyllo-inositol treatment would result in accelerated clearance. TgCRND8 mice expressing both Swedish (KM670/671NL) and Indiana (V717F) APP mutations under the hamster prion (PrP) promoter at 5 months of age were either treated with scyllo-inositol or received an initial MRIgFUS treatment delivering BAM-10 prior to scyllo-inositol treatment for one month. Treated animals and untreated TgCRND8 littermates were then sacrificed at 6 months of age, and their brains were processed for immunohistochemistry and immunofluorescence. Amyloid load was quantified and analyzed through immunohistochemical staining. Astrocyte and microglial activation were quantified and analyzed through immunofluorescent staining. We found that both the scyllo-inositol treatment and combination treatment, MRIgFUS/BAM10+scyllo-inositol, significantly reduced amyloid load and astrocyte activation in the hippocampus and the cortex. Furthermore, in both treatment paradigms microglial activation and phagocytosis was increased in comparison to the untreated mice. There were no differences detected between the two treatment paradigms. We propose that the 30-day scyllo-inositol treatment saturated the early benefit of the MRIgFUS/BAM-10 treatment. In the future, multiple FUS treatments combined with BAM-10 throughout the duration of scyllo-inositol treatment may lead to more effective amyloid clearance.

Amyloid-ß plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound.

Noninvasive, targeted drug delivery to the brain can be achieved using transcranial focused ultrasound (FUS), which transiently increases the permeability of the blood-brain barrier (BBB) for localized delivery of therapeutics from the blood to the brain. Previously, we have demonstrated that FUS can deliver intravenously-administered antibodies to the brain of a mouse model of Alzheimer's disease (AD) and rapidly reduce plaques composed of amyloid-ß peptides (Aß). Here, we investigated two potential effects of transcranial FUS itself that could contribute to a reduction of plaque pathology, namely the delivery of endogenous antibodies to the brain and the activation of glial cells. We demonstrate that transcranial FUS application leads to a significant reduction in plaque burden four days after a single treatment in the TgCRND8 mouse model of AD and that endogenous antibodies are found bound to Aß plaques. Immunohistochemical and western blot analyses showed an increase in endogenous immunoglobulins within the FUS-targeted cortex. Subsequently, microglia and astrocytes in FUS-treated cortical regions show signs of activation through increases in protein expression and changes in glial size, without changes in glial cell numbers. Enhanced activation of glia correlated with increased internalization of Aß in microglia and astrocytes. Together these data demonstrate that FUS improved the bioavailability of endogenous antibodies and led to a temporal activation of glial cells, providing evidence towards antibody- and glia-dependent mechanisms of FUS-mediated plaque reduction.