Selection of an Efficient AAV Vector for Robust CNS Transgene Expression

Adeno-associated virus (AAV) capsid libraries have generated improved transgene delivery vectors. We designed an AAV library construct, iTransduce, that combines a peptide library on the AAV9 capsid with a Cre cassette to enable sensitive detection of transgene expression. After only two selection rounds of the library delivered intravenously in transgenic mice carrying a Cre-inducible fluorescent protein, we flow sorted fluorescent cells from brain, and DNA sequencing revealed two dominant capsids. One of the capsids, termed AAV-F, mediated transgene expression in the brain cortex more than 65-fold (astrocytes) and 171-fold (neurons) higher than the parental AAV9. High transduction efficiency was sex-independent and sustained in two mouse strains (C57BL/6 and BALB/c), making it a highly useful capsid for CNS transduction of mice. Future work in large animal models will test the translation potential of AAV-F

RNA Aptamer Probes as Optical Imaging Agents for the Detection of Amyloid Plaques

Optical imaging using multiphoton microscopy and whole body near infrared imaging has become a routine part of biomedical research. However, optical imaging methods rely on the availability of either small molecule reporters or genetically encoded fluorescent proteins, which are challenging and time consuming to develop. While directly labeled antibodies can also be used as imaging agents, antibodies are species specific, can typically not be tagged with multiple fluorescent reporters without interfering with target binding, and are bioactive, almost always eliciting a biological response and thereby influencing the process that is being studied. We examined the possibility of developing highly specific and sensitive optical imaging agents using aptamer technology. We developed a fluorescently tagged anti-Aβ RNA aptamer, β55, which binds amyloid plaques in both ex vivo human Alzheimer’s disease brain tissue and in vivo APP/PS1 transgenic mice. Diffuse β55 positive halos, attributed to oligomeric Aβ, were observed surrounding the methoxy-XO4 positive plaque cores. Dot blots of synthetic Aβ aggregates provide further evidence that β55 binds both fibrillar and non-fibrillar Aβ. The high binding affinity, the ease of probe development, and the ability to incorporate multiple and multimodal imaging reporters suggest that RNA aptamers may have complementary and perhaps advantageous properties compared to conventional optical imaging probes and reporters

Cromolyn Reduces Levels of the Alzheimer’s Disease-Associated Amyloid β-Protein by Promoting Microglial Phagocytosis

Amyloid-beta protein (Aβ) deposition is a pathological hallmark of Alzheimer’s disease (AD). Aβ deposition triggers both pro-neuroinflammatory microglial activation and neurofibrillary tangle formation. Cromolyn sodium is an asthma therapeutic agent previously shown to reduce Aβ levels in transgenic AD mouse brains after one-week of treatment. Here, we further explored these effects as well as the mechanism of action of cromolyn, alone, and in combination with ibuprofen in APPSwedish-expressing Tg2576 mice. Mice were treated for 3 months starting at 5 months of age, when the earliest stages of β-amyloid deposition begin. Cromolyn, alone, or in combination with ibuprofen, almost completely abolished longer insoluble Aβ species, i.e. Aβ40 and Aβ42, but increased insoluble Aβ38 levels. In addition to its anti-aggregation effects on Aβ, cromolyn, alone, or plus ibuprofen, but not ibuprofen alone, increased microglial recruitment to, and phagocytosis of β-amyloid deposits in AD mice. Cromolyn also promoted Aβ42 uptake in microglial cell-based assays. Collectively, our data reveal robust effects of cromolyn, alone, or in combination with ibuprofen, in reducing aggregation-prone Aβ levels and inducing a neuroprotective microglial activation state favoring Aβ phagocytosis versus a pro-neuroinflammatory state. These findings support the use of cromolyn, alone, or with ibuprofen, as a potential AD therapeutic

Inhibition of the NFAT pathway alleviates amyloid β neurotoxicity in a mouse model of Alzheimer's disease

Amyloid β (Aβ) peptides, the main pathological species associated with Alzheimer’s disease (AD), disturb intracellular calcium homeostasis, which in turn activates the calcium-dependent phosphatase calcineurin (CaN). CaN activation induced by Aβ leads to pathological morphological changes in neurons, and overexpression of constitutively active calcineurin is sufficient to generate a similar phenotype, even without Aβ. Here, we tested the hypothesis that calcineurin mediates neurodegenerative effects via activation of the nuclear transcription factor of activated T-cells (NFAT). We found that both spine loss and dendritic branching simplification induced by Aβ exposure were mimicked by constitutively active NFAT, and abolished when NFAT activation was blocked using the genetically encoded inhibitor VIVIT. When VIVIT was specifically addressed to the nucleus, identical beneficial effects were observed, thus enforcing the role of NFAT transcriptional activity in Aβ-related neurotoxicity. In vivo, when VIVIT or its nuclear counterpart were overexpressed in a transgenic model of Alzheimer’s disease via a gene therapy approach, the spine loss and neuritic abnormalities observed in the vicinity of amyloid plaques were blocked. Overall, these results suggest that NFAT/calcineurin transcriptional cascades contribute to Aβ synaptotoxicity, and may provide a new specific set of pathways for neuroprotective strategies

Amyloid beta induces the morphological neurodegenerative triad of spine loss, dendritic simplification, and neuritic dystrophies through calcineurin activation

Amyloid beta containing plaques are surrounded by dystrophic neurites in the Alzheimer disease (AD) brain, but whether and how plaques induce these neuritic abnormalities remain unknown. We tested the hypothesis that soluble oligomeric assemblies of Aβ, which surround plaques, induce calcium mediated secondary cascades that lead to dystrophic changes in local neurites. We show that soluble Aβ oligomers lead to activation of the calcium-dependent phosphatase CaN (PP2B) which in turn activates the transcriptional factor nuclear factor of activated T cells (NFAT). Activation of these signaling pathways, even in the absence of Aβ, is sufficient to produce a virtual phenocopy of Aβ induced dystrophic neurites, dendritic simplification, and dendritic spine loss in both neurons in culture and in the adult mouse brain. Importantly, the morphological deficits in the vicinity of Aβ deposits in a mouse model of AD are ameliorated by CaN inhibition, supporting the hypothesis that CaN/NFAT are aberrantly activated by Aβ, and that CaN/NFAT activation is responsible for disruption of neuronal structure near plaques. In accord with this, we also detect increased levels of an active form of CaN and NFATc4 in the nuclear fraction from the cortex of patients with AD. Thus, Aβ appears to mediate the neurodegeneration of AD, at least in part, by activation of CaN and subsequent NFAT-mediated downstream cascades

Neuronal Cholesterol Accumulation Induced by Cyp46a1 Down-Regulation in Mouse Hippocampus Disrupts Brain Lipid Homeostasis

Impairment in cholesterol metabolism is associated with many neurodegenerative disorders including Alzheimer's disease (AD). However, the lipid alterations underlying neurodegeneration and the connection between altered cholesterol levels and AD remains not fully understood. We recently showed that cholesterol accumulation in hippocampal neurons, induced by silencing Cyp46a1 gene expression, leads to neurodegeneration with a progressive neuronal loss associated with AD-like phenotype in wild-type mice. We used a targeted and non-targeted lipidomics approach by liquid chromatography coupled to high-resolution mass spectrometry to further characterize lipid modifications associated to neurodegeneration and cholesterol accumulation induced by CYP46A1 inhibition. Hippocampus lipidome of normal mice was profiled 4 weeks after cholesterol accumulation due to Cyp46a1 gene expression down-regulation at the onset of neurodegeneration. We showed that major membrane lipids, sphingolipids and specific enzymes involved in phosphatidylcholine and sphingolipid metabolism, were rapidly increased in the hippocampus of AAV-shCYP46A1 injected mice. This lipid accumulation was associated with alterations in the lysosomal cargoe, accumulation of phagolysosomes and impairment of endosome-lysosome trafficking. Altogether, we demonstrated that inhibition of cholesterol 24-hydroxylase, key enzyme of cholesterol metabolism leads to a complex dysregulation of lipid homeostasis. Our results contribute to dissect the potential role of lipids in severe neurodegenerative diseases like AD

In Vivo Two Photon Imaging of Astrocytic Structure and Function in Alzheimer’s Disease

The physiological function of the neurovascular unit is critically dependent upon the complex structure and functions of astrocytes for optimal preservation of cerebral homeostasis. While it has been shown that astrocytes exhibit aberrant changes in both structure and function in transgenic murine models of Alzheimer’s disease (AD), it is not fully understood how this altered phenotype contributes to the pathogenesis of AD or whether this alteration predicts a therapeutic target in AD. The mechanisms underlying the spatiotemporal relationship between astrocytes, neurons and the vasculature in their orchestrated regulation of local cerebral flow in active brain regions has not been fully elucidated in brain physiology and in AD. As there is an incredible urgency to identify therapeutic targets that are well-tolerated and efficacious in protecting the brain against the pathological impact of AD, here we use the current body of literature to evaluate the hypothesis that pathological changes in astrocytes are central to the pathogenesis of AD. We also examine the current tools available to assess astrocytic calcium signaling in the living murine brain as it has an important role in the complex interaction between astrocytes, neurons and the vasculature. Furthermore, we discuss the altered function of astrocytes in their interaction with neurons in the preservation of glutamate homeostasis and additionally address the role of astrocytes at the vascular interface and their contribution to functional hyperemia within the living murine brain in health and in AD

Intrathecal sc-AAV9-CB-GFP: Systemic Distribution Predominates Following Single-Dose Administration in Cynomolgus Macaques

Biodistribution of self-complementary adeno-associated virus-9 (scAAV9)–chicken β-actin promoter–green fluorescent protein (GFP) was assessed in juvenile cynomolgus macaques infused intrathecally via lumbar puncture or the intracisterna magna (1.0×1013 or 3.0×1013 vg/animal), with necropsy 28 days later. Our results characterized central nervous system biodistribution compared with systemic organs and tissues by droplet digital polymerase chain reaction (ddPCR) for DNA and in situ hybridization. GFP expression was characterized by Meso Scale Discovery electrochemiluminescence immunosorbent assay and immunohistochemistry (IHC). Biodistribution was widespread but variable, with vector DNA and GFP expression greatest in the spinal cord, dorsal root ganglia (DRG), and certain systemic tissues (e.g. liver), with low concentrations detected in many brain regions. Transduction and expression were observed primarily in perivascular astrocytes in the brain, with a paucity of transduction or expression in the neurons. Despite direct administration into the cerebrospinal fluid (CSF), limited distribution to key brain structures identified as targets for monogenic neurologic disease was observed. Greater GFP expression was observed in hepatocytes, striated myocytes (skeletal muscle), cardiomyocytes, spinal cord lower motor neurons, and DRG sensory neurons by IHC. These results suggest caution for use of scAAV9-based intrathecal delivery with the current expression cassette as a modality for neurologic diseases that require widespread brain neuronal expression. This capsid/expression cassette may be better suited for diseases that express a secreted protein and/or do not required widespread brain neuronal transduction

Image_1_In Vivo Two Photon Imaging of Astrocytic Structure and Function in Alzheimer’s Disease.tif

<p>The physiological function of the neurovascular unit is critically dependent upon the complex structure and functions of astrocytes for optimal preservation of cerebral homeostasis. While it has been shown that astrocytes exhibit aberrant changes in both structure and function in transgenic murine models of Alzheimer’s disease (AD), it is not fully understood how this altered phenotype contributes to the pathogenesis of AD or whether this alteration predicts a therapeutic target in AD. The mechanisms underlying the spatiotemporal relationship between astrocytes, neurons and the vasculature in their orchestrated regulation of local cerebral flow in active brain regions has not been fully elucidated in brain physiology and in AD. As there is an incredible urgency to identify therapeutic targets that are well-tolerated and efficacious in protecting the brain against the pathological impact of AD, here we use the current body of literature to evaluate the hypothesis that pathological changes in astrocytes are central to the pathogenesis of AD. We also examine the current tools available to assess astrocytic calcium signaling in the living murine brain as it has an important role in the complex interaction between astrocytes, neurons and the vasculature. Furthermore, we discuss the altered function of astrocytes in their interaction with neurons in the preservation of glutamate homeostasis and additionally address the role of astrocytes at the vascular interface and their contribution to functional hyperemia within the living murine brain in health and in AD.</p