Neuroinflammation and fractalkine signaling in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder, and the most common form of dementia. As the understanding of AD has progressed, it is now believed that AD is an amyloid-initiated tauopathy with neuroinflammation serving as the link between amyloid deposition, tau pathology, and neurodegeneration. As microglia are the main immune effectors in the central nervous system, they have been the focus of attention in studies investigating the neuroinflammatory component of AD. Therefore, recent work has focused on immunomodulators, which can alter microglial activation without suppressing activity, as potential therapeutics for AD. Fractalkine (CX3CL1; FKN), a unique chemokine with a one-to-one relationship with its receptor, signals through its cognate receptor (CX3CR1) to reduce expression of pro-inflammatory genes in activated microglia. Disrupting FKN signaling has opposing effects on the two hallmark pathologies of AD, but over-expressing a soluble FKN has been shown to reduce tau pathology while not altering amyloid pathology. Recently, differential signaling has been reported when comparing two cleavage variants of soluble FKN. These differential effects may explain recent studies reporting seemingly conflicting results regarding the effect of FKN over expression on AD pathologies.

Fractalkine over expression suppresses α-synuclein-mediated neurodegeneration.

In Parkinson's disease, α-synuclein is known to activate microglia and this activation has been proposed as one of the mechanisms of neurodegeneration. There are several signals produced by neurons that have an anti-inflammatory action on microglia, including CX3CL1 (fractalkine). We have shown that a soluble form of CX3CL1 is required to reduce neuron loss in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and that fractalkine agonism can reduce neuron loss in a 6-hydroxydopamine lesion model. Here, we show that fractalkine can reduce α-synuclein-mediated neurodegeneration in rats. Rats that received fractalkine showed abrogated loss of tyrosine hydroxylase and Neu-N staining. This was replicated in animals where we expressed fractalkine from astrocytes with the glial fibrillary acid protein (GFAP) promoter. Interestingly, we did not observe a reduction in MHCII expression suggesting that soluble fractalkine is likely altering the microglial state to a more neuroprotective one rather than reducing antigen presentation.

Induction of tauopathy in a mouse model of amyloidosis using intravenous administration of adeno-associated virus vectors expressing human P301L tau.

INTRODUCTION: Alzheimer's disease (AD) is a progressive neurodegenerative disease in which extracellular aggregates of the amyloid beta (Aß) peptide precede widespread intracellular inclusions of the microtubule-associated protein tau. The autosomal dominant form of AD requires mutations that increase production or aggregation of the Aß peptide. This has led to the hypothesis that amyloid deposition initiates downstream responses that lead to the hyperphosphorylation and aggregation of tau. METHODS: Here we use a novel approach, somatic gene transfer via intravenous adeno-associated virus (AAV), to further explore the effects of pre-existing amyloid deposits on tauopathy. APP+PS1 mice, which develop amyloid deposits at 3 to 6 months of age, and non-transgenic littermates were injected at 8 months of age intravenously with AAV-PHP.eB encoding P301L human tau. Tissue was collected at 13 months and tauopathy was assessed. RESULTS: Total human tau expression was observed to be relatively uniform throughout the brain, reflecting the vascular route of AAV administration. Phospho-tau deposition was not equal across brain regions and significantly increased in APP+PS1 mice compared to non-transgenic controls. Interestingly, the rank order of phospho-tau deposition of affected brain regions in both genotypes paralleled the rank order of amyloid plaque deposits in APP+PS1 mice. We also observed significantly increased MAPT RNA expression in APP+PS1 mice compared to non-transgenic despite equal AAV transduction efficiency between groups. DISCUSSION: This model has advantages over prior approaches with widespread uniform human tau expression throughout the brain and the ability to specify the stage of amyloidosis when the tau pathology is initiated. These data add further support to the amyloid cascade hypothesis and suggest RNA metabolism as a potential mechanism for amyloid-induced tauopathy.

Toward Development of Neuron Specific Transduction After Systemic Delivery of Viral Vectors.

Widespread transduction of the CNS with a single, non-invasive systemic injection of adeno-associated virus is now possible due to the creation of blood-brain barrier-permeable capsids. However, as these capsids are mutants of AAV9, they do not have specific neuronal tropism. Therefore, it is necessary to use genetic tools to restrict expression of the transgene to neuronal tissues. Here we compare the strength and specificity of two neuron-specific promoters, human synapsin 1 and mouse calmodulin/calcium dependent kinase II, to the ubiquitous CAG promoter. Administration of a high titer of virus is necessary for widespread CNS transduction. We observed the neuron-specific promoters drive comparable overall expression in the brain to the CAG promoter. Furthermore, the neuron-specific promoters confer significantly less transgene expression in peripheral tissues compared with the CAG promoter. Future experiments will utilize these delivery platforms to over-express the Alzheimer-associated pathological proteins amyloid-beta and tau to create mouse models without transgenesis.

CNS-Wide over Expression of Fractalkine Improves Cognitive Functioning in a Tauopathy Model.

Accumulating evidence increasingly implicates regulation of neuroinflammation as a potential therapeutic target in Alzheimer's disease and other neurodegenerative disorders. Fractalkine (FKN) is a unique chemokine that is expressed and secreted by neurons and reduces expression of pro-inflammatory genes. To further demonstrate the utility of agents that increase FKN signaling throughout the central nervous system as possible therapies for AD, we assessed the impact of soluble FKN (sFKN) over expression on cognition in tau depositing rTg450 mice after the onset of cognitive deficits. Using adeno-associated virus serotype 4, we infected cells lining the ventricular system with soluble FKN to increase FKN signaling over a larger fraction of the brain than achieved with intraparenchymal injections. We found that soluble FKN over expression by cells lining the ventricles significantly improved cognitive performance on the novel mouse recognition and radial arm water maze tasks. These benefits were achieved without detectable reductions in tau hyperphosphorylation, hippocampal atrophy, or microglial CD45 expression. Utilizing qPCR, we report a significant increase in Vegfa expression, indicating an increase in trophic support and possible neovascularization in AAV-sFKN-injected mice. To our knowledge, this is the first demonstration that FKN over expression can rescue cognitive function in a tau depositing mouse line. Graphical Abstract Regulating neuroinflammation is an attractive therapeutic target for Alzheimer's disease. Microglial activation can not only drive pathology but also accelerate cognitive decline. The chemokine fractalkine regulates the microglial phenotype, increasing trophic support of neurons, and significantly improving cognitive functioning in the rTg4510 mouse model of tauopathy.

Development of a Screening Platform to Identify Small Molecules That Modify ELP1 Pre-mRNA Splicing in Familial Dysautonomia.

Familial dysautonomia (FD) is an autonomic and sensory neuropathy caused by a mutation in the splice donor site of intron 20 of the ELP1 gene. Variable skipping of exon 20 leads to a tissue-specific reduction in the level of ELP1 protein. We have shown that the plant cytokinin kinetin is able to increase cellular ELP1 protein levels in vivo and in vitro through correction of ELP1 splicing. Studies in FD patients determined that kinetin is not a practical therapy due to low potency and rapid elimination. To identify molecules with improved potency and efficacy, we developed a cell-based luciferase splicing assay by inserting renilla (Rluc) and firefly (Fluc) luciferase reporters into our previously well-characterized ELP1 minigene construct. Evaluation of the Fluc/Rluc signal ratio enables a fast and accurate way to measure exon 20 inclusion. Further, we developed a secondary assay that measures ELP1 splicing in FD patient-derived fibroblasts. Here we demonstrate the quality and reproducibility of our screening method. Development and implementation of this screening platform has allowed us to efficiently screen for new compounds that robustly and specifically enhance ELP1 pre-mRNA splicing.