Lowering levels of reelin in entorhinal cortex layer II-neurons results in lowered levels of intracellular amyloid-ß.

Projection neurons in the anteriolateral part of entorhinal cortex layer II are the predominant cortical site for hyper-phosphorylation of tau and formation of neurofibrillary tangles in prodromal Alzheimer's disease. A majority of layer II projection neurons in anteriolateral entorhinal cortex are unique among cortical excitatory neurons by expressing the protein reelin. In prodromal Alzheimer's disease, these reelin-expressing neurons are prone to accumulate intracellular amyloid-ß, which is mimicked in a rat model that replicates the spatio-temporal cascade of the disease. Two important findings in relation to this are that reelin-signalling downregulates tau phosphorylation, and that oligomeric amyloid-ß interferes with reelin-signalling. Taking advantage of this rat model, we used proximity ligation assay to assess whether reelin and intracellular amyloid-ß directly interact during early, pre-plaque stages in anteriolateral entorhinal cortex layer II reelin-expressing neurons. We next made a viral vector delivering micro-RNA against reelin, along with a control vector, and infected reelin-expressing anteriolateral entorhinal cortex layer II-neurons to test whether reelin levels affect levels of intracellular amyloid-ß and/or amyloid precursor protein. We analysed 25.548 neurons from 24 animals, which results in three important findings. First, in reelin-expressing anteriolateral entorhinal cortex layer II-neurons, reelin and intracellular amyloid-ß engage in a direct protein-protein interaction. Second, injecting micro-RNA against reelin lowers reelin levels in these neurons, amounting to an effect size of 1.3-4.5 (Bayesian estimation of Cohen's d effect size, 95% credible interval). This causes a concomitant reduction of intracellular amyloid-ß ranging across three levels of aggregation, including a reduction of Aß42 monomers/dimers amounting to an effect size of 0.5-3.1, a reduction of Aß prefibrils amounting to an effect size of 1.1-3.5 and a reduction of protofibrils amounting to an effect size of 0.05-2.1. Analysing these data using Bayesian estimation of mutual information furthermore reveals that levels of amyloid-ß are dependent on levels of reelin. Third, the reduction of intracellular amyloid-ß occurs without any substantial associated changes in levels of amyloid precursor protein. We conclude that reelin and amyloid-ß directly interact at the intracellular level in the uniquely reelin-expressing projection neurons in anteriolateral entorhinal cortex layer II, where levels of amyloid-ß are dependent on levels of reelin. Since amyloid-ß is known to impair reelin-signalling causing upregulated phosphorylation of tau, our findings are likely relevant to the vulnerability for neurofibrillary tangle-formation of this entorhinal neuronal population.

Combined targeting of pathways regulating synaptic formation and autophagy attenuates Alzheimer's disease pathology in mice.

All drug trials completed to date have fallen short of meeting the clinical endpoint of significantly slowing cognitive decline in Alzheimer's disease (AD) patients. In this study, we repurposed two FDA-approved drugs, Fasudil and Lonafarnib, targeting synaptic formation (i.e., Wnt signaling) and cellular clearance (i.e., autophagic) pathways respectively, to test their therapeutic potential for attenuating AD-related pathology. We characterized our 3xTg AD mouse colony to select timepoints for separate and combinatorial treatment of both drugs while collecting cerebrospinal fluid (CSF) using an optimized microdialysis method. We found that treatment with Fasudil reduced Aß at early and later stages of AD, whereas administration of Lonafarnib had no effect on Aß, but did reduce tau, at early stages of the disease. Induction of autophagy led to increased size of amyloid plaques when administered at late phases of the disease. We show that combinatorial treatment with both drugs was effective at reducing intraneuronal Aß and led to improved cognitive performance in mice. These findings lend support to regulating Wnt and autophagic pathways in order to attenuate AD-related pathology.

In Vivo Microdialysis in Mice Captures Changes in Alzheimer's Disease Cerebrospinal Fluid Biomarkers Consistent with Developing Pathology.

BACKGROUND: Preclinical models of Alzheimer's disease (AD) can provide valuable insights into the onset and progression of the disease, such as changes in concentrations of amyloid-ß (Aß) and tau in cerebrospinal fluid (CSF). However, such models are currently underutilized due to limited advancement in techniques that allow for longitudinal CSF monitoring. OBJECTIVE: An elegant way to understand the biochemical environment in the diseased brain is intracerebral microdialysis, a method that has until now been limited to short-term observations, or snapshots, of the brain microenvironment. Here we draw upon patient-based findings to characterize CSF biomarkers in a commonly used preclinical mouse model for AD. METHODS: Our modified push-pull microdialysis method was first validated ex vivo with human CSF samples, and then in vivo in an AD mouse model, permitting assessment of dynamic changes of CSF Aß and tau and allowing for better translational understanding of CSF biomarkers. RESULTS: We demonstrate that CSF biomarker changes in preclinical models capture what is observed in the brain; with a decrease in CSF Aß observed when plaques are deposited, and an increase in CSF tau once tau pathology is present in the brain parenchyma. We found that a high molecular weight cut-off membrane allowed for simultaneous sampling of Aß and tau, comparable to CSF collection by lumbar puncture in patients. CONCLUSION: Our approach can further advance AD and other neurodegenerative research by following evolving neuropathology along the disease cascade via consecutive sampling from the same animal and can additionally be used to administer pharmaceutical compounds and assess their efficacy.

Bridging the Gap Between Fluid Biomarkers for Alzheimer's Disease, Model Systems, and Patients.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease characterized by the accumulation of two proteins in fibrillar form: amyloid-ß (Aß) and tau. Despite decades of intensive research, we cannot yet pinpoint the exact cause of the disease or unequivocally determine the exact mechanism(s) underlying its progression. This confounds early diagnosis and treatment of the disease. Cerebrospinal fluid (CSF) biomarkers, which can reveal ongoing biochemical changes in the brain, can help monitor developing AD pathology prior to clinical diagnosis. Here we review preclinical and clinical investigations of commonly used biomarkers in animals and patients with AD, which can bridge translation from model systems into the clinic. The core AD biomarkers have been found to translate well across species, whereas biomarkers of neuroinflammation translate to a lesser extent. Nevertheless, there is no absolute equivalence between biomarkers in human AD patients and those examined in preclinical models in terms of revealing key pathological hallmarks of the disease. In this review, we provide an overview of current but also novel AD biomarkers and how they relate to key constituents of the pathological cascade, highlighting confounding factors and pitfalls in interpretation, and also provide recommendations for standardized procedures during sample collection to enhance the translational validity of preclinical AD models.