Aims

Biased G protein-coupled receptor (GPCR) signaling is a comparatively new area of investigation that is transforming the conceptualization of GPCR signaling and its application for disease therapeutics. Surprisingly, biased GPCR signaling is a largely unexplored area in Alzheimer’s disease (AD) research. In AD, the brain reacts to the pathological accumulation of amyloid-β (Aβ) and tau with an early and profound neuroinflammatory response, which includes alterations in the functional profile of microglia, astrocytes, and oligodendrocytes. Although neuroinflammation is an integral component and putative driver of AD pathogenesis, the cell type-specific mechanisms that characterize this response and its impact on cognitive decline are incompletely understood.

Methods

We utilized a CRISPR/Cas9 strategy to develop a G protein-biased GPCR 3 (GPR3) mouse model that lacks GPR3-mediated β-arrestin signaling for immunohistochemical, biochemical, fluorescent activated cell sorting (FACS), and transcriptome analysis.

Results

We determined that biased GPR3 mice exhibit normal cognitive and behavioral functions, which are disrupted in Gpr3-deficient mice. We further determined that biased GPR3 signaling reduces Aβ levels and the Aβ plaque burden. Intriguingly, we found that GPR3 is more abundantly expressed in astrocytes relative to microglia and neurons. Specifically, biased GPR3 signaling leads to alterations in the profile, function, and activation of microglia and astrocytes and reveals a putatively important cell-autonomous role and non-cell-autonomous bridge between microglial activation and neuronal survival in AD.

Conclusions

These studies identify the previously unappreciated cell type-specific functions of GPR3 and biased GPR3 signaling in AD. These studies also establish the in vivo impact of biased GPR3 signaling on neuronal- and glial-dependent pathways and provide proof of concept for the development of safer GPCR-targeting therapeutics with more directed pharmacological action for AD.

Aims

Established transgenic mouse models such as 5xFAD resemble hallmarks of Alzheimer’s disease (AD), such as amyloid-β deposition, chronic neuroinflammation, and behavioural deficits. Activated microglia cluster around β-amyloid deposits, suggesting that phagocytosis by this cell type is important for either the formation or clearance of amyloid plaques. Chronic inflammatory activation of microglia (e.g. by interferon-alpha or -gamma signalling) and associated cytokine production is largely mediated through STAT (“Signal Transducer and Activator of Transcription”)-dependent transcription of cytokine-responsive genes. By crossing 5xFAD with STAT1-deficient (STAT1^-/-) mice, we aimed at studying a presumed inflammation-modulating effect by suppressing interferon-alpha and -gamma signalling, focussing on microglial function and phagocytic activity, amyloid plaque pathology, and behavioural deficits at different time points.

Methods

Following a comprehensive behavioural analysis focussing on motor and recognition memory tasks, immunohistochemical analyses of disease-associated markers were conducted on brain samples from 5xFAD and 5xFAD/STAT1^-/- mice. Extracellular amyloid-β plaque pathology was assessed with 3D lightsheet fluorescence microscopy of brain hemispheres. In addition, electrochemiluminescence assays and gene expression analyses (transcriptome analysis, qPCR) were carried out in hippocampal and cortical brain samples.

Results

A deficiency in interferon-alpha & -gamma signalling significantly ameliorated both memory deficits and motor function pathology in 5xFAD mice. Immunohistochemical analyses and electrochemiluminescence assays revealed an unchanged overall amyloid pathology at later stages. Interestingly, young 5xFAD/STAT1^-/- mice showed a lower Aβ plaque load, linked to an altered phagocytic phenotype of immune cells. A bulk transcriptome analysis of the hippocampus and subsequent qPCR validation demonstrated various differentially expressed genes associated with neurogenesis, protective immune response and reduced cellular damage.

Conclusions

These findings highlight the crucial role of inflammatory mechanisms in the pathogenesis of AD and specifically point to modulating microglia activation and function as potential therapeutic intervention.

Aims

Emerging evidence suggests that peripheral immune cells contribute to Alzheimer's disease (AD) neuropathogenesis. Among these, mast cells are known for their functions in allergic reactions and neuroinflammation; however, little is known about their role in AD.

Methods

We crossed 5XFAD mice with mast cell-deficient strains Cpa3^Cre/+ and Kit^Wv/Wv mice. We performed behavior experiments, single-cell RNA-sequencing, biochemical assays, and immunofluorescence staining to observe the effects on AD-related neuropathology and cognitive impairment.

Results

We found that dural mast cells are able to scan cerebrospinal fluid (CSF) contents and thereby indirectly monitor the brain environment. In addition, mast cell depletion improved contextual fear conditioning in 5XFAD mice without affecting cued fear conditioning, anxiety-like behavior, or amyloid burden. Furthermore, mast cell depletion led to an upregulation of transcriptomic signatures for putatively protective disease-associated microglia and resulted in reduced markers indicative of reactive astrocytes.

Conclusions

We hypothesize a system of bidirectional communication between dural mast cells and the brain, where mast cells respond to signals from the brain environment by expressing immune-regulatory mediators, impacting cognition and glial cell function. These findings highlight mast cells as potential therapeutic targets for AD.

Aims

To induce restoration of degenerated dopaminergic neurons with non-invasive viral vector-mediated neurotrophic factor delivery achieved by targeted blood-brain barrier opening (BBBO) with theranostic ultrasound (ThUS) in vivo.

Methods

BBBO with focused ultrasound in conjunction with systemically administered microbubbles is a safe and reversible technique for targeted drug delivery to the brain, providing a non-invasive alternative to direct intracranial injection. A novel configuration for transcranial BBBO developed by our group, called ThUS, was used to perform simultaneous bilateral delivery of AAV encoding human neurturin (NTRN) to the murine substantia nigra (Fig. 1A-B).

To induce neurodegeneration, 16-week-old male C57BL/6J mice (Charles River, Kingston, NY) underwent a sub-acute dosing scheme of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) consisting of daily intraperitoneal injections (30 mg/kg) for 5 days. After a 21-day period of neurodegeneration, mice were anesthetized with isoflurane anesthesia and underwent 2 minutes of ThUS-mediated BBBO immediately after intravenous co-injection of AAV9-hSynapsin-hNTRN (Vector Biolabs) and house-made polydisperse microbubbles (8e8 microbubbles/mL). 90 days post-BBBO, mice were sacrificed for histology with tyrosine hydroxylase (TH) staining (Fig. 1C).

Results

Quantification of GFP fluorescence area revealed a significant ~96% increase in dendritic network density in the substantia nigra pars reticulata (SNr) in MPTP mice which received ThUS+AAV (Fig. 2A-C). No significant differences in SNr dendrite density were observed between groups receiving either ThUS or AAV alone compared to MPTP mice which did not receive treatment intervention (Fig. 2C). A 2-fold increase in cell body density within the pars compacta (SNc) was observed in MPTP mice treated with ThUS+AAV relative to MPTP mice alone (Fig. 2D).

Conclusions

ThUS-mediated AAV-hNTRN delivery induced histological evidence of neurorestoration in Parkinsonian mice, demonstrating the potential for a more effective and non-invasive option for gene delivery in PD treatment.

Aims

Aberrant low gamma secretase activity is associated with most of the presenilin mutations that underlie familial Alzheimer’s disease (fAD). However, the role of gamma secretase in the more prevalent sporadic AD (sAD) remains unaddressed.

Methods

Using molecular approaches, we studied the cell autonomous function of ApoE in cells, neurons and in mice.

Results

We report that apolipoprotein E (ApoE), the most important genetic risk factor of sAD, is expressed in human and monkey neurons. In addition, ApoE interacts with gamma secretase and inhibits it with substrate specificity in cell-autonomous manners through its conserved C-terminal region (CT). This ApoE CT-mediated inhibitory activity is differentially compromised in different ApoE isoforms, resulting in an ApoE2 > ApoE3 > ApoE4 potency rank order inversely correlating to their associated AD risk. Interestingly, in an AD mouse model, neuronal ApoE CT migrates to amyloid plaques in the subiculum from other regions and alleviates the plaque burden.

Conclusions

Together, our data reveal a hidden role of ApoE as a gamma secretase inhibitor with substrate specificity and suggest that this precision gamma-inhibition by ApoE may protect against the risk of sAD.

Aims

To improve clinical translatability of non-clinical in-vivo Alzheimer’s disease (AD) models, a humanized APP knock-in mouse model (APP^SAA) was recently created (Xia, D. et al.). The genetic modifications lead to increased Aβ42/40 ratios in AD relevant tissues, resulting in an age-dependent amyloid deposition. Here we assess the value of this model for non-clinical efficacy studies of experimental drugs with diverse mechanisms of action to facilitate the development of novel AD therapeutics.

Methods

APP^SAA mice and WT controls were aged and sacrificed at various time points. In addition, APP^SAA mice were treated for 3 months from the age of 3 months onward with an NLRP3-inhibitor, an in-house developed calcium homeostasis modulator or vehicle. The major pathological hallmarks were investigated with biochemical and immunohistological assays. Since APP^SAA mice lack an obvious behavioral phenotype, synaptic plasticity was investigated to serve as a functional readout.

Results

Cortical Aβ40, Aβ42 and pyroglutamate modified Aβ42 (N3pE-42) levels were shown to be affected with age. In addition, cortical amyloid plaques, surrounded by activated microglia, were shown to be present already at an age of 3 months, albeit at a low level. With age, the number of cortical plaques increased. Interestingly, several of the observed hallmarks were counteracted by compound intervention. In addition we were able to confirm the presence of endogenous phospho-Tau positive dystrophic neurites within these plaques, clearly demonstrating the translational relevance of this model. At an age of 4 and 6 months, defects in synaptic plasticity however, were not observed with the current stimulation protocol.

Conclusions

These data lead us to conclude that this knock-in model is a broadly applicable tool to investigate efficacy of disease-modifying drugs with diverse mechanisms of action even without directly targeting protein aggregates.

Aims

Tauopathies elicit different clinical phenotypes depending on the tau strain and regional susceptibility in the brain. CryoEm identification of tau proteoforms have revealed unique structures that classify various strains. Posttranslational modifications (PTMs) may also play a pivotal role in tau structure and conformer identity. We searched for novel PTMs of tau that could significantly impact tau structure that may be enriched in specific tauopathies.

Methods

We discovered that tau could be posttranslationally modified through a process called citrullination (citR), which changes arginine residues within proteins to a citrulline via enzyme peptidyl arginine deiminases (PADs). Structurally, this conversion alters charge, inter/ intramolecular interactions, and promote unfolding. To that end we created 11 highly specific citR tau antibodies that recognize sites within the N-terminal domain, proline-rich region, microtubule binding domain, and c-terminal domain. We tested how citrullination impacts tau phosphorylation, degradation, and aggregation. Further, we measured if citR tau was present in animal models and various human tauopathies.

Results

We found that citrullination impacts phosphorylation, degradation, and fibrillization. We also identified increased citR tau in 3 animal models of tauopathy. Mice showed early activation of PAD4 and increased citR tau in soluble, detergent soluble fractions, but was less abundant in detergent insoluble fractions. Importantly, citR tau increased in AD, PSP, CTE, Pick’s disease, ARTAG, PART, FTLD, and AGD. However, citR tau epitopes and morphology differed in various tauopathies suggesting that PADs may elicit conformer bias for different tau structures. These data suggest that the citrullination may recognize structural identity in certain tauopathies.

Conclusions

Overall, we identified a new and common PTM of tau that significantly impacts structure, function, and potentially stable proteoforms of tau. These results offer new therapeutic opportunities for various anti-tau approaches.

Aims

Methods

Results

Conclusions