Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer's disease, restores synapse integrity and memory in a disease mouse model.

Increasing evidence supports a role for deficient Wnt signaling in Alzheimer's disease (AD). Studies reveal that the secreted Wnt antagonist Dickkopf-3 (DKK3) colocalizes to amyloid plaques in AD patients. Here, we investigate the contribution of DKK3 to synapse integrity in healthy and AD brains. Our findings show that DKK3 expression is upregulated in the brains of AD subjects and that DKK3 protein levels increase at early stages in the disease. In hAPP-J20 and hAPPNL-G-F/NL-G-F mouse AD models, extracellular DKK3 levels are increased and DKK3 accumulates at dystrophic neuronal processes around plaques. Functionally, DKK3 triggers the loss of excitatory synapses through blockade of the Wnt/GSK3ß signaling with a concomitant increase in inhibitory synapses via activation of the Wnt/JNK pathway. In contrast, DKK3 knockdown restores synapse number and memory in hAPP-J20 mice. Collectively, our findings identify DKK3 as a novel driver of synaptic defects and memory impairment in AD.

Alzheimer's disease is the most common form of dementia worldwide. The cognitive decline typically observed in this condition is associated with the weakening and eventually the loss of synapses, the structures that allow neurons to communicate. Increasing evidence points to this deterioration being linked to deficiency in the Wnt signalling pathway, a cascade of molecular events crucial for brain function and development. The DKK protein family helps to tightly regulate the Wnt pathway by dampening its activity. Previous work suggests that DKK proteins could also be connected to Alzheimer's disease. For example, an elevated amount of DKK1 leads to synapse and memory defects in mice, while brain production of DKK1 is increased in individuals with late Alzheimer's. More recent studies show high levels of another DKK protein, DKK3, in Alzheimer's patients. This protein is also present in the harmful amyloid-ß aggregates, named 'plaques', that typically form in the brain in this condition. Despite these findings, how DKK3 participates in synaptic health remains unclear. To address this question, Martin-Flores, Podpolny et al. tracked DKK3 levels in the brains of Alzheimer's patients, revealing that they increase early in the disease. Additional experiments in Alzheimer's mouse models suggested that DKK3 secretion rise before amyloid-ß plaques form, with the protein then accumulating in abnormal neuronal structures present in the surroundings of these toxic deposits. Martin-Flores, Podpolny et al. then examined the impact of DKK3 on the Wnt pathway, and ultimately, on the balance between synapses that control neuronal activity. These experiments showed that elevated DKK3 levels are linked to a loss of synapses which are excitatory, with a concomitant increase in those that are inhibitory. Crucially, reducing DKK3 levels in a mouse model of Alzheimer's restored this synaptic balance and improved memory, highlighting DKK3 as a potential driver of cognitive impairment. Overall, these findings help to refine our understanding of the molecular mechanisms that contribute to synaptic impairment in Alzheimer's disease. They may also be relevant for researchers studying other conditions that involve aberrant activity of the Wnt pathway, such as cancer.

S-acylation of the Wnt receptor Frizzled-5 by zDHHC5 controls its cellular localization and synaptogenic activity in the rodent hippocampus.

Proper localization of receptors for synaptic organizing factors is crucial for synapse formation. Wnt proteins promote synapse assembly through Frizzled (Fz) receptors. In hippocampal neurons, the surface and synaptic localization of Fz5 is regulated by neuronal activity, but the mechanisms involved remain poorly understood. Here, we report that all Fz receptors can be post-translationally modified by S-acylation and that Fz5 is S-acylated on three C-terminal cysteines by zDHHC5. S-acylation is essential for Fz5 localization to the cell surface, axons, and presynaptic sites. Notably, S-acylation-deficient Fz5 is internalized faster, affecting its association with signalosome components at the cell surface. S-acylation-deficient Fz5 also fails to activate canonical and divergent canonical Wnt pathways. Fz5 S-acylation levels are regulated by the pattern of neuronal activity. In vivo studies demonstrate that S-acylation-deficient Fz5 expression fails to induce presynaptic assembly. Our studies show that S-acylation of Frizzled receptors is a mechanism controlling their localization and function.

A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease.

Synapse loss strongly correlates with cognitive decline in Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Deficient Wnt signaling contributes to synapse dysfunction and loss in AD. Consistently, a variant of the LRP6 receptor, (LRP6-Val), with reduced Wnt signaling, is linked to late-onset AD. However, the impact of LRP6-Val on the healthy and AD brain has not been examined. Knock-in mice, generated by gene editing, carrying this Lrp6 variant develop normally. However, neurons from Lrp6-val mice do not respond to Wnt7a, a ligand that promotes synaptic assembly through the Frizzled-5 receptor. Wnt7a stimulates the formation of the low-density lipoprotein receptor-related protein 6 (LRP6)-Frizzled-5 complex but not if LRP6-Val is present. Lrp6-val mice exhibit structural and functional synaptic defects that become pronounced with age. Lrp6-val mice present exacerbated synapse loss around plaques when crossed to the NL-G-F AD model. Our findings uncover a previously unidentified role for Lrp6-val in synapse vulnerability during aging and AD.

Microglial Expression of the Wnt Signaling Modulator DKK2 Differs between Human Alzheimer's Disease Brains and Mouse Neurodegeneration Models.

Wnt signaling is crucial for synapse and cognitive function. Indeed, deficient Wnt signaling is causally related to increased expression of DKK1, an endogenous negative Wnt regulator, and synapse loss, both of which likely contribute to cognitive decline in Alzheimer's disease (AD). Increasingly, AD research efforts have probed the neuroinflammatory role of microglia, the resident immune cells of the CNS, which have furthermore been shown to be modulated by Wnt signaling. The DKK1 homolog DKK2 has been previously identified as an activated response and/or disease-associated microglia (DAM/ARM) gene in a mouse model of AD. Here, we performed a detailed analysis of DKK2 in mouse models of neurodegeneration, and in human AD brain. In APP/PS1 and APPNL-G-F AD mouse model brains as well as in SOD1G93A ALS mouse model spinal cords, but not in control littermates, we demonstrated significant microgliosis and microglial Dkk2 mRNA upregulation in a disease-stage-dependent manner. In the AD models, these DAM/ARM Dkk2+ microglia preferentially accumulated close to ßAmyloid plaques. Furthermore, recombinant DKK2 treatment of rat hippocampal primary neurons blocked WNT7a-induced dendritic spine and synapse formation, indicative of an anti-synaptic effect similar to that of DKK1. In stark contrast, no such microglial DKK2 upregulation was detected in the postmortem human frontal cortex from individuals diagnosed with AD or pathologic aging. In summary, the difference in microglial expression of the DAM/ARM gene DKK2 between mouse models and human AD brain highlights the increasingly recognized limitations of using mouse models to recapitulate facets of human neurodegenerative disease.

Structural Analysis and Development of Notum Fragment Screening Hits.

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer's disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 µM. Analysis of the fragments' diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 µM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.

Design of a Potent, Selective, and Brain-Penetrant Inhibitor of Wnt-Deactivating Enzyme Notum by Optimization of a Crystallographic Fragment Hit.

Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.

Epigenetic repression of Wnt receptors in AD: a role for Sirtuin2-induced H4K16ac deacetylation of Frizzled1 and Frizzled7 promoters.

Growing evidence supports a role for deficient Wnt signalling in Alzheimer's disease (AD). First, the Wnt antagonist DKK1 is elevated in AD brains and is required for amyloid-ß-induced synapse loss. Second, LRP6 Wnt co-receptor is required for synapse integrity and three variants of this receptor are linked to late-onset AD. However, the expression/role of other Wnt signalling components remain poorly explored in AD. Wnt receptors Frizzled1 (Fzd1), Fzd5, Fzd7 and Fzd9 are of interest due to their role in synapse formation/plasticity. Our analyses showed reduced FZD1 and FZD7 mRNA levels in the hippocampus of human early AD stages and in the hAPPNLGF/NLGF mouse model. This transcriptional downregulation was accompanied by reduced levels of the pro-transcriptional histone mark H4K16ac and a concomitant increase of its deacetylase Sirt2 at Fzd1 and Fzd7 promoters in AD. In vitro and in vivo inhibition of Sirt2 rescued Fzd1 and Fzd7 mRNA expression and H4K16ac levels at their promoters. In addition, we showed that Sirt2 recruitment to Fzd1 and Fzd7 promoters is dependent on FoxO1 activity in AD, thus acting as a co-repressor. Finally, we found reduced levels of SIRT2 inhibitory phosphorylation in nuclear samples from human early AD stages with a concomitant increase in the SIRT2 phosphatase PP2C. This results in hyperactive nuclear Sirt2 and favours Fzd1 and Fzd7 repression in AD. Collectively, our findings define a novel role for nuclear hyperactivated SIRT2 in repressing Fzd1 and Fzd7 expression via H4K16ac deacetylation in AD. We propose SIRT2 as an attractive target to ameliorate AD pathology.

A Role for Frizzled and Their Post-Translational Modifications in the Mammalian Central Nervous System.

The Wnt pathway is a key signalling cascade that regulates the formation and function of neuronal circuits. The main receptors for Wnts are Frizzled (Fzd) that mediate diverse functions such as neurogenesis, axon guidance, dendritogenesis, synapse formation, and synaptic plasticity. These processes are crucial for the assembly of functional neuronal circuits required for diverse functions ranging from sensory and motor tasks to cognitive performance. Indeed, aberrant Wnt-Fzd signalling has been associated with synaptic defects during development and in neurodegenerative conditions such as Alzheimer's disease. New studies suggest that the localisation and stability of Fzd receptors play a crucial role in determining Wnt function. Post-translational modifications (PTMs) of Fzd are emerging as an important mechanism that regulates these Wnt receptors. However, only phosphorylation and glycosylation have been described to modulate Fzd function in the central nervous system (CNS). In this review, we discuss the function of Fzd in neuronal circuit connectivity and how PTMs contribute to their function. We also discuss other PTMs, not yet described in the CNS, and how they might modulate the function of Fzd in neuronal connectivity. PTMs could modulate Fzd function by affecting Fzd localisation and stability at the plasma membrane resulting in local effects of Wnt signalling, a feature particularly important in polarised cells such as neurons. Our review highlights the importance of further studies into the role of PTMs on Fzd receptors in the context of neuronal connectivity.

Wnt-Frizzled Signaling Regulates Activity-Mediated Synapse Formation.

The formation of synapses is a tightly regulated process that requires the coordinated assembly of the presynaptic and postsynaptic sides. Defects in synaptogenesis during development or in the adult can lead to neurodevelopmental disorders, neurological disorders, and neurodegenerative diseases. In order to develop therapeutic approaches for these neurological conditions, we must first understand the molecular mechanisms that regulate synapse formation. The Wnt family of secreted glycoproteins are key regulators of synapse formation in different model systems from invertebrates to mammals. In this review, we will discuss the role of Wnt signaling in the formation of excitatory synapses in the mammalian brain by focusing on Wnt7a and Wnt5a, two Wnt ligands that play an in vivo role in this process. We will also discuss how changes in neuronal activity modulate the expression and/or release of Wnts, resulting in changes in the localization of surface levels of Frizzled, key Wnt receptors, at the synapse. Thus, changes in neuronal activity influence the magnitude of Wnt signaling, which in turn contributes to activity-mediated synapse formation.

Striatal Synapse Degeneration and Dysfunction Are Reversed by Reactivation of Wnt Signaling.

Synapse degeneration in the striatum has been associated with the early stages of Parkinson's and Huntington's diseases (PD and HD). However, the molecular mechanisms that trigger synaptic dysfunction and loss are not fully understood. Increasing evidence suggests that deficiency in Wnt signaling triggers synapse degeneration in the adult brain and that this pathway is affected in neurodegenerative diseases. Here, we demonstrate that endogenous Wnt signaling is essential for the integrity of a subset of inhibitory synapses on striatal medium spiny neurons (MSNs). We found that inducible expression of the specific Wnt antagonist Dickkopf-1 (Dkk1) in the adult striatum leads to the loss of inhibitory synapses on MSNs and affects the synaptic transmission of D2-MSNs. We also discovered that re-activation of the Wnt pathway by turning off Dkk1 expression after substantial loss of synapses resulted in the complete recovery of GABAergic and dopamine synapse number. Our results also show that re-activation of the Wnt pathway leads to a recovery of amphetamine response and motor function. Our studies identify the Wnt signaling pathway as a potential therapeutic target for restoring neuronal circuits after synapse degeneration.

Miro1-dependent mitochondrial dynamics in parvalbumin interneurons.

The spatiotemporal distribution of mitochondria is crucial for precise ATP provision and calcium buffering required to support neuronal signaling. Fast-spiking GABAergic interneurons expressing parvalbumin (PV+) have a high mitochondrial content reflecting their large energy utilization. The importance for correct trafficking and precise mitochondrial positioning remains poorly elucidated in inhibitory neurons. Miro1 is a Ca²+-sensing adaptor protein that links mitochondria to the trafficking apparatus, for their microtubule-dependent transport along axons and dendrites, in order to meet the metabolic and Ca2+-buffering requirements of the cell. Here, we explore the role of Miro1 in PV+ interneurons and how changes in mitochondrial trafficking could alter network activity in the mouse brain. By employing live and fixed imaging, we found that the impairments in Miro1-directed trafficking in PV+ interneurons altered their mitochondrial distribution and axonal arborization, while PV+ interneuron-mediated inhibition remained intact. These changes were accompanied by an increase in the ex vivo hippocampal γ-oscillation (30-80 Hz) frequency and promoted anxiolysis. Our findings show that precise regulation of mitochondrial dynamics in PV+ interneurons is crucial for proper neuronal signaling and network synchronization.

Wnt Signaling Through Nitric Oxide Synthase Promotes the Formation of Multi-Innervated Spines.

Structural plasticity of synapses correlates with changes in synaptic strength. Dynamic modifications in dendritic spine number and size are crucial for long-term potentiation (LTP), the cellular correlate of learning and memory. Recent studies have suggested the generation of multi-innervated spines (MIS), in the form of several excitatory presynaptic inputs onto one spine, are crucial for hippocampal memory storage. However, little is known about the molecular mechanisms underlying MIS formation and their contribution to LTP. Using 3D enhanced resolution confocal images, we examined the contribution of Wnt synaptic modulators in MIS formation in the context of LTP. We show that blockage of endogenous Wnts with specific Wnt antagonists supresses the formation of MIS upon chemical LTP induction in cultured hippocampal neurons. Gain- and loss-of-function studies demonstrate that Wnt7a signaling promotes MIS formation through the postsynaptic Wnt scaffold protein Disheveled 1 (Dvl1) by stimulating neuronal nitric oxide (NO) synthase (nNOS). Subsequently, NO activates soluble guanylyl cyclase (sGC) to increase MIS formation. Consistently, we observed an enhanced frequency and amplitude of excitatory postsynaptic currents. Collectively, our findings identify a unique role for Wnt secreted proteins through nNOS/NO/sGC signaling to modulate MIS formation during LTP.

5-Phenyl-1,3,4-oxadiazol-2(3H)-ones Are Potent Inhibitors of Notum Carboxylesterase Activity Identified by the Optimization of a Crystallographic Fragment Screening Hit.

Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of the role Notum plays in disease, supporting the need to discover new small-molecule inhibitors. A crystallographic X-ray fragment screen was performed, which identified fragment hit 1,2,3-triazole 7 as an attractive starting point for a structure-based drug design hit-to-lead program. Optimization of 7 identified oxadiazol-2-one 23dd as a preferred example with properties consistent with drug-like chemical space. Screening 23dd in a cell-based TCF/LEF reporter gene assay restored the activation of Wnt signaling in the presence of Notum. Mouse pharmacokinetic studies with oral administration of 23dd demonstrated good plasma exposure and partial blood-brain barrier penetration. Significant progress was made in developing fragment hit 7 into lead 23dd (>600-fold increase in activity), making it suitable as a new chemical tool for exploring the role of Notum-mediated regulation of Wnt signaling.

Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo.

Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance.

Rational design of a conformation-specific antibody for the quantification of Aß oligomers.

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.

Correction: Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

[This corrects the article DOI: 10.1371/journal.pbio.3000414.].

Single-Cell Quantification of mRNA Expression in The Human Brain.

RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis.

Wnt Signaling Deregulation in the Aging and Alzheimer's Brain.

Growing evidence suggests that synaptic signaling is compromised in the aging brain and in Alzheimer's disease (AD), contributing to synaptic decline. Wnt signaling is a prominent pathway at the synapse and is required for synaptic plasticity and maintenance in the adult brain. In this review, we summarize the current knowledge on deregulation of Wnt signaling in the context of aging and AD. Emerging studies suggest that enhancing Wnt signaling could boost synaptic function during aging, and ameliorate synaptic pathology in AD. Although further research is needed to determine the precise contribution of deficient Wnt signaling to AD pathogenesis, targeting Wnt signaling components may provide novel therapeutic avenues for synapse protection or restoration in the brain.

Deficient Wnt Signaling and Synaptic Vulnerability in Alzheimer's Disease: Emerging Roles for the LRP6 Receptor.

Synapse dysfunction and loss represent critical early events in the pathophysiology of Alzheimer's disease (AD). While extensive research has elucidated the direct synaptotoxic effects of Amyloid-ß (Aß) oligomers, less is known about how signaling pathways at the synapse are affected by Aß. A better understanding of the cellular and molecular mechanisms underlying synaptic vulnerability in AD is key to illuminating the determinants of AD susceptibility and will unveil novel therapeutic avenues. Canonical Wnt signaling through the Wnt co-receptor LRP6 has a critical role in maintaining the structural and functional integrity of synaptic connections in the adult brain. Accumulating evidence suggests that deficient Wnt signaling may contribute to AD pathology. In particular, LRP6 deficiency compromises synaptic function and stability, and contributes to Aß production and plaque formation. Here, we review the role of Wnt signaling for synaptic maintenance in the adult brain and the contribution of aberrant Wnt signaling to synaptic degeneration in AD. We place a focus on emerging evidence implicating the LRP6 receptor as an important modulator of AD risk and pathology.