Introduction Blood-based biomarkers that can aid diagnosis of Parkinson’s Disease (PD) dementia (PDD), and predict PDD onset in people with PD are urgently needed. Plasma small extracellular vesicles (SEV) reflect molecular changes in living human brain. Next-generation RNA-sequencing (RNA-Seq) of PDD plasma SEV can advance our understanding of PDD molecular pathology, and identify blood-based biomarkers. Hence, we conducted the first comprehensive transcriptomic analysis of PDD plasma SEV. Methods We investigated plasma SEV RNA of PDD, PD, and people without PD or dementia (Controls) using RNA-Seq (n=15/group; N=45). SEV were separated by ultracentrifugation, and characterized by cryo-transmission electron microscopy. We identified differentially expressed genes (DEGs) in PDD plasma SEV using an edgeR-based data analysis pipeline and verified them by high-throughput qPCR. We assessed functional implications of identified DEGs using Ingenuity Pathway and causal network analyses. Results We identified 51 transcriptome-wide significant (edgeR q<0.05) DEGs, compared to controls, and 26 transcriptome-wide significant DEGs, compared to PD, in PDD plasma SEV. The identified DEGs, which included WNT5A, MAPT, FOSB, MIR324, MIR574, MIR3161, and MIR6821 were significantly enriched among Tetrahydrofolate salvage, Reelin signalling, tRNA splicing, Wnt signalling, and ERBB signalling pathways. We identified eight potential multiplex plasma SEV RNA biomarker assays that can distinguish PDD from PD with at least 80% sensitivity and specificity using an artificial intelligence-based algorithm. Conclusion Future research on the identified dysfunctional molecular pathways may facilitate discovery of novel therapeutic targets for PDD. Diagnostic biomarker potential of the derived multiplex RNA biomarker assays should be investigated by larger clinical studies.

Synapse loss driven by amyloid-(3 (A(3) is an early event in Alzheimer's disease (AD). Although the mechanism by which A(3 drives synapse loss remain poorly understood data indicate that a disruption of Wnt signalling plays an important part. We have shown that A(3 exerts its effects on synapses through Dickkopf-1 (Dkk1), a secreted protein that acts upon Wnt signalling via a direct interaction with the canonical Wnt pathway co-receptor proteins, LRP5 and LRP6, preventing their interaction with the receptor Frizzled. This antagonises canonical, Wnt/(3-catenin, signalling and allows concomitant activation of non-canonical signalling pathways. We contend that it is the switch from canonical to non-canonical Wnt signalling activity that drives synapse loss and subsequent cognitive impairment in AD, driven by A(3 and mediated by Dkk1. Preventing the Dkk1-LRP5/6 interaction could protect synapses and cognition against A(3 by maintaining canonical Wnt signalling. To test this, we mapped the Dkk1-LRP6 interaction by peptide array and identified a small peptide able to disrupt the Dkk1-LRP6 interaction. This Dkk1-LRP6 'disruptor' peptide dose dependently restores canonical Wnt signalling in the presence of Dkk1; blocks Dkk1-driven dendritic spine loss in primary rat cortical cultures and the accompanying increase in endogenous A(3 production; and when administered intracerebroventricularly to a rat acute A(3 model, blocks A(3-driven cognitive impairment. These data support our contention that the ability of A(3 to induce Dkk1 and the effects of Dkk1 on LRP6 are an important element in AD aetiopathology and establish Dkk1 as a therapeutic target for protecting synapse and cognition in AD.

It has been suggested that a-synuclein (aSyn), a major player in Parkinson's disease (PD), plays a role in Alzheimer's disease (AD). Several reports have also concluded that aSyn and amyloid-beta (Ab) are mechanistically linked, although how is unclear. Synapse loss is an early feature in both PD and AD and held to be the driver of both diseases. We have previously uncovered a signalling pathway required for Ab-driven dendritic spine loss - a non-canonical branch of Wnt signalling known as the Wnt/Planar Cell Polarity (Wnt/PCP) pathway. We asked if a synaptotoxic form of aSyn known to impact dendritic spines, the A53T autosomal dominant PD mutant form of aSyn (A53T-aSyn), might act on synapses through the same pathway. Here, by blocking all Wnt activity with the porcupine inhibitor IWP2, we show that A53T-aSyn driven spine loss is Wnt-dependent. By silencing Daam1, which is unique to Wnt/PCP, we show that A53T-aSyn spine loss is Daam1-dependent. Finally, using the pan-ROCK inhibitor fasudil indicates the mechanism also involves ROCK1/2, which Daam1 signals to via RhoA to modulate actin cytoskeletal dynamics within dendritic spines. Together, these observations indicate that A53T-aSyn-driven spine loss involves the Wnt/PCP pathway, the same pathway that mediates Ab synaptotoxicity. This indicates that Ab and aSyn are mechanistically connected and that a common pathway is responsible for synapse loss in AD and PD. It also begins to explain why this group of neurodegenerative diseases have many features in common and suggests that drugs which target Wnt/PCP could be of benefit for both AD and PD. ### Competing Interest Statement The authors have declared no competing interest.

Synapse loss driven by amyloid-β (Aβ) is an early event in Alzheimer's disease (AD). Although the mechanism by which Aβ drives synapse loss remain poorly understood data indicate that a disruption of Wnt signalling plays an important part. We have shown that Aβ exerts its effects on synapses through Dickkopf-1 (Dkk1), a secreted protein that acts upon Wnt signalling via a direct interaction with the canonical Wnt pathway co-receptor proteins, LRP5 and LRP6, preventing their interaction with the receptor Frizzled. This antagonises canonical, Wnt/β-catenin, signalling and allows concomitant activation of non-canonical signalling pathways. We contend that it is the switch from canonical to non-canonical Wnt signalling activity that drives synapse loss and subsequent cognitive impairment in AD, driven by Aβ and mediated by Dkk1. Preventing the Dkk1-LRP5/6 interaction could protect synapses and cognition against Aβ by maintaining canonical Wnt signalling. To test this, we mapped the Dkk1-LRP6 interaction by peptide array and identified a small peptide able to disrupt the Dkk1-LRP6 interaction. This Dkk1-LRP6 ‘disruptor’ peptide dose dependently restores canonical Wnt signalling in the presence of Dkk1; blocks Dkk1-driven dendritic spine loss in primary rat cortical cultures and the accompanying increase in endogenous Aβ production; and when administered intracerebroventricularly to a rat acute Aβ model, blocks Aβ-driven cognitive impairment. These data support our contention that the ability of Aβ to induce Dkk1 and the effects of Dkk1 on LRP6 are an important element in AD aetiopathology and establish Dkk1 as a therapeutic target for protecting synapse and cognition in AD.

Lithium is prescribed as a mood stabilizer for bipolar disorder and severe depression. However, the mechanism of action of lithium is unknown and there are major side effects associated with prolonged medication. This motivates a search for safer alternative drug repurposing candidates. Given that the drug mechanism may be encoded in transcriptional changes, we generated the gene expression profile for acute lithium treatment of cortical neuronal cultures. We found that the lithium-associated transcription response harbors a significant component that is the reverse of that seen in human brain samples from patients with major depression, bipolar disorder, and a mouse model of depression. Interrogating publicly available drug-driven expression data, we found that cardiotonic steroids drive gene expression in a correlated manner to our acute lithium profile. An analysis of the psychiatric medication cohort of the Norwegian Prescription Database showed that cardiotonic prescription is associated with a lower incidence of lithium prescription. Our transcriptional and epidemiological observations point towards cardiotonic steroids as possible repurposing candidates for lithium. These observations motivate a controlled trial to establish a causal connection and genuine therapeutic benefit in the context of depression.

Alzheimer’s disease (AD) is an escalating global challenge, with more than 40 million people affected, and this number is projected to increase to more than 100 million by 2050. While amyloid-targeting antibody treatments (lecanemab and donanemab) are a significant step forward, the benefits of these therapies remain limited. This highlights the necessity for safe and effective compounds that offer greater therapeutic benefits to the majority of individuals with or at risk of AD. Drug repurposing allows for a cost-effective, time-efficient strategy to accelerate the availability of treatments, owing to the availability of safety information. This study focuses on the third iteration of the Delphi consensus programme aimed at identifying new high-priority drug candidates for repurposing in AD. An international expert panel comprising academics, clinicians and industry representatives was convened. Through a combination of anonymized drug nominations, systemic evidence reviews, iterative consensus rankings, and lay advisory inputs, drug candidates were evaluated and ranked based on rational, non-clinical, and clinical evidence and overall safety profiles. Among the 80 candidates that were nominated by the expert panel, seven underwent review, with only three candidates meeting the following consensus criteria of relevant mechanisms for targeting neurodegenerative pathways, non-clinical efficacy, and tolerability in older individuals. The three agents were: [1] the live attenuated herpes zoster (HZ) vaccine (Zostavax) [2], sildenafil, a phosphodiesterase-5 (PDE-5) inhibitor, and [3] riluzole, a glutamate antagonist. The HZ vaccine additionally offers potential for population-level dementia risk reduction. This Delphi consensus identified three high-priority drug repurposing candidates for AD with favourable safety profiles and mechanistic plausibility, which are considered suitable for pragmatic clinical trials, including remote or hybrid designs. The PROTECT platform, which supports international cohorts in the UK, Norway, and Canada, offers a well-established means to conduct such trials effectively, thus helping to accelerate the evaluation and potential deployment of these drug candidates to benefit individuals with or at risk for AD.

We aim to investigate efficacies of Ras homolog (Rho)-associated kinases (ROCK) inhibitors on Alzheimer’s disease (AD) pathological proteins in human induced pluripotent stem cell (iPSC)-differentiated human neurons and the P301S tau transgenic mouse model (PS19). Quantitative liquid chromatography-mass spectrometry (LC-MS/MS) and targeted ELISA were implemented to investigate the effect of treatment with fasudil or its derivatives on the human neurons and brains from PS19 mice. We explored the efficacy of these ROCK inhibitors in reducing tau phosphorylation, and the brain proteomic profiles after their administration in mice. We found a significant negative correlation between the brain levels of phosphorylated tau (pTau) and both fasudil and its metabolite hydroxyfasudil. Human neuronal culture exposed to selective ROCK inhibitors exhibited similar efficacies. Proteomic profiling of mice brains exposed to fasudil revealed the activation of the mitochondrial tricarboxylic acid (TCA) cycle and blood-brain barrier (BBB) gap junction metabolic pathways. Our results provide evidence to support the development of ROCK inhibitors as AD therapeutics.

Synaptogenesis involves the transformation of dendritic filopodial contacts into stable connections with the exact apposition of synaptic components. Signalling triggered by Wnt/β-catenin and calcium has been postulated to aid this process. However, it is unclear how such a signalling process orchestrates synapse formation to organise the spatial arrangement of synapses along dendrites. We show that WNT7A is loaded on dynamic dendritic filopodia during spine formation in human cortical neurons. WNT7A is present at the tips of the filopodia and the contact sites with dendrites of neighbouring neurons, triggering spatially restricted localisation of the Wnt co-receptor LRP6. Here, we demonstrate that WNT7A at filopodia tips leads to the induction of calcium transients, the clustering of pre- and postsynaptic proteins, and the subsequent transformation into mature spines. Although soluble WNT7A protein can also support synaptogenesis, it fails to provide this degree of spatial information for spine formation and calcium transients, and synaptic markers are induced ectopically along the dendrites. Our data suggest that dendritic filopodia are WNT7A-bearing cytonemes required for focal calcium signalling and initiation of synapse formation, and provide an elegant mechanism for orchestrating the positioning of synapses along dendrites.

Research evidence indicating common metabolic mechanisms through which type 2 diabetes mellitus (T2DM) increases risk of late-onset Alzheimer's dementia (LOAD) has accumulated over recent decades. The aim of this systematic review is to provide a comprehensive review of common mechanisms, which have hitherto been discussed in separate perspectives, and to assemble and evaluate candidate loci and epigenetic modifications contributing to polygenic risk linkages between T2DM and LOAD. For the systematic review on pathophysiological mechanisms, both human and animal studies up to December 2023 are included. For the qualitative meta-analysis of genomic bases, human association studies were examined; for epigenetic mechanisms, data from human studies and animal models were accepted. Papers describing pathophysiological studies were identified in databases, and further literature gathered from cited work. For genomic and epigenomic studies, literature mining was conducted by formalised search codes using Boolean operators in search engines, and augmented by GeneRif citations in Entrez Gene, and other sources (WikiGenes, etc.). For the systematic review of pathophysiological mechanisms, 923 publications were evaluated, and 138 gene loci extracted for testing candidate risk linkages. 3 57 publications were evaluated for genomic association and descriptions of epigenomic modifications. Overall accumulated results highlight insulin signalling, inflammation and inflammasome pathways, proteolysis, gluconeogenesis and glycolysis, glycosylation, lipoprotein metabolism and oxidation, cell cycle regulation or survival, autophagic-lysosomal pathways, and energy. Documented findings suggest interplay between brain insulin resistance, neuroinflammation, insult compensatory mechanisms, and peripheral metabolic dysregulation in T2DM and LOAD linkage. The results allow for more streamlined longitudinal studies of T2DM-LOAD risk linkages.

IntroductionThe goal of this study is to explore the pharmacological potential of the amyloid-reducing vasodilator fasudil, a selective Ras homolog (Rho)-associated kinases (ROCK) inhibitor, in the P301S tau transgenic mouse model (Line PS19) of neurodegenerative tauopathy and Alzheimer's disease (AD).MethodsWe used LC-MS/MS, ELISA and bioinformatic approaches to investigate the effect of treatment with fasudil on the brain proteomic profile in PS19 tau transgenic mice. We also explored the efficacy of fasudil in reducing tau phosphorylation, and the potential beneficial and/or toxic effects of its administration in mice.ResultsProteomic profiling of mice brains exposed to fasudil revealed the activation of the mitochondrial tricarboxylic acid (TCA) cycle and blood-brain barrier (BBB) gap junction metabolic pathways. We also observed a significant negative correlation between the brain levels of phosphorylated tau (pTau) at residue 396 and both fasudil and its metabolite hydroxyfasudil.ConclusionsOur results provide evidence on the activation of proteins and pathways related to mitochondria and BBB functions by fasudil treatment and support its further development and therapeutic potential for AD.

The pan Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor fasudil acts as a vasodilator and has been used as a medication for post-cerebral stroke for the past 29 years in Japan and China. More recently, based on the involvement of ROCK inhibition in synaptic function, neuronal survival, and processes associated with neuroinflammation, it has been suggested that the drug may be repurposed for neurodegenerative diseases. Indeed, fasudil has demonstrated preclinical efficacy in many neurodegenerative disease models. To facilitate an understanding of the wider biological processes at play due to ROCK inhibition in the context of neurodegeneration, we performed a global gene expression analysis on the brains of Alzheimer's disease model mice treated with fasudil via peripheral IP injection. We then performed a comparative analysis of the fasudil-driven transcriptional profile with profiles generated from a meta-analysis of multiple neurodegenerative diseases. Our results show that fasudil tends to drive gene expression in a reverse sense to that seen in brains with post-mortem neurodegenerative disease. The results are most striking in terms of pathway enrichment analysis, where pathways perturbed in Alzheimer's and Parkinson's diseases are overwhelmingly driven in the opposite direction by fasudil treatment. Thus, our results bolster the repurposing potential of fasudil by demonstrating an anti-neurodegenerative phenotype in a disease context and highlight the potential of in vivo transcriptional profiling of drug activity.

SummaryNeuronal circuits evolve as a precisely patterned network. In this context, a growing neuron must locate the appropriate target area on a neurite of a neighbouring cell with which to connect. Controlled target selection involves dendritic filopodial contacts and requires the exact apposition of synaptic components. Calcium signalling has been postulated to trigger the transformation from dendritic filopodia into functional synapses. However, calcium is a rather unspecific signalling system, and it needs to be clarified how the exact development of synaptic connections is controlled. Similarly, Wnt/β-catenin signalling promotes synapse formation; however, how secreted Wnts induce and maintain synapses on neuronal dendrites is not well understood. Here, we show that Wnt-7a is tethered to the tips of dynamic dendritic filopodia during spine formation in human cortical neurons. These filopodia can activate Wnt signalling precisely at the contact sites on the dendrites of an adjacent neuron. Subsequently, local calcium transients can be observed at these Wnt-positive contact sites. Depleting either the filopodial-loaded Wnt or the extracellular calcium pool blocks the clustering of pre- and post-synaptic markers, hence the establishment of stable connections. Therefore, we postulate that local Wnt-7a signalling from the tip of the dendritic filopodia, verified by simultaneous calcium signalling, provides an elegant mechanism for orchestrating focal synapse maturation.

Abstract Introduction Reducing brain levels of both soluble and insoluble forms of amyloid beta (Aβ) remains the primary goal of most therapies that target Alzheimer's disease (AD). However, no treatment has so far resulted in patient benefit, and clinical trials of the most promising drug candidates have generally failed due to significant adverse effects. This highlights the need for safer and more selective ways to target and modulate Aβ biogenesis. Methods Peptide technology has advanced to allow reliable synthesis, purification, and delivery of once‐challenging hydrophobic sequences. This is opening up new routes to target membrane processes associated with disease. Here we deploy a combination of atomic detail molecular dynamics (MD) simulations, living‐cell Förster resonance energy transfer (FRET), and in vitro assays to elucidate the atomic‐detail dynamics, molecular mechanisms, and cellular activity and selectivity of a membrane‐active peptide that targets the Aβ precursor protein (APP). Results We demonstrate that Aβ biogenesis can be downregulated selectively using an APP occlusion peptide (APPOP). APPOP inhibits Aβ production in a dose‐dependent manner, with a mean inhibitory concentration (IC50) of 450 nM toward exogenous APP and 50 nM toward endogenous APP in primary rat cortical neuronal cultures. APPOP does not impact the γ‐secretase cleavage of Notch‐1, or exhibit toxicity toward cultured primary rat neurons, suggesting that it selectively shields APP from proteolysis. Discussion Drugs targeting AD need to be given early and for very long periods to prevent the onset of clinical symptoms. This necessitates being able to target Aβ production precisely and without affecting the activity of key cellular enzymes such as γ‐secretase for other substrates. Peptides offer a powerful way for targeting key pathways precisely, thereby reducing the risk of adverse effects. Here we show that protecting APP from proteolytic processing offers a promising route to safely and specifically lower Aβ burden. In particular, we show that the amyloid pathway can be targeted directly and specificically. This reduces the risk of off‐target effects and paves the way for a safe prophylactic treatment.

BACKGROUND:Alzheimer's disease (AD) is the most predominant form of dementia. Rho-associated coiled coil kinase (ROCK) inhibitor, fasudil, is one of the candidate drugs against the AD progression. OBJECTIVE:We aimed to investigate possible changes of AD associated markers in three-dimensional neuro-spheroids (3D neuro-spheroids) generated from induced pluripotent stem cells derived from AD patients or healthy control subjects (HC) and to determine the impact of pharmacological intervention with the ROCK inhibitor fasudil. METHODS:We treated 3D neuro-spheroids with fasudil and tested the possible effect on AD markers by ELISA, transcriptomic and proteomic analyses. RESULTS:Transcriptomic analysis revealed a reduction in the expression of AKT serine/threonine-protein kinase 1 (AKT1) in AD neuro-spheroids, compared to HC. This decrease was reverted in the presence of fasudil. Proteomic analysis showed up- and down-regulation of proteins related to AKT pathway in fasudil-treated neuro-spheroids. We found an evident increase of phosphorylated tau at four different residues (pTau181, 202, 231, and 396) in AD compared to HC-derived neuro-spheroids. This was accompanied by a decrease of secreted clusterin (clu) and an increase of intracellular clu levels in AD patient-derived neuro-spheroids. Increases of phosphorylated tau in AD patient-derived neuro-spheroids were suppressed in the presence of fasudil. CONCLUSIONS:Fasudil modulates clu protein levels and enhances AKT1 that results in the suppression of AD associated tau phosphorylation.

The birth of new neurons from neural stem cells (NSC)s during developmental and adult neurogenesis arises from a myriad of highly complex signalling cascades. Emerging as one of these is the nuclear factor erythroid 2-related factor (NRF2)-signaling pathway. Regulation by NRF2 is reported to span the neurogenic process from early neural lineage specification and NSC regulation to neuronal fate commitment and differentiation. Here, we re-view these reports selecting only those where NRF2 signaling was directly manipulated to provide a clearer case for a direct role of NRF2 in embryonic and adult neurogenesis. With few studies providing mechanistic insight into this relationship, we lastly discuss key pathways linking NRF2 and stem cell regulation outside the neural lineage to shed light on mechanisms that may also be relevant to NSCs and neurogenesis.

The pan ROCK inhibitor fasudil acts as a vasodilator and has been used as a medication post cerebral stroke for the past 27 years in Japan and China. More recently, on the basis of the involvement of ROCK inhibition on synaptic function, neuronal survival and processes associated with neuroinflammation, it has been suggested that the drug may be repurposed for neurodegenerative diseases. Indeed, fasudil has demonstrated preclinical efficacy in many neurodegenerative disease models. To facilitate an understanding of the wider biological processes at play due to ROCK inhibition in the context of neurodegeneration we performed a global gene expression analysis on the brains of Alzheimer’s disease model mice treated with fasudil via peripheral i.p injection. Our results show that fasudil tends to drive gene expression in a reverse sense to that seen in postmortem neurodegenerative disease brains. The results are most striking in terms of pathway enrichment analysis where pathways regulated in Alzheimer’s disease and by fasudil treatment are overwhelmingly regulated in opposite directions. Thus, our results bolster the repurposing potential of fasudil by demonstrating an anti-neurodegenerative phenotype in a disease context and highlight the potential of in vivo transcriptional profiling of drug activity.

Indium zinc oxide (IZO) is one of the most promising anode materials for organic light-emitting diodes (OLEDs). In the present paper, IZO–Ag–IZO multilayer films were prepared by sputtering and their electrical and optical properties were investigated. The multilayer that consisted of a 30-nm-thick top IZO layer sputtered under Ar, a 14-nm-thick Ag layer, and a 30-nm-thick IZO sputtered under O2 was observed to exhibit the highest figure of merit among the investigated multilayer films because of its low sheet resistance (3.6 Ω/sq) and high transmittance (86.9%). OLEDs fabricated using the developed IZO/Ag/IZO anode exhibited a higher luminance and a greater current density than those fabricated using an anode composed of a single layer of IZO or indium tin oxide. As a result, we observed that the IZO/Ag/IZO multilayer exhibits good electrical and optical properties as a transparent conductive film and is suitable for use as an anode in OLEDs.

Amyloid plaques are a hallmark of Alzheimer's disease (AD) that develop in its earliest stages. Thus, non-invasive detection of these plaques would be invaluable for diagnosis and the development and monitoring of treatments, but this remains a challenge due to their small size. Here, we investigated the utility of manganese-enhanced MRI (MEMRI) for visualizing plaques in transgenic rodent models of AD across two species: 5xFAD mice and TgF344-AD rats. Animals were given subcutaneous injections of MnCl2 and imaged in vivo using a 9.4 T Bruker scanner. MnCl2 improved signal-to-noise ratio but was not necessary to detect plaques in high-resolution images. Plaques were visible in all transgenic animals and no wild-types, and quantitative susceptibility mapping showed that they were more paramagnetic than the surrounding tissue. This, combined with beta-amyloid and iron staining, indicate that plaque MR visibility in both animal models was driven by plaque size and iron load. Longitudinal relaxation rate mapping revealed increased manganese uptake in brain regions of high plaque burden in transgenic animals compared to their wild-type littermates. This was limited to the rhinencephalon in the TgF344-AD rats, while it was most significantly increased in the cortex of the 5xFAD mice. Alizarin Red staining suggests that manganese bound to plaques in 5xFAD mice but not in TgF344-AD rats. Multi-parametric MEMRI is a simple, viable method for detecting amyloid plaques in rodent models of AD. Manganese-induced signal enhancement can enable higher-resolution imaging, which is key to visualizing these small amyloid deposits. We also present the first in vivo evidence of manganese as a potential targeted contrast agent for imaging plaques in the 5xFAD model of AD.