Retinal Müller glia alterations and their impact on ocular glymphatic clearance in an Alzheimer's disease mouse model.

Amyloid beta (Aβ) deposits are well-documented in the retinas of Alzheimer's disease (AD) patients, with retinal Aβ levels closely correlating with brain Aβ deposition. Impaired glymphatic clearance is a key mechanism implicated in Aβ accumulation in the AD brain; however the potential role of glymphatic clearance deficits in driving retinal Aβ accumulation remains poorly understood. Furthermore, alterations in Müller glial cell (MGC) biology, known to play a critical role in retinal homeostasis and neuroinflammatory responses, may exacerbate Aβ pathology. This study investigates whether optic nerve glymphatic clearance impairments and Müller glia cell (MGC) biology alterations collectively contribute to retinal Aβ accumulation. Comparing 3-month-old female 5xFAD vs. wild-type (WT) mice, we compared MGC phenotypic differences and optic nerve glymphatic clearance rates. Immunofluorescence was used to quantify MGC expression of glial fibrillary acidic protein (GFAP) to quantify gliosis and aquaporin-4 (AQP4), which is the main molecular driver of glymphatic clearance in both the brain and the eye. To observe glymphatic clearance in the anterograde direction (from eye toward brain), we performed intravitreal injections of fluorescent human Aβ40 as well as fluorescent cadaverine to observe extracellular fluid transport. AQP4 demonstrated a robust upregulation across all retina layers in 5xFAD mice, marked by enhanced perivascular localization and increased expression in the neuropil. GFAP levels were notably elevated in the peripheral retina of 5xFAD mice, suggesting potential reactive gliosis and glymphatic disruptions originating in these regions. Importantly, the study observed consistent anterograde glymphatic transport of fluorescent Aβ or cadaverine tracers via the optic nerve, indicating maintained transport efficiency in this pathway. These findings reinforce and expand on prior evidence that Müller glial cell (MGC) biology undergoes significant changes in the early stages of AD, potentially preceding clinical symptoms. The identification of the peripheral retina as a critical yet underexplored region in understanding retinal AD pathology highlights its potential as an accessible biomarker and a potential target for early intervention. Further research will dissect the complex interplay between local Aβ production and clearance to clarify the retina's role in AD progression, paving the way for novel diagnostic and therapeutic strategies.

To determine if glial fibrillary acidic protein (GFAP) is associated with brain injury in children with sickle cell disease (SCD), we measured plasma GFAP among cross-sectional groups of unselected children with SCD, subsets of children with SCD and normal brain MRI or MRI evidence of cerebral infarct, healthy pediatric controls, and adults with brain injury. Children with SCD had higher plasma GFAP than healthy pediatric controls (mean concentrations 0.14 ± 0.37 vs. 0.07 ± 0.08 ng/mL; P 5 0.003); also, 16.0% (16/100) of children with SCD and cerebral infarct had GFAP elevations above the 95th percentile of healthy pediatric controls (P 5 0.04). Although not statistically significant, children with SCD and cerebral infarct had more elevated GFAP levels than with SCD and no infarct (16/100, 16.0% vs. 14/168, 8.3%; P 5 0.07). Children with SCD and acute brain ischemia had a higher proportion of elevated GFAP than SCD children with normal MRI (3/6, 50% vs.8.3%; P 5 0.01). GFAP was associated with elevated systolic blood pressure in the preceding year and correlated positively with white blood cell count and negatively with age and performance IQ. Plasma GFAP is elevated among children with SCD and may be associated with subclinical brain injury.

MyD88 Deficiency Ameliorates β-Amyloidosis in an Animal Model of Alzheimer's Disease

Severalreports havestatedthe neuroprotective and learning/memory effects of Tachyspermum ammi seed extract (TASE) and its principal component thymol;however, little is known about its underlying molecular mechanisms and neurogenesis potential. This studyaimedtoprovide insights into TASE and athymol-mediated multifactorial therapeutic approach in a scopolamine-induced Alzheimer's disease (AD) mouse model. TASE and thymol supplementation significantly reduced oxidative stress markers suchas brain glutathione, hydrogen peroxide, and malondialdehyde in mouse whole brain homogenates. Tumor necrosis factor-alpha was significantly downregulated, whereas theelevation of brain-derived neurotrophic factor and phospho-glycogen synthase kinase-3 beta (serine 9) enhanced learning and memory in the TASE- and thymol-treated groups. A significant reduction in the accumulation of Aβ 1-42 peptides was observed in thebrainsofTASE- and thymol-treated mice. Furthermore, TASE and thymol significantly promoted adult neurogenesis, with increased doublecortin positive neurons in the subgranular and polymorphic zones of the dentate gyrus in treated-mice. Collectively, TASE and thymol could potentially actas natural therapeutic agentsforthetreatmentof neurodegenerative disorders, suchas AD.

The prion protein (PrP) is best known for its association with prion diseases. However, a controversial new role for PrP in Alzheimer disease (AD) has recently emerged. In vitro studies and mouse models of AD suggest that PrP may be involved in AD pathogenesis through a highly specific interaction with amyloid-β (Aβ42) oligomers. Immobilized recombinant human PrP (huPrP) also exhibited high affinity and specificity for Aβ42 oligomers. Here we report the novel finding that aggregated forms of huPrP and Aβ42 are co-purified from AD brain extracts. Moreover, an anti-PrP antibody and an agent that specifically binds to insoluble PrP (iPrP) co-precipitate insoluble Aβ from human AD brain. Finally, using peptide membrane arrays of 99 13-mer peptides that span the entire sequence of mature huPrP, two distinct types of Aβ binding sites on huPrP are identified in vitro. One specifically binds to Aβ42 and the other binds to both Aβ42 and Aβ40. Notably, Aβ42-specific binding sites are localized predominantly in the octapeptide repeat region, whereas sites that bind both Aβ40 and Aβ42 are mainly in the extreme N-terminal or C-terminal domains of PrP. Our study suggests that iPrP is the major PrP species that interacts with insoluble Aβ42 in vivo. Although this work indicated the interaction of Aβ42 with huPrP in the AD brain, the pathophysiological relevance of the iPrP/Aβ42 interaction remains to be established.

Delivery of therapeutic substances into the brain poses a significant challenge in the treatment of neurological disorders. This is primarily due to the blood–brain barrier (BBB), which restricts access, alongside the limited stability and distribution of these agents within the brain tissue. Here we demonstrate an efficient delivery of microRNA (miRNA) and antisense RNA preferentially to neurons compared to astroglia in the brain of healthy and Alzheimer's disease mice, via disulfide-linked conjugation with poly(ß-L-malic acid-trileucine)-copolymer a biodegradable, amphiphilic, and multivalent platform. By conjugating a D-configured (D3)-peptide (vector) for specific targeting, highly efficient delivery across the BBB is achieved through the Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1) transcytosis pathway, amyloid beta (Aβ) peptides. Nanodrug distribution was determined by fluorescent labeling and analyzed by microscopy in neurons, astroglia, and in extracellular amyloid plaques typical for Alzheimer's disease. Whereas D-configured BBB-vectors can efficiently target neurons, L-configured (e.g., AP2-peptide) guided vector can only cross BBB but not seem to bind neurons. An analysis of post-injection fluorescence distribution, and RNA-seq followed by real-time PCR validation, confirmed a successful in vivo delivery of morpholino-miRNA-186 nanoconjugates into mouse brain. The size and fluorescence intensity of the intracellular nanodrug particulates were analyzed and verified by a competition with non-fluorescent conjugates. Differentially expressed genes (DEGs) from RNA-seq were identified in the nanodrug injected mice, and the changes of selected DEGs related to Alzheimer's disease were further validated by western blot and real-time PCR. Collectively, these results demonstrated that D3-peptide-conjugated nanopolymer drug is able to achieve neuron-selective delivery of miRNA and can serve as an efficient brain delivery vehicle in Alzheimer's disease (AD) mouse models.

Our recent study has demonstrated that peripheral amylin treatment reduces the amyloid pathology in the brain of Alzheimer's disease (AD) mouse models, and improves their learning and memory. We hypothesized that the beneficial effects of amylin for AD was beyond reducing the amyloids in the brain, and have now directly tested the actions of amylin on other aspects of AD pathogenesis, especially neuroinflammation. A 10-week course of peripheral amylin treatment significantly reduced levels of cerebral inflammation markers, Cd68 and Iba1, in amyloid precursor protein (APP) transgenic mice. Mechanistic studies indicated the protective effect of amylin required interaction with its cognate receptor because silencing the amylin receptor expression blocked the amylin effect on Cd68 in microglia. Using weighted gene co-expression network analysis, we discovered that amylin treatment influenced two gene modules linked with amyloid pathology: 1) a module related to proinflammation and transport/vesicle process that included a hub gene of Cd68, and 2) a module related to mitochondria function that included a hub gene of Atp5b. Amylin treatment restored the expression of most genes in the APP cortex toward levels observed in the wild-type (WT) cortex in these two modules including Cd68 and Atp5b. Using a human dataset, we found that the expression levels of Cd68 and Atp5b were significantly correlated with the neurofibrillary tangle burden in the AD brain and with their cognition. These data suggest that amylin acts on the pathological cascade in animal models of AD, and further supports the therapeutic potential of amylin-type peptides for AD.

There is emerging evidence that amyloid beta (Aβ) aggregates forming neuritic plaques lead to impairment of the lipid-rich myelin sheath and glia. In this study, we examined focal myelin lipid alterations and the disruption of the myelin sheath associated with amyloid plaques in a widely used familial Alzheimer's disease (AD) mouse model; 5xFAD. This AD mouse model has Aβ42 peptide-rich plaque deposition in the brain parenchyma. Matrix-assisted laser desorption/ionization imaging mass spectrometry of coronal brain tissue sections revealed focal Aβ plaque-associated depletion of multiple myelin-associated lipid species including sulfatides, galactosylceramides, and specific plasmalogen phopshatidylethanolamines in the hippocampus, cortex, and on the edges of corpus callosum. Certain phosphatidylcholines abundant in myelin were also depleted in amyloid plaques on the edges of corpus callosum. Further, lysophosphatidylethanolamines and lysophosphatidylcholines, implicated in neuroinflammation, were found to accumulate in amyloid plaques. Double staining of the consecutive sections with fluoromyelin and amyloid-specific antibody revealed amyloid plaque-associated myelin sheath disruption on the edges of the corpus callosum which is specifically correlated with plaque-associated myelin lipid loss only in this region. Further, apolipoprotein E, which is implicated in depletion of sulfatides in AD brain, is deposited in all the Aβ plaques which suggest apolipoprotein E might mediate sulfatide depletion as a consequence of an immune response to Aβ deposition. This high-spatial resolution matrix-assisted laser desorption/ionization imaging mass spectrometry study in combination with (immuno) fluorescence staining of 5xFAD mouse brain provides new understanding of morphological, molecular and immune signatures of Aβ plaque pathology-associated myelin lipid loss and myelin degeneration in a brain region-specific manner. Read the Editorial Highlight for this article on page 7.

Purpose: The increase in butyrylcholinesterase (BChE) activity in the brain of Alzheimer disease (AD) patients and animal models of AD position this enzyme as a potential biomarker of the disease. However, the information on the ability of BChE to serve as AD biomarker is contradicting, also due to scarce longitudinal studies of BChE activity abundance. Here, we report 11C-labeling, in vivo stability, biodistribution, and longitudinal study on BChE abundance in the brains of control and 5xFAD (AD model) animals, using a potent BChE selective inhibitor, [11C]4, and positron emission tomography (PET) in combination with computerised tomography (CT). We correlate the results with in vivo amyloid beta (Aβ) deposition, longitudinally assessed by [18F]florbetaben-PET imaging.Methods: [11C]4 was radiolabelled through 11C-methylation. Metabolism studies were performed on blood and brain samples of female wild type (WT) mice. Biodistribution studies were performed in female WT mice using dynamic PET-CT imaging. Specific binding was demonstrated by ex vivo and in vivo PET imaging blocking studies in female WT and 5xFAD mice at the age of 7 months. Longitudinal PET imaging of BChE was conducted in female 5xFAD mice at 4, 6, 8, 10 and 12 months of age and compared to age-matched control animals. Additionally, Aβ plaque distribution was assessed in the same mice using [18F]florbetaben at the ages of 2, 5, 7 and 11 months. The results were validated by ex vivo staining of BChE at 4, 8, and 12 months and Aβ at 12 months on brain samples.Results: [11C]4 was produced in sufficient radiochemical yield and molar activity for the use in PET imaging. Metabolism and biodistribution studies confirmed sufficient stability in vivo, the ability of [11C]4 to cross the blood brain barrier (BBB) and rapid washout from the brain. Blocking studies confirmed specificity of the binding. Longitudinal PET studies showed increased levels of BChE in the cerebral cortex, hippocampus, striatum, thalamus, cerebellum and brain stem in aged AD mice compared to WT littermates. [18F]Florbetaben-PET imaging showed similar trend of Aβ plaques accumulation in the cerebral cortex and the hippocampus of AD animals as the one observed for BChE at ages 4 to 8 months. Contrarily to the results obtained by ex vivo staining, lower abundance of BChE was observed in vivo at 10 and 12 months than at 8 months of age.Conclusions: The BChE inhibitor [11C]4 crosses the BBB and is quickly washed out of the brain of WT mice. Comparison between AD and WT mice shows accumulation of the radiotracer in the AD-affected areas of the brain over time during the early disease progression. The results correspond well with Aβ accumulation, suggesting that BChE is a promising early biomarker for incipient AD.

The physiological actions of a gut hormone, glucagon-like peptide-1 (GLP-1), in Alzheimer's disease (AD) brain remain poorly understood, although GLP-1 receptor (GLP-1R) expression in this organ has been shown in several experimental studies. Therefore, we explored whether the GLP-1R signaling promotes the clearance of amyloid β (Aβ) (1-42) which is a core pathological hallmark of AD, focusing on the water channel protein aquaporin 4 (AQP4) localized to astrocyte endfeet perivascular membranes in intact brain. First, we confirmed that Glp1r mRNA is predominantly expressed at perivascular site of astrocytes in normal mouse cerebral cortex through in situ hybridization analysis. Next, we observed that 20-week subcutaneous administration of a GLP-1R agonist (GLP-1RA) liraglutide significantly reduced Aβ (1-42) accumulation in the cerebral cortex and improved spatial working memory in an AD mouse model, AppNL-G-F/NL-G-F mice. Furthermore, our current data revealed that the 4-week liraglutide treatment relocalized subcellular AQP4 in morphologically injured reactive astrocytes of AppNL-G-F/NL-G-F mice to the cell surface perivascular site through PKA-mediated AQP4 phosphorylation. Such translocation of phosphorylated AQP4 to astrocyte cell surface following incubation with liraglutide was observed also in the present in vitro study using the cell line in which AQP4 cDNA was introduced into immortalized human astrocyte. These results suggest that enhanced intracerebral GLP-1R signaling following peripheral administration of GLP-1RA restores AQP4 subcellular polarization in reactive astrocytes and would promote Aβ excretion possibly through increasing AQP4-mediated intracerebral water flux in the brain in AD.

Role of the Macrophage Inflammatory Protein-1α/CC Chemokine Receptor 5 Signaling Pathway in the Neuroinflammatory Response and Cognitive Deficits Induced by β-Amyloid Peptide

There is an urgent need to improve the translational validity of Alzheimer's disease (AD) mouse models. Introducing genetic background diversity in AD mouse models has been proposed as a way to increase validity and enable the discovery of previously uncharacterized genetic contributions to AD susceptibility or resilience. However, the extent to which genetic background influences the mouse brain proteome and its perturbation in AD mouse models is unknown. In this study, we crossed the 5XFAD AD mouse model on a C57BL/6J (B6) inbred background with the DBA/2J (D2) inbred background and analyzed the effects of genetic background variation on the brain proteome in F1 progeny. Both genetic background and 5XFAD transgene insertion strongly affected protein variance in the hippocampus and cortex (n = 3,368 proteins). Protein co-expression network analysis identified 16 modules of highly co-expressed proteins common across the hippocampus and cortex in 5XFAD and non-transgenic mice. Among the modules strongly influenced by genetic background were those related to small molecule metabolism and ion transport. Modules strongly influenced by the 5XFAD transgene were related to lysosome/stress responses and neuronal synapse/signaling. The modules with the strongest relationship to human disease—neuronal synapse/signaling and lysosome/stress response—were not significantly influenced by genetic background. However, other modules in 5XFAD that were related to human disease, such as GABA synaptic signaling and mitochondrial membrane modules, were influenced by genetic background. Most disease-related modules were more strongly correlated with AD genotype in the hippocampus compared with the cortex. Our findings suggest that the genetic diversity introduced by crossing B6 and D2 inbred backgrounds influences proteomic changes related to disease in the 5XFAD model, and that proteomic analysis of other genetic backgrounds in transgenic and knock-in AD mouse models is warranted to capture the full range of molecular heterogeneity in genetically diverse models of AD.

Comparative analysis of cortical gene expression in mouse models of Alzheimer's disease

9R, the cholinesterase and amyloid beta aggregation dual inhibitor, as a multifunctional agent to improve cognitive deficit and neuropathology in the triple-transgenic Alzheimer's disease mouse model

Development of chemical isotope labeling LC-MS for tissue metabolomics and its application for brain and liver metabolome profiling in Alzheimer's disease mouse model