Background Prior work in preclinical late-onset Alzheimer's disease (LOAD) focused on neuritic plaque development suggested that intracellular ferritin expression in microglia and extracellular deposition of amyloid-β (Aβ) are innate neuroprotective mechanisms geared specifically towards limiting aging-dependent increases in intracerebral free iron which likely contribute to development of neurofibrillary degeneration (NFD). Objective Improve understanding of LOAD pathogenesis. Methods Immunohistochemical comparison of the extent of NFD with the intensity of ferritin expression and Aβ deposition in three brain regions, including temporal lobe (entorhinal cortex, hippocampus), frontal, and occipital cortex in 34 non-demented human subjects at Braak stages II-III. Results Ferritin-positive microglia are present with similar quantity and intensity in the allo- and isocortices of every individual in the cohort. Extracellular Aβ deposition in the isocortex is observed before substantial NFD develops, but in the allocortex (temporal lobe) there are no Aβ deposits in 50% of subjects despite extensive NFD. Cytoskeletal lesions in the allocortex consist of atrophic grid cells, abundant pretangles, neuropil threads, neurofibrillary tangles, and neuritic plaques; isocortical sites show either no NFD at all or only minimal NFD presenting as solitary pretangles or tangles, neuropil threads, or droplet degeneration spheres from ferroptotic neurons. Presence of degenerating grid neurons in entorhinal cortex coincides with microglial apoptosis. Conclusions Neuroprotection via ferritin expression and Aβ deposition is more effective in the isocortex than in allocortex. Findings support the hypothesis that degeneration or death of neuroprotective microglia promotes neuronal degeneration.

Background: The local availability of thyroid hormones (TH) depends on transporters like monocarboxylate transporter 8 (MCT8). While its importance in fetal brain development is established, a comprehensive regional characterization of MCT8 in the adult human brain remains lacking. This knowledge is essential for understanding MCT8's role in normal physiology, aging, and neurological disease. Methods: We investigated MCT8 distribution in the adult and elderly human brain across multiple structural levels, encompassing distinct cell types, the cellular microenvironment, and cytoarchitecture. Comparing 8 brain regions from 14 donors, we employed immunostaining, whole-slide image analysis, fluorescence in situ hybridization (RNAscope), and stimulated emission depletion (STED) microscopy to assess protein and transcriptome expression. Braak staging was applied to assess MCT8 expression in relation to Alzheimer's disease (AD) neurofibrillary pathology. Findings: MCT8 protein and SLC16A2 mRNA were robustly expressed in glial cells, neurons, and brain barriers across all examined regions. RNAscope confirmed strong concordance between transcript presence and immunoreactivity. STED microscopy localized MCT8 signal to plasma membranes, perinuclear regions, and axonal/dendritic compartments. Braak staging revealed no significant difference in MCT8 expression between donors with high versus low neurofibrillary pathology. Interpretation: MCT8 is broadly expressed throughout the adult and elderly human brain at both macro- and microenvironmental levels. Its distinct localization suggests ongoing functional roles in inter- and intracellular TH transport, and its expression appears to remain stable regardless of AD progression.

Microglia are the resident immune cells of the human central nervous system and play key roles in development, homeostasis, and disease. These functions are mediated by a broad repertoire of cell-surface receptors, including G protein-coupled receptors such as the ADP receptor P2Y12 and GPR34, a receptor for lysophosphatidylserine. While GPR34 deficiency has been linked to impaired microglial phagocytosis, its regulation in relation to amyloid-β (Aβ) and tau pathology in Alzheimer’s disease (AD) remains unclear. We performed a quantitative analysis of microglial density, morphology, and GPR34 expression in the medial temporal lobe cortex (MTLC) of elderly human body and tissue donors across the AD spectrum. Using fluorescence in situ hybridization and immunolabeling, we analyzed 187,670 microglial cells and correlated microglial parameters with the severity and spatial proximity of Aβ plaques and tau inclusions. In parallel, we analyzed human single-nucleus RNA sequencing data from 236,002 cells to assess GPR34 expression across microglial subtypes, brain regions, and neuropathological stages. Microglial density and overall morphology in the MTLC were largely preserved, independent of local Aβ or hyperphosphorylated tau burdens. Apart from a moderate shortening of microglial processes in the immediate vicinity of Aβ plaques, no consistent pathology-associated morphological changes were detected. GPR34 expression showed pronounced cell-to-cell variability and differed across microglial subtypes and brain regions, but neither expression intensity nor the proportion of GPR34-positive microglia correlated consistently with Braak stage or Thal phase. These findings suggest that GPR34 regulation in human microglia is highly context-dependent and shaped by regional and cellular heterogeneity rather than AD-associated pathology alone.

The regulation of appetite by pharmaceuticals has gained significant interest for the treatment of obesity and cachexia. The melanocortin 4 receptor (MC4R) and the ghrelin receptor (GhrR) are known to play a crucial role in the regulation of energy homeostasis. Thus, peptide ligands, which modulate these receptors, have become attractive therapeutic lead structures. A key challenge is the efficient delivery of such peptides to the targeted receptors, which are expressed in the hypothalamus. Therefore, direct nose-to-brain delivery is a compelling strategy. Here, we report on food intake that is modulated by using intranasal applied peptides. We synthesized fluorescently labelled variants of the MC4R agonist setmelanotide, the GhrR agonist ghrelin (Ghr) and the GhrR inverse agonist KbFwLL-NH2 [β-(3-benzothienyl)-D-alanine (b)] and assessed their receptor activity. Further, we measured the permeability and stability of these peptides on Calu-3 cells as a model system for the nasal mucosa. Next, the uptake of peptides after intranasal application was analysed in vivo by quantification of fluorescent signals in the olfactory bulb, cortex and hypothalamus. In addition, we monitored the effects of the two most promising peptides on food intake in vivo. Although no significant changes in body weight were observed, we detected differences in the daily change in food intake: this parameter was reduced for mice treated with setmelanotide variants and increased for mice treated with GhrR agonists compared to a control group. Taken together, our findings clearly underline the high potential of intranasal peptide administration for modulating food intake.

Many diseases, including obesity, have systemic effects that perturb multiple organ systems throughout the body1,2. However, tools for comprehensive, high-resolution analysis of disease-associated changes at the whole-body scale have been lacking. Here we developed MouseMapper, a suite of foundation-model-based deep-learning algorithms enabling multi-system analysis of disease across the entire mouse body. MouseMapper enables whole-body quantitative analysis of nerves and immune cells, resolving fine axonal branches and immune-cell clusters while automatically segmenting 31 organs and tissues. We used MouseMapper to study diet-induced obesity, and identified structural alterations of the infraorbital branch of the trigeminal ganglia. This structural impairment in infraorbital nerves was associated with functional sensory deficits in whisker sensing. Furthermore, we identified proteomic changes in the trigeminal ganglion affecting axon remodelling and complement pathways both in mice and humans. MouseMapper also generated detailed three-dimensional inflammation maps by characterizing immune cell cluster compositions across tissues. The MouseMapper framework demonstrates robust generalizability across different imaging resolutions and datasets. Our study provides a powerful, scalable approach for identifying and quantifying systemic pathologies, bridging molecular insights from animal models to human conditions.

Understanding radiation-induced tissue modifications and cell death within the complex cellular architecture of the brain requires experimental systems that preserve tissue integrity and intercellular crosstalk. At the same time, microglial cells as primary regulators of primary and secondary inflammation in the brain are difficult to study in single cell cultures and it is crucial to investigate their behaviour in their complex tissue microenvironment. Rodent organotypic hippocampal slice cultures (OHSCs) and human patient-derived brain tumor cultures (PDTCs) offer physiologically relevant platforms for investigating the differential effects of X-ray and heavy-ion irradiation on glial as well as immune cell populations. Both models retain native cytoarchitecture, extracellular matrix composition, and functional cell-cell interactions, making them uniquely suited to study the spatial and temporal dynamics of inflammation or cell death in a multicellular context. Here, we present a standardized methodology for preparing, irradiating, and assessing PDTCs, employing multimodal readouts as cytokine measurement of the supernatant, cell death, microglia morphology and function. Novel molecular analyses are possible upon certain considerations and may alter our understanding of species differences and help to distinguish cell death pathways to attribute damage to specific cell types. Standardization of these protocols across model systems is essential for generating reproducible, comparative data while preserving the inherent complexity that underlies intercellular communication and coordinated responses to injury. This approach enables translationally relevant insight into the mechanisms of radiation-induced brain injury to develop strategies for neuroprotection and treatment in clinical settings.

This study investigated the impact of tissue preservation methods on protein profiles analyzed by reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS) using data-independent acquisition (DIA). Proteomic profiles from formalin-fixed, formalin-fixed and paraffin-embedded (FFPE), and fresh-frozen human brain tissues (cortex and hippocampus, n = 6) were compared, including an FFPE-specific protein extraction kit (n = 4). Formalin-fixed samples more closely resembled fresh-frozen profiles than FFPE or FFPE-Kit samples, while still showing high correlation and overlap with FFPE tissues in principal component analyses. A core set of 1753 proteins was consistently detected across all sample preparation methods. A total of 35 proteins were identified exclusively in fresh-frozen samples, but without functional enrichment. Quantitative comparisons to the proteome of fresh-frozen tissue revealed an underrepresentation of cellular processes, energy metabolism, signaling, and transport related to protein properties such as length, location, and hydrophobicity. In contrast, neuronal development and phagosome-related pathways were overrepresented in fixed tissues. In a pilot study comparing low (Braak 0-II, n = 4) and high (Braak IV-VI, n = 4) Alzheimer's disease (AD) stages using formalin-fixed samples, we identified 12 potential protein biomarkers, primarily nucleosomal proteins and carboxypeptidase M (CPM). These findings suggest that formalin-fixed brain tissue provides reliable proteomic information, making it a valuable resource for neurodegenerative disease research. SIGNIFICANCE: Proteomics offers enormous potential for investigating the molecular regulation of the human brain. Valuable tissue samples are often preserved in formalin or additionally with paraffin for later analysis. The potential value of these preserved samples for proteomic analysis has already been recognized. However, tissue preservation poses a challenge for proteome analysis. Consequently, several studies have compared different protein extraction protocols for fixed samples. In addition, studies have been published comparing protein extraction from FFPE samples with fresh-frozen samples. To our knowledge, this is the first study to compare protein extraction across all three tissue preservation methods with subsequent functional analysis using samples obtained from the same donors, thereby eliminating inter-donor variability and enabling a direct comparison of preservation effects. This study validates a protein extraction protocol from formalin-fixed samples, laying the groundwork for future research into potential biomarkers in formalin-fixed samples.

Peptidoglycan (PGN) is a large complex polymer critical to structure and function of all bacterial species. Intact PGN and its fragments are inflammatory, contributing to infectious and autoimmune disease. Recent studies show that PGN physiologically contributes to immune setpoints, and importantly also to mouse brain development and behavior. However, for the human brain, it remains unknown whether PGN and its fragments differentially gain access to distinct brain regions, which cell types accumulate it, and whether PGN brain load varies with age. Therefore, we investigated human postmortem brain samples of donors with an extensive age range, from newborns to nonagenarians. We examined two monoclonal antibodies against PGN which were validated using dot blot analysis, competition assays and immunofluorescence experiments on bacteria sacculi, which jointly showed specific detection of Gram-positive PGN. As positive reference tissue, brain tissue from sepsis patients, and human liver were used, both showing the expected high PGN levels. In adult brain tissue of different age (34- to 94-year-old) and sex, we detected PGN signals in seven different brain regions, with highest loads in the occipital cortex, hippocampal formation, frontal cortex, the periventricular region and the olfactory bulb. Age-dependent increase of signals was not evident by microscopic observations and only weak correlation was found by statistical analysis in this cohort. PGN was found intracellularly in the cytoplasm surrounding the cell nucleus in astrocytes, oligodendrocytes, neurons, and endothelial cells, but not in macrophages like microglia. PGN was absent in brain tissues of three human newborns (stillbirth to four weeks old). For comparison, three brain regions from non-human primates of varying age (newborn to 21 years) were immunohistochemically stained. The highest PGN-load was observed in brain tissue from 18- to 21-year-old macaques. This first systematic evaluation of PGN in human postmortem brain suggests that PGN accumulates during lifetime until it reaches a plateau by homeostatic turnover and highlights the ubiquitous presence of PGN in human brain tissues, and their ability to participate in physiological as well as pathological processes throughout life.

Background: High-fat diet (HFD) consumption is commonly linked to low-grade brain inflammation and increased risk of neurodegeneration. However, in our previous research, HFD exposure for up to 24 weeks did not increase pro-inflammatory cytokine expression or impair learning and spatial memory. To further investigate neuroimmune responses, we examined microglial activation at the transcriptional level. Methods: Male C57BL/6J mice were fed either a normal diet (ND) or HFD for 4, 12, or 24 weeks. Bulk RNA sequencing was performed across four brain regions (cerebellum, hippocampus, hypothalamus, cortex) to assess region-specific transcriptional responses. Results: HFD induced region- and time-dependent transcriptional changes. In the hypothalamus, 0/11/37 differentially expressed genes (DEGs; p-value < 0.05; fold change > 1.5) were detected at 4, 12, and 24 weeks, respectively. In the hippocampus, 2/41/42 DEGS were observed; in the cortex, 1/3/68 DEGS; and in the cerebellum, 27/0/0 DEGS at the respective time points, indicating minimal cerebellar involvement beyond the early time point. Across all conditions, three genes (Lcn2, Ch25h, Gimap9) were consistently regulated. Several DEGs were linked to microglial activation and inflammatory signaling. In the manuscript, we discuss 33 biologically relevant DEGs in detail. Transcriptomic signatures and pathway enrichment analyses suggest potential engagement of NF-κB-related pathways, although this interpretation remains indirect. Conclusions: These findings demonstrate that HFD selectively alters brain homeostasis by inducing region-specific transcriptional changes associated with microglial activation and inflammatory processes. While NF-κB-related pathways emerged as recurrent candidates, direct mechanistic validation is required.

Excessive fat consumption increases the risk of Alzheimer's disease (AD), potentially through diet-induced neuroinflammation. Microglia, the brain's immune cells, are affected by obesity and diet. Phytosterols (PS), plant-derived cholesterol-like compounds, accumulate in the brain with age, and their content correlates with dietary intake. We hypothesize that the accumulation of PS modulates microglial activation and exerts anti-inflammatory effects. We investigated the effects of a normal diet (ND), high-fat diet (HFD), HFD with 2% PS (HFD+2% PS), and HFD with 4% PS (HFD+4% PS) on neuroinflammation in female and male C57BL/6J mice. Flow cytometry (FC) of microglia showed no significant regulation of pro- (IFN-γ, IL-1β, TNF-α) and anti-inflammatory (IL-10) cytokines due to diet, but sex- and age-dependent differences were observed. Immunofluorescence staining showed no TREM2 upregulation, indicating a lack of microglial activation in response to HFD. PS supplementation significantly reduced HFD-induced weight gain, suggesting metabolic effects. Contrary to existing research, we found no evidence of HFD-induced neuroinflammation or microglial activation. However, the reduction in weight gain with PS supplementation suggests potential metabolic benefits, which could have implications for the treatment of obesity. The potential effects on neuroinflammation remain unclear.

Far from being a niche concern, women’s brain health is a global issue, affecting more than half of the world’s population. Despite this, the unique aspects of how the female brain adapts, reorganizes and ages, particularly those shaped by hormonal transitions across the lifespan, have not received proportionate attention in research agendas, funding priorities or clinical guidelines.

Body homeostasis relies on accurate communication between the brain and the periphery. Disruption of this communication can contribute to disease. Tanycytes are located at the interface of the cerebrospinal fluid (CSF), bloodstream, and hypothalamus, where they sense circulating nutrients and regulate neuroendocrine axes and metabolism. However, the mechanisms by which they sense CSF signals remain largely unclear. Recent evidence that tanycytes possess primary cilia — key sensory organelles — led us to hypothesize that tanycytic cilia function as sensory antennae that detect metabolic cues in the CSF. Here, we demonstrate that tanycytic cilia exhibit distinct morphologies across subtypes and physiological states. They respond dynamically to hormonal and nutrient availability; notably, excess oleic acid shortens cilia, promotes lipid droplet accumulation, and reduces Ca2+ responses to ATP and glucose. Disrupting cilia via knockdown of intraflagellar transport (IFT) genes produced similar defects and impaired autophagy. Finally, selective Ift88 knockout in tanycytes increased body weight and reduced thermogenic activity in female mice. These findings identify tanycytic cilia as key sensors regulating energy balance. ### Competing Interest Statement The authors have declared no competing interest.

The neuropeptide Y multireceptor-multiligand system plays an important role in multiple physiological processes. Targeting the neuropeptide Y1 (Y1R) and Y2 (Y2R) receptors has gained interest in treating weight and mental disorders. Nose-to-brain delivery is an effective tool to overcome the challenges of peptide delivery to cerebral structures. In this study, fluorescently labeled peptides that selectively activate either Y1R or Y2R were studied. The permeability of these compounds was evaluated on Calu-3 cells, a model system of the nasal mucosa. Particular attention was paid to the stability of peptides, and translocation of the intact compounds was demonstrated by combining a permeability assay with a receptor activation assay. Two compounds, selectively targeting either Y1R or Y2R, were selected, and their uptake after intranasal application was analyzed in vivo. Two different imaging systems were compared: whole slide scanning and confocal microscopy. Both methods allow detecting specific signals from the fluorescently labeled peptides. While whole slide scanning provides a comprehensive anatomical overview, confocal microscopy offers an improved signal-to-noise ratio. Finally, peptide-specific signals were quantified over time, displaying rapid peptide uptake within the first 15 min and sustained signals for up to 24 h. Overall, cell-based and in vivo assays were combined to select peptides with high pharmacological potential for nasal applications.

Cognitive impairment is common in extracerebral diseases such as chronic kidney disease (CKD). Kidney transplantation reverses cognitive impairment, indicating that cognitive impairment driven by CKD is therapeutically amendable. However, we lack mechanistic insights allowing development of targeted therapies. Using a combination of mouse models (including mice with neuron-specific IL-1R1 deficiency), single cell analyses (single nuclei RNA sequencing and single cell thallium autometallography), human samples and in vitro experiments we demonstrate that microglia activation impairs neuronal potassium homeostasis and cognition in CKD. CKD disrupts the barrier of brain endothelial cells in vitro and the blood-brain barrier in vivo, establishing that the uremic state modifies vascular permeability in the brain. Exposure to uremic conditions impairs calcium homeostasis in microglia, enhances microglial potassium efflux via the calcium-dependent channel KCa3.1, and induces p38-MAPK associated IL-1β maturation in microglia. Restoring potassium homeostasis in microglia using a KCa3.1-specific inhibitor (TRAM34) improves CKD-triggered cognitive impairment. Likewise, inhibition of the IL-1β receptor 1 (IL-R1) using anakinra or genetically abolishing neuronal IL-1R1 expression in neurons prevent CKD-mediated reduced neuronal potassium turnover and CKD-induced impaired cognition. Accordingly, in CKD mice, impaired cognition can be ameliorated by either preventing microglia activation or inhibiting IL-1R-signaling in neurons. Thus, our data suggest that potassium efflux from microglia triggers their activation, which promotes microglia IL-1β release and IL-1R1-mediated neuronal dysfunction in CKD. Hence, our study provides new mechanistic insight into cognitive impairment in association with CKD and identifies possible new therapeutic approaches.

BackgroundClinical progress in form of “total mesometrial resection” (TMMR) in cervical cancer and “total mesorectal excision” (TME) in rectal cancer can be traced to a paradigm-shift regarding the extent and range of resection. More significance is bestowed upon embryologically defined borders which define compartments, “morphogenetic units” and “cancer fields”, that have to be addressed in order to avoid incomplete tumor resection. We want to transfer this rationale on the pancreas and define such borders for pancreatic compartments.Material and methodsWe used 26 unfixed body donors (16 male, 10 female) ranging in age from 64 to 98 years. Manual preparation consisted of performing the Cattell-Braasch maneuver to restore embryologic anatomy and define fascial remnants of the borders of the dorsal and ventral mesogastrium with focus on the pancreatic fusion fasciae and peripancreatic spaces.ResultsWe tracked what used to be the dorsal and ventral mesogastrium and assigned their remnants to the bowel and pancreas. Following avascular embryologic fascial fusion planes along the mesogastria we could demonstrate peripancreatic spaces, which were sealed off from bordering surfaces of presumably different morphogenetic units and possible cancer fields. Reverting embryologic development also seemed possible within the pancreas, demonstrating the embryologic fusion plane between the ventral and dorsal pancreatic buds as two distinct compartments.ConclusionsFollowing pancreatic fusion fasciae by separating embryologic fusion planes enables to define the pancreatic compartments which might play a major role in applying the success of TMMR and TME on pancreatic resection and define pancreatic cancer fields.

Neuritic plaques are pathognomonic and terminal lesions of Alzheimer's Disease (AD). They embody AD pathogenesis because they harbor in one space critical pathologic features of the disease: amyloid deposits, neurofibrillary degeneration (NFD), neuroinflammation, iron accumulation. Neuritic plaques are thought to arise from the conversion of diffuse extracellular deposits of amyloid beta protein (Aβ), and it is believed that during conversion amyloid toxicity creates the dystrophic neurites of neuritic plaques, as well as neurofibrillary tangles (NFTs). However, recent evidence from human post-mortem studies suggests a much different mechanism of neuritic plaque formation where the first step in their creation is neuronal degeneration driven by iron overload and ferroptosis. Similarly, NFTs represent corpses of iron-laden neurons that develop independent of Aβ deposits. In this review, we will focus on the role of free redox-active iron in the development of typical AD pathology, as determined largely by evidence obtained in human temporal lobe during early, preclinical stages of AD. The findings have allowed construction of a scheme of AD pathogenesis where brain iron is center stage and is involved in every step of the sequence of events that produce characteristic AD pathology. We will discuss how the study of preclinical AD has produced a fresh and revised assessment of AD pathogenesis that may be important for reconsidering current therapeutic efforts and guiding future ones. Significance Statement This review offers a novel perspective on AD pathogenesis where elevated brain iron plays a central role and is involved throughout the development of lesions. We review arguments against the amyloid cascade theory and explain how recent findings in humans during early preclinical disease support iron-mediated cell death and endogenous iron containment mechanisms as critical components of neuritic plaque formation and the ensuing dementia.

The melanocortin-4 receptor (MC4R) is a key player in the hypothalamic leptin–melanocortin pathway that regulates satiety and hunger. MC4R belongs to the G protein-coupled receptors (GPCRs), which are known to form heterodimers with other membrane proteins, potentially modulating receptor function or characteristics. Like MC4R, thyroid hormones (TH) are also essential for energy homeostasis control. TH transport across membranes is facilitated by the monocarboxylate transporter 8 (MCT8), which is also known to form heterodimers with GPCRs. Based on the finding in single-cell RNA-sequencing data that both proteins are simultaneously expressed in hypothalamic neurons, we investigated a putative interplay between MC4R and MCT8. We developed a novel staining protocol utilizing a fluorophore-labeled MC4R ligand and demonstrated a co-localization of MC4R and MCT8 in human brain tissue. Using in vitro assays such as BRET, IP1, and cAMP determination, we found that MCT8 modulates MC4R-mediated phospholipase C activation but not cAMP formation via a direct interaction, an effect that does not require a functional MCT8 as it was not altered by a specific MCT8 inhibitor. This suggests an extended functional spectrum of MCT8 as a GPCR signaling modulator and argues for the investigation of further GPCR-protein interactions with hitherto underrepresented physiological functions.

Background We have previously shown that droplet degeneration (DD) signifies the beginning of neuritic plaque formation during Alzheimer's disease (AD) pathogenesis. As microglia associated with neuritic plaques exhibited strong ferritin expression and Perl's iron staining showed iron in microglia, droplet spheres and neuritic plaque cores, we hypothesized that DD is a form of ferroptosis. Objective Detection of molecular markers of ferroptosis in AD brains. Methods Immunohistochemical detection of transferrin receptor (TfR) and ferritin as ferroptosis markers in prefrontal cortex of AD brains, investigation of spatial correlation of these with histopathological hallmarks of AD, visualization of ferroptotic marker genes by in situ hybridization, comparison of expression of ferroptosis genes with snRNAseq analyses and comparison of TfR and ferritin expression in different neurofibrillary tangle (NFT) stages. Results TfR was found on neurons that appeared to be degenerating and exhibited typical features of droplet degeneration. Co-localization with hyperphosphorylated tau (p-tau) was a rare event. TfR-positive neurons increased with higher NFT stages as did ferritin expression in microglia. mRNA of genes linked to ferroptosis was detected in pretangles and p-tau negative neurons, less in DD. snRNAseq analyses support a link between AD, ferroptosis and TfR as a ferroptosis marker. Conclusions Increased expression of TfR and ferritin in high NFT stages, demonstration of ferroptotic marker genes in Alzheimer's lesions, as well as snRNAseq analyses strengthen our hypothesis that DD represents ferroptosis. Because of the morphological similarity between TfR-positive structures and DD, TfR might be an early ferroptosis marker expressed transiently during AD pathogenesis.

Background. Organ transplantation reverses cognitive impairment in chronic kidney disease (CKD), indicating that cognitive impairment driven by CKD is therapeutically amendable. We recently demonstrated that impaired cognition in CKD is linked to interleukin1S (IL-1 S) release from microglia and IL-1 receptor type 1 signalling in neuronal cells, thereby identifying a signalling pathway that can be exploited therapeutically. However, the mechanism of IL-1 S maturation in microglia in CKD remains unknown. We hypothesized that microglia cells require caspase-1 for CKD-driven cognitive impairment. Methods. We used a combination of single-cell analyses, in situ analyses, genetically modified mouse models (including newly generated Cre-LoxP mouse models) and in vitro models. The current study builds on a recently identified intercellular cross-talk between microglia and neurons that impairs cognition in CKD. Results. Here we show that despite NLRP3 inflammasome activation in the brain and protection of mice with constitutive NLRP3 deficiency from CKD-induced cognitive impairment, caspase-1 is not required for IL-1 S maturation in microglia and targeted caspase-1 deficiency in microglia does not improve cognition in CKD mice. These data indicate that IL-1 S maturation in microglia is independent of the NLRP3-caspase-1 interaction in CKD. Indeed, microglia activation in CKD induces noncanonical, cathepsin C-caspase-8- mediated IL-1 S maturation. Depletion of cathepsin C or caspase-8 blocks IL-1 S maturation in microglia. Preliminary analyses suggest that noncanonical microglia IL-1 S maturation occurs also in diabetes mellitus. Conclusion. These results identify a noncanonical IL-1 S-maturation pathway as a potential therapeutic target to combat microgliainduced neuronal dysfunction in CKD and possibly other peripheral diseases.