Abstract Mechanotransduction plays a fundamental role in regulating immune cell function, yet how engineered virus-like nanospikes engage mechanosensitive signaling pathways to modulate innate immunity remains poorly understood. Here, we report virus-like nanotopography as a previously unrecognized regulator of Piezo1-mediated mechanotransduction in macrophages using virus-like mesoporous silica nanoparticles (VLPSi) with tunable rigid nanospike lengths. We demonstrate a direct structure–activity relationship between nanospike geometry and Piezo1-dependent Ca² + influx, with longer nanospikes inducing significantly greater intracellular Ca² + signaling. Building on this mechanistic insight, we developed biomimetic cancer cell membrane (CM)-coated, MSA-2-loaded VLPSi nanoparticle (CM/MSA-2@VLPSi) and investigate the combination of nanospikes-activated Piezo1 with STING signaling and CM antigens presentation in macrophage immune reprogramming. The resulting biomimetic nanoparticles robustly activate the STING–TBK1–IRF3/NF-κB axis, increase IFN-β and pro-inflammatory cytokine production, and promote macrophage polarization toward M1 phenotype in a spike-length-dependent manner. Collectively, the present study provides a biomimetic strategy for enhancing the M1 polarization of macrophage through the coordinated regulation of mechanical, inflammatory, and antigenic signals.

Diagnostic testing and neurosurgical management involving CSF shunting in patients with idiopathic normal pressure hydrocephalus (iNPH) offers a unique, ethically sustainable opportunity to study the aging human brain. The common co-occurrence of neurodegenerative pathologies such as amyloid-β plaques and neurofibrillary tau tangles (NFT) provides a valuable window for research into the Alzheimer’s disease-related mechanisms. Interindividual variability among iNPH patients enables investigation and validation of biomarkers and molecular mechanisms, opening possibilities for new treatments. In vitro techniques and culturing methods for living brain tissue expand the utility of samples for electrophysiological studies, drug testing, and examining genetic risk variants. Careful sectioning and processing are essential for high-quality samples with intact cells for functional analysis. A rigorous pipeline is also needed for preserving samples for transcriptomic, proteomic, and structural analyses, with emphasis on RNA integrity and tissue architecture for single-cell sequencing and spatial analysis. Here, we review the use of small cortical brain biopsies in neurodegenerative diseases research and diagnostics. Furthermore, we share our team’s experience in acquiring and preserving living brain samples during iNPH CSF shunt surgeries, along with our protocols for resection, use, and preservation of biopsies for preclinical research. We highlight the potential of living human tissue for neuroscience, early neurodegenerative disease diagnosis, and targeted therapies.

C9orf72 hexanucleotide repeat expansion (C9-HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (FTD). However, approximately half of the FTD patients are sporadic without a clear genetic background. To compare characteristics of microglia from different FTD subtypes, we generated induced pluripotent stem cell-derived microglia (iMG) from sporadic and C9-HRE-carrying behavioral variant FTD (bvFTD) patients and healthy controls. C9-HRE iMG displayed C9-HRE-associated RNA foci and dipeptide repeat proteins. All bvFTD iMG had fewer LAMP2-A-positive vesicles compared to control iMG. Additionally, C9-HRE iMG showed significantly increased LC3BII/I conversion after bafilomycin A1 treatment and altered phagocytic activity. The gene expression profile of C9-HRE iMG only modestly differed from the control iMG, but was greatly different from the sporadic bvFTD patient iMG. Our data show alterations in phagocytic and autophagosomal/lysosomal pathways and gene expression profiles between C9-HRE and sporadic bvFTD iMG for the first time.

Abstract Alzheimer’s disease (AD) pathology involves amyloid deposition, reactive gliosis, and localized tissue alterations that coexist within the same brain regions, creating heterogeneous microstructural environments within individual imaging voxels. Conventional diffusion MRI averages these environments into aggregate measures, potentially obscuring their distinct contributions. Frequency-dependent multidimensional MRI (ωMD-MRI) resolves distributions of water components with different diffusion length scales, anisotropies, and relaxation properties, providing sensitivity to microstructural restriction, heterogeneity, and shape-size correlations within a voxel. Whether these measurements reveal microstructural complexity associated with AD pathology remains unclear. Here, we performed ωMD-MRI on ex vivo brain specimens from approximately 8-month-old 5xFAD and wild-type mice and interpreted the imaging findings alongside complementary histology. ωMD-MRI revealed widespread but spatially nonuniform differences between 5xFAD and wild-type brains. Measurements sensitive to microstructural restriction, heterogeneity, and shape-size correlations consistently indicated greater microstructural heterogeneity in 5xFAD brains, with the most prominent differences in the hippocampal formation and major cerebral white matter tracts. Complementary qualitative histology demonstrated extensive amyloid deposition and glial activation in affected regions, while overall cytoarchitecture and myelin organization remained largely preserved. Thus, the ωMD-MRI abnormalities occurred in tissue characterized by multiple coexisting pathological and relatively preserved microstructural environments rather than widespread structural degeneration. These findings demonstrate that ωMD-MRI can reveal the spatial and microstructural heterogeneity associated with amyloid pathology and provide a more comprehensive characterization of AD-related tissue alterations.

Abstract Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders, yet the environmental drivers that accelerate its progression remain poorly defined. Traffic-related air pollution is emerging as a modifiable AD risk factor, but how inhaled particles perturb microglial clearance of amyloid beta (Aβ) is unknown. Microglia are the principal Aβ-clearing phagocytes of the brain. Here, we showed that exposure of primary mouse microglia and human induced pluripotent stem cell-derived microglia (iMGLs) to 3–100 µg/mL diesel exhaust particles (DEP) disrupted microglial homeostasis, induced morphological abnormalities, increased reactive oxygen species, impaired lysosomal degradation, and led to a concentration-dependent loss of phagocytic capacity. Importantly, DEP markedly reduces Aβ uptake in both species. Transcriptomic profiling revealed a DEP-induced, non-canonical state characterized by metabolic reprogramming, broad suppression of inflammatory pathways, antigen-presentation, chemokine, and species-specific remodeling during subsequent Aβ challenge, including defective chemotaxis, cell cycle, and cytoskeletal signatures. These data show that DEP profoundly alters microglial transcriptional and metabolic states, leading to impaired Aβ clearance, which could, thereby, further contribute to AD progression.

Abstract Objective Temporal lobe epilepsy (TLE) is characterized by recurrent seizures originating usually from the hippocampus, and approximately one‐third of TLE patients remain refractory to pharmacological interventions. Surgical resection offers a potential cure for refractory TLE cases, with approximately 70% achieving seizure freedom. Still, the pathogenesis of TLE remains poorly understood. Electrophysiological characterization of the resected tissue with microelectrode arrays (MEAs) can help reveal the pathogenesis of TLE, but the commercially available MEAs cover only a small part of the hippocampal cross‐section. The objective here is to develop a MEA that can cover a significant area of a human hippocampal slice to help understand the electrophysiology of TLE pathogenesis. Methods The custom MEA, entitled Hippo‐MEA, was designed to have 60 round electrodes, each 60 μm in diameter, in an area of 5.6 mm × 5.6 mm. The titanium nitride‐coated electrodes were deposited on borosilicate glass using ion beam‐assisted e‐beam deposition (IBAD) process. Hippo‐MEA's sample chamber, named Sample Cup, was designed large enough to hold a human hippocampal slice. Hippocampal samples were obtained from patients undergoing neurosurgical tissue resection for TLE treatment and sliced to 300 μm thick sections for Hippo‐MEA recording. Data from the Hippo‐MEA were recorded using the commercially available MEA2100‐Mini‐system. Cellular composition of the recording area was analyzed with immunohistochemistry (IHC). Results Extracellular action potentials (EAPs) and local field potentials (LFPs) were successfully recorded from acute human hippocampal slices. Activity was mainly localized to the dentate gyrus, whereas the CA regions found sclerotic and gliotic in IHC analysis showed little to no activity. Significance Hippo‐MEA is compatible with a commercially available and widely used data acquisition system and enables recording of EAPs and LFPs across several regions of human hippocampal tissue. This enables Hippo‐MEA, in combination with other methods, to help discover the neurophysiological mechanisms of TLE. Plain Language Summary In some cases of temporal lobe epilepsy (TLE), the only treatment option is to surgically remove a part of the brain, including a structure known as the hippocampus, which is often the source of the electrical epileptic activity. Understanding hippocampal function is important to understanding the reasons behind TLE and designing the surgeries. Microelectrode arrays (MEAs) are a tool for recording the electrical function of tissues. Here we present a new MEA, named Hippo‐MEA, that is big enough to measure a cross‐section of the human hippocampus.

Human-induced pluripotent stem cell-derived midbrain organoids offer a promising platform for modeling Parkinson's disease (PD). Yet, their utility has been limited by the absence of microglia and the development of a necrotic core during maturation. Here, we present an air-liquid interface (ALI) slice culture system for extended cultivation of midbrain organoids (mORGs), enabling efficient microglial integration, improved neuronal viability, and enhanced functional maturation. Compared with conventional mORGs grown in suspension cultures, the ALI method supports more consistent engraftment of microglial progenitors and the development of astrocytes and oligodendrocyte progenitors, as revealed by single-cell RNA sequencing. Functionally, ALI-mORGs exhibited robust and reproducible neural network activity, with N-methyl-D-aspartate (NMDA) stimulation reliably inducing synchronous bursting, as measured by 3D microelectrode array recordings. Importantly, exposure to alpha-synuclein (αSyn) preformed fibrils triggered the progressive accumulation of phosphorylated αSyn inclusions, closely recapitulating key features of PD pathology. The ALI-mORG model addresses major limitations of existing systems and provides a more physiologically relevant platform for investigating cellular mechanisms of PD and for supporting future therapeutic strategies.

Plastic production continues to rise in 2025, despite environmental concerns and growing evidence pointing to its adverse impact on human health. Our knowledge of how micro- and nano-plastics affect brain health is, however, still in its infancy. To understand how neurons respond to stress caused by plastics, we exposed primary neurons to polystyrene nanoplastics (PS-NPs). We evaluated effects of the three different sizes of PS-NPs (50, 100, and 250 nm) at concentrations ranging from 0.05 μg/ml to 1 μg/ml. The exposure, relatively low in the tested concentrations and short in the exposure regimen (24 h), did not lead to impairments in neuronal metabolism. However, confocal imaging confirmed the uptake of 250 nm particles by neurons. Despite the lack of metabolic effects, the treatment contributed to neurite elongation in a size-specific manner: the effects were observed upon 50 nm PS-NPs treatment, but not when challenged by the 250 nm particles. Furthermore, subtle but statistically significant changes in the neuronal transcriptome were induced by the smaller, but not larger particles. Additionally, electrophysiological measurements of firing rate and spike amplitude at the time points of 2 and 24 h were not affected by the PS-NPs treatment. These findings highlight the importance of focusing further research specifically on nanoscale plastic particles in the context of brain health.

BACKGROUND:As the aging population increases, the prevalence of Alzheimer's disease and other memory disorders increases, and emerging neurotherapies offer new hope, yet little is known about nurses' knowledge and perspectives on these innovations, despite their critical role in patient care. This study aimed to investigate nurses' attitudes, expectations, concerns, and perspectives toward innovative therapies for memory disorders. METHODS:A multiple-choice questionnaire (24 items) in Finnish was developed to explore nurses' views. The questionnaire was distributed via email to nursing homes in the Wellbeing Services County of North Savo, Finland, in autumn 2024. Composite scores for perceived benefits, perceived barriers, enabling factors, professional role-related responsibilities, and worries were generated for statistical analyses. RESULTS:A total of 132 nurses responded to the survey, most of whom were practical nurses working in nursing homes. Overall familiarity with innovative neurotherapies was low, and many participants indicated that they were not at all familiar with these treatments. Opinions on the future role of such therapies in managing memory disorders varied, with some considering them significant or moderately important. Several respondents also expressed serious concerns regarding the efficacy and potential side effects of these therapies. Regression analyses showed that lower composite outcome scores were consistently associated with being a social and healthcare student, being somewhat familiar with innovative neurotherapies, and, in some models, with shorter work experience or specific workplace settings. However, adjusted R2 values ranged from 0.09 to 0.18, indicating that demographic and professional background factors explained only a modest proportion of variance in the composite outcomes. CONCLUSION:Nurses currently have limited familiarity with innovative neurotherapies, but there is cautious optimism about their future role in treating memory disorders, including Alzheimer's disease.

Autism spectrum disorder (ASD) is a group of complex neurodevelopmental conditions characterized by persistent deficits in social interaction, communication, and repetitive behavior. Although the etiology of ASD has traditionally been linked to neuronal dysfunctions, emerging evidence highlights the significance of non-neuronal cells, particularly microglia and astrocytes, in the pathophysiology of ASD. Although both glial cell types exhibit diverse functional states, microglia are primarily associated with eliminating unused neurons and synapses during early brain development, whereas astrocytes in the tripartite synapse mainly support synapse formation and actively regulate excitatory and inhibitory neurotransmission. Together, microglia and astrocytes complement each other in shaping a dynamic excitatory/inhibitory (E/I) network in the developing brain. Abnormal glial function during critical neurodevelopmental periods is emerging as a significant, and possibly even a leading factor in the E/I imbalance underlying ASD. In this review we highlight recent findings on how microglia and astrocytes contribute to E/I imbalance and other neuronal dysfunctions in ASD, particularly during the vulnerable prenatal period. GABAergic transmission, with a shift in the polarity of GABA action early in development, represents a particularly vulnerable target for aberrant modulation by glia. Recent transcriptomic and multi-omics studies show that signaling pathways thought to be neuron-specific can also be activated in glia at distinct developmental time points, shaping their unique contributions to ASD. This growing knowledge opens new perspectives for using glia-targeted therapeutic approaches for mitigating debilitating aspects of ASD.

Ischemic stroke triggers a rapid immune infiltration to the brain, reshaping its cell type composition and possibly confounding the analysis of immune cell enriched molecules. MicroRNAs (miRNAs) act as powerful regulators by fine-tuning messenger RNA (mRNA) expression, thereby modulating cell type specific responses to ischemia. Several bulk tissue analyses suggest elevated levels of anti-inflammatory miRNA, miR-223-3p, in ischemic stroke but pathological relevance of this deregulation remains unclear due to lack of single-cell resolution. We show that miR-223-3p is acutely increased in ischemic stroke patients’ blood and selectively expressed in myeloid cells in mice. Due to myeloid specificity of miR-223-3p, it appears massively elevated in bulk ischemic brain lysates, although the elevation primarily reflects increased myeloid cell abundance rather than per-cell upregulation in the brain. Strikingly, brain infiltrating macrophages exhibited acutely reduced miR-223-3p levels. We hypothesize that these macrophages initially acquire a more proinflammatory phenotype than the brain resident macrophages, and re-analysis of single-cell transcriptomics (GSE234052) further supported a proinflammatory macrophage phenotype after acute cerebral ischemia. Our study exemplifies the inherent limitations of bulk tissue analyses and underscores the need to revisit conclusions drawn from the tissue-level data, especially when immune cell infiltration and enrichment of a molecule is suspected. Overcoming the technical challenges of integrated spatial miRNA and mRNA detection at single-cell resolution will be essential to dissect the regulatory mechanisms underlying ischemic stroke pathology and to inform the development of effective immunomodulatory therapies. Increased brain miR-223-3p in ischemic stroke is caused by changes in cell type composition. In healthy brain, miR-223-3p is expressed by microglia and resident macrophages. Ischemia causes infiltration of miR-223-3p-rich peripheral myeloid cells to the brain, increasing the level of miR-223-3p in tissue lysates. In ischemic macrophages, miR-223-3p was decreased while no deregulation was found in microglia.

Age-related macular degeneration (AMD) is a global vision threatening disease affecting the macular region of the retina. AMD is classified into two forms: dry and wet AMD. There are no effective treatment options available for dry AMD (80% of cases). The cellular pathology includes oxidative stress and dysfunctional autophagy challenging the homeostasis of the retinal pigment epithelial (RPE) cells. Clinical findings include the formation of drusen deposits beneath the RPE cells consisting of 42 amino acids long amyloid beta (Aβ) among other components. However, the origin of drusen remains elusive. The 5xFAD (familiar Alzheimer's disease) mouse model of Alzheimer's disease produces abundant levels of Aβ making it an interesting model to study the possible relationship of Aβ to the formation of extracellular deposits and AMD-like pathology. An immunohistology analysis of the 5xFAD mouse model showed accumulation of autophagic markers SQSTM1 (sequestosome 1) and ubiquitin in the RPE. Concurrently, the markers of secretory autophagy enabling the delivery of the intracellular material to the extracellular lumen were upregulated. Aβ, SQSTM1, ubiquitin, catalase, and TRIM16 (tripartite motif containing 16) shifted age-dependently from intracellular origin to drusen-like deposits beneath the RPE cells. Additionally, classical proteins secreted via secretory autophagy, IL-1β (interleukin 1β), HMGB1 (high mobility group box-1), and ferritin showed similar accumulation which became visible in fundus age-dependently. These findings suggest a role for Aβ in the cellular pathogenesis of AMD. Furthermore, this model showed activated secretory autophagy pathway suggesting a role for Aβ in drusen-like deposition formation.

Frontotemporal dementia (FTD) is the second most common cause of dementia in patients under 65 years, characterized by diverse clinical symptoms, neuropathologies, and genetic background. Synaptic dysfunction is suggested to play a major role in FTD pathogenesis. Disturbances in the synaptic function can also be associated with the C9orf72 repeat expansion (C9-HRE), the most common genetic mutation causing FTD. C9-HRE leads to distinct pathological hallmarks, such as C9orf72 haploinsufficiency and development of toxic RNA foci and dipeptide repeat proteins (DPRs). FTD patient brains, including those carrying the C9-HRE, are also characterized by neuropathologies involving accumulation of TDP-43 and p62/SQSTM1 proteins. This study utilized induced pluripotent stem cell (iPSC)-derived cortical neurons from C9-HRE-carrying or sporadic FTD patients and healthy control individuals. We report that the iPSC neurons derived from C9-HRE carriers developed typical C9-HRE-associated hallmarks, including RNA foci and DPR accumulation. All FTD neurons demonstrated increased cytosolic accumulation of TDP-43 and p62/SQSTM1 and changes in nuclear size and morphology. In addition, the FTD neurons displayed reduced number and altered morphologies of dendritic spines and significantly altered synaptic function indicated by a decreased response to stimulation with GABA. These structural and functional synaptic disturbances were accompanied by upregulated gene expression in the FTD neurons related to synaptic function, including synaptic signaling, glutamatergic transmission, and pre- and postsynaptic membrane, as compared to control neurons. Pathways involved in DNA repair were significantly downregulated in FTD neurons. Only one gene, NUPR2, potentially involved in DNA damage response, was differentially expressed between the sporadic and C9-HRE-carrying FTD neurons. Our results show that the iPSC neurons from FTD patients recapitulate pathological changes of the FTD brain and strongly support the hypothesis of synaptic dysfunction as a crucial contributor to disease pathogenesis in FTD.

Traffic-related ultrafine particles (UFPs) are an emerging health concern affecting the brain and increasing the risk of Alzheimer’s disease (AD). PI3K/AKT signaling is known to contribute to neuronal survival and to be altered in AD. The nasal olfactory mucosa (OM) is a sensory tissue exposed directly to ambient air, and a starting point for olfactory neural circuits towards the brain. Evidence of air pollution-induced transcriptional regulation via microRNAs (miRNA) and DNA methylation (DNAmet) is accumulating and air pollutant-mediated disturbances in PI3K/AKT signaling have been reported. By utilizing a highly translational human-based in vitro model of OM, we aimed to investigate possible gene regulatory mechanisms in PI3K/AKT signaling induced by UFPs, and to compare the responses between cognitively healthy and individuals with AD. miRNA expression was analyzed using next-generation sequencing (NGS) and chip-based methylation analysis was performed to detect differentially methylated loci (DML). These data were combined with previously published transcriptomics analysis (mRNA) to construct an mRNA-miRNA-DNAmet-integrative network. Protein level changes were studied by immunoassays. We observed UFP-induced reductions in viability and increases in oxidative stress and DNA damage without eminent cell death. Integrative network analysis revealed multiple connections of miRNAs to differentially expressed genes in the PI3K/AKT pathway, and effects were most prominent in AD cells. Similarly, in AD cells DML were identified in transcription factor and apoptosis genes, downstream of PI3K/AKT signaling. Conclusively, traffic-related UFPs influence gene regulation of PI3K/AKT signaling to modulate OM cell survival, with existing AD pathology resulting in heightened vulnerability to UFP effects.

Aberrant and sustained activation of microglia is implicated in the progression and severity of multiple sclerosis (MS). However, whether intrinsic alterations in microglial function impact the pathogenesis of this disease remains unclear. We conducted transcriptomic and functional analyses of microglia-like cells (iMGLs) differentiated from induced pluripotent stem cells (iPSCs) from patients with MS (pwMS) to answer this question. The pwMS showed increased innate immune cell activity via 18-kDa translocator protein positron emission tomography imaging. After confirming that the differentiated iMGLs transcriptional profile is determined by the microglial cell type, comparative studies were performed to identify the transcriptional and functional differences between iMGLs from pwMS and healthy controls. Importantly, MS iMGLs presented cell-autonomous differences in their regulation of inflammation, both in the basal state and following inflammatory lipopolysaccharide challenge. Through transcriptomic profiling, we showed that MS iMGLs display increased expression of genes upregulated in MS pathology. Furthermore, upregulated genes in MS iMGLs were associated with immune receptor activation, antigen presentation, and the complement system. MS iMGLs demonstrated transcriptional similarities to lesion-specific microglia in MS, marked by upregulation of immune-related genes and pathways, including those involved in antigen presentation. Finally, functional analyses indicated that the transcriptional changes in MS iMGLs corresponded with modulation of cytokine secretion and increased phagocytosis. Together, our results provide evidence of putative cell-autonomous microglial activation in pwMS and identify transcriptomic and functional changes that recapitulate the phenotypes observed in vivo in microglia from pwMS. These findings indicate that MS disease-specific iPSCs are valuable tools for studying disease-specific microglial activation in vitro and highlight microglia as potential therapeutic targets in MS.

Chronic lymphocytic leukemia (CLL) is divided into unmutated (UM-CLL) and mutated (M-CLL) subtypes depending on somatic hypermutation (SHM) frequency in their immunoglobulin heavy chain V (IGHV) region. We previously demonstrated that CD27bright memory B cells (MBCs) are germinal center (GC)-dependent with higher mutation rate, whereas CD27dull MBCs accumulate fewer mutations and originate independently from the GC. We conducted a meta-transcriptomic analysis on bulk RNA data from 116 individuals combining four CLL cohorts and healthy B cell subsets (naïve, CD27dull and CD27bright MBCs) to decipher the transcriptional and mechanistic functions of CLL subtypes. CD27bright MBCs showed more transcriptional similarity to M-CLL rather than UM-CLL. Functional enrichment analysis revealed that LPL, ZNF667 and ZNF667-AS1 are potential informative biomarkers for stratification of CLL subtypes. They are part of the mechanistic regulatory pathways of CLL pathology through cholesterol and Epithelial Mesenchymal Transition (EMT) regulation. We applied markers for the GC B-cell substages to map in silico the CLL cohorts to their potential GC B cell counterpart. UM-CLL represented transcriptional mimicry to an early intermediary GC substage whereas M-CLL mimicked later substages in the GC. This could potentially explain the IGHV mutational status of M-CLL as well as hypothesize that CLL subtypes could derive from a GC-dependent pathway.

Autism spectrum disorder (ASD) presents a range of lifelong challenges in social communication, repetitive behaviors, and restricted interests, affecting over 2% of the preschool population. Early neurodevelopmental disruptions, particularly those affecting microglia, appear to be central to the pathophysiology of ASD, with microglia influencing synaptic development and stability in the brain. However, the neurobiological mechanisms underlying ASD are still not fully understood. Traditional ASD studies, which rely on animal models and postmortem tissues, have limitations in capturing human-specific neurodevelopmental dynamics. Recent advances in human model systems, including induced pluripotent stem cell (iPSC)-derived neural cultures and brain organoids, offer promising insights into microglia-neuron interactions relevant to ASD. This review evaluates current research using human-based models to explore ASD pathophysiology, focusing on the role of microglia in neurodevelopment, and discusses the strengths and future potential of these innovative approaches.

The molecular mechanisms leading to Alzheimer's disease (AD) are poorly known. This is due to the lack of human tissue samples for research representing early changes of AD pathology, raising a need for development of novel human-based preclinical models for AD. Idiopathic normal pressure hydrocephalus (iNPH) is an neurodegenerative disease characterized by an impaired cerebrospinal fluid (CSF) clearance. iNPH is treated by a shunt surger to drain the excess CSF into the abdominal cavity. During the surgery, 10-20mm 3 Broadman area 8 biopsy can be excised by minimally invasive methods for preclinical studies. Due to the early AD-related pathology present in a subpopulation of iNPH patients, the brains of these patients offer a unique window to evaluate cellular events taking place during the course of AD pathology progression. We have set up a pipeline to evaluate, in a layer and cell-type dependent manner, the intrinsic neuronal operational properties in the iNPH biopsies. Our patch clamp and multi electrode array (MEA) studies show that these biopsies are viable and retain the required microcircuit to study network and synaptic function in human cortex. We have carried out integrative analysis of human neuronal electrophysiology at single neuron and network level followed by subsequent cellular morphological reconstructions to register the primary pathological changes in neuronal functions in correlation with existing AD-related pathology. The presence of Aβ deposits induced a decrease in the L1-induced inhibition and led to hyperexcitability in response to application of NMDA in MEA recordings. Interestingly, the global spine density of supraganular pyramidal neurons was increased in biopsies with AD-related pathology. Pyramidal neurons in cases with both Aβ and tau exhibited more consistent deficits in the intrinsic neuronal properties with increase in sodium and potassium currents and a strong propensity to bursting under NMDA stimulation. This is the first study to show that the accumulation of Aβ and tau alters synaptic transmission and consolidation of a hyperexcitable supragranular cortical network. The iNPH biopsies provide a unique opportunity to unravel how AD-related pathology alters neuronal network functionality in humans.

In idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer's disease (AD), clearance of cerebrospinal fluid (CSF) is impaired, leading to the buildup of harmful proteins and metabolic waste in the brain, contributing to disease pathology. A significant number of iNPH patients have AD as a comorbidity, and both diseases display impaired memory and altered levels of amyloid beta (Aβ) and microtubule-associated protein Tau (Tau) in CSF, suggesting intersecting disease mechanisms. Genetic variants may cause loss-of-function mutations that affect protein levels or alter gene expression, potentially revealing novel pathogenic or protective mechanisms. We recently conducted a genome-wide association study (GWAS) for iNPH using FinnGen data and identified the SLCO1A2, MLLT10, AMZ1/GNA12 , and C16orf95 loci (Räsänen et al. 2024). To confirm and identify further genetic variants at the four GWAS-identified loci, we conducted whole genome sequencing (WGS) on ∼300 iNPH patients from the Kuopio iNPH cohort. To test the effects of the discovered variants on phenotype, we associated the genotypes to phenotypes within the Kuopio iNPH cohort, which includes immunohistochemistry from human brain biopsies, CSF biomarkers, and functional and cognitive data from patients in WGS. We will also utilize FinnGen phenotype and genotype data, including patients with NPH and AD diagnoses, to evaluate the variants' effects on onset age and CSF biomarker levels. Finally, we aim to validate the variants' effects on Aβ42 and Tau clearance from CSF to blood in a cell model of the blood-CSF barrier (BCB). Our preliminary results indicate that WGS-confirmed variants in SLCO1A2 loci affect the onset age of iNPH and AD. Additionally, these variants are associated with altered Aβ42 levels in the CSF of iNPH patients. Preliminary analysis indicates a functional proof-of-concept where the analysis of genotype and phenotype data can be used to identify clearance-related variants. We aim to further validate the variants' effects on CSF clearance in a model of the BCB.