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In addition to amyloid and tau pathology in the central nervous system (CNS), inflammatory processes and synaptic dysfunction are highly important mechanisms involved in the development and progression of dementia diseases. In the present study, we conducted a comparative analysis of selected pro-inflammatory proteins in the CNS with proteins reflecting synaptic damage and core biomarkers in mild cognitive impairment (MCI) and early Alzheimer's disease (AD). To our knowledge, no studies have yet compared CXCL12 and CX3CL1 with markers of synaptic disturbance in cerebrospinal fluid (CSF) in the early stages of dementia. The quantitative assessment of selected proteins in the CSF of patients with MCI, AD, and non-demented controls (CTRL) was performed using immunoassays (single- and multiplex techniques). In this study, increased CSF concentration of CX3CL1 in MCI and AD patients correlated positively with neurogranin (r = 0.74; p < 0.001, and r = 0.40; p = 0.020, respectively), ptau181 (r = 0.49; p = 0.040), and YKL-40 (r = 0.47; p = 0.050) in MCI subjects. In addition, elevated CSF levels of CXCL12 in the AD group were significantly associated with mini-mental state examination score (r = −0.32; p = 0.040). We found significant evidence to support an association between CX3CL1 and neurogranin, already in the early stages of cognitive decline. Furthermore, our findings indicate that CXCL12 might be a useful marker for tract severity of cognitive impairment. [ABSTRACT FROM AUTHOR]

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant forms of dementia caused by mutations in the integral membrane protein 2B (ITM2B, also known as BRI2) gene. Secretase processing of mutant BRI2 leads to secretion and deposition of BRI2‐derived amyloidogenic peptides, ABri and ADan that resemble APP/β‐amyloid (Aβ) pathology, which is characteristic of Alzheimer's disease (AD). Amyloid pathology in FBD/FDD manifests itself predominantly in the microvasculature by ABri/ADan containing cerebral amyloid angiopathy (CAA). While ABri and ADan peptide sequences differ only in a few C‐terminal amino acids, CAA in FDD is characterized by co‐aggregation of ADan with Aβ, while in contrast no Aβ deposition is observed in FBD. The fact that FDD patients display an earlier and more severe disease onset than FBD suggests a potential role of ADan and Aβ co‐aggregation that promotes a more rapid disease progression in FDD compared to FBD. It is therefore critical to delineate the chemical signatures of amyloid aggregation in these two vascular dementias. This in turn will increase the knowledge on the pathophysiology of these diseases and the pathogenic role of heterogenous amyloid peptide interactions and deposition, respectively. Herein, we used matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI) in combination with hyperspectral, confocal microscopy based on luminescent conjugated oligothiophene probes (LCO) to delineate the structural traits and associated amyloid peptide patterns of single CAA in postmortem brain tissue of patients with FBD, FDD as well as sporadic CAA without AD (CAA+) that show pronounced CAA without parenchymal plaques. The results show that CAA in both FBD and FDD consist of N‐terminally truncated‐ and pyroglutamate‐modified amyloid peptide species (ADan and ABri), but that ADan peptides in FDD are also extensively C‐terminally truncated as compared to ABri in FBD, which contributes to hydrophobicity of ADan species. Further, CAA in FDD showed co‐deposition with Aβ x‐42 and Aβ x‐40 species. CAA+ vessels were structurally more mature than FDD/FBD CAA and contained significant amounts of pyroglutamated Aβ. When compared with FDD, Aβ in CAA+ showed more C‐terminal and less N‐terminally truncations. In FDD, ADan showed spatial co‐localization with Aβ3pE‐40 and Aβ3‐40 but not with Aβx‐42 species. This suggests an increased aggregation propensity of Aβ in FDD that promotes co‐aggregation of both Aβ and ADan. Further, CAA maturity appears to be mainly governed by Aβ content based on the significantly higher 500/580 patterns observed in CAA+ than in FDD and FBD, respectively. Together this is the first study of its kind on comprehensive delineation of Bri2 and APP‐derived amyloid peptides in single vascular plaques in both FDD/FBD and sporadic CAA that provides new insight in non‐AD‐related vascular amyloid pathology. Cover Image for this issue: https://doi.org/10.1111/jnc.15424 [ABSTRACT FROM AUTHOR]

Background: Microglia are active modulators of Alzheimer's disease but their role in relation to amyloid plaques and synaptic changes due to rising amyloid beta is unclear. We add novel findings concerning these relationships and investigate which of our previously reported results from transgenic mice can be validated in knock-in mice, in which overexpression and other artefacts of transgenic technology are avoided. Methods: AppNL-F and AppNL-G-F knock-in mice expressing humanised amyloid beta with mutations in App that cause familial Alzheimer's disease were compared to wild type mice throughout life. In vitro approaches were used to understand microglial alterations at the genetic and protein levels and synaptic function and plasticity in CA1 hippocampal neurones, each in relationship to both age and stage of amyloid beta pathology. The contribution of microglia to neuronal function was further investigated by ablating microglia with CSF1R inhibitor PLX5622. Results: Both App knock-in lines showed increased glutamate release probability prior to detection of plaques. Consistent with results in transgenic mice, this persisted throughout life in AppNL-F mice but was not evident in AppNL-G-F with sparse plaques. Unlike transgenic mice, loss of spontaneous excitatory activity only occurred at the latest stages, while no change could be detected in spontaneous inhibitory synaptic transmission or magnitude of long-term potentiation. Also, in contrast to transgenic mice, the microglial response in both App knock-in lines was delayed until a moderate plaque load developed. Surviving PLX5266-depleted microglia tended to be CD68-positive. Partial microglial ablation led to aged but not young wild type animals mimicking the increased glutamate release probability in App knock-ins and exacerbated the App knock-in phenotype. Complete ablation was less effective in altering synaptic function, while neither treatment altered plaque load. Conclusions: Increased glutamate release probability is similar across knock-in and transgenic mouse models of Alzheimer's disease, likely reflecting acute physiological effects of soluble amyloid beta. Microglia respond later to increased amyloid beta levels by proliferating and upregulating Cd68 and Trem2. Partial depletion of microglia suggests that, in wild type mice, alteration of surviving phagocytic microglia, rather than microglial loss, drives age-dependent effects on glutamate release that become exacerbated in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Background: The panel of fluid- and imaging-based biomarkers available for neurodegenerative disease research is growing and has the potential to close important gaps in research and the clinic. With this growth and increasing use, appropriate implementation and interpretation are paramount. Various biomarkers feature nuanced differences in strengths, limitations, and biases that must be considered when investigating disease etiology and clinical utility. For example, neuropathological investigations of Alzheimer's disease pathogenesis can fall in disagreement with conclusions reached by biomarker-based investigations. Considering the varied strengths, limitations, and biases of different research methodologies and approaches may help harmonize disciplines within the neurodegenerative disease field. Purpose of review: Along with separate review articles covering fluid and imaging biomarkers in this issue of Alzheimer's Research and Therapy, we present the result of a discussion from the 2019 Biomarkers in Neurodegenerative Diseases course at the University College London. Here, we discuss themes of biomarker use in neurodegenerative disease research, commenting on appropriate use, interpretation, and considerations for implementation across different neurodegenerative diseases. We also draw attention to areas where biomarker use can be combined with other disciplines to understand issues of pathophysiology and etiology underlying dementia. Lastly, we highlight novel modalities that have been proposed in the landscape of neurodegenerative disease research and care. [ABSTRACT FROM AUTHOR]

Until relatively recently, a diagnosis of probable Alzheimer's disease (AD) and other neurodegenerative disorders was principally based on clinical presentation, with post-mortem examination remaining a gold standard for disease confirmation. This is in sharp contrast to other areas of medicine, where fluid biomarkers, such as troponin levels in myocardial infarction, form an integral part of the diagnostic and treatment criteria. There is a pressing need for such quantifiable and easily accessible tools in neurodegenerative diseases. In this paper, based on lectures given at the 2019 Biomarkers in Neurodegenerative Diseases Course, we provide an overview of a range of cerebrospinal fluid (CSF) and blood biomarkers in neurodegenerative disorders, including the 'core' AD biomarkers amyloid β (Aβ) and tau, as well as other disease-specific and general markers of neuroaxonal injury. We then highlight the main challenges in the field, and how those could be overcome with the aid of new methodological advances, such as assay automation, mass spectrometry and ultrasensitive immunoassays. As we hopefully move towards an era of disease-modifying treatments, reliable biomarkers will be essential to increase diagnostic accuracy, allow for earlier diagnosis, better participant selection and disease activity and treatment effect monitoring. [ABSTRACT FROM AUTHOR]

Imaging mass spectrometry (IMS) is a promising new chemical imaging modality that generates a large body of complex imaging data, which in turn can be approached using multivariate analysis approaches for image analysis and segmentation. Processing IMS raw data is critically important for proper data interpretation and has significant effects on the outcome of data analysis, in particular statistical modeling. Commonly, data processing methods are chosen based on rational motivations rather than comparative metrics, though no quantitative measures to assess and compare processing options have been suggested. We here present a data processing and analysis pipeline for IMS data interrogation, processing and ROI annotation, segmentation, and validation. This workflow includes (1) objective evaluation of processing methods for IMS datasets based on multivariate analysis using PCA. This was then followed by (2) ROI annotation and classification through region-based active contours (AC) segmentation based on the PCA component scores matrix. This provided class information for subsequent (3) OPLS-DA modeling to evaluate IMS data processing based on the quality metrics of their respective multivariate models and for robust quantification of ROI-specific signal localization. This workflow provides an unbiased strategy for sensitive annotation of anatomical regions of interest combined with quantitative comparison of processing procedures for multivariate analysis allowing robust ROI annotation and quantification of the associated molecular histology. [ABSTRACT FROM AUTHOR]

Abstract While the molecular mechanisms underlying Alzheimer's disease (AD) remain largely unknown, abnormal accumulation and deposition of beta amyloid (Aβ) peptides into plaques has been proposed as a critical pathological process driving disease progression. Over the last years, neuronal lipid species have been implicated in biological mechanisms underlying amyloid plaque pathology. While these processes comprise genetic features along with lipid signaling as well as direct chemical interaction of lipid species with Aβ mono- and oligomers, more efforts are needed to spatially delineate the exact lipid-Aβ plaque interactions in the brain. Chemical imaging using mass spectrometry (MS) allows to probe the spatial distribution of lipids and peptides in complex biological tissues comprehensively and at high molecular specificity. As different imaging mass spectrometry (IMS) modalities provide comprehensive molecular and spatial information, we here describe a multimodal ToF-SIMS- and MALDI-based IMS strategy for probing lipid and Aβ peptide changes in a transgenic mouse model of AD (tgAPP ArcSwe). Both techniques identified a general AD-associated depletion of cortical sulfatides, while multimodal MALDI IMS revealed plaque specific lipid as well as Aβ peptide isoforms. In addition, MALDI IMS analysis revealed chemical features associated with morphological heterogeneity of individual Aβ deposits. Here, an altered GM1 to GM2/GM3 ganglioside metabolism was observed in the diffuse periphery of plaques but not in the core region. This was accompanied by an enrichment of Aβ1–40arc peptide at the core of these deposits. Finally, a localization of arachidonic acid (AA) conjugated phosphatidylinositols (PI) and their corresponding degradation product, lyso-phosphatidylinositols (LPI) to the periphery of Aβ plaques was observed, indicating site specific macrophage activation and ganglioside processing. Graphical abstract Unlabelled Image Highlights • Imaging mass spectrometry incl. Tof-SIMS and MALDI are emerging tools in neuroscience. • Multimodal imaging allows to probe chemical architecture of Aβ pathology at a single plaque level. • IMS and multivariate analysis reveal plaque morphology associated lipid regulations in transgenic AD mice. • GM1 is spatially associated with Aβ 1–40 both co-localizing to core region of mature plaques. [ABSTRACT FROM AUTHOR]

The environmental toxin β-N-methylamino-L-alanine (BMAA) has been proposed to contribute to neurodegenerative diseases. We have previously shown that neonatal exposure to BMAA results in dose-dependent cognitive impairments, proteomic alterations and progressive neurodegeneration in the hippocampus of adult rats. A high BMAA dose (460 mg/kg) also induced intracellular fibril formation, increased protein ubiquitination and enrichment of proteins important for lipid transport and metabolism. The aim of this study was therefore to elucidate the role of neuronal lipids in BMAA-induced neurodegeneration. By using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS), we characterized the spatial lipid profile in the hippocampus of six month-old rats that were treated neonatally (postnatal days 9–10) with 460 mg/kg BMAA. Multivariate statistical analysis revealed long-term changes in distinct ganglioside species (GM, GD, GT) in the dentate gyrus. These changes could be a consequence of direct effects on ganglioside biosynthesis through the b-series (GM3-GD3-GD2-GD1b-GT1b) and may be linked to astrogliosis. Complementary immunohistochemistry experiments towards GFAP and S100β further verified the role of increased astrocyte activity in BMAA-induced brain damage. This highlights the potential of imaging MS for probing chemical changes associated with neuropathological mechanisms in situ . This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. [ABSTRACT FROM AUTHOR]

Introducing heterozygous humanized tau to App NL-F/NL-F knock-in mice results in the first mouse model of Alzheimer's disease in which age and amyloid-β pathology interact to initiate neurofibrillary tau tangle pathology, not dependent on mutations in MAPT. Gradual progression from amyloid-β to tau pathology in NLFTau m/h mice opens possibilities for understanding processes precipitating clinical stages of Alzheimer's disease and development of translatable therapies to prevent the onset of tau pathology.

In Alzheimer's disease (AD), amyloid-beta (Aβ) peptides are produced by proteolytic cleavage of the amyloid precursor protein (APP), which can occur during synaptic vesicle (SV) cycling at presynapses. Precisely how amyloidogenic APP processing may impair presynaptic proteostasis and how to therapeutically target this process remains poorly understood. Using App knock-in mouse models of early Aβ pathology, we found proteins with hampered degradation accumulate at presynaptic sites. At this mild pathological stage, amyloidogenic processing leads to accumulation of Aβ 42 inside SVs. To explore if targeting SVs modulates Aβ accumulation, we investigated levetiracetam (Lev), a SV-binding small molecule drug that has shown promise in mitigating AD-related pathologies despite its mechanism of action being unclear. We discovered Lev reduces Aβ 42 levels by decreasing amyloidogenic processing of APP in a SV2a-dependent manner. Lev corrects SV protein levels and cycling, which results in increased surface localization of APP, where it favors processing via the non-amyloidogenic pathway. Using metabolic stable isotopes and mass spectrometry we confirmed that Lev prevents the production of Aβ 42 in vivo. In transgenic mice with aggressive pathology, electrophysiological and immunofluorescent microscopy analyses revealed that Lev treatment reduces SV cycling and minimizes synapse loss. Finally, we found that human Down syndrome brains with early Aβ pathology, have elevated levels of presynaptic proteins, confirming a comparable presynaptic deficit in human brains. Taken together, we report a mechanism that highlights the therapeutic potential of Lev to modify the early stages of AD and represent a promising strategy to prevent Aβ 42 pathology before irreversible damage occurs., Competing Interests: Competing interests: The authors declare no competing interests.

It is of critical importance to our understanding of Alzheimer's disease (AD) pathology to determine how key pathological factors are interconnected and implicated in nerve cell death, clinical symptoms, and disease progression. The formation of extracellular beta-amyloid (Aβ) plaques is the major pathological hallmark of AD and Aβ has been suggested to be a critical inducer of AD, driving disease pathogenesis. Exactly how Aβ plaque formation begins and how ongoing plaque deposition proceeds and initiates subsequent neurotoxic mechanisms is not well understood. The primary aim of our research is to elucidate the biochemical processes underlying early Aβ plaque formation in brain tissue. We recently introduced a chemical imaging paradigm based on mass spectrometry imaging (MSI) and metabolic isotope labelling to follow stable isotope labelling kinetics (iSILK) in vivo to track the in vivo build-up and deposition of Aβ. Herein, knock-in Aβ mouse models ( App NL-F ) that develop Aβ pathology gradually are metabolically labeled with stable isotopes. This chemical imaging approach timestamps amyloid plaques during the period of initial deposition allowing the fate of aggregating Aβ species from before and during the earliest events of plaque pathology through plaque maturation to be tracked. To identify the molecular and cellular response to plaque maturation, we integrated iSILK with single plaque transcriptomics performed on adjacent tissue sections. This enabled changes in gene expression to be tracked as a function of plaque age (as encoded in the Aβ peptide isotopologue pattern) distinct from changes due to the chronological age or pathological severity. This approach identified that plaque age correlates negatively with gene expression patterns associated with synaptic function as early as in 10-month-old animals but persists into 18 months. Finally, we integrated hyperspectral confocal microscopy into our multiomic approach to image amyloid structural isomers, revealing a positive correlation between plaque age and amyloid structural maturity. This analysis identified three categories of plaques, each with a distinct impact on the surrounding microenvironment. Here, we identified that older, more compact plaques were associated with the most significant synapse loss and toxicity. These data show how isotope-encoded MS imaging can be used to delineate Aβ toxicity dynamics in vivo. Moreover, we show for the first time a functional integration of dynamic MSI, structural plaque imaging and whole genome-wide spatial transcriptomics at the single plaque level. This multiomic approach offers an unprecedented combination of temporal and spatial resolution enabling a description of the earliest events of precipitating amyloid pathology and how Aβ modulates synaptotoxic mechanisms., Competing Interests: Conflicts of interest HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZPath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, LabCorp, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures sponsored by Alzecure, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, Roche, and WebMD, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work).

Organic dyes typically have electronically excited states of both singlet and triplet multiplicity. Controlling the energy difference between these states is a key factor for making efficient organic light emitting diodes and triplet sensitizers, which fulfill essential functions in chemistry, physics, and medicine. Here, we propose a strategy to shift the singlet excited state of a known sensitizer to lower energies without shifting the energy of the triplet state, thus without compromising the ability of the sensitizer to do work. We covalently connect two to four sensitizers in such a way that their transition dipole moments are aligned in a head-to-tail fashion, but, through steric encumbrance, the delocalization is minimized between each moiety. Exciton coupling between the singlet excited states considerably lowers the first excited singlet state energy. However, the energy of the lowest triplet excited state is unperturbed because the exciton coupling strength depends on the magnitude of the transition dipole moments, which for triplets are very small. We expect that the presented strategy of designed intramolecular exciton coupling will be a useful concept in the design of both photosensitizers and emitters for organic light emitting diodes as both benefits from a small singlet-triplet energy gap., (© 2024. The Author(s).)

Acetylcholine (ACh) is a critical neurotransmitter influencing various neurophysiological functions. Despite its significance, quantitative methods with adequate spatiotemporal resolution for recording a single exocytotic ACh efflux are lacking. In this study, we introduce an ultrafast amperometric ACh biosensor that enables 50 kHz electrochemical recording of spontaneous single exocytosis events at axon terminals of differentiated cholinergic human SH-SY5Y neuroblastoma cells with sub-millisecond temporal resolution. Characterization of the recorded amperometric traces revealed seven distinct current spike types, each displaying variations in shape, time scale, and ACh quantities released. This finding suggests that exocytotic release is governed by complex fusion pore dynamics in these cells. The absolute number of ACh molecules released during exocytosis was quantified by calibrating the sensor through the electroanalysis of liposomes preloaded with varying ACh concentrations. Notably, the largest quantal release involving approximately 8000 ACh molecules likely represents full exocytosis, while a smaller release of 5000 ACh molecules may indicate partial exocytosis. Following a local administration of bafilomycin A1, a V-ATPase inhibitor, the cholinergic cells exhibited both a larger quantity of ACh released and a higher frequency of exocytosis events. Therefore, this ACh sensor provides a means to monitor minute amounts of ACh and investigate regulatory release mechanisms at the single-cell level, which is vital for understanding healthy brain function and pathologies and optimizing drug treatment for disorders.

Introduction: The established cerebrospinal fluid (CSF) phosphorylated tau181 (p-tau181) may not reliably reflect concomitant Alzheimer's disease (AD) and primary age-related tauopathy (PART) found in Creutzfeldt-Jakob disease (CJD) at autopsy., Methods: We investigated CSF N-terminal p-tau181, p-tau217, and p-tau231 with in-house Simoa assays in definite CJD (n = 29), AD dementia (n = 75), mild cognitive impairment (MCI) due to AD (n = 65), and subjective cognitive decline (SCD, n = 28). Post-mortem examination performed in patients with CJD 1.3 (0.3-14.3) months after CSF collection revealed no co-pathology in 10, concomitant AD in 8, PART in 8, and other co-pathologies in 3 patients., Results: N-terminal p-tau was increased in CJD versus SCD (p < 0.0001) and correlated with total tau (t-tau) in the presence of AD and PART co-pathology (rho = 0.758-0.952, p ≤ 001). Concentrations in CJD +AD were indistinguishable from AD dementia, with the largest fold-change in p-tau217 (11.6), followed by p-tau231 and p-tau181 (3.2-4.5)., Discussion: Variable fold-changes and correlation with t-tau suggest that p-tau closely associates with neurodegeneration and concomitant AD in CJD., Highlights: N-terminal phosphorylated tau (p-tau) biomarkers are increased in Creutzfeldt-Jakob disease (CJD) with and without concomitant AD. P-tau217, p-tau231, and p-tau181 correlate with total tau (t-tau) and increase in the presence of amyloid beta (Aβ) co-pathology. N-terminal p-tau181 and p-tau231 in Aβ-negative CJD show variation among PRNP genotypes. Compared to mid-region-targeting p-tau181, cerebrospinal fluid (CSF) N-terminal p-tau has greater potential to reflect post-mortem neuropathology in the CJD brain., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Amyloid plaque deposition is recognized as the primary pathological hallmark of Alzheimer's disease(AD) that precedes other pathological events and cognitive symptoms. Plaque pathology represents itself with an immense polymorphic variety comprising plaques with different stages of amyloid fibrillization ranging from diffuse to fibrillar, mature plaques. The association of polymorphic Aβ plaque pathology with AD pathogenesis, clinical symptoms and disease progression remains unclear. Advanced chemical imaging tools, such as functional amyloid microscopy combined with MALDI mass spectrometry imaging (MSI), are now enhanced by deep learning algorithms. This integration allows for precise delineation of polymorphic plaque structures and detailed identification of their associated Aβ compositions. We here set out to make use of these tools to interrogate heterogenic plaque types and their associated biochemical architecture. Our findings reveal distinct Aβ signatures that differentiate diffuse plaques from fibrilized ones, with the latter showing substantially higher levels of Aβx-40. Notably, within the fibrilized category, we identified a distinct subtype known as coarse-grain plaques. Both in sAD and fAD brain tissue, coarse grain plaques contained more Aβx-40 and less Aβx-42 compared with cored plaques. The coarse grain plaques in both sAD and fAD also showed higher levels of neuritic content including paired helical filaments (PHF-1)/phosphorylated phospho Tau-immunopositive neurites. Finally, the Aβ peptide content in coarse grain plaques resembled that of vascular Aβ deposits (CAA) though with relatively higher levels of Aβ1-42 and pyroglutamated Aβx-40 and Aβx-42 species in coarse grain plaques. This is the first of its kind study on spatial in situ biochemical characterization of different plaque morphotypes demonstrating the potential of the correlative imaging techniques used that further increase the understanding of heterogeneous AD pathology. Linking the biochemical characteristics of amyloid plaque polymorphisms with various AD etiologies and toxicity mechanisms is crucial. Understanding the connection between plaque structure and disease pathogenesis can enhance our insights. This knowledge is particularly valuable for developing and advancing novel, amyloid-targeting therapeutics., Competing Interests: Conflicts of interest HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZPath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures in symposia sponsored by Alzecure, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, and Roche, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). KB has served as a consultant, at advisory boards, or at data monitoring committees for Abcam, Axon, BioArctic, Biogen, JOMDD/Shimadzu. Julius Clinical, Lilly, MagQu, Novartis, Ono Pharma, Pharmatrophix, Prothena, Roche Diagnostics, and Siemens Healthineers, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program, outside the work presented in this paper.

Aggregated species of amyloid-β (Aβ) are one of the pathological hallmarks in Alzheimer's disease (AD), and ligands that selectively target different Aβ deposits are of great interest. In this study, fluorescent thiophene-based ligands have been used to illustrate the features of different types of Aβ deposits found in AD brain tissue. A dual-staining protocol based on two ligands, HS-276 and LL-1, with different photophysical and binding properties, was developed and applied on brain tissue sections from patients affected by sporadic AD or familial AD associated with the PSEN1 A431E mutation. When binding to Aβ deposits, the ligands could easily be distinguished for their different fluorescence, and distinct staining patterns were revealed for these two types of AD. In sporadic AD, HS-276 consistently labeled all immunopositive Aβ plaques, whereas LL-1 mainly stained cored and neuritic Aβ deposits. In the PSEN1 A431E cases, each ligand was binding to specific types of Aβ plaques. The ligand-labeled Aβ deposits were localized in distinct cortical layers, and a laminar staining pattern could be seen. Biochemical characterization of the Aβ aggregates in the individual layers also showed that the variation of ligand binding properties was associated with certain Aβ peptide signatures. For the PSEN1 A431E cases, it was concluded that LL-1 was binding to cotton wool plaques, whereas HS-276 mainly stained diffuse Aβ deposits. Overall, our findings showed that a combination of ligands was essential to identify distinct aggregated Aβ species associated with different forms of AD.

Blood phosphorylated tau (p-tau) biomarkers, including p-tau217, show high associations with Alzheimer's disease (AD) neuropathologic change and clinical stage. Certain plasma p-tau217 assays recognize tau forms phosphorylated additionally at threonine-212, but the contribution of p-tau212 alone to AD is unknown. We developed a blood-based immunoassay that is specific to p-tau212 without cross-reactivity to p-tau217. Here, we examined the diagnostic utility of plasma p-tau212. In five cohorts (n = 388 participants), plasma p-tau212 showed high performances for AD diagnosis and for the detection of both amyloid and tau pathology, including at autopsy as well as in memory clinic populations. The diagnostic accuracy and fold changes of plasma p-tau212 were similar to those for p-tau217 but higher than p-tau181 and p-tau231. Immunofluorescent staining of brain tissue slices showed prominent p-tau212 reactivity in neurofibrillary tangles that co-localized with p-tau217 and p-tau202/205. These findings support plasma p-tau212 as a peripherally accessible biomarker of AD pathophysiology., (© 2024. The Author(s).)

Lipid dysregulations have been critically implicated in Alzheimer's disease (AD) pathology. Chemical analysis of amyloid-β (Aβ) plaque pathology in transgenic AD mouse models has demonstrated alterations in the microenvironment in the direct proximity of Aβ plaque pathology. In mouse studies, differences in lipid patterns linked to structural polymorphism among Aβ pathology, such as diffuse, immature, and mature fibrillary aggregates, have also been reported. To date, no comprehensive analysis of neuronal lipid microenvironment changes in human AD tissue has been performed. Here, for the first time, we leverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) through a high-speed and spatial resolution commercial time-of-light instrument, as well as a high-mass-resolution in-house-developed orbitrap system to characterize the lipid microenvironment in postmortem human brain tissue from AD patients carrying Presenilin 1 mutations (PSEN1) that lead to familial forms of AD (fAD). Interrogation of the spatially resolved MSI data on a single Aβ plaque allowed us to verify nearly 40 sphingolipid and phospholipid species from diverse subclasses being enriched and depleted, in relation to the Aβ deposits. This included monosialo-gangliosides (GM), ceramide monohexosides (HexCer), ceramide-1-phosphates (CerP), ceramide phosphoethanolamine conjugates (PE-Cer), sulfatides (ST), as well as phosphatidylinositols (PI), phosphatidylethanolamines (PE), and phosphatidic acid (PA) species (including Lyso-forms). Indeed, many of the sphingolipid species overlap with the species previously seen in transgenic AD mouse models. Interestingly, in comparison to the animal studies, we observed an increased level of localization of PE and PI species containing arachidonic acid (AA). These findings are highly relevant, demonstrating for the first time Aβ plaque pathology-related alteration in the lipid microenvironment in humans. They provide a basis for the development of potential lipid biomarkers for AD characterization and insight into human-specific molecular pathway alterations.

Introduction: Amyloid-beta (Aβ) pathology is the precipitating histopathological characteristic of Alzheimer's disease (AD). Although the formation of amyloid plaques in human brains is suggested to be a key factor in initiating AD pathogenesis, it is still not fully understood the upstream events that lead to Aβ plaque formation and its metabolism inside the brains. Methods: Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) has been successfully introduced to study AD pathology in brain tissue both in AD mouse models and human samples. By using MALDI-MSI, a highly selective deposition of Aβ peptides in AD brains with a variety of cerebral amyloid angiopathy (CAA) involvement was observed. Results: MALDI-MSI visualized depositions of shorter peptides in AD brains; Aβ1-36 to Aβ1-39 were quite similarly distributed with Aβ1-40 as a vascular pattern, and deposition of Aβ1-42 and Aβ1-43 was visualized with a distinct senile plaque pattern distributed in parenchyma. Moreover, how MALDI-MSI covered in situ lipidomics of plaque pathology has been reviewed, which is of interest as aberrations in neuronal lipid biochemistry have been implicated in AD pathogenesis. Discussion: In this study, we introduce the methodological concepts and challenges of MALDI-MSI for the studies of AD pathogenesis. Diverse Aβ isoforms including various C- and N-terminal truncations in AD and CAA brain tissues will be visualized. Despite the close relationship between vascular and plaque Aβ deposition, the current strategy will define cross talk between neurodegenerative and cerebrovascular processes at the level of Aβ metabolism.

Importance: Blood-based measurements of total tau (T-tau) are commonly used to examine neuronal injury in patients with traumatic brain injury (TBI), but current assays do not differentiate between brain-derived tau (BD-tau) and tau produced in peripheral tissues. A novel assay for BD-tau has recently been reported that selectively quantifies nonphosphorylated tau of central nervous system origin in blood samples., Objectives: To examine the association of serum BD-tau with clinical outcomes in patients with severe TBI (sTBI) and its longitudinal changes over 1 year., Design, Setting, and Participants: This prospective cohort study was conducted at the neurointensive unit at the Sahlgrenska University Hospital, Gothenburg, Sweden, between September 1, 2006, and July 1, 2015. The study included 39 patients with sTBI followed up for up to 1 year. Statistical analysis was performed between October and November 2021., Exposures: Serum BD-tau, T-tau, phosphorylated tau231 (p-tau231), and neurofilament light chain (NfL) measured on days 0, 7, and 365 after injury., Main Outcomes and Measures: Associations of serum biomarkers with clinical outcome and longitudinal change in sTBI. Severity of sTBI was evaluated using the Glasgow Coma Scale at hospital admission, while clinical outcome was assessed with the Glasgow Outcome Scale (GOS) at 1-year follow-up. Participants were classified as having a favorable outcome (GOS score, 4-5) or unfavorable outcome (GOS score, 1-3)., Results: Among the 39 patients (median age at admission, 36 years [IQR, 22-54 years]; 26 men [66.7%]) in the study on day 0, the mean (SD) serum BD-tau level was higher among patients with unfavorable outcomes vs those with favorable outcomes (191.4 [190.8] pg/mL vs 75.6 [60.3] pg/mL; mean difference, 115.9 pg/mL [95% CI, 25.7-206.1 pg/mL]), while the other markers had smaller between-group mean differences (serum T-tau, 60.3 pg/mL [95% CI, -22.0 to 142.7 pg/mL]; serum p-tau231, 8.3 pg/mL [95% CI, -6.4 to 23.0 pg/mL]; serum NfL, -5.4 pg/mL [95% CI, -99.0 to 88.3 pg/mL]). Similar results were recorded on day 7. Longitudinally, baseline serum BD-tau concentrations showed slower decreases in the whole cohort (42.2% on day 7 [from 138.6 to 80.1 pg/mL] and 93.0% on day 365 [from 138.6 to 9.7 pg/mL]) compared with serum T-tau (81.5% on day 7 [from 57.3 to 10.6 pg/mL] and 99.0% on day 365 [from 57.3 to 0.6 pg/mL]) and p-tau231 (92.5% on day 7 [from 20.1 to 1.5 pg/mL] and 95.0% on day 365 [from 20.1 to 1.0 pg/mL]). These results did not change when considering clinical outcome, where T-tau decreased twice as fast as BD-tau in both groups. Similar results were obtained for p-tau231. Furthermore, the biomarker levels on day 365 were lower, compared with day 7, for BD-tau but not T-tau or p-tau231. Serum NfL had a different trajectory to the tau biomarkers, with levels increasing by 255.9% on day 7 compared with day 0 (from 86.8 to 308.9 pg/mL) but decreasing by 97.0% by day 365 vs day 7 (from 308.9 to 9.2 pg/mL)., Conclusions and Relevance: This study suggests that serum BD-tau, T-tau, and p-tau231 have differential associations with clinical outcome and 1-year longitudinal change in patients with sTBI. Serum BD-tau demonstrated utility as a biomarker to monitor outcomes in sTBI and can provide valuable information regarding acute neuronal damage.

Amyloid beta (Aβ) plaques are a major pathological hallmark of Alzheimer's disease (AD) and constitute of structurally heterogenic entities (polymorphs) that have been implicated in the phenotypic heterogeneity of AD pathology and pathogenesis. Understanding amyloid aggregation has been a critical limiting factor to gain understanding of AD pathogenesis, ultimately reflected in that the underlying mechanism remains elusive. We identified a fluorescent probe in the form of a turn-off photoswitchable norbornadiene derivative (NBD1) with several microenvironment-sensitive properties that make it relevant for applications within advanced fluorescence imaging, for example, multifunctional imaging. We explored the application of NBD1 for in situ delineation of structurally heterogenic Aβ plaques in transgenic AD mouse models. NBD1 plaque imaging shows characteristic broader emission bands in the periphery and more narrow emission bands in the dense cores of mature cored plaques. Further, we demonstrate in situ photoisomerization of NBD1 to quadricyclane and thermal recovery in single plaques, which is relevant for applications within both functional and super-resolution imaging. This is the first time a norbornadiene photoswitch has been used as a probe for fluorescence imaging of Aβ plaque pathology in situ and that its spectroscopic and switching properties have been studied within the specific environment of senile Aβ plaques. These findings open the way toward new applications of NBD-based photoswitchable fluorescent probes for super-resolution or dual-color imaging and multifunctional microscopy of amyloid plaque heterogeneity. This could allow to visualize Aβ plaques with resolution beyond the diffraction limit, label different plaque types, and gain insights into their physicochemical composition.

Beta-amyloid (Aβ) plaque pathology is one of the most prominent histopathological feature of Alzheimer's disease (AD). The exact pathogenic mechanisms linking Aβ to AD pathogenesis remain however not fully understood. Recent advances in amyloid-targeting pharmacotherapies highlight the critical relevance of Aβ aggregation for understanding the molecular basis of AD pathogenesis. We developed a novel, integrated, tetramodal chemical imaging paradigm for acquisition of trimodal mass spectrometry imaging (MSI) and interlaced fluorescent microscopy from a single tissue section. We used this approach to comprehensively investigate lipid-Aβ correlates at single plaques in two different mouse models of AD (tgAPP Swe and tgAPP ArcSwe ) with varying degrees of intrinsic properties affecting amyloid aggregation. Integration of the multimodal imaging data and multivariate data analysis identified characteristic patterns of plaque-associated lipid- and peptide localizations across both mouse models. Correlative fluorescence microscopy using structure-sensitive amyloid probes identified intra-plaque structure-specific lipid- and Aβ patterns, including Aβ 1-40 and Aβ 1-42 along with gangliosides (GM), phosphoinositols (PI), conjugated ceramides (CerP and PE-Cer), and lysophospholipids (LPC, LPA, and LPI). Single plaque correlation analysis across all modalities further revealed how these distinct lipid species were associated with Aβ peptide deposition across plaque heterogeneity, indicating different roles for those lipids in plaque growth and amyloid fibrillation, respectively. Here, conjugated ceramide species correlated with Aβ core formation indicating their involvement in initial plaque seeding or amyloid maturation. In contrast, LPI and PI were solely correlated with general plaque growth. In addition, GM1 and LPC correlated with continuous Aβ deposition and maturation. The results highlight the potential of this comprehensive multimodal imaging approach and implement distinct lipids in amyloidogenic proteinopathy.

We present a novel, correlative chemical imaging strategy based on multimodal matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), hyperspectral microscopy, and spatial chemometrics. Our workflow overcomes challenges associated with correlative MSI data acquisition and alignment by implementing 1 + 1-evolutionary image registration for precise geometric alignment of multimodal imaging data and their integration in a common, truly multimodal imaging data matrix with maintained MSI resolution (10 μm). This enabled multivariate statistical modeling of multimodal imaging data using a novel multiblock orthogonal component analysis approach to identify covariations of biochemical signatures between and within imaging modalities at MSI pixel resolution. We demonstrate the method's potential through its application toward delineating chemical traits of Alzheimer's disease (AD) pathology. Here, trimodal MALDI MSI of transgenic AD mouse brain delineates beta-amyloid (Aβ) plaque-associated co-localization of lipids and Aβ peptides. Finally, we establish an improved image fusion approach for correlative MSI and functional fluorescence microscopy. This allowed for high spatial resolution (300 nm) prediction of correlative, multimodal MSI signatures toward distinct amyloid structures within single plaque features critically implicated in Aβ pathogenicity., Competing Interests: The authors declare the following competing financial interest(s): H.Z. has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZPath, Annexon, Apellis, Artery Therapeutics, AZTherapies, CogRx, Denali, Eisai, Nervgen, Novo Nordisk, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures in symposia sponsored by Cellectricon, Fujirebio, Alzecure, Biogen, and Roche, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). K.B. has served as a consultant at advisory boards or at data monitoring committees for Abcam, Axon, BioArctic, Biogen, JOMDD/Shimadzu. Julius Clinical, Lilly, MagQu, Novartis, Ono Pharma, Pharmatrophix, Prothena, Roche Diagnostics, and Siemens Healthineers and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program, outside the work presented in this paper. The other authors declare no competing interests. The authors declare no competing interests., (© 2023 The Authors. Published by American Chemical Society.)

Using spatial cell-type-enriched transcriptomics, we compare plaque-induced gene (PIG) expression in microglia-touching plaques, neighboring plaques, and far from plaques in an aged Alzheimer's mouse model with late plaque development. In 18-month-old APP NL-F/NL-F knockin mice, with and without the Alzheimer's disease risk mutation Trem2 R47H/R47H , we report that expression of 38/55 PIGs have plaque-induced microglial upregulation, with a subset only upregulating in microglia directly contacting plaques. For seven PIGs, including Trem2, this upregulation is prevented in APP NL-F/NL-F Trem2 R47H/R47H mice. These TREM2-dependent genes are all involved in phagocytic and degradative processes that we show correspond to a decrease in phagocytic markers and an increase in the density of small plaques in Trem2-mutated mice. Furthermore, despite the R47H mutation preventing increased Trem2 gene expression, TREM2 protein levels and microglial density are still marginally increased on plaques. Hence, both microglial contact with plaques and functioning TREM2 are necessary for microglia to respond appropriately to amyloid pathology., Competing Interests: Declaration of interests A.V. and S.-L.J.P. were employed by Nanostring Technologies., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Amyloid-β (Aβ) pathology in Alzheimer's disease (AD) is characterized by the formation of polymorphic deposits comprising diffuse and cored plaques. Because diffuse plaques are predominantly observed in cognitively unaffected, amyloid-positive (CU-AP) individuals, pathogenic conversion into cored plaques appears to be critical to AD pathogenesis. Herein, we identified the distinct Aβ species associated with amyloid polymorphism in brain tissue from individuals with sporadic AD (s-AD) and CU-AP. To this end, we interrogated Aβ polymorphism with amyloid conformation-sensitive dyes and a novel in situ MS paradigm for chemical characterization of hyperspectrally delineated plaque morphotypes. We found that maturation of diffuse into cored plaques correlated with increased Aβ1-40 deposition. Using spatial in situ delineation with imaging MS (IMS), we show that Aβ1-40 aggregates at the core structure of mature plaques, whereas Aβ1-42 localizes to diffuse amyloid aggregates. Moreover, we observed that diffuse plaques have increased pyroglutamated Aβx-42 levels in s-AD but not CU-AP, suggesting an AD pathology-related, hydrophobic functionalization of diffuse plaques facilitating Aβ1-40 deposition. Experiments in tgAPP Swe mice verified that, similar to what has been observed in human brain pathology, diffuse deposits display higher levels of Aβ1-42 and that Aβ plaque maturation over time is associated with increases in Aβ1-40. Finally, we found that Aβ1-40 deposition is characteristic for cerebral amyloid angiopathy deposition and maturation in both humans and mice. These results indicate that N-terminal Aβx-42 pyroglutamation and Aβ1-40 deposition are critical events in priming and maturation of pathogenic Aβ from diffuse into cored plaques, underlying neurotoxic plaque development in AD., (© 2019 Michno et al.)

Background: Parathyroid hormone-related protein (PTHrP) is involved in intracellular calcium regulation, neural cell proliferation and synaptic transmission. To date, no studies have been performed to evaluate the potential of PTHrP concentrations in cerebrospinal fluid (CSF) as a biomarker of brain pathophysiology. In this study we evaluated the association between CSF concentrations of PTHrP with the core CSF biomarkers of Alzheimer's disease (AD)., Methods: PTHrP and calcium were analysed using validated mass spectrometry based methods in a set of CSF samples that tested positive (n = 45) and negative (n = 45) for the AD biomarkers, including total tau protein (T-tau), phosphorylated tau protein (P-tau) and amyloid-β 42 (Aβ42). The measured CSF concentrations of PTHrP and calcium (Ca) were evaluated for association with AD CSF biomarkers., Results: PTHrP and Ca concentrations in CSF samples ranged between 25 and 137 pmol/L and 0.92-1.53 mmol/L, respectively. Higher concentrations of PTHrP were observed in association with increased concentrations of T-tau and P-tau in the AD and the control group; while a stronger correlation was observed in the control group (ρ = 0.6, p < 0.0001; and ρ = 0.72, p < 0.0001, for T-tau and P-tau, respectively). Negative correlation was observed between concentrations of PTHrP and Aβ42 in the AD group (ρ = 0.27, p = 0.015). A statistically significantly lower ratio Aβ42/PTHrP was observed in the AD group (p < 0.0001)., Conclusion: In the current study, we observed an association of PTHrP concentrations with concentrations of clinically used CSF biomarkers of AD. Concentrations of PTHrP were positively correlated with concentrations of T-tau and P-tau, suggesting an association with neuronal secretion and function, which is reduced upon progression to AD pathology. Our data suggest potential utility of the Aβ42/PTHrP ratio in assessment of AD progression., (© 2018 The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). Published by Elsevier B.V. All rights reserved.)

During the last decades, imaging mass spectrometry has gained significant relevance in biomedical research. Recent advances in imaging mass spectrometry have paved the way for in situ studies on drug development, metabolism and toxicology. In contrast to whole-body autoradiography that images the localization of radiolabeled compounds, imaging mass spectrometry provides the possibility to simultaneously determine the discrete tissue distribution of the parent compound and its metabolites. In addition, imaging mass spectrometry features high molecular specificity and allows comprehensive, multiplexed detection and localization of hundreds of proteins, peptides and lipids directly in tissues. Toxicologists traditionally screen for adverse findings by histopathological examination. However, studies of the molecular and cellular processes underpinning toxicological and pathologic findings induced by candidate drugs or toxins are important to reach a mechanistic understanding and an effective risk assessment strategy. One of IMS strengths is the ability to directly overlay the molecular information from the mass spectrometric analysis with the tissue section and allow correlative comparisons of molecular and histologic information. Imaging mass spectrometry could therefore be a powerful tool for omics profiling of pharmacological/toxicological effects of drug candidates and toxicants in discrete tissue regions. The aim of the present review is to provide an overview of imaging mass spectrometry, with particular focus on MALDI imaging mass spectrometry, and its use in drug development and toxicology in general.

The nervous system constitutes arguably the most complicated and least understood cellular network in the human body. This consequently manifests itself in the fact that the molecular bases of neurodegenerative diseases remain unknown. The limited understanding of neurobiological mechanisms relates directly to the lack of appropriate bioanalytical technologies that allow highly resolved, sensitive, specific and comprehensive molecular imaging in complex biological matrices. Imaging mass spectrometry (IMS) is an emerging technique for molecular imaging. The technique is characterized by its high chemical specificity allowing comprehensive, spatial protein and peptide profiling in situ. Imaging MS represents therefore a powerful approach for investigation of spatio-temporal protein and peptide regulations in CNS derived tissue and cells. This review aims to provide a concise overview of major developments and applications concerning imaging mass spectrometry based protein and peptide profiling in neurobiological and biomedical research. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is one of the most effective tools for localizing small molecules and compounds directly in thin tissue sections. MALDI IMS should be used when the distribution of molecular species is not known and to localize changes due to a disease process or a treatment. In recent years it has become increasingly clear that many pathological processes are not readily correlated to dramatic changes in protein levels. MALDI IMS can aid the localization of areas where the cellular concentration of proteins may be high enough to play an important biological role, but when the precise location is unknown. Here, we present a MALDI IMS protocol and data analysis of molecular imaging of multiple rat brain sections.

Analysis of proteins in human body fluids is challenging since the composition of the sample often is rather complex. Here we present a method for analysis of proteins in aqueous humor from two groups of cataract patients, with and without pseudoexfoliation (PEX). Aqueous humor is an extracellular fluid contained in the anterior chamber of the eye between the cornea and iris. The limited volume of sample requires sophisticated analysis techniques. Our method is based on a total tryptic digestion of the sample followed by capillary LC-MALDI MS and MS/MS analysis of the peptides. The method is rapid, efficient and suitable as a complement or alternative to more commonly used methods based on gel electrophoretic experiments. With this method we found and unambiguously identified 30 nonredundant proteins. Proteins found include general transport proteins such as albumin and apolipoprotein A1 but also specific proteins involved in immune response, such as complement factors. Cystatin C, clusterin, and crystallins were also found. Although the number of proteins was roughly the same in both groups there was a significant difference in their identities. These findings may give some new insights into the pathophysiology of the PEX syndrome., (Copyright © 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)