Background The clinical phenotype of corticobasal syndrome can be caused by aggregation of various proteins in the brain, including 4-repeat tau in a substantial proportion of the patients. Reliable biomarkers for differentiating corticobasal syndrome with aggregation of 4-repeat tau from related parkinsonian disorders remain limited. We therefore aimed to (i) map syndrome-specific CSF proteomic signatures across various parkinsonian disorders, (ii) identify associated molecular pathways and (iii) explore protein biomarkers capable of differentiating β-amyloid negative corticobasal syndrome. Methods We quantified 127 CSF proteins using the Nucleic acid Linked Immuno-Sandwich Assay (NULISA) on a biomarker-enhanced cohort of clinically characterized patients with Parkinson's disease, multiple system atrophy, progressive supranuclear palsy and corticobasal syndrome. Cohort inclusion required fulfillment of established clinical diagnostic criteria supported by complementary biomarkers indicative of the presumed underlying proteinopathy. Differential protein abundance was assessed using age- and sex-adjusted generalized linear models. Pathway-level alterations were examined using panel-aware gene set enrichment analysis. Biomarker candidates for distinguishing β-amyloid-negative corticobasal syndrome from other parkinsonian disorders were identified using elastic-net feature selection and validated by receiver operating characteristic analysis. Results β-amyloid-negative corticobasal syndrome exhibited the most pronounced proteomic alterations, characterized by widespread increases in protein abundance and enrichment of inflammatory and vascular pathways, including IL-10 associated signaling when compared to controls. A core set of 37 proteins was consistently altered relative to other parrkinsonian syndromes, with a subset of proteins converging across multiple analytical approaches, including inflammatory mediators (CXCL8, IL-18, CX3CL1) and vascular/metabolic proteins (VEGFA, PGK1). These proteins also demonstrated high discriminatory potential, with an area under the curve above 0.85. Conclusions High-plex CSF proteomics suggests that β-amyloid-negative corticobasal syndrome is associated with a distinct vascular-immune molecular profile, indicating molecular differences from clinically similar parkinsonian disorders. These findings provide insight into biological processes linked to corticobasal syndrome and highlight candidate protein signatures that to our knowledge have not yet been systematically associated with β-amyloid-negative CBS and may support molecular stratification in parkinsonian disorders.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a central regulator of microglial activity and loss-of-function coding variants are major risk factors for late onset Alzheimer’s disease (LOAD). To better understand the molecular and functional changes associated with TREM2 signalling in microglia, we generated a TREM2 reporter mouse. In APP transgenic animals, bulk RNA-sequencing of isolated microglia sorted based on reporter expression highlighted TREM2 level-related changes in major immunometabolic pathways, and enrichment of genes in oxidative phosphorylation and cholesterol metabolism in microglia with increased TREM2 expression. Metabolic and lipidomic profiling of sorted microglia showed that, independent of Aβ pathology, TREM2 expression correlated with signatures consistent with increased cellular redox, energetics, and cholesterol homoeostasis. In accordance, metabolic activity correlated with phagocytic capacity. Finally, we performed chronic treatment with a TREM2 agonist antibody and identified a window of TREM2 expression where microglia are most responsive, thereby informing clinical applications of TREM2 agonists. TREM2 is an important AD risk factor playing essential roles in the microglial response to amyloid pathology. Here, authors show using a TREM2 reporter mouse that TREM2 levels are critical for efficacy of TREM2 agonism informing current clinical efforts.

Stem-cell-based in vitro models offer promising potential to elucidate human brain cell functions and interactions under physiological and pathological conditions. However, harnessing this potential is impaired by low reproducibility, maturity, or cell-type diversity of existing models. Especially, prolonged incorporation of mature microglia and studies of neuroinflammation have proven challenging. Here, we developed a 3D cortical brain tissue model (3BTM) containing neurons, astrocytes, and microglia with high reproducibility, maturity, and viability. 3BTMs show morphological, functional, and proteomic maturation of all cell types, leading to high similarity to their in vivo counterparts. Incorporated microglia survive for over 6 months and display mature morphology, functions, and gene expression. Importantly, when engineered to model Alzheimer's disease (AD) pathology, 3BTMs recapitulate key disease hallmarks including amyloid deposition, increased phospho-Tau levels, and neuroinflammation, with microglia shifting their transcriptional landscape to disease-relevant signatures. Treatment of AD 3BTMs with anti-Aβ immunotherapy cleared deposits and largely reversed disease signatures in glia. Together, our model offers unprecedented possibilities for studying physiological and pathological states of human brain tissue and translational applications. ### Competing Interest Statement J.K., C.C.G., and D.P. have filed a patent application covering generation, maintenance, and applications of 3BTMs. D.P. is an advisor to ISAR Bioscience GmbH, Planegg. All other authors declare no competing interests. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, EXC2145, ID 390857198, EXC2151, ID 390873048, TRR 274/1,2 project Z01 – ID 408885537 BrightFocus Foundation, ADR AD2019604S Centers of Excellence in Neurodegeneration, CoEN6005 Bundesministerium für Bildung und Forschung, FKZ: 16LW0473, FKZ: 01ED2402A Ministry of Culture and Science of North Rhine-Westphalia

Dementia with Lewy bodies (DLB) is characterized by marked biological heterogeneity, only partly explained by the frequent presence of Alzheimer's disease (AD) copathology. Whether microglial responses contribute to this heterogeneity and how they are modulated by APOE genotype remain poorly understood. Using a specific immunoassay targeting cleaved soluble TREM2 (sTREM2), we investigated TREM2-dependent microglial responses across two independent DLB cohorts (n = 129) with molecular biomarker profiling and longitudinal follow-up. sTREM2 showed stage-dependent associations with AD-related biomarkers, being associated with amyloid-related changes during prodromal DLB and predominantly to tau-related markers at dementia stage. APOEε4 carriers exhibited approximately two-fold lower sTREM2 levels than non-carriers specifically during prodromal DLB, independently of AD copathology, with more pronounced effects observed in women. Longitudinal analyses showed that higher baseline sTREM2 levels during prodromal DLB were associated with slower cognitive decline independently of AD-related biomarkers. These findings suggest that APOEε4 attenuates early TREM2-mediated microglial responses in DLB through mechanisms beyond AD copathology. Together, our results identify the APOE-TREM2 axis as a potential contributor to disease heterogeneity and support stage-specific, biomarker-guided therapeutic strategies targeting TREM2 signaling in Lewy body disorders.

Neuroinflammation is increasingly recognized as a key modulator of therapeutic response and adverse events in Alzheimer’s disease (AD), especially during anti-amyloid-β (Aβ) monoclonal antibody (Aβ-mAb) treatment. We applied longitudinal translocator protein (TSPO) positron emission tomography (PET) to evaluate TSPO-associated neuroinflammatory responses to chronic Aβ-mAb therapy and their modulation by the peroxisome proliferator-activated receptor γ (PPARγ) agonist pioglitazone. AppNL-G-F knock-in mice underwent TSPO-PET and Aβ-PET imaging at 5, 7.5, and 10 months of age across four treatment arms: placebo, Aβ-mAb, pioglitazone, and combination therapy. TSPO-PET detected early and progressive neuroinflammatory responses to Aβ-mAb that appeared lower with pioglitazone co-treatment. Both mono- and combination therapy were associated with altered temporal and spatial dynamics of the TSPO-PET signal. In addition, we applied a previously validated microglia desynchronization index based on TSPO-PET connectivity, which captured individual variation in regional TSPO-PET organization and correlated with cognitive performance. Together, TSPO-PET and its regional synchronicity can quantify longitudinal, region-specific treatment effects, which may help differentiate harmful from adaptive neuroinflammatory responses. These findings highlight the potential of TSPO-PET as a stratification biomarker to optimize therapeutic interventions. TSPO-PET therefore enables in vivo tracking of treatment-associated neuroinflammatory responses during anti-Aβ immunotherapy and provides a non-invasive framework for evaluating combination strategies targeting amyloid pathology and immune regulation in AD.

Stem-cell-based in vitro models offer promising potential to elucidate human brain cell functions and interactions, but limitations in reproducibility, maturation and cell-type diversity persist. Especially, prolonged incorporation of mature microglia and studies of neuroinflammation have proven challenging. Here, we developed a human induced pluripotent stem cell-based three-dimensional cortical brain tissue model (3BTM) containing neurons, astrocytes and microglia with high reproducibility, maturity and viability. 3BTMs show morphological, functional and proteomic maturation of all cell types, leading to high similarity to their in vivo counterparts. Incorporated microglia survive for over 6 months and display mature morphology, functions and gene expression. Importantly, when engineered to model Alzheimer's disease pathology, 3BTMs recapitulate key disease hallmarks, including amyloid deposition, increased phospho-tau levels and neuroinflammation, with microglia shifting their transcriptional landscape to disease-relevant signatures. Treatment of Alzheimer's disease 3BTMs with anti-Aβ immunotherapy cleared deposits and largely reversed disease signatures in glia. Together, our microglia-containing model provides a platform for studying physiological and pathological states of human brain tissue.

Anti-amyloid β-peptide (Aβ) immunotherapy was developed to reduce amyloid plaque pathology and slow cognitive decline during progression of Alzheimer’s disease. Efficient amyloid plaque clearance has been proven in clinical trials testing anti-Aβ antibodies, with the impact on cognitive endpoints correlating with the extent of plaque removal. However, treatment is associated with adverse side-effects, such as oedema and haemorrhages, which are potentially linked to the induced immune response. To improve the safety profile of these molecules, it is imperative to understand the consequences of anti-Aβ antibody treatment on immune cell function. Here, we investigated the effects of long-term chronic anti-Aβ treatment on amyloid plaque pathology and microglial response in the APP-SAA triple knock-in mouse model. Mice were treated weekly with anti-Aβ antibody from 4-8 months of age. Long-term treatment with anti-Aβ results in a robust and dose-dependent removal of amyloid plaque pathology, with a higher efficiency for removing diffuse over dense-core plaques. Analysis of the CSF proteome indicates a reduction of markers for neurodegeneration including Tau and α-Synuclein, as well as immune cell related proteins. Bulk RNA-seq revealed a dose-dependent decrease in brain-wide disease-associated microglial (DAM) and glycolytic gene expression, which is supported by a parallel decrease of glucose uptake and protein levels of Triggering receptor of myeloid cells 2 (Trem2) protein, a major immune receptor involved in DAM activation of microglia. In contrast, DAM activation around remaining plaques remains high regardless of treatment dose. In addition, microglia surrounding remaining plaques display a dose-dependent increase in microglial clustering and a selective increase in antigen presenting and immune signalling proteins. These findings demonstrate that long-term chronic anti-Aβ mediated removal of Aβ leads to a dose dependent decrease in brain-wide microglial DAM activation and neurodegeneration, while microglia at residual plaques display a combined DAM and antigen presenting phenotype that suggests a continued treatment response. ![Figure][1] Graphical abstract: Schematic overview of the effects of chronic long-term anti-Aβ treatment in APP-SAA mice Schematic was created with BioRender.com ### Competing Interest Statement C.H. and K.S. collaborate with Denali Therapeutics Inc. and C.H. is a member of the advisory boards of AviadoBio, Cure Ventures and Curie.Bio. M.B. is a member of the Neuroimaging Committee of the EANM. M.B. has received speaker honoraria from Roche, GE Healthcare, Iba, and Life Molecular Imaging; has advised Life Molecular Imaging and GE healthcare; and is currently on the advisory board of MIAC. T.S., C.H., S.S.D., V.W., D.X., J.W.L. and K.M.M. are full time employees of Denali Therapeutics Inc. * α-Syn : α-Synuclein Aβ : Amyloid β-peptide AD : Alzheimer’s disease APP : Amyloid precursor protein ARIA : Amyloid-related imaging abnormalities ARIA-E : ARIA-related oedema ARIA-H : ARIA-related haemorrhage BCA : Bicinchoninic acid BSA : Bovine serum albumin CAA : Cerebral amyloid-angiopathy CE : Cholesterol ester COA : Cortico-amygdala area CSF : Cerebrospinal fluid CTF : C-terminal fragment DAM : Disease associated microglia DAPI : 40,6-diamidino-2-phenylindole DEA : Diethylamine DEG : Differentially expressed gene diaPASEF : Data Independent Acquisition Parallel Accumulation–Serial Fragmentation ELISA : Enzyme-linked immunosorbent assay EtOH : Ethanol FA : Formic acid FcγR : Fc gamma receptor FBB : Florbetaben FDG : Fluorodeoxyglucose FDR : False discovery rate Gfap : Glial fibrillary acidic protein GM3 : Ganglioside mannose 3 GSEA : Gene set enrichment analysis HBSS : Hanks’ buffered salt solution hTfR : Human transferrin receptor IFN : Intereferon Il1rn : Interleukin-1 receptor anatagonist i.p. : intraperitoneal KI : Knock-in LAMP1 : Lysosomal-associated membrane protein 1 LC-MS : Liquid chromatography - mass spectrometry LC-MS/MS : Liquid chromatography - tandem mass spectrometry LOAD : Late-onset Alzheimer’s disease MACS : Magnetic-activated cell sorting MBq : Megabecquerel MCI : Mild cognitive impairment MHC : Major histocompatibility complex MMF : Medetomidine-midazolam-fentanyl MR : Magnetic resonance MRI : Magnetic resonance imaging MSD : Meso Scale Discovery MX-04 : Methoxy-04 NaCl : Sodium chloride NDS : Normal donkey serum PBS : Phosphate-buffered saline PET : Positron-emission tomography PFA : Paraformaldehyde RIPA : Radioimmunoprecipitation assay RNA-seq : RNA-sequencing ROI : Region of interest ROS : Reactive oxygen species RT : Room temperature SEM : Standard error of the mean SUV : Standard uptake value TBS : Tris-buffered saline TIMS : Trapped Ion Mobility Spectrometry Trem2 : Triggering receptor expressed on myeloid cells 2 VOI : Voxel of interest VT : Total volume of distribution [1]: pending:yes

Clinical and genetic research links altered cholesterol metabolism with ALS development and progression, yet pinpointing specific pathomechanisms remain challenging. We investigated how cholesterol dysmetabolism interacts with protein aggregation, demyelination, and neuronal loss in ALS. Bulk RNAseq transcriptomics showed decreased cholesterol biosynthesis and increased cholesterol export in ALS mouse models (GA-Nes, GA-Camk2a GA-CFP, rNLS8) and patient samples (spinal cord), suggesting an adaptive response to cholesterol overload. Consequently, we assessed the efficacy of the cholesterol-binding drug 2-hydroxypropyl-β-cyclodextrin (CD) in a fast-progressing C9orf72 ALS mouse model with extensive poly-GA expression and myelination deficits. CD treatment normalized cholesteryl ester levels, lowered neurofilament light chain levels, and prolonged lifespan in female but not male GA-Nes mice, without impacting poly-GA aggregates. Single nucleus transcriptomics indicated that CD primarily affected oligodendrocytes, significantly restored myelin gene expression, increased density of myelinated axons, inhibited the disease-associated oligodendrocyte response, and downregulated the lipid-associated genes Plin4 and ApoD. These results suggest that reducing excess free cholesterol in the CNS could be a viable ALS treatment strategy.

Memory clinic patients typically present with Alzheimer’s disease (AD) and cerebral small vessel disease (SVD) to varying degrees. Therefore, it is crucial to determine the etiology of cognitive deficits for facilitating patient-centered treatment in memory clinics. Plasma biomarkers (ptau 217 , Glial Fibrillary Acidic Protein [GFAP], Neurofilament light chain [NfL]) and fixel-based advanced diffusion MRI markers (fiber density, fiber-bundle cross-section) show potential towards disentangling AD- and SVD-related brain changes (Dewenter et al., Brain, 2023). However, their predictive power in understanding heterogeneous and SVD/AD-specific cognitive deficits in memory clinic patients remains incomplete. We assessed i) how plasma-based and fixel markers explain AD-typical and SVD-typical cognitive deficits and ii) their interrelation to uncover disease-specific mechanisms. We included n=76 in-house memory clinic patients with Simoa-based plasma ptau 217 , GFAP and NfL assessments, comprehensive neuropsychological testing (CERAD-plus battery) and 3T MRI. Global white matter hyperintensity (WMH) volume and average skeletonized mean diffusivity (MD) were included as well-established SVD markers. AD severity was probed through plasma ptau 217 and cortical thickness of the pre-established AD signature ROI. Advanced diffusion MRI was used to assess fiber density and fiber bundle cross-section of key white matter tracts (Figure 1A). Using linear regression, increased ptau 217, reduced cortical thickness in AD signature ROI and fiber-bundle cross-section were highly associated with impaired episodic memory, i.e. a typical AD-related symptom (Figure 1B, Figure 2A). In contrast, GFAP and NfL increases and reductions in MSMD and fiber density were associated with executive dysfunction, a typical sign of SVD-related cognitive impairment (Figure 1B, Figure 2A). Additionally, fiber density was associated with GFAP and NfL levels, while fiber bundle-cross section, a macroscopic marker of tract atrophy, was not associated with any of the plasma markers (Figure 2B). Ptau 217 displayed high sensitivity and specificity for AD-typical memory impairment, whereas GFAP and NfL were associated with SVD-typical processing speed and executive impairment. Additionally, fiber density, a pre-established imaging marker for SVD, was associated with GFAP and NfL. This highlights the effectiveness of these markers in distinguishing and characterizing SVD and AD in memory clinic patients and emphasizes the importance of considering concomitant SVD in patients with elevated GFAP and NfL levels.

Anti-amyloid β-peptide (Aβ) immunotherapy was developed to reduce amyloid plaque pathology and slow cognitive decline during progression of Alzheimer’s disease. Efficient amyloid clearance has been proven in clinical trials testing anti-Aβ antibodies, by their impact on cognitive endpoints correlating with the extent of amyloid removal. However, treatment is associated with adverse side effects, such as oedema and haemorrhages, which are potentially linked to the induced immune response. To improve the safety profile of these molecules, it is imperative to understand the consequences of anti-Aβ antibody treatment on immune cell function. Here, we investigated the effects of long-term chronic anti-Aβ treatment on amyloid plaque pathology and microglial response in the APP-SAA triple knock-in mouse model with an intervention paradigm early during amyloidogenesis. Long-term treatment with anti-Aβ results in a robust and dose-dependent lowering of amyloid plaque pathology, with a higher efficiency for reducing diffuse over dense-core plaque deposition. Analysis of the CSF proteome indicates a reduction of markers for neurodegeneration including Tau and α-Synuclein, as well as immune-cell-related proteins. Bulk RNA-seq revealed a dose-dependent attenuation of disease-associated microglial (DAM) and glycolytic gene expression, which is supported by a parallel decrease of glucose uptake and protein levels of Triggering Receptor Expressed on Myeloid cells 2 (Trem2) protein, a major immune receptor involved in DAM activation of microglia. In contrast, DAM activation around residual plaques remains high, regardless of treatment dose. In addition, microglia surrounding residual plaques display a dose-dependent increase in microglial clustering and a selective increase in antigen-presenting and immune signalling proteins. These findings demonstrate that chronic early intervention by an anti-amyloid immunotherapy leads to a dose-dependent decrease in plaque formation, which is associated with lower brain-wide microglial DAM activation and neurodegeneration. Microglia at residual plaques still display a combined DAM and antigen-presenting phenotype that suggests a continued treatment response.

Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder mainly driven by mutations in the NPC1 gene, causing lipid accumulation within late endosomes/lysosomes and resulting in progressive neurodegeneration. Although microglial activation precedes neuronal loss, it remains elusive whether loss of the membrane protein NPC1 in microglia actively contributes to NPC pathology. In a mouse model with depletion of NPC1 in myeloid cells, we report severe alterations in microglial lipidomic profiles, including the enrichment of bis(monoacylglycero)phosphate, increased cholesterol, and a decrease in cholesteryl esters. Lipid dyshomeostasis was associated with microglial hyperactivity, marked by an increase in translocator protein 18 kDa (TSPO). These hyperactive microglia initiated a pathological cascade resembling NPC-like phenotypes, including a shortened life span, motor impairments, astrogliosis, neuroaxonal pathology, and increased neurofilament light chain (NF-L), a neuronal injury biomarker. As observed in the mouse model, patients with NPC showed increased NF-L in the blood and microglial hyperactivity, as visualized by TSPO-PET imaging. Reduced TSPO expression in blood-derived macrophages of patients with NPC was measured after N -acetyl- l -leucine treatment, which has been recently shown to have beneficial effects in patients with NPC, suggesting that TSPO is a potential marker to monitor therapeutic interventions for NPC. Conclusively, these results demonstrate that myeloid dysfunction, driven by the loss of NPC1, contributes to NPC disease and should be further investigated for therapeutic targeting and disease monitoring.

Background The key pathological signature of ALS/ FTLD is the mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm. However, TDP-43 gain of function in the cytoplasm is still poorly understood since TDP-43 animal models recapitulating mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm are missing.Methods CRISPR/Cas9 technology was used to generate a zebrafish line (called CytoTDP), that mis-locates endogenous TDP-43 from the nucleus to the cytoplasm. Phenotypic characterization of motor neurons and the neuromuscular junction was performed by immunostaining, microglia were immunohistochemically localized by whole-mount tissue clearing and muscle ultrastructure was analyzed by scanning electron microscopy. Behavior was investigated by video tracking and quantitative analysis of swimming parameters. RNA sequencing was used to identify mis-regulated pathways with validation by molecular analysis.Results CytoTDP fish have early larval phenotypes resembling clinical features of ALS such as progressive motor defects, neurodegeneration and muscle atrophy. Taking advantage of zebrafish's embryonic development that solely relys on yolk usage until 5 days post fertilization, we demonstrated that microglia proliferation and activation in the hypothalamus is independent from food intake. By comparing CytoTDP to a previously generated TDP-43 knockout line, transcriptomic analyses revealed that mis-localization of endogenous TDP-43, rather than TDP-43 nuclear loss of function, leads to early onset metabolic dysfunction.Conclusions The new TDP-43 model mimics the ALS/FTLD hallmark of progressive motor dysfunction. Our results suggest that functional deficits of the hypothalamus, the metabolic regulatory center, might be the primary cause of weight loss in ALS patients. Cytoplasmic gain of function of endogenous TDP-43 leads to metabolic dysfunction in vivo that are reminiscent of early ALS clinical non-motor metabolic alterations. Thus, the CytoTDP zebrafish model offers a unique opportunity to identify mis-regulated targets for therapeutic intervention early in disease progression.

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TAR DNA-binding protein 43 (TDP-43) pathology (FTLD- GRN ). Multiple therapeutic strategies are in clinical development to restore PGRN in the CNS, including gene therapy. However, a limitation of current gene therapy approaches aimed to alleviate FTLD-associated pathologies may be their inefficient brain exposure and biodistribution. We therefore developed an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant [AAV(L):bPGRN] in two mouse models of FTLD- GRN , namely, Grn knockout and GrnxTmem106b double knockout mice. This therapeutic strategy avoids potential safety and biodistribution issues of CNS-administered AAVs and maintains sustained concentrations of PGRN in the brain after a single dose. AAV(L):bPGRN treatment reduced several FTLD- GRN –associated pathologies including severe motor function deficits, aberrant TDP-43 phosphorylation, dysfunctional protein degradation, lipid metabolism, gliosis, and neurodegeneration in the brain. The potential translatability of our findings was tested in an in vitro model using cocultured human induced pluripotent stem cell (hiPSC)–derived microglia lacking PGRN and TMEM106B and wild-type hiPSC-derived neurons. As in mice, aberrant TDP-43, lysosomal dysfunction, and neuronal loss were ameliorated after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN). Together, our studies suggest that peripherally administered brain-penetrant PGRN replacement strategies ameliorate FTLD- GRN relevant phenotypes including TDP-43 pathology, neurodegeneration, and behavioral deficits. Our data provide preclinical proof of concept for the use of this AAV platform for treatment of FTLD- GRN and potentially other CNS disorders.

Abstract Background The prion-like spreading of Tau pathology is the leading cause of disease progression in various tauopathies. A critical step in propagating pathologic Tau in the brain is the transport from the extracellular environment and accumulation inside naïve neurons. Current research indicates that human neurons internalize both the physiological extracellular Tau (eTau) monomers and the pathological eTau aggregates. However, similarities or differences in neuronal transport mechanisms between Tau species remain elusive. Method Monomers, oligomers, and fibrils of recombinant 2N4R Tau were produced and characterized by biochemical and biophysical methods. A neuronal eTau uptake and accumulation assay was developed for human induced pluripotent stem cell-derived neurons (iPSCNs) and Lund human mesencephalic cells (LUHMES)-derived neurons. Mechanisms of uptake and cellular accumulation of eTau species were studied by using small molecule inhibitors of endocytic mechanisms and siRNAs targeting Tau uptake mediators. Results Extracellular Tau aggregates accumulated more than monomers in human neurons, mainly due to the higher efficiency of small fibrillar and soluble oligomeric aggregates in intraneuronal accumulation. A competition assay revealed a distinction in the neuronal accumulation between physiological eTau Monomers and pathology-relevant aggregates, suggesting differential transport mechanisms. Blocking heparan sulfate proteoglycans (HSPGs) with heparin only inhibited the accumulation of eTau aggregates, whereas monomers’ uptake remained unaltered. At the molecular level, the downregulation of genes involved in HSPG synthesis exclusively blocked neuronal accumulation of eTau aggregates but not monomers, suggesting its role in the transport of pathologic Tau. Moreover, the knockdown of LRP1, as a receptor of Tau, mainly reduced the accumulation of monomeric form, confirming its involvement in Tau’s physiological transport. Conclusion These data propose that despite the similarity in the cellular mechanism, the uptake and accumulation of eTau Monomers and aggregates in human neurons are regulated by different molecular mediators. Thus, they address the possibility of targeting the pathological spreading of Tau aggregates without disturbing the probable physiological or non-pathogenic transport of Tau Monomers.

ObjectivesNeurodegeneration is considered a relevant pathophysiologic feature in neurologic disorders associated with antibodies against glutamic acid decarboxylase 65 (GAD65). In this study, we investigate surrogates of neuroaxonal damage in relation to disease duration and clinical presentation.MethodsIn a multicentric cohort of 50 patients, we measured serum neurofilament light chain (sNfL) in relation to disease duration and disease phenotypes, applied automated MRI volumetry, and analyzed clinical characteristics.ResultsIn patients with neurologic disorders associated with GAD65 antibodies, we detected elevated sNfL levels early in the disease course. By contrast, this elevation of sNfL levels was less pronounced in patients with long-standing disease. Increased sNfL levels were observed in patients presenting with cerebellar ataxia and limbic encephalitis, but not in those with stiff person syndrome. Using MRI volumetry, we identified atrophy predominantly of the cerebellar cortex, cerebellar superior posterior lobe, and cerebral cortex with similar atrophy patterns throughout all clinical phenotypes.DiscussionTogether, our data provide evidence for early neuroaxonal damage and support the need for timely therapeutic interventions in GAD65 antibody-associated neurologic disorders.

Multiple system atrophy (MSA), an atypical parkinsonian syndrome, is a rapidly progressive neurodegenerative disease with currently no established fluid biomarkers available. MSA is characterized by an oligodendroglial α-synucleinopathy, progressive neuronal cell loss and concomitant astrocytosis. Here, we investigate glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL) as fluid biomarkers for differential diagnosis, assessment of clinical disease severity and prediction of disease progression in MSA. GFAP and NfL levels were analyzed in plasma and CSF samples of 47 MSA patients as well as 24 Parkinson’s disease (PD) and 25 healthy controls (HC) as reference cohorts. In MSA, biomarker levels were correlated to baseline and longitudinal clinical disease severity (UMSARS scores). In MSA, GFAP levels in CSF and plasma predicted baseline clinical disease severity as indicated by UMSARS scores, while NfL levels predicted clinical disease progression as indicated by longitudinal changes in UMSARS scores. Cross-sectionally, NfL levels in CSF and plasma were significantly elevated in MSA compared to both PD and HC. Receiver operating curves (ROC) indicated high diagnostic accuracy of NfL for distinguishing MSA from PD (CSF: AUC = 0.97, 95

Corticobasal syndrome (CBS) with underlying 4-repeat tauopathy is a progressive neurodegenerative disorder characterized by declining cognitive and motor functions. Biomarkers for assessing pathological brain changes in CBS including tau- and microglia-PET or neurofilament light chain (NfL) have recently been evaluated for differential diagnosis and disease staging, yet the prognostic accuracy of these biomarkers for predicting disease trajectories remains unclear. To address this, we performed a head-to-head comparison of neuroimaging (tau-PET, microglia-PET) and plasma biomarker NfL as prognostic tools for future clinical trajectories in 21 CBS patients with longitudinal clinical data. We included 21 clinically diagnosed CBS patients with ∼2-year clinical follow-up data who underwent baseline [ 18 F]PI-2620-PET for assessing tau pathology, [ 18 F]GE-180-PET for microglia activation and plasma-NfL for neurodegeneration. All patients were negative on amyloid biomarkers. To quantify tau and microglia load we assessed summary scores of whole-brain, cortical and subcortical tracer signal. NfL was assessed using immunoassays on the Simoa platform. Symptom progression was determined using a battery of cognitive and motor tests. Using linear mixed models, we tested whether the assessed biomarkers at baseline were associated with faster progression of symptoms over time (i.e. time x biomarker interaction). Overall, there was on average a rapid decline over time on the assessed clinical scores. For tau-PET, CBS patients with higher global tau load showed faster clinical progression. This time x tau-PET interaction was driven by cortical rather than subcortical tau-PET load. Patients with higher [ 18 F]GE-180-PET whole-brain load showed slower clinical progression. Concerning plasma biomarker NfL, higher plasma-NfL was prognostic of faster clinical deterioration. In a subsequent sensitivity analysis, we found that tau-PET, microglia-PET as well as plasma-NfL showed significant (i.e. p<0.05) interaction effects with time on clinical trajectories when tested in the same model, suggesting that these biomarkers explain unique variability in future disease trajectories. [ 18 F]PI-2620 tau-PET, [ 18 F]GE-180 microglia-PET and plasma-NfL show prognostic potential for clinical progression in CBS patients with probable 4-repeat tauopathy, which can be useful for clinical decision making as well as stratifying patients in clinical trials.

Background and ObjectivesCorticobasal syndrome (CBS) with underlying 4-repeat tauopathy is a progressive neurodegenerative disease characterized by declining cognitive and motor functions. Biomarkers for assessing pathologic brain changes in CBS including tau-PET, 18 kDa translocator protein (TSPO)-PET, structural MRI, neurofilament light chain (NfL), or glial fibrillary acidic protein (GFAP) have recently been evaluated for differential diagnosis and disease staging, yet their association with disease trajectories remains unclear. Therefore, we performed a head-to-head comparison of neuroimaging (tau-PET, TSPO-PET, structural MRI) and plasma biomarkers (NfL, GFAP) as prognostic tools for longitudinal clinical trajectories in beta-amyloid (A beta)-negative CBS.MethodsWe included patients with clinically diagnosed A beta-negative CBS with clinical follow-up data who underwent baseline structural MRI and plasma-NfL analysis for assessing neurodegeneration, [F-18]PI-2620-PET for assessing tau pathology, [F-18]GE-180-PET for assessing microglia activation, and plasma-GFAP analysis for assessing astrocytosis. To quantify tau and microglia load, we assessed summary scores of whole-brain, cortical, and subcortical PET signal. For structural MRI analysis, we quantified subcortical and cortical gray matter volume. Plasma NfL and GFAP values were assessed using Simoa-based immunoassays. Symptom progression was determined using a battery of cognitive and motor tests (i.e., Progressive Supranuclear Palsy Rating Scale [PSPRS]). Using linear mixed models, we tested whether the assessed biomarkers at baseline were associated with faster symptom progression over time (i.e., time x biomarker interaction).ResultsOverall, 21 patients with A beta-negative CBS with similar to 2-year clinical follow-up data were included. Patients with CBS with more widespread global tau-PET signal showed faster clinical progression (PSPRS: B/SE = 0.001/0.0005, p = 0.025), driven by cortical rather than subcortical tau-PET. By contrast, patients with higher global [F-18]GE-180-PET readouts showed slower clinical progression (PSPRS: B/SE = -0.056/0.023, p = 0.019). No association was found between gray matter volume and clinical progression. Concerning fluid biomarkers, only higher plasma-NfL (PSPRS: B/SE = 0.176/0.046, p < 0.001) but not GFAP was associated with faster clinical deterioration. In a subsequent sensitivity analysis, we found that tau-PET, TSPO-PET, and plasma-NfL showed significant interaction effects with time on clinical trajectories when tested in the same model.Discussion[F-18]PI-2620 tau-PET, [F-18]GE-180 TSPO-PET, and plasma-NfL show prognostic potential for clinical progression in patients with A beta-negative CBS with probable 4-repeat tauopathy, which can be useful for clinical decision-making and stratifying patients in clinical trials.

Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy (“rNLS8” model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1β, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression. Graphical Abstract