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Benzodiazepines are widely administered drugs to treat anxiety and insomnia. In addition to tolerance development and abuse liability, their chronic use may cause cognitive impairment and increase the risk for dementia. However, the mechanism by which benzodiazepines might contribute to persistent cognitive decline remains unknown. Here we report that diazepam, a widely prescribed benzodiazepine, impairs the structural plasticity of dendritic spines, causing cognitive impairment in mice. Diazepam induces these deficits via the mitochondrial 18 kDa translocator protein (TSPO), rather than classical γ-aminobutyric acid type A receptors, which alters microglial morphology, and phagocytosis of synaptic material. Collectively, our findings demonstrate a mechanism by which TSPO ligands alter synaptic plasticity and, as a consequence, cause cognitive impairment.

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.

Primary spinal cord astrocytomas are rare, hence few data exist about the prognostic significance of molecular markers. Here we analyze a panel of molecular alterations in association with the clinical course. Histology and genome sequencing was performed in 26 spinal astrocytomas operated upon between 2000 and 2020. Next-generation DNA/RNA sequencing (NGS) and methylome analysis were performed to determine molecular alterations. Histology and NGS allowed the distinction of 5 tumor subgroups: glioblastoma IDH wildtype (GBM); diffuse midline glioma H3 K27M mutated (DMG-H3); high-grade astrocytoma with piloid features (HAP); diffuse astrocytoma IDH mutated (DA), diffuse leptomeningeal glioneural tumors (DGLN) and pilocytic astrocytoma (PA). Within all tumor entities GBM (median OS: 5.5 months), DMG-H3 (median OS: 13 months) and HAP (median OS: 8 months) showed a fatal prognosis. DMG-H3 tend to emerge in adolescence whereas GBM and HAP develop in the elderly. HAP are characterized by CDKN2A/B deletion and ATRX mutation. 50% of PA tumors carried a mutation in the PIK3CA gene which is seemingly associated with better outcome (median OS: PIK3CA mutated 107.5 vs 45.5 months in wildtype PA). This exploratory molecular profiling of spinal cord astrocytomas allows to identify distinct subgroups by combining molecular markers and histomorphology. DMG-H3 tend to develop in adolescence with a similar dismal prognosis like GBM and HAP in the elderly. We here describe spinal HAP with a distinct molecular profile for the first time. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01222-6.

MGMT promotor methylation is associated with favourable outcome in high-grade glioma. In glioma WHO grade II, it is unclear whether the extent of MGMT promotor methylation and its prognostic role is independent from other molecular markers. We performed a retrospective analysis of 155 patients with glioma WHO grade II. First, all 155 patients were assigned to three molecular groups according to the 2016 WHO classification system: (1) oligodendroglioma, IDH-mutant and 1p19q co-deleted (n = 81); (2) astrocytoma, IDH-mutant and 1p19q non-codeleted (n = 54); (3) astrocytoma, IDH-wildtype (n = 20). MGMT promotor methylation was quantified using Sanger sequencing of the CpG sites 74–98 within the MGMT promotor region. Highest numbers of methylated CpG sites were found for oligodendroglioma, IDH-mutant and 1p19q co-deleted. When 1p19q co-deletion was absent, numbers of methylated CpG sites were higher in the presence of IDH-mutation. Accordingly, lowest numbers were seen in the IDH-wildtype subpopulation. In the entire cohort, larger numbers of methylated CpG sites were associated with favourable outcome. When analysed separately for the three WHO subgroups, a similar association was only retained in astrocytoma, IDH-wildtype. Collectively, extent of MGMT promotor methylation was strongly associated with other molecular markers and added prognostic information in astrocytoma, IDH-wildtype. Evaluation in prospective cohorts is warranted.

Additional file 1. Supplementary Table 1: Description of study cohort. Supplementary Table 2: Primers used for TP53 amplification and Sanger sequencing. Supplementary Table 3: Common TP53 variants in the study cohort. Supplementary Table 4: Comparison of TP53 mutant versus TP53 wild type group. Supplementary Figure 1. Chromatograms showing the TP53 variants found in the corticotroph tumor of patient #1 and #8 (Table 1). A. The variant c.398T>A was present in homozygocity in the tumor and absent in the blood. B. The variant c.1009C>G is detected in all available surgical specimens in this patient. First and 2nd surgeries were Cushing’s disease tumors and 4th and 5th CTP-BADX/NS.

BACKGROUND: Multiple System Atrophy is a rare neurodegenerative disease with alpha-synuclein aggregation in glial cytoplasmic inclusions and either predominant olivopontocerebellar atrophy or striatonigral degeneration, leading to dysautonomia, parkinsonism, and cerebellar ataxia. One prior genome-wide association study in mainly clinically diagnosed patients with Multiple System Atrophy failed to identify genetic variants predisposing for the disease. OBJECTIVE: Since the clinical diagnosis of Multiple System Atrophy yields a high rate of misdiagnosis when compared to the neuropathological gold standard, we studied only autopsy-confirmed cases. METHODS: We studied common genetic variations in Multiple System Atrophy cases (N=731) and controls (N=2898). RESULTS: The most strongly disease-associated markers were rs16859966 on chromosome 3, rs7013955 on chromosome 8, and rs116607983 on chromosome 4 with P-values below 5 × 10-6 , all of which were supported by at least one additional genotyped and several imputed single nucleotide polymorphisms. The genes closest to the chromosome 3 locus are ZIC1 and ZIC4 encoding the zinc finger proteins of cerebellum 1 and 4 (ZIC1 and ZIC4). INTERPRETATION: Since mutations of ZIC1 and ZIC4 and paraneoplastic autoantibodies directed against ZIC4 are associated with severe cerebellar dysfunction, we conducted immunohistochemical analyses in brain tissue of the frontal cortex and the cerebellum from 24 Multiple System Atrophy patients. Strong immunohistochemical expression of ZIC4 was detected in a subset of neurons of the dentate nucleus in all healthy controls and in patients with striatonigral degeneration, whereas ZIC4-immunoreactive neurons were significantly reduced inpatients with olivopontocerebellar atrophy. These findings point to a potential ZIC4-mediated vulnerability of neurons in Multiple System Atrophy. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Efficient treatment of stress-related disorders, such as depression, is still a major challenge. The onset of antidepressant drug action is generally quite slow, while the anxiolytic action of benzodiazepines is considerably faster. However, their long-term use is impaired by tolerance development, abuse liability and cognitive impairment. Benzodiazepines act as positive allosteric modulators of ɣ-aminobutyric acid type A (GABAA) receptors. 3α-reduced neurosteroids such as allopregnanolone also are positive allosteric GABAA receptor modulators, however, through a site different from that targeted by benzodiazepines. Recently, the administration of neurosteroids such as brexanolone or zuranolone has been shown to rapidly ameliorate symptoms in post-partum depression or major depressive disorder. An attractive alternative to the administration of exogenous neurosteroids is promoting endogenous neurosteroidogenesis via the translocator protein 18k Da (TSPO). TSPO is a transmembrane protein located primarily in mitochondria, which mediates numerous biological functions, e.g., steroidogenesis and mitochondrial bioenergetics. TSPO ligands have been used in positron emission tomography (PET) studies as putative markers of microglia activation and neuroinflammation in stress-related disorders. Moreover, TSPO ligands have been shown to modulate neuroplasticity and to elicit antidepressant and anxiolytic therapeutic effects in animals and humans. As such, TSPO may open new avenues for understanding the pathophysiology of stress-related disorders and for the development of novel treatment options.

Objective: ��-amyloid PET (A��-PET) is an important tool for quantification of amyloidosis in the brain of suspected Alzheimer's disease (AD) patients and transgenic AD mouse models. Despite the excellent correlation of A��-PET with gold standard immunohistochemical assessments, the relative contributions of fibrillar and non-fibrillar A�� components to the in vivo A��-PET signal remain unclear. Thus, we obtained two murine cerebral amyloidosis models that present with distinct A�� plaque compositions and performed regression analysis between immunohistochemistry and A�� PET to determine the biochemical contributions to A��-PET signal in vivo. Methods: We investigated groups of AppNL-G-F and APPPS1 mice at three, six and 12 months of age by longitudinal 18F-florbetaben A��-PET and with immunohistochemical analysis of the fibrillar and total A�� burdens. We then applied group level inter-modality regression models using age and genotype matched sets of fibrillar/ non-fibrillar A�� data (predictors) and A��-PET results (outcome) for both transgenic models. An independent group of double-hit APPPS1 mice with dysfunctional microglia due to knock-out of triggering receptor expression on myeloid cells 2 (Trem2-/-) served for validation and evaluation of translational impact. Results: Neither fibrillar nor non-fibrillar A�� content alone sufficed to explain the A��-PET findings in either transgenic AD model. However, a regression model compiling fibrillar and non-fibrillar A�� together with the estimate of individual heterogeneity and age at scanning could explain a 93% of variance of the A��-PET signal (P

Background O-(2-[18F]-fluoroethyl)-L-tyrosine (18F-FET) is a highly sensitive PET tracer for glioma imaging, and its uptake is suggested to be driven by an overexpression of the L-type amino-acid transporter 1 (LAT1). However, 30% of low- and 5% of high-grade gliomas do not present enhanced 18F-FET uptake at primary diagnosis (“18F-FET-negative gliomas”) and the pathophysiologic basis for this phenomenon remains unclear. The aim of this study was to determine the expression of LAT1 in a homogeneous group of newly diagnosed 18F-FET-negative gliomas and to compare them to a matched group of 18F-FET-positive gliomas. Forty newly diagnosed IDH-mutant astrocytomas without 1p/19q codeletion were evaluated (n = 20 18F-FET-negative (tumour-to-background ratio (TBR) 18F-FET-positive gliomas (TBR > 1.6)). LAT1 immunohistochemistry (IHC) was performed using SLC7A5/LAT1 antibody. The percentage of LAT1-positive tumour cells (%) and the staining intensity (range 0–2) were multiplied to an overall score (H-score; range 0–200) and correlated to PET findings as well as progression-free survival (PFS). Results IHC staining of LAT1 expression was positive in both, 18F-FET-positive as well as 18F-FET-negative gliomas. No differences were found between the 18F-FET-negative and 18F-FET-positive group with regard to percentage of LAT1-positive tumour cells, staining intensity or H-score. Interestingly, the LAT1 expression showed a significant negative correlation with the PFS (p = 0.031), whereas no significant correlation was found for TBRmax, neither in the overall group nor in the 18F-FET-positive group only (p = 0.651 and p = 0.140). Conclusion Although LAT1 is reported to mediate the uptake of 18F-FET into tumour cells, the levels of LAT1 expression do not correlate with the levels of 18F-FET uptake in IDH-mutant astrocytomas. In particular, the lack of tracer uptake in 18F-FET-negative gliomas cannot be explained by a reduced LAT1 expression. A higher LAT1 expression in IDH-mutant astrocytomas seems to be associated with a short PFS. Further studies regarding mechanisms influencing the uptake of 18F-FET are necessary.

Objectives: To identify the clinical characteristics of the subgroup of benign progressive supranuclear palsy with particularly long disease duration; to define neuropathological determinants underlying variability in disease duration in progressive supranuclear palsy. Methods: Clinical and pathological features were compared among 186 autopsy-confirmed cases with progressive supranuclear palsy with ≥10 years and shorter survival times. Results: The 45 cases (24.2%) had a disease duration of ≥10 years. The absence of ocular motor abnormalities within the first 3 years from disease onset was the only significant independent clinical predictor of longer survival. Histopathologically, the neurodegeneration parameters in each survival group were paralleled anatomically by the distribution of neuronal cytoplasmic inclusions, whereas the tufted astrocytes displayed anatomically an opposite severity pattern. Most interestingly, we found significantly less coiled bodies in those who survive longer, in contrast to patients with less favorable course. Interpretation: A considerable proportion of patients had a more ”benign” disease course with ≥10 years survival. They had a distinct pattern and evolution of core symptoms compared to patients with short survival. The inverted anatomical patterns of astrocytic tau distribution suggest distinct implications of these cell types in trans-cellular propagation. The tempo of disease progression appeared to be determined mostly by oligodendroglial tau, where the high degree of oligodendroglial tau pathology might affect neuronal integrity and function on top of neuronal tau pathology. The relative contribution of glial tau should be further explored in cellular and animal models. ANN NEUROL 2022.

Objective: The cIMPACT-NOW update 6 introduced glioblastoma diagnosis based on the combination of IDH-wildtype status and TERT promotor mutation. MGMT promotor methylation is associated with outcome in glioblastoma as defined by histopathology according to the WHO 2016 classification. However, it is[for full text, please go to the a.m. URL], 72. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Polnischen Gesellschaft für Neurochirurgie

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X211018904 for Binding characteristics of [18F]PI-2620 distinguish the clinically predicted tau isoform in different tauopathies by PET by Mengmeng Song, Leonie Beyer, Lena Kaiser, Henryk Barthel, Thilo van Eimeren, Ken Marek, Alexander Nitschmann, Maximilian Scheifele, Carla Palleis, Gesine Respondek, Maike Kern, Gloria Biechele, Jochen Hammes, Gèrard Bischof, Michael Barbe, Özgür Onur, Frank Jessen, Dorothee Saur, Matthias L Schroeter, Jost-Julian Rumpf, Michael Rullmann, Andreas Schildan, Marianne Patt, Bernd Neumaier, Olivier Barret, Jennifer Madonia, David S Russell, Andrew W Stephens, Andre Mueller, Sigrun Roeber, Jochen Herms, Kai Bötzel, Adrian Danek, Johannes Levin, Joseph Classen, Günter U Höglinger, Peter Bartenstein, Victor Villemagne, Alexander Drzezga, John Seibyl, Osama Sabri, Guido Boening, Sibylle Ziegler and Matthias Brendel in Journal of Cerebral Blood Flow & Metabolism

Additional file 1. Full patients characteristics with detailed LAT1 H-score values.

Additional file 2: Source data.

Additional file 1: Supplementary figures.

Progressive accumulation of Amyloid-β (Aβ) deposits in the brain is a characteristic neuropathological hallmark of Alzheimer’s disease (AD). During disease progression, extracellular Aβ plaques undergo specific changes in their composition by the sequential deposition of different modified Aβ species. Microglia are implicated in the restriction of amyloid deposits and play a major role in internalization and degradation of Aβ. Recent studies showed that rare variants of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) are associated with an increased risk for AD. Post-translational modifications of Aβ could modulate the interaction with TREM2, and the uptake by microglia. Here, we demonstrate that genetic deletion of TREM2 or expression of a disease associated TREM2 variant in mice lead to differential accumulation of modified and non-modified Aβ species in extracellular plaques and intraneuronal deposits. Human brains with rare TREM2 AD risk variants also showed altered deposition of modified Aβ species in the different brain lesions as compared to cases with the common variant of TREM2. These findings indicate that TREM2 plays a critical role in the development and the composition of Aβ deposits, not only in extracellular plaques, but also intraneuronally, that both could contribute to the pathogenesis of AD. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01263-x.

Objectives: Autoradiography on brain tissue is used to validate binding targets of newly discovered radiotracers. The purpose of this study was to correlate quantification of autoradiography signal using the novel next-generation tau positron emission tomography (PET) radiotracer [18F]PI-2620 with immunohistochemically determined tau-protein load in both formalin-fixed paraffin-embedded (FFPE) and frozen tissue samples of patients with Alzheimer’s disease (AD) and Progressive Supranuclear Palsy (PSP). Methods: We applied [18F]PI-2620 autoradiography to postmortem cortical brain samples of six patients with AD, five patients with PSP and five healthy controls, respectively. Binding intensity was compared between both tissue types and different disease entities. Autoradiography signal quantification (CWMR = cortex to white matter ratio) was correlated with the immunohistochemically assessed tau load (AT8-staining, %-area) for FFPE and frozen tissue samples in the different disease entities. Results: In AD tissue, relative cortical tracer binding was higher in frozen samples when compared to FFPE samples (CWMRfrozen vs. CWMRFFPE: 2.5-fold, p < 0.001), whereas the opposite was observed in PSP tissue (CWMRfrozen vs. CWMRFFPE: 0.8-fold, p = 0.004). In FFPE samples, [18F]PI-2620 autoradiography tracer binding and immunohistochemical tau load correlated significantly for both PSP (R = 0.641, p < 0.001) and AD tissue (R = 0.435, p = 0.016), indicating a high agreement of relative tracer binding with underlying pathology. In frozen tissue, the correlation between autoradiography and immunohistochemistry was only present in AD (R = 0.417, p = 0.014) but not in PSP tissue (R = −0.115, p = n.s.). Conclusion: Our head-to-head comparison indicates that FFPE samples show superiority over frozen samples for autoradiography assessment of PSP tau pathology by [18F]PI-2620. The [18F]PI-2620 autoradiography signal in FFPE samples reflects AT8 positive tau in samples of both PSP and AD patients.

Additional file 1. Characteristics of spinal high-grade astrocytomas with piloid features. Micropgraphs of H&E-stained sections of cases #3, #4, #16 and #23 are shown alongside Manhattan plots showing their respective copy-number profiles. Cases #3, #4, and #23 had deletions of the CDKN2A/B genes, highlighted by blue arrows. Scale bar, 100 µm

Objectives: Autoradiography on brain tissue is used to validate binding targets of newly discovered radiotracers. The purpose of this study was to correlate quantification of autoradiography signal using the novel next-generation tau positron emission tomography (PET) radiotracer [18F]PI-2620 with immunohistochemically determined tau-protein load in both formalin-fixed paraffin-embedded (FFPE) and frozen tissue samples of patients with Alzheimer's disease (AD) and Progressive Supranuclear Palsy (PSP). Methods: We applied [18F]PI-2620 autoradiography to postmortem cortical brain samples of six patients with AD, five patients with PSP and five healthy controls, respectively. Binding intensity was compared between both tissue types and different disease entities. Autoradiography signal quantification (CWMR = cortex to white matter ratio) was correlated with the immunohistochemically assessed tau load (AT8-staining, %-area) for FFPE and frozen tissue samples in the different disease entities. Results: In AD tissue, relative cortical tracer binding was higher in frozen samples when compared to FFPE samples (CWMRfrozen vs. CWMRFFPE: 2.5-fold, p < 0.001), whereas the opposite was observed in PSP tissue (CWMRfrozen vs. CWMRFFPE: 0.8-fold, p = 0.004). In FFPE samples, [18F]PI-2620 autoradiography tracer binding and immunohistochemical tau load correlated significantly for both PSP (R = 0.641, p < 0.001) and AD tissue (R = 0.435, p = 0.016), indicating a high agreement of relative tracer binding with underlying pathology. In frozen tissue, the correlation between autoradiography and immunohistochemistry was only present in AD (R = 0.417, p = 0.014) but not in PSP tissue (R = -0.115, p = n.s.). Conclusion: Our head-to-head comparison indicates that FFPE samples show superiority over frozen samples for autoradiography assessment of PSP tau pathology by [18F]PI-2620. The [18F]PI-2620 autoradiography signal in FFPE samples reflects AT8 positive tau in samples of both PSP and AD patients.

Microglia activation is the brain's major immune response to amyloid plaques in Alzheimer's disease (AD). Both cerebrospinal fluid (CSF) levels of soluble TREM2 (sTREM2), a biomarker of microglia activation, and microglia PET are increased in AD; however, whether an increase in these biomarkers is associated with reduced amyloid‐beta (Aβ) accumulation remains unclear. To address this question, we pursued a two‐pronged translational approach. Firstly, in non‐demented and demented individuals, we tested CSF sTREM2 at baseline to predict (i) amyloid PET changes over ∼2 years and (ii) tau PET cross‐sectionally assessed in a subset of patients. We found higher CSF sTREM2 associated with attenuated amyloid PET increase and lower tau PET. Secondly, in the App NL‐G-F mouse model of amyloidosis, we studied baseline 18F‐GE180 microglia PET and longitudinal amyloid PET to test the microglia vs. Aβ association, without any confounding co‐pathologies often present in AD patients. Higher microglia PET at age 5 months was associated with a slower amyloid PET increase between ages 5‐to‐10 months. In conclusion, higher microglia activation as determined by CSF sTREM2 or microglia PET shows protective effects on subsequent amyloid accumulation., TREM2 is a protein almost exclusively expressed by microglia in the brain. This study investigates the association between soluble TREM2 (sTREM2) levels in cerebrospinal fluid and the longitudinal Aβ accumulation in human and mouse.

BACKGROUND In vivo assessment of neuroinflammation by 18-kDa translocator protein positron-emission-tomography (TSPO-PET) ligands receives growing interest in preclinical and clinical research of neurodegenerative disorders. Higher TSPO-PET binding as a surrogate for microglial activation in females has been reported for cognitively normal humans, but such effects have not yet been evaluated in rodent models of neurodegeneration and their controls. Thus, we aimed to investigate the impact of sex on microglial activation in amyloid and tau mouse models and wild-type controls. METHODS TSPO-PET (18F-GE-180) data of C57Bl/6 (wild-type), AppNL-G-F (��-amyloid model), and P301S (tau model) mice was assessed longitudinally between 2 and 12 months of age. The AppNL-G-F group also underwent longitudinal ��-amyloid-PET imaging (A��-PET; 18F-florbetaben). PET results were confirmed and validated by immunohistochemical investigation of microglial (Iba-1, CD68), astrocytic (GFAP), and tau (AT8) markers. Findings in cerebral cortex were compared by sex using linear mixed models for PET data and analysis of variance for immunohistochemistry. RESULTS Wild-type mice showed an increased TSPO-PET signal over time (female +23%, male +4%), with a significant sex �� age interaction (T = - 4.171, p���

Microglial dysfunction is a key pathological feature of Alzheimer's disease (AD), but little is known about proteome-wide changes in microglia during the course of AD and their functional consequences. Here, we performed an in-depth and time-resolved proteomic characterization of microglia in two mouse models of amyloid v (Av) pathology, the overexpression APPPS1 and the knock-in APP-NL-G-F (APP-KI) model. We identified a large panel of Microglial Av Response Proteins (MARPs) that reflect heterogeneity of microglial alterations during early, middle and advanced stages of Av deposition and occur earlier in the APPPS1 mice. Strikingly, the kinetic differences in proteomic profiles correlated with the presence of fibrillar Av, rather than dystrophic neurites, suggesting that fibrillar Av may trigger the AD-associated microglial phenotype and the observed functional decline. The identified microglial proteomic fingerprints of AD provide a valuable resource for functional studies of novel molecular targets and potential biomarkers for monitoring AD progression or therapeutic efficacy.

Additional file 1: Supplemental figure 1. Overview of representative images of IBA1 and CD68 immunohistochemistry (IHC) stainings from 2.7-6.6 M: The upper row shows representative images of the cortex (CTX), the lower row shows representative images of the brainstem (BRST). Magnification 40x objective/oil; scale Bar: 30 μm; WT = wild-type; TG = P301S; M = age in months.

BACKGROUND: In 2018-19, Borna disease virus 1 (BoDV-1), the causative agent of Borna disease in horses, sheep, and other domestic mammals, was reported in five human patients with severe to fatal encephalitis in Germany. However, information on case frequencies, clinical courses, and detailed epidemiological analyses are still lacking. We report the occurrence of BoDV-1-associated encephalitis in cases submitted to the Institute of Clinical Microbiology and Hygiene, Regensburg University Hospital, Regensburg, Germany, and provide a detailed description of newly identified cases of BoDV-1-induced encephalitis. METHODS: All brain tissues from 56 encephalitis cases from Bavaria, Germany, of putative viral origin (1999-2019), which had been submitted for virological testing upon request of the attending clinician and stored for stepwise diagnostic procedure, were systematically screened for BoDV-1 RNA. Two additional BoDV-1-positive cases were contributed by other diagnostic centres. Positive results were confirmed by deep sequencing, antigen detection, and determination of BoDV-1-reactive antibodies in serum and cerebrospinal fluid. Clinical and epidemiological data from infected patients were collected and analysed. FINDINGS: BoDV-1 RNA and bornavirus-reactive antibodies were detected in eight newly analysed encephalitis cases and the first human BoDV-1 isolate was obtained from an unequivocally confirmed human BoDV-1 infection from the endemic area. Six of the eight BoDV-1-positive patients had no record of immunosuppression before the onset of fatal disease, whereas two were immunocompromised after solid organ transplantation. Typical initial symptoms were headache, fever, and confusion, followed by various neurological signs, deep coma, and severe brainstem involvement. Seven of nine patients with fatal encephalitis of unclear cause were BoDV-1 positive within one diagnostic centre. BoDV-1 sequence information and epidemiological analyses indicated independent spillover transmissions most likely from the local wild animal reservoir. INTERPRETATION: BoDV-1 infection has to be considered as a potentially lethal zoonosis in endemic regions with reported spillover infections in horses and sheep. BoDV-1 infection can result in fatal encephalitis in immunocompromised and apparently healthy people. Consequently, all severe encephalitis cases of unclear cause should be tested for bornaviruses especially in endemic regions.

Frontotemporal lobar degeneration with neuronal inclusions of the TAR DNA-binding protein 43 (FTLD-TDP) represents the most common pathological subtype of FTLD. We established the international FTLD-TDP whole-genome sequencing consortium to thoroughly characterize the known genetic causes of FTLD-TDP and identify novel genetic risk factors. Through the study of 1131 unrelated Caucasian patients, we estimated that C9orf72 repeat expansions and GRN loss-of-function mutations account for 25.5% and 13.9% of FTLD-TDP patients, respectively. Mutations in TBK1 (1.5%) and other known FTLD genes (1.4%) were rare, and the disease in 57.7% of FTLD-TDP patients was unexplained by the known FTLD genes. To unravel the contribution of common genetic factors to the FTLD-TDP etiology in these patients, we conducted a two-stage association study comprising the analysis of whole-genome sequencing data from 517 FTLD-TDP patients and 838 controls, followed by targeted genotyping of the most associated genomic loci in 119 additional FTLD-TDP patients and 1653 controls. We identified three genome-wide significant FTLD-TDP risk loci: one new locus at chromosome 7q36 within the DPP6 gene led by rs118113626 (p value = 4.82e-08, OR = 2.12), and two known loci: UNC13A, led by rs1297319 (p value = 1.27e-08, OR = 1.50) and HLA-DQA2 led by rs17219281 (p value = 3.22e-08, OR = 1.98). While HLA represents a locus previously implicated in clinical FTLD and related neurodegenerative disorders, the association signal in our study is independent from previously reported associations. Through inspection of our whole-genome sequence data for genes with an excess of rare loss-of-function variants in FTLD-TDP patients (n ≥ 3) as compared to controls (n = 0), we further discovered a possible role for genes functioning within the TBK1-related immune pathway (e.g., DHX58, TRIM21, IRF7) in the genetic etiology of FTLD-TDP. Together, our study based on the largest cohort of unrelated FTLD-TDP patients assembled to date provides a comprehensive view of the genetic landscape of FTLD-TDP, nominates novel FTLD-TDP risk loci, and strongly implicates the immune pathway in FTLD-TDP pathogenesis.

Neuroinflammation may have beneficial or detrimental net effects on the cognitive outcome of Alzheimer's disease patients (AD). 18kDa translocator protein (TSPO) imaging by positron-emission-tomography (PET) enables longitudinal monitoring of microglial activation in vivo. We compiled serial PET measures of TSPO and amyloid with terminal cognitive assessment (water maze) in an AD transgenic mouse model (PS2APP) from eight to 13 months of age, followed by immunohistochemical analyses of microglia, amyloid and synaptic density. Better cognitive outcome and higher synaptic density in PS2APP mice was predicted by higher TSPO expression at eight months. The progression of TSPO activation to 13 months also showed a moderate association with spared cognition, but amyloidosis did not correlate with the cognitive outcome, regardless of the timepoint. This first PET investigation with longitudinal TSPO- and amyloid-PET together with terminal cognitive testing in an AD mouse model indicates that continuing microglial response seems to impart preserved cognitive performance.

Aim: Immunmodulatory treatment with the PPARγ agonist Pioglitazone shows heterogeneous results in Alzheimer`s disease (AD). We aimed to apply longitudinal in-vivo PET monitoring combined with terminal cognitive assessment to investigate PET as a tool for longitudinal monitoring of treatment effects and to establish efficacy determinants.Methods: PS2APP (TG) mice and C57Bl/6 (WT) mice were randomly stratified in Pioglitazone (N=13 TG, N=8 WT) and placebo treatment (N=10 TG, N=7 WT) at the age of 8 months. Therapy was conducted for 5 months (mo). All mice received amyloid-PET (Aβ-PET) scans (F-18-Florbetaben) at baseline and after 5 mo, to investigate progression of plaque load. TSPO-PET (F-18-GE180) was performed at baseline, after 0.5 mo, 2.5 mo and 5 mo to analyze changes in microglial activation. Forebrain PET signals were assessed for each timepoint. Behavioral testing after the terminal PET scan was followed by molecular validation including immunohistochemical (IHC) and profiling of microglia phenotypes, plaque composition and synaptic density.Results: Pioglitazone treatment resulted in a reduction of -13% (p < 0.001) in the TSPO-PET signal as well as an unexpected increase of Aβ-PET in the treatment group compared to placebo (+16% vs. +13%; p < 0.05; supporting Fig. a) for the entire monitoring period. IHC proved Aβ-PET results and showed more fibrillar plaque load in the treatment group (4.2% vs 2.8%, p < 0.05; Fig. b). Nonetheless, we observed improved cognitive performance (escape latency 3.2 vs. 4.8 seconds, p < 0.05) and attenuated synaptic loss (0.43 vs. 0.38 VGLUT1/µm2, p < 0.001) after treatment. Importantly, we discovered a direct correlation between Aβ-PET increase with TSPO decrease (R=0.62; p < 0.05) and improved cognitive outcome (R=0.39 p < 0.05). IHC revealed, that dense proportions of the Aβ plaque raised during treatment, whereas looser parts of Aβ plaques decreased. Genetic analyses indicated a treatment dependent change in microglial gene signature (Fig. c).Conclusions: Reduction of microglial activation by Pioglitazone anti-inflammatory treatment is associated with increasing fibrillar Aβ. However, this shift of loose to more dense proportions of Aβ plaques is accompanied by improved cognitive performance and attenuated synaptic loss after treatment. This finding has strong implications to Pioglitazone treatment monitoring by Aβ-PET as a signal increase over time could be misinterpreted as a treatment failure. Furthermore, this indicates for the first time that increasing fibrillar amyloidosis can preserve cognition.

Comparison of 18F-T807 and 18F-THK5117 PET in a Mouse Model of Tau Pathology.

Figure S3. iMG differentiation and validation. a Schematic of the in vitro differentiation of iPSC-derived microglia-like cells (iMG). (i): Human iPSCs are grown in feeder free conditions with no spontaneous differentiation. Scale bar: 250 μm. (ii): Embryoid bodies are formed in the presence of 3 factors SCF, BMP4, and VEGF; Scale bar: 750 μm. (iii): Myeloid cells are generated after culturing with IL3 and MCSF growth factors for 3–4 weeks, then stained for classic myeloid/macrophage markers CD68. Scale bar: 50 μm. (iv): Further differentiation to microglia-like cells that positive for microglial markers P2RY12. Scale bar: 20 μm. (v): Addition of the two final factors (CD200 and CX3CL1) matures the iMG. Scale bar: 20 μm. b Heat map showing mRNA expression of a microglial gene signature in iMG, human monocyte-derived macrophages (hMac), human primary microglia (hMG), and iPSC samples. Clear clustering is observed between iMG and hMG. (PDF 159 kb)

Figure S2. Sequence of the aberrantly spliced murine Trem2 mRNA. Trem2 gene sequence. Exon in black; intron in green; // indicates splicing sites. (PDF 93 kb)

Table S1. Allele specific quantitative PCR for Trem2 R47H knock-in mice (PDF 92 kb)

Table S2. Allele specific quantitative PCR for iMG and patient brains (PDF 104 kb)

Objectives: We previously established dual tracer PET imaging of β-amyloid (Aβ) and activated microglia in Alzheimer’s disease (AD) mouse models. Associations between both biomarkers during disease progression of AD are still discussed controversially. The aim of the present longitudinal study was to enlighten the longitudinal association between Aβ and microglial response during aging of APPSL70 mice, an amyloid mouse model, in vivo. A variant baseline age enabled us to perform correlation analysis with the longitudinal biomarker progression over seven months by dual tracer PET. Terminal immunohistochemistry served for interpretation of PET results under consideration of findings at individual plaque and cell level.Methods: APPSL70 mice (N=17; starting age: 15-38 weeks) and age-matched C57Bl/6 controls (WT) were investigated in a longitudinal design using amyloid (18F-florbetaben) and 18kDa translocator protein (TSPO) PET (18F-GE180). PET was performed at baseline (0 weeks), intermediate (+10 weeks) and terminal (+26 weeks) time points. A frontal cortical volume-of-interest (VOI) with hindbrain white matter reference served for calculation of standardized-uptake-value ratio (SUVRCTX/WM) values. Terminal percentage differences were calculated relative to baseline (Δ-%). Additionally, Z-scores versus WT were calculated for amyloid-PET and TSPO-PET, and the microglial activation relative to amyloidosis was defined as the Z-score difference (TSPO - Aβ). Immunohistochemistry for activated microglia (Iba-1) and fibrillar Aβ (methoxy-X04) were performed with 3D stacks to analyze brain volumes of microglial activation relative to the plaque size and the distance to plaque borders. Results: Relative to the baseline age, an exponential function of Δ-% SUVRCTX/WM was observed in Aβ-PET of APPSL70 mice. Contrary an inverse U-shape was present for Δ-% SUVRCTX/WM of TSPO-PET with a peak increase at a baseline age of 28 weeks. Longitudinal Z-score differences dropped with aging, meaning that less microglial response was observed relative to ongoing amyloidosis in aging APPSL70 mice. Immunohistochemistry revealed that the fraction of activated microglia at the plaque border decreased with increasing plaque size, while the plaque size was associated with age. IBA-1 positive cells in regions without Aβ plaques were significantly lower in APPSL70 mice when compared to WT. Conclusions: Microglial activity decreases relative to ongoing amyloidosis during aging of APPSL70 mice. The plaque-associated microglial fraction showed saturation and correlated with plaque size and aging, most likely related to limited proliferation of microglia, emptying the peripheral reserve.

Figure S1. Off-target analysis of in-house made Trem2 R47H knock-in mice. a Sanger-sequencing chromatograms of the Trem2 on-target site and the six putative off target sites of animal Trem2 R47H ki ID-7. Mixed peaks in the Trem2 locus show the correct R47H substitution (CGC > CAC) and the three silent mutations for genotyping purposes. Mixed peaks in traces of site #2 reveal a Δ10-Indel mutation at the putative cut site, indicating a true off target event. Underlined: Protospacer; arrow head: putative cut site; green letters: PAM site on shown strand; red letters: PAM site on complementary strand; yellow: Δ10-Indel mutation. b Sanger sequencing results of Trem2 R47H positive off-springs of male ID-7, which was crossed with a C57BL/6 N female. The Δ10-Indel allele was inherited to animals ID-7-3 und ID-7-12 that were excluded from any further breedings and experiments. (PDF 201 kb)

Quantitative study of the neurochemical characteristics of the AxDs. (PDF 351 kb)

Schematic representation of Aβ plaques and AxDs development over time. (PDF 200 kb)

Size ratio of dystrophic segments. (PDF 393 kb)

Re-formation of AxDs and sprouting phenomenon (re-growth) in the APP-PS1 mouse. (PDF 341 kb)

Location of auto-fluorescent spots. (PDF 371 kb)

Correlative light and FIB/SEM microscopy. (PDF 317 kb)

Schematic representation of Aβ plaques and AxDs development over time. (PDF 200 kb)

Relationship between Aβ plaques and AxDs formation and development. Quantitative analysis of different types of Aβ plaques. (PDF 463 kb)

Size ratio of dystrophic segments. (PDF 393 kb)

Behavior of auto-fluorescent spots after the elimination of an AxD. (PDF 204 kb)

Relationship between Aβ plaques and AxDs formation and development. Quantitative analysis of different types of Aβ plaques. (PDF 463 kb)

Quantitative study of the neurochemical characteristics of the AxDs. (PDF 351 kb)

Re-formation of AxDs and sprouting phenomenon (re-growth) in the APP-PS1 mouse. (PDF 341 kb)

Table showing the number of imaging positions that was successfully imaged weekly during near 6 months. (PDF 260 kb)