Human induced pluripotent stem cells (hiPSCs) are a promising tool for studying neurological diseases and developing therapies for neurodegenerative diseases. Differentiation of hiPSCs into neurons can be achieved by dual SMAD inhibition (dSMADi) or by induced neurogenin 2 (NGN2) overexpression ("iNGN2"). Starting directly from hiPSCs, iNGN2 shortens the time to a neuronal stage but leads to neurons partially resembling peripheral or posterior fates while dSMADi more faithfully recapitulates telencephalic development. To modify the iNGN2 approach, we applied an accelerated induction paradigm that is dependent on the inhibition of BMP, MEK, and WNT pathways ("BMWi"), to commit hiPSCs into a telencephalic fate before iNGN2. The resulting neurons showed strong expression of telencephalic markers, with decreased levels of peripheral and posterior marker genes compared to iNGN2 alone. The resulting telencephalic neurons are suitable for a tau aggregation assay. Furthermore, we could demonstrate that during BMWi treatment, the cells are amenable to additional regional patterning cues. This allowed the generation of neurons from different regions of the CNS and peripheral nervous system (PNS), which will significantly facilitate in vitro modeling of a range of neurodevelopmental and neurodegenerative disorders.

Human microglia are critically involved in Alzheimer’s disease (AD) progression, as shown by genetic and molecular studies. However, their role in tau pathology progression in human brain has not been well described. Here, we characterized 32 human donors along progression of AD pathology, both in time—from early to late pathology—and in space—from entorhinal cortex (EC), inferior temporal gyrus (ITG), prefrontal cortex (PFC) to visual cortex (V2 and V1)—with biochemistry, immunohistochemistry, and single nuclei-RNA-sequencing, profiling a total of 337,512 brain myeloid cells, including microglia. While the majority of microglia are similar across brain regions, we identified a specific subset unique to EC which may contribute to the early tau pathology present in this region. We calculated conversion of microglia subtypes to diseased states and compared conversion patterns to those from AD animal models. Targeting genes implicated in this conversion, or their upstream/downstream pathways, could halt gene programs initiated by early tau progression. We used expression patterns of early tau progression to identify genes whose expression is reversed along spreading of spatial tau pathology (EC > ITG > PFC > V2 > V1) and identified their potential involvement in microglia subtype conversion to a diseased state. This study provides a data resource that builds on our knowledge of myeloid cell contribution to AD by defining the heterogeneity of microglia and brain macrophages during both temporal and regional pathology aspects of AD progression at an unprecedented resolution.

In Alzheimer’s disease (AD) progression, amyloid beta load uniformly increases across the brain cortex, while neurofibrillary tangles progressively spread in a stereotypical pattern from entorhinal to visual cortex. AD is known to have a strong genetic link to microglia and recently, expression profiling at single cell resolution has identified disease‐associated microglia populations with differential amyloid‐beta vs. tau pathology association in human AD brain. However, prior human studies focusing on microglia included a limited number of donors and/or cells per donor, and how microglial transcriptomes change with progression of tau pathology remains largely unknown.

Tau pathology is known as a primary driver of neurodegeneration in Alzheimer’s disease (AD). Understanding its underlying molecular mechanism is critical in expanding our knowledge of AD pathogenesis and developing novel AD therapeutic strategies. However, interrogating tau induced neurotoxicity mechanisms has been difficult due to heterogeneous susceptibility of neurons to tau pathology. Here, we aim to identify the most vulnerable neuronal subpopulation to tau pathology in AD and reveal more clear molecular mechanisms of tau induced neuro-toxicity/degeneration by analyzing gene expression changes in the vulnerable population. We performed single nuclei RNA sequencing and tau biochemistry from same tissue blocks of the same AD patients (5 brain regions of 32 AD donors with various Braak stages). About 1.5x10 6 neurons were enriched in total by NeuN antibody-based flow cytometry sorting and these cells were clustered into 15 neuronal subpopulations based on their similarity in gene expression. We tested for association between relative neuronal population abundance and tau pathology readouts (phospho-T231 ELISA, HT7-HT7 SIMOA and HEK seeding). Based on the strength of correlation, we identified the neuronal subpopulation that reduces relative abundance in association with its tau pathology. We identified a tau vulnerable neuronal population that showed strong negative correlation between its relative abundance and tau pathology readouts in BA20 and BA46. The population was one of largest excitatory subpopulation distinguished by marker genes, CBLN2 and LINC00507. This outcome was supported by multiple published transcriptomics studies and histologically validated by multiplexed in situ hybridization and immunohistochemistry. Differential gene expression analysis of the vulnerable neuronal population identified a list of genes that potentially links tau pathology and neuronal death. This study with large number of captured neurons for single cell transcriptomics and quantitative tau pathology readout for direct comparison to transcriptomics data enabled the discovery of a vulnerable neuronal subpopulation in more precise manner and revealed genes related to tau associated neurotoxicity more clearly. This result serves as a great starting point to further interrogate fundamental mechanisms of tau-driven neurodegeneration in AD and accelerate therapeutic target and biomarker discovery.

Chronic neuroinflammation is a pathogenic component of Alzheimer’s disease (AD) that may limit the ability of the brain to clear amyloid deposits and cellular debris. Tight control of the immune system is therefore key to sustain the ability of the brain to repair itself during homeostasis and disease. The immune‐cell checkpoint receptor/ligand pair PD‐1/PD‐L1, known for their inhibitory immune function, is expressed also in the brain. Here, we report upregulated expression of PD‐L1 and PD‐1 in astrocytes and microglia, respectively, surrounding amyloid plaques in AD patients and in the APP/PS1 AD mouse model. We observed juxtamembrane shedding of PD‐L1 from astrocytes, which may mediate ectodomain signaling to PD‐1‐expressing microglia. Deletion of microglial PD‐1 evoked an inflammatory response and compromised amyloid‐β peptide (Aβ) uptake. APP/PS1 mice deficient for PD‐1 exhibited increased deposition of Aβ, reduced microglial Aβ uptake, and decreased expression of the Aβ receptor CD36 on microglia. Therefore, ineffective immune regulation by the PD‐1/PD‐L1 axis contributes to Aβ plaque deposition during chronic neuroinflammation in AD. Neuroinflammation, a hallmark of Alzheimer’s disease (AD), is involved in beta‐amyloid peptide (Aβ) plaque deposition and clearance. Here, the PD‐1/PD‐L1 axis is found as an important pathway for regulating the immune system in the brain, sustaining microglial Aβ uptake and reducing chronic neuroinflammation. The immune‐checkpoint receptor/ligand pair PD‐1/PD‐L1 sustains phagocytic function of microglia to prevent Aβ plaque deposition in APP/PS mouse models and patient tissues.

Alzheimer’s disease (AD) is the most prevalent form of dementia and is characterized by abnormal extracellular aggregates of amyloid-β and intraneuronal hyperphosphorylated tau tangles and neuropil threads. Microglia, the tissue-resident macrophages of the central nervous system (CNS), are important for CNS homeostasis and implicated in AD pathology. In amyloid mouse models, a phagocytic/activated microglia phenotype has been identified. How increasing levels of amyloid-β and tau pathology affect human microglia transcriptional profiles is unknown. Here, we performed snRNAseq on 482,472 nuclei from non-demented control brains and AD brains containing only amyloid-β plaques or both amyloid-β plaques and tau pathology. Within the microglia population, distinct expression profiles were identified of which two were AD pathology-associated. The phagocytic/activated AD1-microglia population abundance strongly correlated with tissue amyloid-β load and localized to amyloid-β plaques. The AD2-microglia abundance strongly correlated with tissue phospho-tau load and these microglia were more abundant in samples with overt tau pathology. This full characterization of human disease-associated microglia phenotypes provides new insights in the pathophysiological role of microglia in AD and offers new targets for microglia-state-specific therapeutic strategies.

Neuroinflammation is a common feature of many neurodegenerative diseases. It fosters a dysfunctional neuron-microglia-astrocyte crosstalk that, in turn, maintains microglial cells in a perniciously reactive state that often enhances neuronal damage. The molecular components that mediate this critical communication are not fully explored. Here, we show that secreted frizzled-related protein 1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. In mouse models of acute and chronic neuroinflammation, SFRP1, largely astrocyte-derived, promotes and sustains microglial activation, and thus a chronic inflammatory state. SFRP1 promotes the upregulation of components of the hypoxia-induced factor-dependent inflammatory pathway and, to a lower extent, of those downstream of the nuclear factor-kappa B. We thus propose that SFRP1 acts as an astrocyte-to-microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation in several neurodegenerative diseases.

Single-nucleus RNA sequencing (sNuc-RNAseq) is an emerging powerful genomics technology that combines droplet microfluidics with next-generation sequencing to interrogate transcriptome changes at single nucleus resolution. Here we developed Abacus, a flexible UMI counter software for sNuc-RNAseq analysis. Abacus draws extra information from sequencing reads mapped to introns of pre-mRNAs (~60% of total data) that are ignored by many single-cell RNAseq analysis pipelines. When applied to our pilot human brain sNuc-RNAseq data, ABACUS nearly doubled the number of nuclei identified by the CellRanger workflow, recovering a large number of nuclei from non-neuronal cells. By incorporating intronic reads into gene expression quantification, we showed that they encoded additional and valid transcription features of individual cells and could be used to improve cluster resolution of different cell types. By separately counting UMIs derived from forward and reverse intronic reads and from exonic reads, Abacus gives users flexibility in representing genes expressed at different abundance levels. In summary, Abacus represents a flexible, improved workflow for sNuc-RNAseq data processing and analysis.

Neuroinflammation is a common feature of many neurodegenerative diseases, which fosters a dysfunctional neuron-microglia-astrocyte crosstalk that, in turn, maintains microglial cells into a perniciously reactive state that often enhance neuronal damage. The molecular components that mediate this critical communication are however not fully explored. Here, we have asked whether Secreted-Frizzled-Related-Protein-1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. We show that in mouse models of acute and chronic neuroinflammation, astrocyte-derived SFRP1 is sufficient to promote and sustain microglial activation, and thus a chronic inflammatory state. SFRP1 allows the upregulation of components of Hypoxia Induced Factors-dependent inflammatory pathway and, to a lower extent, of those downstream of the Nuclear Factor-kappaB. We thus propose that SFRP1 acts as a critical astrocyte to microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation present in several neurodegenerative diseases.### Competing Interest StatementThe authors have declared no competing interest.

The hypothesis that accumulation of beta-amyloid (Aβ) species in the brain represents a major event in Alzheimer's disease (AD) pathogenesis still prevails; nevertheless, an array of additional pathological processes contributes to clinical presentation and disease progression. We sought to identify novel targets for AD within genes related to amyloid precursor protein (APP) processing, innate immune responses, and the catecholamine system. Through a series of bioinformatics analyses, we identified TLR5 and other genes involved in toll-like receptor (TLR) signaling as potential AD targets. It is believed that Aβ species induce activation of microglia and astrocytes in AD, with a negative impact on disease progression. The TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases plays pivotal roles in limiting inflammatory responses upon TLR stimulation, for which we further studied their implication in the TLR5 alterations observed in AD. We validated the up-regulation of TLR5 in the frontal cortex of moderate AD cases. In addition, we observed up-regulation of the TAM ligands protein S (PROS1), galectin-3 (LGALS3), and Tulp-1. Furthermore, we identified an association of the TAM ligand GAS6 with AD progression. In THP-1 cells, co-stimulation with Aβ and flagellin for 24 h induced up-regulation of TYRO3 and GAS6, which could be prevented by neutralization of TLR5. Our results underscore the role of TLR dysregulations in AD, suggesting the presence of an immunosuppressive response during moderate disease stages, and implicate TAM signaling in AD immune dysregulation.

A beta(1-42) is well accepted to be a primary early pathogenic agent in Alzheimer's disease (AD). However, other amyloid peptides are now gaining considerable attention as potential key participants in AD due to their proposed higher neuronal toxicity. Impairment of the glutamatergic system is also widely accepted to be associated with pathomechanisms underlying AD. There is ample evidence that A beta(1-42) affects GLUN2B subunit containing N-methyl-D-aspartate receptor function and abolishes the induction of long term potentiation (LTP). In this study we show that different beta-amyloid species, 1-42 A beta(1-42) and 1-40 (A beta(1-40)) as well as post-translationally modified forms such as pyroglutamate-modified amyloid-(A beta pE3) and nitrated A beta (3NTyr10-A beta), when applied for 90 min to murine hippocampal slices, concentration dependently prevented the development of CA1-LTP after tetanic stimulation of the Schaffer collaterals with IC(50)s of 2, 9, 2 and 35 nM, respectively whilst having no effect on baseline AMPA receptor mediated fEPSPs. A beta(1-43) had no effect. Interestingly, the combination of all A beta species did not result in any synergistic or additive inhibitory effect on LTP - the calculated pooled A beta species IC50 was 20 nM. A low concentration (10 nM) of the GLUN2B receptor antagonist Radiprodil restored LTP in the presence of A beta(1-42), 3NTyr10-A beta, A beta(1-40), but not A beta pE3. In contrast to AMPA receptor mediated fEPSPs, all different beta-amyloid species tested at 50 nM supressed baseline NMDA-EPSC amplitudes. Similarly, all different A beta species tested decreased spine density. As with LTP, Radiprodil (10 nM) reversed the synaptic toxicity of A beta species but not that of A beta pE3. These data do not support the enhanced toxic actions reported for some A beta species such as A beta pE3, nor synergistic toxicity of the combination of different A beta species. However, whilst in our hands A beta pE(3-42) was actually less toxic than A beta(1-42), its effects were not reversed by Radiprodil indicating that the target receptors/subunits mediating such synaptotoxicity may differ between the different A beta species tested. (C) 2018 Elsevier Ltd. All rights reserved.

BACKGROUND:Neuroinflammation has gained increasing attention as a potential contributing factor in Alzheimer's disease (AD) pathology. A clinical cerebrospinal fluid biomarker capable of monitoring this process during the course of the disease has yet to emerge, chiefly owing to contradictory research findings. In this study, we sought to clarify the utility of inflammatory biomarkers in diagnostic procedures of AD in three steps: (1) to screen for proteins that are robustly detectable in cerebrospinal fluid; (2) based on this analysis, to explore any associations between the analytically robust markers and salient pathological features of AD; and (3) to determine the discriminative power of these markers in the clinical diagnosis of AD.METHODS:From a total of 46 proteins, 15 that were robustly detectable in cerebrospinal fluid were identified. A subsequent analysis of these markers in a cohort of 399 patients (nondemented subjects, patients with mild cognitive impairment [MCI], and patients with AD, supplemented by smaller cohorts of other diseases) was conducted. Fluid biomarker data were related to AD pathology and neuropsychological markers and adjusted for confounders such as age, sex, apolipoprotein E genotype, and biobank storage time.RESULTS:Cerebrospinal fluid levels of C-reactive protein and soluble TREM2 differed between nondemented subjects, patients with MCI, or patients with AD and were associated with amyloid and tau pathology. Several markers were associated with tau pathology only or with other neurodegenerative diseases. Correlations between neuropsychological performance and inflammatory markers were weak, but they were most prominent in AD and for the most challenging cognitive tests. All investigated covariates had significant influence, with varying effects across the markers. Still, none of the markers achieved discriminative power of more than 70% to distinguish between patient groups defined by clinical or neuropathological categories.CONCLUSIONS:Basic analytical considerations proved indispensable for this type of study because only one-third of the tested markers were robustly detectable in cerebrospinal fluid. Detectable inflammatory protein markers were associated in multiple ways with AD pathology. Yet, even significantly associated markers were not powerful enough in terms of effect strength, sensitivity, and specificity, and hence they were not suited for direct use in clinical diagnostic practice. Targets other than those most commonly considered in this field of research might provide results with better clinical applicability.

Despite its wide application in studies on memory and disease-related cognitive impairment, the use of the novel object recognition (NOR) test in research on aging has been limited and produced conflicting results. The purpose of this study was to characterize NOR across the lifespan of male C57BL/6 mice (3, 6, 9, 12, 15, and 24 months), the most popular rodent model of aging. NOR, measured after a delay of 24 hours, was analyzed with respect to variability, its dependence on locomotor and exploratory activity, and with an emphasis on its temporal dynamics. The latter has been recognized in rats, but never been investigated in mice. We find that although locomotor activity decreases monotonically above 3 months of age, exploratory activity in our setup remains constant and sufficient for NOR testing up to 12 months. As a major finding, we show that NOR depends on exploration time in the test phase in an age-dependent manner. Whereas NOR in our paradigm remains significant up to at least 20 seconds total exploration time in 3 and 6 months old mice, novel object preference at 9 and 12 months of age is detectable after 10 seconds exploration, but decreases rapidly and is missed when the first 20 seconds are analyzed. Analysis of the first 2 minutes of the test phase does not detect NOR except for 3 months old mice. We conclude that temporal aspects of NOR have been neglected and must be considered when making comparisons between or within mice at different ages.

The spreading of pathology within and between brain areas is a hallmark of neurodegenerative disorders. In patients with Alzheimer’s disease, deposition of amyloid-β is accompanied by activation of the innate immune system and involves inflammasome-dependent formation of ASC specks in microglia. ASC specks released by microglia bind rapidly to amyloid-β and increase the formation of amyloid-β oligomers and aggregates, acting as an inflammation-driven cross-seed for amyloid-β pathology. Here we show that intrahippocampal injection of ASC specks resulted in spreading of amyloid-β pathology in transgenic double-mutant APP Swe PSEN1 dE9 mice. By contrast, homogenates from brains of APP Swe PSEN1 dE9 mice failed to induce seeding and spreading of amyloid-β pathology in ASC-deficient APP Swe PSEN1 dE9 mice. Moreover, co-application of an anti-ASC antibody blocked the increase in amyloid-β pathology in APP Swe PSEN1 dE9 mice. These findings support the concept that inflammasome activation is connected to seeding and spreading of amyloid-β pathology in patients with Alzheimer’s disease.

•Protocol for quantitative proteomics of nitrosylation on synaptosomal proteins.•Identification of endogenous nitrosylation independent of induction by NO donors.•Use of iodoTMT sixplex mass tags for stable labeling, enrichment, identification, and multiplex quantitation.•Applicable on low amounts of sample material of mouse and human brain tissue.

Antecedentes: La evaluación del riesgo preoperatorio en la cirugía oncológica es importante para mejorar el tratamiento y los resultados.El objetivo de este estudio fue analizar el impacto de la tomografía computarizada (TAC) en la evaluación de la sarcopenia en los resultados a corto y largo plazo en pacientes sometidos a resecciones quirúrgicas de neoplasias gastrointestinales y hepatopancreatobiliares. Métodos: Se realizó una búsqueda sistemática en EMBASE, PubMed y Web of Science para identificar estudios relevantes publicados antes de septiembre de 2014.Se siguieron las directrices de la guías PRISMA para revisiones sistemáticas.La inclusión, la validez de los estudios incluidos y la extracción de datos fue realizada de forma independiente por dos investigadores.Resultados: Después de analizar 692 registros, trece estudios observacionales con un total de 2.884 pacientes fueron incluidos en el análisis.Se observó una gran variación en la prevalencia de la sarcopenia (de 17,0 a 78,7%).La sarcopenia se asoció de forma independiente con una menor supervivencia global en siete de diez estudios, independientemente de la localización del tumor.Se describieron cocientes de riesgos instantáneos (hazard ratio, HR) de hasta 3,19 (cáncer hepático), 1,63 (cáncer de páncreas), 1,85 (cáncer colorrectal (CRC)) y 2,89 (metástasis hepáticas colorrectales [CLM]).Para el cáncer de esófago, se demostró un HR de 0,31 por un aumento de la masa muscular.En pacientes con CRC y CLM, la sarcopenia se asoció de forma independiente con la mortalidad postoperatoria (CRC; razón de oportunidades [odds ratio,OR] 43,3), con complicaciones (CRC; OR 0,95 por aumento de la masa muscular, CLM; OR 222) y con complicaciones graves (CLM; OR 3,12).Conclusión: La evaluación de la sarcopenia preoperatoria mediante el análisis de un solo corte del TAC se asocia con una disminución de la supervivencia global en neoplasias gastrointestinales y hepatopancreatobiliares, y con un aumento de la morbilidad postoperatoria en pacientes con cáncer colorrectal con o sin metástasis hepáticas.

BACKGROUND:The pathophysiology of adhesion formation after abdominal and pelvic surgery is still largely unknown. The aim of the study was to investigate the role of macrophage polarization and the effect of peroxisome proliferator-activated receptor (PPAR) γ stimulation on adhesion formation in an animal model.METHODS:Peritoneal adhesion formation was induced by the creation of ischaemic buttons within the peritoneal wall and the formation of a colonic anastomosis in wild-type, interleukin (IL) 10-deficient (IL-10(-/-) ), IL-4-deficient (IL-4(-/-) ) and CD11b-Cre/PPARγ(fl) (/fl) mice. Adhesions were assessed at regular intervals, and cell preparations were isolated from ischaemic buttons and normal peritoneum. These samples were analysed for macrophage differentiation and its markers, and expression of cytokines by quantitative PCR, fluorescence microscopy, arginase activity and pathological examination. Some animals underwent pioglitazone (PPAR-γ agonist) or vehicle treatment to inhibit adhesion formation. Anastomotic healing was evaluated by bursting pressure measurement and collagen gene expression.RESULTS:Macrophage M2 marker expression and arginase activity were raised in buttons without adhesions compared with buttons with adhesions. IL-4(-/-) and IL-10(-/-) mice were not affected, whereas CD11b-Cre/PPARγ(fl) (/fl) mice showed decreased arginase activity and increased adhesion formation. Perioperative pioglitazone treatment increased arginase activity and decreased adhesion formation in wild-type but not CD11b-Cre/PPARγ(fl) (/fl) mice. Pioglitazone had no effect on anastomotic healing.CONCLUSION:Endogenous macrophage-specific PPAR-γ signalling affected arginase activity and macrophage polarization, and counter-regulated peritoneal adhesion manifestation. Pharmacological PPAR-γ agonism induced a shift towards macrophage M2 polarization and ameliorated adhesion formation in a macrophage-dependent manner. Surgical relevance Postoperative adhesion formation is frequently seen after abdominal surgery and occurs in response to peritoneal trauma. The pathogenesis is still unknown but includes an imbalance in fibrinolysis, collagen production and inflammatory mechanisms. Little is known about the role of macrophages during adhesion formation. In an experimental model, macrophage M2 marker expression was associated with reduced peritoneal adhesion formation and involved PPAR-γ-mediated arginase activity. Macrophage-specific PPAR-γ deficiency resulted in reduced arginase activity and aggravated adhesion formation. Pioglitazone, a PPAR-γ agonist, induced M2 polarization and reduced postoperative adhesion formation without compromising anastomotic healing in mice. Pioglitazone ameliorated postoperative adhesion formation without compromising intestinal wound healing. Therefore, perioperative PPAR-γ agonism might be a promising strategy for prevention of adhesion formation after abdominal surgery.