Candidate-based screening via gene modulation in human neurons and astrocytes implicates FERMT2 in Aß and TAU proteostasis.

Large-scale 'omic' studies investigating the pathophysiological processes that lead to Alzheimer's disease (AD) dementia have identified an increasing number of susceptibility genes, many of which are poorly characterized and have not previously been implicated in AD. Here, we evaluated the utility of human induced pluripotent stem cell-derived neurons and astrocytes as tools to systematically test AD-relevant cellular phenotypes following perturbation of candidate genes identified by genome-wide studies. Lentiviral-mediated delivery of shRNAs was used to modulate expression of 66 genes in astrocytes and 52 genes in induced neurons. Five genes (CNN2, GBA, GSTP1, MINT2 and FERMT2) in neurons and nine genes (CNN2, ITGB1, MINT2, SORL1, VLDLR, NPC1, NPC2, PSAP and SCARB2) in astrocytes significantly altered extracellular amyloid-ß (Aß) levels. Knockdown of AP3M2, CNN2, GSTP1, NPC1, NPC2, PSAP and SORL1 reduced interleukin-6 levels in astrocytes. Only knockdown of FERMT2 led to a reduction in the proportion of TAU that is phosphorylated. Further, CRISPR-Cas9 targeting of FERMT2 in both familial AD (fAD) and fAD-corrected human neurons validated the findings of reduced extracellular Aß. Interestingly, FERMT2 reduction had no effect on the Aß42:40 ratio in corrected neurons and a reduction of phospho-tau, but resulted in an elevation in Aß42:40 ratio and no reduction in phospho-tau in fAD neurons. Taken together, this study has prioritized 15 genes as being involved in contributing to Aß accumulation, phosphorylation of tau and/or cytokine secretion, and, as illustrated with FERMT2, it sets the stage for further cell-type-specific dissection of the role of these genes in AD.

Deconvolving the contributions of cell-type heterogeneity on cortical gene expression.

Complexity of cell-type composition has created much skepticism surrounding the interpretation of bulk tissue transcriptomic studies. Recent studies have shown that deconvolution algorithms can be applied to computationally estimate cell-type proportions from gene expression data of bulk blood samples, but their performance when applied to brain tissue is unclear. Here, we have generated an immunohistochemistry (IHC) dataset for five major cell-types from brain tissue of 70 individuals, who also have bulk cortical gene expression data. With the IHC data as the benchmark, this resource enables quantitative assessment of deconvolution algorithms for brain tissue. We apply existing deconvolution algorithms to brain tissue by using marker sets derived from human brain single cell and cell-sorted RNA-seq data. We show that these algorithms can indeed produce informative estimates of constituent cell-type proportions. In fact, neuronal subpopulations can also be estimated from bulk brain tissue samples. Further, we show that including the cell-type proportion estimates as confounding factors is important for reducing false associations between Alzheimer's disease phenotypes and gene expression. Lastly, we demonstrate that using more accurate marker sets can substantially improve statistical power in detecting cell-type specific expression quantitative trait loci (eQTLs).

Microglial immunophenotype in dementia with Alzheimer's pathology.

BACKGROUND: Genetic risk factors for Alzheimer's disease imply that inflammation plays a causal role in development of the disease. Experimental studies suggest that microglia, as the brain macrophages, have diverse functions, with their main role in health being to survey the brain parenchyma through highly motile processes. METHODS: Using the Medical Research Council Cognitive Function and Ageing Studies resources, we have immunophenotyped microglia to investigate their role in dementia with Alzheimer's pathology. Cerebral cortex obtained at post-mortem from 299 participants was analysed by immunohistochemistry for cluster of differentiation (CD)68 (phagocytosis), human leukocyte antigen (HLA)-DR (antigen-presenting function), ionized calcium-binding adaptor molecule (Iba1) (microglial motility), macrophage scavenger receptor (MSR)-A (plaque-related phagocytosis) and CD64 (immunoglobulin Fcγ receptor I). RESULTS: The presence of dementia was associated positively with CD68 (P < 0.001), MSR-A (P = 0.010) and CD64 (P = 0.007) and negatively with Iba1 (P < 0.001). Among participants without dementia, the cognitive function according to the Mini-Mental State Examination was associated positively with Iba1 (P < 0.001) and negatively with CD68 (P = 0.033), and in participants with dementia and Alzheimer's pathology, positively with all microglial markers except Iba1. Overall, in participants without dementia, the relationship with Alzheimer's pathology was negative or not significant, and positive in participants with dementia and Alzheimer's pathology. Apolipoprotein E (APOE) ε2 allele was associated with expression of Iba1 (P = 0.001) and MSR-A (P < 0.001) and APOE ε4 with CD68, HLA-DR and CD64 (P < 0.001). CONCLUSIONS: Our findings raise the possibility that in dementia with Alzheimer's pathology, microglia lose motility (Iba-1) necessary to support neurons. Conversely, other microglial proteins (CD68, MSR-A), the role of which is clearance of damaged cellular material, are positively associated with Alzheimer's pathology and impaired cognitive function. In addition, our data imply that microglia may respond differently to Aß and tau in participants with and without dementia so that the microglial activity could potentially influence the likelihood of developing dementia, as supported by genetic studies, highlighting the complexity and diversity of microglial responses.

Performance and complications of lumbar puncture in memory clinics: Results of the multicenter lumbar puncture feasibility study.

INTRODUCTION: Lumbar puncture (LP) is increasingly performed in memory clinics. We investigated patient-acceptance of LP, incidence of and risk factors for post-LP complications in memory clinic populations. METHODS: We prospectively enrolled 3868 patients (50% women, age 66 ± 11 years, mini mental state examination 25 ± 5) at 23 memory clinics. We used logistic regression analysis using generalized estimated equations to investigate risk factors for post-LP complications, such as typical postlumbar puncture headache (PLPH) and back pain. RESULTS: A total of 1065 patients (31%) reported post-LP complaints; 589 patients (17%) reported back pain, 649 (19%) headache, of which 296 (9%) reported typical PLPH. Only few patients needed medical intervention: 11 (0.3%) received a blood patch, 23 (0.7%) were hospitalized. The most important risk factor for PLPH was medical history of headache. An atraumatic needle and age >65 years were preventive. Gender, rest after LP, or volume of cerebrospinal fluid had no effect. DISCUSSIONS: The overall risk of complications is relatively low. If risk factors shown in this study are taken into account, LPs can be safely performed in memory clinics.

Intermolecular disulfide bonds between nucleoporins regulate karyopherin-dependent nuclear transport.

Disulfide (S-S) bonds play important roles in the regulation of protein function and cellular stress responses. In this study, we demonstrate that distinct sets of nucleoporins (Nups), components of the nuclear pore complex (NPC), form S-S bonds and regulate nuclear transport through the NPC. Kinetic analysis of importin ß demonstrated that the permeability of the NPC was increased by dithiothreitol treatment and reduced by oxidative stress. The permeability of small proteins such as GFP was not affected by either oxidative stress or a reducing reagent. Immunoblot analysis revealed that the oxidative stress significantly induced S-S bond formation in Nups 358, 155, 153 and 62 but not 88 and 160. The direct involvement of cysteine residues in the formation of S-S bonds was confirmed by mutating conserved cysteine residues in Nup62, which abolished the formation of S-S bonds and enhanced the permeability of the NPC. Knocking down Nup62 reduced the stress-inducible S-S bonds of Nup155, suggesting that Nup62 and Nup155 are covalently coupled via S-S bonds. From these results, we propose that the inner channel of the NPC is somehow insulated from the cytoplasm and is more sensitive than the cytoplasm to the intracellular redox state.

Human disease-specific cell signatures in non-lesional tissue in Multiple Sclerosis detected by single-cell and spatial transcriptomics.

Recent investigations of cell type changes in Multiple Sclerosis (MS) using single-cell profiling methods have focused on active lesional and peri-lesional brain tissue, and have implicated a number of peripheral and central nervous system cell types. However, an important question is the extent to which so-called "normal-appearing" non-lesional tissue in individuals with MS accumulate changes over the lifespan. Here, we compared post-mortem non-lesional brain tissue from donors with a pathological or clinical diagnosis of MS from the Religious Orders Study or Rush Memory and Aging Project (ROSMAP) cohorts to age- and sex-matched brains from persons without MS (controls). We profiled three brain regions using single-nucleus RNA-seq: dorsolateral prefrontal cortex (DLPFC), normal appearing white matter (NAWM) and the pulvinar in thalamus (PULV), from 15 control individuals, 8 individuals with MS, and 5 individuals with other detrimental pathologies accompanied by demyelination, resulting in a total of 78 samples. We identified region- and cell type-specific differences in non-lesional samples from individuals diagnosed with MS and/or exhibiting secondary demyelination with other neurological conditions, as compared to control donors. These differences included lower proportions of oligodendrocytes with expression of myelination related genes MOBP, MBP, PLP1, as well as higher proportions of CRYAB+ oligodendrocytes in all three brain regions. Among microglial signatures, we identified subgroups that were higher in both demyelination (TMEM163+/ERC2+), as well as those that were specifically higher in MS donors (HIF1A+/SPP1+) and specifically in donors with secondary demyelination (SOCS6+/MYO1E+), in both white and grey matter. To validate our findings, we generated Visium spatial transcriptomics data on matched tissue from 13 donors, and recapitulated our observations of gene expression differences in oligodendrocytes and microglia. Finally, we show that some of the differences observed between control and MS donors in NAWM mirror those previously reported between control WM and active lesions in MS donors. Overall, our investigation sheds additional light on cell type- and disease-specific differences present even in non-lesional white and grey matter tissue, highlighting widespread cellular signatures that may be associated with downstream pathological changes.

Cellular dynamics across aged human brains uncover a multicellular cascade leading to Alzheimer's disease.

Alzheimer's Disease (AD) is a progressive neurodegenerative disease seen with advancing age. Recent studies have revealed diverse AD-associated cell states, yet when and how they impact the causal chain leading to AD remains unknown. To reconstruct the dynamics of the brain's cellular environment along the disease cascade and to distinguish between AD and aging effects, we built a comprehensive cell atlas of the aged prefrontal cortex from 1.64 million single-nucleus RNA-seq profiles. We associated glial, vascular and neuronal subpopulations with AD-related traits for 424 aging individuals, and aligned them along the disease cascade using causal modeling. We identified two distinct lipid-associated microglial subpopulations, one contributed to amyloid-ß proteinopathy while the other mediated the effect of amyloid-ß in accelerating tau proteinopathy, as well as an astrocyte subpopulation that mediated the effect of tau on cognitive decline. To model the coordinated dynamics of the entire cellular environment we devised the BEYOND methodology which uncovered two distinct trajectories of brain aging that are defined by distinct sequences of changes in cellular communities. Older individuals are engaged in one of two possible trajectories, each associated with progressive changes in specific cellular communities that end with: (1) AD dementia or (2) alternative brain aging. Thus, we provide a cellular foundation for a new perspective of AD pathophysiology that could inform the development of new therapeutic interventions targeting cellular communities, while designing a different clinical management for those individuals on the path to AD or to alternative brain aging.

Multicellular communities are perturbed in the aging human brain and Alzheimer's disease.

The role of different cell types and their interactions in Alzheimer's disease (AD) is a complex and open question. Here, we pursued this question by assembling a high-resolution cellular map of the aging frontal cortex using single-nucleus RNA sequencing of 24 individuals with a range of clinicopathologic characteristics. We used this map to infer the neocortical cellular architecture of 638 individuals profiled by bulk RNA sequencing, providing the sample size necessary for identifying statistically robust associations. We uncovered diverse cell populations associated with AD, including a somatostatin inhibitory neuronal subtype and oligodendroglial states. We further identified a network of multicellular communities, each composed of coordinated subpopulations of neuronal, glial and endothelial cells, and we found that two of these communities are altered in AD. Finally, we used mediation analyses to prioritize cellular changes that might contribute to cognitive decline. Thus, our deconstruction of the aging neocortex provides a roadmap for evaluating the cellular microenvironments underlying AD and dementia.

INPP5D regulates inflammasome activation in human microglia.

Microglia and neuroinflammation play an important role in the development and progression of Alzheimer's disease (AD). Inositol polyphosphate-5-phosphatase D (INPP5D/SHIP1) is a myeloid-expressed gene genetically-associated with AD. Through unbiased analyses of RNA and protein profiles in INPP5D-disrupted iPSC-derived human microglia, we find that reduction in INPP5D activity is associated with molecular profiles consistent with disrupted autophagy and inflammasome activation. These findings are validated through targeted pharmacological experiments which demonstrate that reduced INPP5D activity induces the formation of the NLRP3 inflammasome, cleavage of CASP1, and secretion of IL-1ß and IL-18. Further, in-depth analyses of human brain tissue across hundreds of individuals using a multi-analytic approach provides evidence that a reduction in function of INPP5D in microglia results in inflammasome activation in AD. These findings provide insights into the molecular mechanisms underlying microglia-mediated processes in AD and highlight the inflammasome as a potential therapeutic target for modulating INPP5D-mediated vulnerability to AD.

Cell-type-specific regulation of APOE and CLU levels in human neurons by the Alzheimer's disease risk gene SORL1.

SORL1 is implicated in the pathogenesis of Alzheimer's disease (AD) through genetic studies. To interrogate the roles of SORL1 in human brain cells, SORL1-null induced pluripotent stem cells (iPSCs) were differentiated to neuron, astrocyte, microglial, and endothelial cell fates. Loss of SORL1 leads to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. SORL1 loss induces a neuron-specific reduction in apolipoprotein E (APOE) and clusterin (CLU) and altered lipid profiles. Analyses of iPSCs derived from a large cohort reveal a neuron-specific association between SORL1, APOE, and CLU levels, a finding validated in postmortem brain. Enhancement of retromer-mediated trafficking rescues tau phenotypes observed in SORL1-null neurons but does not rescue APOE levels. Pathway analyses implicate transforming growth factor ß (TGF-ß)/SMAD signaling in SORL1 function, and modulating SMAD signaling in neurons alters APOE RNA levels in a SORL1-dependent manner. Taken together, these data provide a mechanistic link between strong genetic risk factors for AD.

A cortical immune network map identifies distinct microglial transcriptional programs associated with ß-amyloid and Tau pathologies.

Microglial dysfunction has been proposed as one of the many cellular mechanisms that can contribute to the development of Alzheimer's disease (AD). Here, using a transcriptional network map of the human frontal cortex, we identify five modules of co-expressed genes related to microglia and assess their role in the neuropathologic features of AD in 540 subjects from two cohort studies of brain aging. Two of these transcriptional programs-modules 113 and 114-relate to the accumulation of ß-amyloid, while module 5 relates to tau pathology. We replicate these associations in brain epigenomic data and in two independent datasets. In terms of tau, we propose that module 5, a marker of activated microglia, may lead to tau accumulation and subsequent cognitive decline. We validate our model further by showing that three representative module 5 genes (ACADVL, TRABD, and VASP) encode proteins that are upregulated in activated microglia in AD.

BIN1 protein isoforms are differentially expressed in astrocytes, neurons, and microglia: neuronal and astrocyte BIN1 are implicated in tau pathology.

BACKGROUND: Identified as an Alzheimer's disease (AD) susceptibility gene by genome wide-association studies, BIN1 has 10 isoforms that are expressed in the Central Nervous System (CNS). The distribution of these isoforms in different cell types, as well as their role in AD pathology still remains unclear. METHODS: Utilizing antibodies targeting specific BIN1 epitopes in human post-mortem tissue and analyzing mRNA expression data from purified microglia, we identified three isoforms expressed in neurons and astrocytes (isoforms 1, 2 and 3) and four isoforms expressed in microglia (isoforms 6, 9, 10 and 12). The abundance of selected peptides, which correspond to groups of BIN1 protein isoforms, was measured in dorsolateral prefrontal cortex, and their relation to neuropathological features of AD was assessed. RESULTS: Peptides contained in exon 7 of BIN1's N-BAR domain were found to be significantly associated with AD-related traits and, particularly, tau tangles. Decreased expression of BIN1 isoforms containing exon 7 is associated with greater accumulation of tangles and subsequent cognitive decline, with astrocytic rather than neuronal BIN1 being the more likely culprit. These effects are independent of the BIN1 AD risk variant. CONCLUSIONS: Exploring the molecular mechanisms of specific BIN1 isoforms expressed by astrocytes may open new avenues for modulating the accumulation of Tau pathology in AD.

Single cell RNA sequencing of human microglia uncovers a subset associated with Alzheimer's disease.

The extent of microglial heterogeneity in humans remains a central yet poorly explored question in light of the development of therapies targeting this cell type. Here, we investigate the population structure of live microglia purified from human cerebral cortex samples obtained at autopsy and during neurosurgical procedures. Using single cell RNA sequencing, we find that some subsets are enriched for disease-related genes and RNA signatures. We confirm the presence of four of these microglial subpopulations histologically and illustrate the utility of our data by characterizing further microglial cluster 7, enriched for genes depleted in the cortex of individuals with Alzheimer's disease (AD). Histologically, these cluster 7 microglia are reduced in frequency in AD tissue, and we validate this observation in an independent set of single nucleus data. Thus, our live human microglia identify a range of subtypes, and we prioritize one of these as being altered in AD.

PKR modulates abnormal brain signaling in experimental obesity.

Metabolic disorders including obesity and type 2 diabetes are known to be associated with chronic inflammation and are obvious risk factors for Alzheimer's disease. Recent evidences concerning obesity and diabetes suggest that the metabolic inflammasome ("metaflammasome") mediates chronic inflammation. The double-stranded RNA-dependent protein kinase (PKR) is a central component of the metaflammasome. In wild type (WT) and PKR-/- mice, blood glucose, insulin and lipid levels and the brain expression of the phosphorylated components of the metaflammasome-PKR, JNK, IRS1 and IKKbeta-were studied after the induction of obesity by a high fat diet (HFD). The results showed significant increased levels of activated brain metaflammasome proteins in exposed WT mice but the changes were not significant in PKR-/- mice. In addition, gain weight was observed in WT mice and also in PKR-/- mice exposed to HFD. Increased blood insulin level was more accentuated in PKR -/- mice. The modulation of PKR activity could be an appropriate therapeutic approach, aimed at reducing abnormal brain metabolism and inflammation linked to metabolic disorders in order to reduce the risk of neurodegeneration.

A molecular network of the aging human brain provides insights into the pathology and cognitive decline of Alzheimer's disease.

There is a need for new therapeutic targets with which to prevent Alzheimer's disease (AD), a major contributor to aging-related cognitive decline. Here we report the construction and validation of a molecular network of the aging human frontal cortex. Using RNA sequence data from 478 individuals, we first build a molecular network using modules of coexpressed genes and then relate these modules to AD and its neuropathologic and cognitive endophenotypes. We confirm these associations in two independent AD datasets. We also illustrate the use of the network in prioritizing amyloid- and cognition-associated genes for in vitro validation in human neurons and astrocytes. These analyses based on unique cohorts enable us to resolve the role of distinct cortical modules that have a direct effect on the accumulation of AD pathology from those that have a direct effect on cognitive decline, exemplifying a network approach to complex diseases.

Metaflammasome components in the human brain: a role in dementia with Alzheimer's pathology?

Epidemiological and genetic studies have identified metabolic disorders and inflammation as risk factors for Alzheimer's disease (AD). Evidence in obesity and type-2 diabetes suggests a role for a metabolic inflammasome ("metaflammasome") in mediating chronic inflammation in peripheral organs implicating IKKß (inhibitor of nuclear factor kappa-B kinase subunit beta), IRS1 (insulin receptor substrate 1), JNK (c-jun N-terminal kinase), and PKR (double-stranded RNA protein kinase). We hypothesized that these proteins are expressed in the brain in response to metabolic risk factors in AD. Neocortex from 299 participants from the MRC Cognitive Function and Ageing Studies was analysed by immunohistochemistry for the expression of the phosphorylated (active) form of IKKß [pSer176/180 ], IRS1 [pS312 ], JNK [pThr183 /Tyr185 ] and PKR [pT451 ]. The data were analyzed to investigate whether the proteins were expressed together and in relation with metabolic disorders, dementia, Alzheimer's pathology and APOE genotype. We observed a change from a positive to a negative association between the proteins and hypertension according to the dementia status. Type-2 diabetes was negatively related with the proteins among participants without dementia; whereas participants with dementia and AD pathology showed a positive association with JNK. A significant association between IKKß and JNK in participants with dementia and AD pathology was observed, but not in those without dementia. Otherwise, weak to moderate associations were observed among the protein loads. The presence of dementia was significantly associated with JNK and negatively associated with IKKß and IRS1. Cognitive scores showed a significant positive relationship with IKKß and a negative with IRS1, JNK and PKR. The proteins were significantly associated with pathology in Alzheimer's participants with the relationship being inverse or not significant in participants without dementia. Expression of the proteins was not related to APOE genotype. These findings highlight a role for these proteins in AD pathophysiology but not necessarily as a complex.

Tiam1/Rac1 complex controls Il17a transcription and autoimmunity.

RORγt is a master transcription factor of Th17 cells and considered as a promising drug target for the treatment of autoimmune diseases. Here, we show the guanine nucleotide exchange factor, Tiam1, and its cognate Rho-family G protein, Rac1, regulate interleukin (IL)17A transcription and autoimmunity. Whereas Tiam1 genetic deficiency weakens IL-17A expression partially and inhibits the development of experimental autoimmune encephalomyelitis (EAE), deletion of Rac1 in T cells exhibits more robust effects on Th17 cells and EAE. We demonstrate Tiam1 and Rac1 form a complex with RORγt in the nuclear compartment of Th17 cells, and together bind and activate the Il17 promoter. The clinical relevance of these findings is emphasized by pharmacological targeting of Rac1 that suppresses both murine and human Th17 cells as well as EAE. Thus, our findings highlight a regulatory pathway of Tiam1/Rac1 in Th17 cells and suggest that it may be a therapeutic target in multiple sclerosis.

Modulation of oxidative stress and tau phosphorylation by the mTOR activator phosphatidic acid in SH-SY5Y cells.

The mammalian target of rapamycin complex 1 (mTORC1) pathway including p70(S6K) (the 70-kDa p70 S6 kinase) and S6, controls protein synthesis, has anti-apoptotic functions and can phosphorylate tau protein. mTORC1 is triggered by nutrients such as phosphatidic acid (PA). Previous experimental studies have shown that oxidative stress may down-regulate this pathway leading to neuronal death. Our results showed that in human neuroblastoma cells, PA exposure can reduce H(2)O(2)-induced apoptosis and can increase tau protein phosphorylation on Ser214 via p70(S6K) activation. These findings reveal that PA, via the mTOR kinase, can trigger tau phosphorylation on a site known to reduce paired helical filament (PHF) formation.