Osteopontin drives neuroinflammation and cell loss in MAPT-N279K frontotemporal dementia patient neurons.

Frontotemporal dementia (FTD) is an incurable group of early-onset dementias that can be caused by the deposition of hyperphosphorylated tau in patient brains. However, the mechanisms leading to neurodegeneration remain largely unknown. Here, we combined single-cell analyses of FTD patient brains with a stem cell culture and transplantation model of FTD. We identified disease phenotypes in FTD neurons carrying the MAPT-N279K mutation, which were related to oxidative stress, oxidative phosphorylation, and neuroinflammation with an upregulation of the inflammation-associated protein osteopontin (OPN). Human FTD neurons survived less and elicited an increased microglial response after transplantation into the mouse forebrain, which we further characterized by single nucleus RNA sequencing of microdissected grafts. Notably, downregulation of OPN in engrafted FTD neurons resulted in improved engraftment and reduced microglial infiltration, indicating an immune-modulatory role of OPN in patient neurons, which may represent a potential therapeutic target in FTD.

Immune Response following FLASH and Conventional Radiation in Diffuse Midline Glioma.

PURPOSE: Diffuse midline glioma (DMG) is a fatal tumor traditionally treated with radiation therapy (RT) and previously characterized as having a noninflammatory tumor immune microenvironment (TIME). FLASH is a novel RT technique using ultra-high dose rate that is associated with decreased toxicity and effective tumor control. However, the effect of FLASH and conventional (CONV) RT on the DMG TIME has not yet been explored. METHODS AND MATERIALS: Here, we performed single-cell RNA sequencing (scRNA-seq) and flow cytometry on immune cells isolated from an orthotopic syngeneic murine model of brainstem DMG after the use of FLASH (90 Gy/sec) or CONV (2 Gy/min) dose-rate RT and compared to unirradiated tumor (SHAM). RESULTS: At day 4 post-RT, FLASH exerted similar effects as CONV in the predominant microglial (MG) population, including the presence of two activated subtypes. However, at day 10 post-RT, we observed a significant increase in the type 1 interferon α/ß receptor (IFNAR+) in MG in CONV and SHAM compared to FLASH. In the non-resident myeloid clusters of macrophages (MACs) and dendritic cells (DCs), we found increased type 1 interferon (IFN1) pathway enrichment for CONV compared to FLASH and SHAM by scRNA-seq. We observed this trend by flow cytometry at day 4 post-RT in IFNAR+ MACs and DCs, which equalized by day 10 post-RT. DMG control and murine survival were equivalent between RT dose rates. CONCLUSIONS: Our work is the first to map CONV and FLASH immune alterations of the DMG TIME with single-cell resolution. Although DMG tumor control and survival were similar between CONV and FLASH, we found that changes in immune compartments differed over time. Importantly, although both RT modalities increased IFN1, we found that the timing of this response was cell-type and dose-rate dependent. These temporal differences, particularly in the context of tumor control, warrant further study.

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.

Re-convolving the compositional landscape of primary and recurrent glioblastoma reveals prognostic and targetable tissue states.

Glioblastoma (GBM) diffusely infiltrates the brain and intermingles with non-neoplastic brain cells, including astrocytes, neurons and microglia/myeloid cells. This complex mixture of cell types forms the biological context for therapeutic response and tumor recurrence. We used single-nucleus RNA sequencing and spatial transcriptomics to determine the cellular composition and transcriptional states in primary and recurrent glioma and identified three compositional 'tissue-states' defined by cohabitation patterns between specific subpopulations of neoplastic and non-neoplastic brain cells. These tissue-states correlated with radiographic, histopathologic, and prognostic features and were enriched in distinct metabolic pathways. Fatty acid biosynthesis was enriched in the tissue-state defined by the cohabitation of astrocyte-like/mesenchymal glioma cells, reactive astrocytes, and macrophages, and was associated with recurrent GBM and shorter survival. Treating acute slices of GBM with a fatty acid synthesis inhibitor depleted the transcriptional signature of this pernicious tissue-state. These findings point to therapies that target interdependencies in the GBM microenvironment.

Associations of VGF with Neuropathologies and Cognitive Health in Older Adults.

OBJECTIVE: VGF is proposed as a potential therapeutic target for Alzheimer's (AD) and other neurodegenerative conditions. The cell-type specific and, separately, peptide specific associations of VGF with pathologic and cognitive outcomes remain largely unknown. We leveraged gene expression and protein data from the human neocortex and investigated the VGF associations with common neuropathologies and late-life cognitive decline. METHODS: Community-dwelling older adults were followed every year, died, and underwent brain autopsy. Cognitive decline was captured via annual cognitive testing. Common neurodegenerative and cerebrovascular conditions were assessed during neuropathologic evaluations. Bulk brain RNASeq and targeted proteomics analyses were conducted using frozen tissues from dorsolateral prefrontal cortex of 1,020 individuals. Cell-type specific gene expressions were quantified in a subsample (N = 424) following single nuclei RNASeq analysis from the same cortex. RESULTS: The bulk brain VGF gene expression was primarily associated with AD and Lewy bodies. The VGF gene association with cognitive decline was in part accounted for by neuropathologies. Similar associations were observed for the VGF protein. Cell-type specific analyses revealed that, while VGF was differentially expressed in most major cell types in the cortex, its association with neuropathologies and cognitive decline was restricted to the neuronal cells. Further, the peptide fragments across the VGF polypeptide resembled each other in relation to neuropathologies and cognitive decline. INTERPRETATION: Multiple pathways link VGF to cognitive health in older age, including neurodegeneration. The VGF gene functions primarily in neuronal cells and its protein associations with pathologic and cognitive outcomes do not map to a specific peptide. ANN NEUROL 2023;94:232-244.

Multi-omic characterization of brain changes in the vascular endothelial growth factor family during aging and Alzheimer's disease.

The vascular endothelial growth factor (VEGF) signaling family has been implicated in neuroprotection and clinical progression in Alzheimer's disease (AD). Previous work in postmortem human dorsolateral prefrontal cortex demonstrated that higher transcript levels of VEGFB, PGF, FLT1, and FLT4 are associated with AD dementia, worse cognitive outcomes, and higher AD neuropathology. To expand prior work, we leveraged bulk RNA sequencing data, single nucleus RNA (snRNA) sequencing, and both tandem mass tag and selected reaction monitoring mass spectrometry proteomic measures from the post-mortem brain. Outcomes included AD diagnosis, cognition, and AD neuropathology. We replicated previously reported VEGFB and FLT1 results, whereby higher expression was associated with worse outcomes, and snRNA results suggest microglia, oligodendrocytes, and endothelia may play a central role in these associations. Additionally, FLT4 and NRP2 expression were associated with better cognitive outcomes. This study provides a comprehensive molecular picture of the VEGF signaling family in cognitive aging and AD and critical insight towards the biomarker and therapeutic potential of VEGF family members in AD.

FMNL2 regulates gliovascular interactions and is associated with vascular risk factors and cerebrovascular pathology in Alzheimer's disease.

Alzheimer's disease (AD) has been associated with cardiovascular and cerebrovascular risk factors (CVRFs) during middle age and later and is frequently accompanied by cerebrovascular pathology at death. An interaction between CVRFs and genetic variants might explain the pathogenesis. Genome-wide, gene by CVRF interaction analyses for AD, in 6568 patients and 8101 controls identified FMNL2 (p = 6.6 × 10-7). A significant increase in FMNL2 expression was observed in the brains of patients with brain infarcts and AD pathology and was associated with amyloid and phosphorylated tau deposition. FMNL2 was also prominent in astroglia in AD among those with cerebrovascular pathology. Amyloid toxicity in zebrafish increased fmnl2a expression in astroglia with detachment of astroglial end feet from blood vessels. Knockdown of fmnl2a prevented gliovascular remodeling, reduced microglial activity and enhanced amyloidosis. APP/PS1dE9 AD mice also displayed increased Fmnl2 expression and reduced the gliovascular contacts independent of the gliotic response. Based on this work, we propose that FMNL2 regulates pathology-dependent plasticity of the blood-brain-barrier by controlling gliovascular interactions and stimulating the clearance of extracellular aggregates. Therefore, in AD cerebrovascular risk factors promote cerebrovascular pathology which in turn, interacts with FMNL2 altering the normal astroglial-vascular mechanisms underlying the clearance of amyloid and tau increasing their deposition in brain.

Aging disrupts circadian gene regulation and function in macrophages.

Aging is characterized by an increased vulnerability to infection and the development of inflammatory diseases, such as atherosclerosis, frailty, cancer and neurodegeneration. Here, we find that aging is associated with the loss of diurnally rhythmic innate immune responses, including monocyte trafficking from bone marrow to blood, response to lipopolysaccharide and phagocytosis. This decline in homeostatic immune responses was associated with a striking disappearance of circadian gene transcription in aged compared to young tissue macrophages. Chromatin accessibility was significantly greater in young macrophages than in aged macrophages; however, this difference did not explain the loss of rhythmic gene transcription in aged macrophages. Rather, diurnal expression of Kruppel-like factor 4 (Klf4), a transcription factor (TF) well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages. Ablation of Klf4 expression abolished diurnal rhythms in phagocytic activity, recapitulating the effect of aging on macrophage phagocytosis. Examination of individuals harboring genetic variants of KLF4 revealed an association with age-dependent susceptibility to death caused by bacterial infection. Our results indicate that loss of rhythmic Klf4 expression in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie age-associated loss of protective immune responses.

An immune response characterizes early Alzheimer's disease pathology and subjective cognitive impairment in hydrocephalus biopsies.

Early Alzheimer's disease (AD) pathology can be found in cortical biopsies taken during shunt placement for Normal Pressure Hydrocephalus. This represents an opportunity to study early AD pathology in living patients. Here we report RNA-seq data on 106 cortical biopsies from this patient population. A restricted set of genes correlate with AD pathology in these biopsies, and co-expression network analysis demonstrates an evolution from microglial homeostasis to a disease-associated microglial phenotype in conjunction with increasing AD pathologic burden, along with a subset of additional astrocytic and neuronal genes that accompany these changes. Further analysis demonstrates that these correlations are driven by patients that report mild cognitive symptoms, despite similar levels of biopsy ß-amyloid and tau pathology in comparison to patients who report no cognitive symptoms. Taken together, these findings highlight a restricted set of microglial and non-microglial genes that correlate with early AD pathology in the setting of subjective cognitive decline.

Discovering sparse transcription factor codes for cell states and state transitions during development.

Computational analysis of gene expression to determine both the sequence of lineage choices made by multipotent cells and to identify the genes influencing these decisions is challenging. Here we discover a pattern in the expression levels of a sparse subset of genes among cell types in B- and T-cell developmental lineages that correlates with developmental topologies. We develop a statistical framework using this pattern to simultaneously infer lineage transitions and the genes that determine these relationships. We use this technique to reconstruct the early hematopoietic and intestinal developmental trees. We extend this framework to analyze single-cell RNA-seq data from early human cortical development, inferring a neocortical-hindbrain split in early progenitor cells and the key genes that could control this lineage decision. Our work allows us to simultaneously infer both the identity and lineage of cell types as well as a small set of key genes whose expression patterns reflect these relationships.

Hallmarks of molecular action of microtubule stabilizing agents: effects of epothilone B, ixabepilone, peloruside A, and laulimalide on microtubule conformation.

Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, whereas others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Recent studies from our laboratory utilizing hydrogen-deuterium exchange in combination with mass spectrometry (MS) provide new information on the conformational effects of Taxol and discodermolide on microtubules isolated from chicken erythrocytes (CET). We report here a comprehensive analysis of the effects of epothilone B, ixabepilone (IXEMPRA(TM)), laulimalide, and peloruside A on CET conformation. The results of our comparative hydrogen-deuterium exchange MS studies indicate that all MSAs have significant conformational effects on the C-terminal H12 helix of α-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent αß-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, the lateral interactions between the adjacent protofilaments in CET are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site binding drugs are used.

Distinct pose of discodermolide in taxol binding pocket drives a complementary mode of microtubule stabilization.

The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.