Microglia, the brain’s resident macrophages, are dynamic CNS custodians with surprising origins in the extra-embryonic yolk sac. The consequences of their distinct ontogeny are unknown but critical ...to understanding and treating brain diseases. We created a brain macrophage transplantation system to disentangle how environment and ontogeny specify microglial identity. We find that donor cells extensively engraft in the CNS of microglia-deficient mice, and even after exposure to a cell culture environment, microglia fully regain their identity when returned to the CNS. Though transplanted macrophages from multiple tissues can express microglial genes in the brain, only those of yolk-sac origin fully attain microglial identity. Transplanted macrophages of inappropriate origin, including primary human cells in a humanized host, express disease-associated genes and specific ontogeny markers. Through brain macrophage transplantation, we discover new principles of microglial identity that have broad applications to the study of disease and development of myeloid cell therapies.
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•Brain signals induce and sustain homeostatic gene expression in microglia•Hematopoietic stem cell (HSC)-derived macrophages attain a microglia-like identity•Stable markers and gene signatures betray HSC origin•Macrophages with HSC origin markers are found in human neurodegeneration
Bennett et al. create a macrophage transplantation system to measure how origin and brain environment contribute to microglial identity. Although diverse macrophage types survive in the brain, only those sharing developmental origins with microglia express microglial genes normally.
We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass ...spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.
The combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS) and latest-generation mass spectrometry instrumentation provides dramatically improved single-cell proteome profiling.
The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the ...role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267-337). Deletion of a BECN1 ECD subregion (amino acids 285-299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.
Neuritic retraction represents a prominent feature of the degenerative phenotype associated with mutations in leucine rich repeat kinase 2 (LRRK2) that are implicated in autosomal dominant and some ...cases of sporadic Parkinson's disease. Alterations in macroautophagy, the vacuolar catabolism of cytoplasmic constituents, have been described in Parkinson's disease. In this study, we utilized retinoic-acid differentiated SH-SY5Y cells to determine whether autophagy contributes to mutant LRRK2-associated neurite degeneration. Transfection of pre-differentiated SH-SY5Y cells with LRRK2 cDNA containing the common G2019S mutation resulted in significant decreases in neurite length, which were not observed in cells transfected with wild type LRRK2 or its kinase-dead K1906M mutation. G2019S LRRK2 transfected cells also exhibited striking increases in autophagic vacuoles in both neuritic and somatic compartments, as demonstrated by fluorescence and western blot analysis of the autophagy marker green fluorescent protein-tagged microtubule-associated protein Light Chain 3 and by transmission electron microscopy. RNA interference knockdown of LC3 or Atg7, two essential components of the conserved autophagy machinery, reversed the effects of G2019S LRRK2 expression on neuronal process length, whereas rapamycin potentiated these effects. The mitogen activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis2-aminophenylthiobutadiene (U0126) reduced LRRK2-induced neuritic autophagy and neurite shortening, implicating MAPK/ERK-related signaling. These results indicate an active role for autophagy in neurite remodeling induced by pathogenic mutation of LRRK2.
Introduction
Maximizing extent of surgical resection with the least morbidity remains critical for survival in glioblastoma patients, and we hypothesize that it can be improved by enhancements in ...intraoperative tumor detection. In a clinical study, we determined if therapeutic antibodies could be repurposed for intraoperative imaging during resection.
Methods
Fluorescently labeled cetuximab-IRDye800 was systemically administered to three patients 2 days prior to surgery. Near-infrared fluorescence imaging of tumor and histologically negative peri-tumoral tissue was performed intraoperatively and ex vivo. Fluorescence was measured as mean fluorescence intensity (MFI), and tumor-to-background ratios (TBRs) were calculated by comparing MFIs of tumor and histologically uninvolved tissue.
Results
The mean TBR was significantly higher in tumor tissue of contrast-enhancing (CE) tumors on preoperative imaging (4.0 ± 0.5) compared to non-CE tumors (1.2 ± 0.3; p = 0.02). The TBR was higher at a 100 mg dose than at 50 mg (4.3 vs. 3.6). The smallest detectable tumor volume in a closed-field setting was 70 mg with 50 mg of dye and 10 mg with 100 mg. On sections of paraffin embedded tissues, fluorescence positively correlated with histological evidence of tumor. Sensitivity and specificity of tumor fluorescence for viable tumor detection was calculated and fluorescence was found to be highly sensitive (73.0% for 50 mg dose, 98.2% for 100 mg dose) and specific (66.3% for 50 mg dose, 69.8% for 100 mg dose) for viable tumor tissue in CE tumors while normal peri-tumoral tissue showed minimal fluorescence.
Conclusion
This first-in-human study demonstrates the feasibility and safety of antibody based imaging for CE glioblastomas.
Abstract The past decade in Parkinson's disease (PD) research has been punctuated by numerous advances in understanding genetic factors that contribute to the disease. Common to most of the genetic ...models of Parkinsonian neurodegeneration are pathologic mechanisms of mitochondrial dysfunction, secretory vesicle dysfunction and oxidative stress that likely trigger common cell death mechanisms. Whereas presynaptic function is implicated in the function/dysfunction of α-synuclein, the first gene shown to contribute to PD, synaptic function has not comprised a major focus in most other genetic models. However, recent advances in understanding the impact of mutations in parkin and LRRK2 have also yielded insights into synaptic dysfunction as a possible early pathogenic mechanism. Autophagy is a common neuronal response in each of these genetic models of PD, participating in the clearance of protein aggregates and injured mitochondria. However, the potential consequences of autophagy upregulation on synaptic structure and function remain unknown. In this review, we discuss the evidence that supports a role for synaptic dysfunction in the neurodegenerative cascade in PD, and highlight unresolved questions concerning a potential role for autophagy in either pathological or compensatory synaptic remodeling. This article is part of a Special Issue entitled “Autophagy and protein degradation in neurological diseases.”
Amyloid beta (Aβ) plaque is a defining pathologic feature of Alzheimer disease (AD). Aducanumab, a monoclonal IgG1 that selectively binds aggregated species of Aβ, has been shown by amyloid positron ...emission tomography (Amyloid PET) to reduce Aβ plaques in patients with prodromal and mild AD. This is the first autopsy report of the AD neuropathology in a patient previously treated with aducanumab. The patient was an 84-year-old woman who was randomized to the placebo arm of the PRIME Phase 1b study (221AD103). The patient progressed to moderate dementia (MMSE = 14/30), beyond the targeted early AD treatment stage, before receiving aducanumab in the long-term extension (LTE). The patient then received 32 monthly doses of aducanumab, titrated up to 6 mg/kg, for a cumulative dose of 186 mg/kg. In the LTE, Amyloid PET scans demonstrated robust Aβ plaque reduction, from a composite standard uptake value ratio (SUVR) of 1.5 at screening to < 1.1 at 56 weeks post-aducanumab dosing. MRI examinations were negative for amyloid-related imaging abnormalities (ARIA). She passed away in hospice care 4 months after her last dose of aducanumab. The postmortem neuropathologic examination confirmed AD neuropathologic changes. Aβ and IBA1 immunohistochemistry assays demonstrated sparse residual Aβ plaque engaged by amoeboid reactive microglia. Phospho-Tau (pTau) immunohistochemistry demonstrated neocortical neurofibrillary degeneration (Braak stage V, NIA/AA Stage B3). However, the density of pTau neuropathology, including neuritic plaque pTau (NP-Tau), appeared lower in the PRIME LTE Patient compared to a reference cohort of untreated Braak stage V–VI, NIA/AA Stage B3 AD cases. Taken together, this case report is the first to provide Amyloid PET and neuropathologic evidence substantiating the impact of aducanumab to reduce Aβ plaque neuropathology in a patient with AD. Furthermore, this report underscores the critical importance of autopsy neuropathology studies to augment our understanding of aducanumab’s mechanism of action and impact on AD biomarkers.
Parkinson's disease is the second most common neurodegenerative disease after Alzheimer's disease and affects 1% of the population above 60 years old. Although Parkinson's disease commonly manifests ...with motor symptoms, a majority of patients with Parkinson's disease subsequently develop cognitive impairment, which often progresses to dementia, a major cause of morbidity and disability. Parkinson's disease is characterized by α-synuclein accumulation that frequently associates with amyloid-β and tau fibrils, the hallmarks of Alzheimer's disease neuropathological changes; this co-occurrence suggests that onset of cognitive decline in Parkinson's disease may be associated with appearance of pathological amyloid-β and/or tau. Recent studies have highlighted the appearance of the soluble form of the triggering receptor expressed on myeloid cells 2 (sTREM2) receptor in CSF during development of Alzheimer's disease. Given the known association of microglial activation with advancing Parkinson's disease, we investigated whether CSF and/or plasma sTREM2 differed between CSF biomarker-defined Parkinson's disease participant subgroups. In this cross-sectional study, we examined 165 participants consisting of 17 cognitively normal elderly subjects, 45 patients with Parkinson's disease with no cognitive impairment, 86 with mild cognitive impairment, and 17 with dementia. Stratification of subjects by CSF amyloid-β and tau levels revealed that CSF sTREM2 concentrations were elevated in Parkinson's disease subgroups with a positive tau CSF biomarker signature, but not in Parkinson's disease subgroups with a positive CSF amyloid-β biomarker signature. These findings indicate that CSF sTREM2 could serve as a surrogate immune biomarker of neuronal injury in Parkinson's disease.
In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the potential to worsen damage and/or to assist in healing. We define the heterogeneity of ...microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2−/− mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2−/− TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets.
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•TBI elevates distinct phenotypes of microglia, macrophages, and dendritic cells•Ccr2 deficiency alters cell proportions and reduces ISG expression in microglia•TBI induces crosstalk between microglia and circulating monocytes•Preclinical translational studies to target human CCR2 after TBI improves outcomes
By single-cell RNA sequencing of traumatically injured and normal brains from wild-type and Ccr2−/− mice, Somebang et al. define microglia, macrophage, and dendritic cell phenotypes in TBI. Targeting mouse and/or human CCR2 reduces specific TBI brain CNS myeloid compartments, dampens type I interferon responses, and improves cognition after TBI.
A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN proteins. Here, we leverage laser ...capture microdissection and nanoPOTS single-cell mass spectrometry-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control tissues. In a follow-up analysis, we examine the impact of stratification of MNs based on cytoplasmic transactive response DNA-binding protein 43 (TDP-43)+ inclusion pathology on the profiles of 2,238 proteins. We report extensive overlap in differentially abundant proteins identified in ALS MNs with or without overt TDP-43 pathology, suggesting early and sustained dysregulation of cellular respiration, mRNA splicing, translation, and vesicular transport in ALS. Together, these data provide insights into proteome-level changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein dynamics in human neurologic diseases.
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•Single-cell proteomic analysis of human ALS motor neurons directly captured by LMD•ALS motor neuron deficiencies in splicing, translation, and vesicular transport machinery•Stratification of single-neuron proteomes based on pathologic TDP-43 inclusions•Utility of trace sample proteomics in augmenting the study of human neurological diseases
Guise and Misal et al. report the unbiased single-cell proteomic analysis of ALS motor neurons directly captured from human tissues to explore disease-associated protein dynamics across TDP43-pathological strata. This study highlights both important technical challenges accompanying single-cell omics studies and emerging avenues for exploring human disease biology with nanoPOTS.
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