Spatial proteomics reveals human microglial states shaped by anatomy and neuropathology.

Microglia are implicated in aging, neurodegeneration, and Alzheimer's disease (AD). Traditional, low-plex, imaging methods fall short of capturing in situ cellular states and interactions in the human brain. We utilized Multiplexed Ion Beam Imaging (MIBI) and data-driven analysis to spatially map proteomic cellular states and niches in healthy human brain, identifying a spectrum of microglial profiles, called the microglial state continuum (MSC). The MSC ranged from senescent-like to active proteomic states that were skewed across large brain regions and compartmentalized locally according to their immediate microenvironment. While more active microglial states were proximal to amyloid plaques, globally, microglia significantly shifted towards a, presumably, dysfunctional low MSC in the AD hippocampus, as confirmed in an independent cohort (n=26). This provides an in situ single cell framework for mapping human microglial states along a continuous, shifting existence that is differentially enriched between healthy brain regions and disease, reinforcing differential microglial functions overall.

Expanded vacuum-stable gels for multiplexed high-resolution spatial histopathology.

Cellular organization and functions encompass multiple scales in vivo. Emerging high-plex imaging technologies are limited in resolving subcellular biomolecular features. Expansion Microscopy (ExM) and related techniques physically expand samples for enhanced spatial resolution, but are challenging to be combined with high-plex imaging technologies to enable integrative multiscaled tissue biology insights. Here, we introduce Expand and comPRESS hydrOgels (ExPRESSO), an ExM framework that allows high-plex protein staining, physical expansion, and removal of water, while retaining the lateral tissue expansion. We demonstrate ExPRESSO imaging of archival clinical tissue samples on Multiplexed Ion Beam Imaging and Imaging Mass Cytometry platforms, with detection capabilities of > 40 markers. Application of ExPRESSO on archival human lymphoid and brain tissues resolved tissue architecture at the subcellular level, particularly that of the blood-brain barrier. ExPRESSO hence provides a platform for extending the analysis compatibility of hydrogel-expanded biospecimens to mass spectrometry, with minimal modifications to protocols and instrumentation.

Single-cell spatial proteomic imaging for human neuropathology.

Neurodegenerative disorders are characterized by phenotypic changes and hallmark proteopathies. Quantifying these in archival human brain tissues remains indispensable for validating animal models and understanding disease mechanisms. We present a framework for nanometer-scale, spatial proteomics with multiplex ion beam imaging (MIBI) for capturing neuropathological features. MIBI facilitated simultaneous, quantitative imaging of 36 proteins on archival human hippocampus from individuals spanning cognitively normal to dementia. Customized analysis strategies identified cell types and proteopathies in the hippocampus across stages of Alzheimer's disease (AD) neuropathologic change. We show microglia-pathologic tau interactions in hippocampal CA1 subfield in AD dementia. Data driven, sample independent creation of spatial proteomic regions identified persistent neurons in pathologic tau neighborhoods expressing mitochondrial protein MFN2, regardless of cognitive status, suggesting a survival advantage. Our study revealed unique insights from multiplexed imaging and data-driven approaches for neuropathologic analysis and serves broadly as a methodology for spatial proteomic analysis of archival human neuropathology. TEASER: Multiplex Ion beam Imaging enables deep spatial phenotyping of human neuropathology-associated cellular and disease features.

Multiplexed Ion Beam Imaging: Insights into Pathobiology.

Next-generation tools for multiplexed imaging have driven a new wave of innovation in understanding how single-cell function and tissue structure are interrelated. In previous work, we developed multiplexed ion beam imaging by time of flight, a highly multiplexed platform that uses secondary ion mass spectrometry to image dozens of antibodies tagged with metal reporters. As instrument throughput has increased, the breadth and depth of imaging data have increased as well. To extract meaningful information from these data, we have developed tools for cell identification, cell classification, and spatial analysis. In this review, we discuss these tools and provide examples of their application in various contexts, including ductal carcinoma in situ, tuberculosis, and Alzheimer's disease. We hope the synergy between multiplexed imaging and automated image analysis will drive a new era in anatomic pathology and personalized medicine wherein quantitative spatial signatures are used routinely for more accurate diagnosis, prognosis, and therapeutic selection.

Single-synapse analyses of Alzheimer's disease implicate pathologic tau, DJ1, CD47, and ApoE.

Synaptic molecular characterization is limited for Alzheimer's disease (AD). Our newly invented mass cytometry­based method, synaptometry by time of flight (SynTOF), was used to measure 38 antibody probes in approximately 17 million single-synapse events from human brains without pathologic change or with pure AD or Lewy body disease (LBD), nonhuman primates (NHPs), and PS/APP mice. Synaptic molecular integrity in humans and NHP was similar. Although not detected in human synapses, Aß was in PS/APP mice single-synapse events. Clustering and pattern identification of human synapses showed expected disease-specific differences, like increased hippocampal pathologic tau in AD and reduced caudate dopamine transporter in LBD, and revealed previously unidentified findings including increased hippocampal CD47 and lowered DJ1 in AD and higher ApoE in AD with dementia. Our results were independently supported by multiplex ion beam imaging of intact tissue. This highlights the higher depth and breadth of insight on neurodegenerative diseases obtainable through SynTOF.

MIBI-TOF: A multiplexed imaging platform relates cellular phenotypes and tissue structure.

Understanding tissue structure and function requires tools that quantify the expression of multiple proteins while preserving spatial information. Here, we describe MIBI-TOF (multiplexed ion beam imaging by time of flight), an instrument that uses bright ion sources and orthogonal time-of-flight mass spectrometry to image metal-tagged antibodies at subcellular resolution in clinical tissue sections. We demonstrate quantitative, full periodic table coverage across a five-log dynamic range, imaging 36 labeled antibodies simultaneously with histochemical stains and endogenous elements. We image fields of view up to 800 µm × 800 µm at resolutions down to 260 nm with sensitivities approaching single-molecule detection. We leverage these properties to interrogate intrapatient heterogeneity in tumor organization in triple-negative breast cancer, revealing regional variability in tumor cell phenotypes in contrast to a structured immune response. Given its versatility and sample back-compatibility, MIBI-TOF is positioned to leverage existing annotated, archival tissue cohorts to explore emerging questions in cancer, immunology, and neurobiology.

Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency.

Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome-specific mouse and human iPS cell lines with known mutations in MECP2.

Noncanonical Wnt signaling orchestrates early developmental events toward hematopoietic cell fate from human embryonic stem cells.

During human development, signals that govern lineage specification versus expansion of cells committed to a cell fate are poorly understood. We demonstrate that activation of canonical Wnt signaling by Wnt3a promotes proliferation of human embryonic stem cells (hESCs)--precursors already committed to the hematopoietic lineage. In contrast, noncanonical Wnt signals, activated by Wnt11, control exit from the pluripotent state and entry toward mesoderm specification. Unique to embryoid body (EB) formation of hESCs, Wnt11 induces development and arrangement of cells expressing Brachyury that coexpress E-cadherin and Frizzled-7 (Fzd7). Knockdown of Fzd7 expression blocks Wnt11-dependent specification. Our study reveals an unappreciated role for noncanonical Wnt signaling in hESC specification that involves development of unique mesoderm precursors via morphogenic organization within human EBs.

OP9 stroma augments survival of hematopoietic precursors and progenitors during hematopoietic differentiation from human embryonic stem cells.

The cellular mechanism and target cell affected by stromal microenvironments in augmenting hematopoietic specification from pluripotent human embryonic stem cells (hESCs) has yet to be evaluated. Here, in contrast to aorta-gonad-mesonephros-derived S62 stromal cells, OP9 cells inhibit apoptosis and also augment the proliferation of hemogenic precursors prospectively isolated from human embryoid bodies. In addition, OP9 stroma supported cells within the primitive hematopoietic compartment by inhibiting apoptosis of CD45(+)CD34(+) cells committed to the hematopoietic lineage, but have no effect on more mature blood (CD45(+)CD34(-)) cells. Inability of hESC-derived hematopoietic cells cocultured with OP9 stromal cells to engraft in both the adult and newborn NOD/SCID mice after intrafemoral and intrahepatic injection illustrated that although OP9 stromal cells augment hESC-derived hematopoiesis and progenitor output, this optimized environment does not confer or augment repopulating function of specified hematopoietic cells derived from hESCs. OP9 coculture also increases hematopoietic progenitors output from hemogenic precursors overexpressing HOXB4. Our study demonstrates that OP9 cells support both hemogenic precursors and their primitive hematopoietic progeny, thereby providing the first evidence toward understanding the cellular targets and mechanisms underlying the capacity of OP9 stromal cells to support hematopoiesis from ESCs and define the future steps required to achieve the global goal of generating bona fide human hematopoietic stem cells from ESC lines. Disclosure of potential conflicts of interest is found at the end of this article.

IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro.

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.

Regulation of Na v channels in sensory neurons.

Voltage-gated Na(+) channels have an essential role in the biophysical properties of nociceptive neurons. Factors that regulate Na(+) channel function are of interest from both pathophysiological and therapeutic perspectives. Increasing evidence indicates that changes in expression or inappropriate modulation of these channels leads to electrical instability of the cell membrane and the inappropriate spontaneous activity that is observed following nerve injury, and that this might contribute to neuropathic pain. The role of Na(v) channels in nociception depends on modulation by factors such as auxiliary beta-subunits, cytoskeletal proteins and the phosphorylation state of neurons. In this review we describe the modulation of Na(v) channels on sensory neurons by auxiliary beta-subunits, protein kinases and cytoskeletal proteins.

Role of auxiliary beta1-, beta2-, and beta3-subunits and their interaction with Na(v)1.8 voltage-gated sodium channel.

The nociceptive C-fibers of the dorsal root ganglion express several sodium channel isoforms that associate with one or more regulatory beta-subunits (beta1-beta4). To determine the effects of individual and combinations of the beta-subunit isoforms, we co-expressed Nav1.8 in combination with these beta-subunits in Xenopus oocytes. Whole-cell inward sodium currents were recorded using the two-microelectrode voltage clamp method. Our studies revealed that the co-expression beta1 alone or in combination with other beta-subunits enhanced current amplitudes, accelerated current decay kinetics, and negatively shifted the steady-state curves. In contrast, beta2 alone and in combination with beta1 altered steady-state inactivation of Nav1.8 to more depolarized potentials. Co-expression of beta3 shifted steady-state inactivation to more depolarized potentials; however, combined beta1beta3 expression caused no shift in channel availability. The results in this study suggest that the functional behavior of Nav1.8 will vary depending on the type of beta-subunit that expressed under normal and disease states.

The C-terminal region as a modulator of rNa(v)1.7 and rNa(v)1.8 expression levels.

Mammalian cells poorly express rNa(v)1.8 channels. In contrast, rNa(v)1.7 dorsal root ganglion channels have 90-fold higher peak Na(+) current densities. We investigated the role of rNa(v)1.7 and rNa(v)1.8 carboxy-termini in modulating the expression of rNa(v)1.7 and rNa(v)1.8 channels in tsA201 cells. Mutations in the ubiquitination site of the C-terminus did not improve rNa(v)1.8 current levels. However, rNa(v)1.8 chimeras containing the entire or the proximal portion of the rNa(v)1.7 C-terminus expressed 3.2-fold and 4.8-fold higher peak current densities, respectively, than parent rNa(v)1.8 channels. We conclude that the two Na(+) channels may have different endoplasmic reticulum processing signals.

Modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by protein kinase A and protein kinase C.

Voltage-gated Na+ channels (VGSC) are transmembrane proteins that are essential for the initiation and propagation of action potentials in neuronal excitability. Because neurons express a mixture of Na+ channel isoforms and protein kinase C (PKC) isozymes, the nature of which channel is being regulated by which PKC isozyme is not known. We showed that DRG VGSC Nav1.7 (TTX-sensitive) and Nav1.8 (TTX-resistant), expressed in Xenopus oocytes were differentially regulated by protein kinase A (PKA) and PKC isozymes using the two-electrode voltage-clamp method. PKA activation resulted in a dose-dependent potentiation of Nav1.8 currents and an attenuation of Nav1.7 currents. PKA-induced increases (Nav1.8) and decreases (Nav1.7) in peak currents were not associated with shifts in voltage-dependent activation or inactivation. The PKA-mediated increase in Nav1.8 current amplitude was prevented by chloroquine, suggesting that cell trafficking may contribute to the changes in Nav1.8 current amplitudes. A dose-dependent decrease in Nav1.7 and Nav1.8 currents was observed with the PKC activators phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate. PMA induced shifts in the steady-state activation of Nav1.7 and Nav1.8 channels by 6.5 and 14 mV, respectively, in the depolarizing direction. The role of individual PKC isozymes in the regulation of Nav1.7 and Nav1.8 was determined using PKC-isozyme-specific peptide activators and inhibitors. The decrease in the Nav1.8 peak current induced by PMA was prevented by a specific epsilonPKC isozyme peptide antagonist, whereas the PMA effect on Nav1.7 was prevented by epsilonPKC and betaIIPKC peptide inhibitors. The data showed that Nav1.7 and Nav1.8 were differentially modulated by PKA and PKC. This is the first report demonstrating a functional role for epsilonPKC and betaIIPKC in the regulation of Nav1.7 and Nav1.8 Na+ channels. Identification of the particular PKC isozymes(s) that mediate the regulation of Na+ channels is essential for understanding the molecular mechanism involved in neuronal ion channel regulation in normal and pathological conditions.

A tryptophan residue (W736) in the amino-terminus of the P-segment of domain II is involved in pore formation in Na(v)1.4 voltage-gated sodium channels.

Voltage-gated Na channels comprise four homologous domains each consisting of six transmembrane segments (S1-S6) linked by loops. The linkers between segments S5 and S6 in each domain (P-loops), denoted as SS1-SS2, form the pore of the channel. It is believed that the SS1 region of the P-loops dips into, while the SS2 region exits out of the membrane. We have reported previously that residues A728 and D730 (in SS1 of domain II) contribute to the external vestibule of the pore of the rat skeletal muscle Na channel (Na(v)1.4). In this study, we examined the role of a conserved neighbouring tryptophan residue at position 736 (W736) in the pore formation. The W736 residue of Na(v)1.4 was replaced by a cysteine using site-directed mutagenesis. Complementary RNAs encoding the wild-type and mutant channels were injected into Xenopus laevis oocytes and macroscopic Na(+) currents measured using the two-microelectrode voltage-clamp technique. The W736C mutant showed increased channel sensitivity to externally applied Cd(2+) and methanethiosulphonate-ethyltrimethylammonium (MTSET). Furthermore, micromolar concentrations of Cd(2+) reduced single-channel current amplitude in the Na(v)1.4/W736C mutant without affecting its voltage dependence. However, only small differences in tetrodotoxin and micro-conotoxin GIIIA affinity were observed between the wild-type and mutant channels. Replacing Na(+) with other cations - K(+), Li(+), Cs(+) or NH(4)(+) - did not change the ion permeation sequence of the Na(v)1.4/W736C mutant channel. The results suggest that W736 contributes to the formation of the pore, close to the mouth of the channel, but is not part of the selectivity filter.