Markers For Subpopulations Research Articles

Muscat detects subpopulation-specific state transitions from multi-sample multi-condition single-cell transcriptomics data

Single-cell RNA sequencing (scRNA-seq) has become an empowering technology to profile the transcriptomes of individual cells on a large scale. Early analyses of differential expression have aimed at identifying differences between subpopulations to identify subpopulation markers. More generally, such methods compare expression levels across sets of cells, thus leading to cross-condition analyses. Given the emergence of replicated multi-condition scRNA-seq datasets, an area of increasing focus is making sample-level inferences, termed here as differential state analysis; however, it is not clear which statistical framework best handles this situation. Here, we surveyed methods to perform cross-condition differential state analyses, including cell-level mixed models and methods based on aggregated pseudobulk data. To evaluate method performance, we developed a flexible simulation that mimics multi-sample scRNA-seq data. We analyzed scRNA-seq data from mouse cortex cells to uncover subpopulation-specific responses to lipopolysaccharide treatment, and provide robust tools for multi-condition analysis within the muscat R package.

TGF-β2 is a Prognostic Biomarker Correlated with Immune Cell Infiltration in Colorectal Cancer: A STROBE-compliant article.

Transforming growth factor-beta (TGF-β2) is an important cytokine regulating immune cell function. However, whether TGF-β2 controls the invasion of colorectal cancer (CRC) by immune cells is unknown. Therefore, we evaluated the expression of TGF-β2 using multiple databases and determined the relationship between TGF-β2 expression and tumor immune infiltration defined by a set of genetic markers. The analysis demonstrated that the expression of TGF-β2 is closely related to the outcome of many cancers, and this correlation was particularly strong in CRC. In addition, the increased expression of TGF-β2 was significantly associated with the expression of various markers of specific immune cell subpopulations, and overexpression of TGF-β2 was closely related to the prognosis of colon cancer patients. Moreover, TGF-β2 was related to the prognosis and infiltration of the tumor by immune cells in CRC patients. The obtained results indicate that TGF-β2 is a critical factor regulating the recruitment of immune cells and controls their infiltration into colorectal tumors. Thus, high expression of TGF-β2 not only facilitates the prognosis in CRC patients, but also may provide a new target for the treatment of CRC.

Abstract 2757: Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most incurable types of cancer. Cancer that develops in the acinar cells of the pancreas is typically not diagnosed until later stages, such as stage 3 or 4. As such, this form of cancer is particularly lethal with only 9% of patients reaching 5-year survival. PDAC is known to have a particularly dense extracellular matrix composed of fibroblasts, which have been previously shown to play an important role in promoting resistance to drug therapy. Characterization of the stromal networks involved in PDAC tumor development as well as protein markers of fibroblast subpopulations within the tumor stroma are critical to developing new fibroblast-targeted therapeutic approaches as well as understanding key signaling molecules that ultimately promote tumor progression and drug resistance. Single-cell RNA sequencing (scRNAseq) was utilized to analyze cancer-associated fibroblasts (CAFs) from KPC mouse-derived, MT3 subcutaneous murine allografts along with two fibroblast lines derived from human PDAC tumors: CRC-811 and IA-1340. Single cell gene expression profiling and subsequent analysis of MT3-derived CAFs resulted in the identification of three CAF subpopulations including a myofibroblast subpopulation that expressed high levels of smooth muscle actin (Acta2), which was also observed in both human samples. Fibroblast subpopulations enriched in Acta2 expression, expressed high levels of Wnt5a along with several other secreted factors including Tgfb1, Tgfb2 and Ctgf. Wnt5a is a secreted protein that activates non-canonical Wnt signaling pathways and is known to regulate normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However Wnt5a is not natively expressed in MT3 cancer cells derived from syngeneic tumors, but potentially in the stroma. We hypothesize it is exclusively derived from fibroblasts. Previously it has been shown that Wnt5a inhibition suppressed gastric cancer metastasis, therefore further validation of the role of myofibroblast-derived Wnt5a on PDAC disease progression is warranted. This study received funding by LDRD 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM Release Number: LLNL-ABS-798442. Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas R. Hum, Stephen Byers, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2757.

Population Dynamics and Neuronal Polyploidy in the Developing Neocortex.

The mammalian neocortex is composed of different subtypes of projection neurons that are generated sequentially during embryogenesis by differentiation of neural progenitors. While molecular mechanisms that control neuronal production in the developing neocortex have been extensively studied, the dynamics and absolute numbers of the different progenitor and neuronal populations are still poorly characterized. Here, we describe a medium throughput approach based on flow cytometry and well-known identity markers of cortical subpopulations to collect quantitative data over the course of mouse neocortex development. We collected a complete dataset in a physiological developmental context on two progenitor and two neuron populations, including relative proportions and absolute numbers. Our study reveals unexpected total numbers of Tbr2+ progenitors. In addition, we show that polyploid neurons are present throughout neocortex development.

Abstract 455: The Roles of VEGF Receptors in Human Cardiac Progenitor Cell Contribution to New Vascular Formation

Background: Human cardiac progenitor cells (CPCs) have been shown to play a valuable role in myocardial tissue maintenance, including their ability to develop into endothelial and vascular smooth muscle cells. The vascular endothelial growth factor (VEGF) ligand family has been identified as essential for angiogenesis. VEGF receptors (VEGFRs) comprise three main subtypes, VEGFRs1,2,3; our prior data identified CPC expression of both VEGFRs and of pro-angiogenic secreted growth factors. Hypothesis: Human CPCs utilise VEGFR signalling to potentiate CPC-driven angiogenesis, directly via CPC differentiation and indirectly via secrotome. Methods: Human adult myocardial tissue samples were collected during cardiac surgery and c-Kit-positive (c-Kit + ), CD45-negative (CD45 - ) CPCs isolated by immunomagnetic bead sorting, with five CPC lines generated from individual-donor samples. The c-Kit + / CD45 - CPC population was then characterised by clonogenicity assay, immunocytochemistry (ICC), and real-time RT-qPCR. Human CPC lines were FACS sorted into 3 lineage groups: endothelial (CD31 + ), smooth muscle (CD91 + /CD140b + /CD31 - ) and uncommitted (CD91 - /CD140b - /CD31 - ). Expression of VEGFRs and markers (SDF1; TGF-β) in CPC sub-populations was assessed by: qPCR; Western blot; ICC. Impact on signal transduction by VEGF-A stimulation was identified by Western blot and ICC. Results: Human CPCs were sorted into populations of: endothelial linage CD31 + (1.26% of total cells), smooth muscle lineage CD91 + /CD140b + /CD31 - (13.77%) and CD91 - /CD140b - /CD31 - (31.28%) cells. Analysis of gene expression identified VEGFRs 1, 2 and 3 in all three sub-populations, but only VEGFR1 expression was confirmed at protein level, seen in all three sub-populations. High expression levels of growth factors secreted by CPCs (SDF, TGF-β, VEGFs, FGF-2) were identified in human CPCs, also in all three sub-populations. Conclusion: Human CPC lines were isolated and analysed in bulk and sub-populations, identifying VEGFR1 expression at both gene and protein levels, but not VEGFR2 and VEGFR3. Our further work will identify signalling pathways in human CPCs linked to VEGF-A stimulation, along with the impact of VEFG-A stimulation on CPC secretome and linked angiogenic potential.

The BCMA:Tomato Mouse – a new reporter to characterize long-lived plasma cells

Abstract Plasma cells provide humoral protection by secreting large amounts of antibodies. The heterogeneity of the plasma cell compartment has recently been demonstrated by functional studies and subpopulation markers, but the distinct properties of long-lived plasma cells (LLPC) are still unresolved. B Cell Maturation Antigen (BCMA) has been described as an essential receptor for LLPC survival. To elucidate the mechanisms of BCMA-mediated survival we established a reporter mouse line that expresses a tdTomato fluorescent protein together with BCMA under the control of the promoter of Tnfrsf17, the gene that encodes BCMA. In this reporter model, we could demonstrate the restricted expression of Tomato and BCMA in plasma cells making this an ideal system for the identification and tracking of plasma cells. Importantly, we observed an increase in the abundance of BCMA:Tomato along with a gradual loss of CD19 expression, delineating a developmental progression from plasmablasts to mature plasma cell subsets. In contrast, plasmablasts generated in vitro display only minimal BCMA induction, confirming the independence of BCMA expression from the Blimp1 master regulator of PC differentiation. In summary, we have developed a new plasma cell-specific reporter model that, together with an inducible BCMA-KO mouse, will allow us to investigate the BCMA-dependent signals of the generation and maintenance of LLPC. This work was supported, in part, by the Deutsche Forschungsgemeinschaft (DFG) through research grants TRR130 to H.-M.J. and W.S. and GRK1660 to H.-M.J.

Targeting AXL-JAK1 in tumor and immune ecosystem of lung cancer

Abstract Intercellular crosstalk between lung adenocarcinoma cells (LACs) and tumor-associated macrophages (TAMs) has been implicated in promoting cancer metastasis; however, how oncogenic signaling networks corroborate in this tumorigenic process remains unexplored. Our findings suggest that coordinated activation of AXL and JAK1-pSTAT3 signaling engenders a vicious cycle of dependence between LACs and TAMs for tumor progression. AXL-overexpressing LACs polarize TAMs to an M2-like phenotype in a JAK1-STAT3-dependent manner, facilitating the development of a proinflammatory tumor microenvironment. Conversely, M2-like TAMs release Gas6 ligand to enhance AXL-mediated cancer stemness and epithelial-to-mesenchymal transition (EMT) in LACs. Cytometry by time-of-flight (CyTOF) is a single-cell detection technology that allows for measurement of multiple protein markers in diverse cell subpopulations. We designed a 34-antibody CyTOF panel to identify immune cells, stromal cells, epithelial cells, oncogenic signaling, stemness and EMT components for LAC tumor profiling. CyTOF profiling of ~642,241 single cells detected 55 subpopulations among LAC cell lines (n=4 with co-culture conditions) and lung tumors (n=12), which were further differentiated based on their cell types (LACs, TAMs, stromal cells, granulocytes, natural killer, B and T lymphocytes). High AXL expression was noticed in tumor cell subpopulation while high pSTAT3 expression was detected in the TAM subpopulation. Moreover, pSTAT3 expression levels were increased in U937 differentiated macrophage after co-culture with A549 cells. This novel platform may facilitate the optimal selection of lung cancer patients for dual targeting of AXL and JAK1-pSTAT3.

Neural Stem Cell Transplantation for Neurodegenerative Diseases.

Neurodegenerative diseases are disabling and fatal neurological disorders that currently lack effective treatment. Neural stem cell (NSC) transplantation has been studied as a potential therapeutic approach and appears to exert a beneficial effect against neurodegeneration via different mechanisms, such as the production of neurotrophic factors, decreased neuroinflammation, enhanced neuronal plasticity and cell replacement. Thus, NSC transplantation may represent an effective therapeutic strategy. To exploit NSCs’ potential, some of their essential biological characteristics must be thoroughly investigated, including the specific markers for NSC subpopulations, to allow profiling and selection. Another key feature is their secretome, which is responsible for the regulation of intercellular communication, neuroprotection, and immunomodulation. In addition, NSCs must properly migrate into the central nervous system (CNS) and integrate into host neuronal circuits, enhancing neuroplasticity. Understanding and modulating these aspects can allow us to further exploit the therapeutic potential of NSCs. Recent progress in gene editing and cellular engineering techniques has opened up the possibility of modifying NSCs to express select candidate molecules to further enhance their therapeutic effects. This review summarizes current knowledge regarding these aspects, promoting the development of stem cell therapies that could be applied safely and effectively in clinical settings.

Long-term effects of acute cadmium exposure on testis immune privilege

Cadmium (Cd) is a widespread and non-biodegradable pollutant of great concern to human health. This element can affect cellular signal transduction and cell-to-cell interaction in the testis. Immune tolerance towards auto- and alloantigens is an important component of testis immunity. It is involved in spermatogenesis and hormone secretion. Plus, the immune tolerance may help to reveal the changes in testis immunity over a long period after Cd exposure. The current research was aimed at investigating the long-term effects of acute Cd exposure on testis immunity by means of elicitation of testicular immune cell composition shift induced by Cd. Cadmium chloride was intraperitoneally injected at 3 mg Cd/kg to mice. After that testis interstitial cells were stained with surface markers for leukocyte and lymphocyte subpopulations (CD45, CD11b, CD3, CD4, CD8, CD25) and analyzed cytofluorimetrically by week 4, 6, 8 and 12 after Cd administration (Cd group). To identify the delayed effects of cadmium on immune tolerance two groups of animals were subjected to intratesticular allotransplantation of neonatal testis (groups ITT and Cd+ ITT). One of the groups was administered with Cd four weeks before the transplantation (Cd+ITT group). I group served as a control that did not undergo any transplantation or Cd injection. For a better demonstration of the phenomenon of immunological tolerance of the testicles, an additional group (UKT group) was used which got grafts under the kidney capsule (non-immune privileged site).Investigation of the cell population showed that CD45+, CD11b+, CD4+, CD8+ cells were permanently present in testicular interstitial tissue in I group. Intratesticular testis transplantation increased the proportion of CD11b+ but did not have such a pronounced effect on CD8+ cells in ITT group. Moreover, the transplantation elevated CD4+ CD25+ cells known for their immunosuppressive property and promoted graft development by week 2 (histological data). Cd injection resulted in severe inflammation that quenched by week 4 (Cd and Cd+ ITT groups). This time point was chosen for transplantation in Cd+ ITT group. Such Cd pretreatment led to a high CD8+ cell proportion and to the delayed appearance of CD4+ CD25+ cells by week 2 (Cd+ ITTgroup). The finding is consistent with the impairment of graft development in Cd+ ITTgroup pretreated with Cd. Observation suggest that Cd pretreatment was associated with disproportion of interstitial immune cell populations which resulted in the impairment of immunoprotective function of the testis. The impairment of testis immunity showed itself only after several weeks of Cd administration, and only when the recipient testis immunity was provoked by alloantigens of donor testes.

Systematic analysis of expression signatures of neuronal subpopulations in the VTA

Gene expression profiling across various brain areas at the single-cell resolution enables the identification of molecular markers of neuronal subpopulations and comprehensive characterization of their functional roles. Despite the scientific importance and experimental versatility, systematic methods to analyze such data have not been established yet. To this end, we developed a statistical approach based on in situ hybridization data in the Allen Brain Atlas and thereby identified specific genes for each type of neuron in the ventral tegmental area (VTA). This approach also allowed us to demarcate subregions within the VTA comprising specific neuronal subpopulations. We further identified WW domain-containing oxidoreductase as a molecular marker of a population of VTA neurons that co-express tyrosine hydroxylase and vesicular glutamate transporter 2, and confirmed their region-specific distribution by immunohistochemistry. The results demonstrate the utility of our analytical approach for uncovering expression signatures representing specific cell types and neuronal subpopulations enriched in a given brain area.

Etv1 Controls the Establishment of Non-overlapping Motor Innervation of Neighboring Facial Muscles during Development

SUMMARYThe somatotopic motor-neuron projections onto their cognate target muscles are essential for coordinated movement, but how that occurs for facial motor circuits, which have critical roles in respiratory and interactive behaviors, is poorly understood. We report extensive molecular heterogeneity in developing facial motor neurons in the mouse and identify markers of subnuclei and the motor pools innervating specific facial muscles. Facial subnuclei differentiate during migration to the ventral hindbrain, where neurons with progressively later birth dates—and evolutionarily more recent functions—settle in more-lateral positions. One subpopulation marker, ETV1, determines both positional and target muscle identity for neurons of the dorsolateral (DL) subnucleus. In Etv1 mutants, many markers of DL differentiation are lost, and individual motor pools project indifferently to their own and neighboring muscle targets. The resulting aberrant activation patterns are reminiscent of the facial synkinesis observed in humans after facial nerve injury.

Molecular, Morphological, and Functional Characterization of Corticotropin-Releasing Factor Receptor 1-Expressing Neurons in the Central Nucleus of the Amygdala.

The central nucleus of the amygdala (CeA) is a brain region implicated in anxiety, stress-related disorders and the reinforcing effects of drugs of abuse. Corticotropin-releasing factor (CRF, Crh) acting at cognate type 1 receptors (CRF1, Crhr1) modulates inhibitory and excitatory synaptic transmission in the CeA. Here, we used CRF1:GFP reporter mice to characterize the morphological, neurochemical and electrophysiological properties of CRF1-expressing (CRF1+) and CRF1-non-expressing (CRF1–) neurons in the CeA. We assessed these two neuronal populations for distinctions in the expression of GABAergic subpopulation markers and neuropeptides, dendritic spine density and morphology, and excitatory transmission. We observed that CeA CRF1+ neurons are GABAergic but do not segregate with calbindin (CB), calretinin (CR), parvalbumin (PV), or protein kinase C-δ (PKCδ). Among the neuropeptides analyzed, Penk and Sst had the highest percentage of co-expression with Crhr1 in both the medial and lateral CeA subdivisions. Additionally, CeA CRF1+ neurons had a lower density of dendritic spines, which was offset by a higher proportion of mature spines compared to neighboring CRF1– neurons. Accordingly, there was no difference in basal spontaneous glutamatergic transmission between the two populations. Application of CRF increased overall vesicular glutamate release onto both CRF1+ and CRF1– neurons and does not affect amplitude or kinetics of EPSCs in either population. These novel data highlight important differences in the neurochemical make-up and morphology of CRF1+ compared to CRF1– neurons, which may have important implications for the transduction of CRF signaling in the CeA.

Transcriptome analysis of distinct coelomocyte populations in Strongylocentrotus purpuratus

Abstract Coelomocytes are important cellular mediators of immunity in echinoderms. In the purple sea urchin (Strongylocentrotus purpuratus) these cells can readily be divided into phagocytes, vibratile cells, red spherule cells, and colorless spherule cells based on their morphology. While some information has been reported about genes that are expressed in specific types of coelomocytes, a comprehensive analysis of the gene expression pattern in each cell type was not available. Using a density gradient coelomocytes from individual adult urchins were separated into three populations (phagocytes, a mixture of vibratile and colorless spherule cells, and red spherule cells), RNA was isolated, and next generation sequencing was employed to determine the transcriptome of each cell population. All three cell populations showed clear differences in their gene expression patterns, and the differences in transcript levels for selected genes that were found to be uniquely expressed in one cell type were confirmed by qRT-PCR using cDNA obtained from additional sea urchins. This bulk population data was then complemented by single-cell RNAseq to obtain information about suitable population and sub-population markers for subsequent studies of coelomocyte biology.

Calcium-Binding Proteins as Determinants of Central Nervous System Neuronal Vulnerability to Disease.

Neuronal subpopulations display differential vulnerabilities to disease, but the factors that determine their susceptibility are poorly understood. Toxic increases in intracellular calcium are a key factor in several neurodegenerative processes, with calcium-binding proteins providing an important first line of defense through their ability to buffer incoming calcium, allowing the neuron to quickly achieve homeostasis. Since neurons expressing different calcium-binding proteins have been reported to be differentially susceptible to degeneration, it can be hypothesized that rather than just serving as markers of different neuronal subpopulations, they might actually be a key determinant of survival. In this review, we will summarize some of the evidence that expression of the EF-hand calcium-binding proteins, calbindin, calretinin and parvalbumin, may influence the susceptibility of distinct neuronal subpopulations to disease processes.

OR12-2 Obesity Induces Changes in T Cell Metabolism and Function

Low-grade inflammation is responsible for many obesity-associated pathologies such as insulin resistance, type 2 diabetes and reduction in protective immunity. T cells play an important role in mounting an effective immune response and the modulation of inflammation, and have been identified as a key player in obesity-associated inflammation. Altered T cell metabolism can influence T cell differentiation and function; however, it is not yet known if or how obesity influences the T cell metabolic state. We hypothesized that diet induced obesity promotes changes in T cell metabolism leading to increased pro-inflammatory T cells function. To test our hypothesis, we assessed the effect of diet-induced obesity (DIO) on T cell subpopulation number and metabolism in the visceral adipose tissue (VAT) and peripheral lymphoid tissue (spleen). To achieve that, C57BL/6 mice were placed on high fat (DIO mice) or regular chow diet (control mice) at weaning and remained on diet for 18 weeks. VAT and spleen were collected and T cells were isolated and analyzed by flow cytometry for: T cells subpopulation markers, glucose uptake, and cytokine production. In addition, the expression of almost 240 enzymes involved in several metabolic pathways were measured in CD4+ T cells by RT-qPCR. We found that proportions of Treg cells were decreased in the VAT of DIO mice while CD8+ T cells increased, indicating an inflammatory environment in the adipose tissue of the DIO mice. CD4+ T cells isolated from the spleen of DIO mice were also found to be metabolically altered with increased glucose uptake and elevated oxygen consumption rate (OCR; a marker of mitochondrial metabolism). The spare respiratory capacity, an indicator of mitochondrial reserve, of CD4+ T cells from DIO mice was found to be reduced by the fatty acids oxidation inhibitor Etomoxir, while the pyruvate transport inhibitor UK5099 was found to have no significant effect, indicating a preference toward the use of fatty acids as a mitochondrial fuel source. The metabolic qPCR panel showed slight differences in expression between control vs. DIO mice with a tendency toward a decrease in the expression of enzymes involved in oxidative phosphorylation and fatty acid transport along with an increase in enzymes that positively influence glycolysis in DIO mice. Lastly, the antidiabetic drug metformin was found to inhibit OCR and alter the expression of several activation markers in CD4+ cells in vitro and in vivo, indicating that metformin may be an effective candidate to reverse T cell metabolic dysfunction and thereby restore T cell dysfunction in obesity. Research Support: National Institutes of Health grant R01-DK106090 and Derfner Foundation Research Award 2018.

IMMU-20. SINGLE CELL CYTOMICS OF PERIPHERAL BLOOD MONONUCLEAR CELLS REVEALS NEW AVENUES FOR GLIOMA IMMUNOTHERAPY

Glioblastoma is the most common primary brain tumor with a dismal prognosis and a lack of effective treatment options after relapse. Despite striking successes that were achieved in the last decade in other types of cancer, evidence of sustainable responses to T cell directed immunotherapy in glioblastoma remains limited. This might be explained by an emerging hypothesis that glioblastomas are cold tumours with poor lymphocyte infiltration. Therefore, some recent efforts have been focused on increasing cytotoxic lymphocyte infiltration. Here we present our workflow for subpopulation frequency, marker expression, and correlation network (INFERENCE) analysis, in this study applied to screen for possible new targets to enhance anti-glioblastoma immune responses. Using high-dimensional, single-cell mass cytometry (CyTOF) we profiled peripheral blood mononuclear cells derived from 160 patients with glioblastoma, lower grade glioma, or metastasis, meningioma and epilepsy as controls. Based on a CyTOF panel composed mainly of lymphocyte markers and check-point inhibition markers we could effectively dissect blood leukocytes into 100 phenotypically separate subsets of CD4 T cells (35 subsets), CD8 T cells (17 subsets), monocytes (12 subsets), NK cells (14 subsets), and B cells (15 subsets), and map their frequencies, phenotypes and correlation statistics. We thereby created an in depth human atlas of peripheral blood mononuclear cells in patients with different types of intracranial tumors. Interestingly, a subset of B cells characterized by high expression of the Interleukin-2 receptor alpha chain (CD25, a typical marker of regulatory T cells) was significantly increased in glioblastoma patients. This immune suppressive B cell subset has previously been associated with enhancing the suppressive effect of regulatory T cells. Further research is warranted to investigate the role of this aberrant B cell subset in the context of glioblastoma and its potential as target for immunotherapy.

Expression of Regeneration-Associated Proteins in Primary Sensory Neurons and Regenerating Axons After Nerve Injury-An Overview.

Peripheral nerve injury results in profound alterations of the affected neurons resulting from the interplay between intrinsic and extrinsic molecular events. Restarting the neuronal regenerative program is an important prerequisite for functional recovery of the injured peripheral nerve. The primary sensory neurons with their cell bodies in the dorsal root ganglia provide a useful in vivo and in vitro model for studying the mechanisms that regulate intrinsic neuronal regeneration capacity following axotomy. These studies frequently need to indicate the regenerative status of the corresponding neurons. We summarize the critical issues regarding immunohistochemical detection of several regeneration-associated proteins as markers for the initiation of the regeneration program in rat primary sensory neurons and indicators of axon regeneration in the peripheral nerves. This overview also includes our own results of GAP43 and SCG10 expression in different DRG neurons following double immunostaining with molecular markers of neuronal subpopulations (NF200, CGRP, and IB4) as well as transcription factors (ATF3 and activated STAT3) following unilateral sciatic nerve injury. Anat Rec, 301:1618-1627, 2018. © 2018 Wiley Periodicals, Inc.

Combinatorial Expression of Grp and Neurod6 Defines Dopamine Neuron Populations with Distinct Projection Patterns and Disease Vulnerability.

Midbrain dopamine neurons project to numerous targets throughout the brain to modulate various behaviors and brain states. Within this small population of neurons exists significant heterogeneity based on physiology, circuitry, and disease susceptibility. Recent studies have shown that dopamine neurons can be subdivided based on gene expression; however, the extent to which genetic markers represent functionally relevant dopaminergic subpopulations has not been fully explored. Here we performed single-cell RNA-sequencing of mouse dopamine neurons and validated studies showing that Neurod6 and Grp are selective markers for dopaminergic subpopulations. Using a combination of multiplex fluorescent in situ hybridization, retrograde labeling, and electrophysiology in mice of both sexes, we defined the anatomy, projection targets, physiological properties, and disease vulnerability of dopamine neurons based on Grp and/or Neurod6 expression. We found that the combinatorial expression of Grp and Neurod6 defines dopaminergic subpopulations with unique features. Grp+/Neurod6 + dopamine neurons reside in the ventromedial VTA, send projections to the medial shell of the nucleus accumbens, and have noncanonical physiological properties. Grp+/Neurod6- dopamine neurons are found in the VTA as well as in the ventromedial portion of the SNc, where they project selectively to the dorsomedial striatum. Grp-/Neurod6 + dopamine neurons represent a smaller VTA subpopulation, which is preferentially spared in a 6-OHDA model of Parkinson’s disease. Together, our work provides detailed characterization of Neurod6 and Grp expression in the midbrain and generates new insights into how these markers define functionally relevant dopaminergic subpopulations.

Calcium-binding protein, secretagogin, specifies the microcellular tegmental nucleus and intermediate and ventral parts of the cuneiform nucleus of the mouse and rat

Secretagogin (SCGN) is a recently discovered calcium binding protein of the EF hand family, cloned from β cells of pancreatic island of Langerhans and endocrine cells of the gastrointestinal gland. SCGN characterizes some particular neuron groups in various regions of the nervous system and is considered as one of the useful neuron subpopulation markers. In the present study we reported that SCGN specifically labelled a particular neuronal cluster in the brainstem of the mice and rats. The comparison of the SCGN immunostaining with the choline acetyltransferase immunostaining and acetylcholinesterase staining clearly indicated that the particular cluster of SCGN positive neurons corresponded to the microcellular tegmental nucleus (MiTg) and the ventral portion of the cuneiform nucleus (CnF), both of which are components of the isthmus. The analyses in mice indicated that SCGN positive neurons in the MiTg and CnF were homogeneous in size and shape, appearing to compose a single complex: their somata were small comparing with the adjacent cholinergic neurons in the pedunculotegmantal nucleus, 10.5 vs 16.0 μm in diameter, and extended 2–3 slender smooth processes. SCGN might be one of significant markers to reconsider the delineations of the structures of the mouse, and presumably rat, brainstem.

Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation.

Functional heterogeneity within the lipid droplet (LD) pool of a single cell has been observed, yet the underlying mechanisms remain enigmatic. Here, we report on identification of a specialized LD subpopulation characterized by a unique proteome and a defined geographical location at the nucleus-vacuole junction contact site. In search for factors determining identity of these LDs, we screened ∼6,000 yeast mutants for loss of targeting of the subpopulation marker Pdr16 and identified Ldo45 (LD organization protein of 45 kD) as a crucial targeting determinant. Ldo45 is the product of a splicing event connecting two adjacent genes (YMR147W and YMR148W/OSW5/LDO16). We show that Ldo proteins cooperate with the LD biogenesis component seipin and establish LD identity by defining positioning and surface-protein composition. Our studies suggest a mechanism to establish functional differentiation of organelles, opening the door to better understanding of metabolic decisions in cells.