PLoS One. 2015 Mar 20;10(3):e0120120.
Grabinski TM, Kneynsberg A, Manfredsson FP, Kanaan NM.
PMID: 25794171 | DOI: 10.1371/journal.pone.0120120.
In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.
Resolving the cellular specificity of TSPO imaging in a rat model of peripherally-induced neuroinflammation
Brain, behavior, and immunity
Vicente-Rodríguez, M;Singh, N;Turkheimer, F;Peris-Yague, A;Randall, K;Veronese, M;Simmons, C;Karim Haji-Dheere, A;Bordoloi, J;Sander, K;Awais, RO;Årstad, E;Consortium, N;Cash, D;Parker, CA;
PMID: 34052363 | DOI: 10.1016/j.bbi.2021.05.025
the increased expression of 18kDa Translocator protein (TSPO) is one of the few available biomarkers of neuroinflammation that can be assessed in humans in vivo by positron emission tomography (PET). TSPO PET imaging of the central nervous system (CNS) has been widely undertaken, but to date no clear consensus has been reached about its utility in brain disorders. One reason for this could be because the interpretation of TSPO PET signal remains challenging, given the cellular heterogeneity and ubiquity of TSPO in the brain. the aim of the current study was to ascertain if TSPO PET imaging can be used to detect neuroinflammation induced by a peripheral treatment with endotoxin lipopolysaccharide (LPS) in a rat model (ip LPS), and investigate the origin of TSPO signal changes in terms of their cellular sources and regional distribution. An initial pilot study utilising both [18F]DPA-714 and [11C]PK11195 demonstrated [18F]DPA-714 to exhibit a significantly higher lesion-related signal in the intracerebral LPS rat model (ic LPS) than [11C]PK11195. Subsequently, [18F]DPA-714 was selected for use in the ip LPS study. twenty-four hours after ip LPS, there was an increased uptake of [18F]DPA-714 across the whole brain. Further analyses of regions of interest, using immunohistochemistry and RNAscope Multiplex fluorescence V2 in situ hybridization technology, showed TSPO expression in microglia, monocyte derived-macrophages, astrocytes, neurons and endothelial cells. The expression of TSPO was significantly increased after ip LPS in a region-dependent manner; with microglia, monocyte-derived macrophages and astrocytes in the substantia nigra, in contrast to the hippocampus where TSPO was mostly confined to microglia and astrocytes. in summary, our data demonstrate the robust detection of peripherally-induced neuroinflammation in the CNS utilizing the TSPO radioligand [18F]DPA-714, and importantly, confirm that the TSPO signal increase arises mostly from a combination of microglia, astrocytes and monocyte-derived macrophages.
Translatomic analysis of regenerating and degenerating spinal motor neurons in injury and ALS
Shadrach, J;Stansberry, W;Milen, A;Ives, R;Fogarty, E;Antonellis, A;Pierchala, B;
| DOI: 10.1016/j.isci.2021.102700
The neuromuscular junction is a synapse critical for muscle strength and coordinated motor function. Unlike CNS injuries, motor neurons mount robust regenerative responses after peripheral nerve injuries. Conversely, motor neurons selectively degenerate in diseases such as amyotrophic lateral sclerosis (ALS). To assess how these insults affect motor neurons in vivo, we performed ribosomal profiling of mouse motor neurons. Motor neuron-specific transcripts were isolated from spinal cords following sciatic nerve crush, a model of acute injury and regeneration, and in the SOD1G93A ALS model. Of the 267 transcripts upregulated after nerve crush, 38% were also upregulated in SOD1G93A motor neurons. However, most upregulated genes in injured and ALS motor neurons were context specific. Some of the most significantly upregulated transcripts in both paradigms were chemokines such as Ccl2 and Ccl7, suggesting an important role for neuroimmune modulation. Collectively these data will aid in defining pro-regenerative and pro-degenerative mechanisms in motor neurons.
Castro, RW;Lopes, MC;Settlage, RE;Valdez, G;
PMID: 37154159 | DOI: 10.1172/jci.insight.168448
Spinal motor neurons have been implicated in the loss of motor function that occurs with advancing age. However, the cellular and molecular mechanisms that impair the function of these neurons during aging remain unknown. Here, we show that motor neurons do not die in old female and male mice, rhesus monkeys, and humans. Instead, these neurons selectively and progressively shed excitatory synaptic inputs throughout the soma and dendritic arbor during aging. Thus, aged motor neurons contain a motor circuitry with a reduced ratio of excitatory to inhibitory synapses that may be responsible for the diminished ability to activate motor neurons to commence movements. An examination of the motor neuron translatome (ribosomal transcripts) in male and female mice reveals genes and molecular pathways with roles in glia-mediated synaptic pruning, inflammation, axonal regeneration, and oxidative stress that are upregulated in aged motor neurons. Some of these genes and pathways are also found altered in motor neurons affected with amyotrophic lateral sclerosis (ALS) and responding to axotomy, demonstrating that aged motor neurons are under significant stress. Our findings show mechanisms altered in aged motor neurons that could serve as therapeutic targets to preserve motor function during aging.
De Schepper, S;Ge, JZ;Crowley, G;Ferreira, LSS;Garceau, D;Toomey, CE;Sokolova, D;Rueda-Carrasco, J;Shin, SH;Kim, JS;Childs, T;Lashley, T;Burden, JJ;Sasner, M;Sala Frigerio, C;Jung, S;Hong, S;
PMID: 36747024 | DOI: 10.1038/s41593-023-01257-z
Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-β oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.
Krawczyk, MC;Pan, L;Zhang, AJ;Zhang, Y;
PMID: 36827449 | DOI: 10.1371/journal.pone.0279736
Though the brain was long characterized as an immune-privileged organ, findings in recent years have shown extensive communications between the brain and peripheral immune cells. We now know that alterations in the peripheral immune system can affect the behavioral outputs of the central nervous system, but we do not know which brain cells are affected by the presence of peripheral immune cells. Glial cells including microglia, astrocytes, oligodendrocytes, and oligodendrocyte precursor cells (OPCs) are critical for the development and function of the central nervous system. In a wide range of neurological and psychiatric diseases, the glial cell state is influenced by infiltrating peripheral lymphocytes. However, it remains largely unclear whether the development of the molecular phenotypes of glial cells in the healthy brain is regulated by lymphocytes. To answer this question, we acutely purified each type of glial cell from immunodeficient Rag2-/- mice. Interestingly, we found that the transcriptomes of microglia, astrocytes, and OPCs developed normally in Rag2-/- mice without reliance on lymphocytes. In contrast, there are modest transcriptome differences between the oligodendrocytes from Rag2-/- and control mice. Furthermore, the subcellular localization of the RNA-binding protein Quaking, is altered in oligodendrocytes. These results demonstrate that the molecular attributes of glial cells develop largely without influence from lymphocytes and highlight potential interactions between lymphocytes and oligodendrocytes.
Shi, Z;Yu, P;Lin, WJ;Chen, S;Hu, X;Chen, S;Cheng, J;Liu, Q;Yang, Y;Li, S;Zhang, Z;Xie, J;Jiang, J;He, B;Li, Y;Li, H;Xu, Y;Zeng, J;Huang, J;Mei, J;Cai, J;Chen, J;Wu, LJ;Ko, H;Tang, Y;
PMID: 36603584 | DOI: 10.1016/j.neuron.2022.12.009
The crosstalk between the nervous and immune systems has gained increasing attention for its emerging role in neurological diseases. Radiation-induced brain injury (RIBI) remains the most common medical complication of cranial radiotherapy, and its pathological mechanisms have yet to be elucidated. Here, using single-cell RNA and T cell receptor sequencing, we found infiltration and clonal expansion of CD8+ T lymphocytes in the lesioned brain tissues of RIBI patients. Furthermore, by strategies of genetic or pharmacologic interruption, we identified a chemotactic action of microglia-derived CCL2/CCL8 chemokines in mediating the infiltration of CCR2+/CCR5+ CD8+ T cells and tissue damage in RIBI mice. Such a chemotactic axis also participated in the progression of cerebral infarction in the mouse model of ischemic injury. Our findings therefore highlight the critical role of microglia in mediating the dysregulation of adaptive immune responses and reveal a potential therapeutic strategy for non-infectious brain diseases.
The Journal of clinical investigation
Yan, P;Kim, KW;Xiao, Q;Ma, X;Czerniewski, LR;Liu, H;Rawnsley, DR;Yan, Y;Randolph, GJ;Epelman, S;Lee, JM;Diwan, A;
PMID: 35511433 | DOI: 10.1172/JCI152565
Microglia, the parenchymal tissue macrophages in the brain, surround amyloid plaques in brains of individuals with Alzheimer's disease (AD) but are ineffective at clearing amyloid to mitigate disease progression. Recent studies in mice indicate that microglia are derived exclusively from primitive yolk sac hematopoiesis and self-renew without contribution from ontogenically distinct monocytes/macrophages of definitive adult hematopoietic origin. Using a genetic fate-mapping approach to label cells of definitive hematopoietic origin throughout life span, we discovered that circulating monocytes contribute 6% of plaque-associated macrophages in aged AD mice. Moreover, peripheral monocytes contributed to a higher fraction of macrophages in the choroid plexus, meninges, and perivascular spaces of aged AD mice versus WT control mice, indicating enrichment at potential sites for entry into the brain parenchyma. Splenectomy, which markedly reduced circulating Ly6Chi monocytes, also reduced abundance of plaque-associated macrophages of definitive hematopoietic origin, resulting in increased amyloid plaque load. Together, these results indicate that peripherally derived monocytes invade the brain parenchyma, targeting amyloid plaques to reduce plaque load.
Matson, KJE;Russ, DE;Kathe, C;Hua, I;Maric, D;Ding, Y;Krynitsky, J;Pursley, R;Sathyamurthy, A;Squair, JW;Levi, BP;Courtine, G;Levine, AJ;
PMID: 36163250 | DOI: 10.1038/s41467-022-33184-1
After spinal cord injury, tissue distal to the lesion contains undamaged cells that could support or augment recovery. Targeting these cells requires a clearer understanding of their injury responses and capacity for repair. Here, we use single nucleus RNA sequencing to profile how each cell type in the lumbar spinal cord changes after a thoracic injury in mice. We present an atlas of these dynamic responses across dozens of cell types in the acute, subacute, and chronically injured spinal cord. Using this resource, we find rare spinal neurons that express a signature of regeneration in response to injury, including a major population that represent spinocerebellar projection neurons. We characterize these cells anatomically and observed axonal sparing, outgrowth, and remodeling in the spinal cord and cerebellum. Together, this work provides a key resource for studying cellular responses to injury and uncovers the spontaneous plasticity of spinocerebellar neurons, uncovering a potential candidate for targeted therapy.
Stogsdill, JA;Kim, K;Binan, L;Farhi, SL;Levin, JZ;Arlotta, P;
PMID: 35948630 | DOI: 10.1038/s41586-022-05056-7
Microglia are specialized macrophages in the brain parenchyma that exist in multiple transcriptional states and reside within a wide range of neuronal environments1-4. However, how and where these states are generated remains poorly understood. Here, using the mouse somatosensory cortex, we demonstrate that microglia density and molecular state acquisition are determined by the local composition of pyramidal neuron classes. Using single-cell and spatial transcriptomic profiling, we unveil the molecular signatures and spatial distributions of diverse microglia populations and show that certain states are enriched in specific cortical layers, whereas others are broadly distributed throughout the cortex. Notably, conversion of deep-layer pyramidal neurons to an alternate class identity reconfigures the distribution of local, layer-enriched homeostatic microglia to match the new neuronal niche. Leveraging the transcriptional diversity of pyramidal neurons in the neocortex, we construct a ligand-receptor atlas describing interactions between individual pyramidal neuron subtypes and microglia states, revealing rules of neuron-microglia communication. Our findings uncover a fundamental role for neuronal diversity in instructing the acquisition of microglia states as a potential mechanism for fine-tuning neuroimmune interactions within the cortical local circuitry.
PLoS One. 2015 May 20;10(5):e0127768.
Kanaan NM, Collier TJ, Cole-Strauss A, Grabinski T, Mattingly ZR, Winn ME, Steece-Collier K, Sortwell CE, Manfredsson FP, Lipton JW.
PMID: 26003047 | DOI: 10.1016/j.ydbio.2015.05.002.
We hypothesized that the study of gene expression at 1, 2, 4, 6 and 16 weeks in the substantia nigra (SN) after intrastriatal 6-OHDA in the Sprague-Dawley rat (rattus norvegicus) would identify cellular responses during the degenerative process that could be axoprotective. Specifically, we hypothesized that genes expressed within the SN that followed a profile of being highly upregulated early after the lesion (during active axonal degeneration) and then progressively declined to baseline over 16 weeks as DA neurons died are indicative of potential protective responses to the striatal 6-OHDA insult. Utilizing a κ-means cluster analysis strategy, we demonstrated that one such cluster followed this hypothesized expression pattern over time, and that this cluster contained several interrelated transcripts that are classified as regeneration-associated genes (RAGs) including Atf3, Sprr1a, Ecel1, Gadd45a, Gpnmb, Sox11, Mmp19, Srgap1, Rab15,Lifr, Trib3, Tgfb1, and Sema3c. All exemplar transcripts tested from this cluster (Sprr1a, Ecel1, Gadd45a, Atf3 and Sox11) were validated by qPCR and a smaller subset (Sprr1a, Gadd45a and Sox11) were shown to be exclusively localized to SN DA neurons using a dual label approach with RNAScope in situ hybridization and immunohistochemistry. Upregulation of RAGs is typically associated with the response to axonal injury in the peripheral nerves and was not previously reported as part of the axodegenerative process for DA neurons of the SN. Interestingly, as part of this cluster, other transcripts were identified based on their expression pattern but without a RAG provenance in the literature. These "RAG-like" transcripts need further characterization to determine if they possess similar functions to or interact with known RAG transcripts. Ultimately, it is hoped that some of the newly identified axodegeneration-reactive transcripts could be exploited as axoprotective therapies in PD and other neurodegenerative diseases.
Pluvinage JV, Haney MS, Smith BAH, Sun J, Iram T, Bonanno L, Li L, Lee DP, Morgens DW, Yang AC, Shuken SR, Gate D, Scott M, Khatri P, Luo J, Bertozzi CR, Bassik MC, Wyss-Coray T.
PMID: 30944478 | DOI: 10.1038/s41586-019-1088-4
Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNA sequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical B cell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of α2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-β oligomers and α-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.
