RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Chabrat, Audrey; Doucet-Beaupré, Hélène; Lévesque, Martin

doi:10.3791/52510

Cited by 8 publications

(8 citation statements)

References 15 publications

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“…A combination of rapid TH immunochemistry and LCM was performed using P1 mice (21). A model AS-LMD LCM system (Leica Microsystems) was used to outline and capture the region of the sections with TH-immunoreactive neurons.…”

Section: Methodsmentioning

confidence: 99%

“…Next, we validated each candidate gene as an Lmx1a/b target by RT quantitative PCR (RT-qPCR) from laser-microdissected mDA neurons from P1 control and Lmx1a/b cKO mice (Fig. 5A) (21). mRNA levels of the mitochondrial complex I genes Ndufa3, Ndufa2, and Ndufa4, the mitochondrial complex III gene Uqcrq, and the mitochondrial complex IV gene Cox6a, all of which are nuclear-encoded subunits of the respiratory chain, were significantly lower in LCM-dissected tissue from Lmx1a/b cKO mice (Fig.…”

Section: Lmx1a/b Coordinate the Expression Of Mitochondrial-associatedmentioning

confidence: 99%

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Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons

Doucet-Beaupré

Gilbert

Profes

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson's disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein + inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons. M idbrain dopaminergic (mDA) neurons control key functions in the mammalian brain, including voluntary movement, associative learning, and motivated behaviors. Dysfunctions of the dopaminergic (DA) system underlie a wide variety of neurological and psychiatric disorders. The progressive and rather selective degeneration of mDA neurons is one of the principal pathological features of Parkinson's disease (PD) (1). In PD, neuronal loss is accompanied by the appearance of α-synucleinenriched intraneuronal inclusions called "Lewy bodies" and "Lewy neurites." The etiologies of PD remain unsolved, but mitochondrial dysfunction emerges as a central mechanism in inherited, sporadic, and toxin-induced PD (2).Specification of the subtype identities of mDA neurons begins during embryonic development. The combinatory activation of transcription factors (TFs) and their target genes allows the progenitors to mature progressively and terminally differentiate into postmitotic neuron subtypes. Tremendous efforts have been made to describe the complex spatiotemporal expression of TFs during mDA neuronal development (see refs. 3 and 4 for reviews). After mDA neuron maturation, a large number of developmentally expressed TFs remain active throughout adulthood. Our knowledge of the functional roles of these TFs in mature neurons remains rudimentary. Accumulating evidence shows that transcription factors including the nuclear receptor related 1 protein (Nurr1), En1, Pitx3, Otx2, and Foxa2, which are recognized for their role in the early development of mDA neurons, are also required for the maintenance of phenotypic neuronal identity in the adult (5).The LIM homeodomain genes Lmx1a/b are early determinants of the fate of mDA progenitors (6), and their actions are essential at each step of DA neuronal generation (7,8). The murine Lmx1a and Lmx1b proteins are closely related and share an overall amino acid identity of 64%, with 100% identity in their homeodomain and 67% and 83% identity in each LIM domain (9). These neuron...

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Section: Methodsmentioning

confidence: 99%

Section: Lmx1a/b Coordinate the Expression Of Mitochondrial-associatedmentioning

confidence: 99%

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons

Doucet-Beaupré

Gilbert

Profes

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Sections of 16 μm were dissected with a Zeiss Observer microscope equipped with Palm MicroBeam device for laser-capture microdissection. Total RNAs were extracted from these dissected tissues as described59.…”

Section: Methodsmentioning

confidence: 99%

Lymphocytic Microparticles Modulate Angiogenic Properties of Macrophages in Laser-induced Choroidal Neovascularization

Tahiri

Omri

Yang

et al. 2016

Sci Rep

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Pathological choroidal neovascularization (CNV) is the common cause of vision loss in patients with age-related macular degeneration (AMD). Macrophages possess potential angiogenic function in CNV. We have demonstrated that human T lymphocyte-derived microparticles (LMPs) exert a potent antiangiogenic effect in several pathological neovascularization models. In this study, we investigated the alteration of proangiogenic properties of macrophages by LMPs treatment in vitro and in vivo models. LMPs regulated the expression of several angiogenesis-related factors in macrophages and consequently stimulated their antiangiogenic effects evidenced by the suppression of the proliferation of human retinal endothelial cells in co-culture experiments. The involvement of CD36 receptor in LMPs uptake by macrophages was demonstrated by in vitro assays and by immunostaining of choroidal flat mounts. In addition, ex vivo experiments showed that CD36 mediates the antiangiogenic effect of LMPs in murine and human choroidal explants. Furthermore, intravitreal injection of LMPs in the mouse model of laser-induced CNV significantly suppressed CNV in CD36 dependent manner. The results of this study suggested an ability of LMPs to alter the gene expression pattern of angiogenesis-related factors in macrophages, which provide important information for a new therapeutic approach for efficiently interfering with both vascular and extravascular components of CNV.

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“…However, similar to FACS, this technique requires specialized equipment. Additionally, this technique requires extensive training and expertise, as the individual user identifies cells for tracing and microdissection based on their morphological appearance, a limitation that may be circumvented by immunolabeling cells prior to LCM (Chabrat, Doucet‐Beaupre, & Levesque, ).…”

Section: Commentarymentioning

confidence: 99%

Magnetic Cell Sorting for In Vivo and In Vitro Astrocyte, Neuron, and Microglia Analysis

2019

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Interest in evaluating individual cellular populations in the central nervous system has prompted the development of several techniques enabling the enrichment of single‐cell populations. Herein we detail a relatively inexpensive method to specifically isolate neurons, astrocytes, and microglia from a mixed homogenate utilizing magnetic beads conjugated to cell‐type specific antibodies. We have used this technique to isolate astrocytes across development and into late adulthood. Finally, we detail the utilization of this technique in novel astrocyte and astrocyte/neuron co‐culture paradigms. © 2019 by John Wiley & Sons, Inc.

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RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Cited by 8 publications

References 15 publications

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons

Lymphocytic Microparticles Modulate Angiogenic Properties of Macrophages in Laser-induced Choroidal Neovascularization

Magnetic Cell Sorting for In Vivo and In Vitro Astrocyte, Neuron, and Microglia Analysis

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