Development of a versatile LCM-Seq method for spatial transcriptomics of fluorescently tagged cholinergic neuron populations

Rumpler, Éva; Göcz, Balázs; Skrapits, Katalin; Sárvári, Miklós; Takács, Szabolcs; Farkas, Imre; Póliska, Szilárd; Papp, Márton; Solymosi, Norbert; Hrabovszky, Erik

doi:10.1016/j.jbc.2023.105121

Cited by 3 publications

(5 citation statements)

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“…A laser capture microdissection/RNA-sequencing (‘LCM-Seq’) method developed in our laboratory (42) has enabled us to study and identify the estrogen-regulated transcripts of KP ARC (43) and KP RP3V (25) neurons. Here we characterized the transcriptome landscape of KP LS neurons using the same animal models and protocols to allow the comparative analysis of the different KP cell types.…”

Section: Resultsmentioning

confidence: 99%

“…The LS was sectioned serially with a cryostat and ZsGreen-tagged KP neurons were isolated with LCM (25, 43). As the low number of KP LS cells in individual mice may result in suboptimal detection sensitivity and quantification precision/accuracy of the LCM-Seq protocol (42), ∼360 fluorescently-tagged neurons were pooled proportionally from 3 brains to prepare each RNA sample and cDNA library, followed by Illumina sequencing (25, 43). Meta-analysis of the two hypothalamic KP neuron transcriptomes was carried out from open-access public repositories associated with the original publications (25, 43).…”

Section: Resultsmentioning

confidence: 99%

“…While much attention has been paid to the reproductive significance of the two hypothalamic KP neuron populations, functions of the extra-hypothalamic KP neurons are still relatively unexplored. Here we have used an LCM-Seq method developed in our laboratory (42) to study the transcriptome profile of KP LS neurons and its estrogen-dependent regulation. This method allowed us to identify more than 15,000 transcripts which could then be compared to the transcripts of KP ARC and KP RP3V neurons we studied earlier using the same methodologies and animal models (25, 43).…”

Section: Discussionmentioning

confidence: 99%

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Estrogen-dependent development and transcriptome regulation of the lateral septal kisspeptin system

Szentkirályi-Tóth,

Göcz,

Takács

et al. 2023

Preprint

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While hypothalamic kisspeptin (KP) neurons play well-established roles in puberty and reproduction, kisspeptin neurons in the lateral septum (KPLS neurons) and other extrahypothalamic sites received less attention. We found that the onset of LS kisspeptin expression was linked to pubertal development and estrogen receptor signaling. Cell numbers were higher in females vs. males, continued to increase in adulthood and exogenous estrogen administered to adult mice was able to switch on the Kiss1 gene promoter in new sets of septal neurons without de novo neurogenesis. Using RNA-Seq studies of laser-microdisected neurons from ovariectomized mice treated with estradiol vs. vehicle, we found that KPLS neurons largely differ from hypothalamic KP neurons in their transcriptome profile which included 571 estrogen-regulated transcripts. Notably, Kiss1 expression in the LS was considerably lower than in hypothalamic KP neurons, being undetectable in ovariectomized mice and inducible by E2 supplementation. Finally, immunohistochemical detection of septal kisspeptin neurons and their fibers in the human brain suggested that the role they play is evolutionarily conserved. Ontogeny, sexual dimorphism and estrogenic regulation raise the intriguing possibility that the KPLS system is a new central player in the estrogen-dependent control of fertility in mice, with possible human correlates.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Estrogen-dependent development and transcriptome regulation of the lateral septal kisspeptin system

Szentkirályi-Tóth,

Göcz,

Takács

et al. 2023

Preprint

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“…Our efforts to detect GnRH immunoreactivity with immunohistochemistry or mRNA expression with RT-PCR and in situ hybridization at this site also failed using samples from either neonatal and adult mice, indicating that, if present, Gnrh is expressed at very low levels only in the murine striatum. The transcriptome database of ChINs available from adult mice contains neither Gnrh nor Gnrhr ( 23 ), but similar high-throughput studies are currently unavailable to indicate the presence/absence of the authentic GnRH and its receptor in ChINs of neonatal mice. Thus, the possibility remains that developmental GnRH-GFP expression in ChINs of this specific transgenic strain represents an ectopic expression.…”

Section: Discussionmentioning

confidence: 99%

Functional GnRH receptor signaling regulates striatal cholinergic neurons in neonatal but not in adult mice

Farkas,

Skrapits,

Sárvári

et al. 2024

Front. Endocrinol.

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Reproduction in mammals is controlled by hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Recent studies from our laboratory established that the basal ganglia of the human brain contain additional large populations of GnRH synthesizing neurons which are absent in adult mice. Such extrahypothalamic GnRH neurons mostly occur in the putamen where they correspond to subsets of the striatal cholinergic interneurons (ChINs) and express GnRHR autoreceptors. In an effort to establish a mouse model for functional studies of striatal GnRH/GnRHR signaling, we carried out electrophysiological experiments on acute brain slices from male transgenic mice. Using PN4-7 neonatal mice, half of striatal ChINs responded with transient hyperpolarization and decreased firing rate to 1.2 µM GnRH, whereas medium spiny projection neurons remained unaffected. GnRH acted on its specific receptor because no response was observed in the presence of the GnRHR antagonist Antide. Addition of the membrane-impermeable G protein-coupled receptor inhibitor GDP-β-S to the internal electrode solution eliminated the effect of GnRH. Further, GnRH was able to inhibit ChINs in presence of tetrodotoxin which blocked action potential mediated events. Collectively, these data indicated that the receptor underlying the effects of GnRH in neonatal mice is localized within ChINs. GnRH responsiveness of ChINs was transient and entirely disappeared in adult mice. These results raise the possibility to use neonatal transgenic mice as a functional model to investigate the role of GnRH/GnRHR signaling discovered earlier in adult human ChINs.

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“…Some spatial omics techniques involve imaging the tissue to be tested, followed by cutting out the region of interest (ROI), such as with LCM [25] and UV photolysis [24] technology. These methods are then combined with sequencing technologies to yield the corresponding omics data.…”

Section: Spatial Transcriptomic Techniquesmentioning

confidence: 99%

Spatial Multi-Omics in Alzheimer’s Disease: A Multi-Dimensional Approach to Understanding Pathology and Progression

Ma,

Shi,

Dong

et al. 2024

CIMB

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Alzheimer’s Disease (AD) presents a complex neuropathological landscape characterized by hallmark amyloid plaques and neurofibrillary tangles, leading to progressive cognitive decline. Despite extensive research, the molecular intricacies contributing to AD pathogenesis are inadequately understood. While single-cell omics technology holds great promise for application in AD, particularly in deciphering the understanding of different cell types and analyzing rare cell types and transcriptomic expression changes, it is unable to provide spatial distribution information, which is crucial for understanding the pathological processes of AD. In contrast, spatial multi-omics research emerges as a promising and comprehensive approach to analyzing tissue cells, potentially better suited for addressing these issues in AD. This article focuses on the latest advancements in spatial multi-omics technology and compares various techniques. Additionally, we provide an overview of current spatial omics-based research results in AD. These technologies play a crucial role in facilitating new discoveries and advancing translational AD research in the future. Despite challenges such as balancing resolution, increasing throughput, and data analysis, the application of spatial multi-omics holds immense potential in revolutionizing our understanding of human disease processes and identifying new biomarkers and therapeutic targets, thereby potentially contributing to the advancement of AD research.

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Development of a versatile LCM-Seq method for spatial transcriptomics of fluorescently tagged cholinergic neuron populations

Cited by 3 publications

References 58 publications

Estrogen-dependent development and transcriptome regulation of the lateral septal kisspeptin system

Estrogen-dependent development and transcriptome regulation of the lateral septal kisspeptin system

Functional GnRH receptor signaling regulates striatal cholinergic neurons in neonatal but not in adult mice

Spatial Multi-Omics in Alzheimer’s Disease: A Multi-Dimensional Approach to Understanding Pathology and Progression

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