Analysis of Primary Cilia in Rodent Brain By Immunofluorescence Microscopy v1

Khan, Shahzad S.

doi:10.17504/protocols.io.bnwimfce

Cited by 2 publications

(4 citation statements)

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“…Mouse genotyping was performed by PCR using genomic DNA isolated from tail clips or ear biopsies. For the experiment shown in Figure 5 Mouse brain processing: Homozygous LRRK2-mutant (R1441C or G2019S of various ages as indicated) or heterozygous Ppm1h -/+ mice (164 days old) and age-matched wild-type controls were fixed by transcardial perfusion using 4% paraformaldehyde (PFA) in PBS as described in Khan et al (2020). Next, whole brain tissue was extracted, post-fixed in 4% PFA for 24 hrs and then immersed in 30% (w/v) sucrose in PBS until the tissue settled to the bottom of the tube (~48 hrs).…”

Section: Methodsmentioning

confidence: 99%

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Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

Khan

Sobu

Dhekne

et al. 2021

eLife

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Activating LRRK2 mutations cause Parkinson's disease, and pathogenic LRRK2 kinase interferes with ciliogenesis. Previously, we showed that cholinergic interneurons of the dorsal striatum lose their cilia in R1441C LRRK2 mutant mice (Dhekne et al., 2018). Here, we show that cilia loss is seen as early as 10 weeks of age in these mice and also in two other mouse strains carrying the most common human G2019S LRRK2 mutation. Loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases yields the same cilia loss phenotype seen in mice expressing pathogenic LRRK2 kinase, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. Moreover, astrocytes throughout the striatum show a ciliation defect in all LRRK2 and PPM1H mutant models examined. Hedgehog signaling requires cilia, and loss of cilia in LRRK2 mutant rodents correlates with dysregulation of Hedgehog signaling as monitored by in situ hybridization of Gli1 and Gdnf transcripts. Dopaminergic neurons of the substantia nigra secrete a Hedgehog signal that is sensed in the striatum to trigger neuroprotection; our data support a model in which LRRK2 and PPM1H mutant mice show altered responses to critical Hedgehog signals in the nigrostriatal pathway.

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Section: Methodsmentioning

confidence: 99%

“…Immunofluorescence staining and microscopy: Brain primary cilia detection and analyses were performed as previously described (Khan et al, 2020). Briefly, frozen slides were thawed at RT for 10 min then gently washed with PBS for 5 min.…”

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

Khan

Sobu

Dhekne

et al. 2021

eLife

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show abstract

“…Mice were euthanized by cervical dislocation 2hr following treatment and the collected tissues were rapidly snap frozen in liquid nitrogen. Mouse brain processing Homozygous LRRK2-mutant (R1441C or G2019S of various ages as indicated) or heterozygous PPM1H -/+ KO mice (164 days old) and age-matched wild-type controls were fixed by transcardial perfusion using 4% paraformaldehyde (PFA) in PBS as described in Khan et al (2020). Next, whole brain tissue was extracted, post-fixed in 4% PFA for 24 hrs and then immersed in 30% (w/v) sucrose in PBS until the tissue settled to the bottom of the tube (~48 hrs).…”

Section: Transgenic Micementioning

confidence: 99%

“…Sections were mounted with ProLong™ Gold Antifade Mountant with DAPI and glass coverslips. Immunofluorescence staining and microscopy Brain primary cilia detection and analyses were performed as previously described (Khan et al, 2020). Briefly, frozen slides were thawed at RT for 10 min then gently washed with PBS for 5 min.…”

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

Khan

Sobu

Dhekne

et al. 2021

Preprint

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Previously, we showed that cholinergic interneurons of the dorsal striatum lose cilia in mice harboring the Parkinson's-disease associated, kinase activating, R1441C LRRK2 mutation (Dhekne et al., 2018). Here we show that this phenotype is also seen in two mouse strains carrying the most common human G2019S LRRK2 mutation. Heterozygous loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases (Berndsen et al., 2019) yields the same cilia loss phenotype, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. In addition, astrocytes throughout the striatum show a ciliation defect in LRRK2 and PPM1H-/+ mutant models. Hedgehog signaling requires cilia, and loss of cilia correlates here with a loss in induction of Hedgehog signaling as monitored by in situ hybridization of Gli1 transcripts. These data support a model in which LRRK2 and PPM1H mutant mice struggle to receive and respond to critical Hedgehog signals in the nigral-striatal pathway.

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Analysis of Primary Cilia in Rodent Brain By Immunofluorescence Microscopy v1

Abstract: We describe here our method for immunostaining of primary cilia in brain sections from wild type and LRRK2 or other mutant mice. Included are procedures for perfusion, tissue harvesting, sectioning and staining, image acquisition and analysis.

Cited by 2 publications

References 1 publication

Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

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