Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2 methods
Aim. Evidence-backed execution summary for Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2 methods from Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Generation of LRRK2 transgenic mice
reagent used in the protocol.
- Use
- For RT-PCR, total RNA was purified from mouse hemi-brains using an RNeasy kit (Qiagen, Valencia, CA, USA) and digested with DNase I (Qiagen). RNA was reverse transcribed using a SuperScript III First-Strand Synthesis system (Invitrogen, Carlsbad, CA, USA) with oligo(dT) 20. cDNA derived from 50 ng of total RNA was...
Western blot analysis
reagent used in the protocol.
- Use
- Protein extracts were prepared from hemi-brains of 2-4 month-old mice as previously described by homogenization in TNE buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 X Complete protease inhibitor cocktail [Roche], 1 X phosphatase inhibitor cocktail I and II [Sigma-Aldrich, St. Louis, MO, USA])....
TH immunohistochemistry and stereological assessments
reagent used in the protocol.
- Use
- Mice were anesthetized with sodium pentobarbital (150 mg/kg) before intracardial perfusion with cold PBS and 4% paraformaldehyde (PFA). Brains were removed, post-fixed with PFA overnight and cryopreserved in 30% sucrose in PBS at 4°C for 48 h. Coronal midbrain sections (40-µm) were prepared and processed f...
Immunohistochemistry
reagent used in the protocol.
- Use
- Coronal sections (30-µm) from cortex, striatum and midbrain were prepared and processed for immunohistochemistry for various pathological markers as previously described,. Briefly, sections were quenched of endogenous peroxidase activity, permeabilized, and incubated with primary antibodies for α-synucle...
Immunofluorescence and confocal microscopy
reagent used in the protocol.
- Use
- Coronal midbrain sections (30 µm) were prepared from 4-5 month G2019S LRRK2 transgenic mice (TG; line 340) and non-transgenic littermate mice (NTG). Sections were processed for immunohistochemistry with rabbit monoclonal anti-LRRK2 antibody (clone c41-2, Epitomics; refer to www.pdonlineresearch.org for detailed...
Supporting Information
reagent used in the protocol.
- Use
- Quantitation of LRRK2 protein levels in LRRK2 transgenic mice. Western blot analysis of soluble brain extracts (75 µg protein) derived from hemi-brains of 3-4 month G2019S (line 340) and R1441C (line 574) LRRK2 transgenic mice (TG) and their non-transgenic littermates (NTG). Blots were probed with a pan-LRRK2 a...
Stereological assessment of TH+/Nissl+ neurons
Unbiased stereological methodology was employed as described previously, to count TH+ and Nissl+ neurons in the left and right pars compacta region of every fourth section throughout the ventral midbrain. Stereological counts were obtained using a computer-assisted image analysis system consisting of an Axiophot 2...
- Use
- Unbiased stereological methodology was employed as described previously, to count TH+ and Nissl+ neurons in the left and right pars compacta region of every fourth section throughout the ventral midbrain. Stereological counts were obtained using a computer-assisted image analysis system consisting of an Axiophot 2...
Normal Locomotor Activity and Prepulse Inhibition in G2019S LRRK2 Transgenic Mice
To investigate whether G2019S mutant LRRK2 expression influences motor performance, we measured locomotor activity in the open field quadrant ( ). G2019S-LRRK2 mice display normal locomotor activity in the open field at 6 and 15 months of age ( ). In contrast, R1441C-LRRK2 mice exhibit a significant reduction in hor...
- Use
- To investigate whether G2019S mutant LRRK2 expression influences motor performance, we measured locomotor activity in the open field quadrant ( ). G2019S-LRRK2 mice display normal locomotor activity in the open field at 6 and 15 months of age ( ). In contrast, R1441C-LRRK2 mice exhibit a significant reduction in hor...
Normal Locomotor Activity and Prepulse Inhibition in G2019S LRRK2 Transgenic Mice
A-D, In the open field G2019S LRRK2 TG mice (line 340) exhibit normal horizontal ( A ) and vertical ( B ) locomotor activity compared to NTG littermates at 6 and 15 months ( n = 7-9 mice/genotype). R1441C LRRK2 TG mice (line 574) reveal normal locomotor activity at 6 months but deficits at...
- Use
- A-D, In the open field G2019S LRRK2 TG mice (line 340) exhibit normal horizontal ( A ) and vertical ( B ) locomotor activity compared to NTG littermates at 6 and 15 months ( n = 7-9 mice/genotype). R1441C LRRK2 TG mice (line 574) reveal normal locomotor activity at 6 months but deficits at...
Generation of LRRK2 transgenic mice
For RT-PCR, total RNA was purified from mouse hemi-brains using an RNeasy kit (Qiagen, Valencia, CA, USA) and digested with DNase I (Qiagen). RNA was reverse transcribed using a SuperScript III First-Strand Synthesis system (Invitrogen, Carlsbad, CA, USA) with oligo(dT) 20. cDNA derived from 50 ng of total RNA was...
- Use
- For RT-PCR, total RNA was purified from mouse hemi-brains using an RNeasy kit (Qiagen, Valencia, CA, USA) and digested with DNase I (Qiagen). RNA was reverse transcribed using a SuperScript III First-Strand Synthesis system (Invitrogen, Carlsbad, CA, USA) with oligo(dT) 20. cDNA derived from 50 ng of total RNA was...
Western blot analysis
Protein extracts were prepared from hemi-brains of 2-4 month-old mice as previously described by homogenization in TNE buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 X Complete protease inhibitor cocktail [Roche], 1 X phosphatase inhibitor cocktail I and II [Sigma-Aldrich, St. Louis, MO, USA])....
- Use
- Protein extracts were prepared from hemi-brains of 2-4 month-old mice as previously described by homogenization in TNE buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 X Complete protease inhibitor cocktail [Roche], 1 X phosphatase inhibitor cocktail I and II [Sigma-Aldrich, St. Louis, MO, USA])....
Immunofluorescence and confocal microscopy
Coronal midbrain sections (30 µm) were prepared from 4-5 month G2019S LRRK2 transgenic mice (TG; line 340) and non-transgenic littermate mice (NTG). Sections were processed for immunohistochemistry with rabbit monoclonal anti-LRRK2 antibody (clone c41-2, Epitomics; refer to www.pdonlineresearch.org for detailed...
- Use
- Coronal midbrain sections (30 µm) were prepared from 4-5 month G2019S LRRK2 transgenic mice (TG; line 340) and non-transgenic littermate mice (NTG). Sections were processed for immunohistochemistry with rabbit monoclonal anti-LRRK2 antibody (clone c41-2, Epitomics; refer to www.pdonlineresearch.org for detailed...
Transmission electron microscopy (TEM)
For TEM analysis, mice were perfused with fixative (3% PFA, 1.5% glutaraldehyde, 100 mM cacodylate, 2.5% sucrose, pH 7.4) and post-fixed for 1 h. Cortex and striatal sections were processed as described. Sections were post-fixed in Palade's OsO 4, en bloc stained in Kellenberger's uranyl acetate, dehydrated, and f...
- Use
- For TEM analysis, mice were perfused with fixative (3% PFA, 1.5% glutaraldehyde, 100 mM cacodylate, 2.5% sucrose, pH 7.4) and post-fixed for 1 h. Cortex and striatal sections were processed as described. Sections were post-fixed in Palade's OsO 4, en bloc stained in Kellenberger's uranyl acetate, dehydrated, and f...
Transmission electron microscopy (TEM)
For quantitation of cytopathology, images were collected on a Philips FEI CM10 TEM equipped with a 11-megapixel Morada soft imaging system camera (Olympus) at the Interdisciplinary Centre for Electron Microscopy (CIME) at EPFL. TEM images were sampled at random over equivalent areas of 800 × 800 µm from repr...
- Use
- For quantitation of cytopathology, images were collected on a Philips FEI CM10 TEM equipped with a 11-megapixel Morada soft imaging system camera (Olympus) at the Interdisciplinary Centre for Electron Microscopy (CIME) at EPFL. TEM images were sampled at random over equivalent areas of 800 × 800 µm from repr...
Supporting Information
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Striatal dopaminergic nerve terminals in G2019S LRRK2 transgenic mice. TH+ immunoreactivity in the striatum of 19-20 month G2019S LRRK2 mice (TG, line 340) compared to their non-transgenic littermate mice (NTG). The optical density of TH+ immunoreactivity was quantified by densitometric analysis of every fourth sect...
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Normal Locomotor Activity and Prepulse Inhibition in G2019S LRRK2 Transgenic Mice
To investigate whether G2019S mutant LRRK2 expression influences motor performance, we measured locomotor activity in the open field quadrant ( ). G2019S-LRRK2 mice display normal locomotor activity in the open field at 6 and 15 months of age ( ). In contrast, R1441C-LRRK2 mice exhibit a significant reduction in horizontal and vertical locomotor activity at 15 months compared to their non-transgenic littermate mice that is not evident at 6 months ( ). We also assessed prepulse inhibition of the acoustic startle reflex, a measure of sensorimotor gating that can be modulated in part by dopaminergic neurotransmission. However, G2019S- and R1441C-LRRK2 transgenic mice do not perform differently from their non-transgenic littermates when tested at 6 months (data not shown) and 15 months of age ( ). Our data demonstrate that G2019S-LRRK2 expression does not influence locomotor activity or...
Normal Locomotor Activity and Prepulse Inhibition in G2019S LRRK2 Transgenic Mice
A-D, In the open field G2019S LRRK2 TG mice (line 340) exhibit normal horizontal ( A ) and vertical ( B ) locomotor activity compared to NTG littermates at 6 and 15 months ( n = 7-9 mice/genotype). R1441C LRRK2 TG mice (line 574) reveal normal locomotor activity at 6 months but deficits at 15 months ( C and D ) compared to NTG mice ( n = 6-9 mice/genotype). Data represent the number of beam breaks during the first 15 min period. E-F, Normal pre-pulse inhibition (PPI) of the acoustic startle response in LRRK2 transgenic mice. E and F, G2019S and R1441C LRRK2 TG mice display normal PPI of the acoustic startle reflex at 15 months compared to their NTG littermates, with increasing pre-pulse tones of 74-90 dB ( n = 6-8 mice/genotype). Data are expressed as % PPI relative to no pre-pulse tone. Bars present the mean ...
Reduced Complexity of Dopaminergic Neurites in Primary Midbrain Cultures from G2019S LRRK2 Transgenic Mice
LRRK2 has been shown to regulate the morphology of neuronal processes in cultured primary cortical neurons,. To further explore this phenotype in our LRRK2 transgenic mice, primary midbrain cultures were prepared from the ventral mesencephalon of G2019S-LRRK2 transgenic mice (line 340) and their non-transgenic littermates. These cultures typically contain 5-10% of TH+ dopaminergic neurons (unpublished observation). Sholl analysis was conducted to provide a measure of neuritic complexity ( and ). At days-in-vitro (DIV) 3, developing dopaminergic neurons from G2019S-LRRK2 mice exhibit significantly reduced overall neurite complexity manifesting as shorter neurites but with modestly increased neurite branching compared to non-transgenic neurons ( and ). However, at DIV 7 when dopaminergic neurite outgrowth is fully established, G2019S-LRRK2 dopaminergic neurons reveal a dramatic...
Western blot analysis
Protein extracts were prepared from hemi-brains of 2-4 month-old mice as previously described by homogenization in TNE buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 X Complete protease inhibitor cocktail [Roche], 1 X phosphatase inhibitor cocktail I and II [Sigma-Aldrich, St. Louis, MO, USA]). Protein concentration was determined by BCA method (Pierce Biotechnology, Rockford, IL, USA) and 75 µg of protein was resolved by SDS-PAGE, transferred to nitrocellulose and probed with rabbit anti-LRRK2 antibodies recognizing human/mouse (JH5514 or monoclonal c81-8, Epitomics, Inc, Burlingame, CA) or human-specific (NB300-267, Novus Biologicals, Littleton, CO, USA ) epitopes, or mouse β-tubulin (TUB 2.1, Sigma-Aldrich) antibody. Refer to www.pdonlineresearch.org for detailed characterization of rabbit monoclonal LRRK2 antibody (clone c81-8, Epitomics, Inc). Quan...
Measurement of biogenic amines by HPLC
HPLC with electrochemical detection was employed to measure the concentration of the biogenic amines, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine (NE) as previously described. Samples were prepared from dissected brain regions and injected onto a C-18 80 × 4.6 mm column (ESA Inc., Chelmsford, MA, USA) with detection using a 2-channel Coulochem II electrochemical detector (ES Inc.). Data were processed on an EZChrome Elite Client Workstation (ESA Inc.) with biogenic amine concentration expressed as ng per mg protein.
TH immunohistochemistry and stereological assessments
Mice were anesthetized with sodium pentobarbital (150 mg/kg) before intracardial perfusion with cold PBS and 4% paraformaldehyde (PFA). Brains were removed, post-fixed with PFA overnight and cryopreserved in 30% sucrose in PBS at 4°C for 48 h. Coronal midbrain sections (40-µm) were prepared and processed for immunohistochemistry with rabbit anti-TH antibody (Novus Biologicals) and Nissl counterstain as previously described.
Transmission electron microscopy (TEM)
For TEM analysis, mice were perfused with fixative (3% PFA, 1.5% glutaraldehyde, 100 mM cacodylate, 2.5% sucrose, pH 7.4) and post-fixed for 1 h. Cortex and striatal sections were processed as described. Sections were post-fixed in Palade's OsO 4, en bloc stained in Kellenberger's uranyl acetate, dehydrated, and flat-embedded in epon. 80 nm en face sections were prepared on a Leica UCT ultramicrotome, collected onto 400 mesh high transmission nickel grids, and post-stained with lead and uranyl acetate. Images were collected on a Philips EM 410 TEM equipped with a Soft Imaging System Megaview III digital camera.
Primary midbrain cultures and analysis of neuritic complexity
Cultures were fixed with 4% PFA and processed for immunocytochemistry with rabbit anti-TH (Novus Biologicals) and mouse anti-MAP2 (Sigma-Aldrich) antibodies and anti-rabbit IgG-AlexaFluor-546 and anti-mouse IgG-AlexaFluor-488 antibodies (Invitrogen). Fluorescent images were captured using a Leica DMI 4000 inverted fluorescence microscope (Leica) at 10x magnification. Sholl analysis was performed on TH+/MAP+ neurons using NIH ImageJ software with a Sholl analysis plug-in (v1.0) developed by the laboratory of Anirvan Ghosh ( http://www.biology.ucsd.edu/labs/ghosh/software/index.html ) to quantify neuritic complexity. Briefly, we constructed continuous concentric circles centered upon the neuronal soma to measure the number of intersections of dendrites with circles of increasing radii. Sholl analysis was performed using the semi-log method and measurements are shown in.
Measurement outputs
What raw and processed outputs should exist?
Unbiased stereological methodology was employed as described previously, to count TH+ and Nissl+ neurons in the left and right pars compacta region of every fourth section thro...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
To determine whether the expression of G2019S-LRRK2 in mice induces the degeneration of nigrostriatal dopaminergic neurons with age, cohorts of LRRK2 transgenic mice were aged t...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
To further examine the impact of G2019S mutant LRRK2 expression on the nigrostriatal dopaminergic pathway, the levels of striatal dopamine and its metabolites, 3,4-dihydroxyphen...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
A-D, Levels of ( A ) dopamine (DA) and its metabolites, ( B ) DOPAC and ( C ) HVA, and ( D ) dopamine turnover ([DOPAC+HVA]/DA) in the striatum, olfactory bulb and cerebr...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
To determine whether the expression of G2019S-LRRK2 in mice induces the degeneration of nigrostriatal dopaminergic neurons with age, cohorts of LRRK2 transgenic mice were aged to 19-21 months.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Unbiased stereological methodology was employed as described previously, to count TH+ and Nissl+ neurons in the left and right pars compacta region of every fourth section thro...; To determine whether the expression of G2019S-LRRK2 in mice induces the degeneration of nigrostriatal dopaminergic neurons with age, cohorts of LRRK2 transgenic mice were aged t...; To further examine the impact of G2019S mutant LRRK2 expression on the nigrostriatal dopaminergic pathway, the levels of striatal dopamine and its metabolites, 3,4-dihydroxyphen...; A-D, Levels of ( A ) dopamine (DA) and its metabolites, ( B ) DOPAC and ( C ) HVA, and ( D ) dopamine turnover ([DOPAC+HVA]/DA) in the striatum, olfactory bulb and cerebr....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
To determine whether the expression of G2019S-LRRK2 in mice induces the degeneration of nigrostriatal dopaminergic neurons with age, cohorts of LRRK2 transgenic mice were aged t...; A, Example of TH and Nissl staining in the substantia nigra of NTG and G2019S LRRK2 TG mice (line 340) at 19-20 months. B and C, Stereological counts for TH+ and Nissl+...; To further examine the impact of G2019S mutant LRRK2 expression on the nigrostriatal dopaminergic pathway, the levels of striatal dopamine and its metabolites, 3,4-dihydroxyphen...; A-D, Levels of ( A ) dopamine (DA) and its metabolites, ( B ) DOPAC and ( C ) HVA, and ( D ) dopamine turnover ([DOPAC+HVA]/DA) in the striatum, olfactory bulb and cerebr...
from paperReporting output
Report representative outputs alongside summary comparisons for Unbiased stereological methodology was employed as described previously, to count TH+ and Nissl+ neurons in the left and right pars compacta region of every fourth section thro..., To determine whether the expression of G2019S-LRRK2 in mice induces the degeneration of nigrostriatal dopaminergic neurons with age, cohorts of LRRK2 transgenic mice were aged t..., To further examine the impact of G2019S mutant LRRK2 expression on the nigrostriatal dopaminergic pathway, the levels of striatal dopamine and its metabolites, 3,4-dihydroxyphen..., A-D, Levels of ( A ) dopamine (DA) and its metabolites, ( B ) DOPAC and ( C ) HVA, and ( D ) dopamine turnover ([DOPAC+HVA]/DA) in the striatum, olfactory bulb and cerebr....
inferred from protocolStructured statistical methods
To determine whether the expression of G2019S-LRRK2 in mice induces the degeneration of nigrostriatal dopaminergic neurons with age, cohorts of LRRK2 transgenic mice were aged t...; A, Example of TH and Nissl staining in the substantia nigra of NTG and G2019S LRRK2 TG mice (line 340) at 19-20 months. B and C, Stereological counts for TH+ and Nissl+...; To further examine the impact of G2019S mutant LRRK2 expression on the nigrostriatal dopaminergic pathway, the levels of striatal dopamine and its metabolites, 3,4-dihydroxyphen...; A-D, Levels of ( A ) dopamine (DA) and its metabolites, ( B ) DOPAC and ( C ) HVA, and ( D ) dopamine turnover ([DOPAC+HVA]/DA) in the striatum, olfactory bulb and cerebr...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
To investigate whether G2019S mutant LRRK2 expression influences motor performance, we measured locomotor activity in the open field quadrant ( ). G2019S-LRRK2 mice display normal locomotor activity in the open field at 6 and 15 months of age ( ). In contrast, R1441C-LRRK2 mice exhibit a significant reduction in horizontal and vertical locomotor activity at 15 months compared to their non-transgenic littermate mice that is not evident at 6 months ( ). We also assessed prepulse inhibition of the acoustic startle reflex, a measure of sensorimotor gating that can be modulated in part by dopaminergic neurotransmission. However, G2019S- and R1441C-LRRK2 transgenic mice do not perform differently from their non-transgenic littermates when tested at 6 months (data not shown) and 15 months of age ( ). Our data demonstrate that G2019S-LRRK2 expression does not influence locomotor activity or prepulse inhibition of the acoustic startle reflex in aged mice. Furthermore, our data suggests that R1441C-LRRK2 mice exhibit a progressive impairment of locomotor activity.
A-D, In the open field G2019S LRRK2 TG mice (line 340) exhibit normal horizontal ( A ) and vertical ( B ) locomotor activity compared to NTG littermates at 6 and 15 months ( n = 7-9 mice/genotype). R1441C LRRK2 TG mice (line 574) reveal normal locomotor activity at 6 months but deficits at 15 months ( C and D ) compared to NTG mice ( n = 6-9 mice/genotype). Data represent the number of beam breaks during the first 15 min period. E-F, Normal pre-pulse inhibition (PPI) of the acoustic startle response in LRRK2 transgenic mice. E and F, G2019S and R1441C LRRK2 TG mice display normal PPI of the acoustic startle reflex at 15 months compared to their NTG littermates, with increasing pre-pulse tones of 74-90 dB ( n = 6-8 mice/genotype). Data are expressed as % PPI relative to no pre-pulse tone. Bars present the mean ± SEM. * P <0.05 comparing TG with NTG as indicated.
LRRK2 has been shown to regulate the morphology of neuronal processes in cultured primary cortical neurons,. To further explore this phenotype in our LRRK2 transgenic mice, primary midbrain cultures were prepared from the ventral mesencephalon of G2019S-LRRK2 transgenic mice (line 340) and their non-transgenic littermates. These cultures typically contain 5-10% of TH+ dopaminergic neurons (unpublished observation). Sholl analysis was conducted to provide a measure of neuritic complexity ( and ). At days-in-vitro (DIV) 3, developing dopaminergic neurons from G2019S-LRRK2 mice exhibit significantly reduced overall neurite complexity manifesting as shorter neurites but with modestly increased neurite branching compared to non-transgenic neurons ( and ). However, at DIV 7 when dopaminergic neurite outgrowth is fully established, G2019S-LRRK2 dopaminergic neurons reveal a dramatic reduction of neurite length and branching with an overall reduction in neurite complexity (, ). We did not perform similar measurements on cultured dopaminergic neurons derived from R1441C-LRRK2 mice (line 574) since these transgenic mice do not exhibit transgene expression within the nigrostriatal...
Protein extracts were prepared from hemi-brains of 2-4 month-old mice as previously described by homogenization in TNE buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 X Complete protease inhibitor cocktail [Roche], 1 X phosphatase inhibitor cocktail I and II [Sigma-Aldrich, St. Louis, MO, USA]). Protein concentration was determined by BCA method (Pierce Biotechnology, Rockford, IL, USA) and 75 µg of protein was resolved by SDS-PAGE, transferred to nitrocellulose and probed with rabbit anti-LRRK2 antibodies recognizing human/mouse (JH5514 or monoclonal c81-8, Epitomics, Inc, Burlingame, CA) or human-specific (NB300-267, Novus Biologicals, Littleton, CO, USA ) epitopes, or mouse β-tubulin (TUB 2.1, Sigma-Aldrich) antibody. Refer to www.pdonlineresearch.org for detailed characterization of rabbit monoclonal LRRK2 antibody (clone c81-8, Epitomics, Inc). Quantitation of total LRRK2 and β-tubulin protein levels by densitometry was conducted using LabImage 1D software (Kapelan Bio-Imaging Solutions, Leipzig, Germany) on Western blot images captured using a FujiFilm LAS-4000 Luminescent Image Analysis system.
HPLC with electrochemical detection was employed to measure the concentration of the biogenic amines, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine (NE) as previously described. Samples were prepared from dissected brain regions and injected onto a C-18 80 × 4.6 mm column (ESA Inc., Chelmsford, MA, USA) with detection using a 2-channel Coulochem II electrochemical detector (ES Inc.). Data were processed on an EZChrome Elite Client Workstation (ESA Inc.) with biogenic amine concentration expressed as ng per mg protein.
Mice were anesthetized with sodium pentobarbital (150 mg/kg) before intracardial perfusion with cold PBS and 4% paraformaldehyde (PFA). Brains were removed, post-fixed with PFA overnight and cryopreserved in 30% sucrose in PBS at 4°C for 48 h. Coronal midbrain sections (40-µm) were prepared and processed for immunohistochemistry with rabbit anti-TH antibody (Novus Biologicals) and Nissl counterstain as previously described.
For TEM analysis, mice were perfused with fixative (3% PFA, 1.5% glutaraldehyde, 100 mM cacodylate, 2.5% sucrose, pH 7.4) and post-fixed for 1 h. Cortex and striatal sections were processed as described. Sections were post-fixed in Palade's OsO 4, en bloc stained in Kellenberger's uranyl acetate, dehydrated, and flat-embedded in epon. 80 nm en face sections were prepared on a Leica UCT ultramicrotome, collected onto 400 mesh high transmission nickel grids, and post-stained with lead and uranyl acetate. Images were collected on a Philips EM 410 TEM equipped with a Soft Imaging System Megaview III digital camera.
Cultures were fixed with 4% PFA and processed for immunocytochemistry with rabbit anti-TH (Novus Biologicals) and mouse anti-MAP2 (Sigma-Aldrich) antibodies and anti-rabbit IgG-AlexaFluor-546 and anti-mouse IgG-AlexaFluor-488 antibodies (Invitrogen). Fluorescent images were captured using a Leica DMI 4000 inverted fluorescence microscope (Leica) at 10x magnification. Sholl analysis was performed on TH+/MAP+ neurons using NIH ImageJ software with a Sholl analysis plug-in (v1.0) developed by the laboratory of Anirvan Ghosh ( http://www.biology.ucsd.edu/labs/ghosh/software/index.html ) to quantify neuritic complexity. Briefly, we constructed continuous concentric circles centered upon the neuronal soma to measure the number of intersections of dendrites with circles of increasing radii. Sholl analysis was performed using the semi-log method and measurements are shown in.
Machine-readable layer
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"name": "Normal Locomotor Activity and Prepulse Inhibition in G2019S LRRK2 Transgenic Mice",
"text": "To investigate whether G2019S mutant LRRK2 expression influences motor performance, we measured locomotor activity in the open field quadrant ( ). G2019S-LRRK2 mice display normal locomotor activity in the open field at 6 and 15 months of age ( ). In contrast, R1441C-LRRK2 mice exhibit a significant reduction in horizontal and vertical locomotor activity at 15 months compared to their non-transgenic littermate mice that is not evident at 6 months ( ). We also assessed prepulse inhibition of the acoustic startle reflex, a measure of sensorimotor gating that can be modulated in part by dopaminergic neurotransmission. However, G2019S- and R1441C-LRRK2 transgenic mice do not perform differently from their non-transgenic littermates when tested at 6 months (data not shown) and 15 months of age ( ). Our data demonstrate that G2019S-LRRK2 expression does not influence locomotor activity or..."
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"name": "Normal Locomotor Activity and Prepulse Inhibition in G2019S LRRK2 Transgenic Mice",
"text": "A-D, In the open field G2019S LRRK2 TG mice (line 340) exhibit normal horizontal ( A ) and vertical ( B ) locomotor activity compared to NTG littermates at 6 and 15 months ( n = 7-9 mice/genotype). R1441C LRRK2 TG mice (line 574) reveal normal locomotor activity at 6 months but deficits at 15 months ( C and D ) compared to NTG mice ( n = 6-9 mice/genotype). Data represent the number of beam breaks during the first 15 min period. E-F, Normal pre-pulse inhibition (PPI) of the acoustic startle response in LRRK2 transgenic mice. E and F, G2019S and R1441C LRRK2 TG mice display normal PPI of the acoustic startle reflex at 15 months compared to their NTG littermates, with increasing pre-pulse tones of 74-90 dB ( n = 6-8 mice/genotype). Data are expressed as % PPI relative to no pre-pulse tone. Bars present the mean \u0011..."
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"name": "Reduced Complexity of Dopaminergic Neurites in Primary Midbrain Cultures from G2019S LRRK2 Transgenic Mice",
"text": "LRRK2 has been shown to regulate the morphology of neuronal processes in cultured primary cortical neurons,. To further explore this phenotype in our LRRK2 transgenic mice, primary midbrain cultures were prepared from the ventral mesencephalon of G2019S-LRRK2 transgenic mice (line 340) and their non-transgenic littermates. These cultures typically contain 5-10% of TH+ dopaminergic neurons (unpublished observation). Sholl analysis was conducted to provide a measure of neuritic complexity ( and ). At days-in-vitro (DIV) 3, developing dopaminergic neurons from G2019S-LRRK2 mice exhibit significantly reduced overall neurite complexity manifesting as shorter neurites but with modestly increased neurite branching compared to non-transgenic neurons ( and ). However, at DIV 7 when dopaminergic neurite outgrowth is fully established, G2019S-LRRK2 dopaminergic neurons reveal a dramatic..."
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"name": "Western blot analysis",
"text": "Protein extracts were prepared from hemi-brains of 2-4 month-old mice as previously described by homogenization in TNE buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 X Complete protease inhibitor cocktail [Roche], 1 X phosphatase inhibitor cocktail I and II [Sigma-Aldrich, St. Louis, MO, USA]). Protein concentration was determined by BCA method (Pierce Biotechnology, Rockford, IL, USA) and 75 µg of protein was resolved by SDS-PAGE, transferred to nitrocellulose and probed with rabbit anti-LRRK2 antibodies recognizing human/mouse (JH5514 or monoclonal c81-8, Epitomics, Inc, Burlingame, CA) or human-specific (NB300-267, Novus Biologicals, Littleton, CO, USA ) epitopes, or mouse β-tubulin (TUB 2.1, Sigma-Aldrich) antibody. Refer to www.pdonlineresearch.org for detailed characterization of rabbit monoclonal LRRK2 antibody (clone c81-8, Epitomics, Inc). Quan..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Measurement of biogenic amines by HPLC",
"text": "HPLC with electrochemical detection was employed to measure the concentration of the biogenic amines, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine (NE) as previously described. Samples were prepared from dissected brain regions and injected onto a C-18 80 × 4.6 mm column (ESA Inc., Chelmsford, MA, USA) with detection using a 2-channel Coulochem II electrochemical detector (ES Inc.). Data were processed on an EZChrome Elite Client Workstation (ESA Inc.) with biogenic amine concentration expressed as ng per mg protein."
},
{
"@type": "HowToStep",
"position": 6,
"name": "TH immunohistochemistry and stereological assessments",
"text": "Mice were anesthetized with sodium pentobarbital (150 mg/kg) before intracardial perfusion with cold PBS and 4% paraformaldehyde (PFA). Brains were removed, post-fixed with PFA overnight and cryopreserved in 30% sucrose in PBS at 4°C for 48 h. Coronal midbrain sections (40-µm) were prepared and processed for immunohistochemistry with rabbit anti-TH antibody (Novus Biologicals) and Nissl counterstain as previously described."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Transmission electron microscopy (TEM)",
"text": "For TEM analysis, mice were perfused with fixative (3% PFA, 1.5% glutaraldehyde, 100 mM cacodylate, 2.5% sucrose, pH 7.4) and post-fixed for 1 h. Cortex and striatal sections were processed as described. Sections were post-fixed in Palade's OsO 4, en bloc stained in Kellenberger's uranyl acetate, dehydrated, and flat-embedded in epon. 80 nm en face sections were prepared on a Leica UCT ultramicrotome, collected onto 400 mesh high transmission nickel grids, and post-stained with lead and uranyl acetate. Images were collected on a Philips EM 410 TEM equipped with a Soft Imaging System Megaview III digital camera."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Primary midbrain cultures and analysis of neuritic complexity",
"text": "Cultures were fixed with 4% PFA and processed for immunocytochemistry with rabbit anti-TH (Novus Biologicals) and mouse anti-MAP2 (Sigma-Aldrich) antibodies and anti-rabbit IgG-AlexaFluor-546 and anti-mouse IgG-AlexaFluor-488 antibodies (Invitrogen). Fluorescent images were captured using a Leica DMI 4000 inverted fluorescence microscope (Leica) at 10x magnification. Sholl analysis was performed on TH+/MAP+ neurons using NIH ImageJ software with a Sholl analysis plug-in (v1.0) developed by the laboratory of Anirvan Ghosh ( http://www.biology.ucsd.edu/labs/ghosh/software/index.html ) to quantify neuritic complexity. Briefly, we constructed continuous concentric circles centered upon the neuronal soma to measure the number of intersections of dendrites with circles of increasing radii. Sholl analysis was performed using the semi-log method and measurements are shown in."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Stereological assessment of TH+/Nissl+ neurons"
},
{
"@type": "HowToTool",
"name": "Normal Locomotor Activity and Prepulse Inhibition in G2019S LRRK2 Transgenic Mice"
},
{
"@type": "HowToTool",
"name": "Normal Locomotor Activity and Prepulse Inhibition in G2019S LRRK2 Transgenic Mice"
},
{
"@type": "HowToTool",
"name": "Generation of LRRK2 transgenic mice"
},
{
"@type": "HowToTool",
"name": "Western blot analysis"
},
{
"@type": "HowToTool",
"name": "Immunofluorescence and confocal microscopy"
},
{
"@type": "HowToTool",
"name": "Transmission electron microscopy (TEM)"
},
{
"@type": "HowToTool",
"name": "Transmission electron microscopy (TEM)"
}
],
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"@type": "HowToSupply",
"name": "Generation of LRRK2 transgenic mice"
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{
"@type": "HowToSupply",
"name": "Western blot analysis"
},
{
"@type": "HowToSupply",
"name": "TH immunohistochemistry and stereological assessments"
},
{
"@type": "HowToSupply",
"name": "Immunohistochemistry"
},
{
"@type": "HowToSupply",
"name": "Immunofluorescence and confocal microscopy"
},
{
"@type": "HowToSupply",
"name": "Supporting Information"
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"headline": "Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2",
"datePublished": "2011",
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"name": "Jaekwang Kim"
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"name": "Deborah A. Swing"
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