Motor neuron intrinsic and extrinsic mechanisms contribute to the pathogenesis of FUS -associated amyotrophic lateral sclerosis methods
Aim. Evidence-backed execution summary for Motor neuron intrinsic and extrinsic mechanisms contribute to the pathogenesis of FUS -associated amyotrophic lateral sclerosis methods from Motor neuron intrinsic and extrinsic mechanisms contribute to the pathogenesis of FUS -associated amyotrophic lateral sclerosis.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Subcellular fractionation and western blotting
reagent used in the protocol.
- Use
- Nuclear and cytoplasmic fractions were prepared from fresh spinal cord tissue using the NE-PER ® Nuclear and Cytoplasmic Extraction reagents (Thermo Scientific) according to the manufacturer. Protein concentration was quantitated using the BCA protein assay kit (Pierce). Equal amounts of protein (10 µ...
Subcellular fractionation and western blotting
reagent used in the protocol.
- Use
- For western blot on protein extracts from spinal cord of Fus + / - and Fus + / + mice, rabbit anti-FUS (Bethyl A300-294A) was used as a primary antibody in a 1:800 dilution. A mouse monoclonal anti-beta-tubulin antibody (clone E7, DSHB, 1:2000) was used as a loading control. As secondary antibodies, HRP-conjug...
Spinal cord histology
reagent used in the protocol.
- Use
- Animals were anesthetized with ketamine (Imalgene 80 mg/kg; Merial, Lyon, France) and xylazine (Rompun 20 mg/kg; Bayer, Lyon, France) and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4. Spinal cord were dissected and fixed by immersion in 4% paraformaldeh...
Spinal cord histology
reagent used in the protocol.
- Use
- Spinal cord sections were stained using rabbit anti-FUS antibody against the FUS N-terminal part (ProteinTech 11570, 1:100), goat anti-ChAT (Millipore AB144-P, diluted 1:50), and Hoechst (Sigma 33342, 1:1000) followed by fluorescently labeled secondary antibodies donkey anti-rabbit Alexa 488 (Jackson A21206), goat a...
Electromyography
reagent used in the protocol.
- Use
- Electromyography was performed as previously described [, ]. Mice at 10 and 22 month of age were anesthetized with a solution of ketamine/xylazine (100 mg/kg; 5 mg/kg) and electrical activity was recorded using a monopolar needle electrode (diameter 0.3 mm; 9013R0312; Medtronic, Minneapolis, MN,...
Electromyography
reagent used in the protocol.
- Use
- Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously [ ]. Briefly, CMAPs were elicited by supramaximal square pulses, of 0.2 ms duration, delivered with a monopolar needle electrode to the sciatic nerve at the sciatic notch level. CMAPs were measured by a monopo...
Spinal cord motor neurons quantifications
reagent used in the protocol.
- Use
- To quantify lower motor neurons, spinal cord cryostat sections of 16 µm were stained with 0.1% Cresyl violet acetate (Certistain ®, MERK#5235) and anti-ChAT (Millipore, AB144-P; diluted 1:50) followed by biotinylated donkey anti-goat IgG (Jackson, 705-066-147; 1:250) as secondary antibody. The staini...
RNAseq
reagent used in the protocol.
- Use
- Total RNA from spinal cords (including dorsal and ventral roots) of Fus ΔNLS/ + ( n = 4) and their control littermates ( n = 4) were extracted with TRIzol (Invitrogen). RNA quality was measured using the Agilent Bioanalyzer system or RNA screen Tape (Agilent technologies) according to the...
Subcellular fractionation and western blotting
Nuclear and cytoplasmic fractions were prepared from fresh spinal cord tissue using the NE-PER ® Nuclear and Cytoplasmic Extraction reagents (Thermo Scientific) according to the manufacturer. Protein concentration was quantitated using the BCA protein assay kit (Pierce). Equal amounts of protein (10 µ...
- Use
- Nuclear and cytoplasmic fractions were prepared from fresh spinal cord tissue using the NE-PER ® Nuclear and Cytoplasmic Extraction reagents (Thermo Scientific) according to the manufacturer. Protein concentration was quantitated using the BCA protein assay kit (Pierce). Equal amounts of protein (10 µ...
Imaging
Single-layer images were acquired using a laser-scanning microscope (confocal Leica SP5 Leica Microsystems CMS GmbH) equipped with × 63 oil objective (NA1.4). Excitation rays are sequential: Hoechst 33342 was excited using diode 405 nm, Alexa 488 by the argon laser 488 nm, Alexa 594 by diode 561 n...
- Use
- Single-layer images were acquired using a laser-scanning microscope (confocal Leica SP5 Leica Microsystems CMS GmbH) equipped with × 63 oil objective (NA1.4). Excitation rays are sequential: Hoechst 33342 was excited using diode 405 nm, Alexa 488 by the argon laser 488 nm, Alexa 594 by diode 561 n...
Motor coordination and muscle strength analysis
Mice were followed weekly for general health, neurological symptoms, body weight, grip test and accelerating rotarod performances starting from weaning (4 weeks of age) until 22 months of age as described previously [ ]. Briefly, mouse motor performance was assessed using rotarod (Ugobasile model 7650). Ea...
- Use
- Mice were followed weekly for general health, neurological symptoms, body weight, grip test and accelerating rotarod performances starting from weaning (4 weeks of age) until 22 months of age as described previously [ ]. Briefly, mouse motor performance was assessed using rotarod (Ugobasile model 7650). Ea...
Inverted grid test
The four limbs hang test uses a wire grid system to non-invasively measure the ability of mice to use sustained limb tension to oppose their gravitational force. The procedure measures 4 limbs hang time in seconds as well as the minimal holding impulse. Each mouse was placed at the simple cage grid and was allowed t...
- Use
- The four limbs hang test uses a wire grid system to non-invasively measure the ability of mice to use sustained limb tension to oppose their gravitational force. The procedure measures 4 limbs hang time in seconds as well as the minimal holding impulse. Each mouse was placed at the simple cage grid and was allowed t...
Gait analysis
Gait parameters of freely moving mice were measured using the CatWalk gait analysis system (Noldus Information Technology, The Netherlands). The CatWalk instrument consists of a hardware system of a long, enclosed glass walkway plate, illuminated with green light, a high-speed video camera, and a software package fo...
- Use
- Gait parameters of freely moving mice were measured using the CatWalk gait analysis system (Noldus Information Technology, The Netherlands). The CatWalk instrument consists of a hardware system of a long, enclosed glass walkway plate, illuminated with green light, a high-speed video camera, and a software package fo...
Gait analysis
The recordings were carried out when the room was completely dark, except for computer screen. Each mouse was placed individually in the CatWalk walkway and allowed to walk freely, in an unforced manner and traverse from side to side the walkway glass plate. Mouse tracks that were straight without any interruption o...
- Use
- The recordings were carried out when the room was completely dark, except for computer screen. Each mouse was placed individually in the CatWalk walkway and allowed to walk freely, in an unforced manner and traverse from side to side the walkway glass plate. Mouse tracks that were straight without any interruption o...
Electromyography
Electromyography was performed as previously described [, ]. Mice at 10 and 22 month of age were anesthetized with a solution of ketamine/xylazine (100 mg/kg; 5 mg/kg) and electrical activity was recorded using a monopolar needle electrode (diameter 0.3 mm; 9013R0312; Medtronic, Minneapolis, MN,...
- Use
- Electromyography was performed as previously described [, ]. Mice at 10 and 22 month of age were anesthetized with a solution of ketamine/xylazine (100 mg/kg; 5 mg/kg) and electrical activity was recorded using a monopolar needle electrode (diameter 0.3 mm; 9013R0312; Medtronic, Minneapolis, MN,...
Electromyography
Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously [ ]. Briefly, CMAPs were elicited by supramaximal square pulses, of 0.2 ms duration, delivered with a monopolar needle electrode to the sciatic nerve at the sciatic notch level. CMAPs were measured by a monopo...
- Use
- Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously [ ]. Briefly, CMAPs were elicited by supramaximal square pulses, of 0.2 ms duration, delivered with a monopolar needle electrode to the sciatic nerve at the sciatic notch level. CMAPs were measured by a monopo...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- For the animal experiments with two groups, the values from each animal were averaged for each genotype group and analyzed by unpaired Student's t test, two-tailed. Comparison of three or four groups was performed using one-way ANOVA and Tukey post hoc test. Data were analyzed by using the Graphics Prism Progr...
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Inverted grid test
The four limbs hang test uses a wire grid system to non-invasively measure the ability of mice to use sustained limb tension to oppose their gravitational force. The procedure measures 4 limbs hang time in seconds as well as the minimal holding impulse. Each mouse was placed at the simple cage grid and was allowed to accommodate to this environment for 3-5 s before the grid was inverted and held approximately 35 cm over a mouse cage containing 5-6 cm of bedding (wood chips). Each of these holding periods began with all four paws of the mouse grasping the grid. The wire grid hanging time (or "hang time") was defined as the amount of time that it takes the mouse to fall down from the inverted grid and was measured visually with a stop watch. In each session, the procedure was repeated three times with approximately 10 min between each assessme...
Gait analysis
The recordings were carried out when the room was completely dark, except for computer screen. Each mouse was placed individually in the CatWalk walkway and allowed to walk freely, in an unforced manner and traverse from side to side the walkway glass plate. Mouse tracks that were straight without any interruption or hesitation were treated as successful runs. Runs with any wall climbing, grooming, and staying on the walkway were not analyzed. An average number of 3 replicate crossings made by each mouse were recorded. The CatWalk software was used to analyze crossings that had at least five cycles of complete steps. The software automatically labeled all areas containing pixels above the set threshold. These areas were identified and assigned to the respective paws. Analysis of the recording generated a wide range of parameters from which the following gait and coordination parameter...
Electromyography
Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously [ ]. Briefly, CMAPs were elicited by supramaximal square pulses, of 0.2 ms duration, delivered with a monopolar needle electrode to the sciatic nerve at the sciatic notch level. CMAPs were measured by a monopolar needle electrode inserted in the gastrocnemius, and the system was grounded by subcutaneously inserted monopolar needle electrodes in the back and the tail of the animal. Amplitudes (mV) from the left and right muscle-evoked responses were measured and averaged, resulting in one average CMAP amplitude per animal, which was used for statistical analysis. The latency was measured as the time from the given electrical stimulus to the appearance of a muscle response-the initial CMAP deflection from the baseline.
Cytoplasmic mislocalization of FUS leads to a mild motor deficit in Fus ΔNLS/ + mice
Indeed, despite normal performance on rotarod and grip strength, Fus ΔNLS/ + mice displayed a significantly shorter hanging time in an inverted grid test (Fig. a), both at 10 and 22 months of age. Holding impulse, which represents the total sustained force exerted by the mouse to oppose the gravitational force [ ], was also significantly decreased in Fus ΔNLS/ + mice (Fig. b). The evaluation of gait performance by CatWalk analysis further confirmed this motor defect. Fus ΔNLS/ + mice demonstrated an irregular walking pattern characterized by phases of fast walking interrupted with stance phases (Fig. c). We also observed a reduction of hind limb stride length (Fig. d) associated with an increase in body speed variation (i.e., the variation in the speed of the walking mouse) for both ages compared to the control mice (Fig. g). In additi...
Oligodendrocytic alterations in Fus ΔNLS/ + spinal cord
To evaluate the molecular mechanisms underlying the phenotypes in Fus ΔNLS/ + mice, we performed RNAseq on spinal cord RNA extracts from 22-month-old Fus ΔNLS/ + and control littermates. Among the genes showing differential expression, several of them encoded for proteins related to myelination (Fig. a). Indeed, mRNA levels of myocilin, Ncmap, Pmp2, Pmp22, Cldn19 and Prx, were all downregulated in RNAseq from Fus ΔNLS/ + mice (Fig. a), and this was confirmed using RT-qPCR on samples obtained from an independent cohort of mice (Fig. b). For instance, Myocilin is required for peripheral myelination [ ], as is Pmp2 [ ] and Prx [ ]. Pmp22 [ ] and Prx [ ] are involved in the morphology of myelinating Schwann cells. Interestingly, we did not observe altered expression of other major myelin genes such as Mbp, Plp1 or Abca1 (Supplementary Fig. 7). Increa...
Measurement outputs
What raw and processed outputs should exist?
Wild type and heterozygous Fus ∆NLS/+ mice and heterozygous Chat -CRE mice, generated as described previously [ ], were bred and housed in the central animal facility of 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
For western blot on protein extracts from spinal cord of Fus + / - and Fus + / + mice, rabbit anti-FUS (Bethyl A300-294A) was used as a primary antibody in a 1:800 dilutio...
- 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
Animals were anesthetized with ketamine (Imalgene 80 mg/kg; Merial, Lyon, France) and xylazine (Rompun 20 mg/kg; Bayer, Lyon, France) and perfused transcardially with...
- 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
Mice were followed weekly for general health, neurological symptoms, body weight, grip test and accelerating rotarod performances starting from weaning (4 weeks of age) unt...
- 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 run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously [ ].
from paperScoring or quantification
Quantify the primary readouts for this experiment: Wild type and heterozygous Fus ∆NLS/+ mice and heterozygous Chat -CRE mice, generated as described previously [ ], were bred and housed in the central animal facility of t...; For western blot on protein extracts from spinal cord of Fus + / - and Fus + / + mice, rabbit anti-FUS (Bethyl A300-294A) was used as a primary antibody in a 1:800 dilutio...; Animals were anesthetized with ketamine (Imalgene 80 mg/kg; Merial, Lyon, France) and xylazine (Rompun 20 mg/kg; Bayer, Lyon, France) and perfused transcardially with...; Mice were followed weekly for general health, neurological symptoms, body weight, grip test and accelerating rotarod performances starting from weaning (4 weeks of age) unt....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously [ ]. Briefly, CMAPs were elicited by supramaximal square pulses, of 0.2...; Total RNA from spinal cords (including dorsal and ventral roots) of Fus ΔNLS/ + ( n = 4) and their control littermates ( n = 4) were extracted with TR...; For the animal experiments with two groups, the values from each animal were averaged for each genotype group and analyzed by unpaired Student's t test, two-tailed. Compar...; To determine whether this mild motor phenotype could be due to an underlying motor neuron disease, we performed electromyography analysis (EMG) on Fus + / + and Fus ΔNLS/ +...
from paperReporting output
Report representative outputs alongside summary comparisons for Wild type and heterozygous Fus ∆NLS/+ mice and heterozygous Chat -CRE mice, generated as described previously [ ], were bred and housed in the central animal facility of t..., For western blot on protein extracts from spinal cord of Fus + / - and Fus + / + mice, rabbit anti-FUS (Bethyl A300-294A) was used as a primary antibody in a 1:800 dilutio..., Animals were anesthetized with ketamine (Imalgene 80 mg/kg; Merial, Lyon, France) and xylazine (Rompun 20 mg/kg; Bayer, Lyon, France) and perfused transcardially with..., Mice were followed weekly for general health, neurological symptoms, body weight, grip test and accelerating rotarod performances starting from weaning (4 weeks of age) unt....
inferred from protocolStructured statistical methods
Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously [ ]. Briefly, CMAPs were elicited by supramaximal square pulses, of 0.2...; Total RNA from spinal cords (including dorsal and ventral roots) of Fus ΔNLS/ + ( n = 4) and their control littermates ( n = 4) were extracted with TR...; For the animal experiments with two groups, the values from each animal were averaged for each genotype group and analyzed by unpaired Student's t test, two-tailed. Compar...; To determine whether this mild motor phenotype could be due to an underlying motor neuron disease, we performed electromyography analysis (EMG) on Fus + / + and Fus ΔNLS/ +...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
The four limbs hang test uses a wire grid system to non-invasively measure the ability of mice to use sustained limb tension to oppose their gravitational force. The procedure measures 4 limbs hang time in seconds as well as the minimal holding impulse. Each mouse was placed at the simple cage grid and was allowed to accommodate to this environment for 3-5 s before the grid was inverted and held approximately 35 cm over a mouse cage containing 5-6 cm of bedding (wood chips). Each of these holding periods began with all four paws of the mouse grasping the grid. The wire grid hanging time (or "hang time") was defined as the amount of time that it takes the mouse to fall down from the inverted grid and was measured visually with a stop watch. In each session, the procedure was repeated three times with approximately 10 min between each assessment of holding time. The mouse body weight was obtained shortly before the test. The physical impulse (holding impulse) is the hanging time multiplied by the gravitational force of the mouse [body mass (g) × 0.00980665 N/g × hanging time (s)]. This parameter represe...
The recordings were carried out when the room was completely dark, except for computer screen. Each mouse was placed individually in the CatWalk walkway and allowed to walk freely, in an unforced manner and traverse from side to side the walkway glass plate. Mouse tracks that were straight without any interruption or hesitation were treated as successful runs. Runs with any wall climbing, grooming, and staying on the walkway were not analyzed. An average number of 3 replicate crossings made by each mouse were recorded. The CatWalk software was used to analyze crossings that had at least five cycles of complete steps. The software automatically labeled all areas containing pixels above the set threshold. These areas were identified and assigned to the respective paws. Analysis of the recording generated a wide range of parameters from which the following gait and coordination parameters were analyzed: Stride length (distance between two consecutive paw placements of the same paw in pixel), swing speed (distance between two consecutive paw placements of the same paw per second), body speed (distance that the animal walks per second) and body speed variation (regularity of body spe...
Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously [ ]. Briefly, CMAPs were elicited by supramaximal square pulses, of 0.2 ms duration, delivered with a monopolar needle electrode to the sciatic nerve at the sciatic notch level. CMAPs were measured by a monopolar needle electrode inserted in the gastrocnemius, and the system was grounded by subcutaneously inserted monopolar needle electrodes in the back and the tail of the animal. Amplitudes (mV) from the left and right muscle-evoked responses were measured and averaged, resulting in one average CMAP amplitude per animal, which was used for statistical analysis. The latency was measured as the time from the given electrical stimulus to the appearance of a muscle response-the initial CMAP deflection from the baseline.
Indeed, despite normal performance on rotarod and grip strength, Fus ΔNLS/ + mice displayed a significantly shorter hanging time in an inverted grid test (Fig. a), both at 10 and 22 months of age. Holding impulse, which represents the total sustained force exerted by the mouse to oppose the gravitational force [ ], was also significantly decreased in Fus ΔNLS/ + mice (Fig. b). The evaluation of gait performance by CatWalk analysis further confirmed this motor defect. Fus ΔNLS/ + mice demonstrated an irregular walking pattern characterized by phases of fast walking interrupted with stance phases (Fig. c). We also observed a reduction of hind limb stride length (Fig. d) associated with an increase in body speed variation (i.e., the variation in the speed of the walking mouse) for both ages compared to the control mice (Fig. g). In addition, 22-month-old Fus ΔNLS/ + mice showed impaired swing and body speed (Fig. e-f). Thus, expression of mutant FUS at a physiological level is associated with partial cytoplasmic mislocalization of the protein and a mild motor deficit in mice. Fig. 3 Fus ΔNLS /+ mice displa...
To evaluate the molecular mechanisms underlying the phenotypes in Fus ΔNLS/ + mice, we performed RNAseq on spinal cord RNA extracts from 22-month-old Fus ΔNLS/ + and control littermates. Among the genes showing differential expression, several of them encoded for proteins related to myelination (Fig. a). Indeed, mRNA levels of myocilin, Ncmap, Pmp2, Pmp22, Cldn19 and Prx, were all downregulated in RNAseq from Fus ΔNLS/ + mice (Fig. a), and this was confirmed using RT-qPCR on samples obtained from an independent cohort of mice (Fig. b). For instance, Myocilin is required for peripheral myelination [ ], as is Pmp2 [ ] and Prx [ ]. Pmp22 [ ] and Prx [ ] are involved in the morphology of myelinating Schwann cells. Interestingly, we did not observe altered expression of other major myelin genes such as Mbp, Plp1 or Abca1 (Supplementary Fig. 7). Increased cytoplasmic FUS staining was observed in Fus ΔNLS/ + oligodendrocytes using double immunofluorescence for FUS and oligodendrocyte specific markers CNPase and carbonic anhydrase II (Fig. c; supplementary Fig. 8). Oligodendrocytes were more numerous in ventral horn white matter of...
Machine-readable layer
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"name": "Motor neuron intrinsic and extrinsic mechanisms contribute to the pathogenesis of FUS -associated amyotrophic lateral sclerosis methods",
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"text": "The four limbs hang test uses a wire grid system to non-invasively measure the ability of mice to use sustained limb tension to oppose their gravitational force. The procedure measures 4 limbs hang time in seconds as well as the minimal holding impulse. Each mouse was placed at the simple cage grid and was allowed to accommodate to this environment for 3-5 s before the grid was inverted and held approximately 35 cm over a mouse cage containing 5-6 cm of bedding (wood chips). Each of these holding periods began with all four paws of the mouse grasping the grid. The wire grid hanging time (or \"hang time\") was defined as the amount of time that it takes the mouse to fall down from the inverted grid and was measured visually with a stop watch. In each session, the procedure was repeated three times with approximately 10 min between each assessme..."
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"text": "The recordings were carried out when the room was completely dark, except for computer screen. Each mouse was placed individually in the CatWalk walkway and allowed to walk freely, in an unforced manner and traverse from side to side the walkway glass plate. Mouse tracks that were straight without any interruption or hesitation were treated as successful runs. Runs with any wall climbing, grooming, and staying on the walkway were not analyzed. An average number of 3 replicate crossings made by each mouse were recorded. The CatWalk software was used to analyze crossings that had at least five cycles of complete steps. The software automatically labeled all areas containing pixels above the set threshold. These areas were identified and assigned to the respective paws. Analysis of the recording generated a wide range of parameters from which the following gait and coordination parameter..."
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"text": "Compound muscle action potentials (CMAP) were recorded in gastrocnemius muscle as described previously [ ]. Briefly, CMAPs were elicited by supramaximal square pulses, of 0.2 ms duration, delivered with a monopolar needle electrode to the sciatic nerve at the sciatic notch level. CMAPs were measured by a monopolar needle electrode inserted in the gastrocnemius, and the system was grounded by subcutaneously inserted monopolar needle electrodes in the back and the tail of the animal. Amplitudes (mV) from the left and right muscle-evoked responses were measured and averaged, resulting in one average CMAP amplitude per animal, which was used for statistical analysis. The latency was measured as the time from the given electrical stimulus to the appearance of a muscle response-the initial CMAP deflection from the baseline."
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"name": "Cytoplasmic mislocalization of FUS leads to a mild motor deficit in Fus ΔNLS/ + mice",
"text": "Indeed, despite normal performance on rotarod and grip strength, Fus ΔNLS/ + mice displayed a significantly shorter hanging time in an inverted grid test (Fig. a), both at 10 and 22 months of age. Holding impulse, which represents the total sustained force exerted by the mouse to oppose the gravitational force [ ], was also significantly decreased in Fus ΔNLS/ + mice (Fig. b). The evaluation of gait performance by CatWalk analysis further confirmed this motor defect. Fus ΔNLS/ + mice demonstrated an irregular walking pattern characterized by phases of fast walking interrupted with stance phases (Fig. c). We also observed a reduction of hind limb stride length (Fig. d) associated with an increase in body speed variation (i.e., the variation in the speed of the walking mouse) for both ages compared to the control mice (Fig. g). In additi..."
},
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"name": "Oligodendrocytic alterations in Fus ΔNLS/ + spinal cord",
"text": "To evaluate the molecular mechanisms underlying the phenotypes in Fus ΔNLS/ + mice, we performed RNAseq on spinal cord RNA extracts from 22-month-old Fus ΔNLS/ + and control littermates. Among the genes showing differential expression, several of them encoded for proteins related to myelination (Fig. a). Indeed, mRNA levels of myocilin, Ncmap, Pmp2, Pmp22, Cldn19 and Prx, were all downregulated in RNAseq from Fus ΔNLS/ + mice (Fig. a), and this was confirmed using RT-qPCR on samples obtained from an independent cohort of mice (Fig. b). For instance, Myocilin is required for peripheral myelination [ ], as is Pmp2 [ ] and Prx [ ]. Pmp22 [ ] and Prx [ ] are involved in the morphology of myelinating Schwann cells. Interestingly, we did not observe altered expression of other major myelin genes such as Mbp, Plp1 or Abca1 (Supplementary Fig. 7). Increa..."
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