Motor neuron intrinsic and extrinsic mechanisms contribute to the pathogenesis of FUS -associated amyotrophic lateral sclerosis methods
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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
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Gait recovery and locomotor function
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- Per-run gait capture with paw placement, timing, and stride features for each animal
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- Cleaned gait metrics table and recovery trend summary across timepoints
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- Group comparisons of gait indices, stride metrics, or recovery curves
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Quantify the primary readouts for this experiment: Gait recovery and locomotor function.
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Report representative outputs alongside summary comparisons for Gait recovery and locomotor function.
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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...
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