Mitigating the Functional Deficit after Neurotoxic Motoneuronal Loss by an Inhibitor of Mitochondrial Fission

Abstract

Amyotrophic lateral sclerosis (ALS) is an extremely complex neurodegenerative disease involving different cell types, but motoneuronal loss represents its main pathological feature. Moreover, compensatory plastic changes taking place in parallel to neurodegeneration are likely to affect the timing of ALS onset and progression and, interestingly, they might represent a promising target for disease-modifying treatments. Therefore, a simplified animal model mimicking motoneuronal loss without the other pathological aspects of ALS has been established by means of intramuscular injection of cholera toxin-B saporin (CTB-Sap), which is a targeted neurotoxin able to kill motoneurons by retrograde suicide transport. Previous studies employing the mouse CTB-Sap model have proven that spontaneous motor recovery is possible after a subtotal removal of a spinal motoneuronal pool. Although these kinds of plastic changes are not enough to counteract the functional effects of the progressive motoneuron degeneration, it would nevertheless represent a promising target for treatments aiming to postpone ALS onset and/or delay disease progression. Herein, the mouse CTB-Sap model has been used to test the efficacy of mitochondrial division inhibitor 1 (Mdivi-1) as a tool to counteract the CTB-Sap toxicity and/or to promote neuroplasticity. The homeostasis of mitochondrial fission/fusion dynamics is indeed important for cell integrity, and it could be affected during neurodegeneration. Lesioned mice were treated with Mdivi-1 and then examined by a series of behavioral test and histological analyses. The results have shown that the drug may be capable of reducing functional deficits after the lesion and promoting synaptic plasticity and neuroprotection, thus representing a putative translational approach for motoneuron disorders.

Keywords: Mdivi-1; behavioral test; cholera toxin-B saporin; gastrocnemius muscle; mouse; neurodegeneration; spinal cord; synaptic plasticity.

Figure 1

Schematic representation of the experimental timeline.

Figure 2

Functional analyses of control and lesioned animals. (A) Measurements of the average body weight before and after CTB-Sap lesion and drug administration, in comparison to control (CTRL) animals; asterisks indicate significant differences from pre-lesion levels. (B) Clinical score measurements of CTB-Sap mice with or without the administration of Mdivi-1; asterisks indicate significant differences between groups. (C,D) Number of footfalls relative to all limbs (C) or to the right hindlimb (RHL) only (D) during the weekly execution of the grid walk test; values are expressed as fold increase from pre-lesion levels. In (E,F), the number of footfalls counted during grid walk test at all time points are pooled together and expressed in % of the CTRL values to compare the groups in relation to the total number of errors relative to all limbs (E) or the RHL only (F). DPL: days post-lesion. In all graphs, asterisks indicate significant difference from CTRL animals while (†) indicates significant difference between vehicle-treated and Mdivi-1-treated CTB-Sap animals.

Figure 3

Functional analyses of control and lesioned animals by gait analysis. (A–E) Average position of feet during stance in relation to the body axis in control (CTRL) and lesioned animals (with or without Mdivi-1 treatment) at 7 and 49 days post-lesion; coordinates refer to the average position of tail base (lower red dot), representing the origin of the coordinates system; the upper red dot represents the average position of the neck, while the middle one represents the center of the body; blue dots indicate the average positions of feet for the forelimbs (upper ones) and hindlimbs (lower ones) at the beginning of stance phase (AEP: anterior extreme position), while orange dots indicate the position of feet at the end of stance phase (PEP: posterior extreme position); for each limb, the distance between AEP and PEP is indicated as stance trace length; coordinates are expressed in cm. (F–H) Mean values of stance trace length relative to right hindlimb (RHL), left hindlimb (LHL), and both forelimbs (FLs) together. (I,J) Average limb distance from the body axis, relative to RHL or the other limbs together (non-RHL). In all graphs, asterisks indicate significant differences from CTRL values. All values are normalized to control levels and expressed as mean ± SEM; actual values can be found in the main text.

Figure 4

Functional analyses of control and lesioned animals by gait analysis. (A,B) Mean values of the stride length (distance between two consecutive foot positions at the beginning of the stance phase) relative to the right hindlimb (RHL) or the other limbs together (non-RHL). (C,D) Mean values of the ratio between swing and stance duration for RHL and non-RHL. (E) Average values of the parameter named claudication, which is the ratio between the LHL and RHL swing speed. In all graphs, asterisks indicate significant differences from CTRL values. All values are normalized to control levels and expressed as mean ± SEM; actual values can be found in the main text.

Figure 5

Graphic illustration of motoneuron numbers (A) and their soma size (B) in the lumbar spinal cord of CTB-Sap-lesioned mice treated with either Mdivi-1 or vehicle alone. (C–E) Representative images taken with the fluorescence microscope, showing ChAT-positive MN profiles from a normal (C) spinal cord (contralateral to the lesion side) or from lesioned spinal cord side of vehicle-treated (D) and Mdivi-1-treated mice (E). The asterisks indicate significant difference (p-value < 0.05).

Figure 6

Quantification of Synapsin-I expression by optical density measurement (A) in the lamina IX of the lumbar spinal region containing the motoneurons innervating the gastrocnemius muscle. (B–G) Representative fluorescence images showing the expression of Synapsin-I (green) surrounding the ChAT-positive MN profiles (red) in normal (B,C), lesioned and vehicle-treated (D,E) or Mdivi-1-treated (F,G) spinal cord. Arrowheads indicate synaptic contacts with the motoneuronal membrane. The asterisks indicate significant difference (p-value < 0.05).

Figure 7

Graphic illustration of muscle atrophy and denervation after CTB-Sap lesion. The graphs represent the measurement of muscle fiber cross-sectional area (A) and the muscle weight (B) of the right (CTB-Sap-injected) and left (intact) gastrocnemius muscles, after treatment with either Mdivi-1 or vehicle alone; asterisks (*) indicate significant difference from contralateral side, while (†) indicates significant difference from vehicle-treated mice. (C) Representative images of hematoxylin–eosin stained sections showing the evident reduction of fiber diameter after lesion, compared to the intact side (right panel); arrows indicate centrally located nuclei in muscle fibers of lesioned animals. (D–F) Examples of EMG signals showing the absence of spontaneous muscle activity in normal muscles of anesthetized mice (D) compared to the presence of fibrillations (E) and positive sharp waves (F) at six weeks after CTB-Sap injection.