Dynamic neuromuscular remodeling precedes motor-unit loss in a mouse model of ALS
Amyotrophic lateral sclerosis ( ALS ) is a fatal neurodegenerative disease characterized by upper and lower motor neuron ( MN ) loss . Loss of neuromuscular junctions ( NMJs ) is a key pathological event in ALS patients ( Fischer et al . , 2004; Killian et al . , 1994 ) and in animal models of the disease ( Clark et al . , 2016; Fischer et al . , 2004; Frey et al . , 2000; Pun et al . , 2006; Tallon et al . , 2016 ) . Denervation of NMJs is observed prior to symptom onset ( Clark et al . , 2016; Fischer et al . , 2004; Pun et al . , 2006; Tallon et al . , 2016; Vinsant et al . , 2013 ) and before any significant MN axonal or cell body loss ( Fischer et al . , 2004; Vinsant et al . , 2013 ) . However , whether early NMJ denervation arises from local branch-specific degeneration ( ‘dying-back hypothesis’ ) or is merely the manifestation of a diseased and globally degenerating motor neuron remains ill-defined . There is considerable evidence which suggests that a subset of degenerating motor-units ( MU , a motor neuron and the muscle fibers it innervates ) swiftly lose their NMJs while a distinct , yet unaffected , population compensates via axonal sprouting ( Pun et al . , 2006; Schaefer et al . , 2005 ) , somewhat akin to a nerve injury . Indeed , NMJs innervated by fast-fatigable MNs are lost early , followed by fast-fatigue resistant and slow MNs ( Frey et al . , 2000; Pun et al . , 2006 ) . This is further supported by electromyographic ( EMG ) recordings showing that the number of MUs declines in parallel with muscle strength ( Azzouz et al . , 1997; Dantes and McComas , 1991; Hegedus et al . , 2007; Hegedus et al . , 2008; Kennel et al . , 1996; McComas et al . , 1971 ) and that surviving MUs increase in size in animal models and in patients ( Dantes and McComas , 1991; Dengler et al . , 1990; McComas et al . , 1971; Schmied et al . , 1999 ) . Taken together , these results strongly suggest that the loss of NMJs within a single MU is a dichotomous and mostly synchronous event and that local synaptic events have a limited impact on the denervation process . Under this monotonic paradigm , degenerating MUs are irrevocably caught in a degenerative cascade while surviving MUs attempt to compensate , consequently undermining the relevance of the NMJ as a disease-modifying therapeutic target . However , the rescue of neuronal loss in SOD1 mice does not necessarily improve NMJ denervation ( Gould et al . , 2006; Parone et al . , 2013; Suzuki et al . , 2007 ) suggesting that local branch-specific events may play a key role in NMJ denervation . In ALS , local alterations of synaptic function and molecular signaling take place very early at the NMJ ( Arbour et al . , 2015; De Winter et al . , 2006; Jokic et al . , 2006; Rocha et al . , 2013; Taetzsch et al . , 2017; Tremblay et al . , 2017 ) , implying that local , MU-independent , structural changes could occur . Finally , ALS is a non-cell autonomous disease where cell types other than motor neurons contribute to the disease pathogenesis ( Boillée et al . , 2006a; Ilieva et al . , 2009 ) . Contribution of various cell types known to interact with NMJs and motor axons has been suggested ( Arbour et al . , 2015; Chiu et al . , 2009; Keller et al . , 2009; Lobsiger et al . , 2009; Loeffler et al . , 2016; Nardo et al . , 2016; Turner et al . , 2010; Wang et al . , 2012 ) , which could contribute to NMJ loss . In this scenario , asynchronous branch-specific synaptic changes would take place over time within single MUs . Altogether , these divergent paradigms highlight that the precise sequence of events taking place at the NMJ in ALS remains ill-defined . From a therapeutic viewpoint , this distinction is of high importance since it could pinpoint the NMJ as an accessible target that can be exploited within a time window between the onset of structural changes and the global degeneration of the MU , as previously suggested ( Cantor et al . , 2018; Miyoshi et al . , 2017; Pérez-García and Burden , 2012 ) . Here , we used longitudinal repeated in vivo imaging of single motor neuron axonal arbors and their NMJs to directly assess the time course and dynamics of structural changes during ALS progression . We predict that a MU-dependent mechanism would result in the more or less synchronous degeneration of the whole MU over a brief period , while a contribution of local neuromuscular changes would result in the asynchronous loss of NMJs within a single MU .
SOD1G37R mice ( loxSOD1G37R ) are well known for their slow disease progression , making it an ideal model to follow single MU changes over time ( Boillée et al . , 2006b; Lobsiger et al . , 2009 ) . To visualize and follow single MUs in control and ALS mice , we crossed SOD1G37R mice to animals expressing a cytoplasmic fluorescent marker in a small random subset of MNs ( Thy1-YFP line H , Figure 1A ) ( Feng et al . , 2000 ) . Repeated in vivo imaging was performed on the Tibialis anterior muscle since numerous NMJs can be observed near the surface when exposed following a minimally invasive surgery ( WT/YFP: N = 5; SOD1G37R/YFP: N = 11 ) . Postsynaptic sites were identified in vivo by staining nicotinic receptors ( nAChR ) with fluorescently labeled α-bungarotoxin ( Li et al . , 2011; Turney et al . , 2012 ) . Except for two cases ( animal #23 and #21 ) , all imaging sessions were performed at two--week intervals for up to 5–6 sessions ( 56–76 days ) . The same MU arbor ( axon and its NMJs ) was identified on every imaging session through the unique ‘pretzel’ shape of its NMJs , the branching pattern of the motor axon , and the position of nearby postsynaptic sites ( Figure 1A , landmarks , Δ , * , ● ) . NMJs were classified based on their innervation status over time ( Figure 1—figure supplement 1 ) . We first confirmed that the same MU arbor and its NMJs were reliably imaged over several weeks without inducing any structural damage . We followed single MUs in WT/YFP animals and found that YFP-labeled axons stayed precisely overlapped with nAChR-rich postsynaptic sites over all imaging sessions performed ( 36/36 NMJs , 5/5 MU arbors , N = 5; Figure 1C ) . Importantly , very few changes in axonal branching pattern , postsynaptic nAChRs and presynaptic nerve terminal organization were observed . Consistent with previous reports ( Balice-Gordon and Lichtman , 1990; Li et al . , 2011; Schaefer et al . , 2005; Turney et al . , 2012 ) , our results show that NMJs of WT mice remained innervated by the same MU and were very stable over long periods of time . We next performed imaging in symptomatic SOD1G37R/YFP animals ( all animals at or past disease onset ) to investigate whether the pattern of NMJ denervation in ALS mice was synchronous or asynchronous . Of note , SOD1G37R/YFP mice were phenotypically and histologically indistinguishable from SOD1G37R mice ( Figure 2—figure supplement 1 ) , showing that YFP expression did not alter the disease course and that YFP-expressing MNs are not more susceptible to the disease . We found that YFP-labeled nerve terminals gradually retracted from some nAChR-rich postsynaptic sites ( Figure 2A–C , for example , NMJ A1 , A3 , A4 ) while other axonal branches remained perfectly overlapped ( e . g . NMJ A2 ) . Some NMJs were even re-innervated by the MU ( Figure 2A , A4 inset , arrowhead , see below for more details ) further suggesting that the MU arborization was not globally degenerating at this stage . Strikingly , most MUs in SOD1 mutant mice ( 16/19 arbors , 10/11 animals ) behaved in a similar fashion , that is , losing NMJs over several imaging sessions rather than abruptly . These include partial ( 29/253 imaged NMJs , 12/19 arbors ) and complete NMJ losses ( 38/253 imaged NMJs , 10/19 arbors ) . Thus , NMJ loss within a single MU starts as a slow asynchronous process , occurring independently of axonal degeneration . A number of visualized motor axons and their remaining NMJs were eventually lost in a synchronous manner ( 7/19 MU arbors , 5/11 mice; Figure 3A ) showing that global MU degeneration ultimately occurs . This phenomenon likely reflects an axonal or central event , such as MN death . Importantly , signs of NMJ denervation preceded most of these MU degenerations ( Figure 3B , 6/7 MU arbors ) . In several cases ( 11/19 MU arbors ) , asynchronous NMJ dismantling started more than a month ( ≥ 3 sessions ) prior to global degeneration of the MU , again showing that NMJ loss is initially a slow asynchronous process ( Figure 3B ) . Hence , we identified two phases of NMJ loss within a MU: an initial slow local branch-specific dismantlement , followed by a sudden global axonal degeneration . These different patterns of MU degeneration are unlikely to be due to differences in MU types ( Frey et al . , 2000; Hegedus et al . , 2007; Hegedus et al . , 2008; Pun et al . , 2006 ) . Indeed , fiber type ( indicative of MU type ) composition was examined on Tibialis anterior cross sections and revealed that nearly all superficial fibers in SOD1G37R/YFP were fast-fatigable during the period of imaging ( myosin heavy-chain isoforms type IIb or IIx; Figure 3—figure supplement 1 ) . Surprisingly , a vast majority of MUs in SOD1G37R/YFP also sprouted toward nearby nAChR-rich postsynaptic sites , forming new synaptic contacts on muscle fibers that they did not innervate initially ( MU expansions or ‘heterologous re-innervation’ , Figure 4A , B; 76/253 NMJs , 14/19 arbors , 9/11 animals ) . These numerous expansions resulted in a net increase in MU size which masked the contemporaneous NMJ dismantlement ( Figure 4C; 26 . 0 ± 10 . 2% loss vs 80 . 7 ± 32 . 2% expansions on session 6 ) . Importantly , we found that over half of analyzed MUs both lost and formed new NMJs throughout the imaging process ( Figure 4—figure supplement 1 , 13/19 arbors , 8/11 animals ) , showing that these opposite processes can occur simultaneously within a single MU . Interestingly , however , there was an inverse relationship between the propensity of axonal branches to be lost or to expand ( Figure 4D ) . Indeed , NMJs arising from distal axonal branches were more susceptible to being lost ( Figure 4D , red dots , 38 events , n = 253 , N = 11 , logistic regression , p=0 . 010 ) , while MU expansions were more likely to occur on proximal branches ( mainly first-order branches ) ( Figure 4D , blue dots , 76 events , n = 253 , N = 11 , logistic regression , p=0 . 001 ) . Hence , proximal branches preserve their capacity to re-innervate heterologous NMJs despite the ongoing degeneration of distal branches at the single MU level . Regardless of this extensive compensation , few MUs re-innervated their own lost NMJs ( ‘autologous re-innervation’ ) and most partial losses were not repaired ( Figure 4C , purple; 2/21 and 15/40 possibilities , respectively ) . Immunolabeling of all motor axons after the last in vivo session showed that some of these asynchronously lost NMJs had been reinnervated by other MUs ( Figure 4—figure supplement 2 , N = 4 ) , confirming that these vacated postsynaptic sites were not refractory to reinnervation in general . This process led the initial NMJ pool of surviving MUs to gradually recede ( Figure 4C , green ) even though their total size remained stable or increased in most cases due to heterologous expansions ( Figure 4C , blue ) . Altogether , these results show that rather than being exclusively degenerating or compensating , MUs are highly dynamic during disease progression , with specific branches either expanding or disassembling . To gain further insight into the mechanisms underlying NMJ dismantlement , we next examined the spatial and temporal relationship between denervation events taking place within a MU . Indeed , local signals emanating from muscle fibers , glial cells or a damaged axonal branch ( e . g . chemorepulsive molecules ( De Winter et al . , 2006; Maimon et al . , 2018 ) or misfolded proteins ( Ayers et al . , 2016; Grad et al . , 2014 ) could potentially propagate through the extracellular space and contribute to NMJ loss in ALS . If such local signals in the neuromuscular environment influence NMJ dismantlement , one would predict that these events may tend to propagate from one or several focal points . To test this possibility , we performed a Sholl-like analysis on all MU arbors bearing multiple complete NMJ losses ( 37 events out of 141 NMJs over 9 MU arbors , N = 6; see Materials and methods ) . Interestingly , asynchronous NMJ dismantling tended to propagate from the first lost NMJ ( Figure 5A and B; multivariate repeated-measures GLM with logistic distribution , interaction between distance and time: p<0 . 001 ) . Indeed , adjacent events ( <300 μm away ) occurred preferentially , but not exclusively , within the next two sessions ( T2 ) , while remote events ( >300 μm ) occurred exclusively three or more sessions later ( T3+ ) . These results unravel a propagation pattern and suggest that factors extrinsic to motor axons ( i . e . the local neuromuscular environment ) interact with the intrinsic properties of motor axons ( e . g . susceptibility of specific branches ) , to shape the pattern of NMJ loss in ALS .
Overall , this detailed in vivo analysis reveals a previously unsuspected dynamism at the NMJ in an animal model of ALS . Our findings highlight a long temporal window between early branch-specific dismantling and global motor axon degeneration which could be therapeutically exploited in ALS ( Arbour et al . , 2017; Moloney et al . , 2014 ) .
WT/YFP ( SOD1-/-;YFP+/- ) and SOD1G37R/YFP ( SOD1+/-;YFP+/- ) double transgenic mice were obtained by crossing transgenic males heterozygote for the human SOD1G37R transgene ( flanked by LoxP sites; loxSOD1G37R ) to transgenic females heterozygote for the Thy1-YFP transgene ( line H; [B6 . Cg-Tg ( Thy1-YFP ) HJrs/J]; The Jackson laboratories , Bar Harbor , ME , stock number 003782 ) ( Feng et al . , 2000 ) . LoxSOD1G37R mice have been described previously ( Boillée et al . , 2006b; Lobsiger et al . , 2009 ) . Single transgenic lines were maintained on a C57BL/6J background . Progenies were genotyped for the SOD1 and the YFP transgenes by PCR performed on a tail sample or an ear punch extract . Mice of both sexes were used . Disease progression was monitored through weekly weighing and all-limb grip strength measurements ( BioSeb , FL; BIO-GS3 ) . Animals were sacrificed using a lethal dose of isoflurane . All experiments were performed in accordance with the guidelines of the Canadian Council on Animal Care , the Comité de Déontologie sur l’Expérimentation Animale of Université de Montréal ( protocol #18 – 040 ) and the CRCHUM Institutional Committee for the Protection of Animals ( protocol #N16008CVV and #N15047ADPs ) . Procedures for repeated in vivo imaging of the Tibialis anterior muscle were adapted from previous reports ( Li et al . , 2011; Schaefer et al . , 2005; Turney et al . , 2012 ) . Mice were anaesthetized with isoflurane ( 2 – 3% in 98 – 97% O2 ) in an induction chamber and maintained under anesthesia using a breathing mask . Mice were placed on their side and a hind limb was immobilized on a custom-made stand . Eye drying was prevented by applying a lubricant ( Vaseline ) . The Tibialis anterior muscle was exposed through an incision made on the external side of the leg ( from ~5 – 10 mm proximal to the knee to ~5 mm proximal the ankle ) . The skin was drawn tight using four hooks ( Guthrie Retractors; #17021 – 13; FST , Canada ) mounted on custom-made blocks around the leg . The muscle was immersed in a physiological solution by regular irrigation with sterile lactated Ringer ( B . Braun Medical , CDMV , Canada ) . Postsynaptic nAChRs were labeled with a non-blocking concentration of Alexa594-conjuguated α-Bungarotoxin ( BTX , 5 μg/mL in sterile lactated Ringer for 10 min; Molecular Probes , Fischer Scientific , Canada ) . α-BTX was applied on the first session and was reapplied once ( if necessary ) only when the labeling was too dim , usually on session 5 or 6 . Muscle contractions and synaptic transmission are known to be preserved at this concentration of α-BTX ( Arbour et al . , 2015; Li et al . , 2011; Turney et al . , 2012; Zuo et al . , 2004 and unpublished observations ) . NMJs near the surface were imaged using a 20X water immersion objective ( 0 . 4 NA , Nikon , Japan ) mounted on an upright epifluorescent microscope ( Nikon , Optiphot-2 ) equipped with a Neo-sCMOS camera ( Andor , UK ) . Fluorescence excitation and emission were filtered using a Brightline Pinkel filter set optimized for CFP/YFP/HcRed ( CFP/YFP/HcRed-3X-A-000; Semrock , NY ) . Images of the whole superficial MU axonal arborization at multiple focal planes were acquired using the Metafluor software ( Molecular Devices , CA ) . After the imaging session , the wound was sutured using 5 – 0 or 6 – 0 vicryl suture ( Johnson and Johnson ) , followed by tissue glue ( GLUture; Abbot Laboratories , WPI , FL ) . Buprenorphine ( 3 μg/10 g body weight; Temgesic , CEVA Animal Health Ltd , UK ) was administered by subcutaneous injections three times a day for 24 hr following the surgery . Antibiotics ( 0 . 1 mg/10 g body weight; Baytril; Bayer . Inc , Canada ) were administered subcutaneously every 24 hr for 72 hr . Mice were given a small treat after each surgery ( Nutella ) to reduce stress and improve recovery . Except for two cases , imaging sessions were performed 2 weeks apart . All available SOD1 animals with YFP-positive surface motor axons , were included in the in vivo analysis ( 16/44 mice tested ) . Only mice in which the MU arbor could be reliably followed for at least three sessions , with at least three NMJs near the surface , were included in the analysis ( 11/16 mice ) . NMJs which were too deep to be fully resolved were discarded . Images were analyzed using the Fiji software . First , focal planes from the same region were aligned using the ‘StackReg’ pluggin . MU branches were traced using the ‘Simple Neurite Tracer’ plugin . The clearest session ( generally session 1 ) was chosen for tracing . All segments of the MU arbor ( from each region imaged ) were merged in Photoshop ( Adobe ) and BTX labelling ( in the overlap between the regions ) was used as a reference for alignment . Tracings were used as a map to identify each NMJ on subsequent imaging sessions . For the purpose of this analysis , two labeled branches that could not be undoubtedly linked to the same main motor axon were considered as independent MNs . Then , the state of innervation of each NMJ was determined and a MU dynamic diagram showing the state of each NMJ within the arborization on each session was created using Visio2013 ( Microsoft ) . NMJs were then classified based on their dynamic changes ( Figure 1—figure supplement 1 ) . For figure representations , contrast was linearly adjusted to facilitate observations . For the spatio-temporal analysis ( Sholl-like analysis ) of asynchronous NMJ dismantling ( Figure 5 ) , distance between NMJs and the initial loss was measured on the full MU tracings or , for a few cases , directly on the raw aligned images . The initial event ( center ) was defined as the first completely lost NMJ . When two or more NMJs were lost simultaneously , the one which first showed signs of denervation ( partial loss ) was defined as the center . If they could not be distinguished in this manner , the analysis was carried out with multiple centers ( 2/9 MU arbors ) . For one animal ( #34 ) , several branches were fairly close to each other , that is in the same field of view ( branches A and B , C and D ) . To more accurately assess the presence of a spreading pattern , a single center per field of view was determined . Branch order was determined using the motor-unit dynamic diagrams . MU expansions were assigned the same branch order as the branch from which they originated from , to determine likelihood of sprouting of axonal branches . To create the heat map ( Figure 4—figure supplement 1 ) , the % of change was calculated by counting the number of gained or lost NMJs over the number of initial NMJs . Partial gains/losses counted as half an event . When an NMJ could not be observed , the % of change was spread across the sessions where it could have occurred . Procedures for tissue preparation and immunostaining were done as previously described ( Tremblay et al . , 2017 ) Briefly , the Tibialis anterior was dissected in an oxygenated ( 95% O2 , 5% CO2 ) Ringer REES solution and mounted in 10% Tragacanth ( Sigma-Aldrich , Canada ) . Muscles were frozen in isopentane cooled to −80°C in liquid nitrogen . Transverse cryosections ( 10 μm ) were incubated in blocking solution ( 10% normal donkey serum in PBS; Jackson Immunoresearch , PA ) for 30 min , then with primary antibodies for 1 hr , and then with secondary antibodies for 1 hr . Sections were finally mounted in Prolong Gold antifade reagent ( Molecular Probes , Fischer Scientific ) . Sections were rinsed three times with PBS 1X for 5 min between each step . Primary antibodies , all from the Developmental Studies Hybridoma Bank ( DSHB , IA ) , were either mouse IgG1 α -MHC type IIa ( SC-71c , 1:200 ) , mouse IgG2b α-MHC type I ( BA-D5c , 1:100 ) and mouse IgM α -MHC type IIb ( BF-F3c , 1:200 ) or mouse IgM α -MHC type IIx ( 6H1s; 1:10 ) and mouse IgG1 α -MHC all but IIx ( BF-35c , 1:200 ) . Secondary antibodies were goat α-mouse IgG1 Alexa 594 ( #115-585-205 ) , goat α-mouse IgG2b Alexa 488 ( #115-545-207 ) and goat α-mouse IgM Alexa 647 ( #115-605-020 ) , all from Jackson Immunoresearch ( all 1:500 ) . Observations were made using an Olympus FV1000 or a Zeiss LSM880 confocal microscope with a 20X water immersion objective ( N . A 0 . 95 or 1 . 0 , respectively ) . Whole muscle sections were reconstructed by superimposing maximum intensity projections of each stack in Photoshop ( Adobe ) . Procedures for whole-mount muscle preparation , spinal cord sectioning and immunostaining were performed as previously described ( Tremblay et al . , 2017 ) , with some minor adjustments . Mice were transcardially perfused with cold PBS 1X and 4% formaldehyde . Both Tibialis anteriors were then dissected and post-fixed for 20 min in 4% formaldehyde while the whole mouse was further fixed overnight at 4°C . Lumbar spinal cords were dissected , post-fixed for 2 hr , cryoprotected in a 30% sucrose-PBS solution for 72 hr at 4°C and frozen in cooled isopentane ( −40 to −50°C ) . Floating 30-μm-thick spinal cord cryosections were washed twice in PBS 1X , incubated in a donkey blocking solution ( 10% NDS , 0 . 3% Triton X-100 in PBS 1X ) for 1 hr and then incubated overnight with primary antibodies against Choline Acetyl-Transferase ( ChAT; 1:100; Goat; Millipore , Canada; AB144P ) and Neuronal Nuclei ( NeuN; 1:300; Mouse IgG1; Millipore; MAB377 ) in donkey blocking solution , then incubated with the secondary antibody donkey anti-goat Alexa 594 ( 1:500; #705-585-147; Jackson Immunoresearch ) in donkey blocking solution for 1 hr , then incubated in a goat blocking solution ( 10% Normal Goat Serum , 0 . 3% Triton X-100 in PBS 1X ) for 1 hr , then incubated with the goat anti-mouse IgG1 DyLight 405 secondary antibody ( 1:500; #115-475-205; Jackson Immunoresearch ) in goat blocking solution for 1 hr and then finally mounted with Prolong Diamond antifade reagent ( Molecular Probes ) . Sections were washed thrice with PBS 1X ( 5 min each ) after each antibody incubation . Tibialis anterior muscles were permeabilized in 100% cold methanol at −20°C for 6 min , then incubated for 1 hr in blocking solution ( 10% NDS , 1% of Triton-X100 diluted in PBS 1X ) . Muscles were then incubated with a rabbit anti-S100β antibody ( 1:250 , Z0311 , Dako ) in blocking solution for 23 hr at 4°C , then with chicken anti-neurofilament M , ( NF-M , 1:2000 , Rockland Immunochemicals Inc ) and mouse IgG1 anti-synaptic vesicular protein 2 ( SV2 , 1:2000 , Developmental Studies Hybridoma Bank ) for 23 hr at 4°C , then with the secondary antibodies goat anti-mouse IgG1 Alexa 647 ( #115-605-205 ) , donkey anti-chicken Alexa 647 ( #703-605-155 ) and donkey anti-rabbit DyLight 405 ( #711-475-152 ) ( all 1:500; Jackson Immunoresearch ) simultaneously with Alexa594-conjugated-α-BTX ( 1 . 33 µg/ml , Molecular Probes , Fischer Scientific , Canada ) for 2 hr . Finally , whole muscle preparations were mounted in Prolong Diamond antifade reagent ( Molecular Probes ) . Muscles were washed six times ( 10 min each ) in PBS 1X-Triton 0 . 01% . Observations and image acquisition were performed on a Zeiss LSM 880 confocal microscope with a 20X water immersion objective ( N . A . 1 . 0 ) or a 63X oil immersion objective ( N . A . 1 . 4 ) . No image manipulations were performed after acquisition , except for linear contrast adjustments for figure presentation . For motor neuron counts , MNs in both ventral horns were counted from 15 to 20 sections per animal , at least 90 μm apart . ChAT- and NeuN-positive cells in the ventral horn were counted as α-MNs while ChAT-positive and NeuN-negative cells were counted as γ-MNs as previously described ( Lalancette-Hebert et al . , 2016; Tremblay et al . , 2017 ) . The Allan Brain Atlas Mouse Spinal cord reference set was used as a reference to ensure that all analyzed sections were in the lumbar spinal cord . Results are expressed as the average number of cells counted per ventral horn in each animal . For the Sholl-like analysis , each lost NMJ represented an event in time ( time after initial loss ) and space ( distance from the initial loss ) occurring over a given number of trials ( total number of NMJs ) . This type of data ( i . e . number of observations over a number of trials as a % of events ) follows a logistic distribution rather than a Gaussian distribution . Hence , a multivariate repeated-measures Generalized linear models ( GLM ) using a logistic distribution was created to test the effects of time and space on the likelihood of observing an event . The time after the initial loss was set as a ‘within-NMJ’ factor ( the repeated measure ) while distance from the initial loss was set as a continuous ‘between-NMJs’ variable . The raw measured distance was used rather than the range bins presented in Figure 5 . For the branch order analysis , a similar approach was used . Each lost or gained NMJ represented an event occurring over a given number of trials ( number of NMJs within that branch order ) in eight conditions ( branch orders ) . Hence , a logistic regression was used to test the effect of the branch order on the likelihood of an NMJ being loss ( using the univariate GLM with logistic distribution command ) . The branch order was set as an ordinal variable ( continuous ) . For these analyses , ‘n’ ( the number of trials , i . e . the sample size ) represent the number of NMJs observed while ‘N’ ( the biological replicates ) represents the number of animals . p-Values smaller than 0 . 05 ( α = 5% ) were considered statistically significant . Analyses were performed in the SPSS 24 . 0 . 0 . 0 ( IBM ) software . When two independent groups were compared , unpaired two-tailed t-tests were performed using Welch’s correction for unequal variance or a Mann-Withney test when data did not follow the assumption of a Gaussian distribution . Variances were compared using the F-test . When the effect of one variable was compared over time ( motor behavior , Figure 2—figure supplement 1 ) , two-way ANOVAs with repeated measures ( RM ) were used . For the post-test , Holm-Sidak’s correction was used . For NMJ innervation ( Figure 2—figure supplement 1 ) , the effect of genotype was analyzed using a GLM with a logistic distribution and Holm-Sidak’s correction was applied for the post-test as previously described ( Tremblay et al . , 2017 ) . Importantly , a GLM with a logistic distribution was used for NMJ innervation instead of a t-test ( as for motor neuron counts ) because NMJ innervation data ( # of denervated NMJs/ # of total NMJs for each animal ) does not follow a Gaussian distribution . Unless otherwise stated , data are presented as mean ± SEM in the histograms and in the text . For these analyses , ‘N’ represents the number of biological replicates ( animals , i . e . the sample size ) while “n’ represents the number of observations ( number of NMJs unless otherwise stated ) . p-Values smaller than 0 . 05 ( α = 5% ) were considered statistically significant . These analyses were performed in the GraphPad Prism 7 . 0 software , with the exception of the analysis for the NMJ innervation was made in SPSS 24 . 0 . 0 . 0 . No sample size estimations were performed before the experiments . All available SOD1 animals which met our inclusion/exclusion criteria were included ( see in vivo images and data analysis ) . For the NMJ innervation and motor neuron counts experiments , sample sizes similar to previous experiments were used ( Tremblay et al . , 2017 ) . No test to detect outliers was performed .
Despite being an early event in ALS , it remains unclear whether the denervation of neuromuscular junctions ( NMJ ) is simply the first manifestation of a globally degenerating motor neuron . Using in vivo imaging of single axons and their NMJs over a three-month period , we identify that single motor-units are dismantled asynchronously in SOD1G37R mice . We reveal that weeks prior to complete axonal degeneration , the dismantling of axonal branches is accompanied by contemporaneous new axonal sprouting resulting in synapse formation onto nearby NMJs . Denervation events tend to propagate from the first lost NMJ , consistent with a contribution of neuromuscular factors extrinsic to motor neurons , with distal branches being more susceptible . These results show that NMJ denervation in ALS is a complex and dynamic process of continuous denervation and new innervation rather than a manifestation of sudden global motor neuron degeneration .
Amyotrophic lateral sclerosis ( ALS ) , also known as Lou Gehrig’s disease , is a fatal neurodegenerative disorder . It occurs when the neurons that control muscles – the motoneurons – disconnect from their target muscles and die . This causes the muscles to weaken and waste away . More and more muscles become affected over time until eventually the muscles that control breathing also become paralyzed . Most patients die within two to five years of diagnosis . Motoneurons consist of a cell body plus a cable-like structure called the axon . The cell body of each motoneuron sits within the spinal cord , and the axon extends out of the spinal cord to the motoneuron’s target muscle . Within the muscle the axon divides into branches , each of which connects with multiple muscle fibers . The breakdown of these connections , known as neuromuscular junctions , is one of the first signs of ALS . Does a motoneuron lose all of its connections with muscle fibers at once , or do the connections break down a few at a time ? This distinction is important as it will help to identify the events that lead to muscle paralysis in ALS . To find out , Martineau et al . studied mice that had two genetic mutations: one that causes ALS and another that produces fluorescent molecules in some motoneurons . This allowed the branches of the motoneurons to be tracked over time with a fluorescence microscope . Martineau et al . found that individual neurons lose their connections to muscle fibers gradually . Moreover , motoneurons grow new branches and form new connections even while losing their old ones . This dual process of pruning and budding lasts for several weeks , until eventually the motoneuron dies . Developing drugs to stabilize neuromuscular junctions during the period when motoneurons gradually disconnect from muscles could be a promising avenue to explore to improve the quality of life of ALS patients . One advantage of this treatment strategy is that neuromuscular junctions in muscles are easier to access than motoneurons inside the spinal cord . To identify potential drugs , future studies will need to focus on the proteins and signals that cause the neuromuscular junctions to break down .