Type selective ablation of postnatal slow and fast fatigue-resistant motor neurons in mice induces late onset kinetic and postural tremor following fiber-type transition and myopathy

Animals on Earth need to hold postures and execute a series of movements under gravity and atmospheric pressure. VAChT-Cre is a transgenic Cre driver mouse line that expresses Cre recombinase selectively in motor neurons of S-type (slow-twitch fatigue-resistant) and FR-type (fast-twitch fatigue-resistant). Sequential motor unit recruitment is a fundamental principle for fine and smooth locomotion; smaller-diameter motor neurons (S-type, FR-type) first contract low-intensity oxidative type I and type IIa muscle fibers, and thereafter larger-diameter motor neurons (FInt-type, FF-type) are recruited to contract high-intensity glycolytic type IIx and type IIb muscle fibers. To selectively eliminate S-and FR-type motor neurons, VAChT-Cre mice were crossbred with NSE-DTA mice in which the cytotoxic diphtheria toxin A fragment (DTA) was expressed in Cre-expressing neurons. The VAChT-Cre;NSE-DTA mice were born normally but progressively manifested various characteristics, including body weight loss, kyphosis, kinetic and postural tremor, and muscular atrophy. The progressive kinetic and postural tremor was remarkable from around 20 weeks of age and aggravated. Muscular atrophy was apparent in slow muscles, but not in fast muscles. The increase in motor unit number estimation was detected by electromyography, reflecting compensatory re-innervation by remaining FInt-and FF-type motor neurons to the orphaned slow muscle fibers. The muscle fibers gradually manifested fast/slow hybrid phenotypes, and the remaining FInt-and FF-type motor neurons gradually disappeared. These results suggest selective ablation of S-and FR-type motor neurons induces progressive muscle fiber-type transition, exhaustion of remaining FInt-and FF-type motor neurons, and late-onset kinetic and postural tremor in mice.


Introduction
The human body consists of approximately 400 skeletal muscles having different shapes and sizes, and each muscle has a specific fiber type composition to fulfill respective functional demands.Skeletal muscles are grossly classified as fast/slow muscles (also called white/red muscles) based on excitability, contraction speed, force, and resistance to fatigue.The physical nature of each muscle is determined by different fiber type compositions and distributions.Adult muscle fibers are classified into type I and type II; the type II fibers are further subdivided into type IIa, type IIx, and type IIb.These muscle fibers are characterized by the specific expression of myosin heavy chain (MyHC) isoforms; type I Abbreviations: ACh, acetylcholine; BSA, bovine serum albumin; ChAT, choline acetyltransferase; CHT1, high-affinity choline transporter 1; CMAP, compound muscle action potential; DTA, diphtheria toxin A fragment; ER, endoplasmic reticulum; MMP, matrix metalloproteinase; MN, motor neuron; MyHC, myosin heavy chain; NSE, neuron-specific enolase; OPN, osteopontin; SMUP, single motor unit potential; VAChT, vesicular acetylcholine transporter.. with MyHC Iβ (MYH7), type IIA with MyHC IIa (MYH2), type IIX with MyHC IIx (MYH1), and type IIB with MyHC IIb (MYH4), and are innervated independently by four types of motor neurons (MNs) classified as S (slow-twitch fatigue-resistant), FR (fast-twitch fatigueresistant), FInt (fast-twitch fatigue-intermediate), and FF (fast-twitch fatigable), respectively.The one-to-one correspondence of motor neuron types to muscle fiber types (S to type I, FR to type IIa, FInt to type IIx, FF to type IIb) is called "motor unit" and constitutes a basis for motor control (Schiaffino and Reggiani, 2011;Greising et al., 2012).The low threshold S units are recruited first, secondly FR units, thirdly FInt units, and finally high threshold FF units.The orderly recruitment of the motor units from smallest to largest in MN diameter is called "size principle" and is needed to perform various voluntary or reflex movements (Henneman E., 1957).
VAChT-Cre is a Cre driver mouse line that expresses Cre recombinase postnatally and selectively in S-type and FR-type MNs (Misawa et al., 2003;Misawa et al., 2016).The Cre-expression specificity in S-type and FR-type MNs was systematically and extensively examined in our previous publication (Misawa et al., 2016).Although the reason for directing the Cre-expression specificity only in the small subset of VAChT-expressing cholinergic neurons is unknown, possibly because of the nature of the human VAChT genomic fragment used and the transgene copy number and location, VAChT-Cre provides a unique opportunity to analyze subtype-specific MN physiology in adult mice.To selectively ablate S-and FR-type MNs, VAChT-Cre mice were crossbred with NSE-DTA mice in which diphtheria toxin A fragment (DTA) was expressed after Cre-mediated loxP-site excision (Kobayashi et al., 2013).The VAChT-Cre;NSE-DTA mice (designated as "ΔSlowMN mice") were born normally but progressively manifested kinetic and postural tremor, along with body weight loss, muscular atrophy, kyphosis, and short life span.The tremor first appeared in the head and neck around 20 weeks of age and was progressive.We analyzed alterations in the composition of fiber types in calf muscles and found that the tremor appearance coincided with muscle atrophy and fiber type transition mainly in type I and IIa muscle fibers.We report here for the first time that selective elimination of S-and FR-type MNs induces late-onset myopathies and progressive kinetic and postural tremor in mice.

Mice
VAChT-Cre.Early (formally designated VAChT-Cre.Fast) is a transgenic Cre-driver line that selectively expresses Cre recombinase in slowtwitch fatigue-resistant (S) and fast-twitch fatigue-resistant (FR) motor neurons (Misawa et al., 2003;Misawa et al., 2016).Eno2-DTA mice (also denoted as NSE-DTA mice) carry the IRES-loxP-STOP-loxP-DTA cassette in the 3′-untranslated region of the mouse Eno2 gene.In Eno2 mRNAexpressing neurons of mice harboring this construct, Cre-mediated recombination induces DTA expression and apoptotic cell death (Kobayashi et al., 2013).CAG-Syp/tdTomato (B6;129S-Gt(ROSA) 26Sortm34.1(CAG-Syp/tdTomato)HZE/J)mice were purchased from Jackson Laboratory.Mice were weaned at 21 days of age.Mice genotyping was performed as described (Kobayashi et al., 2013;Misawa et al., 2003) or by following the standard PCR protocol provided by Jackson Laboratory (https://www.jax.org/strain/012570).All experiments were conducted in accordance with Japanese national guidelines and regulations and were reviewed and approved by the Animal Care and Use Committee of Keio University.

Tremor measurements
Mice were placed in a suspended plastic box to which a wireless accelerometer (MVP-RF8-HC, MicroStone) was attached at the bottom by a cloth tape as described (Yamanaka et al., 2016).The motion of the box was recorded for 3 min at a sampling rate of 1 kHz.The recorded data were subjected to fast Fourier transformation and transformed to the amplitude at frequencies (0-50 Hz) by waveform analysis software (MVP-RF8-S, MicroStone).Harmaline-induced tremor was recorded at min after intraperitoneal injection of harmaline (15 mg/kg in saline).Each anti-tremor agent (propranolol, 20 mg/kg; phenobarbital, 10 mg/ kg; ethanol, 1.5 g/kg; all dissolved in saline) was intraperitoneally injected 30 min prior to the harmaline injection or 40 min prior to the tremor measurement.

Muscle fiber type identification
A one-step multi-colored immunostaining method (Sawano et al., 2016) was used to visualize muscle fiber types using a mixture of four rat monoclonal antibodies each specific for adult myosin heavy chain (MyHC) isoforms: MyHC1, 2 A, 2× and 2B.The anti-MyHC1, anti-MyHC2A, anti-MyHC2X, and anti-MyHC2B antibodies were conjugated with Alexa Fluor 647, Alexa Fluor 405, Fluorescein, and Alexa Fluor 594, respectively.Freshly prepared muscle cross-sections (20 μm) were collected onto glass slides and processed as described (Sawano et al., 2016).Briefly, the sections on glass slides were steamed (heat treatment) for 5 min, permeabilized with 1.0% Triton X-100 in PBS, incubated in blocking buffer (1% BSA, 0.1% cold fish skin gelatin, 0.1% Triton X-100, 0.05% Tween-20, and 100 mM glycine in PBS) and then incubated with the mixture of the fluorescence-conjugated antibodies in the blocking buffer.The labeled specimens were washed and mounted by coverslips with Fluorescent Mounting Medium (DAKO) and examined using an Olympus FV-1000 confocal microscope system or an Olympus FV-3000 confocal microscope system.The one-step immunostaining method can distinguish the MyHC isoforms unbiasedly at a single fiber level within individual tissue sections and identify hybrid H. Misawa et al. fibers which simultaneously express two or more MyHC isoforms.

Motor unit number estimation
Compound muscle action potential (CMAP) and single motor unit potential (SMUP) were recorded, and motor unit number estimation was calculated as described by Arnold et al. (2015).Briefly, mice were anesthetized with isoflurane inhalation (4% introduction, 1.5% maintenance) and placed in the prone position with body temperature maintained at 37 • C by a heating pad.The recording electrodes (ring electrodes; 1.5 cm apart) and the stimulating electrodes (needle electrodes) were placed on the shaved skin as shown in Fig. 5A.The ground electrode (ring electrode) was placed on the tail.Stimuli were given with monophasic pulses (0.1 ms duration, 0.5 Hz frequency) from an electrostimulator (NIHONKODEN SEN-3301) with fine intensity control (NIHONKODEN SS-203 J).Recordings were made with a Power lab data acquisition system (ADInstruments PL2604) at 100 Hz and analyzed with LabChart Pro v7 software (ADInstruments).Filter settings were 10 Hz/5 KHz.

Statistical analyses
The data analyses were done using GraphPad Prism 8 (MDF).The results were analyzed using the Student's t-test, one-way analysis of variance (ANOVA) with Tukey's multiple comparison tests or two-way ANOVA with Sidak's multiple comparison test.Log-rank test was used for survival analysis.The data are presented as means +/− standard error of the mean (SEM) unless otherwise indicated.Differences were considered statistically significant when P values were <0.05.

Genetic ablation of S-and FR-type MNs in mice
To verify specific cell ablation, we crossed three mouse lines: VAChT-Cre.Early, CAG-Syp/tdTomato and NSE-DTA.VAChT-Cre.Early selectively express Cre recombinase in S-and FR-type MNs; the Cre expression begins 1 week after birth and reaches 80% of S -and FR-type MNs within 5 weeks (Misawa et al., 2003;Misawa et al., 2016).CAG-Syp/ tdTomato is a reporter line in which a synaptophysin-tdTomato fusion protein is expressed following Cre-mediated DNA recombination.NSE-DTA (also called Eno2-DTA) contains the IRES-loxP-STOP(pgk-neo-polyA)-loxP-DTA cassette at the 3′-untranslated region of the mouse Eno2 gene (Kobayashi et al., 2013).The Cre-mediated recombination induces the DTA expression and apoptotic cell death in Eno2-expressing neurons.In VAChT-Cre.Early(+):CAG-Syp/tdTomato(+) mice (double mutants), ca.30% were positive for Syp/tdTomato within ChAT-positive MNs (within all MNs including both α and γ MNs) in the cervical, thoracic, or lumbar spinal cord (Fig. 1A, B, C, D).In VAChT-Cre.Early(+):CAG-Syp/ tdTomato(+):NSE-DTA(+) mice (triple mutants), most of Syp/ tdTomato-positive MNs disappeared, whereas the number of Syp/ tdTomato-negative MNs were stable, suggesting efficient and accurate cell ablation by the Cre-mediated DNA recombination (Fig. 1A, B, C, D).Although the total number of MNs is small, the proportion of Syn/ tdTomato-positive MNs is higher at the thoracic level than at the cervical or lumbar level, probably reflecting dominant innervation of the trunk muscles.The number of γ MNs (determined as ChAT-positive and NeuN-negative cells) was slightly decreased but not reached statistically significant in the triple mutant mice compared with the double mutant mice (Fig. 1E).

Progressive body weight loss, short survival, kyphosis, kinetic and postural tremor in ΔSlowMN mice
Next, we analyzed gross phenotypes of VAChT-Cre.Early(+):NSE-DTA(+) mice (designated as "ΔSlowMN") and compared with those of VAChT-Cre.Early(− ):NSE-DTA(+) mice ("Control").ΔSlowMN mice were viable and visually indistinguishable from control littermates at birth and until the weaning period.At the weaning period (21 days old), MN innervation to muscle fibers was compared between Control and ΔSlowMN mice by imaging pre-synaptic (high-affinity choline transporter 1; CHT1) and post-synaptic (nicotinic acetylcholine receptor labeled by α-bungarotoxin) markers (Fig. 2A).Almost 100% of neuromuscular junctions (NMJs) are preserved in Control mice.In contrast, in ΔSlowMN mice, >70% of NMJs were denervated in the soleus muscle (dominant in type I-and type IIa-fibers), and only 5% in the gastrocnemius muscles (dominant in type IIx-and type IIb-fibers) (Fig. 2B).Although Control mice showed sustained body weight gain, that of ΔSlowMN mice became plateaued around 15 weeks and then decreased thereafter; at 40 weeks ΔSlowMN mice have an average 70% body weight compared with Control mice (Fig. 3A).ΔSlowMN mice exhibited a shorter life span with an average 50% mortality rate at 38-40 weeks (Fig. 3B).Kyphosis was evident in ΔSlowMN mice from 20 weeks onward (Fig. 3C).Visible kinetic and postural tremor was first observed around 20 weeks and gradually aggravated thereafter, especially in the head and neck (Supplementary Videos 1 and 2).A footprint analysis revealed shortened strides but no ataxia in ΔSlowMN mice compared with Control mice (Fig. 3D).

Kinetic and postural tremor inΔSlowMN mice is different from the harmaline-induced tremor
Spectral analyses of the mouse tremor were performed using an accelerometer attached to a hanging box.ΔSlowMN mice showed enhanced tremor power in a broad frequency range (3-50 Hz) with a small peak at 19 Hz compared with Control mice (Fig. 4A).Intraperitoneal (IP) injection of harmaline, a well-known mouse model of essential tremor (Wilms et al., 1999), robustly induced 14-Hz tremor in Control mice (Fig. 4B and Supplementary Video 3).In ΔSlowMN mice, IP injection of harmaline produced a new 14-Hz tremor along with the 19-Hz kinetic and postural tremor (Fig. 4C and Supplementary Video 4).The low tremor power amplitude in ΔSlowMN mice was possibly due to gross muscle atrophy compared with Control mice.Immunohistochemical analysis of brain sections detected intense c-Fos-positive neurons in the inferior olive nucleus both in ΔSlowMN and Control mice after the harmaline injection (Fig. 4D).Phenobarbital (PB), propranolol (PPL) and ethanol (EtOH) are known to be effective to patients with essential tremor.These reagents all suppressed the harmaline-induced tremor in Control mice, however, showed no effects on the kinetic and postural tremor voluntary observed in ΔSlowMN mice (Supplementary Fig. 1).

Electromyographic motor unit number estimation
In order to analyze the possible compensatory re-innervation on the denervated muscle fibers by remaining MNs, compound muscle action potential (CMAP) recording and motor unit number estimation (MUNE) were performed in the hindlimb muscles, and the results were compared between ΔSlowMN and Control mice at 20-22 weeks of age.Our experimental configuration (Fig. 5A) was adapted from the method described by Arnold et al. (2015).The CMAP, total muscle output induced by supramaximal sciatic nerve stimulation, was the same between ΔSlowMN and Control mice (Fig. 5B).The single motor unit potential (SMUP), the average output of single motor units, was increased (1.66-fold) in the ΔSlowMN mice compared with Control mice (Fig. 5C).The MUNE, estimated motor unit number calculated by CMAP/average SMUP, was decreased (0.68-fold) in ΔSlowMN mice compared with Control mice (Fig. 5D), suggesting collateral sprouting and compensatory re-innervation of the remaining Fint-or FF-MNs on the vacant type I or type IIa muscle fibers.

Muscle analyses
The soleus muscle (mostly slow fibers), the gastrocnemius muscle (mixed slow and fast fibers), or the extensor digitorum longus muscle (mostly fast fibers) were dissected out from ΔSlowMN or Control mice at 25 weeks of age and the macroscopic morphology was compared (Fig. 6A).The soleus muscle from ΔSlowMN mice looked pale and thin compared with that from Control mice, suggesting apparent muscle atrophy.Though the gastrocnemius muscle from ΔSlowMN mice looked slightly smaller than that from Control mice, the colour is nearly the same between them.There was no discernible difference in the extensor digitorum longus muscle between ΔSlowMN and Control mice.To delineate muscle fiber type composition, transverse sections of calf muscles, containing the soleus, gastrocnemius, and plantaris muscles (Fig. 6B) were stained by the one-step quadruple-immunofluorescence method which can visualize four individual MyHC isoforms simultaneously (MyHC1, 2 A, 2× and 2B; Sawano et al., 2016).At 38 weeks of age, the atrophied soleus muscle mostly disappeared in ΔSlowMN mice (Fig. 6C).Considerable difference in the fiber type composition was observed in the plantaris muscle and the bone-side portion of the gastrocnemius muscle, the two portions contained both slow and fast muscle fibers (Fig. 6C).In the skin-side portion of gastrocnemius muscle, mostly consisted of fast muscle fibers, the difference in the fiber type composition looked marginal between ΔSlowMN or Control mice (Fig. 6C).
Cre expression in VAChT-Cre.Early mice begins at 1 week after birth and reaches maximal expression after 5 weeks (Misawa et al., 2003;Misawa et al., 2016).On the other hand, the visible tremor phenotype in ΔSlowMN mice was detected much later around 20 weeks of age and  thereafter.We analyzed alternations in muscle fiber types in the calf muscles at 10 weeks, 20 weeks, and 40 weeks of age in ΔSlowMN and Control mice (Fig. 7).A minimum number of 200 muscle fibers were quantified in each muscle portions and classified into five types as MyHC1, 2 A, 2×, 2B and Hybrid.The hybrid fibers, which are barely detectable in normal adult muscles, express two or more MyHC isoforms.In the soleus muscle of ΔSlowMN mice, the number of countable fibers was below 100 at 20 weeks and 40 weeks of age, therefore, we showed the result with the mark "atrophy" in Fig. 7A.Large difference was observed in the plantaris and the bone-side portion of the lateral gastrocnemius at 20 and 40 weeks of age (Fig. 7B and D).The most of hybrid fiber types observed in these portions were MyHC2A/MyHC2X or MyHC2X/MyHC2B, suggesting possible compensatory reinnervation of denervated muscles by FInt or FF MNs.

Discussion
VAChT-Cre is a transgenic Cre-driver mouse line, in which Cre expression is driven by the 11.3 kb human vesicular acetylcholine transporter (VAChT) promoter (Misawa et al., 2003).The line exhibited Cre expression restricted to approximately half of the postnatal somatic MNs.Our initial analysis of VAChT-Cre lines suggested that Cre expression in each MN pool was a stochastic event, with no clear preference for a particular MN subtype (Misawa et al., 2003).However, reevaluation using newly identified MN subtype markers revealed that the line has specificity for Cre expression in S-and FR-type MNs innervating type I and IIa muscle fibers (Misawa et al., 2016).VAChT is expressed in all cholinergic neurons and is commonly used as a reliable cholinergic marker.Although the exact mechanisms are unknown, the 11.3 kb human VAChT genomic fragment used to create VAChT-Cre has the ability to induce restricted Cre expression in S-and FR-type MNs (Misawa et al., 2016), providing a unique Cre-driver mouse line for subtype-selective gene modification in adult MNs.The VAChT-Cre lines have been used so far in various research projects for introducing MNspecific gene modifications (Misawa et al., 2006;Yamanaka et al., 2008;Haramanti et al., 2010;Hideyama et al., 2010;Zhai et al., 2011;Tashiro et al., 2012;Iguchi et al., 2013;Lecomte et al., 2014;Yamanaka et al., 2016;Yamamotoya et al., 2022).Phenotypes that are thought to be abnormalities of the slow-type MNs, such as abnormalities in red muscles, have often been reported in previous studies (Lecomte et al., 2014;Yamamotoya et al., 2022).
Selective innervation by each MN type to the corresponding muscle fiber type is a basis of "size principle" of motor unit recruitment (Henneman, 1957).Smaller diameter MNs (S-and FR-type MNs) first discharge and produce small amounts of force in slow muscle fibers (oxidative type I and IIa fibers), and then larger diameter MNs (FInt-and FF-type MNs) are recruited to produce greater force in fast muscle fibers (glycolytic type IIx and IIb fibers) to accomplish each motor tasks.The orderly contraction of muscle fibers in the order of type I, type IIa, type IIx, and type IIb allows for the maintenance of posture and smooth movement.In this study, VAChT-Cre was crossed with NSE-DAT to selectively resect postnatal S-and FR-type MNs.Initially, it was hypothesized that the disordered "size principle" would cause instant motility defects in ΔSlowMN mice.However unexpectedly, the motor phenotype of ΔSlowMN mice was subtle over a long period, with a pronounced tremor phenotype becoming evident much later (around weeks) when muscle atrophy and compensatory fiber type transition were detected.
Tremor phenotype in ΔSlowMN mice exhibited a postural and kinetic nature (Supplementary Video 1), which is reminiscent of patients with essential tremor.Harmalin-induced tremor, which induces rhythmic and synchronized activation of inferior olive neurons, is known as a mouse model of essential tremor.In the present study, harmalinetreatment inΔSlow MN mice induced robust 19-Hz tremor upon kinetic and postural tremor (Fig. 4C, Supplementary Video 4) as well as c-Fos expression in the inferior olive nucleus (Fig. 4D), suggesting that the tremor induced by harmaline differs from that spontaneously observed in ΔSlowMN mice in its mechanism of tremor.It is also noteworthy that the tremor intensity of ΔSlowMN mice after harmaline administration is smaller than that of Control mice.This indicates an overall decrease in tremor intensity primarily due to atrophy of the trunk muscles.In recent years, attention has focused on the relationship between abnormalities of functional proteins in the slow muscle fibers and the occurrence of tremors."Myogenic tremor" is a recently emerging disease entity that is associated with several congenital myopathies affecting either thick or thin filament-associated proteins expressed in the type I or type IIa muscle fibers (Stavusis et al., 2020;Schaefer et al., 2021).Although most pathological tremor syndromes have been linked to oscillatory activity in the central and/or peripheral nervous system, myogenic tremor is thought to have its tremor origin in muscle (Schaefer et al., 2021).The myogenic tremor-associated thick filament proteins include MyHC Iβ (MYH7), MyHC IIa (MYH2), myosin regulatory light chain (MYL2), and slow myosin binding protein-C (MYBPC1).The thin filament-associated proteins causing myogenic tremor include slow skeletal troponin T (TNNT1), nebulin (NEB), and tropomyosinα-3 chain (TPM3).Besides patients, the gene mutations in these sarcomeric proteins are also reported in pigs (Richter et al., 1995;Tammen et al., 1999;Murgiano et al., 2012) and calves (Wiedemar et al., 2015), which are linked to myopathy with relatively high-frequency, kinetic and postural tremor.
Presently it is unknown how the abnormalities in the specific fiber types containing slow-twitch or fatigue-resistant characteristics eventually induce "myopathy with tremor" phenotype.Potential etiologies of pathological tremors are divided into three types which are linked to a pacemaker (also called oscillator) in the central nervous system, the peripheral nervous system, or the muscle itself.The abnormalities in ΔSlow MN mice originate from the death of S/FR MN, which induces abnormalities in muscle fibers.Given the coincidence of the onset of tremor with the timing of the observation of these changes, it is possible that the mechanism of myogenic tremor is involved in the tremor of ΔSlow MN.In addition, a general increase in tremor power was  observed not only at a specific frequency (19 Hz) but also over a wide frequency range, suggesting the presence of multiple tremor pacemakers and enhanced physiological tremor.In normal adult muscles, individual muscle fibers are characterized by the specific expression of single myosin heavy chain (MyHC) isoforms ("pure fiber types") but, though at low frequency, some muscle fibers are shown to express two or more MyHC isoforms known as "hybrid fiber types".In ΔSlowMN mice, we detected aberrant "hybrid" muscle fibers expressing MyHC2A/MyHC2X or MyHC2X/MyHC2B at 20 or 40 weeks of age (Fig. 7B and C).Further analysis of the structure and function of sarcomere in abnormal fibers (hybrid fibers) is needed.Although central or peripheral oscillators have been suggested as the cause of tremor, ΔSlow MN mice may share the presence of a tremor oscillation involving muscles as well.ΔSlow MN mice may provide a new model of tremor involving both muscles and nerves and may contribute to the elucidation of the as-yet unexplained mechanism of tremor.
ΔSlowMN mice exhibited a shorter life span (Fig. 3B) but the cause of death is unknown.Lecomte et al. (2014) created mice deficient in ACh synthesis in MNs by crossing VAChT-Cre and ChAT-floxed mice and reported late-onset and progressive deficits similar to patients with postpolio neuropathy.Post-polio syndrome (PPS) is a disease affecting longterm survivors of poliomyelitis, beginning decades after an acute attack of paralytic poliomyelitis (Trojan and Cashman, 2005).Although the exact mechanism leading to the delayed recurrence of MN disease symptoms is enigmatic, a possible explanation is that long-term overuse of remaining MNs which need to compensate for and re-innervating onto orphaned muscle fibers become overused and unable to support the expanded motor units, resulting in chronic fatigue and muscle weakness.In ΔSlow MN mice, the function of the lost S-and FR-type MNs is compensated for by the sprouting and reinnervation of the remaining FInt-and FF-type MNs, resulting in motor unit expansion.At the muscle level, this expanded motor unit leads to the conversion of muscle fiber type to a "hybrid" form.MNs and muscles influence each other in both directions, and this change also alters the properties of MNs, converting the properties of MNs to the "remodeled" form which expresses both Fast and Slow MN markers (Fig. 8).Remodeled MNs are known to express ER stress markers and are known to be in a state of exhaustion like that seen in aging MNs (Morisaki et al., 2016).ΔSlow MN mice may provide a model that reflects the long-term motoneuron overuse states and their consequences.
There are inherent limitations to this study.First, to ensure neuronspecific expression of the lethal protein, we used the NSE-DTA mouse line; since DTA expression is regulated using the 3′-untranslated region of NSE mRNA, DTA expression is not only dependent on Cre-induced recombination but also on the expression level of the NSE gene (NSE mRNA level).Therefore, the interpretation that the toxin showed effects due to increased expression of the NSE gene cannot be ruled out.To resolve this issue, it is necessary to analyze the results of crosses with ROSA-DTA mice, for example, which use a strong and ubiquitous promoter, and compare them with the present results.Second, although the primary properties of each type of muscle fiber are known to be determined during the embryonic period, rapid muscle growth occurs during the first 3 weeks of life after birth.In our ΔSlowMN mice, MN death is known to begin 1 week after birth and progress after that.Thus, the present results can be interpreted not merely as a direct effect of S-and FR-type motor nerve death in mature motor units but also as a possible differential growth in the number and size of muscle fibers in the early postnatal period.

Fig. 1 .
Fig. 1.Selective ablation of S-and FR-type motor neurons in ΔSlow MN mice.(A) VAChT-Cre.Early:CAG-Syp/tdTomato mice (double mutants) at 8 weeks old expressed Syp/tdTomato fluorescence in a group (ca.30%) of ChAT-positive motor neurons in the cervical spinal cord.The Syp/tdTomato-positive motor neurons mostly disappeared in NSE-DTA:VAChT-Cre.Early:CAG-Syp/tdTomato mice (triple mutants) at the same age.(B, C, and D) Quantitative comparison of the number of tdTomato-positive and tdTomato-negative motor neurons between the double and triple mutants in the cervical (B), thoracic (C), or lumbar (D) spinal cord.n = 3, 40 sections were analyzed per animal.(D) Comparison of the number of γ motor neurons (classified as ChAT-positive and NeuN-negative cells) between the double and triple mutants.Data are pooled from the cervical and lumber spinal cord (n = 3, 40 sections were counted per animal).**p < 0.01.NS, no significant, Student's t-test.Scale bar, 100 μm.

Fig. 3 .
Fig. 3. Phenotype of ΔSlow MN mice.(A) Both male and female ΔSlowMN mice showed significantly reduced body weight compared with Control mice.Each mouse was weighed twice a week starting P21 through 40 weeks of age.**p < 0.01 (from 20 to 40 weeks), two-way repeated measure ANOVA with Sidak's multiple comparison test.(B) Survival curve showing the reduced lifespan of ΔSlowMN mice compared with Control mice.Overall survival was determined for both sexes from birth through 60 weeks of age.***p < 0.001, log-rank test.(C) Representative photomicrographs and X-ray radiographs of 35-weeks-old ΔSlowMN mice show spinal deformity (kyphosis) and smaller body mass compared to Control mice.(D) Representative footprint patterns of a 35-weeks-old Control or ΔSlowMN mice (hindlimbs) without signs of ataxia.Walking direction, left to right.

Fig. 4 .
Fig. 4. Power spectral analysis of tremor in ΔSlowMN mice.(A) Amplitude of tremor-like movements analyzed by the accelerometer following a fast Fourier transformation from ΔSlowMN or Control mice.n = 6 for each genotype (30-35 weeks of age).(B) Harmaline-induced tremor in Control mice.Animal movements were recorded before and after injection.n = 3 (30-35 weeks of age).(C) Harmaline-induced tremor in ΔSlowMN mice.Animal movements were recorded before and after injection.n = 4 (25-30 weeks of age).(D) Harmaline-induce c-Fos immunoreactivity in the inferior olive of Control and ΔSlowMN mice.Scale bar, 100 μm.

Fig. 5 .
Fig. 5. Electromyographic indexes in ΔSlowMN mice.(A) Experimental set-up showing the position of recording and stimulation electrodes.(B) Compound muscle action potential (CMAP) induced by supramaximal stimulation of the sciatic nerve.(C) Average single motor unit potential (SMUP) is determined by an incremental stimulation technique.(D) Motor unit number estimation (MUNE) is calculated by dividing the CAMP amplitude by the SMUP amplitude.*p < 0.05 (n = 3), one-way ANOVA with Tukey's multiple comparison tests.

Fig. 6 .
Fig. 6.Changes in muscle gross morphology and fiber types in ΔSlowMN mice.(A) The soleus muscle (SOL) from ΔSlowMN mice is pale and thin compared with that from Control mice (25 weeks of age).No such differences were seen in the gastrocnemius muscle (GAS) and extensor digitorum longus muscle (EDL).Scale bar, 2 mm.(B) Schematic illustration of a transverse section of the calf muscle, with the location of "bone side" or "skin side" in the lateral gastrocnemius muscle (LG).MG, medial gastrocnemius muscle; P, the plantaris muscle; S, the soleus muscle.(C) Immunopositive MyHC isoforms were visualized in calf muscle transverse-sections from ΔSlowMN mice or Control mice at 38 weeks of age (MyHC1, white; MyHC2A, blue; MyHC2X, green; MyHC2B, red).The soleus muscle was mostly atrophied and disappeared in ΔSlowMN mice.Scale bar, 1 mm.

Fig. 7 .
Fig. 7. Representative transverse sections and graphs of each portion of calf muscles visualized by the one-step four-colour immunostaining method.(A) Soleus muscle (left to the dotted line) shows massive atrophy in ΔSlowMN mice at 20 and 40 weeks of age.(B) Plantaris muscle shows a decrease in MyHC IIa fibers and the appearance of hybrid fibers in ΔSlowMN mice at 20 and 40 weeks of age.(C) The bone side of the lateral gastrocnemius muscle exhibits a decrease in MyHC IIa fibers and the appearance of hybrid fibers in ΔSlowMN mice at 20 and 40 weeks of age.(D) There are no apparent changes in fiber types in the skin side of the gastrocnemius muscle between ΔSlowMN and Control mice.Scale bar, 100 μm.Muscle fiber types were analyzed by counting >200 muscle fibers at each portion.n = 3 at each age of the week.*p < 0.05, **P < 0.01, Student's t-test.