No significant sex differences in incidence or phenotype for the SMNΔ7 mouse model of spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disease that affects 1 out of every 6,000–10,000 individuals at birth, making it the leading genetic cause of infant mortality. In recent years, reports of sex differences in SMA patients have become noticeable. The SMNΔ7 mouse model is commonly used to investigate pathologies and treatments in SMA. However, studies on sex as a contributing biological variable are few and dated. Here, we rigorously investigated the effect of sex on a series of characteristics in SMA mice of the SMNΔ7 model. Incidence and lifespan of 23 mouse litters were tracked and phenotypic assessments were performed at 2-day intervals starting at postnatal day 6 for every pup until the death of the SMA pup(s) in each litter. Brain weights were also collected post-mortem. We found that male and female SMA incidence does not differ significantly, survival periods are the same across sexes, and there was no phenotypic difference between male and female SMA pups, other than for females exhibiting lesser body weights at early ages. Overall, this study ensures that sex is not a biological variable that contributes to the incidence ratio or disease severity in the SMNΔ7 mouse model.

Introduction

Spinal Muscular Atrophy (SMA), the leading genetic cause in infant mortality, is an autosomal recessive disease that affects 1 out of every 6,000–10,000 individuals at birth [1, 2]. In recent years, there have been three treatments developed and approved by the U.S. Food and Drug Administration that have alleviated symptoms for some SMA patients. These treatments have exhibited short-term efficacy but are highly expensive and even ineffective for some patients. Moreover, the long-term response to these treatments remains inconclusive [3, 4]. These new treatments, while promising, do not represent cures for the disease, and show the need for a continued investigation into the barriers that hold back treatment effectiveness.

The deletion or mutation of survival motor neuron 1 (SMN1) gene causes lower motor neuron dysfunction and degeneration, leading to progressive muscle atrophy, weakness, and paralysis in ~96% of SMA patients [5, 6]. SMN1 is the disease-determining gene for SMA, but an additional gene, SMN2, can partially make up for the reduced protein levels when SMN1 is dysfunctional. The number of SMN2 copies carried by SMA patients is inversely correlated to symptom severity, which is defined by age of onset, level of motor function, and patient survival time [2, 7]. These factors are used clinically to classify patients into SMA types 0 through 4, with type 0 being most severe and type 4 being the least [8–11]. Generally, the higher the SMN2 copy number, the less symptom severity SMA patients experience. However, even within each SMA type, there is substantial heterogeneity across patients [12]. This heterogeneity can be explained, at least in part, by several other disease-modifying genes that are suggested to also contribute to disease severity, both SMN-dependent and -independent. Among these contributing proteins identified thus far, several of them are exclusively encoded by genes on the X chromosome, such as Plastin 3 (PLS3) [13, 14], Ubiquitin Specific Peptidase 9 X-Linked (USP9X) [15], and Ubiquitin Like Modifier Activating Enzyme (UBA1) [16]. Moreover, there is accumulating evidence for an important sex-specific response bias that impacts the structure and function of SMN-diminished tissues, and that this likely stems from maternally inherited mitochondrial adaptations and the aforementioned X chromosome-linked modifying factors (for review, see [17]).

Reports of sex differences in SMA patients are largely inconsistent or incomplete. One study reported a male to female ratio of 2.0 in SMA type I patients, with no difference among sexes in age of onset or life expectancy [18]. Another study reported the infantile form of SMA is more severe in males [19]. However, in studies with type I and II SMA patients, no sex differences in ratio or symptom severity were reported [20–23]. For the less severe forms of SMA, studies report milder symptoms and slower clinical progression for females compared to males [19, 20, 22, 24–27], although there are some studies that contradict these findings [21, 23]. Nonetheless, these studies indicate the role of sex in the development and severity of SMA pathology is worthy of more attention. Resolving the inconsistencies observed across studies in SMA patients is of increasing importance as evidence suggests that biological sex is a factor in the development and phenotypical expression of other neurodegenerative diseases, such as Parkinson’s, Alzheimer’s, and amyotrophic lateral sclerosis [28]. These sex differences may reflect common biological mechanisms across neurological disease and further research may aid in developing new therapies and applying effective clinical care. Another study in mouse brains showed that young adult females have lower oxidative stress and a more reduced nicotinamide adenine dinucleotide (NADH)-linked respiration rate [29], suggesting that female mice may be more resistant to certain mechanisms of pathological neurodegeneration.

Animal models for SMA have proved extremely useful in developing our understanding of the disease and are widely accepted as a viable mode of investigation [30–32]. Mouse models are the predominant choice in SMA research [33, 34]. Mice possess a single copy of the homologous human SMN gene known as Smn. Smn knock-out mice do not survive past embryonic stages [35, 36]. Introduction of human SMN2 rescues Smn knockout mice from embryonic lethality, providing mice a lifespan of about 5 postnatal days [36, 37]. Mouse models of SMA have been used extensively to understand SMA pathology but have rarely been used in analyzing sex-specific incidence or symptom severity. One recent study reported that neuromuscular junction defects and muscle atrophy were more prominent in male Smn^2B/−;SMN2^+/− mice [38].

The SMA-affected mice of the SMNΔ7 mouse model transgenically express the human SMN2 gene and SMNΔ7 isoform, which lacks exon 7, with a knockout of the murine Smn gene. This provides enough functional SMN protein to allow for a short-term survival period averaging about 13 postnatal days, making this mouse model a powerful and commonly used tool to assess phenotypes that represent type 1 and type 2 SMA [39]. One study developed a battery of quantitative behavioral tests in the SMNΔ7 mouse model for the identification, evaluation, and development of therapeutic candidates for SMA. In this study, the authors compared phenotypes between male and female SMA mice and mainly found no significant differences other than for females exhibiting lower body weights [40]. However, the sample size of this study was rather small, and sex differences in this model have not been thoroughly revisited since. Given the importance of this SMA mouse model and the recently developing question of sex-specific vulnerabilities in SMA patients, the study described in this paper was performed to revisit and expand on the question of sex differences in the SMNΔ7 mouse model by taking a close look at both incidence and phenotypic discrepancies.

To investigate sex-specific phenotypes in SMNΔ7 mice, we document the progression of disease severity and motor task ability for both male and female pups from postnatal day 6 (P6) after the symptom onset at around P5 in SMA pups [39]. We expanded on previous studies [40, 41] by assessing not only body weight and survival with respect to sex, but also the likelihood of male and female SMA pups, as well as the physical ability of SMA pups, quantified by a series of locomotor ability tasks. The righting reflex and negative geotaxis tasks were used to assess motor and sensory development of neonate mice across sexes [42–44]. Moreover, we also analyzed for any sex-specific differences in the progression of these physical abilities. We report that male and female SMA pups are born at the same incidence rate, and that sex as a biological factor may contribute to differences in body weight, but does not significantly affect lifespan, physical ability, or phenotype severity in SMNΔ7 mice. This study provides evidence that there are no significant sex differences in SMA phenotypes in the SMNΔ7 model, and that sex-specific phenotypic differences should not be a major concern when using this mouse model to study SMA.

Materials and Methods

Animals

All animal procedures were approved by the Institutional Animal Care and Use Committee of Delaware State University. Mice were maintained under a 14/10 h light/dark photoperiod with PMI rodent diet (Animal Specialties and Provisions) and water available ad libitum. Heterozygote male and female mice of the FVB.Cg-Grm7 < Tg(SMN2)89Ahmb > Smn1<tm1Msd>Tg(SMN2*delta7) 4299 strain obtained from Jackson Laboratory (stock#: 005025, Bar Harbor, ME) were mated to produce pups for experiments. Full litters were used for experimentation and were tracked for the lifespan of the SMA pup(s). At this point, all mice in the litter were euthanized and brains were collected for weighing. Mouse genotyping was done by Transnetyx (Germantown, TN) after experimentation and data analyses, but before performing statistical tests for significance. Wild type and heterozygous pups were pooled as controls, and pups with the homozygous mutation formed the SMA group as described previously [32, 45]. A total of 7 breeding pairs of SMNΔ7 mice were used to collect pups. 23 litters were tracked, totaling to 158 pups with 35 genetically confirmed SMA pups being used for this study.

Body weights and righting reflex task

After the birth of a litter, locomotor testing began on post-natal day (P) 6. On this day, and subsequently (two-day intervals), a series of experiments were performed. For every mouse in the litter, body weight was acquired. Every mouse was also subject to two tasks that measure disease phenotype severity including the righting reflex task and the negative geotaxis task. These tasks were routinely performed during the dark period between 11:00 and 15:00 on a Tecniplast changing station (Buguggiate, Italy). On a few days of experimentation, no researcher was available for testing. This caused a peak in biological and technical replicate sizes at P10–14, rather than P6 (Table 1–4).

Table 1.

Mouse numbers (n) of body weight measurements for male and female control (CTL) and SMA pups at each postnatal day of testing.

Sample sizes (n)			P6	P8	P10	P12	P14	P16	P18	P20
Body Weight	CTL	M	39	41	50	43	45	27	12	2
		F	40	44	50	44	48	29	10	1
	SMA	M	14	14	14	14	11	6	3	3
		F	14	15	18	12	14	8	4	1

Table 4.

The mouse numbers (n) of the negative geotaxis task for male and female control (CTL) and SMA pups at each postnatal day of testing.

Sample sizes (n)			P6	P8	P10	P12	P14	P16	P18	P20
Negative Geotaxis Task	CTL	M	35	37	45	39	39	26	11	5
		F	41	45	51	51	47	32	13	3
	SMA	M	13	13	13	13	11	6	3	2
		F	13	15	18	12	14	8	4	1

On a testing day, each mouse was weighed and subject to the right reflex task and the negative geotaxis task, in that order. On the changing station, body weight was recorded one at a time, then a single trial of the righting reflex task was performed. For this task, mice were held on their backs for 5 seconds. After this period, the experimenter removed contact and started the timer. The mouse was then allowed to right itself to a prone position. Once the mouse was upright and in a natural prone position, the time is recorded in seconds. Mice were considered to have failed the task if it took longer than 60 seconds to complete. It typically takes healthy mice about 5 seconds to complete this task, but 70–90% of SMA mice cannot complete this task in that time [41]. Mice were allowed a 2-minute resting period before the negative geotaxis task.

Negative geotaxis task

For the negative geotaxis task, a 45° inclined platform was used, with microfiber paper towel covering the surface. The pup was placed ⅔ up the platform with its head pointing downwards and the timer began. Mice will naturally turn so their heads face upwards. The time it took for the pup to turn 180° was tracked. The pup was considered to have completed the task once all four paws had been stepped into a natural standing position. Once the fourth paw was placed, the time was recorded in seconds. Healthy mice, usually P12 or older, did not always turn themselves upwards. This was thought to be due to a lack of a sense of danger, rather than a locomotive deficit. For this reason, the experimenter would flick the platform to initiate movement from the mouse. There were two conditions for failure. The pup was considered to have failed if they could not turn 180° with all four paws within 60 seconds, or if the pup could not remain in a righted prone position on the inclined platform and fell. In the latter case, the latency for failure was recorded. The last test response observed was no attempt. A pup was considered to not attempt the task if they did not make any motion towards turning upwards for the entire 60 second trial. This test was performed in 3 trials, with a short rest in between trials. Trials were averaged, as well as kept separate for analysis. These tests were performed for every pup in a litter and repeated at 2-day intervals throughout the lifespan of the SMA pup(s). After the SMA pup(s) lifespan, the remaining littermates were euthanized and every brain was collected, weighed, and recorded with age. In a few cases, pups did not survive because they were rejected by the breeding dams. Brain weights were not collected for SMA pups in this scenario.

Statistical analysis

Statistical analyses were planned and performed in the Graphpad Prism 9 software (graphpad.com), which was then used to generate figures. Fisher’s exact contingency test was used where proportions or percentages were being compared across male and female. This included the proportions of female SMA pups versus male SMA pups, as well as the failure percentage, the no attempt to attempt ratio, and the success to failure ratio for the negative geotaxis task between female SMA and male SMA pups. Multiple unpaired Student’s t-tests with Welch correction were used to compare measurements across male and female SMA and control pups at each postnatal day of testing for body weights and brain weights. Šídák-Bonferroni’s method of correction for multiple comparisons was used for the righting times with and without including the failed trials, as well as the time to success and time to failure for the negative geotaxis task. To assess any difference in lifespan, a Kaplan Meier survival curve was used with the Mantel-Cox log-rank test, logrank test for trend, and Gehan-Beslow-Wilcoxon test.

Results

No significant difference was observed in SMA incidence between male and female SMA pups

To investigate whether the occurrence rate of male and female SMA pups are significantly different, SMNΔ7 mice breeding pairs were tracked. Once a female was notably pregnant, we examined the mice daily to record date of birth. At the end of the suspected SMA pup(s) lifespan, littermate controls were euthanized, and the tail of every pup was collected to be genotyped for SMA and for sex. A total of 7 breeding cages of SMNΔ7 mice were used to collect pups, with each cage containing 1 male heterozygous and 2 female heterozygous mice. Altogether, 23 litters were tracked, totaling to 158 pups. Out of all 23 litters, 11 of them contained one or more male SMA pups and 15 contained one or more female SMA pups; of which a contingency test revealed no significant difference between the proportions (p = 0.63; Fig. 1a). Out of the 158 pups tracked, 82 were female, 75 were male, and 1 was undetermined. The undetermined pup was not included in statistical analysis. Of the 82 female pups born, 21 of them were genetically confirmed to be SMA (25.6%). Of the 75 male pups born, 14 of them were genetically confirmed to be SMA (18.7%). A contingency test revealed no significant difference between the proportions of female and male SMA pups (p = 0.34; Fig. 1b). These results suggest that the likelihood of a male pup versus a female pup being born with SMA are not significantly different in the SMNΔ7 mouse model.

Fig. 1. The proportion of female and male SMA pups born are not significantly different.

a) Of 23 litters (black), 11 litters contained one or more male SMA pups (dark gray) and 15 litters contained one or more female SMA pups (light gray). b) 21 of 82 (25.6%) female pups and 14 of 75 (18.7%) male pups were genetically confirmed to be SMA. Fisher’s exact contingency tests were applied to statistically compare proportions.

The progression of brain and body weights, as well as survival, are identical between male and female SMA pups

To compare changes in body mass over time, the body weights of SMA pup(s) and littermates were acquired and tracked at postnatal day (P) 6 and at subsequent 2-day intervals over the lifespan of the SMA pup(s) in the litter. P22 was the longest survival time of an SMA pup in this study. The body weights for control pups were consistent with reports for this mouse line from Jackson Laboratories (www.jax.org; Fig. 2a). Consistent with a previous study [40], male SMA body weights were significantly higher than female SMA pups at P6 (p = 0.006) and P8 (p = 0.011) but showed no statistical significance at any other time points (Fig. 2b). The rate of body mass deterioration was also compared across sexes using a linear regression analysis and a comparison of lines of best fit. The regression slope for male SMA pups was −0.180 and for female SMA pups was −0.106, with the statistical difference being p = 0.14 (Fig. 2c). Sample sizes for each experimental group at each postnatal day of testing are reported in Table 1.

Fig. 2. Male SMA body weights are higher at younger ages, but decay with a rate comparable to female pups.

a) Body weight progression curves of SMA pups SMA weights, as well as control pups. b) Body weight progression curves of just SMA pups with a smaller y-axis range to emphasize relative differences. Individual data points are plotted, and significant differences are shown. Multiple unpaired Student’s t-tests with Welch correction. c) Linear regression of the average body weights from P8 (peak body weight) to P16, to indicate the linear decrease in body weight over time between male and female SMA pups. Lines of best fit and their slopes are shown for both male (blue) and female (red). *p < 0.05, **p < 0.01; linear regression analysis, comparison of line of best fits. All sample sizes (n) are shown in Table 1.

The lifespans of SMA pups were recorded and the brain weights of SMA and littermate control pups were collected. The majority of the SMA pups died from disease complications. Across 7 litters, 6 female and 4 male SMA pups were rejected by the breeding dam; all of these occurred within the range of postnatal day 12 to 21. Tails were still collected from these 10 pups for genotyping and included in the sex difference analysis of incidence. There were 3 cases where the brain was still completely intact, thus the weights were measured and included in the analysis. SMA brain weights of both male and female pups were lower than littermate controls. Every age at which intact brain weights could be statistically compared showed no significant difference between male and female SMA pups. This was also the case when brain weights for all ages were pooled for comparison (data not shown). Sample sizes for each day where brains were collected are reported in Table 2. Additionally, a Kaplan Meier survival curve for comparison between male and female SMA pups was generated. Since we cannot be sure whether the pup died from ingestion by a breeding parent or from the consequences of SMA, none of the 10 rejected mice were included in the lifespan assessment. Although the n numbers for SMA pups are low (Table 2), there were no significant differences shown for the Mantel-Cox log-rank test, logrank test for trend, or Gehan-Beslow-Wilcoxon test. The median survival for male and female SMA pups were both 18 (Fig. 3). These data suggest that the body weight progression is slightly different between male and female SMA pups at early-stage but body weights reach the same level by the late-stage of the disease. We also observe that the brain weight progression is the same rate between the two groups and there is no difference in the lifespan between male and female pups in the SMNΔ7 model.

Table 2.

Mouse numbers (n) of brain weight measurements for male and female control (CTL) and SMA pups at each postnatal day that brains were collected.

Sample sizes (n)			P14	P15	P16	P17	P18	P19	P20	P21	P22
Brain Weight	CTL	M	10	8	12	0	2	3	4	1	0
		F	6	8	5	0	7	4	3	1	0
	SMA	M	2	1	0	1	3	1	3	1	1
		F	2	0	1	1	1	2	3	0	0

Fig. 3. The lifespans of male and female SMA-affected pups are comparable.

Kaplan Meier survival curves for female SMA pups (red) and male SMA pups (blue) depicting the probability of survival over the course of 22 postnatal days. Mantel-Cox log-rank test (p = 0.108), logrank test for trend (p = 0.533), and Gehan-Beslow-Wilcoxon test (p = 0.148).

Motor function deficiency is not significantly different between male and female SMA pups

Righting Reflex

To quantify the severity of motor function deficiencies for male and female SMA pups, we performed righting reflex and negative geotaxis tasks, both of which require proper motor function from neonates. The righting reflex task was performed for all mice in a litter at 2-day intervals starting from P6 for the lifespan of the SMA pup(s). Statistical comparisons of righting time between male and female SMA pups were performed with and without including the failed trials (Fig. 4a and Fig. 4b, respectively). The number of biological replicates is equal to the number of technical replicates when the failed trials are included (n = N; Fig. 4a, Table 3), since each pup performed just one trial of the righting reflex task each day of testing. Righting time generally decreased as the mice of both sexes got older, yet neither the comparison including the failed trials nor the comparison excluding them displayed significant differences between male and female SMA pups at any age (Fig. 4a,b). The progression of failed trial percentages were compared across sexes as well. As mice got older, the ability to complete the task generally improved for both sexes, however no significant differences between sexes were observed at any age (Fig. 4c). Technical replicate sizes (N) are shown in Table 3. These results suggest that sex is not a biological variable that affects a particular mouse’s ability to perform the righting reflex task.

Fig. 4. Male and female SMA-affected pups exhibit similar righting ability.

Righting reflex times, in seconds (s), at 2-day intervals starting at P6 until P16 are not significantly different when a) including failed trials (>60s task time) and b) excluding failed trials. Multiple unpaired Student’s t-tests with Šídák-Bonferroni method of correction for multiple comparisons. c) Failure rate as a percentage, for male (blue) and female (red) SMA pups at 2-day intervals from P6 to P16 are not significantly different. Fisher’s exact contingency tests to statistically compare proportions. Biological and technical replicate sizes are shown in Table 3.

Table 3.

Replicate numbers (N) of both the righting reflex and negative geotaxis tasks for each analytical strategy employed.

Sample sizes (N)				P6	P8	P10	P12	P14	P16	P18	P20
Righting Reflex Task	w/out Failures	SMA	M	3	4	5	8	8	3	2	1
			F	3	3	4	5	8	7	3	1
	w/Failures		M	14	14	14	13	12	6	3	2
			F	14	15	18	12	14	9	4	1
		CTL	M	37	39	48	42	41	26	12	4
			F	41	44	50	44	46	29	10	3
Negative Geotaxis Task	No Attempt	SMA	M	2	2	2	1	1	1	0	0
			F	2	4	6	6	4	5	1	0
	Time to Failure		M	18	18	16	16	11	7	3	0
			F	15	16	10	9	9	4	6	2
	Time to Success		M	1	6	7	8	14	6	5	5
			F	4	1	11	5	8	9	5	1
		CTL	M	97	105	138	119	117	75	33	9
			F	95	118	146	129	135	87	30	9

Negative Geotaxis Task

We also performed the negative geotaxis task for each mouse at 2-day intervals starting at P6 for the lifespan of the SMA pup(s). Three trials were performed for all mice. Multiple measurements were tracked and compared to completely assess the physical ability of SMA mice. The latency of finishing a successful trial (Fig. 5a) and the latency of a failed trial (Fig. 5b) were compared. Both latencies were relatively consistent over all time points where the test was administered. When comparing between male and female, neither the latency for success or the latency for failure showed statistical significance at any age for SMA pups or control pups (Fig. 5a,b). There were also scenarios where the mice would not make any physical attempt at completing the task, albeit this was less common. The no attempt to attempt ratio was compared across sexes at each post-natal day of testing as well. A higher ratio means that the mice more often did not attempt the task. We observed a trend that female SMA pups were less likely to attempt the task, particularly as the days went on. But no statistical significance was observed (Fig. 5c). Statistical analyses were performed to determine whether trial number had a significant effect on task latency. There was no significant difference across any group, indicating that fatigue did not affect task performance (not shown). Another comparison of the success to failure ratio was performed, where a higher ratio equates to a higher likelihood of successfully completing the negative geotaxis task for SMA pups. At P8 and P14, male SMA pups were significantly more successful at performing the task compared to female SMA pups (Fig. 5d). Control mice were 100% successful at completing the task and attempted it in 100% of the trials. Technical replicate sizes and biological replicate sizes for each postnatal day of testing are reported in Table 3 and Table 4, respectively. These results suggest that female SMA pups may be worse than male SMA pups at performing the negative geotaxis task, however this difference is minimal.

Fig. 5. Comparable negative geotaxis task ability between male and female SMA pups.

Negative geotaxis task ability is comparable between male (blue) and female (red) SMA pups at each 2-day interval from based on measures of a) time (in seconds) to successful trial and b) time (in seconds) to fail the trial. c) The no attempt to attempt ratio for male and female SMA pups at each 2-day interval from P6 to P16 was not significantly different. A higher ratio equates to less likelihood to attempt the task. d) The success to failure ratio for male and female SMA pups at each 2-day interval from P6 to P16 was significantly higher for males at P8 and P14. A higher ratio equates to a higher success rate. *p < 0.05. For data in panels a and b, multiple unpaired Student’s t-tests with Šídák-Bonferroni method of correction for multiple comparisons were used, and technical replicate sizes are shown in Table 4. For the table in panels c and d, Fisher’s exact contingency tests were used to statistically compare proportions, and biological replicate sizes for c and d are shown in Table 4.

Discussion

The data presented in this study provide a rigorous analysis of incidence and phenotypic differences between male and female SMA-affected mice using the SMNΔ7 mouse model. To date, no studies have focused solely on investigating sex as a biological variable in SMNΔ7 mice. However, clinical data suggests that there are sex-based biases in SMA disease incidence and severity. In this study, we tracked SMA incidence in 23 litters and used phenotypic assessments, consisting of a battery of locomotor tests, to assess whether sex is a biological variable that contributes to SMA symptom severity. The findings in this study indicate that sex does not conclusively contribute to any phenotypic variation in the SMNΔ7 mouse model for SMA.

Mouse models for SMA are null for the murine Smn gene and typically have an insertion of human SMN2 genes [30]. The SMNΔ7 mouse model was used in this study due to its practical timeline and it being among the most used animal models in SMA research. This model has two gene insertions, human centromeric SMN2 and human SMNΔ7, which is a major product of SMN2, and aberrantly splices exon 7. Expressing these two genes in Smn null mice initially extended survival to about 13.3 days. However, 2013 reports from Jackson Laboratories report a mean survival of 17.7 days (Jackson Laboratories: stock no. 005025). This is consistent with the reported median survival times reported in this study, which were 18 days for both male and females. The extension of lifespan in the same mouse model over time may be due to the improvement of facilities, genetic drift, or other causes. Furthermore, the level of full length SMN protein present among SMN2^+/+;SMNΔ7^+/+;Smn^−/− mice causes their phenotypes to more accurately mimic that of human patients [39]. However, genetic backgrounds, as well as disease and neuroprotective mechanisms, are different between mouse models and humans. Moreover, SMA phenotypes and motor deficits in mice do not perfectly reflect that of SMA patients. Thus, it’s become important to revisit and enhance our documentation of sex-specific phenomena in SMA mouse models due to the commonplace usage of this model [30, 31].

Sex-specific differences in incidence and symptom severity have been observed in clinical studies for many neurodevelopmental and neurodegenerative diseases [46], such as amyotrophic lateral sclerosis (ALS) [47], Alzheimer’s disease (AD) [48], and Parkinson’s disease (PD) [49]. ALS is more prevalent in males, but survival is worse for females [47, 50]. In Alzheimer’s, females have higher prevalence and greater cognitive deterioration [48, 51]; and in Parkinson’s males are preferentially affected whereas females experiences faster disease progression and greater mortality [49]. In SMA patients, accumulating evidence also supports a sex-specific bias. Males have higher incidence and more severe phenotypes across different SMA subtypes, populations, and ethnicities [12, 20, 52], and a recent clinical study investigating male and female sibling pairs with SMA found that males experience more severe motor deficits [53].

Incidence was not significantly different between male and female SMNΔ7 mice in this study; however, the question remains open for other mouse models of SMA. For studies investigating phenotypic variation across sexes in mice, very few have been performed to date. In a study using a battery of tests for SMNΔ7 mice reported that female SMA mice have lesser body weights at all ages, but no significance was found [40]. In both sexes, reduced body weight is common in SMA mice due to the abnormal metabolism caused by SMN depletion [30]. At P6 and P8, we observed significantly decreased body weight in female SMA-affected mice compared to male SMA-affected mice. Controls at the same ages showed no sex-specific effect, which is consistent with sex-specific reports from Jackson Laboratories for healthy albino mice that start at three weeks (Jackson Laboratories: stock no. 000651). Other studies have observed a reduction in body weight being more prominent in female SMA mice as well [54, 55]. This effect may be related to the asymmetric inheritance of the mitochondrial genome and the X chromosome [17], which are better optimized in females than in males [56, 57]. In humans, studies addressing phenotypes such as body weight across male and female SMA patients are hard to perform due to extreme heterogeneity unrelated to disease phenotypes. The cause of this early-stage weight discrepancy between sexes is difficult to pinpoint exactly, but it is likely attributable to mitochondrial, genetic, immune, and metabolic differences [17, 56–59], as well as the severity in which SMN depletion affects the relevant mechanisms.

To investigate motor ability in SMNΔ7 mice, we performed two tests: the righting reflex and the negative geotaxis task. Healthy neonatal mice can right themselves to a prone position within 5 seconds after being placed on their backs. This is referred to as the righting reflex response, and this ability is seen as early as P2 [41]. In a previous study on SMNΔ7 mice which did not control for sex, only 10–30% of SMA mice possessed this reflexive ability. From the head-down position, a healthy pup will typically turn its body so that its head faces. This is referred to as the negative geotaxis task, and the average age for this reflex to appear in rodents is P7 with a range from P3–15 [41, 60]. In a study on neuromotor transmission, male Smn^2B/−;SMN2^+/− mice displayed greater denervation and lesser muscle fiber usage compared to females [38]. However, results from the locomotor tests performed in our study did not convincingly display a sex-specific vulnerability in males or females. It is possible that other locomotor tests may reveal sex differences in SMA mouse phenotypes, but the righting reflex and negative geotaxis tasks are among the most used for SMA mouse models.

Conclusion

In recent years, sex differences in SMA patients have become an important area of study, with females exhibiting lesser incidence and symptom severity compared to males. It has become prudent to make efforts to document sex-specific differences in SMA patients, as well as revisit and develop our understanding in animal models. Here, we investigated sex differences in SMNΔ7 mice by documenting incidence for the first time. We also revisited sex-specific vulnerabilities in survival, phenotypic severity, and locomotor ability, which hasn’t been investigated in nearly two decades. While some studies indicate an important role of sexually dimorphic systems in combating disease pathology in SMA mice, we report that SMA incidence is not significantly different across sexes and that phenotypes and locomotion in SMA-affected SMNΔ7 mice remain mostly consistent across sexes. Understanding sex differences in SMA patients and mouse models has been elusive given the ubiquitous and diverse nature and function of the SMN protein. Research on male vulnerability in other neurodegenerative diseases has provided hints, helping to direct SMA research. X-linked modifying factors, disproportionate mitochondrial genetic potency, differential sex hormones, and sexually dimorphic immune response and cellular metabolism may all be relevant in mediating SMA pathology. These differences do not appear to lead to significant sex differences in incidence or phenotype for the SMNΔ7 mouse model, underscoring that the genetic divergence between mice and humans makes it difficult to make conclusions about sex-specific vulnerabilities in SMA patients based on studies in mice. However, our study does not rule out that there may be sex differences in SMNΔ7 mouse model cellular pathology or in other less severe SMA mouse models. Future studies aimed at characterizing differences in cellular and molecular pathologies across male and female SMNΔ7 mice will be valuable. In addition, characterizing sex differences in both phenotype and pathology at later points in the murine lifespan using less severe SMA mouse models may reveal important effects as well and is worth investigating in the future.

Highlights.

• Male patients with less severe SMA subtypes have higher incidence ratio and symptom vulnerability.

• The SMNΔ7 mouse model does not exhibit sex differences in incidence ratio.

• The SMNΔ7 mouse model continues to show consistency in phenotypic severity across sexes.

• Sex-specific vulnerabilities in SMA patients are not present in one of the most common mouse models of SMA.