The potential benefit of leptin therapy against amyotrophic lateral sclerosis (ALS)

Abstract Background Targeting leptin could represent a rational strategy to treat amyotrophic lateral sclerosis (ALS), as previously clinical studies have shown its levels to be associated with a lower risk of ALS disease. However, very little is known about the potential influence of leptin in altering disease progression in ALS, as it has thus far been correlated with the protection exerted by increased fat mass stores. Methods We studied the impact of leptin treatment beginning at 42‐days of age (asymptomatic stage of disease) in the TDP‐43 (TDP43A315T) transgenic (Tg) ALS mouse model. Results Our study shows that leptin treatment was associated with altered expression of adipokines and metabolic proteins in TDP43A315T mice. We also observed that weight loss decline was less prominent after leptin treatment in TDP43A315T mice relative to vehicle‐treated animals. In TDP43A315T mice treated with leptin the disease duration lasted longer along with an improvement in motor performance relative to vehicle‐treated animals. Conclusions Collectively, our results support leptin as a potential novel treatment approach for ALS.

condition. ALS therefore represents one of the most challenging socioeconomic problems of our future. There is an urgent need to improve upon current approaches to ALS.
Leptin is a polypeptide hormone primarily secreted by adipocytes that exerts its main biological function in the brain (Stephens et al., 1995;Zhang et al., 1994). Leptin acts by binding to receptors that are structurally related to the cytokine receptor class I family, while only its long isoform (Ob-Rb) is thought to transmit the majority of leptin's biological signals (Friedman & Halaas, 1998). However, in addition to its classical role in the neuroendocrine regulation of food intake, both clinical and epidemiological data show leptin to be a promising neuroprotective drug for progressive neurological conditions. Accumulating in vivo and in vitro studies have suggested that leptin has notable effects on neuroprotection (Bahor et al., 2017;Begley & Ellis, 2012;Fernandez-Martos et al., 2017;Liu et al., 2020;Marwarha et al., 2010) as well as on improving learning and cognitive function in Alzheimert's disease (AD) (Farr et al., 2006;Sato et al., 2011;Searcy et al., 2012). Many of the neurological beneficial properties of leptin are currently being experimentally probed in order to get a strong foothold of the therapeutic implications for other neurological disorders as Parkinson's disease (Rahnemayan et al., 2021;Zou et al., 2019). In the context of ALS, leptin levels are inversely associated with ALS risk (Nagel et al., 2017). A positive correlation of plasma leptin and body mass index (BMI) was observed in ALS patients (Ngo et al., 2015). This is of interest because patients with ALS are unable to maintain their body weight (Bouteloup et al., 2009;Desport et al., 2001), and rapid weight loss is clinically associated with worse disease outcomes (Ahmed et al., 2019;Kuraszkiewicz et al., 2020). Indeed, weight loss is a predictor of shorter survival in ALS (Lee et al., 2021), raising the possibility that leptin could be exploited to derive therapeutic effects.
Given the evidence showing leptin levels related with overall survival and prognosis of ALS patients (Nagel et al., 2017), this study aimed to determine the impact of leptin treatment in the well validated TAR DNA binding protein (TDP-43) A315T murine model of ALS TDP-43 proteinopathy (Hatzipetros et al., 2014;Wegorzewska et al., 2009), which recapitulates several aspects of the human ALS, providing, to our knowledge, the first insights into the potential benefit of leptin to ALS.

Monitoring and behavioral assessments
To monitor disease progression and onset (defined as the last day of individual peak body weight before gradual loss occurs) determination, body weight lost was measured and motor performance was evaluated using rotarod test. All mice were weighed and assessed three times per week until the disease onset-stage. After that mice were then checked daily in the morning until the disease end-stage (defined as the weight below 20% of the initial weight on each of 3 consecutive days). The rotarod motor test was performed on all mice once a week (Dang et al., 2014), starting from the 7 weeks of age until the day of euthanasia.
Animals were previously trained for three consecutive days and three times a day to promote the learning of the task. The accelerated protocol was applied for this motor monitoring as described previously by Mandillo et al. (Mandillo et al., 2008). In brief, mice were placed on a rotarod apparatus (Model 7650, Ugo Basile) at a speed of 4 rpm with acceleration up to 40 rpm over 300 s. Three tests were performed for each mouse with a minimal interval of 20 min, and the average of the longest two performances was taken as the final result for analysis.

Sample preparation
At disease end-stage, animals were terminally anesthetized with sodium pentobarbitone (140 mg/kg) and transcardially perfused with room temperature 0.01 M phosphate buffered saline (PBS; pH 7.4).
Blood was collected and processed as previously described (Rodriguez et al., 2021). Plasma samples were immediately frozen on dry ice and stored at -80 • C for later analysis.

Measurement of metabolic markers in plasma
Total ghrelin, the adipokines, resistin, and leptin, and metabolic

Statistical analysis
All data are presented as means ± standard error of the mean (SEM).
Differences between means were assessed by two-way ANOVA fol-

Leptin treatment modifies disease progression and improves motor performance in TDP-43 A315T mice
The biological impact of leptin treatment during the progression of ALS is unknown, even though this hormone has previously been shown to be associated with a lower risk of ALS disease and to confer a survival advantage in ALS patients (Nagel et al., 2017;Ngo et al., 2015). Thus, as it have been previously reported that TDP-43 A315T mice exhibit weight loss during disease progression (Esmaeili et al., 2013;Guo et al., 2012;Hatzipetros et al., 2014;Medina et al., 2014;Rodriguez et al., 2021), we assessed the capacity of leptin treatment to modify weight changes over time in TDP-43 A315T

Leptin treatment altered peripheral levels of ghrelin, resistin and leptin in plasma of TDP-43 A315T mice
We next studied how metabolic markers in plasma were affected by leptin treatment in TDP-43 A315T mice compared to age-matched WT littermates, as metabolic homeostasis is unbalanced in ALS patients (Ioannides et al., 2016), and this hormone is historically known for its important role in regulating body weight (Friedman, 2011). Plasma immunoassay analysis indicated circulating total ghrelin concentrations were increased in both WT and TDP-43 A315T mice at the endstage of disease, being significantly increased in Lep-treated TDP-43 A315T mice compared to VH-treated WT (p = .02; Figure 3a).
Resistin and leptin levels also showed genotype-specific differences (Figure 3b,c). Dunnett's post hoc test demonstrated significant differences in circulating levels of resistin VH-treated TDP-43 A315T mice F I G U R E 1 Leptin treatment alters body weight and changes disease onset and duration in TDP-43 A315T mice. (a) Body weight was monitored over time in WT controls and TDP-43 A315T mice treated with 0.03 mg/kg/day IN with leptin or VH (PBS-TDM; pH 7.2) daily for 14 consecutive days, beginning at the asymptomatic state, at 42 days of age. Starting weight on week 7. Although there was a trend, no significant differences were observed between Lep-treated TDP-43 A315T mice or VH-treated TDP-43 A315T mice. (b) Average disease onset and (c) disease duration were determined in WT controls and TDP-43 A315T mice treated with leptin or VH using body weight as a physiological parameter. Average disease duration of the animal was calculated as the time between the onset of disease (defined as the last day of individual peak bodyweight before gradual loss occurs) and the day of death. Comparatively the disease duration was higher in Lep-treated TDP-43 A315T mice vs. VH-treated TDP-43 A315T mice. Values are expressed as mean ± SEM. Comparison between groups was performed by two-way ANOVA followed by Dunett's post hoc test to compare all groups with VH-treated WT mice, while Tukey's post hoc test was used for multiple comparisons between all groups, where (a) * p < .05, ** p < .01, *** p < .001 versus VH-treated WT mice; # p < .05, ## p < .01, ### p < 0.001 versus Lep-treated WT mice; (b) ** p < .01 versus VH-treated TDP-43 A315T mice; and (c) ** p < .01 versus Lep-treated TDP-43 A315T mice. Corresponding graphs as per (a), that is, VH-treated WT mice (n = 4, white square and solid line), Lep-treated WT mice (n = 4, blue square and dashed line), VH-treated TDP-43 A315T mice (n = 3, white circles and solid line), and Lep-treated TDP-43 A315T mice (n = 3, orange circles and dashed line) compared to age-matched WT littermates (p = 0.02; Figure 3b). In addition, circulating resistin and leptin concentrations were lower in TDP-43 A315T mice compared to age-matched WT littermates, with this reaching statistical significance at the end-stage of disease in both groups compared to Lep-treated WT mice (p = .0019 and p = .007, respectively; Figure 3b,c). Additionally, a positive linear correlation was found using Spearman's test among the plasmatic levels of ghrelin and leptin compared to resistin in WT mice (Figure 4a; r = 0.81, p < .05; r = 0.762, p < .05, respectively), while no linear correlation was found in any of the adipocytokines studied for the TDP-43 A315T mice (Figure 4b).

DISCUSSION
A growing body of evidence shows disturbances in energy metabolism in ALS (Blasco et al., 2020;Tefera & Borges, 2016;Tefera et al., 2021;Vandoorne et al., 2018), suggesting that targeting metabolism could represent a rational strategy to treat this disease. Mild obesity appears to improve survival in ALS patients (Paganoni et al., 2011), and obesity affects leptin, which is associated with a lower risk of developing ALS

F I G U R E 2
Leptin treatment significantly improves motor performance in TDP-43 A315T mice. Behavioral assessment of motor function was performed in WT controls and TDP-43 A315T mice treated with leptin or VH over time. Significant differences between Lep-and VH-treated mice were seen. Values are expressed as mean ± SEM. Comparison between groups was performed by two-way ANOVA followed by Dunett's post hoc test to compare all groups with VH-treated WT mice, while Tukey's post hoc test was used for multiple comparisons between all groups, where * p < .05, ** p < .01, *** p < .001 versus VH-treated WT mice; # p < .05, ## p < .01, ### p < .001 1 versus Lep-treated WT mice; $ p < .05 versus VH-treated TDP-43 A315T mice. Corresponding graphs, VH-treated WT mice (n = 4, white square and solid line), Lep-treated WT mice (n = 4, blue square and dashed line), VH-treated TDP-43 A315T mice (n = 3, white circles and solid line), and Lep-treated TDP-43 A315T mice (n = 3, orange circles and dashed line) (Nagel et al., 2017). Indeed, plasma levels of leptin may be used as prog- also be worthwhile to consider other potential disease-relevant effects of leptin treatment in the TDP-43 A315T mice, in which a progressive motor impairment has been described (Stallings et al., 2010). In addition, we also found lower circulating levels of the adipokine resistin in TDP-43 A315T mice compared to controls WT animals, in contrast to clinical data, which have shown no differences in plasmatic levels of resistin between controls and ALS patients (Ngo et al., 2015). This result support previous unpublished data from our lab showing a downregulation of peripheral protein resistin levels in TDP-43 A315T mice (Rodriguez et al., 2021). In addition to the marked decrease of circulat-  (Singh et al., 2010). This observation is of interest because PAI-1 is a metabolic protein associated with an increase in adiposity and body mass index (BMI) (Kahn et al., 2006;Ngo et al., 2015). The majority of ALS patients have low BMI (Dardiotis et al., 2018), and a positive correlation of plasma leptin and BMI was observed in human ALS (Ngo et al., 2015). Thus, it is plausible that the  (Ferri & Coccurello, 2017). This protein stimulates insulin secretion in response to food intake (Elliott et al., 1993), and it has been proposed as an obesity-promoting hormone (Holst & Rosenkilde, 2020). Thus, it is conceivable that the up-regulation of circulating levels of GIP in Lep-treated TDP-43 A315T mice would help to reduce disturbances in energy metabolism associated with the progression of ALS in TDP-43 A315T mice (Shan et al., 2010;Wang et al., 2013). However, future experiments should try to corroborate this hypothesis.
Finally, a positive linear correlation was found among ghrelin and leptin relative to resistin levels in the WT animals, which is related to insulin resistance. Likewise, a positive correlation was found among the plasma levels of GIP and GLP-1 relative to insulin concentrations in the WT genotype, consistent with their role as incretins (Seino et al., 2010).
However, no correlations were determined in the TDP-43 A315T genotype, indicating a possible development of insulin resistance in this Tg mouse model of ALS. It should be noted that a positive correlation was found between levels of GIP and PAI-1 as well as between GLP-1 and glucagon in TDP-43 A315T mice.

CONCLUSIONS
In summary, our research provides the first preliminary experimental evidence for a potential therapeutic effect of leptin treatment in motor function as well as in some metabolic disturbances present in

ACKNOWLEDGMENTS
The authors would like to gratefully acknowledge the Animal Facility and Experimental Surgery Unit of the UDI-HNP for their excellent technical support. This work was supported by the funding from the Consejería de Educación, Cultura y Deportes, Fondo Europeo de Desarrollo Regional (FEDER), Junta de Comunidades de Castilla-la Mancha (SBPLY/17/180501/000303).

AUTHOR CONTRIBUTION
A.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study is available from the corresponding author upon reasonable request.

PEER REVIEW
The peer review history for this article is available at https://publons.