The Effects of Diet and Sex in Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with no known cure. Approximately 90% of ALS cases are sporadic, suggesting there are multiple contributing factors that influence the disease risk, onset, and progression. Diet and sex are two factors that have been reported to alter ALS risk, onset and progression in humans and in animal models, providing potential modifiers of disease. Several epidemiological studies have identified diets that positively affect ALS patients, including various high-calorie fat- or sugar-based diets, while animal models have been developed to test how these diets are working on a molecular level. These diets may offset the metabolic alterations that occur in ALS, such as hypermetabolism, lowered body mass index, and hyperlipidemia. Sex-dependent differences have also come forth from large-scale epidemiological studies as well as mouse-model studies. In addition, sex hormones have been shown to affect disease risk or progression. Herein, studies on the effects of diet and sex on ALS risk, onset, and progression will be reviewed. Understanding these sex- and diet-dependent outcomes may lead to optimized patient-specific therapies for ALS.

1. Introduction

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a devastating neurodegenerative disease, with approximately fifteen new cases being diagnosed every day (1). ALS is caused by progressive degeneration of the motor neurons in the brain and spinal cord that eventually leads to an inability to perform voluntary movements, typically resulting in death by respiratory paralysis within 2 to 5 years. ALS gained attention in the early 1940s when the legendary Yankee baseball player Lou Gehrig was diagnosed and eventually succumbed to the disease. Although ALS has been an important topic of scientific research for almost a century, ALS has proven to be a very complex disease, with safe and effective therapies slow in the making. In fact, only one drug has been FDA-approved for ALS patients (riluzole) and it has the potential to extend life by only a few months (2).

Approximately 10% of ALS cases are familial (fALS) while the remaining 90% of cases are sporadic (sALS). This means that there is likely not a single causative genetic mutation of the disease. In 1993, a mutation in superoxide dismutase 1 (SOD1) was identified in about 20% of fALS cases, allowing for the development of a model to represent the disease (3). Since this discovery of SOD1 alleles, many other ALS-associated alleles have been identified, including but not limited to alleles of TARDBP, FUS, TDP-43, ATXN2, and C9orf72. Further study of these alleles has provided a major breakthrough in ALS understanding because they display functional overlap with sALS pathways, suggesting there are common disease pathways for sALS and fALS and providing models for the study of theses molecular pathways.

Due to the largely sporadic nature of most ALS cases, researchers have sought to identify common factors that contribute to ALS risk, onset, and progression. One common factor observed in most ALS patients is altered metabolism. ALS patients struggle to sustain a healthy weight and display altered lipid and/or respiratory metabolism, leading to exacerbated disease symptoms and shorter lifespan (4). These metabolic symptoms have brought diet to the forefront of treatment. Several researchers have set out to identify diet-based therapies to ameliorate these metabolic alterations and restore healthy metabolism in ALS patients. By implementing specific dietary programs, particularly high-calorie diets, ALS patients have displayed slower disease progression and improved quality of life. Furthermore, altered metabolic states have been detected in ALS mouse models before physical symptoms are noticeable (5), suggesting metabolic changes may be a cause of the disease, not just a symptom. This finding has led to increased focus on how the patient’s metabolic health before and after disease onset affects ALS.

In addition to metabolism-based risk factors, sex-dependent differences in ALS development have been well documented, with men reported to be two-three times more susceptible. In 2016, the NIH established a requirement for sex to be reported as an important biological variable. As such, many studies conducted before this time were only in one gender, more often males than females, potentially overshadowing key findings and disease pathways that may only be present in one gender. As diet and metabolism have been more extensively studied, sex-specific differences have also come forth with certain therapies. It is important to understand why ALS presents differently in males and in females, which may lead to a greater molecular understanding. In addition, discovering how different therapies behave within each sex will help in the development of optimized, patient-specific therapies. This review is focused on highlighting the metabolic phenotypes associated with ALS as well as how diet and sex factor into the risk, onset, progression of ALS patients.

2. The role of hypermetabolism and body mass index in ALS

2.1. Epidemiological studies reveal hypermetabolism in ALS patients

ALS patients often present with metabolic abnormalities that increase the energy required for their body to function, even though they have decreased mobility. Between 25–68% of all ALS patients present with the hypermetabolic phenotype of increased energy expenditure, especially at rest ((6–10) (for a recent review of these hypermetabolic changes see Ferri and Coccurello (11)). Briefly, the metabolic rate for a whole animal (the O₂ uptake, CO₂ output and heat given off) is largely dependent on the metabolic rate of the skeletal muscle, which is abundant and highly active. Several studies have investigated the contributions of skeletal muscle metabolism and degeneration to ALS progression. Early neuromuscular degeneration occurs in ALS prior to motor neuron loss (12, 13) and alterations to mitochondrial function are common in ALS patients, including altered mitochondrial dynamics (fragmentation) and mitochondrial aggregation (14–24). Although it is clear that skeletal muscle hypermetabolism and degeneration does coexist with motor-neuron degeneration, the magnitude of motor-neuron contribution to disease is still under debate. In addition, the cause of the hypermetabolism is uncertain. What is known is that skeletal muscle hypermetabolism combined with physical deterioration, including the inability to swallow and changes in appetite, can lead to a vicious cycle of malnutrition and further deterioration in ALS patients.

2.2. Epidemiological studies reveal a protective effect of increased body mass index in ALS patients

Increased body mass index (BMI) corresponds to an increased survival time for ALS patients as revealed in several studies (25–29), while a decreased BMI may increase disease severity (4). How BMI acts as a protecting factor is largely unknown, however it may have a protective effect during malnutrition. As researchers have delved into the relationship between BMI and ALS, sex-dependent differences have been revealed. A study conducted in Western Europe including over five-hundred thousand individuals showed that increased pre-diagnostic body fat reduced the risk of ALS mortality (30). These risk factors varied between the sexes, with underweight women three times more likely to die from ALS.

Diet is a modifiable factor that can potentially compensate for the increased energy demand or a lower BMI in ALS patients, influencing how the disease progresses in the individual. The effects of lipid- and sugar-based diets on ALS progression from both human and mouse studies are discussed in the following sections (3–4).

3. Lipid-rich diets and hyperlipidemia appear to play a protective role in ALS

3.1. Epidemiological studies reveal a lipid-rich diet and hyperlipidemia as positive prognostic factors in ALS

Epidemiologic studies have shown that a high-fat diet can reduce the risk of developing ALS by at least 34% and potentially delay the onset of disease (31, 32), presumably by protecting against the hypermetabolic phenotype. Elevated levels of both fasting triglycerides and cholesterol have been shown to be positive prognostic factors in ALS. Serum triglycerides above median levels have been shown to extend patient lifespan by 14 months (33) and elevated cholesterol levels correlated with 3.25 times improved survival time (34). In a seeming paradox, hyperlipidemia is also correlated with ALS. A study done in 655 humans revealed that hyperlipidemia was present in two-times as many ALS patients compared to controls, with a high LDL/HDL ratio significantly extending patient lifespan (35). This suggests that hyperlipidemia (high LDL) is causal, or that it is an adaptive response to the onset of disease. In support of these findings, a recent study conducted to determine risk factors associated with ALS found that LDL cholesterol and coronary heart disease were both causally linked to ALS, and further analysis showed that the link between ALS and coronary heart disease is due to elevated LDL cholesterol (36). Therefore, hyperlipidemia (high LHL) is typically associated with ALS, but elevated triglyceride and cholesterol levels provide a positive prognostic factor.

Another study in support of a high-lipid diet for ALS patients considered biological changes that accompany percutaneous endoscopic gastrostomy (gastrostomy), a process beneficial for ALS patients who have trouble swallowing or eating enough to meet their nutritional needs. They measured levels of total cholesterol and low-density lipoprotein in patients before, at the time of, and after percutaneous endoscopic gastrostomy. Their results indicate that increased variation of total cholesterol between the three time points were indicative of decreased survival. They suggest that a diet focused on healthy cholesterol supplementation, especially in patients with gastrostomy, may prove beneficial in extending patient lifespan (37).

The paradoxical predictive and protective nature of hyperlipidemia in ALS begs the question of the chicken and the egg. Is hyperlipidemia a factor that is detectable before neurodegeneration and hence a causative factor in ALS development, or a biological adaptation to meet the altered energy demands of the pre-ALS cell? How does a high-fat diet protect against ALS progression, is it simply high caloric intake that is protective, or are specific lipids preferred? These are questions that should be explored in animal models.

3.2. Animal models of ALS support altered lipid metabolism as well as the protective effects of a lipid-rich diet

Both hyperlipidemia and the protective nature of a high-fat diet have been observed in ALS mouse models as well, confirming the results of human studies. In SOD1 mutant mice, lipids were absorbed and triglyceride-rich lipoproteins cleared both in greater frequency, resulting in decreased levels of triglyceride-rich lipoproteins after eating, also known as postprandial lipemia. When supplemented with a high-fat diet, these alterations were ameliorated and neuroprotection occurred (38). In addition to postprandial lipemia, a high-fat diet also protected against postprandial cholesterolemia, suggesting an increased lipid intake for ALS patients to compensate for the increased energy needs. Another study looked at the effect of TDP-43 knockout versus overexpression in mice (39). They observed that when TDP-43 is knocked out post-natal (the knockout is embryonic lethal), the mice exhibit weight loss, fat depletion, and rapid death whereas when TDP-43 is overexpressed, mice exhibit increased fat accumulation, adipocyte hypertrophy, and altered responses to insulin in skeletal muscle. This supports TDP-43 as a regulator of body mass composition and glucose homeostasis, suggesting that mutations in TDP-43 may be a contributing factor to the metabolic abnormalities seen in ALS (39). In addition, ATXN2-deficient mice also display hyperlipidemia (40). Future studies could include an investigation on the specific fat and lipid composition in these mice compared to healthy controls as well as how a diet rich in specific lipids could affect fat composition and ALS disease progression.

In agreement with these studies on altered lipid metabolism in mice deficient in ALS-associated genes, it has been shown that a high-fat diet can extend lifespan by increasing fat stores and improving motor function in TDP-43^A315T and SOD1-G93A ALS mouse models (41, 42). In contrast, one report did find that caloric restriction improved motor function in SOD-G93A mice (43), however it also hastened clinical onset. The contrasting diets and the resulting lifespan show that a high-fat diet can act as an essential element in relieving hypermetabolic stress in ALS mouse models. However, this simple statement must be taken with caution because different fats may have different effects in ALS patients. For examples, a study done in a SOD1^G93A transgenic mouse model showed exposure to the omega-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid upon onset of disease did not affect how the disease presented or progressed. In fact, pre-symptomatic exposure to eicosapentaenoic acid significantly decreased lifespan (44). This suggests further studies are needed to understand how specific fatty acids are working in the metabolic pathways of ALS and which ones will be most beneficial for ALS patients. It also suggests the need for careful understanding of how different diets are affecting ALS patients prior to disease onset because it is still uncertain whether hyperlipidemia contributes to the onset of disease, or whether it is solely a protective adaptation.

3.3. The ketogenic diet (high-fat, low-carb) restores healthy mitochondrial function and energy metabolism in both humans and animals with a SOD1 mutation

The ketogenic diet has been considered as one type of high-fat diet for ALS patients (a high-fat, low-carb diet) that works to mimic carbohydrate starvation, replacing carbohydrates with ketone bodies as the main source of energy. Initially, the ketogenic diet was employed to help those with pharmacoresistant epilepsy. It was later applied to neurodegenerative diseases like ALS due to common metabolic abnormalities seen in the diseases (45). Typically, when the SOD1 mutation is present, complex I activity of the electron transport chain and ATP production are decreased (46). When on a ketogenic diet, the principle ketone body, D-β-hydroxybutyrate (DBH) can prevent rotenone-mediated inhibition of complex I in a SOD1^G93A transgenic mouse model (47). Furthermore, the diet was shown to increase total body weight and spinal cord motor neurons. This suggests that a ketogenic diet may be working to protect against ALS by restoring the function of complex I and promoting ATP synthesis.

A second study performed by the same group was conducted using caprylic triglyceride, also known as fractioned coconut oil, a substance metabolized into ketone bodies. This is an attractive substance because it could be used as an easily distributed supplement for ALS patients. They found that caprylic triglyceride restored healthy energy metabolism, thus improving motor function in SOD1^G93A ALS mice (48). Combined, these studies suggest that a ketogenic diet may be beneficial to patients with a SOD1 mutation. However, there are few data exploring how the ketogenic diet could affect other forms of the disease, such as patients with FUS or ATXN2 mutations.

3.4. Animal models reveal additive effects of high-fat diet and drug therapies in ALS

High-calorie diets may prove pivotal for disease treatment both alone or in effective combination with drug therapies. One study reported benefits when using a calorie energy supplemented diet (CED, a normal chow diet supplemented with fat and cholesterol) combined with a multifunctional monoamine oxidase (MAO). The effects were additive and positively affected motor performance as well as mouse survival in a SOD1^G93A model (49). This study suggests that an optimized diet may be combined with a drug therapy to significantly improve patient life.

3.5. Animal models reveal a hypermetabolic state and hypolipidemia in early ALS

In an effort to determine if hyperlipidemia is causal or an adaptive, protective effect in ALS, studies were conducted in a SOD1^G86R mouse model (5). Before neurodegeneration was detectable, skeletal muscles were in a hypermetabolic state, and they remained in a hypermetabolic state after neurodegeneration occurred. To offset this energy imbalance, a fat-enriched high-energy diet was fed to the mice after disease onset, effectively increasing body mass and adipose tissue and extending the mean survival time by 20%. However, it is still possible that hyperlipidemia is not causal but rather the result of a compensatory mechanism used to meet the altered energy demands, even in early ALS. To address this issue, SOD1^G86R mice were studied in the asymptomatic stage (65 days of age) and glycolytic muscles were shown to preferentially utilize lipids over glucose (50). This lipid metabolism may prove to be detrimental to the cell as an increase in lipid by-products can contribute to lipotoxicity and ROS production, eventually leading to denervation and having toxic effects on mitochondria. Interestingly, oxidative muscles such as the soleus showed no change in this study. Another study confirmed hypolipidemia in the pre-symptomatic stage in SOD1^G93A mice, with hypolipidemia being significantly greater in males than in females (51). Thus, increased lipid metabolism appears to cause hypolipidemia early in ALS models, with a high-fat diet helping to improve caloric intake and extending life. However, this diet may also be feeding the high lipid metabolism, having toxic effects on mitochondria. Thus, high-calorie diets that do not feed lipid metabolism may prove to be even more effective in long-term treatment and survival.

4. The sweet benefit of sugars: providing healthy weight gain and protection against TDP-43 proteotoxicity

4.1. Epidemiological studies suggest a sugar-rich diet could be more beneficial than a lipid-rich diet

Recently, a study was conducted comparing two very different high-calorie diets in ALS progression: sugar- and fat-based. This study included 24 ALS patients and used both a high-fat hypercaloric diet (HF/HC) and a high-carbohydrate hypercaloric diet (HC/HC). The results indicated that patients on a HF/HC diet were unable to gain weight, even when consuming 174% of their estimated energy requirements. In addition, patients on the HF/HC diet noticed uncomfortable gastrointestinal side-effects. In contrast, patients on a HC/HC diet gained weight. Overall, they suggested a HC/HC diet as a safe and beneficial diet for ALS patients over a HF/HC diet (52). It is important to note that this study had a very small sample size, making it necessary to conduct this study in a larger population to solidify conclusions. However, the data are promising and suggest that a high-carbohydrate diet may be even more beneficial than a high-fat diet and bring with it fewer negative side effects, perhaps by avoiding the excess lipid metabolism discussed above.

4.2. A C. elegans model of neurodegeneration provides insight into the protective role of sugar in ALS

The molecular mechanisms behind the role of sugars in ALS remain largely unstudied, however C. elegans models of neurodegeneration, including mutant polyglutamine, TDP-43, FUS, and amyloid-β toxicity have been developed to study the function of both glucose and sucrose as neuroprotectants. It was found that glucose and sucrose could extend lifespan of mutant C. elegans in a dose-dependent manner by reducing the amount of misfolded proteins. Importantly, excessive glucose in healthy controls still exhibited its typical negative effects on lifespan, fertility, and dauer formation, highlighting the necessity of applying the aforementioned diets only in regards to ALS therapy (53). Furthermore, a second study was conducted testing the benefits of maple syrup in C. elegans due to its high sugar content as well as its abundance of other compounds such as antioxidants and phenols. This study showed that maple syrup could in fact protect against TDP-43 proteotoxicity and that sucrose contributed to this protection. In addition to sucrose, the authors found two specific phenols protective against hypoxic stress as well as amyloid-B and alpha-synuclein proteotoxicity (54). These phenols are found in other natural products in addition to maple syrup.

5. Foods with high phenol content may protect against neurodegeneration

5.1. The high phenol content in fruits and vegetables may be contributing to their reported benefits for ALS patients

Studies have not been conducted looking at the benefit of specific phenols from fruits and vegetables in ALS, but fruits, vegetables, antioxidants and beta-carotenes were shown to be associated with increased ALS function in a study including over 302 ALS patients (55). When looking at the benefits of fruits and beta-carotenes specifically, increased intake was associated with a decreased risk of sporadic ALS in a small study done with seventy-seven Koreans (56). Overall, these are very limited, small-scale studies that do not specifically address the phenol content of fruit and vegetables. Therefore, animal studies lead the way in considering phenols in ALS treatment.

5.2. Animal models of ALS show improved motor function and neuroprotection when fed a diet high in phenols

Natural phenolic compounds have been identified as potential amyloid aggregation inhibitors (57), and those studied in association with ALS are discussed herein. For a review of additional phenolic compounds that have been tested in other degenerative diseases, see (57).

A known diet that is high in phenols is the Mediterranean diet, which has also been shown to reduce neurodegeneration through its high olive oil content (58). SOD1^G93A mice exposed to a diet high in extra virgin olive oil showed an extended lifespan and increased motor performance (59). A second supportive study showed that an extra virgin olive oil extract acted as a neuroprotective agent in cultures obtained from a SOD1^G93A mouse model (60). The extract reduced neurodegeneration by downregulating the amount of nitric oxide released from activated glia stimulated by the SOD1 mutation. In addition, the TLR4 signaling pathway, a known pathogenic pathway in ALS, was inhibited by the olive oil extract.

Another group focused on an anthocyanin-enriched extract from strawberries, a compound known for its antioxidant, anti-inflammatory, and anti-apoptotic properties (61). Anthocyanins are of the flavanoid group, a type of plant phenolic (62). They found that hSOD^G93A mice supplemented with the extract exhibited delayed disease onset and extended survival. Improved motor function was also observed in these mice through increased grip strength as well as histological analysis, which revealed healthier neuromuscular junctions and reduced astrogliosis in the spinal cord. Given these initial results from animal studies in response to phenol exposure, it is not unwise to consider phenol as a neuroprotective agent.

6. Other nutritional interventions

6.1. The Deanna Protocol, although promising, lacks substantial evidence to support its benefit in ALS

The Deanna Protocol is a nutritional supplement reported to prevent glutamate excitotoxicity and preserve healthy metabolic function. The supplement is essentially composed of arginine-alpha-ketoglutarate, gamma-aminobutyric acid, a medium chain triglyceride high in caprylic acid and Coenzyme Q10. In a SOD1^G93A mouse model, mice with Deanna Protocol supplementation exhibited improved neurological function, improved motor performance, and extended survival time when compared to controls (63). This suggests that using the Deanna Protocol may extend patient lifespan as well as improve their quality of life. However, this is the only study published on the Deanna Protocol in an ALS model, and has yet to be replicated by another group or in humans.

6.2. A Gluten-Free diet is likely only beneficial for ALS patients who also have gluten sensitivity or celiac disease

A somewhat controversial diet thought to benefit some patients with ALS is a gluten-free diet. Initial studies reported a link between celiac disease or gluten insensitivity and ALS (64, 65). However, many other studies have shown no link between the two diseases, and thus no benefit of a gluten-free diet (66, 67). In addition, many neurologic manifestations that present with celiac disease may be misdiagnosed as ALS, suggesting that the link between ALS and celiac disease may not really exist (68). A substantial amount of data will need to be presented for a gluten-free diet to be considered beneficial for those with ALS, unless the patient is diagnosed with gluten sensitivity or celiac disease in addition to ALS.

6.3. Vitamin supplementation is dependent on the individual and the state of the disease

The study of how vitamins affect ALS patients is somewhat limited. Many ALS patients present with insufficient vitamin D levels, requiring vitamin D supplementation. Vitamin D supplementation has been associated with improved gross motor function both in human and mouse studies (69–71). Vitamin D deficiency has had negative effects in patients and has been shown to significantly accelerate disease progression and shorten patient lifespan (72–75). Thus, vitamin D supplementation may prove beneficial for ALS patients, especially when below a healthy level.

Vitamin E supplementation has been associated with a decreased risk for ALS, but studies have not shown it to slow disease progression or extend patient lifespan (76, 77). As such, it has been recommended to those with a history of familial ALS as a preventative measure (69). For a more in-depth review of these vitamins as well as additional vitamins and their effect on ALS development or progression see (69).

6.4. L-serine as a potential therapeutic treatment for ALS

Dietary supplementation of the amino acid L-serine may also act as a neuro-protectant. Initial findings of Paul Cox correlated cyanobacterial toxin B-N-methylamino-L-alanine (BMAA), with the development of ALS in Guam populations (78). This finding led to further research that implicated BMAA in the formation of neurofibrillary tangles and beta-amyloid deposits in vivo, and suggested other at-risk populations that live near cyanobacterial reservoirs. These studies are one of the few on environmental factors, and the levels of BMAA required for these effects have remained controversial. However, through this research, the supplementation of L-serine was identified as a cellular protectant against BMAA poisoning and has led to L-serine supplementation as a potential therapeutic treatment (79). Phase 1 clinical trials were published in 2018 reporting a 34% reduction in progression slope (80, 81)

7. Epidemiological studies show ALS presents differently in males and females

7.1. Gender-dependent differences are possibly due to sex hormones

When reviewing studies of how diet affects ALS pathogenesis, it may be noticed that many of the in vivo experiments were performed in only one sex, usually male, with any sex-dependent differences remaining unstudied. In studies that do include both sexes, sex-specific differences were often seen (30, 51, 82). Many studies state that because males are more susceptible to the disease and sometimes develop more severe disease symptoms, they are a better model to study. However, differences between the sexes suggest that there is an underlying factor protecting females that could be exploited if identified. In addition, the application of various therapies may differ between sexes. These differences highlight both the need for understanding the mechanisms behind sex-dependent changes in ALS as well as the need for studies to be conducted in both sexes when possible.

Sex-dependent differences in ALS point to sex hormones as being strong contributing factors. Many epidemiological studies have shown that women are less susceptible to developing ALS and exhibit less severe disease progression (83–86). However, these differences become less significant as patients age, with reports showing post-menopausal women being just as likely to develop ALS as men (87). These findings point towards sex hormones as the strong protective factor to sex-dependent differences. Sex-dependent differences are also seen in the effects of BMI on ALS development, with underweight women three times more likely to die from ALS, while women with increased waist/hip ratio have a decreased risk. Men, on the other hand, show a significant linear relationship between increased BMI and decreased risk (30). Furthermore, sex differences were seen when body fat distribution was studied to determine the protective effects of visceral and subcutaneous fat (82). Overall fat content was unchanged between ALS patients and controls, but the composition of the fat was different. Both males and females with ALS showed an increase in visceral fat and a decrease in subcutaneous fat when compared to healthy controls. Visceral fat did not impact ALS clinical severity or survival. On the other hand, subcutaneous fat did predict survival in males, but not females, with an increase in fat corresponding to increased survival.

The data from humans are mirrored in mouse models in that ALS disease development and progression are also varied amongst the sexes (88–90). Males typically exhibit an earlier onset of disease and females typically live longer, despite having similar symptomatic stage durations. In addition, it has been reported that male TDP-43 transgenic mice develop a stronger phenotype than females (91). Males will exhibit abrupt onset of the disease around day 14–18, while females won’t exhibit symptoms until around day 30, with only gradual development of disease beginning with a small tremor. It is believed that this difference is due to an approximate 2–3-fold increase in TDP-43 accumulation in the male. These sex-dependent differences seen in disease onset and progression in both humans as well as mice strongly suggest a role of sex hormones in ALS.

7.2. Estrogen and progesterone are protective against ALS in epidemiological studies

Estrogen and progesterone are the two most abundant female sex hormones. Estrogen is primarily an ovarian sex hormone, yet it has many functions outside of those related to reproduction. It has been reported to play a significant role in lipid and carbohydrate metabolism, electrolyte balance, and the central nervous system (92). The higher prevalence of ALS in males points towards a protective effect of female sex hormones, primarily estrogen and progesterone. In epidemiological studies, a longer exposure to endogenous estrogen combined with a longer reproductive time-span significantly increases survival time in post-menopausal women with ALS (93). In both men and women, elevated endogenous progesterone levels show a positive correlation with survival time and time to diagnosis (94).

In addition to endogenous hormones affecting ALS, exogenous hormones are also an important factor to consider, as approximately 80% of women in the United States use hormonal contraceptives to prevent pregnancy (95). However, there is limited research into how exogenous hormones affect ALS onset and progression in females. In one epidemiological study consisting of 653 patients and 1,217 controls, exogenous estrogens and progestogens were shown to decrease the chance of developing ALS in the female population (96). In contrast, a study of 193 postmenopausal women reported oral contraceptive use was not associated with ALS risk (97). It is important to note that this study did not consider the other various hormonal contraceptives, such as intrauterine devices or vaginal rings. Although inconclusive, the results of these epidemiological studies have pushed for a deeper understanding of the function of exogenous sex hormones in ALS development and progression.

7.3. Estrogen and progesterone are protective against ALS in animal models

Decreasing the amount of endogenous estrogen has proven to have deleterious effects on female mice. In SOD1^G93A transgenic mice, ovariectomy led to acceleration of the disease, making disease progression and lifespan comparable to male mice (98, 99). In addition, ovariectomy attenuated the anti-inflammatory and anti-apoptotic actions of estrogen (100). When supplemented with a high dose of 17beta-estradiol (E2), a form of estrogen, ovariectomized females exhibited extended lifespan (98, 99). In addition, male mice treated with E2 exhibited significantly improved motor function in addition to inflammasome activity being ameliorated (101). Combined, these data strongly suggest that estrogen is having a neuroprotective effect on disease onset and progression.

Few studies have been performed examining the effect of progesterone in an in vivo model. One study hypothesized that progesterone may be acting to delay neurodegeneration by activating autophagy in a SOD1^G93A transgenic mouse model (102). Although the onset of disease was not shown to be affected by progesterone, the progression of motor dysfunction was significantly delayed. After histological examination, it was clear that the progesterone-treated group exhibited reduced spinal motor neuron death.

The effects of progesterone are reflected in the Wobbler mouse model of ALS, where progesterone is upregulated likely as a neuroprotective response to neurodegeneration (103). There have been many studies conducted in Wobbler mice looking at the protective effects of progesterone (see (103–108)). However, it is important to note that the VPS54 mutation responsible for the Wobbler mouse has not been found in ALS patients, as such the specifics on these studies have not been included in this review (109). It would be very interesting to replicate the experiments performed in Wobbler mice in different ALS mouse models.

7.4. Epidemiological studies of the effect of testosterone

Testosterone is primarily a male sex hormone, but altered levels of testosterone have been reported in females with ALS, however with conflicting results. In one study, testosterone levels were elevated in females, and as the patients aged they remained elevated instead of declining with age as they do in healthy controls (110). A second study done in 92 patients reported no change in total testosterone in either males or females with ALS. However, they did report a significant decrease in free testosterone in those with ALS. They suggest that this difference is a result of testosterones inability to cross the blood brain barrier in its unbound form and that it is free testosterone that impacts ALS development (111).

Interestingly, prenatal levels of testosterone may be an influencing factor in the development of sporadic ALS. There is some evidence suggesting that by measuring and comparing the lengths of the index finger and the ring finger (2D:4D ratio) you can crudely estimate the exposure of testosterone in utero for both males and female, with a reduced ratio associating with increased testosterone. In ALS patients, the 2D:4D ratio was lower than controls, suggesting increased prenatal testosterone levels were influencing ALS development (112). However, another large-scale study was unable to verify this association (113).

7.5. Male and female ALS mice respond differently to exercise, perhaps due to estrogen levels

Sex-dependent differences have also been seen in response to exercise. Exercise is a somewhat controversial topic in ALS, with some reports claiming benefits to the patient, others claiming harm, and yet others reporting no change (for a recent review, see (114)). These conflicting reports are in part due to the short lifespan following diagnosis, making the effects of exercise difficult to measure in humans. A few groups have overcome this issue by studying the effect of exercise in mice. In SOD1^G93A mice, high-intensity endurance training was beneficial for females only, with females having an increased survival time (115). Male mice exhibited a hastened decrease in motor performance and death following clinical onset. It is unknown why the females are protected from the deleterious effects of exercise, but it is hypothesized that estrogen could be playing a role. It is also possible that female SOD1^G93A mice live longer regardless of exercise, as has been reported (88, 89). A second study measuring the effects of exercise in low-copy and high-copy human SOD1^G93A mice found that exercise hastened clinical onset in males only (116). In agreement with the previous study, exercise extended the lifespan of high-copy hSOD1 female mice only. They suggest that because non-exercising females had more irregular estrous cycles, they were exposed to less estrogen, and as such estrogen was acting as a protective factor in exercising female mice.

7.6. The hormone leptin alters metabolism in a sex-specific manner in mice

As mentioned previously, metabolism is altered in ALS. The effect of sex-hormones on metabolism in ALS is supported by an article showing leptin-dependent changes in SOD1 mice. Leptin is a hormone that regulates satiety and energy expenditure, changing the overall metabolic state of the organism. SOD1 mice typically exhibit hypermetabolism, but when placed in a leptin-deficient background they exhibit sex-dependent improvements in energy homeostasis and decelerated disease progression (117). Females exhibited a significant increase in survival and motor function and a decreased energy expenditure, while males showed similar changes but that were much less drastic. Leptin is known to be affected by various hormones, including sex hormones, suggesting that the changes seen in this study are dependent upon the sex hormones present in the mice (117). Further testing on how specific hormones are affecting these changes have not been conducted but would provide significant insight into how sex hormones are affecting metabolism in the SOD1 mouse model of ALS.

8. Concluding remark

ALS is a devastating neurodegenerative disease with few treatments and no cure. Although the cause of ALS is largely unknown, different factors have been studied to reduce disease risk, slow disease progression, and extend survival time (Figure 1). Two potentially modifiable risk factors have been discussed in this review: diet and sex hormones.

Figure 1.

Diet and sex are important contributing factors to ALS onset, progression, and/or survival time. It is important to understand that these factors are closely linked to one another and that by altering one factor it may cause repercussions in the other. This suggests mindfulness when developing models for the study of ALS.

Diet and metabolic health go hand in hand. ALS patients often present with altered metabolic phenotypes, including hypermetabolism and altered fatty acid metabolism as well as hyperlipidemia. The data regarding hyperlipidemia are both consistent and paradoxical. Consistent because several large-scale studies report hyperlipidemia associated with the presence of ALS in humans, yet both hyperlipidemia and a corresponding higher BMI have been reported as protective in disease progression. These findings suggest hyperlipidemia may be a protective physiological response to the disease rather than causal. In support of this hypothesis, SOD1^G93A mice were shown to be hypermetabolic in the pre-symptomatic stage with preference for lipid metabolism (50). This lipid metabolism produces toxic lipid by-products that can contribute to lipotoxicity and ROS production, eventually leading to denervation and mitochondrial damage. Another study confirmed hypolipidemia in the pre-symptomatic stage in SOD1^G93A mice, suggesting hyperlipidemia is an adaptive state (51), however further investigation is needed.

Diets that increase weight (such as high-fat or high-sugar high calorie diets) have been shown to decrease disease progression both in humans and mice in several studies, presumably by combating the malnutrition due to hypermetabolism and disease progression. However, it is conceivable that supplying more lipids to feed the lipid hypermetabolism present in ALS may not be the optimal high-calorie diet. Identifying high-calorie diets that avoid lipotoxicity, ROS production and mitochondrial damage while stimulating healthy metabolic pathways may prove to be a more beneficial long-term treatment. Of interest for further study are high-carbohydrate (52) or sucrose-based (53) diets, certain lipid-rich diets (47, 48)(64), and diets targeting particular phenols with anti-oxidative effects that may reduce ROS production (58)–(62). Combined, these studies solidify both current holistic therapies that can be applied to benefit ALS patients as well as identify molecular pathways that can be targeted for future treatment development.

Sex is the second factor discussed in this review. Sex hormones play an important role in ALS independent of diet, with females protected from ALS development, disease severity and disease progression. In addition, this review highlighted sex discrepancies seen in many studies involving diet and metabolic health in ALS. Unfortunately, most mouse studies involving diet and metabolic health only include the male population, and even human studies are often male-biased due to the rate of ALS being higher in males. In cases where both males and females are included, differences between the sexes were seen which could be obstructing clear results from studies if males and females respond differently, contributing to contradictory reports. These findings are consistent with recent studies that show dramatic differences in male/female response to a high-fat diet in wild type mice (118), suggesting this diet may behave differently in male/female ALS patients. In addition, estrogen has been posited to play a protective role in ALS. The impact of these findings is three-fold: first, it suggests that the metabolic abnormalities seen in ALS may differ between sexes; second, it suggests estrogen-related pathways as potential therapeutic targets; and third, it highlights the importance of studying potential molecular pathways for drug development in both sexes, as they may be influenced by sex-dependent factors. Thus, ALS studies should be performed on both males and females, with sex being an additional variable analyzed statistically. By considering diet and sex factors individually, and together, it will allow for more effective and safe therapies for ALS patients.

Table 1.

A summary of reported dietary effects on ALS onset, progression or development.

Diet	Model	Sex	Outcome	Survival	Time to onset	Other notable results	Reference(s)
High fat	SOD1G86R mice	M	+	↑	↑	↑ body mass, ↑ adipose tissue	(5)
High fat	Humans	M/F	+	n.r.	n.r.	↓ risk (34%)	(31)
High fat (PUFA)	Humans	M/F	+	n.r.	n.r.	↓ risk (50–60%)	(32)
High fat	SOD1G93A mice	M	+	↑	↑	↑ fat storage, ↓ AMPK activity, ↑ motor function	(41)
High fat	TDP-43A315T mice	M	+	↑	n.r.	Delayed AMPK activation	(42)
Caloric restriction	SOD1G93A mice	M	−	↓	↓	↓ fat storage, ↑ AMPK activity, ↓ motor function	(41)
Caloric restriction	TDP-43G93A mice	M/F	−	n.c.	↓	↑ motor function	(43)
Eicosapentaenoic acid	SOD1G93A mice	F	−	↓	↑	EPA administered before disease onset was detrimental to the animal	(44)
Ketogenic	SOD1G93A mice	M	+	n.r.	n.r.	↑ complex I, ↑ ATP synthesis	(47)
Ketogenic	SOD1G93A mice	M	+	n.c.	n.r.	↑ healthy energy metabolism and ↓ disease progression	(48)
HC/HC	Humans	M/F	+	n.r.	n.r.	↑ weight gain	(52)
HF/HC	Humans	M/F	+/−	n.r.	n.r.	↓ weight gain	(52)
Glucose	C. elegans ALS models	n/a	+	↑	n.c.	↓ autophagy, ER stress, and muscle damage	(53)
Maple syrup	C. elegans (TDP-4)	n/a	+	↑	n.r.	Phenols GA and CA were responsible	(54)
Antioxidants, carotenes, fruits, vegetables	Humans	M/F	+	n.r.	n.r.	↑ function	(55)
Fruits and beta-carotene	Humans	M/F	+	n.r.	n.r.	↓ risk	(56)
Extra virgin olive oil	SOD1G93A mice	F	+	↑	n.c.	↓ autophagy, ER stress and muscle damage	(59)
Extra virgin olive oil extract	HEK cells/motoneurons/glia cultures from SOD1G93A mice	n.r.	+	n.r.	n.r.	Neuroprotective and prevented nitric oxide release	(60)
The Deanna Protocol	SOD1G93A mice	M	+	↑	n.r.	↑ survival and motor function	(63)

(M) male, (F) female, (+) positive, (−) negative, (↑) increased, (↓) decreased, (n.r.) not reported, (n.c.) no change, (HC/HC) high carbohydrate/high caloric, (HF/HC) high fat/high calorie

Table 2.

A summary of reported sex-related differences associated with ALS onset, progression or development.

Hormone	Treatment	Sex	Model	Outcome	Survival	Time to Onset	Other notable results	Reference(s)
General sex hormones	Observational	M/F	TDP-43 transgenic	+F −M	n.r.	n.r.	Abrupt disease onset in M ↑ TDP-43 accumulation	(91)
Exogenous estrogen and progesterone	Observational	F	Humans	+	n.r.	n.r.	Exogenous estrogens and progestogens contributed to a ↓ risk	(96)
Exogenous sex hormones	Oral contraceptive use	F	Post-menopausal women	n.c.	n.r.	n.r.	Oral contraceptive use did not impact ALS risk	(97)
Estrogen	Lifelong exposure and reproductive timespan	F	Post-menopausal humans	+	↑	n.c.	↑ lifelong exposure ↑ reproductive timespan associated with ↓ risk	(93)
Estrogen	Ovariectomy	F	SOD1G93A mice	−	↓	n.c.	↓ lifespan	(98, 99)
Estrogen	Ovariectomy + estrogen supplementation	F	SOD1G93A mice	+	↑	n.c	↓ ALS motor neuron progression, ↑ lifespan	(98, 99)
Estrogen	Estrogen supplementation	M	SOD1G93A mice	+	↑	n.c	↑ motor function ↓ neurodegeneration and ↓ inflammasome activity	(101)
N/A	Ovariectomy	F	hSOD1G93A mice	−	↓	↑	↓ estrogen-dependent anti-inflammation and anti-apoptosis	(100)
Progesterone	Elevated progesterone	M/F	Humans	+	↑	↓	↑ hypothalamic-pituitary-adrenal axis activation?	(94)
Progesterone	Progesterone supplementation	M	SOD1G93A mice	+	↑	n.c.	↓ disease progression ↓ spinal motor neuron death	(102)
Testosterone	Observational	M/F	Humans	n.r.	n.r.	n.r.	↑ testosterone levels in F ALS	(110)
Testosterone	Observational	M/F	Humans	n.r.	n.r.	n.r.	↓ free testosterone in ALS	(111)
In utero testoster one	Observational	M/F	Humans	−	n.r.	n.r.	↓ lower 2D:4D ratio in ALS, suggesting ↑ exposure to testosterone in utero contributed	(112)
General sex hormones	High-endurance exercise	M/F	SOD1G93A mice	+F −M	↑ F ↓ M	↑ F ↓ M	Exercise beneficial for F, deleterious for M	(115)
General sex hormones	Exercise	M/F	hSOD1 mice	+F −M	n.r.	↑ F ↓ M	Exercise benefit for F likely due to ↑ estrogen	(116)
Leptin	Leptin deficiency	M/F	SOD1 mice	+F +M	+F +M	↑ F ↑ M	↑ survival and motor function ↓ energy, stronger phenotypes in F	(117)

(M) male, (F) female, (+) positive, (−) negative, (↑) increased, (↓) decreased, (n.r.) not reported, (n.c.) no change, (N/A) not applicable