Therapeutic effects of hirsutella sinensis on the disease onset and progression of amyotrophic lateral sclerosis in SOD1G93A transgenic mouse model

Abstract Aims Although the pathophysiology of amyotrophic lateral sclerosis (ALS) is still not completely understood, the deregulated microglia polarization and neuroinflammation have been shown to contribute to the pathogenesis and progression of this disease. In the present study, we aimed to determine whether hirsutella sinensis (HS) could reduce neuroinflammatory and pathological changes in the spinal cord of SOD1G93A model mice of ALS and consequently ameliorate disease onset and progression. Methods SOD1G93A mice were chronically treated with HS by gavage. Their lifespan was recorded, and motor behavior was evaluated by rotarod test. The pathological changes in skeletal muscles and motor neurons in spinal cords were assessed by immunofluorescent staining and hematoxylin‐eosin staining. The microglia activation and neuroinflammation were determined by immunofluorescent staining and RT‐PCR. Results Our data suggested that repeated HS administration prolonged the lifespan and extended disease duration of ALS mice without significant delay on disease onset. HS ameliorated the pathological changes in the motor neurons and gastrocnemius muscles. Moreover, HS promoted the transition of microglia from pro‐inflammatory M1 to anti‐inflammatory M2 phenotype in the spinal cord of ALS mice. Conclusion All these findings indicate that HS may serve as a potential therapeutic candidate for the treatment of ALS.


| INTRODUC TI ON
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease, characterized by the selective deterioration of upper and lower motor neurons in the motor cortex, brain stem, and spinal cord, leading to muscle paralysis, respiratory deficiency, and death. 1 The majority of ALS cases (over 90%) are sporadic (sALS), and approximately 10% cases are familial (fALS). 2 Mutations of the superoxide dismutase-1 (SOD1) gene account for approximately 20% of fALS. SOD1 G93A (glycine 93 to alanine) is one of the common diseases causing gene mutations in ALS, and the SOD1 G93A transgenic mouse is the most used experimental model. 2 Several pathogenic events, including protein misfolding/aggregation, oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation, have been found to be involved in ALS pathogenesis. However, the exact pathophysiology of ALS is still not fully understood.
Consequently, till now, only two drugs, riluzole and edaravone, have been approved by FDA and applied clinically for the treatment of ALS. These two drugs moderately prolong the lifespan of patients with ALS. However, riluzole benefits only a subset of patients, and its side effects are difficult to predict. Edaravone, as a free radical scavenger, appears to show modest benefits on slowing disease progression in patients with short disease duration and good respiratory function. 3,4 Therefore, new therapeutic approaches are still urgently needed.
Recently, the microglia-mediated inflammation has been recognized as a neuropathological hallmark of ALS. 5 Microglia cells, the resident tissue macrophages of the central nervous system (CNS), belong to the innate immune system. During the development, adulthood and aging of CNS, microglia play many critical roles in not only the mediation of immune response, but also the elimination of apoptotic cells, production of growth factors, maintenance of synapse structure and function. However, the excessive production of pro-inflammatory cytokines from over-activated microglia contributes to the pathophysiology progression in many neurodegenerative diseases, including ALS. 6,7 Therefore, suppression of microglia-mediated inflammation has been considered as an important strategy in ALS treatment.
It has been reported that traditional Chinese medicine might be beneficial to improve symptoms and prolong the survival of patients with ALS. [8][9][10] Hirsutella sinensis (HS, also known as Cordyceps sinensis, Chinese caterpillar fungus or "Dong Chong Xia Cao" in Chinese, Figure 1A) is a unique fungus growing on caterpillars and is a highly valued tonic medicine claimed to treat a wide range of disorders, such as asthma, 11 chronic kidney disease, 12 and kidney transplant recipients. 13 Cordycepin (3′-deoxyadenosine), one of the major chemical components of HS ( Figure 1B), has been identified to exert anti-oxidative, anti-inflammation, antitumor, and neuroprotective effects. [14][15][16][17] Moreover, previous studies have further revealed a suppressive role of cordycepin on LPS-induced microglia activation F I G U R E 1 The effects of hirsutella sinensis (HS) on body weight, disease onset, lifespan, and duration in SOD1 G93A mice. A, Raw material of HS; B, chemical structure of main component of HS, cordycepin; C, body weight curves of three groups. The probability of disease onset (D) and lifespan (E) was analyzed by Kaplan-Meier survival analysis; F, the data of disease duration. G, Body weight gain during disease progression (between 64 and 124 days). P values were analyzed by one-way ANOVA. Data are presented as mean ± SEM. **P < 0.01 vs TG + H 2 O group; ### P < 0.0001 vs wild-type (WT) group;*P < 0.05 vs TG + H 2 O group. N = 10 in each group and inflammation in vitro. 18,19 Adenosine, another active component of HS, is not only an energy transfer and signal conversion agent in cells, but also plays a role in cell protection and tissue damage prevention. It has been shown that adenosine could regulate neuroimmunity and treat mental disorders. 20 HS is rich in a variety of chemical components that have medicinal activities, 21 suggesting a therapeutic potential of HS for ALS. To confirm this hypothesis, in this study, we evaluated the impacts of repeated HS administration on disease onset and progression of ALS in SOD1 G93A mouse model.
The potential ameliorating activities of HS on various disease phenotypes, especially the microglia activation and neuroinflammation, were further investigated. Twenty-eight age-matched WT littermates were administered with drinking water by gavage (once daily, 10 mL/kg body weight). All mice were weighed every 4 days from 64 days of the age to death.

| Animals and treatments
Eighteen mice per group were sacrificed at the age of 120 days for fresh spinal cords and muscles sampling or were perfused with PFA for the histological analysis of spinal cords.

| Rotarod test for the assessment of disease onset
From the age of 70 days, all animals were subjected to rotarod training using an IITC rotarod apparatus (4 cm diameter, 20 rpm; IITC Life Science Inc). After 7 days adaptation training (once daily, 5 minutes/d), the mice were tested every 2 days with three trials (5 minutes/trial). The rotarod performance was recorded to assess whether animals could complete one of all three trials. While the mice could not stay on the rotating rod for 5 minutes for all three trials, the date of the age was recorded and defined as disease onset. 24

| Assessment of animal's lifespan
The date was considered as the day of death, when the mice could not right itself within 30 seconds after being placed on its back. 25 Then, the lifespan was calculated as the duration between the day of birth and the day of death, and the disease duration was defined as the duration between the day of disease onset and the day of death.

| Pathological analysis of skeletal muscles
Fresh gastrocnemius muscle (5 × 5 × 10 mm 3 ) of mice at 120 days of age was dissected, embedded with bassora gum, and immersed immediately in liquid nitrogen. The muscle tissues were sectioned at 10 μm thickness and stained with hematoxylin-eosin (HE) for morphological analysis or with nicotinamide adenine dinucleotide hydrogen (NADH) to determine muscle fiber types.

| Real-time PCR
Mice at the age of 120 days were sacrificed for spinal cord tissue sampling. The total RNA was extracted using RNAiso Plus (Total RNA extraction reagent; Takara). Then, the total RNA was synthesized for cDNA by Revertra Ace qPCR RT kit (Takara). Real-time PCR was performed with TransStart Top Green qPCR SuperMix (TransGen Biotech) and measured by Applied Biosystems 7500 Real-Time PCR System (Life Technologies Corporation). The primer sequences used were summarized in Table 1. The relative expression levels were analyzed by the 2 −∆∆Ct algorithm normalizing to GAPDH and relative to the control groups (n = 6 in each group).

| Impacts of HS on body weight, disease onset, lifespan, and disease duration in SOD1 G93A mice
Body weight loss is a frequent feature of ALS. As shown in Figure 1C,G, while the body weight of WT mice was gradually increased, SOD1 G93A mice showed an clearly decreased body weight during disease progression (between 64 and 124 days). Repeated HS treatment ameliorated this body weight loss in SOD1 G93A mice ( Figure 1G).

| HS ameliorated motor neuron loss in spinal cord of SOD1 G93A mice
Nissl staining was performed to investigate the effects of HS on motor neuron loss in the L4-5 spinal cord of SOD1 G93A mice. As shown in Figure 2A Figure 2D).

| HS relieved pathological changes in gastrocnemius muscles of SOD1 G93A mice
As shown in the Figure 2, at the age of 120 days, the structures of muscle fibers changed apparently in SOD1 G93A mice as compared with those in WT littermates. The HE-stained cryosections of gastrocnemius muscles in SOD1 G93A mice illustrated typical pathological features of ALS including atrophic muscle fibers, hematoxylin inclusion, and central nuclei ( Figure 2E). Our data clearly showed that HS treatment ameliorated the muscle atrophy by increasing fiber area ( Figure 2G).
Furthermore, we also performed NADH staining to quantify the grouped type I or type II myofibers and to evaluate the oxidative metabolism in gastrocnemius muscles. Consistent with HE staining, the NADH staining showed more dark blue oxidative muscle fibers and grouped myofibers in SOD1 G93A mice than WT mice ( Figure 2F). We found that HS treatment ameliorated these pathological changes ( Figure 2F,H).

| Effects of HS on microglial and astrocytic activation in the spinal cord of SOD1 G93A mice
In order to investigate the effects of HS on microglial and astrocytic activation in spinal cords of mice, the immunofluorescence staining was performed using anti-Iba-1 antibody and anti-GFAP antibody, whereas the integrated density in HS-treated SOD1 G93A mice had no significant difference as compared with those control SOD1 G93A mice treated with water ( Figure 3C). GFAP-positive staining and quantitative analysis indicated a similar tendency as Iba-1, although no statistical significance was reached ( Figure 3B,D). These imaging data were further confirmed by Western blotting (supplementary Figure S1).

| HS rebalanced microglia M1/M2 polarization in spinal cord of SOD1 G93A mice
To investigate whether HS affected the phenotype of microglial activation, double staining was performed in the L4-5 spinal cord of SOD1 G93A mice using CD86 (M1 marker) or Arg-1 (M2 marker) with Iba-1. As expected, the counts of CD86 + /Iba-1 + cells increased in the spinal cord of SOD1 G93A mice as compared with WT mice, indicating a stimulated M1 polarization ( Figure 4A). Moreover, the total counts of Arg-1 + /Iba-1 + cells were also increased in SOD1 G93A mice as compared with WT mice (Figure 5A), suggesting a compensatory change in M2 polarization in response to toxic stimuli. Interestingly, while HS treatment significantly inhibited the M1 polarization ( Figure 4B), HS further strengthened the M2 polarization of microglia ( Figure 5B).
These results indicated that HS treatment might promote the transition of microglial polarization from M1 to M2 phenotype.
We further performed RT-PCR to evaluate the mRNA level of M1 and M2 markers in the L4-5 spinal cord of SOD1 G93A mice. Consistent with the data of immunofluorescent staining, the mRNA level of M1 marker CD86 was significantly increased in SOD1 G93A mice as compared with WT mice and was clearly reduced by HS treatment F I G U R E 3 Effects of Hirsutella sinensis on the activation of microglia and astrocytes. Iba-1 and GFAP antibodies were used to detect the microglia (A) and astrocytes (B), respectively, in the L4-5 spinal cords, scale bar = 50 μm. The integrated density of Iba-1-positive staining (C) and GFAP-positive staining (D) was quantitatively analyzed by one-way ANOVA. N = 6 in each group. Values are presented as mean ± SEM. ### P < 0.0001 vs wild-type (WT) group; # P < 0.05 vs WT group ( Figure 6A). The mRNA level of M2 marker Arg-1 was increased in SOD1 G93A mice, which was further promoted by HS administration ( Figure 6B). The mRNA level of CD206, another M2 marker, shared the similar trend as Arg-1 ( Figure 6C).

| HS partially reversed the elevation of mRNA levels of cytokines in the spinal cord of SOD1 G93A mice
The mRNA levels of both pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and anti-inflammatory cytokines (Ym1 and IL-10) were evaluated in the spinal cord of SOD1 G93A mice by RT-PCR. Our results indicated that, as compared with WT group, the mRNA levels of pro-inflammatory cytokines were significantly increased in TG mice treated with water and were partially reversed by HS treatment (Figure 6D-F).
Additionally, although no statistical significance was found, the mRNA levels of Ym1 and IL-10 were also upregulated in those HS-treated SOD1 G93A mice as compared with control TG mice ( Figure 6G,H).

| D ISCUSS I ON
It has now become evident that neuroinflammation is a promi-  Previous studies found that HS regulated inflammatory by inhibiting the expression of TNF-α, IL-6, and IL-1β, 27 and its main component cordycepin suppressed the release of TNF-α and IL-1β and exerted neuroprotective effects. 19 Consistent with these previous findings, we found that the mRNA levels of TNF-α and IL-6 were clearly decreased in HS-treated SOD1 G93A mice compared with SOD1 G93A mice treated with water. Interestingly, the mRNA levels of Ym1 and IL-10 had an upregulated tendency after HS administration. These data further validated that HS might inhibit the activation of M1 microglia and promoted the M2 microglia polarization.
Skeletal muscle atrophy is another characteristic of ALS, as the consequence of the motor neuron loss. Our dater showed that HS treatment ameliorated the atrophy of gastrocnemius muscles in SOD1 G93A mice. NADH staining is usually used to distinguish muscle fiber types. Type I fibers normally exerted dark blue NADH staining, whereas the type II fibers have the light NADH staining. At the F I G U R E 6 Effects of hirsutella sinensis (HS) on microglia activation and cytokines levels. The relative expression of mRNA of CD86 (A), Arg-1 (B), and CD206 (C) was analyzed by real-time PCR by one-way ANOVA. N = 6 in each group. ### P < 0.0001 vs wild-type (WT) group; ## P < 0.001 vs WT group; ** P < 0.01 vs TG + H 2 O group; * P < 0.05 vs TG + H 2 O group. The relative expression of TNF-α (D), IL-6 (E), IL-1β (F), Ym1 (G), and IL-10 (H) in spinal cords was analyzed by real-time PCR by one-way ANOVA. N = 6 in each group. Data are presented as mean ± SEM. ### P < 0.0001 vs wild-type (WT) group; *P < 0.05 vs TG + H 2 O group; # P < 0.05 vs WT group later stage of ALS, the type II fibers usually either atrophy or convert to the type I fibers. Consistent with the previous study, our data showed that HS treatment regulated the dark blue staining in the gastrocnemius muscles of SOD1 G93A mice.
In conclusion, HS prolonged the lifespan and extended the disease duration in SOD1 G93A mouse model of ALS. HS also decreased the motor neuron loss and ameliorated the gastrocnemius muscles atrophy. The therapeutic effects of HS against ALS might be mediated by its activities of anti-oxidative stress and anti-inflammation through promoting transition of microglia polarization from M1 to M2 phenotype. All these findings predicted a promising therapeutic potential of HS for ALS.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.