Could ozone become a complementary therapeutic approach to improve metabolic and endocrine response of ALS patients?

doi:10.21203/rs.3.rs-1616097/v1

Download PDF

Article

Could ozone become a complementary therapeutic approach to improve metabolic and endocrine response of ALS patients?

https://doi.org/10.21203/rs.3.rs-1616097/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Amyotrophic lateral sclerosis (ALS), a devastating progressive neurodegenerative disease, has no effective treatment. Recent evidence supports a strong metabolic component in ALS pathogenesis, raising additional therapeutic targets against ALS. At this respect, improvements in motor deficits and disease-associated weight loss after repeated exposures to ozone (O₃) in the mouse model of ALS based on TDP-43 proteinopathy (TDP-43^A315T mice) have been reported. Here, the underlying molecular mechanisms to determine whether O₃ exposure induces metabolic changes in ALS have been investigated. Molecular biology analysis demonstrated that O₃ significantly modified the expression profile of hypothalamic neuropeptides, altering phosphorylation levels of the signal transducer and activator transcription 3 (STAT3) and protein kinase B (Akt), concomitantly to increase the expression of genes involved in metabolism and thermogenesis in the brown adipose tissue (BAT) of TDP-43^A315T mice. Composition of fecal gut microbiome of exposed TDP-43^A315T mice varied significantly compared to wild type (WT) controls. Densitometric analysis of neuromuscular junction, indicated that O₃ does not impaired the progression of disease in the skeletal muscle. Our findings suggest the effectiveness of O₃ exposure to induce metabolic effects in the hypothalamus and BAT of this ALS mouse model and may be a new complementary non-pharmacological approach for ALS therapy.

Amyotrophic Lateral Sclerosis (ALS)

TDP-43 transgenic mouse

Metabolic disturbances

Ozone (O3)

Signaling pathways

Gut microbiome

Amyotrophic Lateral Sclerosis (ALS) is considered the third most common neurodegenerative disease worldwide ¹, and is becoming a disease with a significant impact and much community awareness for many countries across the globe. Over 60% of patients die within 3–5 years of diagnosis. There is no cure for ALS. Although much effort has been made in the past two decades to understand the complexity and heterogeneity of ALS, the mechanisms by which progressive degeneration and death of motor neurons occurs have not yet been elucidated. Indeed, both genetic and risk factors from gene-environment interactions, including metabolic alterations, contribute to the disease progression and pathogenesis.

Not until a couple of years ago that both clinical and epidemiological studies provided for the first time evidence that ozone (O₃), a highly oxidative gas, may be a potential novel therapeutic strategy in the treatment of degenerative disorders ^2–4. In this context, clinical trials evidenced the effectiveness of O₃ therapy in the treatment of multiple sclerosis ^2,3,5, due to its potential to induce controlled oxidative stress ⁶, through mechanism of action involving its interaction with the nuclear factor erythroid-derived 2–like 2 (Nrf2) ³, a key regulator of inducible antioxidant responses ⁷. Additionally, clinical data indicated the immunomodulatory effect of O₃ therapy ⁸, and animal work demonstrated the effectiveness of this gas to activate the hypothalamic-pituitary-adrenal axis, which has the potential to alter the composition of the gut microbiota and increase gastrointestinal permeability ^{9, 10}, suggesting the potential role of O₃ to induce controlled metabolic effects.

We have previously reported that repeated exposure to O₃ significantly reduce metabolic disturbances in a transgenic (Tg) mouse model of ALS ¹¹ expresses human transactive response DNA-binding protein 43 (TDP-43) with an A315T mutation (hTDP-43^A315T) ¹². However, the underlying molecular mechanisms and pathways that link O₃ exposures to disease-associated weight loss in TDP-43^A315T mice were not determined. Therefore, in the current study, we sought to replicate our previous study in a new cohort of TDP-43^A315T mice to examine relationships between O₃ exposure and induced metabolic effects in ALS disease. To this end, we assessed the mRNA and protein expression profile of molecular pathways with a main role in the regulation of the metabolic homeostasis on the hypothalamus and the brown adipose tissue (BAT) at the disease end-stage, defined as when weight is 20% below the initial weight on three consecutive days (~ 95–100 ± 2 days), in TDP-43^A315T mice compared to age-matched wild type (WT) littermate controls. At the selected time-point, the analysis of faecal bacterial community diversity with 16S rDNA sequencing were also carried out to determine the impact of O₃ exposure on gut microbiota. Additionally, by confocal imaging we analysed tibialis anterior (TA) hind limb muscle using immunohistochemistry (IHC) analysis, to investigate the effect of O₃ exposure on innervation of neuromuscular junction (NMJ).

Animals

Male TDP43^A315TTg mice (Strain No. 010700, Bar Harbor, ME, USA) and the genetic background–matched WT littermate control mice were used in this study ¹². Animals expressing the hTDP-43 transgene were confirmed via PCR according to the distributor’s protocol. Animals were group-housed under standard housing conditions with a 12 h light–dark cycle, and food and water ad libitum. The maintenance and use of mice and all experimental procedures were approved by the Animal Ethics Committee of the Hospital Nacional de Parapléjicos (HNP) (Approval No 26/OH 2018), in accordance with the Spanish Guidelines for the Care and Use of Animals for Scientific Purposes. All analyses were conducted by personnel blinded to the animal genotype.

Experimental design and O₃ exposure

Mice at 42 days of age (asymptomatic stage of disease) underwent two consecutive weeks of O₃vs. FA (Filter Air) exposure following the protocol described ^11,46. Each genotype of mice was divided into two subgroups (n = 3-7 TDP-43^A315T mice/subgroup and n = 5-8 WT mice/subgroup) according to the exposure. During the 15 consecutive days of exposure (4 hours/day), animals had food and water ad libitum, and their general health was regularly checked. During the procedure no differences in body weight gain between groups (O₃group vs. FA group) were displayed. Then, to monitor disease onset (defined as the last day of individual peak body weight before a gradual loss occurs) and progression, all mice were weighed and assessed three times per week until the disease onset-stage, after which they were checked daily in the morning until the disease end-stage, at which the mice were euthanized.

Tissue preparation

Animals were terminally anesthetized with sodium pentobarbitone (140 mg/kg), and transcardially perfused with 0.01 M phosphate buffered saline (PBS; pH 7.4). Tissue samples of hypothalamus and BAT were processed to extract both mRNA and proteins for real time PCR and Western-blotting analysis. Samples were immediately frozen on dry ice and stored at -80°C for later analysis. For IHC analysis, TA samples were dissected, post-fixed overnight in 4% paraformaldehyde (PFA in 0.01M PBS), and then transferred to 18% and then 30% sucrose solutions overnight. Serial cryosections (50 µm thick) were cut on a cryostat (Leica CM 1850).

RNA isolation and RT-qPCR

Total RNA was isolated from hypothalamus and BAT using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Absorbance at 260 nm was measured using a NanoDrop (ThermoFisher, Waltham, MA; USA) to determine RNA concentration. Complementary DNA (cDNA) (0.5 µg of total RNA for hypothalamus and 1 µg for BAT, respectively) was synthesized following the procedure described ⁴⁷. Relative quantification of each gene was performed using 25 ng of reverse transcribed total RNA, 20 pmol/ml of both sense and antisense primers and the SYBR Green PCR Master Mix (Applied Biosystems) in a final reaction volume of 20 µl. Each sample was run in duplicate and 18S rRNA ⁴⁸ was used as a control to normalize gene expression. The reactions were run on an ABI PRISM 7900 Fast Sequence Detection System instrument and software (AppliedBiosystem) according to the manufacturer's protocol. Primers were designed using NCBI/Primer-BLAST software and the sequences are detailed in Tables 1 and 2. In all cases relative quantification for each gene was performed by the ∆∆Ct method ⁴⁹.

Protein extraction and western blot analysis

Proteins from the hypothalamus were extracted using RIPA buffer (Sigma Aldrich) containing a cocktail of protease inhibitors (Roche). Protein concentration was measured using the BioRad Protein assay based on the Bradford method. Samples were measured at 595 nm on an automatic microplate analyser (Tecan Infinite M200, Grödig, Austria). Western blotting was performed as previously described ⁴⁸ with some modifications. Denatured hypothalamus protein samples (30 µg) were separated using 10% Bolt Bis-Tris Plus gels (Invitrogen). After that, the gels were transferred onto a polyvinylidene fluoride (PVDF) membranes (Bio-Rad) and blocked with Tris-Buffered Saline (TBS) with 0.1% Tween-20 (Bio-Rad), and 5% Bovine Serum Albumin (BSA) or non-fat dried milk (blocking buffer) for 1 hour at room temperature (RT). Membranes were then incubated with the corresponding primary antibody in blocking buffer at 4˚C overnight. The primary antibodies used were rabbit anti-long-form of the leptin receptor (Ob-Rb) (1:500; Abcam), mouse anti-Akt (1:250; Santa Cruz), rabbit anti-phospho Akt (Ser473) (1:500; Cell Signaling), mouse anti-STAT3 (1:250; Santa Cruz Biotechnology), rabbit anti-phospho STAT3 (Tyr705) (1:500; Santa Cruz Biotechnology), rabbit anti-supressor of cytokine signaling 3 (SOCS-3) (1:500; Cell Signaling), and rabbit anti-neuropeptide Y (NPY) (1:200; Abcam). A corresponding anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Vector Laboratories) were used. Mouse anti-GAPDH (1:5000, Millipore) and/or mouse anti-actin (1:1000; Cell Signaling) were used as the loading controls. Peroxidase activity was visualized by using Luminata Forte Western horseradish peroxidase (HRP) substrate (Millipore) and determined by densitometry using an ImageQuant LAS 4000 mini system (GE Healthcare Little Chalfont, United Kingdom). Band intensity was measured as the integrated intensity using ImageJ software (v1.4; NIH). All data were normalized to control values on each membrane.

Muscle histology and analysis of NMJ

TA samples were processed as is previously described. The stain of NMJs were conducted following the protocol described ⁵⁰, with slight modifications. Briefly, slides were brought up to RT and incubated for 1 hour with alpha(α)-bungarotoxin (α-BTX) (Invitrogen) at 1:500 in TBS. Then, slides were incubated in blocking solution (Dako) for 1 hour. Primary antibodies against betha(β)-III-tubulin (Promega) and synaptophysin (SYN) (GeneTex) were applied at 1:150 and 1:250, respectively, in blocking solution, and incubated at 4˚C overnight. Sections were then incubated with their corresponding mouse Alexa488 and rabbit Alexa647-conjugated secondary antibodies (Thermo Fisher Scientific), both diluted 1:1000 in blocking solution, for 1 hour at RT. Finally, the slides were coverslipped in “Immumount” (Thermo Scientific) and confocal images were collected at RT with x20 objective, and LAS-AF software (Leica).

NMJs from flattened z stacks of TA were analysed (ImageJ software v1.4; NIH) by personnel blinded to the animal genotype. Brightness and contrast thresholds were set to optimize the signal-to-noise ratio of the presynaptic staining (β-III-tubulin and SYN). Innervated NMJs were defined as having observed overlap of staining for pre- and post-synaptic elements. Partial denervated NMJs were defined as α-BTX signal in the presence of incomplete presynaptic staining. Denervated NMJs were defined as α-BTX signal in the absence of presynaptic staining ^50-53

Fecal collection, DNA extraction and Illumina sequencing and analysis

Two to three freshly evacuated fecal pellets were collected from each mouse in the morning, prior to the end-stage of disease. Briefly, mice temporarily were placed individually into empty autoclaved cages and allowed to defecate. Fecal pellets were collected aseptically from each mouse and placed into a sterile tube. Faecal samples were stored at –80 °C prior to DNA extraction.

Genomic DNA was extracted with a Purelink Microbiome DNA purification kit (Invitrogen) according to the manufacturer's instructions, and subjected to high throughput sequencing. DNA yields were determined through fluorometry (Qubit, Life Technologies, Carlsbad, CA) by using a reagent kit (Quant-iT BR dsDNA Kit, Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Amplicons purification and Illumina sequencing were performed commercially by Macrogen, Inc., Korea (www.macrogen.com).

A total of 3,484,598 reads were obtained. Raw sequences were processed using CUTADAPT ⁵⁴ to remove primers. Then, forward and reverse reads were trimmed at 250 and 210 bp, respectively, using DADA2 ⁵⁵. After being denoised and dereplicated, the remaining forward and reverse reads were assembled. Chimeras were removed using the ‘removeBiomeraDenova’ function in the DADA2 pipeline, which resulted in the final taxon table based on 1,053,851 reads. Taxonomic annotation was performed using the SILVA 132 database ⁵⁶.

Analyses and graphical representations were performed in R version 4.1.0 (R Core Team., 2013) ⁵⁷ using vegan ⁵⁸, phyloseq ⁵⁹, Microbiome ⁶⁰, DESeq2 ⁶¹, MASS ⁶², ggplot2 ⁶³, ampvis2 ⁶⁴ and VennDiagram ⁶⁵ packages. Sample metadata, taxonomic units as amplicon sequencing variants (ASV) matrix and taxa information were imported into a phyloseq object. Then, alpha diversity measures were calculated (i.e. ASV observed, Chao1, Shannon, Gini-Simpson and Pielou’s Eveness indexes). Bray–Curtis distances were used for calculating beta diversity and non-metric multidimensional scaling (NMDS). Permutational multivariate analysis of variance (PerMANOVA) was carried out based on the Bray-Curtis dissimilarity matrix by using permutest and adonis functions from vegan package.

Statistical analysis

All data are presented as mean ± standard error of the mean (SEM). Differences between means were assessed by two-way ANOVA followed by Dunett’s post hoc test, to compare all groups with control WT-FA mice, and Tukey's post hoc test were used for multiple comparisons between all groups. For all statistical tests, a p value of < 0.05 (CI 95%) was assumed to be significant. Statistical analysis was performed using GraphPad Prism software (version 8.3.1).

O₃ exposure modified the expression profile of hypothalamic neuropeptides altering STAT3 and Akt pathways in TDP-43^A315T mice

Since metabolic abnormalities have been reported in both ALS patients ¹³ and mouse models of ALS ^14-17, and we previously reported a progressive decline in body weight in TDP-43^A315T mice compared to WT mice ¹¹, we evaluated if genes involved in metabolism were affected in the hypothalamus of TDP-43^A315T mice compared to WT controls in responses to FA or O₃exposure (Fig. 1). RT-qPCR analysis demonstrated that there was a significant effect of genotype on the expression profile of POMC (p < 0.0001) and AgRP (p = 0.01) transcripts in the hypothalamus (Fig. 1A-B). Tukey´s post hoc test demonstrated a significant increase in POMC mRNA expression in both TDP-43^A315T FA and O₃-exposed mice compared to WT controls in response to FA (p = 0.0003 and p = 0.003, respectively; Fig. 1A). Conversely, Tukey´s post hoc test demonstrated a significant upregulation of AgRP mRNA levels in the hypothalamus of TDP-43^A315T O₃-exposed compared to WT controls in response to O₃ (p = 0.01). Additionally, immunoblotting analysis demonstrated differences in the protein expression levels of NPY between TDP-43^A315T and WT mice, with a significant effect of genotype (p = 0.01) and exposure (p = 0.02) across groups (Fig. 1C). Tukey´s post hoc test demonstrated a significant hypothalamic upregulation of NPY protein levels in TDP-43^A315T FA-exposed relative to WT controls in responses to FA or O₃ (p = 0.04 and p = 0.01, respectively).

We next investigated the protein levels of Ob-Rb, the long isoform of leptin receptor, SOCS3, a main inhibitor of the leptin signaling, and the phosphorylation levels of Akt (pSer⁴⁷³-Akt) and STAT3 (pTyr⁷⁰⁵-STAT3) proteins (Fig. 2), as they are important targets in the regulation of glucose and energy metabolism ¹⁸. Although, we found no significant effect of either genotype or O₃ exposure on Ob-Rb and SOCS3 protein expression levels (Fig. 2A-B). Dunnett´s post hoc test demonstrated a statistically significant increase on the phosphorylation levels of STAT3 protein in the hypothalamus of TDP-43^A315T mice FA-exposed compared to WT controls in response to FA (p = 0.04) (Fig. 2C). There was a significant effect of the O₃ exposure (p = 0.0035) on the phosphorylation levels of Akt protein in the hypothalamus (Fig. 2D). Additionally, Tukey's post hoc test demonstrated a statistically significant hypothalamic increase on the phosphorylation levels of Akt protein in TDP-43^A315T mice FA-exposed compared to WT controls (p = 0.03) and TDP-43^A315T mice (p = 0.008) in responses to O₃ (Fig. 2D).

O₃ exposure leads to increase the expression of genes involved in metabolism and thermogenesis in the BAT of TDP-43^A315Tmice

BAT is a thermogenic organ with an important role in controlling energy expenditure and the regulation of body weight ¹⁹. Interestingly, animal work has shown that acute O₃ exposure causes endocrine and metabolic changes, increasing food intake and body fat mass ²⁰. Thus, to determine the metabolic impact of O₃ exposure on altering BAT thermogenic activity in TDP-43^A315Tmice compared to age-matched WT littermate controls at the end-stage of disease, we first conducted PCR analysis to examine the expression profile of genes involved in brown adipocyte differentiation (Fig. 3). RT-qPCR analysis demonstrated a significant effect of O₃ exposure in the pattern of expression of AdipoQ, Fabp4, Prdm16 and PPARγ genes between TDP-43^A315T mice and WT controls. There were significant effects of genotype and exposure (p < 0.05) on the expression profile of AdipoQ, Fabp4, Prdm16 and PPARγ transcripts in BAT across groups (Fig. 3A-D). In TDP-43^A315T FA-exposed, AdipoQ, Fabp4, Prdm16 and PPARγ mRNA levels were significantly upregulated compared to WT controls in responses to FA (p = 0.0002, p < 0.0001, p = 0.04 and p < 0.0001, respectively), with this increase being significant at the end-stage of disease compared to AdipoQ, Fabp4 and PPARγ mRNA levels in WT controls in responses to O₃ exposure (p = 0.0007, p < 0.0001 and p < 0.0001, respectively). Tukey's post hoc test demonstrated an overall statistically significant downregulation of all genes (AdipoQ: p = 0.01, Fig. 3A; Fabp4: p < 0.0001, Fig. 3B; Prdm16: p = 0.01, Fig. 3C; and PPARγ: p = 0.0001, Fig. 3D) in the BAT of TDP-43^A315T mice in responses toO₃ exposure.

We next examined the expression of genes involved in BAT-mediated thermogenesis, including Ucp1 and Pgc-1a (Fig. 3E-F). There were significant effects of genotype (p < 0.0001) and exposure (p = 0.008 and p = 0.009, respectively) on the expression profile of both genes in the BAT. In TDP-43^A315T mice FA-exposed, Ucp1 and Pgc-1a mRNA levels were significantly upregulated compared to WT controls in responses to either FA and O₃ exposure (p < 0.0001; Fig. 3E-F), while a different pattern of expression was observed in TDP-43^A315T mice exposed to O₃. Tukey's post hoc test demonstrated a significantly upregulation of Ucp1 and Pgc-1a transcripts in the BAT compared to WT controls in responses to FA (p = 0.003 and p = 0.0001, respectively), with this increase being significantly downregulated compared to WT controls in responses to O₃ exposure (p = 0.0009 and p = 0.005). In addition, RT-qPCR analysis demonstrated that O₃exposure upregulated Ucp1 and Pgc-1a mRNA levels in TDP-43^A315T mice and age-matched WT controls, with a statistically significant downregulation on their expression (p < 0.0001).

To further examine the effect of O₃ exposure on BAT, we next analysed the expression profile of genes known to mediate glucose uptake, particularly Glut4, which is significantly expressed in BAT ²¹ (Fig. 3G). Interestingly, although RT-qPCR analysis demonstrated a similar pattern of expression of Glut4 gene between TDP-43^A315T mice and WT controls, Tukey's post hoc test demonstrated a statistically significant upregulation of Glut4 mRNA levels in the BAT of TDP-43^A315T mice in responses to O₃exposure (p < 0.0015), which is of interest as we previously reported plasma glucose levels were highest at disease end-stage after O₃ exposure in TDP-43^A315T mice ¹¹. Finally, since leptin modulates body weight gain and energy expenditure ^22,23, at least in part, by modulating thermogenesis ²⁴, and, as it has been provided that leptin treatment increase body temperature in leptin deficient mice (ob/ob mice) ²⁵, we next asked how leptin and Ob-Rb were affected in the BAT of TDP-43^A315Tmice in response to O₃ exposure (Fig. 4). We have been previously reported ²⁸ an upregulation of leptin mRNA levels in the white adipose tissue (WAT) of TDP-43^A315T mice, however, although there was no effect of genotype, RT-qPCR analysis demonstrated that there was a significant effect of exposure (p = 0.02) on the expression profile of leptin transcript in the BAT (Fig. 4A). In contrast, RT-qPCR analysis demonstrated a statistically significant BAT upregulation of Ob-Rb mRNA levels in TDP-43^A315T mice FA-exposed compared to WT controls in responses to FA or O₃exposure (p < 0.0001; Fig. 4B). In addition, Tukey's post hoc test demonstrated a statistically significant downregulation of Ob-Rb mRNA levels (p < 0.0001; Fig. 4B) in the BAT of TDP-43^A315T mice in responses to O₃exposure.

O₃ exposure did not induce fecal microbiota compositional changes in TDP-43^A315Tmice

We next studied fecal gut microbiome composition in TDP-43^A315Tmice compared to age-matched WT littermate controls in response to FA or O₃exposure. Microbial-mediated effects on ALS neuropathology were assessed through 16S rRNA amplicon sequencing in fecal samples from TDP-43^A315T and WT controls prior to the disease end-stage. The rarefaction curve of Chao1 index suggested that the diversity of ASVs is not affected by either genotype or exposure, since any trend is observed as indicated by the mix higher curves thereof under the same sequencing depth (Fig. 6A). Additionally, all the curves tend to be smooth and reach the plateau, which indicates that the amount of sequencing data was sufficient and reasonable, and that more data would only produce a few new ASVs. The total number of observed genera (alpha diversity) was similar in both TDP-43^A315T mice and WT controls whether they were exposed to FA or O₃. However, evenness was higher in WT mice than in TDP-43^A315Tmice (p = 0.03), suggesting that a more even distribution of taxa can be found in WT controls compared to TDP-43^A315Tmice. The Venn diagram exhibited the exact number of ASVs common for the four groups (174). This number was higher than the number of unique ASVs in WT controls exposed to O₃, but lower than the unique ASVs observed in the other groups (Fig. 6B). Actually, the gut microbiome composition of TDP-43^A315T mice and WT controls yielded significantly different compositions (Fig. 7). TDP-43^A315T mice showed a significant increase in ASVs within Proteobacteria phyla like Parasutterella and Escherichia/Shigella genus. In addition, several ASVs within specific genera were observed in TDP-43^A315T mice but not in WT controls (e.g., Ruminococcus, Parabacteroides, Marvinbryantia, Blautia) (Fig. S1).

O₃ exposure does not impaired the NMJs in the skeletal muscle of TDP-43^A315T mice

We next studied alterations in NMJs in TDP-43^A315Tmice compared to age-matched WT littermate controls at the end-stage of disease in response to FA or O₃exposure, as NMJ pathology is well-known in mouse models of ALS ²⁶. To this aim we examined the TA muscle by IHC analysis (Fig. 5). Densitometric analysis demonstrated a significant effect of genotype on the percentage of innervated and denervated (p < 0.0001, respectively) NMJs in the TA muscle of TDP-43^A315T mice compared to WT controls (Fig. 5E-G). Tukey´s post hoc test demonstrated a significant decrease on the percentage of innervated NMJs in the TA muscle of TDP-43^A315T mice exposed to FA and O₃relative to WT mice in response to FA (p = 0.0013 and p = 0.007, respectively) and O₃ exposure (p = 0.0007 and p = 0.003, respectively) (Fig. 5E). Similarly, IHC analysis showed a significant decrease on the percentage of denervated NMJs in the TA muscle of TDP-43^A315T mice exposed to FA and O₃compared to WT in response to FA and O₃ exposure (p = 0.002 in all cases; Fig. 5F). There was a slight increase in partially denervation of NMJs in the TA muscle of TDP-43^A315T mice O₃-exposed above to FA-exposed, however, the differences were not statistically significant (Fig. 5G).

Although the cellular basis for motor neuron degeneration in ALS is not yet fully understood, it is increasingly recognized that energy homeostasis (the balance between energy intake and expenditure) appears to be compromised in ALS ²⁷. ALS patients develop prominent changes in weight and eating behavior that result from and mediate the underlying neurodegenerative process. Emerging research suggests that these alterations may be mediated through changes in the hypothalamic function ²⁸. Indeed, neuropeptides derived from POMC provide a strong anorexigenic effect (i.e., decreases food intake), while NPY/AgRP neurons have a potent orexigenic effect (i.e., increase food intake). Here, we report a downregulation of AgRP mRNA levels and an upregulation of POMC transcripts in TDP-43^A315T mice compared to WT controls, which is in accordance with the progressive decline in body weight in TDP-43^A315T mice ^11,29−32. We also found that O₃ exposure reduces NPY protein expression levels in TDP-43^A315T mice in response to either FA or O₃ exposure, which could partly be due to modifications in food intake. This observation is of interest because in a situation of negative energy balance, such as the malnutrition observed in ALS, the expression of NPY would be normally increased ^33,34, and thus, this result is in agreement with previously data showing how O₃ exposure causes endocrine and metabolic changes increasing food intake and body fat mass ²⁰. In addition, besides sensitive genes involved in metabolism, we also examined leptin signaling in the hypothalamus of TDP-43^A315T mice, as central hypothalamic leptin signaling has a critical role in promoting energy homeostasis via modulation of food intake and energy expenditure ³⁵. Nevertheless, although no differences in Ob-Rb mRNA levels between groups were observed, a significant decrease in serine phosphorylation of Akt was found in the hypothalamus of TDP-43^A315T mice in response to O₃ compared with FA exposure. This data is of particular interest as Akt is an important target in the regulation of glucose and energy metabolism ¹⁸, which is in accordance with previous data from our group showing how O₃ exposure resulted in higher plasma glucose levels at later time points ¹¹.

Considering that BAT has profound effects on body weight and metabolism in rodents ³⁶, it is conceivable that the reported improvement on loss of body weight in TDP-43^A315T mice in response to O₃ exposure, could be due to the effect of this gas to modify its thermogenic function. Indeed, the ability of this adipose tissue to expend energy has increased interest in stimulating thermogenesis to treat metabolic diseases such as obesity and diabetes type II. In this context, our results confirm a significant upregulation of genes involved in brown adipocyte differentiation, including AdipoQ, Fabp4, Prdm16 and PPARγ mRNAs in BAT of TDP-43^A315T mice in response to FA compared with O₃ exposure. We also found significant effects of O₃ exposure on the expression profile of genes involved in BAT-mediated thermogenesis such as Ucp1 and Pgc-1a. These data are of interest because evidence supports that the decrease in thermogenesis is likely associated with a greater predisposition to body weight gain ³⁷, which is in accordance with previous data from our group showing a less severe decline disease-associated weight loss in TDP-43^A315T mice exposed to O₃. Indeed, we also found a significant upregulation of Glut4 mRNA levels in the BAT of TDP-43^A315T mice in responses to O₃ exposure, which is an interesting data as we previously reported plasma glucose levels were highest at the end-stage of disease after O₃ exposure in TDP-43^A315T mice ¹¹. Finally, concomitantly to the decrease in the expression profile of AdipoQ, Fabp4, Prdm16, PPARγ, Ucp1 and Pgc-1a in the BAT of TDP-43^A315T mice, our results confirm a significant downregulation of Ob-Rb mRNA expression levels in responses to O₃ exposure, which is in accordance with experimental data showing how obese mice that lack of Ob-Rb receptor showed a decreased BAT thermogenic capacity ³⁸.

In attempt to have a better knowledge on the potential major role of O₃ exposure to induce controlled metabolic effects, we examined fecal microbiome composition in TDP-43^A315T mice. Gut microbiome has been implicated in ALS development ³⁹, and thus, it is conceivable that the metabolic changes determined in TDP-43^A315T mice in responses to O₃ exposure could be also due to a compensatory effect of O₃ on gut dysbiosis (an imbalance in the gut microbiota community). Indeed, in ALS disease gut dysbiosis affects the central nervous system via pro-inflammatory mediators (i.e., cytokines and hormone-like molecules), thus, impacting gut-brain communications ⁴⁰. In this context, our data showed how the gut microbiome composition of TDP-43^A315T mice yielded significantly different composition compared with WT controls. Although no statistical changes were found on gut bacterial communities in TDP-43^A315T mice in response to O₃ compared with FA exposure, an increase in the relative abundance of genera like Bacteroides, Parabacteroides or Marvinbryantia was observed as a result of such exposure (Fig. S1). In addition, gut microbiome composition of WT mice showed a significant increase in Lachnospiraceae NK4A136 group as a result of O₃ exposure, and, this data is of interest because this group is a potential butyrate-producer associated with probiotic activity in mice ⁴¹. Indeed, it has been found that butyrate is one of the main short-chain fatty acids (SCFAs) produced by microbiota helping to maintain gut barrier integrity and inhibit inflammation ⁴². This result might indicate the capacity of this gas to restore physiological homeostasis and potentially improve dysbiosis.

Finally, as metabolic alterations in ALS are associated with the progression of disease pathology, we examined potential changes in NMJ innervation in TA muscles of TDP-43^A315T mice, as degenerative processes in the skeletal muscle, particularly involving NMJs, are observed throughout disease progression in ALS ^43,44. Our densitometric analysis showed no significant changes on NMJs in TA muscles in TDP-43^A315T mice in response to O₃ exposure compared with FA exposure, although a significant impairment of NMJs in TDP-43^A315T mice compared to WT controls in response to either FA or O₃ exposures was found, which is in agreement with previous data reported in mutant TDP-43 mice (i.e., Tg NEFH-hTDP-43ΔNLS or TDP-43 rNLS bigenic mice) showing a progressive NMJs denervation followed by spinal cord motor neuron loss ⁴⁵.

In summary, we have previously shown that repeated exposure to O₃ in TDP-43^A315T mice at the pre-symptomatic stage of pathology, significantly improved motor function and disease-associated weight loss compared to TDP-43^A315T mice exposed to FA ¹¹. Our present replication study has confirmed those findings, and provided a mechanistic link between O₃ exposure and the improvement of the metabolic disturbances present in TDP-43^A315T mice.

Data availability

Some or all data used to support the findings of this study are available from the corresponding author upon request.

1 Pfrieger, F. W. Neurodegenerative Diseases and Cholesterol: Seeing the Field Through the Players. Front Aging Neurosci 13, 766587, doi:10.3389/fnagi.2021.766587 (2021).

2 Izadi, M. et al. Changes in Th17 cells frequency and function after ozone therapy used to treat multiple sclerosis patients. Mult Scler Relat Disord 46, 102466, doi:10.1016/j.msard.2020.102466 (2020).

3 Scassellati, C., Galoforo, A. C., Bonvicini, C., Esposito, C. & Ricevuti, G. Ozone: a natural bioactive molecule with antioxidant property as potential new strategy in aging and in neurodegenerative disorders. Ageing Res Rev 63, 101138, doi:10.1016/j.arr.2020.101138 (2020).

4 Mašán, J., Sramka, M. & Rabarova, D. The possibilities of using the effects of ozone therapy in neurology. Neuro endocrinology letters 42, 13-21 (2021).

5 Delgado-Roche, L. et al. Medical ozone promotes Nrf2 phosphorylation reducing oxidative stress and pro-inflammatory cytokines in multiple sclerosis patients. Eur J Pharmacol 811, 148-154, doi:10.1016/j.ejphar.2017.06.017 (2017).

6 Braidy, N. et al. Therapeutic relevance of ozone therapy in degenerative diseases: Focus on diabetes and spinal pain. J Cell Physiol 233, 2705-2714, doi:10.1002/jcp.26044 (2018).

7 Gupte, A. A., Lyon, C. J. & Hsueh, W. A. Nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2), a key regulator of the antioxidant response to protect against atherosclerosis and nonalcoholic steatohepatitis. Curr Diab Rep 13, 362-371, doi:10.1007/s11892-013-0372-1 (2013).

8 Diaz-Luis, J., Menéndez-Cepero, S. & Macias-Abrahan, C. Ozone therapy immunomodulatory effect in the selective immunoglobulin A deficiency. Journal of Ozone Therapy 1, doi:10.7203/jo3t.1.1.2015.12161 (2015).

9 Tashiro, H. & Shore, S. A. The Gut Microbiome and Ozone-induced Airway Hyperresponsiveness. Mechanisms and Therapeutic Prospects. Am J Respir Cell Mol Biol 64, 283-291, doi:10.1165/rcmb.2020-0288TR (2021).

10 T, A. et al. Exposure to Ambient Air Pollutants is Associated with the Composition of the Gut Microbiome in Adolescents from Southern California. Environmental Epidemiology 3, 6-7, doi:10.1097/01.EE9.0000605664.50459.e7 (2019).

11 Rodriguez, A. et al. Repeated Exposure to Ozone Produces Changes in Metabolic Disturbances Present in the TDP-43A315T Transgenic Model of Amyotrophic Lateral Sclerosis. Diagn Pathol Open 2021 6:S8, doi:10.1101/2021.02.12.430915 (2021).

12 Wegorzewska, I., Bell, S., Cairns, N. J., Miller, T. M. & Baloh, R. H. TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci U S A 106, 18809-18814, doi:10.1073/pnas.0908767106 (2009).

13 D'Amico, E. et al. Metabolic Abnormalities, Dietary Risk Factors and Nutritional Management in Amyotrophic Lateral Sclerosis. Nutrients 13, doi:10.3390/nu13072273 (2021).

14 Lim, M. A. et al. Genetically altering organismal metabolism by leptin-deficiency benefits a mouse model of amyotrophic lateral sclerosis. Hum Mol Genet 23, 4995-5008, doi:10.1093/hmg/ddu214 (2014).

15 Shan, X., Chiang, P. M., Price, D. L. & Wong, P. C. Altered distributions of Gemini of coiled bodies and mitochondria in motor neurons of TDP-43 transgenic mice. Proc Natl Acad Sci U S A 107, 16325-16330, doi:10.1073/pnas.1003459107 (2010).

16 Wang, W. et al. The ALS disease-associated mutant TDP-43 impairs mitochondrial dynamics and function in motor neurons. Hum Mol Genet 22, 4706-4719, doi:10.1093/hmg/ddt319 (2013).

17 Ferrer-Donato, A., Contreras, A., Frago, L. M., Chowen, J. A. & Fernandez-Martos, C. M. Alterations in Leptin Signaling in Amyotrophic Lateral Sclerosis (ALS). Int J Mol Sci 22, doi:10.3390/ijms221910305 (2021).

18 Varela, L. & Horvath, T. L. Leptin and insulin pathways in POMC and AgRP neurons that modulate energy balance and glucose homeostasis. EMBO Rep 13, 1079-1086, doi:10.1038/embor.2012.174 (2012).

19 Scotney, H. et al. Glucocorticoids modulate human brown adipose tissue thermogenesis in vivo. Metabolism 70, 125-132, doi:10.1016/j.metabol.2017.01.024 (2017).

20 Nappi, F. et al. Endocrine Aspects of Environmental "Obesogen" Pollutants. Int J Environ Res Public Health 13, doi:10.3390/ijerph13080765 (2016).

21 Shimizu, Y. et al. Increased expression of glucose transporter GLUT-4 in brown adipose tissue of fasted rats after cold exposure. Am J Physiol 264, E890-895, doi:10.1152/ajpendo.1993.264.6.E890 (1993).

22 Pan, W. W. & Myers, M. G., Jr. Leptin and the maintenance of elevated body weight. Nat Rev Neurosci 19, 95-105, doi:10.1038/nrn.2017.168 (2018).

23 Pandit, R., Beerens, S. & Adan, R. A. H. Role of leptin in energy expenditure: the hypothalamic perspective. Am J Physiol Regul Integr Comp Physiol 312, R938-R947, doi:10.1152/ajpregu.00045.2016 (2017).

24 Barrios, V. et al. Leptin Modulates the Response of Brown Adipose Tissue to Negative Energy Balance: Implication of the GH/IGF-I Axis. Int J Mol Sci 22, doi:10.3390/ijms22062827 (2021).

25 Fischer, A. W. et al. Leptin Raises Defended Body Temperature without Activating Thermogenesis. Cell Rep 14, 1621-1631, doi:10.1016/j.celrep.2016.01.041 (2016).

26 Mejia Maza, A. et al. NMJ-Analyser identifies subtle early changes in mouse models of neuromuscular disease. Sci Rep 11, 12251, doi:10.1038/s41598-021-91094-6 (2021).

27 Dupuis, L., Pradat, P. F., Ludolph, A. C. & Loeffler, J. P. Energy metabolism in amyotrophic lateral sclerosis. Lancet Neurol 10, 75-82, doi:10.1016/S1474-4422(10)70224-6 (2011).

28 Vercruysse, P., Vieau, D., Blum, D., Petersen, A. & Dupuis, L. Hypothalamic Alterations in Neurodegenerative Diseases and Their Relation to Abnormal Energy Metabolism. Front Mol Neurosci 11, 2, doi:10.3389/fnmol.2018.00002 (2018).

29 Esmaeili, M. A., Panahi, M., Yadav, S., Hennings, L. & Kiaei, M. Premature death of TDP-43 (A315T) transgenic mice due to gastrointestinal complications prior to development of full neurological symptoms of amyotrophic lateral sclerosis. Int J Exp Pathol 94, 56-64, doi:10.1111/iep.12006 (2013).

30 Guo, Y. et al. HO-1 induction in motor cortex and intestinal dysfunction in TDP-43 A315T transgenic mice. Brain Res 1460, 88-95, doi:10.1016/j.brainres.2012.04.003 (2012).

31 Hatzipetros, T. et al. C57BL/6J congenic Prp-TDP43A315T mice develop progressive neurodegeneration in the myenteric plexus of the colon without exhibiting key features of ALS. Brain Res 1584, 59-72, doi:10.1016/j.brainres.2013.10.013 (2014).

32 Medina, D. X., Orr, M. E. & Oddo, S. Accumulation of C-terminal fragments of transactive response DNA-binding protein 43 leads to synaptic loss and cognitive deficits in human TDP-43 transgenic mice. Neurobiol Aging 35, 79-87, doi:10.1016/j.neurobiolaging.2013.07.006 (2014).

33 Caron, A. et al. POMC neurons expressing leptin receptors coordinate metabolic responses to fasting via suppression of leptin levels. Elife 7, doi:10.7554/eLife.33710 (2018).

34 Pedroso, J. A. et al. Changes in Leptin Signaling by SOCS3 Modulate Fasting-Induced Hyperphagia and Weight Regain in Mice. Endocrinology 157, 3901-3914, doi:10.1210/en.2016-1038 (2016).

35 Munzberg, H., Singh, P., Heymsfield, S. B., Yu, S. & Morrison, C. D. Recent advances in understanding the role of leptin in energy homeostasis. F1000Res 9, doi:10.12688/f1000research.24260.1 (2020).

36 Svensson, P. A. et al. Gene expression in human brown adipose tissue. Int J Mol Med 27, 227-232, doi:10.3892/ijmm.2010.566 (2011).

37 Tremblay, A., Royer, M. M., Chaput, J. P. & Doucet, E. Adaptive thermogenesis can make a difference in the ability of obese individuals to lose body weight. Int J Obes (Lond) 37, 759-764, doi:10.1038/ijo.2012.124 (2013).

38 Trayhurn, P., Thurlby, P. L. & James, W. P. A defective response to cold in the obese (obob) mouse and the obese Zucker (fafa) rat [proceedings]. Proc Nutr Soc 35, 133A (1976).

39 Boddy, S. L. et al. The gut microbiome: a key player in the complexity of amyotrophic lateral sclerosis (ALS). BMC Med 19, 13, doi:10.1186/s12916-020-01885-3 (2021).

40 Rowin, J., Xia, Y., Jung, B. & Sun, J. Gut inflammation and dysbiosis in human motor neuron disease. Physiol Rep 5, doi:10.14814/phy2.13443 (2017).

41 Wu, M.-R., Chou, T.-S., Huang, C.-Y. & Hsiao, J.-K. A potential probiotic- Lachnospiraceae NK4A136 group: Evidence from the restoration of the dietary pattern from a high-fat diet. (2020).

42 Ma, L. et al. Spermidine improves gut barrier integrity and gut microbiota function in diet-induced obese mice. Gut Microbes 12, 1-19, doi:10.1080/19490976.2020.1832857 (2020).

43 Dupuis, L. & Loeffler, J. P. Neuromuscular junction destruction during amyotrophic lateral sclerosis: insights from transgenic models. Curr Opin Pharmacol 9, 341-346, doi:10.1016/j.coph.2009.03.007 (2009).

44 Dadon-Nachum, M., Melamed, E. & Offen, D. The "dying-back" phenomenon of motor neurons in ALS. J Mol Neurosci 43, 470-477, doi:10.1007/s12031-010-9467-1 (2011).

45 Walker, A. K. et al. Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43. Acta Neuropathol 130, 643-660, doi:10.1007/s00401-015-1460-x (2015).

46 Bello-Medina, P. C., Prado-Alcala, R. A. & Rivas-Arancibia, S. Effect of Ozone Exposure on Dendritic Spines of CA1 Pyramidal Neurons of the Dorsal Hippocampus and on Object-place Recognition Memory in Rats. Neuroscience 402, 1-10, doi:10.1016/j.neuroscience.2019.01.018 (2019).

47 Fernandez, C. M. et al. The expression of rat resistin isoforms is differentially regulated in visceral adipose tissues: effects of aging and food restriction. Metabolism 58, 204-211 (2009).

48 Fernandez-Martos, C. M. et al. Differential expression of Wnts after spinal cord contusion injury in adult rats. PLoS One 6, e27000, doi:10.1371/journal.pone.0027000

PONE-D-11-06684 [pii] (2011).

49 Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001).

50 White, M. A. et al. TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in mouse model of ALS-FTD. Nat Neurosci 21, 552-563, doi:10.1038/s41593-018-0113-5 (2018).

51 Blizzard, C. A. et al. Identifying the primary site of pathogenesis in amyotrophic lateral sclerosis - vulnerability of lower motor neurons to proximal excitotoxicity. Dis Model Mech 8, 215-224, doi:10.1242/dmm.018606 (2015).

52 Cappello, V. et al. Analysis of neuromuscular junctions and effects of anabolic steroid administration in the SOD1G93A mouse model of ALS. Mol Cell Neurosci 51, 12-21, doi:10.1016/j.mcn.2012.07.003 (2012).

53 Clark, J. A., Southam, K. A., Blizzard, C. A., King, A. E. & Dickson, T. C. Axonal degeneration, distal collateral branching and neuromuscular junction architecture alterations occur prior to symptom onset in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. J Chem Neuroanat 76, 35-47, doi:10.1016/j.jchemneu.2016.03.003 (2016).

54 Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. 2011 17, 3, doi:10.14806/ej.17.1.200 (2011).

55 Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13, 581-583, doi:10.1038/nmeth.3869 (2016).

56 Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41, D590-596, doi:10.1093/nar/gks1219 (2013).

57 Team, R. C. R Core Team (2013) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna. (2013).

58 Oksanen, J. et al. Vegan: community ecology package [online]. R Package version 2.0-5. (2011).

59 McMurdie, P. J. & Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8, e61217, doi:10.1371/journal.pone.0061217 (2013).

60 Lahti L, S. S., et al. Tools for microbiome analysis in R. Microbiome package version 1.15.1. . Bioconductor, 2017; http://microbiome.github.com/microbiome/. (2017).

61 Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550, doi:10.1186/s13059-014-0550-8 (2014).

62 Ripley, B. e. a. Package “mass”. Cran r 538, 113-120. (2013).

63 Wickham, H. ggplot2. WIREs Computational Statistics 3, 180-185, doi:https://doi.org/10.1002/wics.147 (2011).

64 Andersen, K. S., Kirkegaard, R. H., Karst, S. M. & Albertsen, M. ampvis2: an R package to analyse and visualise 16S rRNA amplicon data. bioRxiv, 299537, doi:10.1101/299537 (2018).

65 Dusa, A. CRAN—Package VennDiagram. https://cran.r-project.org/web/packages/VennDiagram/. (2021).

Funding: This work was supported by 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).

Acknowledgements: The authors would like to gratefully acknowledge Agueda Ferrer-Donato and Surgery Unit of the UDI-HNP for their excellent technical support. Sara Rodríguez-Sánchez is supported by a PhD Fellowship from the University of Castilla-La Mancha research plan (2019-PREDUCLM-10801).

Conflicts of Interest: The authors declare no conflict of interest.

Conceptualization: CM. F.-M; Data curation: S.R., N.V., S.S., M.C.-P; Formal analysis: S.R., N.V., S.S., M.C.-P., A.R., CM.F.-M.; Software: S.R., N.V., S.S., CM.F.-M.; Funding acquisition: CM. F.-M.; Investigation: S.R., N.V., S.S., CM. F.-M.; Methodology: S.R., N.V., S.S., M.C.-P., A.R.,CM.F.-M.; Project administration: CM. F.-M. Resources: CM. F.-M; Supervision: A.A., LL.P., CM. F.-M. Validation: A.A., LL.P., CM. F.-M. Writing - original draft: S.R., N.V., S.S., CM.F.-M. Writing - review & editing: S.R., N.V., S.S., A.R., A.A., LL.P., CM. F.-M.

Table 1. Primers used for quantitative real time reverse transcription-polymerase chain reaction (RT-qPCR) of genes implicated in regulation of energy homeostasis in the hypothalamus.

Gene name	Forward primer	Reverse primer
POMC	5’-AGAACGCCATCATCAAGAAC-3’	5’-AAGAGGCTAGAGGTCATCAG-3’
AgrP	5’-TGGCCTCAAGAAGACAACTG-3’	5’-CATTGGCTAGGTGCGACTAC-3’

Table 2. Primers used for RT-qPCR of genes involved in the metabolism of BAT.

Gene name	Forward primer	Reverse primer
Prdm16	5’-ATGGGCTTTGACCATACCCG-3’	5’-TCGGCTCCAAAGCTAACAGG-3’
PGC1α	5’-GCACGCAGCCCTATTCATTG-3’	5’-TGAGTCTCGACACGGAGAGT-3’
PPARγ	5’-TGAAAGAAGCGGTGAACCACTG-3’	5’-TGGCATCTCTGTGTCAACCATG-3’
AdipoQ	5’-TCCCAATGTACCCATTCGCT-3’	5’-AGAGTCCCGGAATGTTGCAG-3’
Fabp4	5’-CCTTCAAACTGGGCGTGGAA-3’	5’-CCCCGCCATCTAGGGTTATG-3’
Glut4	5’-GCTCTGACCGATGGGGAACC-3’	5’-AAACTGAAGGGAGCCAAGCA-3’
UCP1	5’-AGTACCCAAGCGTACCAAGC-3’	5’-GACCCGAGTCGCAGAAAGA-3’
Leptin	5’-TTCACACACGCAGTCGGTAT-3’	5’-GAAGTCCAAGCCAGTGACCC-3’
Ob-Rb	5’-CCCCCACTGAAAGACAGCTT-3’	5’-TTCACATCCCCGAAGACTGC-3’

No competing interests reported.

FigureS1.jpg
Figure S1. Log2 fold-change analysis of the ASVs obtained from the TDP-43^A315T mice compared to WT controls in response to FA or O₃ exposures at the end-stage of disease. Bacterial ASVs grouped by genus and coloured by phylum (legends). Log2 fold-change values indicate the strength and direction of the association to WT (<0) and TG (>0) mice. Genera observed in this figure were statistically significantly different between locations at p<0.01 corrected. Abbreviations: WT, Wild-type mice; TG, TDP-43^A315Tmice; FA, filtered air; O₃, ozone.

Download PDF

Editorial decision: Major revision
04 Aug, 2022
Reviews received at journal
21 Jul, 2022
Reviewers agreed at journal
11 Jul, 2022
Reviewers invited by journal
30 May, 2022
Editor assigned by journal
30 May, 2022
Editor invited by journal
16 May, 2022
Submission checks completed at journal
13 May, 2022
First submitted to journal
02 May, 2022

You are reading this latest preprint version

Could ozone become a complementary therapeutic approach to improve metabolic and endocrine response of ALS patients?

Status:

Version 1

Abstract

Figures

Introduction

Methods

Results

Discussion

References

Declarations

Tables

Additional Declarations

Supplementary Files

Status:

Version 1