Mitochondrial dysregulation occurs early in ALS motor cortex with TDP-43 pathology and suggests maintaining NAD+ balance as a therapeutic strategy

Mitochondrial defects result in dysregulation of metabolomics and energy homeostasis that are detected in upper motor neurons (UMNs) with TDP-43 pathology, a pathology that is predominantly present in both familial and sporadic cases of amyotrophic lateral sclerosis (ALS). While same mitochondrial problems are present in the UMNs of ALS patients with TDP-43 pathology and UMNs of TDP-43 mouse models, and since pathologies are shared at a cellular level, regardless of species, we first analyzed the metabolite profile of both healthy and diseased motor cortex to investigate whether metabolomic changes occur with respect to TDP-43 pathology. High-performance liquid chromatography, high-resolution mass spectrometry and tandem mass spectrometry (HPLC–MS/MS) for metabolite profiling began to suggest that reduced levels of NAD+ is one of the underlying causes of metabolomic problems. Since nicotinamide mononucleotide (NMN) was reported to restore NAD+ levels, we next investigated whether NMN treatment would improve the health of diseased corticospinal motor neurons (CSMN, a.k.a. UMN in mice). prpTDP-43A315T-UeGFP mice, the CSMN reporter line with TDP-43 pathology, allowed cell-type specific responses of CSMN to NMN treatment to be assessed in vitro. Our results show that metabolomic defects occur early in ALS motor cortex and establishing NAD+ balance could offer therapeutic benefit to UMNs with TDP-43 pathology.

www.nature.com/scientificreports/ motor cortices of 90 days old (P90) prpTDP-43 A315T and healthy wildtype (WT) control mice were dissected out and metabolites were isolated by methanol extraction method followed by high performance mass spectrometry profiling of metabolites (Fig. 1b). We chose P90 as the time of investigation, because this is a symptomatic stage with profound CSMN loss, and it coincides to the disease stage, when patients show symptoms and TDP-43 pathology begins to occur. Analyses of metabolites resulted in the identification of distinct set of metabolites (Supplementary Table 1), some of which are upregulated (Fig. 1c), and some downregulated ( Fig. 1d) with respect to healthy WT control motor cortex. Enrichment analysis was performed using Metaboanalysis software 28 . Heatmap of hierarchical clustering analysis of top 50 highly enriched metabolites revealed differential presence of key metabolites (Fig. 2a). Pyruvate metabolism, tyrosine and nicotinamide metabolism, glycine and serine metabolism, citric acid cycle, glutathione metabolism, and carnitine synthesis were among important metabolite pathways that were different between healthy and diseased motor cortex (Fig. 2b,c). Glutamate is precursor for glutathione biosynthesis. Purine and glutamate metabolism were the top hits, suggesting perhaps ATP and glutathione biology are impacted. (Fig. 2a-c).
The ratio of GSH and its oxidative derivative GSSG directly corelates with cellular oxidative stress 29 . GSH scavange reactive oxygen species (ROS) and converts into GSSH 30 . Thus, higher GSSG/GSH ratio indicates increased oxidative stress. We find that GSSG/GSH ratio is significantly higher in the motor cortex of prpTDP-43 A315T mice even at P90 (WT: 0.02 ± 0.002, n = 4; prpTDP-43 A315T : 0.051 ± 0.001, n = 4, p = 0.0005, Fig. 3c). Thus, these altered metabolomic changes and perturbed balance in the motor cortex of prpTDP-43 A315T mice suggest a deficit in energy homeostasis and increased oxidative stress, events that are associated with problems with mitochondrial integrity and function.   31 . Interestingly, CSMN also retain their eGFP expression in culture. Therefore, UCHL1-eGFP mice allow precise visualization and cellular assessment of UMNs in vitro 31 .
Crossbreeding of UCHL-eGFP with prpTDP-43 A315T mouse model of ALS, generated prpTDP-43 A315T -UeGFP mice (diseased) and WT-UeGFP mice (healthy control). When dissociated cortical cultures are established from these mice, CSMN are distinguished among many different cortical cells and neurons, as they retain their eGFP expression (Fig. 4a,b). Motor cortex is isolated from P3 pups of WT-eGFP and and prpTDP-43 A315T -UeGFP mice, and dissociated cells are plated on glass coverslips to establish motor cortex cultures in vitro 32 . CSMN of WT-UeGFP mice express eGFP and were healthy with large pyramidal cell bodies, prominent apical dendrites and long axons (Fig. 4a). In contrast, CSMN of prpTDP-43 A315T -UeGFP mice failed to extend long axons and lacked branching and arborization, even though they retained eGFP expression (Fig. 4b). There was significant reduction in diseased CSMN axon length (WT: 371.7 ± 10.96 μm, n = 3 mice; prpTDP-43 A315T : 203.9 ± 13.9 μm, n = 3 mice, p = 0.0007, Fig. 4c). Percent distribution of axon length also confirmed that majority of CSMN in prpTDP-43 A315T -UeGFP mice were shorter when compared to the CSMN of WT-UeGFP mice (Fig. 4d). Diseased CSMN had significantly reduced branching and arborization, as revealed by Sholl analyses (Fig. 4e). Since average CSMN axon length and the extent of neuronal branching and arborization enabled quantitative assessment of differences between healthy and diseased CSMN, these outcome measures were utilized to assess CSMN response to treatment with a cellular precision that was not possible before ( Fig. 4c-e).   16,17 . Metabolomics analyses revealed NAD + deficiency in the motor cortex. After having established a robust in vitro model for evaluating CSMN health, we next investigated whether increasing NAD + levels could improve their health.
Since NMN was previously reported to be an NAD + precursor 27 and NMN led to the production of NAD +26 , we tested the hypothesis that NMN treatment would help maintain the NAD + balance in diseased CSMN, and thus improve their health. NMN was added to the culture medium (1 µM) of both healthy (WT-UeGFP) and diseased (prpTDP-43 A315T -UeGFP ) motor cultures in vitro, and CSMN responses to treatment was quantitatively assessed by measuring changes in average axon length and the extent of branching and arborization (Fig. 5). CSMN of prpTDP-43 A315T -UeGFP mice had short axons and did not display branching and arborization (Fig. 5a). However, upon NMN treatment (1 µM), there were significant improvements (Fig. 5b). For example, the average axon length increased in diseased CSMN (prpTDP-43 A315T -UeGFP: 203.9 ± 13.9 μm, n = 3 mice, prpTDP-43 A315T -UeGFP + NMN: 372.8 ± 15.8 μm, n = 3 mice; adjusted p value = 0.0002). In addition, the percent distribution of CSMN based on their axon length also revealed an overall and significant shift towards longer axons, such that about 80% of treated CSMN had axons longer than 300 μm, whereas about 90% of untreated CSMN had maximum axon lenth of 200 μm (Fig. 5f).
NIMN treatment was not toxic to WT-UeGFP CSMN (Fig. 5c,d). Regardless of their NMN treatment, they had similar axon lenth and arborization, and were comparable (WT-eGFP: 371.7 ± 10.96 μm, n = 3 mice; WT-eGFP + NMN: 350.3 ± 18.7 μm, n = 3 mice, adjusted p value = 0.753, Fig. 5e). www.nature.com/scientificreports/ In addition to axon length, diseased CSMN treated with NMN also displayed improved branching and arborization especially within the 50-200 μm radius, as revealed by Sholl analyses (Fig. 5g NMN treatment improved mitochondrial stability. We used dissociated motor cortex cultures isolated from prpTDP-43 A315T -UeGFP mice to investigate the basis of NMN-mediated improvement in CSMN health. To visualize the precise ultrastructural changes that occur at an organnelle level, we utilized correlative light electron microscopy (CLEM), on a total of 4 CSMN (untreated (n = 2) and NMN treated (n = 2)). CSMN were evident with their eGFP expression in vitro (Fig. 6a) and they were prepared for single cell EM analyses to reveal potential changes, especially at the site of mitochondrion. Healthy CSMN from WT-UeGFP mice were used as internal controls with similar treatment paradigms. Most of mitochondria in CSMN of prpTDP-43 A315T -UeGFP mice had defects especially in their inner mitochondrial membrane; cristae structures were disintegrated and were not detectable ( Fig. 6b; CSMN#1: 59%, n = 46 mitochondria; CSMN #2: 60%, n = 40 mitochondria). However, upon NMN treatment, there were significant improvements in the overall architecture of mitochondria and they had a more intact inner membrane with visible cristae structures. CSMN of prpTDP-43 A315T -UeGFP mice had fewer diseased mitochondria ( Fig. 6c; CSMN #3: 27%, n = 55 mitochondria; CSMN # 4: 45%, n = 49 mitochondria). The CLEM results were obtained from 4 independent CSMN isolated from four different mice, revealed the impact of NMN treatment on mitochondrial structure and integrity (Fig. 6b,c).

Discussion
Mitochondrial problems are mainly studied within the context of energy depletion. However, since mitochondria are also a major site for metabolomic regulation, problems or dysregulation of metabolites are now gaining attention, especially for motor neuron diseases, which display mitochondrial problems as one of the early cellular www.nature.com/scientificreports/ pathologies. We previously showed the presence of mitochondrial dysfunction as early as P15 in the motor cortex, but more precisely in the CSMN of TDP-43 A315T mice 17 , one of the best characterized mouse models of TDP-43 pathology 16,19,33,34 . Numerous other studies, utilizing different animal models [35][36][37] or cell lines 38 , all agreed on the presence of mitochondrial problems especially with respect to TDP-43 pathology. Therefore, we investigated potential metabolomic dysregulation that may occur in the motor cortex of ALS with TDP-43 pathology. We have chosen prpTDP-43 A315T mice for our analyses, because this mouse model of TDP-43 pathology was generated based on the A315T mutation detected in the TARDP gene of ALS patients, who had TDP-43 pathology in their cortex 19 , and because the mouse model strictly recapitulated many aspects of the disease, including progressive CSMN degeneration 16 . Numerous other studies have also chosen this mouse model as one of the best characterized and most informative mouse models for TDP-43 pathology [39][40][41][42][43] . More importantly, when detailed cellular analyses were performed with electron microscopy (EM), exact same cellular problems were detected both in the UMNs of ALS patients with TDP-43 pathology and the CSMN of this mouse model of TDP-43 pathology 16 .
TDP-43 pathology was originally defined with the accumulation of proteins that include phosphorylated TDP-43 (pTDP-43) in the cytoplasm of neurons that undergo degeneration 10 , and this pathology was broadly observed in a wide spectrum of ALS cases 44 . However, recent evidence also reveals TDP-43 accumulations in the nucleus, not only in the cytoplasm 11,12 . Especially in the case of some mutated forms of TDP-43, the protein aggregations are vastly ubiquitinated, but the presence of pTDP-43 is not dominant 14,15,45 . Regardless, patients with TDP-43 mutations have TDP-43 pathology, and the mouse model that have A315T mutation has TDP-43 accumulations mostly in the nucleus, have extensive ubiquitinated protein accumulations in the cytoplasm, and based on the antibody used, pTDP-43 are also detected in the cytoplasm 19,46 . Therefore, in line with the current definition of TDP-43 pathology, this mouse model has been considered one of the most reliable models for TDP-43 pathology, especially in the cortex 16,19,[39][40][41][42][43] .
Metabolomic dysregulations in the plasma samples isolated from ALS patients were previously reported [47][48][49][50][51][52][53][54] . These studies showed significant alterations in ceramide levels as well as benzoate, creatine and fatty acid metabolisms and sphingomyelins pathways, with respect to ALS progression 49 , and these changes in metabolite levels correlated with patient's ALS Functional Rating Scale (ALSFRS-R) scores 55 . Likewise, alteration in glucose and lipid metabolisms were reported to contribute to neuronal vulnerability in diseased motor neurons 50 . Components of lipid metabolism such as β-hydroxybutyric acid and medium-chain fatty acids were also found to be dysregulated 56 . www.nature.com/scientificreports/ Metabolomic changes begin to reveal the some of the underlying causes of vulnerability in the motor cortex. In this study, we report that a select set of metabolites, such as phosphoenol pyruvate (PEP), glutathione (GSH), 2-hydroxyglutarate (2-HG), and NAD + , display significant differences between the healthy and diseased motor cortex with TDP-43 pathology at a symptomatic stage.
Oxidative stress is a prominent physiological feature of ALS, and mechanisms responsible for reducing oxidative stress are compromised 22 . In the motor cortex of ALS patients, glutathione (GSH) levels were reported to be reduced when compared to healthy controls 57 . Likewise, in hSOD1 G93A mouse models of ALS, depletion of GSH resulted in degeneration of spinal motor neurons, dysfunctional mitochondria, and neuron loss 58 . GSH is critically important for scavenging reactive oxygen species (ROS) generated by mitochondria 30 , and is also a substrate for antioxidant enzymes that detoxify hydrogen peroxide and lipid peroxides 59 . Importantly, early studies demonstrate a correlation between GSH deficiency and mitochondrial damage 60 . GSH normally exists in its reduced from and is converted into its oxidized form, glutathione disulfide (GSSG), due to oxidative stress. To diminish the effects of oxidative stress, the enzyme glutathione reductase reverts GSSG into GSH. Thus, the ratio of GSH/GSSG is a marker of neuronal oxidative stress and is often used as a marker of cellular toxicity 30 . In resting state, GSSG/GSH ratio were maintained at lower levels, however, in a cell that is experiencing oxidative stress this ratio shifts toward more GSSG 29 . In our study, the motor cortex with TDP-43 pathology displayed increased GSSG/GSH ratio indicating ongoing oxidative stress.
Perturbation of NAD + /NADH ratio indicates energy deficiency and is associated with many diseases, including ALS 22 . NAD + accepts H+ released from various substrates and converts into NADH, releasing ATP. A balanced NAD + /NADH ratio is critical for continuous generation of ATP. Metabolic dysregulation leads into depletion of NAD + and that in turn affects energy homeostasis. NAD + levels can be restored by compounds such as NR (nicotinamide riboside), NAM (nicotinamide), NA (niacin), and NMN (nicotinamide mononucleotide) 61 . These precursors convert into NAD + that is ready to be used in energy generation pathways. Restoration of NAD + levels had been beneficial and neuroprotective in Alzheimer's disease and intracerebral hemorrhage [62][63][64][65] . Moreover, NAD + enhancement has shown attenuation of astrocyte mediated neurotoxicity in vitro 66,67 . Treatment of hSOD1 G93A mouse model of ALS with NR slowed down degeneration of spinal motor neurons and reduced neuroinflammation 68 . In an in vitro model of TDP-43 deficiency in spinal motor neurons, loss of TDP-43 is accompanied by axonal defects. Lack of TDP-43 leads to perturbed transcriptome in axon and dendrites and dysregulated transcripts code for genes that are key component of mRNA translation, cytoskeleton, and mitochondria mediated energy production and metabolism 35 .
Building evidence, including the current study, suggests that mitochondria mediated metabolite disturbances are emerging as prevalent feature in ALS, and restoration of NAD + levels by NAD+ precursor compounds could in fact be a potential therapeutic strategy for improving neuron health. Therefore, we tested whether NMN treatment would help improve the health of CSMN that are diseased due to TDP-43 pathology.
In an effort to investigate whether NMN treatment would have an impact on the health of UMNs diseased with TDP-43 pathology, we utilized dissociated motor cortex cultures established from prpTDP-43 A315T -UeGFP mice, in which CSMN are genetically labeled with eGFP expression that is stable and long-lasting, and when in culture retain their CSMN identity and eGFP expression. These diseased reporter mice were exceptionally valuable for directly assessing the cellular responses of healthy and diseased CSMN to treatments in vitro and in vivo [69][70][71] . Here, we utilized them to investigate their response to NMN treatment. We find that acute and shortterm treatment of diseased CSMN with NMN not only improve their neuronal health, but also help mitochondria to regain the integrity of their inner membrane. Our results reveal early metabolomic dysfunction in the motor cortex of prpTDP-43 A315T mice and begin to suggest that reestablishing the perturbed balance may help UMNs regain their health and stability.
Even though long-term exposure to NMN may not be favorable for axonal stability or neuron health due to its interaction with Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1), and by modulating its activity 72,73 , maintaining NAD + / NADH balance appears to be an important contributor to UMN health, especially the ones that are diseased due to TDP-43 pathology. Therefore, our results suggest the unique importance of understanding the metabolomic problems that occur early in the diseased motor cortex, and encourages for the development of novel therapeutic strategies by reestablishing the perturbed NAD + levels in the brain.

Conclusions
Metabolomic defects begin to occur early in the motor cortex with TDP-43 pathology. There is altered energy balance and reduced levels of ATP. Since metabolomic analyses reveal reduced levels of NAD + as a potential converging outcome of altered metabolomic imbalance, NMN treatment is applied to upregulate NAD + levels and to investigate the relationship between increased NAD + and CSMN health. Our findings, which utilize a novel CSMN reporter line with TDP-43 pathology, not only reveal the presence of metabolomic defects at an early stage of the disease, but also suggest that proper modulation of metabolomic imbalance could offer therapeutic strategies to improve the integrity of mitochondria, and thus the health of diseased UMNs.

Materials and methods
Animals. All animal experiments were performed in compliance with the standards set by National Institutes of Health (NIH) and were reviewed and approved by the Northwestern University Institutional Animal Care and Use committee (IACUC approval number # IS00004844). All experiments were conducted in compliance with the ARRIVE guidelines. The following mouse strains were used in this study: prp-TDP-43 A315T (procured from Jackson Laboratory, stock no. 010700), and UCHL1-eGFP (generated by the Ozdinler Laboratory at Northwestern Targeted Mutagenesis Core Facility, now also available at Jackson Laboratory, stock no. 022476). Gastrointestinal obstruction have been reported in the prp-TDP-43 A315T mouse line 74  www.nature.com/scientificreports/ mice with high fat jelly diet prevented their gastrointestinal problems and their death was reported to be due to TDP-43 pathology mediated degeneration, and not due to gastrointestinal problems 75,76 . In this study, all TDP-43 mouse models were fed with the jelly diet. Hemizygous UCHL1-eGFP females were bred to hemizygous prp-TDP-43 A315T males to generate prp-TDP-43 A315T -UeGFP mice. All the mice used in this study were on C57/BL6 background and were P90 at the time of analyses. Animals were housed at a controlled temperature (23 °C) and controlled light-dark cycle (12-12 h), with free access to water and food 16,17 .
Tissue preparation and metabolite extraction. Mice were deeply anesthetized with intraperitoneal injection of Ketamine (90 mg/kg, and Xylazine (10 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA, USA) 16 . Intact brain was removed and motor cortex was dissected out, weighed and immediately frozen in liquid nitrogen. The tissue was homogenized in 1 ml chilled 80% chromatography grade methanol and vigorously vortexed three times. 200 µL tissue homogenate was mixed into a tube pre-added with 800 µL of ice-cold methanol/water 80% (vol/vol) followed by vortexing rigorously for 1 min, and then centrifuge at ~ 20,160g for 15 min in a refrigerated centrifuge. The metabolite-containing supernatant was separated into a fresh tube and stored at − 80 °C until metabolite profiling was performed 77 .
Method for sample reconstitution after extraction. Extraction solution was dried using SpeedVac.
50% acetonitrile was added to the tube for reconstitution following by vigorously shaking for 30 s. Samples solution was then centrifuged for 15 min @ 20,000g, 4 °C. Supernatant was collected for LCMS analysis 77 .
Hydrophilic metabolites profiling. Comprehensive metabolomics was performed as described earlier 77 .
Briefly, samples were analyzed by High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry and Tandem Mass Spectrometry (HPLC-MS/MS). Specifically, system consisted of a Thermo Q-Exactive in line with an electrospray source and an Ultimate3000 (Thermo) series HPLC consisting of a binary pump, degasser, and auto-sampler outfitted with a Xbridge Amide column (Waters; dimensions of 4.6 mm × 100 mm and a 3.5 µm particle size Motor cortex cultures. P3 motor cortices isolated from WT-UeGFP, and prpTDP-43 A315T -UeGFP mice, dissected, dissociated, and cultured on glass coverslips (4 × 10 4 cells per 18 mm diameter coverslip, Fisherbrand) coated with poly-L-lysine (10 mg/mL, Sigma). Neurons were cultured in serum-free medium [SFM; 0.034 mg/L BSA, 1 mM L-glutamine, 25 U/mL penicillin, 0.025 mg/mL streptomycin, 35 mM glucose, and 0.5% B27 in Neurobasal-A medium (Life Technologies)] in a humidified tissue culture incubator in the presence of 5% CO 2 at 37 °C as previously described 28 . NMN (1 µM) was added at the start of the culture. Cultures were fixed after 3 days in vitro.
Immunocytochemistry. Anti-GFP (1:1000, Abcam) antibody was applied in blocking solution (PBS, 0.05% BSA, 2% FBS, 1% Triton X-100, and 0.1% saponin) for 2 h at room temperature. After incubation, and washes with PBS, appropriate secondary fluorescent antibodies (1:500, AlexaFluor-conjugated, Invitrogen) were added to the blocking solution at room temperature for 2 h in the dark. Nuclei were counterstained with DAPI. www.nature.com/scientificreports/ Data analysis. Measurement of axon length. CSMN from WT-UeGFP, and prp-TDP-43 A315T -UeGFP mice (n = 3) were culture in vitro for 3 days. Immunocytochemistry was performed with eGFP to enhance visualization of GFP+ neurons. Images were acquired using epifluorescent microscope. Neurite length was measure using FIJI ImageJ software. CSMN processes were traced using the Simple Neurite Tracer plugin from FIJI (NIH), which enables semi-autonomous tracing of neuron morphology. The longest neurite was selected to measure the length of the axon in µm.
Sholl analyses. The aggregation of the neurite tracings centered at the soma generates a profile available for Sholl analysis to quantify number of intersections at 5 µm radius intervals for each neuron included in analysis. Minimum 15 neurons per treatment from 3 independent experiments were used for analysis.
Statistics. Statistical analyses were performed using Prism software (GraphPad Software Inc., La Jolla, CA, USA) 16 . For each quantification at least n = 3 mice were used for each genotype and group. D' Agostino and Pearson normality tests were performed on all data sets. Student's t test was used to determine statistical differences between two experimental groups depending on the genotype, experimental conditions and the disease group. One way ANOVA was used for analysis of more than two groups. Data are shown as mean ± SEM of at least three replicates and are representative of three independent experiments unless otherwise stated and statistically significant differences were taken at p < 0.05, and p values are reported in the text.