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

Abstract

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-43^A315T-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.

Figure 1

Metabolite profiling of motor cortex revealed dysregulated metabolite due to TDP-43 pathology. (a) representative electron microscopy images of mitochondria in WT healthy CSMN and in CSMN that are diseased due to TDP-43 pathology (b) schematic representation of workflow of study design. (c) list of selected upregulated metabolites. (d) List of selected downregulated metabolites. Scale bar = 200 nm.

Figure 2

Enrichment analysis of metabolites. (a) Hierarchical clustering heat map of top 50 differentially present metabolites, (b) overview of enriched metabolites, and (c) analysis of enriched metabolites.

Figure 3

The ratio of key metabolites that are involved in energy homeostasis and oxidative stress are dysregulated in the motor cortex that is diseased due to TDP-43 pathology. (a) ratio of ATP/ADP; (b) ratio of NAD⁺/NADH and (c) ratio of GSSG/GSH.

Figure 4

CSMN that express mutated from of human TDP-43^A315T extend shorter neurites in vitro. (a) representative images of CSMN from WT-UeGFP mouse; (b) representative images of CSMN from prpTDP-43^A315T-UeGFP mouse model of ALS; (c) bar graph representation of average neurite length; (d) percent distribution of axon length; and (e) Sholl analysis of arborization of neurites. Scale bar = 20 µm.

Figure 5

Nicotinamide mononucleotide (NMN) improves the health of CSMN that become diseased due to TDP-43 pathology. (a) a representative image of CSMN from prpTDP-43^A315T-UeGFP mouse; (b) a representative image of CSMN from prpTDP-43^A315T-UeGFP mouse treated with 1 µM NMN; (c) a representative image of CSMN from WT-UeGFP mouse; (d) a representative image of CSMN from WT-UeGFP mouse treated with NMN. (e) Bar graph representation of average neurite length; (f) Percent distribution of axon length; and (g) Sholl analysis of arborization of neurites. Scale bar = 20 µm.

Figure 6

Correlative light electron microscopy shows improvement of mitochondrial ultrastructure upon NMN treatment. (a) a representative image of motor cortex neurons cultured on a gridded glass bottom 35 mm dish; boxed area enlarged to highlight presence of GFP expressing CSMN; (b) representative electron micrograph of an individual CSMN from prpTDP-43^A315T-UeGFP mouse showing mitochondrial ultrastructural defects, images of mitochondrion are enlarged to right; and (c) representative electron micrograph of an individual CSMN from prpTDP-43^A315T-UeGFP treated with 1 µM NMN showing improved mitochondrial ultrastructure, images of mitochondrion are enlarged to right. Scale bar = 1 µm.