Neuroprotective Effects of RNS60 in TDP ‐43 Pathology‐Associated Amyotrophic Lateral Sclerosis

Introduction TDP‐43 pathology is broadly observed in the cerebral cortex of patients with amyotrophic lateral sclerosis (ALS). RNS60, an experimental treatment for acute ischemic stroke and ALS, enhanced mitochondrial biogenesis and function in other preclinical models. We investigated whether RNS60 improved mitochondrial stability and upper motor neuron (UMN) health in a TDP‐43 mouse model of ALS. Methods prpTDP‐43 A315T ‐UeGFP mice, in which UMNs express green fluorescent protein (eGFP), and WT‐UeGFP mice were treated with RNS60 or placebo intraperitoneally every other day from post‐natal day (P) 30 until P90. Astrogliosis and microgliosis in brain and spinal cord were quantified by immunocytochemistry. Mitochondrial ultrastructure was studied via electron microscopy, and mitochondrial function was assessed using flow cytometry. Neuromuscular junction (NMJ) integrity was assessed in gastrocnemius, tibialis, and diaphragm muscles. Results RNS60 treatment reduced defective mitochondria in UMNs (prpTDP‐43 A315T + vehicle: 53.2% ± 0.71%; prpTDP‐43 A315T + RNS60: 19.6% ± 1.4%, p = 0.0001) and spinal motor neurons (prpTDP‐43 A315T + vehicle: 70.1% ± 0.4.48%; prpTDP‐43 A315T + RNS60: 33.5% ± 4.43%, p = 0.001). It increased mitochondrial membrane polarization (prpTDP‐43 A315T ‐UeGFP + vehicle: 7184 ± 1689 mean intensity; prpTDP‐43 A315T ‐UeGFP+RNS60: 22120 ± 4818 mean intensity, p = 0.032), reduced the extent of astrogliosis and microgliosis in motor cortex and spinal cord, protected UMNs compared to placebo, and enhanced the proportion of intact NMJs in leg and diaphragm muscles (prpTDP‐43 A315T ‐UeGFP + vehicle: 29.6% ± 3.6%; prpTDP‐43 A315T ‐UeGFP + RNS60: 64.3% ± 4.4%, p = 0.0002). Discussion These results suggest that RNS60 treatment promotes motor neuron health in ALS by protecting mitochondrial structure and function, preserving NMJ integrity, and reducing gliosis.