Project Summary Abstract Amyotrophic lateral sclerosis (ALS) is a fatal disease of widespread motor neuron degeneration in the brain and spinal cord. Progressive impairment of the blood-CNS--barrier (B-CNS-B) represents an additional disease mechanism. Capillary endothelial cell (EC) damage in the CNS has been shown in ALS rodent models and in ALS patients. We demonstrated benefits of intravenously (iv) transplanted human bone marrow-derived endothelial progenitor cells (hBM-EPCs) on functional disease outcomes and motor neurons in an SOD1 mouse model of ALS by attenuating damage to the compromised barrier. Transplanted hBM-EPCs may also exert positive effects by release of extracellular vesicles (EVs) and facilitate restoration of degenerated ECs through delivery of cargo proteins. Apolipoprotein A1 (ApoA1) was determined as the most abundant high-expression protein in EVs and may represent a therapeutically active component for EC-targeted regeneration. We showed that ApoA1 enhanced EC survival in an ALS-like pathologic condition in vitro. The purpose of this project is to determine whether ApoA1 facilitates endothelium homeostasis leading to B-CNS-B repair in ALS. The significant scientific advance of this project is the demonstration that ApoA1 administration elucidates reparative processes in B-CNS-B restoration and promotes motor neuron survival in G93A SOD1 mutant mice. Also, the determination of reparative mechanisms underlying B-CNS-B restoration by assessing ApoA1 effects is the novelty of this project. An important aspect of the proposed study is B-CNS-B restoration in a symptomatic mouse model of ALS with existing barrier damage. Aim 1 will establish therapeutic efficacy of a single iv administration of ApoA1 into symptomatic ALS mice of both genders on B-CNS-B repair by examining behavioral disease outcomes (Aim 1A), functional (Aim 1B) barrier repair, glial cells status (Aim 1C), and motor neuron survival (Aim 1D). Aim 2 will determine the mechanism(s) of ApoA1-mediated vascular repair in symptomatic ALS mice by examining the pathway of this protein on endothelium integrity. This aim will address activity of the ApoA1 protein by impeding the downstream signaling through the cytosolic PI3K/Akt pathway. The effects of inhibiting intracellular signaling will be examined with the same outcomes as described in Aim 1 Sub-aims. Our experimental design to determine the efficacy of ApoA1 administration is a highly translational and innovative mechanism-based approach for repairing the damaged B-CNS-B. Positive project outcomes will evidence the mechanistic role of ApoA1 protein in restoring EC function towards repair of the altered B-CNS-B in ALS. Even if Aim 1 results are negative, probing ApoAI in Aim 2 via inhibitory paradigm will reveal novel ApoAI-based approaches to optimize protein treatment. This study represents a relatively low-risk, but high-reward and innovative protein-mediated therapy for vascular repair in ALS, thereby facilit...