APO-FERRITIN AS A THERAPEUTIC TREATMENT FOR AMYOTROPHIC LATERAL SCLEROSIS

Background: Iron accumulation and deposition have been reported in patients with amyotrophic lateral sclerosis (ALS). It is known that iron misregulation can induce oxidative stress and that oxidative stress has been implicated in the pathogenesis of the disease. ALS is a multifaceted disease, with lines of research implicating both neurons and glia. Motor neuron loss is associated with elevated pro-inflammatory agents, which are mediators of microgliosis. Microgliosis also has been demonstrated in mutant SOD1 mice where microglial activation precedes observable weakness and motor neuron loss and in studies showing that chronic stimulation of microglia exacerbates the disease. Therapeutic intervention strategies have been disappointing in ALS. Previous work in mouse models of ALS have indicated that iron chelation with a chemical agent extends lifespan. Therefore, we propose the use of apo-ferritin, molecules of the iron-storage protein ferritin that are not iron-laden, as an ionophore to sequester excess iron. The major advantage of apo-ferritin is that it is a naturally occurring protein and is part of the innate mechanism to redistribute iron from the brain to the periphery where it can be stored safely or used to make red blood cells. The ability of cells to sequester iron in ferritin affords cellular protection and has been shown in multiple systems, both in vitro and in vivo.Hypothesis: The overall hypothesis is that infusion of apo-ferritin protein into the brain will provide neuroprotection by limiting the availability of excess iron to catalyze free-radical production.Specific Aims: In this proposal, we propose to infuse apo-ferritin, a naturally occurring iron-specific ionophore, into ALS mouse models to study the effects of binding and redistribution of iron. The first aim is to test the hypothesis that intracerebroventricular infusion of apo-ferritin into the established SOD1G93A mouse model of ALS provides neuroprotection leading to delayed disease onset, slower disease progression, and longer lifespan. The second aim will use a novel mouse line that carries both the SOD1[G93A] and HFE[wt/H67D] mutations. The HFE gene variant is a risk factor for ALS and alters iron metabolism and leads to accelerated disease progression when combined with the SOD1 mutation in mice. Thus, we will test the hypothesis that intracerebroventricular infusion of apo-ferritin into these mice leads to delayed disease onset, slower disease progression, and longer lifespan. Embedded in this aim is the hypothesis that pharmacogenetics will impact treatment strategies in ALS. The third aim is to test the hypothesis that targeting apoferritin directly to microglia is the most effective method for improving clinical and histological outcomes in the ALS mouse model. For this aim, the apo-ferritin will be encapsulated by liposomes that are conjugated with lipopolysaccharide (LPS) for targeting to microglia via the Toll-Like Receptor 4 (TLR4). The mice will receive intracerebroventricular injections of these targeted liposomes.Study Design: Mice from the established SOD1[G93A] mouse model of ALS will be used in Aims 1 and 3. In Aim 2, these SOD1[G93A] mice will be crossbred with mice bearing the H67D genetic polymorphism in the HFE gene to yield SOD1[G93A]/HFE[wt/H67D] mice that have both the SOD1 toxicity attributes and aberrant iron acquisition due to the HFE[wt/H67D] mutation. In Aims 1 and 2, apo-ferritin will be continually infused intracerebroventricularly under the control of an osmotic pump. In Aim 3 we will examine the effectiveness and applicability of targeted liposomal delivery of apo-ferritin to the microglia. We anticipate that this approach will supplement the function of microglia leading to increased clearance of iron from the site of injury.Impact: The work described in this proposal will directly impact the development of ALS therapeutics by providing the naturally occurring protein ferritin to serve as an iron ionophore to redirect iron from accumulating and promoting neurotoxicity in the damaged site. The ultimate goal is to go directly from the proposed preclinical studies to intrathecal infusion of apo-ferritin into human patients. Apo-ferritin is advantageous over chemical chelators that have been shown to have positive impact on the course of the disease because ferritin is a selective, natural ionophore, not an indiscriminate chelator that will deplete iron necessary for normal metabolic reactions as well as excess iron. Moreover, as a natural protein, our approach takes advantage of the body's innate method for recycling iron. In addition, the use of our nanotechnology platform to encapsulate ferritin in nanovesicles and to target the delivery of ferritin to select cells within the central nervous system will be investigated. These targeted liposomes allow for cell-specific delivery of therapeutic agents. There is the potential for tremendous flexibility with targeted liposomes that would allow agents to be delivered to other cell types with different targeting moieties or payload for delivery. Thus, the proposed technology in this application provides a novel treatment strategy and a novel delivery system.