Lucia Notterpek - US grants

Abstract Heritable demyelinating neuropathies, including Charcot-Marie-Tooth disease type 1A (CMT1A), account for a significant portion of peripheral nerve disorders leading to muscle atrophy and functional impairment. Peripheral myelin protein 22 (PMP22) is a hydrophobic integral membrane protein within Schwann cells, whose abnormal expression is associated with the majority of CMT1A cases. In most patients with demyelinating neuropathy, the PMP22 gene is duplicated, while in a smaller fraction of CMT1A and in Dejerine-Sottas Syndrome, single amino acid substitutions in PMP22 are present. Studies of nerve biopsies from neuropathic patients revealed abnormal retention of PMP22 within the Schwann cell cytosol, and the lack of correct myelin protein expression. To gain understanding into the subcellular pathogenesis of PMP22- associated neuropathies, we have characterized the posttranslational processing of PMP22 and found slowed degradation and abnormal intracellular accumulation of the protein within Schwann cells from neuropathic mice, including the point mutant Trembler J and the PMP22 overexpressor models. Since cytosolic PMP22 is only detected in nerve tissue from neuropathic and not normal mice, the abnormal intracellular accumulation of PMP22 likely contributes to the disease pathogenesis. Indeed, upon overwhelming the ubiquitin-proteasome pathway, cytosolic aggregates of PMP22 form and recruit essential Schwann cell molecules, including chaperones and myelin proteins, which alter the protein balance of the cell. Under permissive conditions, Schwann cells isolated from neonatal nerves have the ability to clear these abnormal cytosolic protein aggregates by a mechanism that is assisted by chaperones and autophagy. During the current cycle of this project, we stimulated the chaperone and autophagic responses within samples from Trembler J and PMP22 overexpressor mice, and found that the abnormal cytosolic aggregation of PMP22 can be suppressed and myelin production improved. Furthermore, we have shown that dietary stimulation of these pathways has proven beneficial to these neuropathic mice. The success of these proof-of-principle experiments sets the stage to move forward with specific pharmacologic treatment paradigms in neuropathic mice, and evaluate treatment outcome on neuromuscular function, nerve morphology and associated subcellular mechanisms. The overall aim of this project is to determine if pharmacologic enhancement of chaperones and autophagic protein degradation can slow or halt the progression of the neuropathy in young mice, and to investigate the response of samples from advanced disease stages to this approach. We will use pharmacologically characterized, known small molecules to stimulate the chaperone and autophagy pathways in young neuropathic mice and in ex vivo samples from advanced disease state mice. These studies will determine if improving the subcellular processing of PMP22 by stimulation of protein homeostatic mechanisms within Schwann cells could provide a viable approach for therapy in CMT1A and related neuropathies.

? DESCRIPTION (provided by applicant): While there has been good progress in understanding the genetic causes of hereditary neuropathies, treatment options for affected individuals are limited to the management of symptoms and surgery in severe cases. The majority of these neuropathies are linked with Peripheral Myelin Protein 22 (PMP22) and include Charcot-Marie-Tooth disease type 1A (CMT1A) and Dejerine-Sottas Syndrome (DSS). CMT1A is most frequently associated with duplication of the PMP22 gene, whereas less common forms of CMT1A and DSS are associated with single amino acid substitutions in PMP22. Importantly, both of these neuropathies are genotypically and phenotypically faithfully modeled in mice. The transgenic C22 mouse models CMT1A, while the spontaneous Trembler J (TrJ) represents severe CMT1A and early-onset DSS. Although therapies for CMT1A and DSS neuropathies are not available, the mouse models have been critical in the identification of pathogenic mechanisms and initial preclinical assessments of novel therapies. Studies from my laboratory with these models and human nerve biopsies revealed the presence of both mutant TrJ-PMP22 and overproduced wild type (Wt) PMP22 within the Schwann cell cytoplasm, indicating altered trafficking of this myelin protein. This discovery led us to hypothesize that protein quality contrl mechanisms, including the chaperone pathway could be compromised in the pathogenesis of PMP22-linked neuropathies. Indeed, we demonstrated that enhancing the heat shock response with the compound EC137, an HSP90 inhibitor, dramatically improves myelination in models of CMT1A and DSS neuropathies. The goal of the current proposal is to obtain in vivo efficacy data on two identified FDA-approved enhancers of the chaperone pathway (AUY922 and BIIB021) in C22 and TrJ neuropathic mice. Preliminary studies indicate that both compounds support Schwann cell viability in vitro and enhance chaperone expression when used at 100-250 nM. In myelinating explant cultures from neuropathic mice both BIIB021 and AUY922 enhanced myelin synthesis. Furthermore, AUY922 significantly improved rotarod performance and muscle force contraction in C22 neuropathic mice when administered at 2 mg/kg, twice a week for 18 weeks. To substantiate these preliminary results in the C22 model, and to establish whether this approach is viable for DSS, additional rigorous preclinical studies are necessary. Aims 1-4 of the proposal define rigorous preclinical studies with AUY922 and BIIB021 in both the C22 and TrJ mice. Working with two distinct genetic forms of the disease will determine if targeting the chaperone pathway could provide benefits for a broad group of CMT patients. Since the proposed candidate therapeutics are already FDA-approved for indications in cancer at much higher doses than our studies predict would be needed in neuropathic patients, if positive, the current study will provide the foundation for subsequent clinical studies in humans.