Dr. Butler’s Contributions to Science
Dr. Butler has worked on the development of multifunctional intra-cellular antibodies as immunotherapeutics for the treatment of Parkinson’s disease (PD). The pathology associated with PD includes the progressive loss of dopaminergic neurons in the substantia nigra region of the brain and the development of Lewy body inclusions that are highly immunopositive for alpha-synuclein (α-syn). Misfolding and accumulation of α-syn are known to trigger PD. Human-derived single domain intracellular antibodies (intrabodies) selected against the hydrophobic core of α-syn, were able to partially reduce toxicity and aggregation in a cellular model of PD. Unfortunately, these intrabodies were poorly soluble when expressed in the cytosol. A colleague in his postdoctoral lab, Eric Kvam, developed a series of physical and chemical determinants that can predict intrabody solubility. They hypothesized that the negatively charged proteasomal targeting motif that was used in their Huntington’s disease studies would confer solubility to our anti-synuclein intrabodies. As expected, anti-synuclein intrabody VH-14PEST was able to clear pathological α-synuclein (α-syn). They have extended these findings in vivo in a recent collaboration with Dr. Kordower.
Dr. Butler also worked on the development of multifunctional intra-cellular antibodies as immunotherapeutics for the treatment of Huntington’s disease (HD). HD is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), the formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD, but have been limited by inefficient methods for delivering protein therapeutics to the brain. To overcome this problem, he explored enhanced intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half-life. In ST14A cells, the fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduced huntingtin exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ~80–90% when compared to scFv-C4 alone.
Dr. Butler has explored the role of Id2 phosphorylation at serine 5 in C2C12 myoblasts: myogenesis and apoptosis. Apoptosis has been implicated in mediating the process of muscle loss that occurs during muscle disuse and with aging. Skeletal muscle is a multi-nucleated post-mitotic tissue that relies on satellite cells, adult myogenic stem cells, to maintain a constant nuclear to cytoplasmic ratio. Inhibitor of differentiation protein-2 (Id2) is a dominant negative helix-loop-helix (HLH) protein, and a positive regulator of proliferation, in various cells. In rodent and bird models of unloading-induced atrophy, the cytosolic content of Id2 is increased, and positively correlated to apoptosis. The N-terminal region of Id2 contains a consensus cdk2 phosphorylation sequence SPVR, which may be involved with the induction of apoptosis. Because the role of Id2 phosphorylation at serine 5 in skeletal muscle cells was unknown. The objective of his work was to determine if the phosphorylation of Id2 at serine 5 alters its cellular localization and role in apoptosis in C2C12 myoblasts. Overexpression of wild type Id2 decreased MyoD protein expression, which corresponded to the increased binding of Id2 to basic HLH proteins E47 and E12. Bromodeoxyuridine incorporation was significantly decreased by the overexpression of phospho-ablated Id2 (S5A); conversely, overexpression of wild type Id2 increased cellular proliferation. The subcellular localization of Id2 and S5D were predominantly nuclear compared to S5A. The decreased nuclear localization of S5A, corresponded to a decrease in cellular proliferation, and an increase in apoptosis. These data suggest that unphosphorylated Id2 is primarily localized in the cytosol where it is growth suppressive, and potentially pro-apoptotic.