Toxic Elk-1 Phosphoform Found in AD, HD, Lewy Body Disease Inclusions

12 February 2010. The protein aggregates that brew discord in several neurodegenerative diseases may have some troublemakers in common. One could be Elk-1, a transcription factor that has been shown to trigger cell death when expressed within neuronal dendrites. Reporting in this month’s PLoS ONE, James Eberwine of the University of Pennsylvania, Philadelphia, and colleagues determined that Elk-1 initiates neuronal death when phosphorylated at a particular site. Furthermore, the researchers found this Elk-1 phosphoform associated with inclusions in Alzheimer, Huntington, and Lewy body disease postmortem tissue.

Part of the ternary complex factor (TCF) family of transcription factors, brain Elk-1 appears in numerous areas such as the cortex, hippocampus, striatum, and cerebellum. Curiously, the protein not only shows up in the nucleus but also in other parts of the neuron, including dendrites. "The real question for us was, why would you have a transcription factor present in the dendrites of the neuron rather than the cell body?" he told ARF. To probe the significance of this localization, his lab developed a phototransfection method that allowed them to introduce tiny amounts of Elk-1 mRNA into specific regions of primary rat neurons (Barrett et al., 2006). "We found that we could put as few as 10 copies of the mRNA in the dendrite, and it would cause cell death," Eberwine said. Those studies revealed that Elk-1 moves from the dendrites into the nucleus as part of the cell death process. Eberwine and colleagues had also shown that Elk-1 in rat brain associates with the mitochondrial permeability transition pore complex in both cell bodies and dendrites, and that pro-apoptotic stimuli enhance this association (Barrett et al., 2006).

To tease out how Elk-1 is regulated, joint first authors Anup Sharma, Linda Callahan, Jai-Yoon Sui, and colleagues generated a series of Elk-1 mRNAs with mutations at known post-translational modification sites. The researchers transfected the mutants into dendrites of rat hippocampal neurons and assessed cell viability six hours later. Most brought on death just as well as wild-type Elk-1, suggesting that those sites are not critical for apoptosis. However, a few mutants were inactive, with effects comparable to mock transfection. These mutants left more than 60 percent of hippocampal neurons intact, about double the viability rate for cells injected with wild-type Elk-1 mRNA. Transfection with the T417A mutant most consistently left the neurons unharmed, demonstrating that phosphorylation at the 417 site is essential for Elk-1-mediated neuronal death.

Given that phosphoproteins appear in pathological protein aggregates of various neurodegenerative diseases, Eberwine’s team looked for Elk-1 in postmortem brain tissue from patients with Lewy body disease (LBD), HD, or AD, and from age-matched healthy seniors. The samples were stained with antibodies to T417+ Elk-1 (i.e., the neurotoxic Elk-1 phosphoform) or, as negative controls, antisera recognizing S383+ Elk-1 (a mutant with little effect on cell death) or T368+ Elk-1 (a phosphoform with unknown functional relevance). The researchers found T417+ Elk-1, but not the other two phosphoforms, colocalizing with Lewy body and pale body inclusions in substantia nigra neurons of LBD tissue. The T417+ phosphoform also showed up in ubiquitin-positive inclusions in HD tissue, and in plaque and tangle inclusions in AD hippocampal samples. "The 417 staining was as strong or stronger than AT8 (which recognizes hyperphosphorylated tau)," Eberwine said. "The colocalization of T417+ Elk-1 with multiple neuronal inclusions suggests a common mechanism of pathogenesis and neuronal loss among distinct neurodegenerative diseases," the authors write. They note that the inclusions could serve as subcellular pathogenic sites that enhance the local enrichment and activation of T417+ Elk-1, which could then dissociate from these sites and initiate cell death.

Potential connections with PD and AD have surfaced in prior studies of Elk-1. The protein forms a complex with α-synuclein in co-transfected cells (Iwata et al., 2001), and seems to inhibit transcription of presenilin 1 (Pastorcic and Das, 2003).

Eberwine and colleagues have taken several directions to extend the current findings. To explore Elk-1’s clinical relevance, they are analyzing additional disease samples and hope to cross Elk-1 knockout mice with various neurodegenerative disease models. On the mechanistic side, Eberwine’s team is working to develop an in vivo assay to identify the kinase that regulates Elk-1’s post-translational state. And from chromatin immunoprecipitation studies done in collaboration with Shelley Russek at Boston University, the scientists have preliminary data identifying putative Elk-1 target genes.—Esther Landhuis.

Sharma A, Callahan LM, Sul JY, Kim TK, Barrett L, Kim M, Powers JM, Federoff H, Eberwine J. A Neurotoxic Phosphoform of Elk-1 Associates with Inclusions from Multiple Neurodegenerative Diseases. PLoS ONE. Feb 2010;5(2):e9002. Abstract

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