Merely overexpressing α-synuclein, even truncated or mutant forms, fails to produce PD-like Lewy body inclusions in cells. However, it is relatively easy to make synuclein fibrils from large batches of purified recombinant protein. Years ago, Lee wondered whether supplying some of that pre-formed material could jumpstart the aggregation process in cells. “It’s templated recruitment. You put in some seeds to recruit normal protein to adopt an abnormal conformation. That’s the overarching theory behind what we were trying to do,” Lee said. The UPenn researchers could indeed use pre-formed fibrils to drive formation of Lewy body-like aggregates in cells (Luk et al., 2009), but the system was extremely artificial—the host was a non-neuronal cell line engineered to express whopping levels of α-synuclein.

In the current study, first author Laura Volpicelli-Daley and colleagues tried something similar in neurons. They generated fibrils from full-length recombinant human α-synuclein and added these, or control saline, to cultures of primary hippocampal neurons from wild-type mice. Two weeks later, immunostaining and biochemistry showed that endogenous α-synuclein in fibril-treated neurons formed insoluble Lewy body-like inclusions with characteristic post-translational modifications, i.e., hyperphosphorylation and ubiquitination. The researchers found that small aggregates formed only in neurites at first, then accumulated in axons, and finally, in cell bodies. This process took one to two weeks and went faster in older neuron cultures, which have accumulated more endogenous α-synuclein.

The aggregates seemed to have functional ramifications. Relative to controls, neurons exposed to pre-formed fibrils had less of key proteins (Snap25, VAMP2, CSPα, synapsin II) in presynaptic areas, as judged by immunoblotting and immunofluorescence. Using calcium imaging to track patterns of the networks neurons established in cultures, the researchers found network changes that reflect compromised synaptic activity. The fibril-treated neurons died faster as well, with 40 percent fewer surviving to day 14 of treatment than control cells.

The researchers recapitulated these findings in primary cultures of cortical and midbrain dopaminergic neurons, and in neurons from other mouse strains as well as from rats. The inclusions and functional deficits failed to develop in neurons from α-synuclein knockout mice, indicating that endogenous α-synuclein did, in fact, form the insoluble intracellular deposits.

“This is a nice expansion of prior work on α-synuclein transmission,” Seung-Jae Lee of Konkuk University, Seoul, Korea, wrote in an e-mail to ARF. “The most important contribution of the work, to me, is the description of the temporal progression of aggregates from the axons to the somatodendrites, and the association of aggregate formation with impaired neural activity and connectivity.”

However, because the study used artificial fibrils rather than neuron-released α-synuclein, “the physiological relevance of the results remains to be determined,” wrote Lee, who has done cell biology studies characterizing the endocytosis of α-synuclein fibrils and oligomers (Lee et al., 2008), and the release of α-synuclein by neurons (Lee et al., 2005; Jang et al., 2010). Lee noted that while prior research has shown smaller species of α-synuclein, namely soluble oligomers and monomers, getting released by neurons and wreaking havoc on neighboring cells (see Danzer et al., 2009; Danzer et al., 2011; Desplats et al., 2009; Emmanouilidou et al., 2010), there is no evidence to date demonstrating this sort of propagation for fibrils.

Though the current study moves toward physiological relevance in its use of primary neurons expressing normal amounts of α-synuclein, the fibrils used to seed aggregation in these cells were made in vitro from recombinant human α-synuclein. Researchers studying PD and other neurodegenerative diseases grapple with how to study physiologically relevant species of protein aggregates (see upcoming ARF Webinar).

To Virginia Lee, the most important aspect of the present work is “the production of a simple and powerful system for studies of sporadic PD.” Her lab is adapting the new model for high-throughput screening for genes or small molecules that eliminate or modify the pathology.—Esther Landhuis.

Reference:
Volpicelli-Daley LA, Luk KC, Patel TP, Tanik SA, Riddle DM, Stieber A, Meaney DF, Trojanowski JQ, Lee VM. Exogenous Alpha-Synuclein Fibrils Induce Lewy Body Pathology Leading to Synaptic Dysfunction and Neuron Death. Neuron. 6 Oct 2011;72:57-71. Abstract