29 May 2006

Cell culture studies notwithstanding, in the brain, the presynaptic terminals of neurons are thought to be a main source of Aβ, and a controversy is simmering in the field about exactly how it gets there. One view, advanced by Larry Goldstein at University of California, San Diego, proposes that some is generated in transport vesicles traveling down the axon to the nerve terminal. In a nutshell, this research suggests that APP anchors cargo vesicles to the motor protein kinesin, either directly or via linker proteins in a complex, and that one and the same vesicle tends to contain all necessary components of APP γ cleavage. Axonal transport blockages would drive up Aβ generation because the components would dwell together in the vesicles longer (see Stokin and Goldstein, 2006; Stokin et al., 2005; Kamal et al., 2001; Alzforum axonal transport discussion). A collaboration of scientists from several groups has been unable to reproduce some of the data, leaving the issue open (see Lazarov et al., 2005; Zheng comment there, and Goldstein reply).

At the Eibsee, two speakers reported that they tested Goldstein’s hypothesis with various cell-based approaches but found no support for it. Stefan Kins, at Heidelberg University, Germany, was a coauthor of Lazarov et al., 2005, and has since run further experiments. In the paper, the authors had suggested that a reported interaction between APP’s cytoplasmic tail and the light chain of kinesin might be nonspecific. At the conference, Kins added that he searched for APP/kinesin complexes with coimmunoprecipitation of mouse brain lysates to examine the idea that APP might interact indirectly with kinesin through a cytoplasmic linker, but detected no such complexes. These approaches still leave open the possibility of an indirect association between APP and kinesin mediated through scaffolding proteins such as JIP. JIP1 has been suggested to couple APP-containing vesicles to kinesin motors (see, e.g., Matsuda et al., 2003), and could conceivably do so through its interaction with the NPTY internalization motif on APP’s cytoplasmic tail. To test this directly, Kins used siRNA to knock down JIP1 in cultures of cortical primary neurons, and found that the neurons transported most of their APP from the ER out to neurites regardless of how much JIP1 they had available. JIP might link a small pool of APP to kinesin, but for the bulk of APP the kinesin linker remains elusive, Kins said. Its linker need not even interact with its cytoplasmic tail, as an APP mutant lacking that piece still traveled out to neurites unperturbed.

Finally, Kins noted that APP, APLP1, and APLP2 are transported to different sections of the cell membrane. APLP1 resides primarily in dendritic, postsynaptic membranes, and APLP2 in presynaptic areas and growth cones, where each forms dimers with APP (Soba et al., 2006). For this reason, Kins suggested that each family member may travel in its own type of vesicle, using a different linker protein to a different subset of kinesin motor, and that this helps direct their specific route of transport.

Claire Goldsbury, who works with Eva-Maria Mandelkow at the Max-Planck Unit for Structural Molecular Biology in Hamburg, approached the issue from a different angle. She is interested in the relationship between tau and APP in axonal transport. Goldsbury tested the proposal that APP/BACE/γ-secretase travel in the same vesicles and are cleaved en route. First, she asked whether blocking APP transport in cultured neurons would increase Aβ production. She cultured rat primary neurons, transfected them with a Swedish APP-YFP construct, imaged the neurons, and measured how much Aβ they contained and secreted. As expected based on prior work from Mandelkow’s group, Goldsbury saw that overexpressing tau gummed up the microtubules and blocked the transport of APP from the nucleus out to the neuron’s tips. As planned, this lengthened the time APP vesicles spent in transit, yet Aβ levels never increased, indicating the APP does not get cleaved in transit. This was shown directly by double-labeling APP at both ends with YFP and CFP and watching the ratio of colors during vesicle movement. The ration did not change, indicating that APP molecules remained intact (Goldsbury et al., Traffic, 2006, in press).

Next, Goldsbury reported that vesicles containing APP and BACE move quite differently in culture. APP vesicles move swiftly down the neuron’s processes, whereas BACE vesicles tended to “dawdle,” moving forward a bit, then back, and taking lots of stationary breaks in between. Cotransfection of both APP (labeled yellow) and BACE (labeled red) showed little overlap in vesicles. At the Keystone conference earlier this year, Mandelkow already reported that APP and BACE reside in different vesicles (see ARF conference story).—Gabrielle Strobel.