Roy Weller of University of Southampton School of Medicine, England. Weller proposes that sporadic AD develops as the aging brain gradually loses its ability to clear Aβ along perivascular fluid drainage pathways.
Several mechanisms act in parallel to rid the brain of excess Aβ: Carrier proteins mediate its uptake into blood (see ARF Live Discussion), microglia and astrocytes ingest it, and enzymes such as neprilysin, IDE and others degrade Aβ locally. In fact, Hasan Mojaheri of the University of Zurich, presented experiments extending Takaomi Saido’s original work with neprilysin. Mojaheri told the audience that mice upregulate neprilysin in response to challenge with Aβ, but that the enzyme can only degrade non-aggregated forms of the peptide, and neprilysin induction makes a difference only in young, pre-symptomatic mice.
But back to Weller, who studies a fourth clearance mechanism, namely transport along blood vessel walls. Vascular amyloid usually accompanies AD, and the anatomical pattern of amyloid within artery and capillary walls in the brain suggests that Aβ deposition may begin with its entrapment in the narrow drainage pathways there, Weller hypothesizes. To visualize these pathways, the researchers injected soluble dextran tracers roughly the size of Aβ into adult mouse brain and then colocalized them with laminin to the basement membrane of capillary and artery walls. The resulting pattern of deposition and drainage models Aβ drainage, Weller said.
Cerebrovascular disease induces an age-related congestion of interstitial flow of Ab because arteriosclerosis reduces the amplitude of arterial pulsations, which generate the putative motive force for the perivascular drainage of Ab, Weller said. Evidence for this is that thrombotic occlusion of cortical arteries results in accumulation of Ab in vessel walls upstream of the thrombus, he added. Weller and James Nicoll contributed a synopsis of this hypothesis to a recent on vascular factors in AD, see Alzforum discussion. See also Weller and Nicoll, 2003 and Preston et al., 2003).
How to Study a Folding Protein? Grab It, Watch It.
If indeed the misfolding of an accumulating protein marks a crucible for pathogenesis, how is one to study it? On this question, Harald Janovjak stood in for principal investigator Daniel Mueller, both at the Max-Planck-Institute of Molecular Cell Biology and Genetics in Dresden, Germany, to highlight what the developing field of atomic force microscopy could do for neurodegeneration researchers.
The Mueller lab has spent the last few years working out protein imaging and structure analysis methods with the atomic force microscope (AFM), and some may just be ready for prime time.
This desktop instrument offers two general advantages. First, that it allows one to image proteins in relatively native conditions, such as in physiologic solutions or embedded in artificial membranes, and do so at a handsome resolution of up to 0.5 nm. Secondly, it’s possible to physically grab a single folded protein, pull it taut, release it, and measure the tiny, sequential forces with which it resists the unfolding and then snaps back into shape. These force traces then enable the scientist to make inferences about structural characteristics of the protein.
Having a crystal or NMR solution structure of the protein at hand is helpful but not necessary, Janovjak said. In fact, the AFM can predict the outline of a protein’s secondary structure, and such predictions have been confirmed by atomic structures elucidated later on.
Janovjak illustrated some new AFM capabilities. For example, he showed images of gap junctions that visualized individual connexon units. Moreover, the scientists imaged a conformational change as the gap junctions closed in response to an increase in calcium (Muller et al., 2002).
Furthermore, the AFM allows the researcher to image intramolecular forces. For example, consider pull-and-release experiments on bacteriorhodopsin, a complex, well-studied intramembrane protein. The tip of the AFM stylus is attached to a flexible cantilever. Mueller and colleagues retracted the cantilever, measured the force-extension spectra, and correlated these spectra to the extraction of that single protein. This analysis told them that pairs of two transmembrane helices unfold pairwise and in this way act as one structural unit. The German scientists also detected particular secondary structure elements, and studied differences in how they unfold and then refold into their configuration when released (Muller et al., 2002). Finally, Janovjak showed examples of how tinkering with the experimental conditions—temperature, pH, ion concentration—can affect the folding of a given protein (Janovjak et al., 2003).
This ability to modify fairly native conditions experimentally is but one reason why the AFM might lend itself to the study of Alzheimer’s and prion diseases, Janovjak suggested. Another is that many of the proteins of interest are located in membranes. In AD, the role of cholesterol and its metabolites, as well as their possible effect on APP processing, has become a research priority. What’s more, APP proteolysis may well occur in lipid rafts, and their composition is thought to change with age. Could one, for example, insert APP into lipid membranes of different composition and see how such environmental changes affect its folding? The Muller lab invites suggestions for research collaborations in the area of neurodegenerative diseases at (Mueller@mpi-cbg.de).
Retro Traffic Reigns in Protein Renegades?
Weller and Jucker study events occurring after Aβ has left the neurons. But some protein misfolding and oligomerization may well begin inside neurons, see for example Takahashi et al., 2004). How does the neuron try to defuse this ticking time bomb of misfolding and aggregating proteins? This question got attention from Wiep Scheper at the Academic Medical Center in Amsterdam, the Netherlands. Scheper studies Rab6A, a small GTPase that functions in the trafficking of proteins through the ER/Golgi membrane network. Previous work on APP trafficking and presenilin had pointed Scheper’s attention to Rab6, and in St. Moritz she reported data on more direct experiments testing its possible role in AD. Working with Frank Baas and colleagues, Scheper found that, in AD post-mortem brains, Rab6A is upregulated and predominantly expressed in pyramidal neurons of the temporal cortex and hippocampus.
Five years ago, German researchers had found that Rab6 travels along a then-newly discovered retrograde transport route from the Golgi back to the ER (White et al., 1999, Girod et al., 1999). Normally, misfolded proteins are caught in the act in the ER, expelled through the Sec61 channel and fed to the proteasome for degradation. Yet some proteins elude this quality control, and make off into the Golgi on their way to the plasma membrane. Scheper suspects that this Rab6-dependent retro route serves to return such escapees to the ER. She cited prior indications that Rab6 might function in quality control (Luo and Gallwitz, 2003, including of the PrP protein (Beranger et al., 2002). Scheper studies Rab6A in relation to the ER chaperone BiP and reported a close correlation between the two in human brain, possibly because Rab6 induces BiP. This work is ongoing, but Scheper suggested that aging, mutations, or proteasome inhibition may overwhelm the cell’s capacity to retrieve and degrade misfolded proteins. —Gabrielle Strobel