Combining their individual fortés, the laboratories of Dave Holtzman at Washington University, St. Louis, and Brian Bacskai and Brad Hyman at Massachusetts General Hospital, Charlestown, treat us to the sight of neurons being treated before our eyes. In a study published online January 20, 2005, in the Journal of Clinical Investigation, fluorescently labeled dystrophic neurites in the cortex of PDAPP mice appear to shrink or disappear just days after Aβ antibodies are applied to the cortical surface.
For several years, Bacskai and Hyman have been wowing people with their "windows on the brain," openings in the skulls of mice through which they apply drugs to the cortex and then observe the results with two-photon microscopy. They have already visualized how plaques grow in AD transgenic mice, and have shown that amyloid plaques are cleared days after the application of Aβ antibodies to the cortical surface (Bacskai et al., 2001; Bacskai et al., 2002). Bacskai and Hyman have also assessed neuritic pathology in PDAPP mice, finding postmortem evidence that the Aβ antibodies can rapidly reduce neuritic pathology (Lombardo et al., 2003), though the researchers were unable to visualize the neurites through the windows.
Enter the glowing yellow mouse from Holtzman's lab. Holtzman and colleagues have crossed PDAPP mice with transgenic mice that express yellow fluorescent protein (YFP) throughout the neuronal cytosol, reaching into distal neurite tips. With these mice, the researchers have found evidence that neuritic dystrophy is much more extensive than was previously thought (Brendza et al., 2003).
Neurite stakeout detects disappearing pathology
The image on the left shows yellow fluorescent protein (YFP)-labeled neurites—imaged through a cranial window with two-photon microscopy—in a living PDAPP;thy-1:YFP double-transgenic mouse. Enlarged, bulbous, dystrophic neurites surrounding an amyloid plaque (not visualized in this image) can be seen. On the right, the same plaque three days after the cortical surface was treated with 10D5 anti-Aβ antibody. There is a reduction in YFP-labeled dystrophic neurites, and the arrows show two dystrophic areas that are present at day 0 and absent at day 3. (Scale bar = 10μm) [images courtesy of Bob Brendza, Washington University]
Bob Brendza in Holtzman's lab led the current collaboration, which also included Bill Klunk and Chet Mathis at the University of Pittsburgh, and Steve Paul and Kelly Bales at Eli Lilly. First, the researchers just watched, and noted that the numbers of dystrophic neurites surrounding individual plaques did not change over a period of three days. Similarly, individual dystrophic neurites under scrutiny were stable during this period. However, when they applied 10D5 Aβ antibodies to the cortical surface, the researchers observed significant morphological changes within only three days. They witnessed a substantial reduction, or disappearance altogether, of smaller dystrophic neurites, as well as the reduction or elimination of dystrophic regions on otherwise normal-looking dendrites or axons. Some of these neurites were followed for up to a week, with no return of the pathology. At the same time, however, larger dystrophic neurites appeared impervious to the antibody treatment, leading the authors to suggest that additional doses, or time, might be needed to get a more widespread benefit.
This method did not allow the researchers to quantify changes in amyloid burden. In a separate series of experiments, they found that the passive immunization reduced total Aβ and thioflavin S-positive plaques but had no effect on levels of PBS-soluble Aβ. This, Brendza and colleagues suggest, would lead to the conclusion that fibrillar Aβ is what the antibodies are targeting.
"Based on these data, it appears that axonal and dendritic structural damage associated with amyloid deposits is not permanent and is, at least in part, reversible over a relatively short time frame. Further, the specificity of the antibody shows that Aβ itself causes these reversible structural changes," write the authors. —Hakon Heimer
Q&A with Bob Brendza.
Q: How do your results extend those of your collaborators at MGH, who looked at effects on neurites following cortical administration of Aβ antibodies about a year and a half ago?
A: The Lombardo et al. paper was a postmortem study. It reported the very important finding that mice receiving antibody treatment had a significant reduction in abnormal neurite geometry that correlated with Aβ clearance. They didn't see much of a change in neuritic dystrophy when assessing it with APP immunoreactivity. YFP expression is a very sensitive marker for assessing neuritic dystrophy and is actually more sensitive than silver or APP staining in detecting dystrophic areas (especially smaller dystrophic swellings). Our 2003 J. Comp. Neurology paper actually compared the different methods. So, the three major differences between our JCI paper and the Lombardo et al. paper was that our study was in vivo, we tracked neuritic dystrophy in individual neuritic plaques, and we used a more sensitive method to monitor neuritic dystrophy and neurite morphology.
The other advantages of using YFP are that YFP requires no exogenous cofactors or substrates to fluoresce and diffuses freely throughout the cytoplasm; thus, it fluorescently labels all neuronal processes including axons, dendrites, and dendritic spines. These features of YFP allow neurites to be easily visualized in live tissue without any potentially disruptive manipulations. Since cytoplasmic YFP labels all neuronal processes indiscriminately, the morphology and dynamics of normal and dystrophic neurites of labeled neurons in PDAPP; YFP double Tg mice can be observed without concerns regarding tissue penetration or alteration of target molecules by the AD pathogenic environment, which are potential problems associated with the use of traditional histological probes.