Taking away a samurai’s sword may prevent destruction, but it also limits his prowess. That’s one way to look at a new study on presenilins. These mighty enzymes deliver the final cut to unleash the Aβ peptides that form the amyloid plaques that gum up the brains of Alzheimer disease patients. Yet curbing their power seems to cut off other vital functions—namely, promoting learning and memory and helping neurons survive to old age—according to recent work by Jie Shen, Brigham and Women’s Hospital, Boston, and colleagues. Reported in this week’s Journal of Neuroscience, the findings also underscore the challenges of developing inhibitors of γ-secretase, the protein unit within which presenilins operate, as possible AD treatments.
The view of presenilins has widened since the membrane proteins were pigeonholed as villains on the business end of γ-secretase. Recent work has placed presenilins in a different light—as guardians of neuronal health. That research suggested new roles for presenilins in maintaining proper intracellular calcium signaling. Calcium dysregulation has garnered increased attention for its possible role in AD and other neurodegenerative disorders (for review, see Bezprozvanny, 2009). Last summer, scientists reported that presenilins interact with and activate sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump proteins, and that SERCA activity seems to influence Aβ production (Green et al., 2008 and ARF related news story). Other work has suggested that presenilins function as ER calcium leak channels (Tu et al., 2006 and ARF related news story), and that several familial AD mutations specifically disrupt this Ca2+ leak function of presenilin 1 (Nelson et al., 2007). Some of these activities could be independent of presenilins’ γ-secretase activity.
Previous work from Shen’s group has reinforced the idea that presenilins are indispensable for cognition and neuronal integrity. Her team conditionally knocked out the two presenilin genes (PS1 and PS2) in mouse postnatal forebrain, and saw a progressive increase in memory impairment and neurodegeneration as these mice got older (Saura et al., 2004 and ARF related news story). But this study left the researchers pondering what, mechanistically, was responsible for presenilin’s essential roles in memory and neuronal survival. The current study tackles one aspect of this issue—whether these functions of presenilin depend on its γ-secretase-dependent or -independent activities.
Led by Shen and first author Katsuhiko Tabuchi, the researchers chose a similar knockout strategy. “Rather than chase after 20 published substrates, many of which are probably not physiological, we decided to first do a genetic dissection because it would be very conclusive,” Shen told ARF. Using the same promoter that drove postnatal forebrain-specific inactivation of PS1/PS2 in their previous study, her team engineered a conditional knockout (cKO) mouse whose cortical excitatory neurons lacked expression of nicastrin, one of three other γ-secretase subunits besides presenilin. (The remaining two are presenilin enhancer 2 [Pen-2] and anterior pharynx defective 1 [Aph-1].)
By and large, the nicastrin cKO mice reproduced the striking phenotype of the lab’s PS1/PS2 animals. At two months of age, both mouse strains had sharply reduced PS1 and Pen-2 levels, though Aph-1 expression had hardly changed. In addition, cortical lysates from each mouse line had normal levels of full-length amyloid precursor protein (APP) but whopping amounts of the C-terminal APP fragment, as predicted by the reduced γ-secretase-mediated cleavage of APP in those cells. These data suggest that the absence of nicastrin destabilizes most components of the γ-secretase complex, compromises its activity, and reduces its APP-cleaving ability.
The biochemical changes in the nicastrin cKO mice were associated with learning and memory deficits that showed up as early as two to three months of age, when the mice still lacked detectable changes in brain volume or cortical neuron numbers relative to the control group. At six to nine months, the nicastrin cKO animals’ performance on memory tests continued to plummet, and the researchers found evidence of neurodegeneration (white and gray matter loss in Nissl-stained sagittal brain sections) and synapse dysfunction (decreased MAP2 and synaptophysin immunoreactivity in neocortex and hippocampus). Neurodegeneration often comes with heightened inflammation, and signs of this also appeared in the nicastrin cKO mice. In Western and immunostaining analyses, their cortical lysates had higher levels of the reactive astrocyte marker GFAP (glial fibrillary acidic protein), as well as elevated Iba-1 (ionized calcium-binding adapter molecule 1), a protein specifically expressed in brain microglia, compared with control mice. The nicastrin cKO animals also had increased apoptosis (greater numbers of TUNEL-positive and caspase-3-positive cells) in the neocortex—measurable at two months of age, more severe at six months—and higher levels of hyperphosphorylated tau at six months.
All together, the data convincingly argue that presenilins support memory formation and neuronal survival through γ-secretase-dependent mechanisms, Shen said. The findings may also offer insight into ongoing discussions about the dearth of neurodegeneration in many APP-overexpressing mouse strains. “People have argued in the past that the reason APP transgenic mice don't have significant neurodegeneration is because the mice are resistant to neuronal loss,” Shen said. However, the new study shows that “the mouse brain is not very resistant to neurodegeneration if you have targeted the right gene. It highlights the importance of presenilins and nicastrin in neuronal survival,” she said. Furthermore, the authors write that their “data—including the increase in tau phosphorylation, and the widespread apoptosis—are consistent with the notion that the neurodegeneration induced by inactivation of γ-secretase subunits resembles the neurodegeneration observed in AD.”
Other scientists are not as convinced that the γ-secretase models exhibit AD-like neurodegenerative pathways. “Accumulation of amyloid has been postulated to play a critical role in AD pathogenesis and abnormal neuronal Ca2+ signaling has also been implicated as one of the pathogenic pathways involved in AD,” wrote Ilya Bezprozvanny of University of Texas Southwestern Medical Center, Dallas, in an e-mail to ARF. He noted that PS1/PS2 conditional double knockout and nicastrin conditional knockout mice do not produce amyloid, and the current study does not reveal Ca2+ signaling dysfunction in the nicastrin cKO mice. “Thus, it remains an open question whether these γ-secretase knockout mice are a faithful model for the neuronal cell death in AD,” he wrote (see full comment below).
Bart De Strooper, at K.U. Leuven in Belgium, and Bezprozvanny raised the possibility that presenilin expression levels in aging neurons of the nicastrin cKO mice could be reduced enough to affect non-proteolytic functions of presenilin. De Strooper pointed out, though, that “the Ca2+ leakage function is maintained in Aph-1 deficient cells (Tu et al., 2006), indicating that presenilin can exert that function outside of the [γ-secretase] complex.” (See full comment below.)
Recent work from De Strooper’s lab added to the emerging picture of γ-secretase as a multi-functional complex. Published in Science several months ago, that study showed that selectively knocking out the B/C isoforms of the Aph-1 component of γ-secretase rescues cognitive defects and neurodegeneration in an AD mouse model (Serneels et al., 2009 and ARF related news story). The data suggest that selective inhibition of γ-secretase components may produce the desired therapeutic benefits without the side effects from inactivating the entire complex.
Scientists seem to agree that unraveling the mechanisms underlying presenilin-mediated neuronal survival remains a top priority for future studies. “The challenge will be now to determine which signaling pathways, downstream from γ-secretase, are involved in neuronal death in the aging brain,” wrote Philippe Marambaud, Feinstein Institute for Medical Research, Manhasset, New York, in an e-mail to ARF (see full comment below). He gives a vote of confidence to signal transduction by cadherins—adhesion proteins that are expressed at mature synapses, are critical for synaptic plasticity, and are cleaved by γ-secretase in neurons upon NMDA receptor stimulation (Marambaud et al., 2003).—Esther Landhuis
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