The reelin protein plays a starring role in neural development, and new findings suggest the neuromodulator does an important job in older animals, too. Researchers led by Joachim Herz at the University of Texas Southwestern Medical Center in Dallas generated mice that lack reelin in the brain when they are adults. As reported July 7 in Science Signaling, these mice appeared normal—unless crossed with a mouse strain that overproduces Aβ. In the face of amyloid toxicity, reelin-deficient mice had accelerated learning and memory problems. The findings suggest that reelin protects neurons from the scourge of AD pathology. Because the AD risk factor ApoE4 thwarts reelin signaling, the study also supports the idea that disease risk and severity in ApoE4 carriers relate to poor reelin signaling.
“In this elegant set of experiments, Herz and colleagues have continued to shed light on the role of reelin in synaptic function and provide further evidence for the protective nature of reelin against amyloid-associated synaptic plasticity and memory dysfunction,” commented Edwin Weeber of the University of South Florida in Tampa.
Reelin is a large secreted protein first discovered for crafting central nervous system (CNS) architecture during development. “Reeler” mice, which lack the reelin gene, suffer a slew of neurodevelopmental problems, including inverted cortical layering and wonky hippocampal and cerebellar structures (see D’Arcangelo et al., 1995). They have severe movement problems and die young. Animals expressing only a single copy of reelin have learning and memory problems despite seemingly normal brain structure (see Tueting et al., 1999; Qiu et al., 2006).
In adults, reelin plays a role in synaptic signaling and plasticity by latching onto Apoer2, a receptor that binds none other than ApoE. Herz and colleagues previously reported that reelin’s engagement of Apoer2 activates the cytosolic receptor disabled-1 (Dab1), leading to tau phosphorylation, as well as the phosphorylation of the NR2 subunit of NMDA receptors (see Hiesberger et al., 1999; Aug 2005 news). Phosphorylated NR2 elevates NMDAR levels at the synaptic surface and boosts calcium influx when these receptors are turned on, raising long-term potentiation (LTP), a hallmark of synaptic plasticity. On the other hand, ApoE4 (but not ApoE2 or E3) promotes cell uptake of Apoer2, limiting reelin effects, including LTP (see Weeber et al., 2002; May 2012 conference news). Aβ also takes a toll on synaptic plasticity, and some studies suggest it may block Apoer2 function as well.
It has been difficult for researchers to tease out reelin’s synaptic effects from its developmental ones, and thus to home in on its potential role in AD. First author Courtney Lane-Donovan and colleagues addressed this issue by generating conditional knockout (cKO) mice that lose reelin expression in the brain after treatment with tamoxifen. The researchers allowed the mice to develop normally, and then turned off reelin expression. In all experiments they treated the animals with tamoxifen when they reached 2 months of age.
One month after switching off reelin, the researchers noticed that the mice had more Dab1 in their brains than did controls. This made sense because reelin is known to phosphorylate Dab1 and target it for degradation. Surprisingly, this elevation of Dab1 did not result in any of the downstream changes previously seen in reelin heterozygous or knockout mice: Glutamate receptor levels were normal, as was the phosphorylation of several key signaling molecules, including tau. CNS architecture did not change. The researchers attributed the more drastic effects of germline reelin removal to developmental deficits that did not occur in the cKO mice.
Despite their normal glutamate receptor levels, hippocampal slices taken from cKO mice at approximately 7 months of age displayed elevated LTP. Oddly, previous studies indicated that mice deficient in reelin had a reduction in LTP from the get-go, while injecting reelin directly into the brain boosted it (see Weeber et al., 2002). Herz speculated that the elevated LTP in the reelin cKO mice represented a compensatory mechanism unique to the loss of reelin in adulthood.
Turning reelin off for one month had little behavioral effect. Compared to untreated mice, the 3-month-old tamoxifen-fed cKO mice had a slight reduction in anxiety: They were less apt to shy away from open spaces in one test, yet displayed a normal propensity to explore the exposed arms of a maze. The treated mice learned the location of submerged platforms in a water maze just as well as untreated mice, and responded by freezing in place when put into a context similar to one in which they had previously received a shock. The researchers concluded that unlike mice deficient in reelin throughout development, loss of reelin in adulthood had few to no behavioral or learning consequences.
The authors next tested the effects of reelin loss in adult mice that overexpress mutant human amyloid precursor protein. They crossed the reelin cKO mice to Tg2576 mice, which start to accumulate Aβ in the brain at around 4 months of age but don’t develop plaques until around 11 months of age. The researchers found no significant differences in the concentrations of soluble or insoluble Aβ between Tg2576 mice with or without reelin expression at 7 months, when both strains had elevated Aβ in the brain compared to wild-type mice. Though neither strain harbored detectable plaques in the cortex, the tamoxifen-treated Tg2576/reelin cKO mice performed poorly in the Morris water maze, while the Tg2576 mice still performed just as well as normal mice. Seven-month-old reelin cKO mice also performed normally on the test, indicating that the combination of mutant APP overexpression and reelin deficiency caused learning difficulties. Hippocampal slices taken from the Tg2576/reelin cKO mice at approximately 7 months of age displayed none of the LTP boost that reelin cKO mice did.
Some commentators pointed out that it is unclear whether Aβ itself caused the effects seen in Tg2576/reelin cKO mice. “Without a reversal by Aβ immunization, for example, it is not possible to conclude that the memory deficits seen here reflect a cooperative effect of reelin loss and Aβ,” wrote Steven Barger of the University of Arkansas in Little Rock. Barger added that APP has been reported to reduce Dab1 signaling, so elevated levels of APP in the Tg2576 mice could have effected reelin signaling independently of Aβ. In the paper, the authors acknowledged that Dab1 could indeed be doused by APP or other binding partners. However, Herz told Alzforum that preliminary results from his lab indicate that removal of Aβ abolishes the early learning and memory problems in the Tg2576/reelin cKO mice.
Herz hypothesized that to make up for lack of reelin signaling in the synapse, neurons produce more AMPA receptors, which would explain the elevated LTP in reelin cKO mice. His lab is investigating this. While additional AMPA receptors may maintain synaptic signaling when everything is running smoothly, Herz proposed that Aβ oligomers, which have been reported to appear by 7 months of age in Tg2576 mice, derail the system by reducing glutamate receptors at the synapse (see Westerman et al., 2002).
Interestingly, ApoE4 knock-in mice, which respond sluggishly to reelin due to reduced Apoer2 surface expression, also have elevated LTP (see Korwek et al., 2009). This compensatory mechanism could explain why ApoE4 carriers are cognitively normal at first, Herz said, but once Aβ comes into play, the compensation is rapidly thrown off.
Herz also speculated that boosting reelin signaling could protect against cognitive decline in the face of AD pathology in some people. One genome-wide association study reported that certain variants of reelin were enriched in cognitively normal people with a high burden of AD pathology, hinting that those variants may be protective. Similarly, some normal older people with a high level of neurofibrillary tangles had high reelin levels (see Kramer et al., 2011). Herz speculated that protective variants might increase reelin levels.—Jessica Shugart
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Research Models Citations
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