Healthy aging relies on proper function of the transcription factor p53. Known as “guardian of the genome,” this protein regulates key physiological events such as the cell cycle, DNA repair, and apoptosis. Research published in this week’s Neuron suggests that a p53 family member could play a similar protective role in the nervous system. Led by David Kaplan and Freda Miller at the University of Toronto, Canadian researchers report that older mice with halved levels of p73, a p53 homolog expressed primarily in the brain, display behavioral deficits characteristic of Alzheimer’s and related diseases. Accompanying these cognitive and motor problems were numerous brain abnormalities. Most notably, neurons degenerated and phospho-tau filaments accumulated throughout relevant brain areas. Furthermore, p73 haploinsufficiency triggered early and robust tau tangle formation and neurodegeneration in a separate mouse model of AD amyloidosis that typically lacks these features. Based on these findings, p73 levels appear to “determine whether your brain is going to be susceptible to injury as you get older,” Kaplan told ARF. But the story may be more complex. P73 isoforms can be pro- or anti-apoptotic, with adult brain primarily expressing the latter. While the new data highlight a protective role for p73, other studies—including several published recently by Alejandra Alvarez and colleagues at Universidad Católica de Chile in Santiago—suggest that Aβ-induced neuronal damage can stabilize pro-apoptotic p73 isoforms and lead to increased cell death.
In the nervous system, p73 is expressed primarily as a dominant-inhibitory isoform (DNp73) that lacks the transactivation domain and thereby opposes the pro-death activity of p53 and full-length p73 (TAp73). Previous work by Kaplan, Miller and colleagues has shown that DNp73 prevents neuronal apoptosis during development (Pozniak et al., 2000; Pozniak et al., 2002) and helps adult neurons resist injury (Walsh et al., 2004). More recent studies have found a weak association between decreased p73 expression and AD susceptibility (Li et al., 2004) and shown that some 10 percent of the human population has lost one copy of a genomic region encompassing the p73 gene (Wong et al., 2007).
First author Monica Wetzel and colleagues put these findings to the test by looking for signs of neurodegeneration in p73-deficient mice (Yang et al., 2000). At three to four months of age, wild-type and p73+/- mice appeared fine behavior-wise. “But when they were old, they started to get all the behavioral and anatomical problems that we see in people who age, or get dementia or AD,” Kaplan said. Specifically, 16- to 18-month-old p73 heterozygotes fared poorly on various neurological and sensory-motor assays including open-field exploration, paw grip endurance, grid walking, and gait analysis. They also did worse than wild-type and young p73+/- animals in the Morris water maze that tests learning and memory.
Using 3D magnetic resonance imaging, the researchers found evidence of neurodegeneration that might underlie these outward abnormalities. Relative to their wild-type counterparts, aged p73+/- mice had 5 to 16 percent reduced volume in the motor cortex, dentate gyrus, posterior cerebellum, and other brain areas important for the behavioral tests. In the motor cortex of old p73+/- mice, neuronal density was lower, and total neuron numbers were down 29 percent compared with aged controls. Sure enough, silver staining revealed dying neurons in aged p73+/- motor cortex but not in young p73+/- or wild-type brains.
As it turns out, the aged p73+/- mice harbored a laundry list of abnormalities typical of neurodegenerative disorders. The list included increased microglial density in the brain, greater numbers of reactive astrocytes in the motor cortex, doubling of the number of neurons aberrantly re-entering the cell cycle, and more brain cells expressing senescence-associated β-galactosidase (SA-β-Gal), a marker of cell aging.
Perhaps the biggest surprise came when the researchers found phospho-tau-containing paired helical filaments (P-PHF-tau) in immunostained sections of aged p73+/- brains—a result established using three P-PHF-tau antibodies and confirmed by the disappearance of the immunostaining upon phosphatase treatment.
What happened when p73-deficient mice were crossed with transgenic CRND8 mice (an AD model that expresses double-mutant amyloid precursor protein (APP) and develops plaques, but not tangles, by three months, and does not show neurodegeneration)? At just 1.5 to two months of age, the researchers saw whopping amounts of P-PHF-tau filaments in CRND8 mice with hemizygous but not wild-type p73 levels. They also observed a 22 percent drop in neuron numbers in the motor cortex of p73+/- CRND8 animals relative to p73+/+ CRND8 littermates.
"These remarkable findings provide an AD model that not only exhibits P-PHF-tau positive filaments and amyloid plaques but, more importantly, demonstrates obvious neurodegeneration that seems to underlie behavioral alterations observed at similar time points,” wrote AD researcher Yong Shen in an e-mail to ARF. Shen is an expert in neuronal cell death signaling at Sun Health Research Institute in Sun City, Arizona.
As for how p73 might regulate neuronal survival, Kaplan and colleagues considered several possibilities—the first being that it antagonizes pro-death family members. This explanation was dismissed, however, when levels of p53 and p63 mRNA and several of their downstream targets were found to be equal in aged p73+/- and p73+/+ cortices. Another possibility is that p73’s effects involve JNK, a death protein that gets upregulated during nerve injury and that has been shown to bind and be inhibited by DNp73 (Lee et al., 2004). JNK also appears associated with AD, as it directly phosphorylates tau on residues important for tangle formation.
To see if p73 might be linked to AD via JNK, the researchers measured JNK activity in cultured p73+/+, p73+/-, and p73-/- cortical neurons. As p73 levels dropped, levels of activated, phosphorylated JNK rose, and neurons became more vulnerable to death in mildly excitotoxic glutamate concentrations. Furthermore, p73-deficient neonatal cortices showed increased tau phosphorylation at two JNK target sites, and treatment with a JNK inhibitor decreased tau phosphorylation in p73+/+ and p73-/- neurons. Further support for the idea that p73 exerts its neuroprotective effects via JNK came from immunocytochemistry studies that revealed in p73+/- CRND8 brains a 20-fold increase in phospho-JNK-positive cells—many of which were also positive for the two phospho-tau residues examined in the neonatal cortices.
All told, Kaplan said, these data suggest that normally “p73 binds to JNK and keeps it from phosphorylating tau.” He said that large-scale genetic analyses could help tease out whether p73 is a susceptibility factor for AD and/or other neurodegenerative diseases—by showing that individuals with lower p73 levels indeed have higher rates of such disorders, as suggested previously in a smaller study (Li et al., 2004). Studies with p73-inducing drugs also could test the claim that p73 prevents age-related neurodegeneration. In other words, Kaplan said, “Can we increase levels of p73 and in doing so protect the brain as we age?”
While the new work points to an overall protective function for p73 in the aging brain, recent reports by Alvarez and others suggest that the protein may have a more nuanced role. Full-length (TAp73) isoforms are regularly removed in healthy cells by the ubiquitin-proteasome system; however, c-Abl kinase activated in response to cell damage has been shown to phosphorylate and stabilize p73 (Tsai and Yuan, 2003). In this month’s issue of the journal Brain, Alvarez, first author Gonzalo Cancino, and colleagues report that the c-Abl/p73 system is activated in cultured neuronal cells exposed to β amyloid, as well as in rats given hippocampal β amyloid injections and AD transgenic mice (APPsw/PSEN1deltaE9).
In these models, intraperitoneal administration of the c-Abl inhibitor STI571 (imatinib mesylate or Gleevec, an FDA-approved treatment for chronic myelogenous leukemia and gastrointestinal stromal tumor) not only reduced Aβ-induced behavioral defects but also apoptosis and tau phosphorylation (Cancino et al., 2008). For previous data on neurodegeneration-relevant effects of Gleevec, see Netzer et al., 2003; Bantscheff et al., 2007. In Alvarez’s hands, Gleevec also reduced neurological symptoms and cerebellar apoptosis in a mouse model for Niemann-Pick type C (NPC) disease, another disorder marked by progressive neuronal loss (Alvarez et al., 2008). Alvarez proposed that cellular damage signals, which appear to stabilize and induce increased expression of full-length p73, might change the balance between anti-apoptotic and pro-apoptotic p53 family members. The new data from Kaplan’s team “highlight the central role of the p53 family in the nervous system—not only in development but also in neurodegeneration—and raise new questions about the complex functions and crosstalk between these proteins,” she wrote to ARF (see full comment below).
Along similar lines, Richard Killick of King's College London, U.K., told ARF via e-mail that the work by Kaplan and colleagues clearly implicates the p53 family in the hyperphosphorylation of tau tangle formation. Though the new data (showing more P-PHF-tau filaments in mice with less p73) would appear at odds with earlier evidence of reduced tau phosphorylation in p53-deficient mice (Ferreira and Kosik, 1996), Killick argues that the findings could be reconciled if “p53 is the main driver of tau phosphorylation and that in brain the dominant-negative forms of p73 repress this (see full comment below).”—Esther Landhuis