6 January 2009. The final issue of the Journal of Neuroscience for 2008 brings two papers that put a new twist on current thinking about amyloid-β (Aβ). The first, from the lab of Ottavio Arancio at Columbia University in New York, presents evidence that very low concentrations of Aβ, in the range that might be released during normal neuronal activity, promote the synaptic changes associated with learning and improve memory in mice. That is precisely the opposite of what happens with high concentrations of Aβ, which block synaptic strengthening and memory function (see ARF related news story and ARF news story). The results suggest a physiological role for released Aβ in synaptic plasticity and memory, and raise questions about the implications of reducing Aβ levels as a therapeutic approach to Alzheimer disease.
The second report adds to a growing body of work on the Mint/X11 family of adaptor proteins and their role in the processing of amyloid precursor protein to release Aβ. The proteins are clearly involved, but different labs have come up with conflicting results about their exact action. In the latest study, Thomas Sudhof, formerly of the University of Texas Southwestern in Dallas and now at Stanford, reports that knocking out the Mint proteins in transgenic AD mice delays the age-dependent accumulation of Aβ peptides and plaques, apparently by decreasing APP processing by β-secretase. The work stands beside previous studies that show opposite effects, where Mint knockout mice have increased Aβ production, and that Mint proteins regulate γ-secretase. The Mint proteins most likely regulate Aβ production by affecting APP trafficking, but clearly much remains to be learned about the interaction.
Aβ in Tiny Doses
Aβ, produced in high amounts in AD, forms soluble oligomers that at nanomolar concentrations impair synaptic long-term potentiation and memory in mice. Aβ is present in normal brain at much lower levels, and Arancio and colleagues looked at the effects of nearer physiological levels on activity-dependent synaptic plasticity and memory in mice. Lead author Daniela Puzzo, who is also at the University of Catania in Italy, first looked at the effects of Aβ on hippocampal slices during induction of LTP. As previously reported, Aβ at 200 nM elicited impairment of LTP. Dialing back the concentration of Aβ revealed a reversal of that action such that at 200 pM Aβ, there was a significant enhancement in LTP. The effect was specific for activity-dependent potentiation and Aβ: 200 pM Aβ did not enhance basal transmission, and a scrambled peptide did not enhance LTP.
To look at the effects of Aβ in vivo, the investigators injected pM concentrations of Aβ bilaterally into the hippocampus of mice, then tested the animals 20 minutes later for spatial learning in the Morris water maze or for contextual association in a fear-conditioning test. In both cases, the mice performed better after Aβ injection.
How does Aβ enhance LTP? Treating hippocampal slices with 200 pM Aβ did not alter postsynaptic AMPA or NMDA glutamate receptor currents, which can be responsible for LTP. Neither did Aβ appear to affect spontaneous release of neurotransmitters. However, Aβ did enhance stimulated neurotransmitter release during prolonged activation (tetanic potentiation). As with other synaptic effects of Aβ, the potentiation depended on α7 nicotinic receptor activation. It was abolished by selective α7 receptor blocker, and was not seen in hippocampal slices from α7 knockout mice. Moreover, the α7 knockout mice showed no enhancement of memory after hippocampal injection of 200 pM Aβ42.
“These findings strongly support a model for Aβ effects in which low concentrations play a novel positive, modulatory role on neurotransmission and memory, whereas high concentrations play the well-known detrimental effect, culminating in dementia,” the authors conclude. Aβ levels have been reported to be regulated by synaptic activity, suggesting a model where the peptide, released during high-frequency stimulation via vesicle exocytosis, then acts through α7 receptors to modulate glutamate neurotransmitter release.
The results suggest that Aβ can positively affect LTP, both in vitro and in vivo, but the active form of Aβ—soluble oligomeric or other—and its concentration at synapses remains to be clarified. Estimates place in vivo concentrations of Aβ in normal brain in the pM range, and the investigators measured similar levels in the mice by ELISA. If Aβ in low concentrations is a pro-memory molecule, this could have ramifications for efforts to treat AD by lowering amyloid load. “Our work does not challenge the amyloid hypothesis,” the authors write. “However, our lack of understanding of the physiologic role of Aβ may present important issues when designing effective and safe approaches to AD therapy.”
Sudhof and colleagues neatly summarize an extensive literature behind Mint/X11 proteins and APP processing in their paper, using a supplementary table to outline 29 previous studies on the subject. Most of the studies, using Mint overexpressing cells, report that the protein suppresses Aβ production. In agreement with the idea that Mint proteins put a brake on Aβ, knocking out Mint1 or Mint2 in mice has been reported to increase amyloidogenic processing of APP (Sano et al., 2006; Saito et al., 2008).
The current study turns that data on its head. Coauthors Angela Ho and Xinran Liu crossed Mint knockout mice into two different AD transgenic mice lines bearing mutated human APP and presenilin genes. In this model, deletion of any of three Mint isoforms delayed early plaque accumulation in hippocampus and cortex, but the magnitude of the effect was isoform-dependent. Mint2 deletion had the greatest impact, and Mint3 the least. A similar pattern was seen for cortical plaques, and for Aβ40 and 42 peptide levels.
The authors attribute the differential effects of the isoforms to their expression pattern in the brain. Staining of brain sections revealed that Mint2 and APP share a similar location in the soma of excitatory pyramidal neurons in the CA3 region of hippocampus. Mint1, on the other hand, did not colocalize with APP. Mint3 is barely expressed in the brain, they write, citing previous work.
To look at the effect of Mint on APP processing, the researchers made neurons lacking all three Mint isoforms. Mice with floxed Mint alleles were crossed with AD mice, and neurons isolated from the resulting animals were infected with a Cre recombinase-expressing virus. These cells made less Aβ40 and 42 compared to Mint expressing cells. The effect on APP processing was to decrease the accumulation of the C-terminal fragments produced by β-secretase and α-secretase. The accumulation of CTFs was only seen in the presence of a γ-secretase inhibitor which prevented their further processing. When the inhibitor was washed out, cleavage of the CTFs proceeded normally, suggesting that deletion of the Mint proteins do not affect γ-secretase activity. This is in contrast to previous results from multiple labs using overexpression, and one report of RNAi knockdown (Xie et al., 2005) that suggested Mint proteins regulate APP processing by γ-secretase.
What do the Mint proteins do? They associate with APP via its intracellular domain, and may play a role in APP trafficking. The knockout mouse phenotype is consistent with an effect on APP trafficking, the authors conclude, because changes in where APP sits in the cell and for how long could influence processing by β-secretase, which occurs intracellularly in endosomes. It will be interesting to see how the details of Mint/APP interactions unfold going into 2009 and beyond.—Pat McCaffrey.
Puzzo D, Privitera L, Leznik E, Fa M, Staniszewski A, Palmeri A, Arancio O. Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus. J. Neurosci. 2008 Dec 31;28(53):14537-14545. Abstract
Ho A, Liu X, Sudhof TC. Deletion of Mint proteins decreases amyloid production in transgenic mouse models of Alzheimer’s disease. J. Neurosci. 2008 Dec 31;28(53):14392-14400. Abstract