Presenilin mutations cause familial Alzheimer disease, but just how they do their damage is not understood. Do mutations lend the proteins an extra, pathogenic function related to cleavage of amyloid precursor protein, or do they ablate some critical role of presenilins in neurons? Work with knockout mice suggests the latter, since loss of presenilins results in age-dependent neurodegeneration. Now, an elegant use of conditional knockouts adds further support to that idea, and strengthens the argument for aberrant calcium fluxes as an underlying defect in AD. In a paper published in Nature July 30, Jie Shen, Harvard Medical School, Boston, and Thomas Sudhof, Stanford University, Palo Alto, California, selectively deleted presenilins from either CA3 or CA1 hippocampal neurons, and find that loss of presynaptic (CA3), but not postsynaptic (CA1) presenilins leads to lower glutamate release, and defects in long-term potentiation and other synaptic responses. Furthermore, they found that the effects on neurotransmitter release appear to be due to decreased calcium flow out of the endoplasmic reticulum (ER) via the ryanodine receptor channel.
It was Shen’s group that first showed how loss of presenilins causes memory deficits and neurodegeneration (see ARF related news story on Saura et al., 2004). “Here we are showing that loss of presenilins cause neurotransmitter release impairment, so our logical conclusion would be that the impaired neurotransmitter release may be the earliest pathogenic change before leading to neurodegeneration and memory impairment.” Shen told ARF.
A knockout is not the same as a mutation, though, and the relevance of the current data to FAD remains to be proven. In that regard, Shen says that they are currently working on three different mouse lines to look at the effects of PS FAD mutant knock-ins on synaptic function. However, the finding that loss of presenilins causes an impairment in calcium release from the endoplasmic reticulum via the ryanodine receptor (RYR) release channel does not fit exactly with a number of earlier studies demonstrating an increase in ER calcium release in mutant PS1-expressing cells, at least some of which appear to be mediated by the RYR. In the July 29 Journal of Neuroscience, Grace (Beth) Stutzmann, Rosalind Franklin University/The Chicago Medical School in North Chicago, Illinois, and colleagues tie the overactivity of the RYR to subtle synaptic dysfunctions in PS1 mutant transgenic mice, and suggest the elevated fluxes are associated with increased expression of RYR receptor mRNA. Together, the work from Stutzmann and Shen answers some questions, but leaves the feeling that there is much left to learn about the role of the RYR and calcium in the earliest stages of AD.
Knowing that presenilin knockouts have synaptic problems, Shen, first author Chen Zhang, and colleagues aimed to look more closely at where exactly those issues arise. They conditionally inactivated presenilin1 (PS1) in either presynaptic (CA3) or postsynaptic (CA1) neurons of the Schaeffer-collateral pathway, and looked at effects on synapse function. They find that presynaptic (CA3) but not postsynaptic inactivation decreases LTP, and alters short-term plasticity and synaptic facilitation in the neurons. The effects were not due to changes in postsynaptic AMPA or NMDA receptors, but instead could be attributed to a decreased probability of glutamate release. Since neurotransmitter release is regulated by calcium, Zhang and coworkers next looked at the effects of blocking calcium fluxes, and found that they could mimic the effects of presenilin loss on synaptic function by depleting ER calcium stores or by blocking the ryanodine receptor ER release channel. In addition, potassium chloride 1 (KCl)-induced calcium release was reduced in PS double knockout hippocampal cells in culture compared to normal neurons, and ryanodine treatment had no further effect. The results suggest that presenilins modulate calcium-induced calcium release via the RYR.
The results were surprising, Shen said, because postsynaptic mechanisms involving NMDA receptors would be an obvious mechanism for memory impairment. “But NMDA receptors are normal in both knockout lines, and only when you knock out presenilins presynaptically do you have LTP deficits,” she said. Postsynaptic changes in NMDAR and AMPAR have also been reported in AD (see ARF related news story), but the new results should raise the interest in presynaptic changes as well.
The next step, Shen said, is to figure out how presenilin is regulating the RYR at the molecular level, and whether γ-secretase activity is involved. “We are just looking at presenilin function, so we don’t know whether presenilin regulates neurotransmitter release in a γ-secretase-dependent or -independent fashion,” Shen says. But, she points out another recent paper from her group (Tabuchi et al., 2009) in which they reported a conditional knockout of nicastrin (another component of γ-secretase) that causes neurodegeneration and memory impairment very similar to the presenilin conditional knockouts. “This means that inactivating either of two γ-secretase components is causing memory impairment and neurodegeneration. The simplest explanation would be that γ-secretase activity is required for memory and neuronal survival.”
Shen also raises the intriguing prospect that defects in neurotransmitter release could be a common theme in neurodegenerative disease generally. Her lab has examined four genes related to familial Parkinson disease and found that in each case, the disease-causing mutations regulate dopamine release. (Three of the studies, on DJ-1 [Goldberg et al., 2005], Pink-1 [Kitada et al., 2007], and Parkin [Kitada et al., 2009] are published, with a fourth on LRRK2 in press.) “The logical question that comes up is whether impaired neurotransmitter release is the pathogenic precursor to neurodegeneration,” Shen says.
Previous work from Stutzmann and Frank LaFerla at the University of California, Irvine, established that even very young mice expressing the FAD mutant PS1 M146V (either as a single gene knock-in or together with mutated APP and tau in the 3xTg mouse) showed elevated ER calcium fluxes through ryanodine channels compared to non-transgenic mice (Stutzmann et al., 2007). But the researchers were stymied to find that the animals had no apparent changes in LTP or other measures of synaptic function until later, when amyloid pathology set in. “We see these very profound increases in RYR-evoked calcium releases from the ER, and the relative increases are greatest in synapse-rich regions in cortical hippocampal neurons. We know that ER calcium release and signaling in general is critical to synaptic physiology, so there had to be some effect,” Stutzmann told ARF.
Therefore, she decided to take another look, and this time first author Shreaya Chakroborty compared young 3xTg mice with non-transgenic mice under conditions of ryanodine receptor blockade. “When we blocked the ryanodine receptor with dantrolene or ryanodine, we now saw a very profound difference between Alzheimer and non-transgenic mice,” Stutzmann says. They found that in 3xTg mice LTP was reduced, and paired pulse facilitation, a measure of short-term potentiation, was increased by dantrolene, which had no such effects in non-transgenic mice. The results suggest that in normal mice, the RYR does not contribute much to either pre- or postsynaptic functions, while in AD mice it is aberrantly involved. In the AD mice, the researchers found a fivefold increase in RYR messenger RNA, which might explain the hyperactivity of the channel in those animals. All of these effects precede any apparent AD pathology.
Stutzmann concludes, “The calcium signaling in the PS1 expressing cells is having a profound effect on cell physiology, but there is some kind of compensation going on by the neurons and synapses to maintain the appearance of homoeostasis.” She is now trying to understand what the compensation involves, but speculates it may come at some cost to the cells. “I’m guessing that whatever is maintaining this homeostasis early is going to be metabolically expensive, and we’re interested in how this calcium phenotype might lead to other pathways associated with later AD.”
While the results between the two papers are not entirely consistent (e.g., Stutzmann sees postsynaptic effects, while Shen does not), that may come down to the differences in the experimental systems (one is a conditional knockout, the other a mutant overexpression) and the questions asked. As someone who has been working on the RYR for some time, Stutzmann says she is “very glad to see other groups studying the RYR-mediated ER calcium release from different angles. At the least, it points to some internal consistency that RYR-mediated calcium signaling is playing a critical role in these changes. Now tying it to the later stages of AD will be the interesting part.”—Pat McCaffrey
- The Senility-Presenilin Connection Turned Upside Down
- Amyloid-β Zaps Synapses by Downregulating Glutamate Receptors
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- Tabuchi K, Chen G, Südhof TC, Shen J. Conditional forebrain inactivation of nicastrin causes progressive memory impairment and age-related neurodegeneration. J Neurosci. 2009 Jun 3;29(22):7290-301. PubMed.
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- Zhang C, Wu B, Beglopoulos V, Wines-Samuelson M, Zhang D, Dragatsis I, Südhof TC, Shen J. Presenilins are essential for regulating neurotransmitter release. Nature. 2009 Jul 30;460(7255):632-6. PubMed.
- Chakroborty S, Goussakov I, Miller MB, Stutzmann GE. Deviant ryanodine receptor-mediated calcium release resets synaptic homeostasis in presymptomatic 3xTg-AD mice. J Neurosci. 2009 Jul 29;29(30):9458-70. PubMed.