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.

Presynaptic Presenilins
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.

Ryanodine Revelations
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.

References:
Zhang C, Wu B, Beglopoulos V, Wines-Samuelson M, Zhang D, Dragatsis I, Sudhof T, Shen J. Presenilins are essential for regulating neurotransmitter release. Nature. 2009 July 30; 460:632-637. Abstract

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. Abstract

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  1. A number of previous studies indicated that FAD mutations and genetic deletions of presenilins are linked with abnormal ER Ca2+ homeostasis (reviewed in 1,2). These two new papers provide a first look at changes in synaptic function resulting from such ER Ca2+ alterations. Chakrobotry at al. demonstrate that PS1-M146V mutation results in dramatically (12-fold) enhanced Ca2+ responses mediated by ryanodine receptors (RyanR) in the spines of hippocampal neurons from 3xTg mice. These effects are much more dramatic than the approximately twofold increase in RyanR-mediated responses in the soma of 3xTg neurons. The potential explanation of these results may be related to increased expression of RyanR2 in 3xTg mice (as suggested by the authors) or to overfilled ER Ca2+ stores due to loss of ER Ca2+ leak function resulting from PS1-M146V mutation (3).

    Perhaps the most interesting findings in the paper resulted from studies of hippocampal synaptic plasticity in 3xTg mice. The short-term form of presynaptic plasticity (paired-pulsed facilitation, PPF) was normal in 3xTg mice when compared to wild-type mice. Long-term potentiation (LTP) was also normal. At the first glance, these results seem to indicate that changes in ER Ca2+ homeostasis induced by PS1-M146V mutation have no effect on short-term and long-term plasticity. However, when the RyanR was blocked by application of dantrolene, very significant differences emerged. Dantrolene had no effect on PPF and LTP in wild-type mice, but in 3xTg mice application of dantrolene increased PPF and reduced LTP. These observations suggest that the RyanR-mediated ER Ca2+ release is playing a much more important role in presynaptic and postsynaptic spines in 3xTg mice than in WT mice.

    In the paper by Zhang at al., sophisticated genetic methods were used to dissect the contribution of presenilins to ER Ca2+ signaling and synaptic function on presynaptic and postsynaptic sides of the hippocampal synapse. Using CA1-specific (postsynaptic) and CA3-specific (presynaptic) Cre-driver lines crossed with PS1floxed/floxed; PS2-/- mice, these investigators discovered that hippocampal LTP, PPF, and high-frequency facilitation are impaired when presenilins are deleted presynaptically (CA3-Cre deletion), but not postsynaptically (CA1-Cre deletion). It should be noted here that PPF and high-frequency facilitation are presynaptic forms of short-term plasticity, so it is not surprising that the phenotype in these experiments has been observed in “presynaptic” but not in “postsynaptic” presenilin knockouts. Deficiency in high-frequency facilitation could be rescued by an increase in extracellular Ca2+ concentration and occluded in the presence of ER SERCA Ca2+ pump inhibitor thapsigargin or RyanR inhibitors ryanodine or dantrolene, but not in the presence InsP3R inhibitor Xestospongin C. These results indicate that defects in high-frequency facilitation in these mice are related to changes in RyanR-mediated Ca2+ release from the presynaptic ER.

    In summary, both papers indicate that PS1-M146V FAD point mutation in presenlin-1 or genetic knockout of presenlin-1 in presynaptic neurons results in imbalance in ER Ca2+ handling and changes in RyanR-mediated Ca2+ release. Resulting differences in ER Ca2+ signaling are manifested as changes in short-term (PPF) and long-term (LTP) form of hippocampal synaptic plasticity. The mechanistic basis for these changes is not well understood, but may be linked to a loss of ER Ca2+ leak function of presenilins (3) resulting from PS1-M146V mutation (Chakrobotry et al.) or genetic deletion of PS1 (Zhang et al.) and resulting ER Ca2+ overload during stimulation.

    References:

    . Calcium dysregulation in Alzheimer's disease: recent advances gained from genetically modified animals. Cell Calcium. 2005 Sep-Oct;38(3-4):427-37. PubMed.

    . Neuronal calcium mishandling and the pathogenesis of Alzheimer's disease. Trends Neurosci. 2008 Sep;31(9):454-63. PubMed.

    . Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations. Cell. 2006 Sep 8;126(5):981-93. PubMed.

  2. Another example that loss of function of APP is likely the basic mechanism of neurodegeneration.

    View all comments by Andre Delacourte
  3. In a new study from Jie Shen’s lab, C. Zhang et al. provide compelling evidence that presenilins (PS) located at presynaptic terminals regulate neurotransmitter release during synaptic transmission. By using new engineered mice with specific deletion of both presenilin genes in presynaptic (CA3) or postsynaptic (CA1) hippocampal neurons, the authors conclusively demonstrate that PS regulate normal neurotransmitter release by controlling activity-induced ryanodine receptor-evoked Ca2+ release. Although the exact mechanism(s) by which presenilin regulates ryanodine receptor function need further investigation, this is the first report showing that regulation of Ca2+ signaling by PS is crucial for glutamate release and synaptic facilitation. These findings are important because they may explain, at least in part, the deficits on LTP and memory previously observed in mutant mice lacking both PS in excitatory neurons of the forebrain (Saura et al., 2004). Previous studies have demonstrated that FAD-linked mutations in presenilin-1 increase calcium levels through different mechanisms and cellular sources (Chan et al., 2000; Stutzmann et al., 2006; Tu et al., 2006; Cheung et al., 2008). In agreement with this idea, Beth Stutzmann’s group report elevated ryanodine receptor-evoked calcium release in CA1 hippocampal neurons of 3xTg-AD mice, a phenotype previously attributed to presenilin-1 mutation (Stutzmann et al., 2006). Notably, Chakroborty et al. found that aberrant calcium release from ER results in enhanced presynaptic (PPF) and reduced postsynaptic (LTP) plasticity in presymptomatic, not yet memory-impaired, 3xTg-AD mice. Together, these new findings suggest that PS dysfunction results in synaptic plasticity and memory deficits by altering calcium signaling.

    One of the interesting points of J. Shen´s paper is the previously unappreciated or underestimated role of presenilin on presynaptic function. Indeed, the presynaptic localization of presenilins and the precursors of Aβ APP CTFs, in glutamatergic synapses (Saura et al., 2005; Zhang et al., 2009), strongly supports a role of PS/γ-secretase on presynaptic function. Inoue et al. demonstrated recently that PS localized at synapses are essential for synapse formation by regulating the γ-secretase cleavage of EphA4 (see Inoue et al., 2009 and ARF related news story). It is possible that besides regulating neurotransmitter release, presenilins may regulate the formation, maturation, and function of excitatory synapses by regulating the processing and/or function of EphA4 as well as other unidentified synaptic proteins. Since presenilins are also present in postsynaptic compartments, studies on postsynaptic presenilin/γ-secretase function need to be carefully evaluated in the future.

    An important question related to AD pathology that has not been addressed in Shen’s present study is whether the hippocampal presynaptic and postsynaptic plasticity deficits caused by loss of presynaptic PS function result in neurodegeneration, such as that observed by loss of PS in the forebrain (Feng et al., 2004; Saura et al., 2004). While inherited mutations in the presenilin genes cause synaptic dysfunction and neurodegeneration in familial cases of AD, in general, PS transgenic mice characterized by presynaptic and/or postsynaptic plasticity deficits do not show signs of neurodegeneration, which suggests that plasticity deficits may not be directly linked to neuron death in PS mutant mice. This raises the possibility for alternative molecular mechanisms regulated by presenilins that impact neuron survival. Although the biological result of loss of PS function may not be strictly the same as FAD mutations, especially for APP processing and Aβ generation, the emerging new roles of PS on synaptic and memory function and neuronal survival may explain why these pathogenic mutations are so aggresive during the disease process.

    References:

    . Deviant ryanodine receptor-mediated calcium release resets synaptic homeostasis in presymptomatic 3xTg-AD mice. J Neurosci. 2009 Jul 29;29(30):9458-70. PubMed.

    . Presenilin-1 mutations increase levels of ryanodine receptors and calcium release in PC12 cells and cortical neurons. J Biol Chem. 2000 Jun 16;275(24):18195-200. PubMed.

    . Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor channel gating. Neuron. 2008 Jun 26;58(6):871-83. PubMed.

    . Forebrain degeneration and ventricle enlargement caused by double knockout of Alzheimer's presenilin-1 and presenilin-2. Proc Natl Acad Sci U S A. 2004 May 25;101(21):8162-7. PubMed.

    . Synaptic activity prompts gamma-secretase-mediated cleavage of EphA4 and dendritic spine formation. J Cell Biol. 2009 May 4;185(3):551-64. PubMed.

    . Conditional inactivation of presenilin 1 prevents amyloid accumulation and temporarily rescues contextual and spatial working memory impairments in amyloid precursor protein transgenic mice. J Neurosci. 2005 Jul 20;25(29):6755-64. PubMed.

    . Loss of presenilin function causes impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Neuron. 2004 Apr 8;42(1):23-36. PubMed.

    . Enhanced ryanodine receptor recruitment contributes to Ca2+ disruptions in young, adult, and aged Alzheimer's disease mice. J Neurosci. 2006 May 10;26(19):5180-9. PubMed.

    . Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations. Cell. 2006 Sep 8;126(5):981-93. PubMed.

    . Presenilins are essential for regulating neurotransmitter release. Nature. 2009 Jul 30;460(7255):632-6. PubMed.

  4. Dr. Saura discussed that FAD presenilin mutation showed neurodegeneration in humans, but not in mice. Why this difference? We believe that homocysteic acid (HA) may be a pathogenic compound for Alzheimer disease. In our hands, HA induced neurodegeneration. We found recently that there is a big difference in urinary HA excretion between humans and mice. Human HA excretion into urine is 1,000 times higher than in mice. From our observation, humans show more severe HA-induced neurodegeneration than do mice, and HA production is related to presenilin activation (submitted for publication).

    View all comments by Tohru Hasegawa

References

News Citations

  1. The Senility-Presenilin Connection Turned Upside Down
  2. Amyloid-β Zaps Synapses by Downregulating Glutamate Receptors

Paper Citations

  1. . Loss of presenilin function causes impairments of memory and synaptic plasticity followed by age-dependent neurodegeneration. Neuron. 2004 Apr 8;42(1):23-36. PubMed.
  2. . Conditional forebrain inactivation of nicastrin causes progressive memory impairment and age-related neurodegeneration. J Neurosci. 2009 Jun 3;29(22):7290-301. PubMed.
  3. . Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial Parkinsonism-linked gene DJ-1. Neuron. 2005 Feb 17;45(4):489-96. PubMed.
  4. . Impaired dopamine release and synaptic plasticity in the striatum of PINK1-deficient mice. Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11441-6. PubMed.
  5. . Impaired dopamine release and synaptic plasticity in the striatum of parkin-/- mice. J Neurochem. 2009 Jul;110(2):613-21. PubMed.
  6. . Enhanced ryanodine-mediated calcium release in mutant PS1-expressing Alzheimer's mouse models. Ann N Y Acad Sci. 2007 Feb;1097:265-77. PubMed.
  7. . Presenilins are essential for regulating neurotransmitter release. Nature. 2009 Jul 30;460(7255):632-6. PubMed.
  8. . Deviant ryanodine receptor-mediated calcium release resets synaptic homeostasis in presymptomatic 3xTg-AD mice. J Neurosci. 2009 Jul 29;29(30):9458-70. PubMed.

Further Reading

Papers

  1. . Presenilins are essential for regulating neurotransmitter release. Nature. 2009 Jul 30;460(7255):632-6. PubMed.
  2. . Deviant ryanodine receptor-mediated calcium release resets synaptic homeostasis in presymptomatic 3xTg-AD mice. J Neurosci. 2009 Jul 29;29(30):9458-70. PubMed.

Primary Papers

  1. . Presenilins are essential for regulating neurotransmitter release. Nature. 2009 Jul 30;460(7255):632-6. PubMed.
  2. . Deviant ryanodine receptor-mediated calcium release resets synaptic homeostasis in presymptomatic 3xTg-AD mice. J Neurosci. 2009 Jul 29;29(30):9458-70. PubMed.