Many scientists now believe that Alzheimer’s disease attacks first at synapses. Mounting evidence shows that Aβ oligomers can wreak havoc on these vulnerable neuronal connections. But early synaptic damage remains a puzzle with many missing pieces. In particular, no one knows exactly how the process begins. To fill in this picture, researchers led by Francesco Cecconi at the University of Rome and the Santa Lucia Foundation in Italy examined synaptic changes in the brains of young AD mice. Their results, reported in the December 12 Nature Neuroscience, implicate an apoptotic protein, caspase-3, in the earliest Aβ-mediated synaptic and learning deficits. Significantly, Cecconi and colleagues showed that they could reverse Aβ-induced damage by inhibiting caspase-3, suggesting a novel avenue for therapeutic exploration. Meanwhile, researchers led by Sergio Ferreira at the Federal University of Rio de Janeiro, Brazil, took a different approach to restoring synaptic function. In the November 29 Journal of Biological Chemistry, they show that activation of dopamine receptors can prevent some of the same sorts of initial synaptic changes seen by Cecconi’s group. These findings complement a November 28 Nature paper that describes a novel mechanism by which Aβ oligomers dampen signaling through NMDA-type glutamate receptors (see ARF related news story). Together, these papers imply that focusing on the molecular mechanisms of synapse damage may help open up new therapeutic paths for early intervention.

Although caspase-3 is best known for its role in programmed cell death, evidence has been growing that it acts on synapses as well. Researchers led by Mark Mattson at the National Institute on Aging in Bethesda, Maryland, saw synaptic caspase-3 activation in cultured hippocampal neurons when they stressed them with glutamate, and showed that the protease can cleave AMPA-type glutamate receptors, potentially modulating neuronal excitability (see Mattson et al., 1998 and Lu et al., 2002). A recent study tied the protease more directly to synaptic function, finding that hippocampal neurons need caspase-3 activity to internalize AMPA receptors and achieve long-term depression (see ARF related news story on Li et al., 2010).

Cecconi and colleagues wondered whether caspase-3 might play a role in Aβ-mediated synaptic failure. To investigate this, first author Marcello D’Amelio used Tg2576 mice, which carry the Swedish APP mutation. These mice develop amyloid plaques and AD-like symptoms at six to eight months of age, but D’Amelio and colleagues discovered that at three months of age, these mice already demonstrated some dendritic spine loss and shrinkage as well as learning deficits in fear conditioning tests. The young mice also displayed altered electrical synaptic properties and increased long-term depression (LTD), which weakens synapses. Looking for molecular changes to explain this, the authors found that an AMPA receptor subunit, GluR1, showed less phosphorylation at serine 845 (pS845-GluR1). Dephosphorylation at this site is known to lead to internalization of AMPA receptors, and, indeed, D’Amelio and colleagues saw reduced levels of AMPA receptors at the synapse.

The authors connected these changes to Aβ by showing that injection of a γ-secretase inhibitor, which reduced Aβ levels, also increased synaptic levels of GluR1, improved spine morphology, and rescued memory problems. This finding agrees with results from other groups, as researchers have shown that Aβ induces internalization of AMPA-type glutamate receptors (see ARF related news story on Hsieh et al., 2006) and can cause reductions in surface expression of GluR1 (see Almeida et al., 2005).

Enter caspase-3. Activated caspase-3 decorated synaptosomes of three-month-old Tg2576 mice more heavily than those of two-month-old AD or wild-type animals, as visualized by several methods including immunoelectron microscopy. Application of a caspase inhibitor to three-month-old mice restored shrunken spines to their normal size and improved memory in vivo, and restored pS845-GluR1 levels in slice cultures. Since caspase-3 is known to activate the phosphatase calcineurin A, D’Amelio and colleagues also blocked calcineurin in slices, and saw a similar restoration of pS845-GluR1 levels.

The data fit with work from Tara Spires-Jones and Brad Hyman at Massachusetts General Hospital in Charlestown, which showed that inhibition of calcineurin rescues spine loss in AD mice (see Rozkalne et al., 2010 and ARF related news story on Wu et al., 2010). Spires-Jones and Hyman have also seen caspase activation in AD mice, although in their work on older mice that overexpress tau, the activated protease led to tau cleavage and formation of neurofibrillary tangles. The data from Cecconi’s group would correspond to an earlier stage of AD, Spires-Jones said, perhaps analogous to a human who was pre-symptomatic. Spires-Jones speculated that in human disease, activated synaptic caspase-3 might ultimately cleave tau as well, leading both to synapse collapse and tangle formation.

Echoing a common theme among commentators, Gunnar Gouras of Weill Cornell Medical College in New York City praised the thoroughness of the work. “This is an impressive study. It brings a new perspective to early synaptic damage.” Gouras said the data throw more pieces into the complex puzzle of early synaptic changes, in which diverse proteins have now been implicated, for example, the Fyn kinase fingered by Lennart Mucke’s group (see Chin et al., 2005) and the mammalian target of rapamycin pathway uncovered by Gouras and colleagues (see Ma et al., 2010). One of the next challenges, Gouras said, is to integrate all these studies and figure out what key players are mediating Aβ’s effect on synapses, and crucially, which one acts first.

Cecconi agrees that it is essential to look at the earliest changes in AD to discover the root cause of the disease and open up more promising targets for early intervention. His group is now looking at events upstream of caspase-3 activation. For example, Aβ oligomers may cause oxidative stress on mitochondria, Cecconi said, and mitochondrial damage might lead to caspase activation. This idea fits with other work showing that Aβ can cause mitochondrial damage (e.g., see ARF related news story on Cho et al., 2009).

Cecconi is also interested in the therapeutic possibilities of caspase-3. He is now studying the effects of long-term treatment of Tg2576 mice with caspase inhibitor to see if this can prevent the development of the disease. Other points in this pathway, such as calcineurin and calcium flux, might also make good therapeutic targets, Cecconi suggested. For people with AD, caspase inhibition might be problematic, however. In healthy mice, Cecconi and colleagues found that such inhibition caused problems with synaptic plasticity. “When thinking about a molecule that can interfere in this process, we should consider a modulator [of caspase-3] rather than a complete inhibitor. This would be a completely new class of molecules that does not yet exist,” he told ARF. In the nearer future, however, Cecconi believes that caspase-3 (or molecules related to its pathway) could make a good diagnostic tool, because its activation occurs so early in the disease. If it turns out that levels of caspase-3 or related molecules are elevated in the cerebrospinal fluid or blood of people with incipient AD, Cecconi said, then potentially a simple test could identify these people years before symptoms start. This would allow people to take preventative measures such as lifestyle changes, Cecconi suggested.

Mattson agrees that caspase-3 inhibition is a chancy strategy in people, pointing out that when the protease is inactivated, neurons become more susceptible to excitotoxicity, and will undergo necrosis instead of apoptosis (see Glazner et al., 2000). Necrosis causes more brain inflammation and is more damaging to surrounding cells than apoptosis. Mattson suggested that more work should be done in human postmortem AD brains to determine whether activated caspase-3 plays a role at synapses in people. Some studies have already found caspase-3 enriched at human synapses (see Louneva et al., 2008).

In the second paper, Ferreira and colleagues described a quite different system for investigating Aβ’s early impact. They treated hippocampal cultures from wild-type rats with soluble Aβ oligomers, yet they saw similar effects of Aβ on synapses. These included a drop in pS845-GluR1, increased internalization of AMPA and NMDA receptors, impaired long-term potentiation (LTP) and enhanced LTD, more activated calcineurin, and spine loss. Since activation of the D1/D5-type dopamine receptors has been shown to increase AMPA receptor insertion and improve memory, first author Sofia Jürgensen treated the cultures with a D1/D5 receptor agonist. The agonist maintained LTP in the face of Aβ treatment, kept the level of pS845-GluR1 high, and restored AMPA and NMDA insertion at the synapse. The results suggest that stimulating the dopamine system might be another potential intervention against memory loss.

Ferreira said that he will next use animal models to discover if dopamine receptor activation has the same beneficial effects in vivo. A new Annals of Neurology paper by Jun-ichi Kira and colleagues at Kyushu University in Fukuoka, Japan, suggests it might: These scientists found that a different dopamine receptor agonist, apomorphine, improved memory and reduced levels of Aβ and hyperphosphorylated tau in the 3xTgAD mouse (see also comment by Ferreira). Moving the dopamine strategy to people is problematic, Ferreira said, even though dopamine agonists are already used in people for some psychiatric disorders and Parkinson’s disease. Mattson points out, for example, that dopaminergic drugs can have adverse affects in normal people, such as causing hallucinations or psychotic behaviors. Ferreira notes, however, that the dopamine system is also activated by behaviors such as exercise and cognitive stimulation, both of which are believed to help delay the onset of AD. The new data may provide a molecular explanation for this phenomenon, Ferreira speculated, and add to the evidence that lifestyle changes can be helpful in AD.—Madolyn Bowman Rogers.

References:
D’Amelio M, Cavallucci V, Middei S, Marchetti C, Pacioni S, Ferri A, Diamantini A, De Zio D, Carrara P, Battistini L, Moreno S, Bacci A, Ammassari-Teule M, Marie H, Cecconi F. Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer’s disease. Nat Neurosci. 2010 Dec 12. Abstract

Jurgensen S, Antonio LL, Mussi GE, Brito-Moreira J, Bomfim TR, De Felice FG, Garrido-Sanabria ER, Cavalheiro EA, Ferreira ST. Activation of D1/D5 dopamine receptors protects neurons from synapse dysfunction induced by amyloid-{beta} oligomers. J Biol Chem. 2010 Nov 29. Abstract

Himeno E, Ohyagi Y, Ma L, Nakamura N, Miyoshi K, Sakae N, Motomura K, Soejima N, Yamasaki R, Hashimoto T, Tabira T, LaFerla FM, Kira J. Apomorphine treatment for Alzheimer’s mice promoting amyloid-{beta} degradation. Ann Neurol. 2010. Abstract

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  1. Despite the significant increase in our understanding of mechanisms that lead to neuronal damage and memory loss in Alzheimer’s disease (AD), to date there are no effective ways to prevent or treat this devastating disease. Thus, identifying and validating novel therapeutic targets in AD remain major research goals. In this paper just published in Annals of Neurology, Himeno and coworkers have investigated the effects of apomorphine (APO) on neuropathological alterations and memory impairment in 3xTg mice. APO is a non-specific dopamine receptor agonist, exhibiting submicromolar affinities for both D1-type and D2-type dopamine receptors. In addition to its dopaminergic action, APO has been shown to protect neurons from oxidative stress in experimental models of Parkinson’s disease and stroke. Because oxidative stress is a prominent feature in AD brains, Himeno et al. tested the hypothesis that APO might protect 3xTg mice from amyloid-induced brain pathology and memory deficits. In line with their expectations, they found that Tg mice treated for one month with weekly injections of APO exhibited significantly improved performance in memory tasks compared to untreated Tg animals. Interestingly, they also found that treatment with APO stimulates the degradation of Aβ by insulin-degrading enzyme (IDE), thus reducing neuronal levels of amyloid peptide. As an added bonus, neuronal levels of phosphorylated tau (p-tau), a hallmark of AD pathology, were also reduced in APO-treated Tg mice.

    Even though Himeno’s study suggests that neuroprotection by APO is largely due to its ability to block neuronal oxidative stress and reduce intraneuronal amyloid, there may be more to this story than first meets the eye. In a recently published study (Jurgensen et al., 2010), we showed that a specific D1-type dopamine receptor agonist blocked the removal of surface AMPA and NMDA receptors from synapses in cultured hippocampal neurons. Redistribution of AMPA and NMDA receptors from synapses is instigated by soluble Aβ oligomers, increasingly recognized as the proximal neurotoxins in AD, and is related to dephosphorylation of critical serine residues responsible for membrane insertion of the receptors. In the specific case of AMPA receptors, we showed that Aβ oligomers induce calcineurin-mediated dephosphorylation of Ser845 of the GluR1 subunit, causing the receptor to be removed from the membrane.

    Because AMPA and NMDA receptors play key roles in synaptic plasticity, our findings provide a direct molecular mechanism to explain the inhibition of synaptic long-term potentiation (LTP) and the facilitation of long-term depression (LTD) induced by Aβ oligomers. Directly supporting and extending these findings, a paper just out in Nature Neuroscience (D’Amelio et al., 2010) showed a calcineurin-dependent decrease in phosphorylation of GluR1 at Ser845, followed by removal of AMPA receptors from synapses in Tg2576-APPSwe mice. Interestingly, both Jurgensen et al. and D’Amelio et al. showed that neither AMPA nor NMDA receptors are readily degraded upon their removal from the surface, suggesting that mechanisms capable of stimulating reinsertion of the receptors into the neuronal membrane could potentially counteract the negative impact of Aβ oligomers. To test this hypothesis, we asked whether a specific D1-receptor agonist could rescue neurons from Aβ oligomer-induced loss of surface receptors and inhibition of plasticity. The rationale for this was that D1 receptors are coupled to stimulatory G proteins that activate adenylate cyclase, leading to increased production of cAMP. In turn, cAMP activates protein kinase A (PKA), which phosphorylates AMPA and NMDA receptors at the serine residues that control membrane insertion.

    Remarkably, we found that the D1 agonist effectively blocked dephosphorylation and removal of AMPA and NMDA receptors from the neuronal membrane, and prevented oligomer-induced inhibition of LTP in hippocampal slices. This suggests that specific activation of D1 dopamine receptors could be a novel therapeutic approach to prevent Aβ oligomer-induced synapse failure and memory loss in the early stages of AD.

    Thus, in addition to the possible antioxidant action and the ability to increase Aβ degradation (as suggested by Himeno’s study), direct neurochemical effects of dopamine receptor agonists may be beneficial in AD. From a therapeutic point of view, however, APO would probably not be the best choice. As mentioned above, APO is a non-specific dopamine receptor agonist, acting on both D1 and D2 families of receptors (in fact, APO has higher affinities for D2-like than for D1-like receptors). Contrary to D1-like receptors, D2-like receptors are coupled to inhibitory G proteins, thus leading to reduced cAMP production and reduced PKA-dependent phosphorylation of AMPA or NMDA receptor subunits. Moreover, because D2 receptors are particularly enriched in the nigrostriatal pathway involved in motor control, use of a drug that activates D2 receptors in this brain region may lead to undesirable, or perhaps unacceptable, side effects. Yet another side effect of APO, related to activation of D2 receptors, is that it is a potent emetic. On the other hand, D1 receptors are enriched in projections from the ventral tegmental area to the hippocampus, a circuitry that is known to play important roles in learning and memory.

    In conclusion, the possibility to combat synapse failure and memory loss by selective activation of D1 receptors should be further investigated as an approach to develop more effective treatments in AD.

    View all comments by Sergio Ferreira
  2. Very nice studies of how a classic small molecule with action at numerous receptors could be therapeutic for AD.

    View all comments by Lon Schneider

References

News Citations

  1. Aβ Downs EphB2 Kinase, Disrupts Glutamate Receptors
  2. Cutting Both Ways—Besides Death, Caspases Mediate Neurons' LTD
  3. AMPA Receptors: Going, Going, Gone in Aβ-exposed Synapses, PSD95 Knockouts
  4. Calcium Hypothesis—Studies Beef Up NFAT, CaN, Astrocyte Connections
  5. NO Kidding? Mitochondria Fission Protein Linked to Neurodegeneration

Paper Citations

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  2. . Direct cleavage of AMPA receptor subunit GluR1 and suppression of AMPA currents by caspase-3: implications for synaptic plasticity and excitotoxic neuronal death. Neuromolecular Med. 2002;1(1):69-79. PubMed.
  3. . Caspase-3 activation via mitochondria is required for long-term depression and AMPA receptor internalization. Cell. 2010 May 28;141(5):859-71. PubMed.
  4. . AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss. Neuron. 2006 Dec 7;52(5):831-43. PubMed.
  5. . Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses. Neurobiol Dis. 2005 Nov;20(2):187-98. PubMed.
  6. . Calcineurin inhibition with FK506 ameliorates dendritic spine density deficits in plaque-bearing Alzheimer model mice. Neurobiol Dis. 2011 Mar;41(3):650-4. PubMed.
  7. . Amyloid beta induces the morphological neurodegenerative triad of spine loss, dendritic simplification, and neuritic dystrophies through calcineurin activation. J Neurosci. 2010 Feb 17;30(7):2636-49. PubMed.
  8. . Fyn kinase induces synaptic and cognitive impairments in a transgenic mouse model of Alzheimer's disease. J Neurosci. 2005 Oct 19;25(42):9694-703. PubMed.
  9. . Dysregulation of the mTOR pathway mediates impairment of synaptic plasticity in a mouse model of Alzheimer's disease. PLoS One. 2010;5(9) PubMed.
  10. . S-nitrosylation of Drp1 mediates beta-amyloid-related mitochondrial fission and neuronal injury. Science. 2009 Apr 3;324(5923):102-5. PubMed.
  11. . Caspase-mediated degradation of AMPA receptor subunits: a mechanism for preventing excitotoxic necrosis and ensuring apoptosis. J Neurosci. 2000 May 15;20(10):3641-9. PubMed.
  12. . Caspase-3 is enriched in postsynaptic densities and increased in Alzheimer's disease. Am J Pathol. 2008 Nov;173(5):1488-95. PubMed.
  13. . Apomorphine Treatment for Alzheimer’s Mice Promoting Amyloid-β Degradation. Annals of Neurology. 2010 Dec 1;
  14. . Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer's disease. Nat Neurosci. 2011 Jan;14(1):69-76. PubMed.
  15. . Activation of D1/D5 dopamine receptors protects neurons from synapse dysfunction induced by amyloid-beta oligomers. J Biol Chem. 2011 Feb 4;286(5):3270-6. PubMed.
  16. . Apomorphine treatment in Alzheimer mice promoting amyloid-β degradation. Ann Neurol. 2011 Feb;69(2):248-56. PubMed.

Other Citations

  1. Tg2576 mice

Further Reading

Papers

  1. . Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer's disease. Nat Neurosci. 2011 Jan;14(1):69-76. PubMed.
  2. . Activation of D1/D5 dopamine receptors protects neurons from synapse dysfunction induced by amyloid-beta oligomers. J Biol Chem. 2011 Feb 4;286(5):3270-6. PubMed.
  3. . Apomorphine treatment in Alzheimer mice promoting amyloid-β degradation. Ann Neurol. 2011 Feb;69(2):248-56. PubMed.

Primary Papers

  1. . Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer's disease. Nat Neurosci. 2011 Jan;14(1):69-76. PubMed.
  2. . Activation of D1/D5 dopamine receptors protects neurons from synapse dysfunction induced by amyloid-beta oligomers. J Biol Chem. 2011 Feb 4;286(5):3270-6. PubMed.
  3. . Apomorphine treatment in Alzheimer mice promoting amyloid-β degradation. Ann Neurol. 2011 Feb;69(2):248-56. PubMed.