1354 VOLUME 9 | NUMBER 11 | NOVEMBER 2006 NATURE NEUROSCIENCE

N E W S A N D V I E W S

Presenilins and Alzheimer disease: the calcium conspiracyGopal Thinakaran and Sangram S Sisodia

Most early-onset familial Alzheimer disease is caused by presenilin mutations. A recent paper reports that the presenilins act as calcium leak channels in the endoplasmic reticulum and thus may regulate intracellular calcium homeostasis.

Alzheimer disease (AD) affects over 4 million people in the United States alone. Most cases of AD are sporadic, but about 10% of all AD cases are inherited. These forms of familial AD (FAD) are usually early-onset (that is, the individual develops the disease before 65 years of age or so) and inherited as an autosomal dominant trait. The discovery that mutations in the PSEN1 and PSEN2 genes cause FAD spawned a wealth of molecular and cellular investigations aimed at unraveling the mechanisms by which the encoded polypeptides, termed presenilin 1 (PS1) and presenilin 2 (PS2), respectively, cause presenile dementia. PS are ubiquitously expressed, multipass membrane proteins that are known to function as the catalytic subunit of a large protease complex, termed γ- secretase, that promotes intramembranous proteolysis of a variety of type I membrane protein substrates, including the β-amyloid precursor protein (APP) and Notch receptors1. γ-Secretase processing of membrane-tethered APP-related derivatives leads to production of β-amyloid peptides (Aβ) that accumulate in the brains of aged individuals and patients with AD, while γ-secretase– mediated proteolysis of Notch and production of an intracellular derivative, NICD, is critical for cell fate specification during development and in proliferating cell populations in the adult. Now, in a recent issue of Cell, Tu and colleagues2 offer the proposal that PS1 dysfunction can contribute to AD pathogenesis independent of Aβ production or deposition. They report that PS1 and PS2 can form passive Ca2+ leak channels in the endoplasmic reticulum (ER), thus implicating defective Ca2+ signaling in AD.

However, our understanding of the mechanisms by which FAD-linked PS variants drive the pathogenesis of disease is still rather

limited. Newly synthesized PS holoproteins are subject to endoproteolysis to generate stable N- and C-terminal fragments (NTF and CTF)3. These PS derivatives assemble into a high-molecular-weight complex, γ-secretase, that consists of at least three other transmembrane proteins: nicastrin, PEN-2 and APH-1 (Fig. 1). The biogenesis, maturation and stability of each of the γ-secretase components are cross-regulated4. For example, the stability and accumulation of the PS NTF and CTF are regulated by the stoichiometric availability of nicastrin, APH-1 and PEN-2. Similarly, PS1 deficiency affects the stability and intracellular trafficking of nicastrin, PEN-2 and APH-1. Expression of FAD-linked PS1 variants is thought to cause disease by elevating concentrations of highly fibrillogenic and neurotoxic 42–amino acid Aβ (Aβ42) peptides—a

‘gain of aberrant function’5; however, this notion has been challenged (see discussion in Alzheimer Research Forum; ref. 6). Studies in cultured cells and transgenic mice have revealed that although expression of FAD-linked PS1 variants elevates production of Aβ42 peptides, there is also a reduction of 40–amino acid Aβ (Aβ40) peptides, a species that appears to inhibit nucle-ation and subsequent fibrillization of the Aβ42 peptides. Significantly, the alterations in Aβ42/Aβ40 ratios are due entirely to the presence of the FAD-linked substitutions. However, PS mutations (over 85 in number) are scattered throughout the PS polypeptide, and occur some distance from the putative catalytic site.

In the new paper, Tu and colleagues explore a nonproteolytic function of PS1. They report that PS1 and PS2 function

The authors are at the Department of Neurobiology,

Pharmacology & Physiology, University of Chicago,

Chicago, Illinois 60637, USA.

e-mail: gopal@uchicago.edu or

ssisodia@bsd.uchicago.edu

Figure 1 Two major functions of PS1. The vast majority of newly-synthesized PS1 holoprotein assembles with nicastrin, PEN-2 and APH-1 in the ER to form γ-secretase complexes. During this assembly process, PS1 holoprotein undergoes endoproteolysis within a prominent cytosolic loop domain (red asterisks). PS1 holoprotein is relatively short-lived (t1/2 < 1 h), whereas processed PS1-derivatives are extremely stable (t1/2 > 24 h). γ-Secretase is found localized in multiple subcellular organelles, where it facilitates proteolysis of a variety of type I membrane protein substrates. Tu et al.2 propose that PS1 holoprotein functions as a low-conductance, cation-permeable ion channel in the ER. TGN, trans-Golgi network; PM, plasma membrane.

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NATURE NEUROSCIENCE VOLUME 9 | NUMBER 11 | NOVEMBER 2006 1355

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as passive ER Ca2+ leak channels and that FAD-linked PS variants fail to exhibit this property2. First, using recombinant PS reconstituted in planar lipid bilayers, they demonstrate that PS1 holoprotein by itself can form low-conductance, divalent cation–permeable ion channels capable of transporting Ba2+, Cs+ and Na+ ions. PS-mediated currents were typically noisy, and the authors did not resolve individual single-channel opening events. Nevertheless, much smaller ion conductance was observed when recombinant PS that contained clinical PSEN-encoded mutations were tested. Second, Fura-2 Ca2+ imaging studies showed abnormal elevations of ER Ca2+ stores in PSEN1−/−PSEN2−/− fibroblasts; expression of wild-type but not clinical PS mutants attenuated ER Ca2+ levels. However, this was not the case in fibroblasts with targeted deletions of all three APH1 genes, indicating that full-length PS polypeptides function as ER Ca2+ leak channels in the absence of APH-1, and probably in the absence of other γ-secretase components. Third, ER PS-dependent Ca2+ leak channel function was not affected by an experimental mutant PS1 variant harboring a mutation of an aspartate residue in transmembrane domain 6 (D257) that is indispensable for γ-secretase activity. Tu et al. therefore propose that full-length PS polypeptides function as ER Ca2+ leak channels independent of association with other γ-secretase components. The direct consequence of impairment in passive ER Ca2+ leak function is an increase in ER luminal Ca2+ ion stores.

This study implicates defective Ca2+ ion conductance as the culprit that provokes disease in patients with mutant PSEN alleles2. Considering our current understanding of PS biology, several features of the study by Tu et al. make the findings all the more remarkable. First, the novel Ca2+ function is proposed to be associated with the full-length PS ‘holoprotein’, a species that is present at virtually undetectable steady-state levels in neuronal and non-neuronal tissue in vivo or in cultured cells3. Therefore, it remains to be seen if this PS holoprotein really has a significant role in vivo. Second, the Ca2+ leak channel function of PS is independent of γ-secretase activity, making this hard to reconcile with the data demonstrating that Aβ production and deposition correlate with neurodegeneration and cognitive impairments in AD. Third, although PS1 has been localized to multiple secretory and endocytic organelles and the plasma membrane, the Ca2+ leak channel function appears to be restricted to ER membranes,

and the significance of this is still unclear. Nonetheless, Tu et al. provide provocative

information on how PS may regulate intracellular Ca homeostasis. FAD-linked PS variants expressed in Xenopus oocytes result in a significant potentiation of inositol-1,4,5-trisphosphate (IP3)-evoked Ca2+ release7. Moreover, there is a lower threshold IP3 level for the generation of Ca2+ puffs in mutant PS1 cells, and mutant PS1 increases the rate and amount of Ca2+ release from the ER. These findings are consistent with abnormal elevation of oocyte ER Ca2+ stores, rather than altered number or activity of IP3- activated cal-cium release channels8. Exaggerated Ca2+ responses were also reported in PC12 cells, SH-SY5Y neuroblastoma cells and neurons derived from transgenic mice expressing FAD-linked mutant PS1 or PS2 (ref. 9). These earlier studies, however, provided limited information on how mutant PS caused overfilling of the ER Ca2+ stores.

PS also has a role in modulating capacitative calcium entry (CCE)—a refill-ing mechanism that regulates the coupled process of IP3-mediated release of ER Ca2+ and the replenishment of intracellular Ca2+ through plasma membrane channels. Cells that lack PS1 or express a dominant- negative PS1 mutant show a potentiation of CCE, whereas FAD-linked PS variants attenuate CCE10–12. Ca2+ influx by way of CCE in PS1−/− but not in wild-type neurons is sufficient to trigger long-term potentiation in hippocampal slice preparations, suggesting a physiological role for PS1 regulation of CCE in neuronal synaptic transmission13. In the Ca2+ imaging experiments reported by Tu et al., the fibroblasts were maintained in Ca2+-deficient buffers, essentially precluding any CCE activity. Therefore, it will be critical to carefully delineate the specific mechanisms by which PS regulates ER Ca2+ leak channel function and Ca2+ influx by way of the CCE pathway and the coordination of these processes that are critical for maintaining neuronal Ca2+ homeostasis.

Although the results reported by Tu et al. would now seem to provide direct evidence that fulfills the long-sought mechanism linking PS function to ER Ca2+ homeostasis, establishing the significance of these findings to neurodegeneration in AD remains a daunting task. There is some evidence linking defective Ca2+ homeostasis to AD: intracellular Ca2+ levels are elevated in embryonic fibroblasts, PC12 cells and primary neurons expressing PS1 mutants following trophic factor withdrawal or

exposure to Aβ peptide. Moreover, cells expressing mutant PS were vulnerable to cell death or more susceptible to β- amyloid toxicity, suggesting perturbed Ca2+ homeostasis as a potential mechanism by which mutant PS1 might sensitize neurons to apoptotic death14. Overexpression of the calcium-binding protein calbindin D28K along with mutant PS1 was sufficient to block cell death, strongly implying that a proapoptotic mechanism involving destabilization of Ca2+ homeostasis renders cells expressing mutant PS1 vulnerable to apoptosis15.

How can the FAD-linked PS- mediated defect in passive ER Ca2+ leak channel function explain the selective loss of vulnerable neurons resulting in the specific cognitive impairment seen in AD? Although the FAD-linked PS1 variants are impaired in Ca2+ leak channel function when expressed in mouse fibroblasts and in membranes prepared from Sf9 cells, it is a huge leap of faith to suggest, as do Tu et al., that their findings argue in support of a “Ca2+ hypothesis of AD”2. The present results only begin to offer a framework for understanding the potential role of PS in Ca2+ homeostasis. Future investigations in mutant PS1 neurons are necessary to understand how impaired ER Ca2+ leak channel function may contribute to short-term physiological consequences of altered synaptic transmission, and to define the relevance of these alterations to AD-related pathologies, neuronal loss and behavioral sequelae in transgenic mouse models.

1. Sisodia, S.S. & St. George-Hyslop, P.H. Nat. Rev. Neurosci. 3, 281–290 (2002).

2. Tu, H. et al. Cell 126, 981–993 (2006).3. Thinakaran, G. et al. Neuron 17, 181–190 (1996).4. Iwatsubo, T. Curr. Opin. Neurobiol. 14, 379–383

(2004).5. Price, D.L. & Sisodia, S.S. Annu. Rev. Neurosci. 21,

479–505 (1998).6. Davis, P. & De Strooper, B. http://www.alzforum.org/

res/for/journal/detail.asp?liveID=46 (2006).7. Leissring, M.A., Paul, B.A., Parker, I., Cotman, C.W.

& LaFerla, F.M. J. Neurochem. 72, 1061–1068 (1999).

8. Leissring, M.A., LaFerla, F.M., Callamaras, N. & Parker, I. Neurobiol. Dis. 8, 469–478 (2001).

9. Smith, I.F., Green, K.N. & LaFerla, F.M. Cell Calcium 38, 427–437 (2005).

10. Leissring, M.A. et al. J. Cell Biol. 149, 793–798 (2000).

11. Yoo, A.S. et al. Neuron 27, 561–572 (2000).12. Herms, J. et al. J. Biol. Chem. 278, 2484–2489

(2003).13. Ris, L., Dewachter, I., Reverse, D., Godaux, E. &

Van Leuven, F. J. Biol. Chem. 278, 44393–44399 (2003).

14. Mattson, M.P., Guo, Q., Furukawa, K. & Pedersen, W.A. J. Neurochem. 70, 1–14 (1998).

15. Guo, Q., Christakos, S., Robinson, N. & Mattson, M.P. Proc. Natl. Acad. Sci. USA 95, 3227–3232 (1998).

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