Scheper W, Hol EM. Protein quality control in Alzheimer's disease: a fatal saviour. Curr Drug Targets CNS Neurol Disord. 2005 Jun;4(3):283-92. Review. Abstract

View all comments by Jeroen Hoozemans
View all comments by Wiep Scheper

Ghribi O, Herman MM, Pramoonjago P, Spaulding NK, Savory J. GDNF regulates the A beta-induced endoplasmic reticulum stress response in rabbit hippocampus by inhibiting the activation of gadd 153 and the JNK and ERK kinases. Neurobiol Dis. 2004;16(2):417-27. Abstract

Ghribi O, Herman MM, Savory J. Lithium inhibits Abeta-induced stress in endoplasmic reticulum of rabbit hippocampus but does not prevent oxidative damage and tau phosphorylation. J Neurosci Res. 2003;71(6):853-62. Abstract

View all comments by Othman Ghribi

Calcium dysregulations have long been considered as a part of neuronal toxicity in AD, as also shown by mutations in presenilins. Likewise, infected cells in prion disease show calcium elevation but the mechanisms causing cell death have remained elusive. This paper shows a possible mechanism by which disturbed calcium regulation causes cell death through a crosstalk between the ER and mitochondria leading ultimately to caspase activation. The paper is highly recommended.

View all comments by Dan Lindholm

However, these important findings do not seem to have a direct application in Alzheimer disease. ER stress, and the consequent UPR, are not implicated in β amyloid production, or in APP processing, as shown by my and other's groups (Siman et al, JBC, 2001; Piccini et al, Neurobiology of disease, 2004). Instead, inhibition of the effects of ER stress may be potentially beneficial in neurodegenerative disorders characterized by intracellular toxic aggregates, such as Parkinson disease and tauopathies, in which ER stress may contribute to the creation of misfolded peptides.

View all comments by Massimo Tabaton

Discrepancies in Two Recent Papers on ER Ca2+-leak Channels in Presenilin1, -2 Double Knockout Cells
This paper describes presenilin (PS)-related mechanisms that affect Ca2+ leak from the endoplasmic reticulum (ER). However, it points to a very different mechanism—Ca2+-channel leak properties of presenilin—to that which we have recently published: upregulation of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) (Kasri et al., 2006). Although these two conclusions are not mutually exclusive, the niggling point is that both papers report very different and even sometimes opposing experimental findings. There is no obvious explanation for these discrepancies, but it is clear that all methodologies currently applied to evaluate ER Ca2+ concentrations and ER Ca2+ leak are imperfect and often lead to contradictory results. This was extensively discussed by Clark Distelhorst and Gordon Shore in their recent review of the conflicting findings...  Read more

Discrepancies in Two Recent Papers on ER Ca2+-leak Channels in Presenilin1, -2 Double Knockout Cells
This paper describes presenilin (PS)-related mechanisms that affect Ca2+ leak from the endoplasmic reticulum (ER). However, it points to a very different mechanism—Ca2+-channel leak properties of presenilin—to that which we have recently published: upregulation of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) (Kasri et al., 2006). Although these two conclusions are not mutually exclusive, the niggling point is that both papers report very different and even sometimes opposing experimental findings. There is no obvious explanation for these discrepancies, but it is clear that all methodologies currently applied to evaluate ER Ca2+ concentrations and ER Ca2+ leak are imperfect and often lead to contradictory results. This was extensively discussed by Clark Distelhorst and Gordon Shore in their recent review of the conflicting findings regarding the effects of Bcl-2 proteins on ER Ca2+ (Distelhorst and Shore 2004).

Before analyzing potential reasons why different experimental findings have been obtained in these two presenilin papers, I will first summarize the findings in each that are not disproved by findings in the other.

The most important observation in our paper is that there is an isoform-specific fourfold upregulation of IP3R1 in murine embryonic fibroblast (MEF) PS double knockout (dko) cells. This observation was very solid, as it was done not only using isoform-specific antibodies against both isoforms of IP3R, but also using a common antibody that allows a simultaneous detection of both receptor isoforms. The latter allows a determination of the relative expression of IP3R-isoforms and is therefore independent of load controls that are problematic when comparing different cell types. One such control, actin, can be especially problematic because the dko cells are characterized by quite a different cellular morphology. Moreover, the fact that the enhanced Ca2+ leak could be reversed using siRNA-mediated downregulation of specifically IP3R1 demonstrates that the increased level in IP3R1 is the primary cause of this leak. The role of IP3R1 as an ER Ca2+-leak channel is in agreement with findings from other groups (Oakes et al., 2005).

The major observation in the paper by Tu et al. is that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. These channel properties of presenilins were confirmed using lipid bilayer reconstitution of the purified proteins, and it suggests a Ca2+ signaling function for presenilins which would provide further support for the “Ca2+ hypothesis of AD.”

While the basic observations of both papers may point to two of the potential mechanisms for ER Ca2+ leak, it should be clear that many other leak pathways may coexist, and, as was adequately discussed by Tu et al., the exact identity of ER Ca2+ leak channels still largely remains an “enigma of Ca2+ signaling” (Camello et al., 2002).

This is where the deviating observations and conflicting results come in. Even worse, the basic observation about the ER Ca2+ level is exactly the opposite in both papers. Although essentially the same immortalized mouse embryonic cell lines (MEF and MEF dko fibroblasts) were used, we found that MEF PS dko cells had a lower ER Ca2+ level, explained by increased IP3R1-mediated leak, whereas in the Tu et al. paper the opposite was found—as may be expected if the leak occurs via presenilins, which have been knocked out. There is no explanation for this discrepancy except that different techniques were used to measure ER (Ca2+) and the Ca2+ leak. In our hands, targeted aequorins were used to estimate ER (Ca2+) and saponin-permeabilized monolayers were used for estimating the leak rate. In the Tu et al. paper, Mag-Fura was used for ER Ca2+ measurement, and Fura-2 fluorescence was used for evaluating Ca2+ fluxes in microsomes.

It is very clear that all these methods have their own drawbacks, and the main problem would appear to be what fraction of the ER is actually measured. In each of the methods used there are uncertainties as to whether only the ER is targeted and whether it may be disturbed by the preparation procedures. As a result it may very well be that different subfractions of the ER have been evaluated. The depletion of the ER in the aequorin method or the use of saponin may have affected the structure of the ER. The preparation of microsomes, on the other hand, certainly results in a mixture of membrane fractions, the distribution and purity of which may also be different for different cell types. A second drawback in these studies is the means used to deplete the ER: ionomycin is not specific and will deplete all Ca2+-containing compartments, whereas thapsigargin will target only those compartments filled up by the SERCA-type Ca2+ pump. In our work, the saponin-permeabilized monolayers largely reflect the thapsigargin-dependent Ca2+ stores, and the small thapsigargin-independent part was subtracted in the calculations. For the preparations in the Tu et al. paper, both for intact cells and for microsomes, large differences were observed between ionomycin-releasable and thapsigargin-releasable Ca2+. These data are interpreted as a measure of the total Ca2+ content and the rate of Ca2+ release, respectively. It is, however, not established that the ionomycin-derived Ca2+ content only reflects the ER. Furthermore, the thapsigargin-induced Ca2+ release rate in intact cells may not only reflect ER Ca2+ release but also the rate of Ca2+ efflux driven by PMCA or Na+/Ca2+ exchanger and Ca2+ uptake by the thapsigargin-independent stores (Golgi, mitochondria). Moreover, in microsomal preparations, the Ca2+-release rate will not only depend on the distribution of presenilin in the different microsomal fractions but also on their diameter, composition, and aggregation, and these parameters may be variable if preparations have to be made from different cell types. One should keep in mind that the MEF dko cells are defective cells that grow more slowly and have different morphology as compared to the wild-type cells. This may result in microsomal fractions with different biochemical and physical properties.

In conclusion, both papers have provided evidence for new mechanisms of ER Ca2+ control, and these mechanisms are clearly related to presenilin expression and may therefore play a role in the pathology of AD. However, the quantitative significance of these leak pathways in intracellular compartments, and particularly in different ER fractions, is very difficult to evaluate. This is largely because there are no fool-proof methods for obtaining preparations that truly reflect and measure the properties of the ER in a real cellular context. Moreover, the cellular heterogeneity of the ER and the existence of other membrane compartments, where IP3Rs or presenilins may operate, remain difficult to fully appreciate. Finally, the molecular tools to evoke ER-related Ca2+ fluxes are imperfect and not equally reliable in all conditions. Appreciation of the significance of the above-mentioned mechanisms for neuronal function and dysfunction will have to wait until more adequate techniques for measuring cellular Ca2+ signals are available.

View all comments by Humbert De Smedt

The planar lipid bilayer approach was, therefore, an ideal preparation to start addressing presenilin function in membranes and its relation to the Ca2+ signaling dysregulation seen with certain AD-linked presenilin mutations. This technique allows one to insert specific channels of interest into a...  Read more

The planar lipid bilayer approach was, therefore, an ideal preparation to start addressing presenilin function in membranes and its relation to the Ca2+ signaling dysregulation seen with certain AD-linked presenilin mutations. This technique allows one to insert specific channels of interest into a modified “model membrane” in order to observe and manipulate their function. Given that wild-type presenilin can form cation-permeable channels in these lipid bilayer models—and that the PS1-M146V and PS2-N141I mutants are impaired in this function—the extension to biological models using murine embryonic fibroblasts (MEFs) and rescue experiments in PS double knockouts (PS-DKOs) becomes easier to interpret and certainly more powerful. Hypothesizing that ER stores overfill due to impaired Ca2+ leak current through presenilin channels is a novel proposition, and it is backed up by clear mechanistic evidence in both model membranes and biological systems.

This study is particularly elegant in that it contributes to our understanding of presenilin function at several levels. At the basic science level, we have new insight into the role of presenilin in the ER—why it is even located there (addressing the spatial paradox)—and a novel candidate for the leak channel—which has been inferred but never really seen. And, since the leak function is separate from its role in the γ-secretase complex, these results also imply an additional, separate, and parallel role of the presenilins in maintaining Ca2+ homeostasis. At the neuropathology level, this is the first real mechanistic study that can point to how AD-linked presenilin mutations can result in increased ER Ca2+ stores through a loss of function.

At a more global level, there is still much to be explored regarding how mutant PS and ER Ca2+ signaling dysregulations are linked to the pathophysiology of AD. Primarily, can impaired Ca2+ leak channels be linked to Aβ plaque formation and neurofibrillary tangles that are diagnostic of AD, or are they a separate and independent phenomenon in the disease process? Interestingly, in Tu’s study, not all PS mutations generated the same channel phenotype, and this will ultimately need reconciling. The PS1-δE9 mutation resulted in an apparent gain of function with increased cation conductance—yet in humans, the M146V and δE9 mutations ultimately result in the same disease state. In the basic research realm, an additional point that needs reconciling is the conflicting data regarding SERCA pump blockers (e.g., thapsigargin). In several studies examining effects of mutant PS1, application of SERCA blockers results in enhanced Ca2+ release into the cytosol (Guo et al., 1997; Leissring et al., 2000; Herms et al., 2003; Stutzmann, personal observation in brain slice preparations), which is at odds with the proposed reduction in the PS-leak channel conductance and the raw data presented in the Tu et al., study.

Determining if/how presenilin interacts with other ER Ca2+ channels such as the IP3 and ryanodine receptors is an important next step, particularly in light of several recent studies demonstrating an increase in ryanodine receptor number and function in PS1-M146V expressing neurons (Chan et al., 2000; Smith et al., 2005; Stutzmann et al., 2006). So, there is likely still more to the PS story that has yet to be uncovered, but this study provides vital information to both the basic science and AD fields, and infuses new life into the Ca2+ hypothesis of AD. And, perhaps most importantly, it provides a clear new direction with which to focus future PS-Ca2+ signaling studies.

References:
Guo Q, Sopher BL, Furukawa K, Pham DG, Robinson N, Martin GM, Mattson MP. Alzheimer's presenilin mutation sensitizes neural cells to apoptosis induced by trophic factor withdrawal and amyloid beta-peptide: involvement of calcium and oxyradicals. J Neurosci. 1997 Jun 1 ; 17(11):4212-22. Abstract

Leissring MA, Akbari Y, Fanger CM, Cahalan MD, Mattson MP, LaFerla FM. Capacitative calcium entry deficits and elevated luminal calcium content in mutant presenilin-1 knockin mice. J Cell Biol. 2000 May 15 ; 149(4):793-8. Abstract

Herms J, Schneider I, Dewachter I, Caluwaerts N, Kretzschmar H, Van Leuven F. Capacitive calcium entry is directly attenuated by mutant presenilin-1, independent of the expression of the amyloid precursor protein. J Biol Chem. 2003 Jan 24 ; 278(4):2484-9. Abstract

Chan SL, Mayne M, Holden CP, Geiger JD, Mattson MP. 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. Abstract

Smith IF, Green KN, LaFerla FM. Calcium dysregulation in Alzheimer's disease: recent advances gained from genetically modified animals. Cell Calcium. 2005 Sep-Oct ; 38(3-4):427-37. Abstract

Stutzmann GE, Smith I, Caccamo A, Oddo S, LaFerla FM, Parker I. 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. Abstract

View all comments by Grace (Beth) Stutzmann

The first finding obtained by Bezprozvanny and colleagues, showing that PSs are leak channels, does not contradict our published data: we have repeatedly demonstrated that overexpression of wt-PS2 and, to a lesser extent, also of wt-PS1, reduces the ER Ca2+ level in different cell models (Zatti et al., 2004; Giacomello et al., 2005;...  Read more

The first finding obtained by Bezprozvanny and colleagues, showing that PSs are leak channels, does not contradict our published data: we have repeatedly demonstrated that overexpression of wt-PS2 and, to a lesser extent, also of wt-PS1, reduces the ER Ca2+ level in different cell models (Zatti et al., 2004; Giacomello et al., 2005; Zatti et al., 2006). Surprisingly, what is not consistent with our findings is the fact that, in our models, the expression of various FAD-linked PS mutants often results in a “gain of function” if considering the effect of PSs on the ER leakage. In fact, the ability of wt PSs to reduce ER Ca2+ release is also shared by different PS mutants: PS2-M239I (Zatti et al., 2004); PS2-T122R (Giacomello et al., 2005); PS2-N141I, PS2-D366A, PS1-A246E, PS1-M146L, PS1-P117L (Zatti et al., 2006). Notably, these mutations include two mentioned by Bezprozvanny and colleagues (PS2-N141I and PS1-M146L/V), as well as one devoid of γ-secretase activity (PS2-D366A).

We have published data (Zatti et al., 2006) showing that the store Ca2+ content is unchanged or even reduced when PSs are expressed in different cell models either stably (such as in human FAD fibroblasts, HEK293, and SH-SY5Y clones) or transiently (such as in HeLa and SH-SY5Y cells, MEFs, and primary cultures of rat neurons). These results were obtained by using two different methodological approaches, that is, by cytosolic Ca2+ imaging with fura-2 (as described by Bezprozvanny and colleagues) and by recombinant ER-targeted aequorin (as described by Kasri et al., 2006). No evidence of an exaggerated Ca2+ release was found in cells expressing any of the investigated PS2 (M239I, -T122R, -N141I, -D366A) or PS1 (-A246E, -L286V, -M146L, -P117L) mutations. Similarly, no Ca2+ overload was found when directly measuring ER and Golgi apparatus Ca2+ levels (using appropriately targeted aequorins) in HeLa and SH-SY5Y cells overexpressing the above-mentioned PS mutants (Zatti et al., 2006), as well as in the stable clones HEK293/PS1-M146L and SH-SY5Y/PS2-T122R and in DKO MEF cells (our unpublished data, and see also Kasri et al., 2006). Consistently, DKO MEFs did not show an increased Ca2+ store content if compared to MEFs expressing only the wt-PS1 when measuring the cytosolic Ca2+ changes induced by store depletion with cyclopiazonic acid (Zatti et al., 2006).

Thus, the reasons for these discrepancies cannot merely be due to differences in the methodology employed for Ca2+ measurements. The true reasons for such discrepancies should indeed be sought if one wishes to shed light on this complex phenomenon. Conversely, ignoring them does not help the AD community and, more importantly, hinders scientific progress.

We believe that, among the different models employed in this type of investigation, human fibroblasts from FAD patients should be given at least the same weight as MEFs, not least because we are interested in the human pathology. A reduced and not an exaggerated Ca2+ release was detected by cytosolic fura-2 measurements in human FAD-fibroblasts carrying the PS1-M146L (two patients) or the PS1-P117L (one patient), whose donors were presenting a devastating early-age-of-onset AD (30 years for the PS1-P117L-carrying subject; Zatti et al., 2006). A stronger reduction in ER Ca2+ content was inferred with the same technique in human FAD-fibroblasts carrying the PS2-M239I (two patients) or the PS2-T122R (two patients) when compared to healthy age-matched control subjects (Zatti et al., 2004; Giacomello et al., 2005).

It is also worth noting that the “abnormal Ca2+ signaling” usually reported for human FAD fibroblasts not always means an increased Ca2+ load since a reduced Ca2+ release was also observed (Peterson et al., 1988; McCoy et al., 1993). The discussion on this issue is further complicated by the fact that the large majority of the studies with AD fibroblasts were carried out in the 1980s-1990s when Alzheimer donors were not genetically characterized. Interestingly, by using aequorin, McCoy et al. (1993) showed a reduced Ca2+release in human early-onset FAD fibroblasts from a Canadian family which was recently shown to carry the PS1-A246E mutation (Huang et al., 2005).

Furthermore, we have to consider that, in FAD fibroblasts, at variance with the rescued DKO MEFs, the PS mutant exerts its effect in the presence of the endogenous wild-type proteins, as occurring also in the majority of the cell models tested. This fact makes the comparison even more problematic.

Given the suggested protective role exerted by a low ER Ca2+ level (Scorrano et al., 2003), we proposed that PS mutations which strongly reduce the ER Ca2+ content (such as those in PS2) should attenuate the pathology, whereas other mutations that leave the ER Ca2+ content unchanged or mildly reduced (such as those in PS1) could be unable to compensate for other defects due to the mutations themselves. Indeed, oxidative stress induces a pronounced Ca2+ overload in PS1-A246E-FAD fibroblasts compared to aged controls (Huang et al., 2005). Our hypothesis is thus consistent with the later ages of onset and milder AD phenotypes observed in patients carrying PS2 mutations with respect to those carrying PS1 ones.

In summary, as far as the physiological role of PSs is concerned, our results are in agreement with those reached by Bezprozvanny and colleagues. However, the presence of contrasting findings with the pathological mutations shows the limitations of the simple definition “gain or loss of function” for multifaceted proteins such as PSs, especially when considering how different are the backgrounds in which their effects are evaluated.

References:
Giacomello M, Barbiero L, Zatti G, Squitti R, Binetti G, Pozzan T, Fasolato C, Ghidoni R, Pizzo P. Reduction of Ca2+ stores and capacitative Ca2+ entry is associated with the familial Alzheimer's disease presenilin-2 T122R mutation and anticipates the onset of dementia. Neurobiol Dis. 2005 Apr;18(3):638-48. Abstract

Kasri NN, Kocks SL, Verbert L, Hebert SS, Callewaert G, Parys JB, Missiaen L, De Smedt H. Up-regulation of inositol 1,4,5-trisphosphate receptor type 1 is responsible for a decreased endoplasmic-reticulum Ca2+ content in presenilin double knock-out cells. Cell Calcium. 2006 Jul;40(1):41-51. Epub 2006 May 3. Abstract

Huang HM, Chen HL, Xu H, Gibson GE. Modification of endoplasmic reticulum Ca2+ stores by select oxidants produces changes reminiscent of those in cells from patients with Alzheimer disease. Free Radic Biol Med. 2005 Oct 15;39(8):979-89. Abstract

McCoy KR, Mullins RD, Newcomb TG, Ng GM, Pavlinkova G, Polinsky RJ, Nee LE, Sisken JE. Serum- and bradykinin-induced calcium transients in familial Alzheimer's fibroblasts. Neurobiol Aging. 1993 Sep-Oct;14(5):447-55. Abstract

Peterson C, Ratan RR, Shelanski ML, Goldman JE. Altered response of fibroblasts from aged and Alzheimer donors to drugs that elevate cytosolic free calcium. Neurobiol Aging. 1988 May-Jun;9(3):261-6. Abstract

Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T, Korsmeyer SJ. BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science. 2003 Apr 4;300(5616):135-9. Epub 2003 Mar 6. Abstract

Smith IF, Green KN, LaFerla FM. Calcium dysregulation in Alzheimer's disease: recent advances gained from genetically modified animals. Cell Calcium. 2005 Sep-Oct;38(3-4):427-37. Review. Abstract

Zatti G, Ghidoni R, Barbiero L, Binetti G, Pozzan T, Fasolato C, Pizzo P. The presenilin 2 M239I mutation associated with familial Alzheimer's disease reduces Ca2+ release from intracellular stores. Neurobiol Dis. 2004 Mar;15(2):269-78. Abstract

Zatti G, Burgo A, Giacomello M, Barbiero L, Ghidoni R, Sinigaglia G, Florean C, Bagnoli S, Binetti G, Sorbi S, Pizzo P, Fasolato C. Presenilin mutations linked to familial Alzheimer's disease reduce endoplasmic reticulum and Golgi apparatus calcium levels. Cell Calcium. 2006 Jun;39(6):539-50. Epub 2006 Apr 18. Abstract

View all comments by Giuliano Binetti
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View all comments by Sandro Sorbi

I performed aluminum neurotoxicity experiments on hippocampal rat neurons several years ago and found dantrolene and dimethylsulfoxide reduced cell death from aluminum toxicity, indicating aluminum toxicity may be mediated through release of calcium from intracellular stores and oxidative stress (1).

There may be multiple mechanisms disrupting calcium metabolism in the endoplasmic reticulum, including metals such as aluminum and other metals potentially capable of oxidation such as copper and iron. Oxidative...  Read more

I performed aluminum neurotoxicity experiments on hippocampal rat neurons several years ago and found dantrolene and dimethylsulfoxide reduced cell death from aluminum toxicity, indicating aluminum toxicity may be mediated through release of calcium from intracellular stores and oxidative stress (1).

There may be multiple mechanisms disrupting calcium metabolism in the endoplasmic reticulum, including metals such as aluminum and other metals potentially capable of oxidation such as copper and iron. Oxidative stress might also be implicated as well.

I am not sure how β amyloid could effect calcium metabolism within the endoplasmic reticulum and other intracellular stores, but amyloid precursor protein could be implicated as well, since it may be capable of forming ion channels.

Disruption of calcium metabolism and β amyloid toxicity may act synergistically in causing cellular dysfunction and Alzheimer disease.

If intracellular structures such as endoplasmic reticulum and sarcoplasmic reticulum are effected by disturbed calcium metabolism, protein assembly in intracellular structures may result in dysfunctional proteins unable to perform intracellular processes normally with subsequent cellular death and resulting Alzheimer disease.

References:
Brenner S. Aluminum neurotoxicity is reduced by dantrolene and dimethylsulfoxide in cultured rat hippocampal neurons. Biol Trace Elem Res. 2002 Apr; 86 (1) 85-89. Abstract

View all comments by Steven Brenner

Under resting conditions, steady-state [Ca2+]L is determined by the dynamic equilibrium of two components: an active Ca2+ uptake mediated by ATP-dependent Ca2+ pumps of the SERCA family and passive Ca2+ efflux via leak channels. Even though this pump-leak cycle appears to be a common property of Ca2+-storing organelles, little is known about the proteins controlling the Ca2+ leak pathway. Several...  Read more

Under resting conditions, steady-state [Ca2+]L is determined by the dynamic equilibrium of two components: an active Ca2+ uptake mediated by ATP-dependent Ca2+ pumps of the SERCA family and passive Ca2+ efflux via leak channels. Even though this pump-leak cycle appears to be a common property of Ca2+-storing organelles, little is known about the proteins controlling the Ca2+ leak pathway. Several mechanisms involving quite different proteins have been previously suggested to explain the basal Ca2+ leak from ER, namely: 1) reverse Ca2+ flux through the pumps (Toyoshima et al., 2002); 2) Ca2+ leak in neutral complexes with small molecules by translocon channels (Lomax et al., 2002; Van Coppenolle et al., 2004); 3) the fluxes of Ca2+ through “natural” ionophores, such as bile acids (Zimniak et al., 1991); 4) an anti-apoptotic protein Bcl-2–mediated Ca2+ leak (Bassik et al., 2004); 5) IP3R- or RYR-mediated Ca2+ leak (Oakes et al., 2005) and, more recently, 6) pannexin 1-mediated calcium leak (Vanden Abeelle et al., 2006). However, “the drawing of these mechanisms is only a fantasy map of the leak terra incognita, and discovery of the exact mechanisms of calcium leak remains a challenge to scientists working in the calcium signaling field.” (Camello et al., 2002).

The team of Ylya Bezprozvanny, using a multidisciplinary approach, clearly demonstrates that the nine transmembrane domain ER proteins, presenilins, account for almost 80 percent of passive calcium leak from the ER. The results of their study strongly suggest that presenilins can form calcium-permeable ion channels and, moreover, that the genetic deletion of presenilins (in double knockout, DKO, mice) resulted in a sixfold reduction in the rate of calcium leak across ER membrane. Heterologous expression of presenilins in DKO mouse embryonic fibroblasts was able to rescue calcium leakage defects observed in DKO cells, which is consistent with an ion “leak-channel” function of presenilins in the ER membrane.

Even more intriguing is the finding that presenilin mutants associated with familial Alzheimer disease (FAD) were not able to form calcium leak channels. Thus, the results of this study strongly support the hypothesis of a crucial role of calcium homeostasis in Alzheimer disease, pointing out the specific function of presenilins.

References:
Bassik MC, Scorrano L, Oakes SA, Pozzan T, Korsmeyer SJ. Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis. EMBO J. 2004 Mar 10;23(5):1207-16. Epub 2004 Mar 4. Abstract

Camello C, Lomax R, Petersen OH, Tepikin AV. Calcium leak from intracellular stores--the enigma of calcium signalling. Cell Calcium. 2002 Nov-Dec;32(5-6):355-61. Review. Abstract

Lomax RB, Camello C, Van Coppenolle F, Petersen OH, Tepikin AV. Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons. J Biol Chem. 2002 Jul 19;277(29):26479-85. Epub 2002 May 6. Abstract

Toyoshima C, Nomura H. Structural changes in the calcium pump accompanying the dissociation of calcium. Nature. 2002 Aug 8;418(6898):605-11. Abstract

Vanden Abeele F, Skryma R, Shuba Y, Van Coppenolle F, Slomianny C, Roudbaraki M, Mauroy B, Wuytack F, Prevarskaya N. Bcl-2-dependent modulation of Ca(2+) homeostasis and store-operated channels in prostate cancer cells. Cancer Cell. 2002 Mar;1(2):169-79. Abstract

Vanden Abeele F, Bidaux G, Gordienko D, Beck B, Panchin YV, Baranova AV, Ivanov DV, Skryma R, Prevarskaya N. Functional implications of calcium permeability of the channel formed by pannexin 1. J Cell Biol. 2006 Aug 14;174(4):535-46. Abstract

Van Coppenolle F, Vanden Abeele F, Slomianny C, Flourakis M, Hesketh J, Dewailly E, Prevarskaya N. Ribosome-translocon complex mediates calcium leakage from endoplasmic reticulum stores. J Cell Sci. 2004 Aug 15;117(Pt 18):4135-42. Epub 2004 Jul 27. Abstract

Zimniak P, Little JM, Radominska A, Oelberg DG, Anwer MS, Lester R. Taurine-conjugated bile acids act as Ca2+ ionophores. Biochemistry. 1991 Sep 3;30(35):8598-604. Abstract

View all comments by Natalia Prevarskaya

Binetti and colleagues raise interesting questions about the effects of presenilin FAD mutations on ER Ca2+ content and on inositol trisphosphate receptor (InsP3R)-mediated Ca2+ release. We attempted to reconcile our results with that of Zatti at al.; however, we ran into significant difficulties in interpreting their data.

Let us consider an example of two PS1 FAD mutants for which extensive datasets are available from several laboratories. Zatti et al. reported that expression of PS1-M146L resulted in reduced Ca2+ response to cyclopiazonic acid (CPA) + histamine (Fig. 1C), no change in response to CPA + bradykinin (BK) (Fig. 1B), ...  Read more

Binetti and colleagues raise interesting questions about the effects of presenilin FAD mutations on ER Ca2+ content and on inositol trisphosphate receptor (InsP3R)-mediated Ca2+ release. We attempted to reconcile our results with that of Zatti at al.; however, we ran into significant difficulties in interpreting their data.

Let us consider an example of two PS1 FAD mutants for which extensive datasets are available from several laboratories. Zatti et al. reported that expression of PS1-M146L resulted in reduced Ca2+ response to cyclopiazonic acid (CPA) + histamine (Fig. 1C), no change in response to CPA + bradykinin (BK) (Fig. 1B), no change for response to CPA + carbamylcholine (CaCh) in HEK293 stable lines (Fig. 3B), no change in ER Ca2+ levels (Fig. 4C) and reduced Golgi Ca2+ levels (Fig. 5C). They also report that CPA response was reduced in human PS1-M146L fibroblasts (Fig. 1D). The response in neurons transfected with PS1-M146L was not tested in Zatti at al. paper.

It is hard to reach a conclusion from these results about the effect of PS1-M146L on ER Ca2+ signaling. Most data in Zatti at al. suggest that PS1-M146L has either no effect or reduces the ER Ca2+ content and the InsP3R-mediated Ca2+ release. This conclusion directly contradicts our results with transfected DKO MEF cells [2] and the extensive characterization of the effects of PS1-M146V mutant on the InsP3R-mediated Ca2+ signals in Xenopus oocytes and in hippocampal neurons by Parker and La Ferla’s laboratories [3,4].

A similar situation exists for PS1-A246E FAD mutant. Zatti et al. reported that expression of PS1-A246E resulted in reduced Ca2+ response to CPA + histamine (Fig. 1C), reduced Ca2+ response to CPA + BK (Fig. 2B), reduced ER Ca2+ levels in HeLa cells, but unchanged ER Ca2+ levels in SH-SY5Y cells (Fig. 4C), reduced Golgi Ca2+ levels in both HeLa and SH-SY5Y (Fig. 5C), and no change in CaCh-induced response in transfected rat cortical neurons (Fig. 6D).

Once again, most of these results seem to indicate that PS1-A246E has either no effect or reduces the ER Ca2+ content and the InsP3R-mediated Ca2+ release. This conclusion directly contradicts our results with transfected DKO MEF cells and human A246E fibroblasts [5], and results from studies of the InsP3R-mediated Ca2+ signals in hippocampal neurons from A246E transgenic mouse performed by Jochen Herms’s laboratory [6].

In summary, we agree with Giuliano Binetti and colleagues that effects of FAD mutations in presenilins on ER Ca2+ homeostasis is a very exciting and important area of AD research. However, as it is clear from the above discussion, much additional work by many laboratories will be required to clarify the exact nature of this interesting phenomenon.

References:
1. Zatti G, Burgo A, Giacomello M, Barbiero L, Ghidoni R, Sinigaglia G, Florean C, Bagnoli S, Binetti G, Sorbi S, Pizzo P, Fasolato C. Presenilin mutations linked to familial Alzheimer's disease reduce endoplasmic reticulum and Golgi apparatus calcium levels. Cell Calcium. 2006 Jun;39(6):539-50. Epub 2006 Apr 18. Abstract

2. Tu H, Nelson O, Bezprozvanny A, Wang Z, Lee SF, Hao YH, Serneels L, De Strooper B, Yu G, Bezprozvanny I. Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations. Cell. 2006 Sep 8;126(5):981-93. Abstract

3. Leissring MA, Paul BA, Parker I, Cotman CW, LaFerla FM. Alzheimer's presenilin-1 mutation potentiates inositol 1,4,5-trisphosphate-mediated calcium signaling in Xenopus oocytes. J Neurochem. 1999 Mar;72(3):1061-8. Abstract

4. Stutzmann GE, Caccamo A, LaFerla FM, Parker I. Dysregulated IP3 signaling in cortical neurons of knock-in mice expressing an Alzheimer's-linked mutation in presenilin1 results in exaggerated Ca2+ signals and altered membrane excitability. J Neurosci. 2004 Jan 14;24(2):508-13. Abstract

5. Nelson O, Tu H, Lei T, Bentahir M, de Strooper B and Bezprozvanny I. (2006) Familial Alzheimer's disease mutations in presenilin 1 disrupt endoplasmic reticulum calcium leak function. Submitted.

6. Schneider I, Reverse D, Dewachter I, Ris L, Caluwaerts N, Kuiperi C, Gilis M, Geerts H, Kretzschmar H, Godaux E, Moechars D, Van Leuven F, Herms J. Mutant presenilins disturb neuronal calcium homeostasis in the brain of transgenic mice, decreasing the threshold for excitotoxicity and facilitating long-term potentiation. J Biol Chem. 2001 Apr 13;276(15):11539-44. Epub 2001 Jan 23. Abstract

View all comments by Ilya Bezprozvanny

In my opinion, the research by Bezprozvanny and colleagues emphasizes the importance of the fundamental role of free calcium modifications in cells undergoing biochemical changes underlying AD. While not questioning the key role of Aβ peptides in this disease, the data add another possible dimension to the key role performed by calcium in cellular stress and death following the biochemical modifications characterizing AD. Hence, some presenilin mutations affecting γ-secretase activity can impair cell viability by increasing Aβ peptide production or by shifting the latter towards the more amyloidogenic Aβ42, resulting in Aβ oligomerization and cell membrane(s) permeabilization. Other mutations that do...  Read more

In my opinion, the research by Bezprozvanny and colleagues emphasizes the importance of the fundamental role of free calcium modifications in cells undergoing biochemical changes underlying AD. While not questioning the key role of Aβ peptides in this disease, the data add another possible dimension to the key role performed by calcium in cellular stress and death following the biochemical modifications characterizing AD. Hence, some presenilin mutations affecting γ-secretase activity can impair cell viability by increasing Aβ peptide production or by shifting the latter towards the more amyloidogenic Aβ42, resulting in Aβ oligomerization and cell membrane(s) permeabilization. Other mutations that do not affect γ-secretase activity can disrupt calcium leakage from the ER, resulting in increased calcium stores in the ER and subsequent ER stress. It cannot be excluded that, for some specific PS mutations, the two effects may act synergistically with more severe cellular stress.

View all comments by Massimo Stefani

Recently, reports from two independent labs (Tsuda et al. and Ratnaparkhi et al.) have provided new and exciting insight on the normal function of VAP proteins and their possible role in the pathogenesis of VAP-induced ALS. Comments on these papers can be summarized as follows.

The paper by Tsuda et al. reports that VAP proteins are cleaved, and an N-terminal fragment of a size compatible with the size of the MSP domain is secreted and binds to the Eph receptors. The pathogenic allele induces the accumulation of the mutant and the wild-type (wt) protein into the ER and a failure to secrete the cleaved MSP domain. Non cell-autonomous effects of the mutant and wt proteins have been reported both at the level of the Drosophila nervous system and the nematode reproductive system. The ability of dVAP to be cleaved and secreted has been shown with an elegant experiment in which the expression of dVAP has been driven in a subset of cells in the wing imaginal discs. A diffusion of dVAP MSP beyond the protein expressing cells was observed. However, there is no direct evidence that this process of cleavage and secretion of VAP proteins is occurring in neurons, in muscles, or in any other tissue that would be more relevant to the human disease.

The ability of the pathogenic allele to induce the formation of aggregates has been previously reported in cell culture (1,4,5) and Drosophila model systems (3). Tsuda and colleagues report that expression of the mutant protein in a null background induces the formation of detergent-insoluble aggregates. Despite the mutant allele being inherited in a dominant manner in humans, these data lead to the important conclusion that the wild-type protein is not necessary for the formation of aggregates. However, an intriguing question arises: how can the presence of these aggregates be reconciled with the ability of the mutant protein to rescue the phenotypes associated with null mutations in dVAP as shown by three independent studies (3, Ratnaparkhi et al., Tsuda et al.). Are these aggregates different from the ones observed when the mutant protein is expressed in the presence of the wt protein?

Other outstanding questions will need to be addressed: which is the protease or proteases responsible for the cleavage? Is the secretion of the MSP domain of VAP proteins occurring through an unconventional mechanism as already proposed for the MSP proteins in C. elegans? Which is the subcellular compartment in which the cleavage occurs?

The paper by Ratnaparkhi et al. focuses on another important aspect, which is the determination of the disease mechanism. In humans, the pathogenic mutation is inherited in a dominant manner. Dominant mutations are due to a gain of function (hypermorphs and neomorphs), dominant-negative interactions (antimorphs) or haplo-insufficiency. Understanding the patho-mechanism of the disease is important as it can indicate new possible strategies for therapeutic interventions. Several lines of evidence support a possible dominant-negative effect of the pathogenic allele. The formation of aggregates, the depletion of the wild-type protein from its normal localization (3,4,5), and the sequestration of the wt protein in the aggregates clearly suggest a dominant-negative effect (4). Moreover, the fact that the pathogenic allele acts as a dominant-negative can be proven if the overexpression of the mutant protein in the presence of the wt protein leads to a phenotype similar to the loss-of-function mutation. Indeed, it has been reported that transgenic expression of the mutant protein induces a reduction in number of boutons (3), a disruption of the presynaptic cytoskeleton (Ratnaparkhi et al.) and a reduction in miniature excitatory post-synaptic potentials (Tsuda et al.). Ratnaparkhi et al. attempt to further support this statement by performing a systematic analysis of mutant phenotypes in different functional contexts. They compared the effect of overexpressing the wt protein with the overexpression of the mutant protein in transgenic lines expressing comparable amounts of transgenes. The expression levels of the proteins were estimated only for the full-length VAP. Although the mutant allele impairs the secretion of the MSP domain, the cleaved product is still produced as shown in several Western blots reported by Tsuda et al. The same Western blots suggest that the levels of the full-length protein and the cleaved MSP domain are not stoichiometrically similar; therefore, restricting the analysis to the expression levels of the full-length protein may be misleading. A cleaved, non-secreted MSP domain could still be responsible for the intracellular, cell-autonomous effects of the protein.

Although there are several lines of evidence supporting a possible dominant-negative effect, there is other evidence suggesting different mechanisms for the disease. Mutant VAP proteins still retain some functional properties of the wt protein such as the ability to self-oligomerize (3,4) and the ability to rescue, at least in part, the mutant phenotype due to the loss of the endogenous protein. The mutant allele has also acquired new functional properties that are not shared by the normal version of the protein such as the propensity to form aggregates and the “floating active zones” phenotype reported by Ratnaparkhi et al. In one report it has also been shown that the mutant protein has an increased ability of inhibiting the activity of ATF6, a transcription factor involved in UPR (6).

We propose that the mutant allele may cause the disease by a combination of mechanisms that include dominant-negative interactions and toxic effects due to gain of new functions. Although a lot still remains to be done, studies published over the last six months have convincingly shown that the variety of genetic tools available in Drosophila can now be exploited to foster our understanding of the patho-mechanisms responsible for motor neuron diseases in humans.

References:
1. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004 Nov;75(5):822-31. Abstract

2. Pennetta G, Hiesinger PR, Fabian-Fine R, Meinertzhagen IA, Bellen HJ. Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner. Neuron. 2002 Jul 18;35(2):291-306. Abstract

3. Chai A, Withers J, Koh YH, Parry K, Bao H, Zhang B, Budnik V, Pennetta G. hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction. Hum Mol Genet. 2008 Jan 15;17(2):266-80. Abstract

4. Kanekura K, Nishimoto I, Aiso S, Matsuoka M. Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8). J Biol Chem. 2006 Oct 6;281(40):30223-33. Abstract

5. Teuling E, Ahmed S, Haasdijk E, Demmers J, Steinmetz MO, Akhmanova A, Jaarsma D, Hoogenraad CC. Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates. J Neurosci. 2007 Sep 5;27(36):9801-15. Abstract

6. Gkogkas C, Middleton S, Kremer AM, Wardrope C, Hannah M, Gillingwater TH, Skehel P. VAPB interacts with and modulates the activity of ATF6. Hum Mol Genet. 2008 Jun 1;17(11):1517-26. Abstract

View all comments by Giuseppa Pennetta

One reason is due to the fact that from a clinical point of view, familial and sporadic ALS cases are virtually identical. As such, it is not unreasonable to postulate that although ALS may be caused by different genetic factors, they all may lead to common sets of pathways that eventually result in the ALS phenotype. Thus, a high level of importance should be placed on understanding the common features of all known ALS genes since they may shed light on these pathways. Therefore, even though VAPB mutations are indeed rare, characterizing their effects may provide insight on how cases of ALS develop overall.

In both of the papers presented (6,7), the authors have each developed a Drosophila model of ALS which expresses mutant VAPB. The use of these models will undoubtedly be beneficial in future experiments to further decipher the ALS phenotype. Of great significance, though, is that each study observes in vivo that mutant VAPB is capable of inducing intracellular aggregates. This work reinforces previously published observation that in vitro expression of mutant human P56S protein results in cellular aggregates (4,5). The fact that the aggregation phenotype of this mutation is conserved down to Drosophila is quite interesting. Aggregates are commonly observed within ALS cases, as well as other neurodegenerative diseases, although whether these aggregates are pathogenic is still up for debate. The formation of intracellular aggregates has also been observed via expression of mutant SOD1 and mutant TDP-43 (3). Taken together, the observation that three different familial ALS genes all are capable of inducing intracellular aggregates reinforces the notion that understanding the activation of pathways by protein misfolding is key to understanding the pathogenic nature of ALS.

References:
1. Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, Ackerley S, Durnall JC, Williams KL, Buratti E, Baralle F, de Belleroche J, Mitchell JD, Leigh PN, Al-Chalabi A, Miller CC, Nicholson G, Shaw CE. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008 Mar 21;319(5870):1668-72. Abstract

2. Kabashi E, Valdmanis PN, Dion P, Spiegelman D, McConkey BJ, Vande Velde C, Bouchard JP, Lacomblez L, Pochigaeva K, Salachas F, Pradat PF, Camu W, Meininger V, Dupre N, Rouleau GA. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat Genet. 2008 May;40(5):572-4. Abstract

3. Winton MJ, Van Deerlin VM, Kwong LK, Yuan W, Wood EM, Yu CE, Schellenberg GD, Rademakers R, Caselli R, Karydas A, Trojanowski JQ, Miller BL, Lee VM. A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro. FEBS Lett. 2008 Jun 25;582(15):2252-6. Abstract

4. Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M. A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004 Nov;75(5):822-31. Abstract

5. Landers JE, Leclerc AL, Shi L, Virkud A, Cho T, Maxwell MM, Henry AF, Polak M, Glass JD, Kwiatkowski TJ, Al-Chalabi A, Shaw CE, Leigh PN, Rodriguez-Leyza I, McKenna-Yasek D, Sapp PC, Brown RH Jr. New VAPB deletion variant and exclusion of VAPB mutations in familial ALS. Neurology. 2008 Apr 1;70(14):1179-85. Abstract

6. Tsuda H, Han SM, Yang Y, Tong C, Lin YQ, Mohan K, Haueter C, Zoghbi A, Harati Y, Kwan J, Miller MA, Bellen HJ. The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors. Cell. 2008 Jun 13;133(6):963-77. Abstract

7. Ratnaparkhi A, Lawless GM, Schweizer FE, Golshani P, Jackson GR. A Drosophila model of ALS: human ALS-associated mutation in VAP33A suggests a dominant negative mechanism. PLoS ONE. 2008 Jun 4;3(6):e2334. Abstract

View all comments by John Landers

Several findings are really surprising: 1) the clear division between resistant motor neurons (RES) and vulnerable ones (VUL); 2) the predictability on development of the disease that the pathogenic scheme described by authors allows; 3) the dissociation between ubiquitination—often considered a pathological hallmark for this disease and other neurodegenerative diseases—and real axonal pathology; 4) the very early changes at a cellular level (as early as postnatal 5 in some markers) that preclude pathological changes; 5) the presence of novel markers of the disease at an immunological level (such as ATF3, PERK, and similar); 6) the distinctive patterns of expression between RES...  Read more

Several findings are really surprising: 1) the clear division between resistant motor neurons (RES) and vulnerable ones (VUL); 2) the predictability on development of the disease that the pathogenic scheme described by authors allows; 3) the dissociation between ubiquitination—often considered a pathological hallmark for this disease and other neurodegenerative diseases—and real axonal pathology; 4) the very early changes at a cellular level (as early as postnatal 5 in some markers) that preclude pathological changes; 5) the presence of novel markers of the disease at an immunological level (such as ATF3, PERK, and similar); 6) the distinctive patterns of expression between RES and VUL neurons; 7) the interplay between growth factor treatment (CTNF) and rescue in ER stress terms; and 8) the positive effect of salubrinal in ALS development and the negative effects of crushing schemes

The findings fit quite well with some of the "usual suspects" theories of ALS, such as the involvement of mitochondria and glia. Mitochondrial impairment (due to unfolded SOD or to other events) would lead to lower ATP levels or to Ca homeostasis dysregulation, which would affect the ER, increasing the unfolded protein response (UPR). Additionally, and pertinent to our case since we linked ER stress to oxidative stress, ER folding capacities are strongly influenced by oxidative milieu and the findings reported here (including participation of hypoxia and NRf2 dependent pathways) agree with the potentially increased oxidative stress in VUL neurons. Most interestingly, many data (as recently reviewed by Cleveland et al. in the last Cell volume—see Lagier-Tourenne et al., 2009) point out the importance of RNA processing in ALS. The hypothetical interplay between alterations in RNA splicing and ER stress in VUL neurons is in agreement with the high structural and energetical requirements of those cells. It seems that long axons and the structural and functional needs that those "near to pathology" cells (VUL motor neurons) exhibit, make them extremely prone to pathology.

It is also somewhat surprising that ER stress, which may be considered a logical and physiological consequence of UPR, is followed by cellular demise. It would be also be very interesting, as apoptosis seems not involved, to characterize the distal events of ER stress; i.e., what is the link between ER stress and denervation. This is because although the authors define that salubrinal treatment is useful at preclinical stages, it seems that its efficiency would be much lower at a clinical stage.

It would be nice to confirm these results in human samples. Though it could be difficult to find VUL and RES motor neurons in samples from human disease specimens, but this would also be useful to extend those findings to the more common form of the disease (sporadic ALS, by far, is commoner than the familial form). Further experiments would have to prove, by in vitro transfection with some of the factors described in the papers, that RESistance to disease is acquired by VULnerable neurons (or vice versa, by using RNAi or similar techniques).

View all comments by Manuel Portero

Ebneth A, Godemann R, Stamer K, Illenberger S, Trinczek B, Mandelkow E. Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease. J Cell Biol. 1998 Nov 2;143(3):777-94. Abstract

View all comments by P.F. Jennings

The data that are most convincing for us are the studies in transgenic mice: there is a clear correlation between the expression levels of Nogo-A and mouse survival and motor ability; this underlines the critical role of Nogo-A in protecting neurons from SOD1-dependent toxicity.

The PDI redistribution upon Nogo-A overexpression is also interesting, but we think that the pathway that leads to this effect is not clear. Is it mediated by a direct interaction or are other proteins involved? What is the biological significance of PDI puncta within the cell?

The main problem for us is that the link between PDI redistribution and the protective role of Nogo-A in ALS is purely correlative. Although it is possible that Nogo-A protects motor neurons by redistributing PDI, this has not been demonstrated. We would like to know more on how redistributed PDI can prevent motor neuron degeneration.

View all comments by Elisa Fasana
View all comments by Matteo Fossati

Nogo-A’s role in ALS is not clearly understood. Is it just a bystander, does it play a role in aggravating the disease, or does it actually help protect against ALS? A previous report (Jokic et al., 2006) has suggested that Nogo-A may be a causative factor or has a role in disease progression, as the authors found that Nogo-A knockout could increase the survival period of ALS SOD(G86R) mice, while its overexpression destabilized neuromuscular junctions, which would eventually result in motor neuron death.

The paper by Yang et al. (2009) provides a contrasting and interesting role for Nogo-A in ALS. The authors showed that Nogo-A may function to enhance survival in ALS mice by redistributing the endoplasmic reticulum (ER) chaperone, protein...  Read more

Nogo-A’s role in ALS is not clearly understood. Is it just a bystander, does it play a role in aggravating the disease, or does it actually help protect against ALS? A previous report (Jokic et al., 2006) has suggested that Nogo-A may be a causative factor or has a role in disease progression, as the authors found that Nogo-A knockout could increase the survival period of ALS SOD(G86R) mice, while its overexpression destabilized neuromuscular junctions, which would eventually result in motor neuron death.

The paper by Yang et al. (2009) provides a contrasting and interesting role for Nogo-A in ALS. The authors showed that Nogo-A may function to enhance survival in ALS mice by redistributing the endoplasmic reticulum (ER) chaperone, protein disulfide isomerise (PDI), to a subcellular compartment of uncertain identity. In contrast to the earlier report, Yang et al. found that deletion of Nogo-A/B accelerated axonal degeneration of another ALS mutant SOD model, the SOD(G93A) mice. Walker’s commentary (2010) on the Yang paper is very comprehensive, and the author has made several cogent and insightful comments on several aspects of the Yang paper.

In furthering the discussion, I feel that there are two important and interesting aspects to the Yang paper that warrant further investigation by workers in the field. Firstly, from a cell biological perspective, it would be exciting to find out exactly to which subcellular compartment PDI is redistributed. Based on a rather limited marker profile, the authors have ruled out Golgi, endosomes, and vesicles in the autophagy pathway. I doubt that the spots are simply protein aggregates. One possibility is that PDI has been redistributed to specific parts of the ER, such as the ER exit sites or the ER-Golgi intermediate compartment (ERGIC) (Appenzeller-Herzog and Hauri, 2006). PDI has been shown to be functionally inactivated in ALS by S-nitrosylation and as such could no longer be protective against misfolded proteins in disease conditions (Walker et al., 2010). Therefore, could Nogo-A aid in the removal of dysfunctional PDI from the ER, and in doing so, eventually enhance protein folding in ER and hence survival? This, of course, begs the question of how Nogo-A could affect PDI’s redistribution without directly interacting or colocalizing with the latter. It should be noted that all Nogo isoforms are primarily ER residents, and Nogo-A and B have been implicated to act in the modulation of ER morphology and shape (Voeltz et al., 2006). Therefore, Nogo isoform expression levels are likely to influence the dynamics and distribution of ER residents, such as the KDEL signal-containing proteins. How this influence is connected to pathological conditions like ALS should be an interesting line of investigation.

The second, more clinically relevant point is the contrasting results between the studies by Jokic et al. and Yang et al. It would be interesting, if only on a speculative basis, to try to understand how such a discrepancy could arise. There are two notable differences between the mouse models used by the different group of authors. Firstly, the nature of the SOD1 mutation is different (G86R for Jokic et al. and G93A for Yang et al.). Secondly, and perhaps connected to a controversy in the Nogo field (Teng and Tang, 2005), the two studies differ in the Nogo knockout mice used. The model used by Jokic et al. is based on the Nogo knockout generated by Simonen et al. (2003), with parts of nogo exons 2 and 3 and the intron between them deleted. The model used by Yang et al., on the other hand, is based on one reported by Kim et al. (2003), generated by a gene trap insertion that maps near the 5′ end of exon 3. The mice used by Simonen et al. no longer expressed the Nogo-A isoform, but both Nogo-B and Nogo-C, the other two major Nogo isoforms, remained expressed. In fact, there appears to be a compensatory upregulation of Nogo-B in the CNS of the mice used by Simonen et al. The mice used by Kim et al., on the other hand, have both Nogo-A and Nogo-B isoforms deleted. In the initial reports on effects of the respective knockouts on axonal regeneration, the mice used by Kim et al. appeared to have a better enhancement in regenerative capacity. The differences in the nature of SOD1 mutant and Nogo isoform deletion could potentially contribute towards the contrasting conclusions reached by the different authors on the role of Nogo in ALS disease onset and progression. For reasons yet unclear, the Jokic/Simonen mice were less prone to mutant SOD1-induced ALS motor neuron degeneration compared to wild-type control, while the Yang/Kim mice were more disease susceptible compared to wild-type. Further comparative investigations would shed light on the differences between these animals.

Many questions still loom ahead with regard to Nogo-A’s actual role and importance in ALS. Efforts in resolving these questions could make crucial contributions toward the prevention and treatment of the disease.

References:
Appenzeller-Herzog C, Hauri HP (2006) The ER-Golgi intermediate compartment (ERGIC): in search of its identity and function. J Cell Sci. 119: 2173-2183. Abstract

Jokic N, Gonzalez de Aguilar JL, Dimou L, Lin S, Fergani A, Ruegg MA, Schwab ME, Dupuis L, Loeffler JP (2006) The neurite outgrowth inhibitor Nogo-A promotes denervation in an amyotrophic lateral sclerosis model. EMBO reports 7:1162–1167. Abstract

Kim JE, Li S, GrandPré T, Qiu D, Strittmatter SM (2003) Axon regeneration in young adult mice lacking Nogo-A/B. Neuron 38:187-199. Abstract

Simonen M, Pedersen V, Weinmann O, Schnell L, Buss A, Ledermann B, Christ F, Sansig G, van der Putten H, Schwab ME (2003) Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury. Neuron 38: 201-211. Abstract

Teng FY and Tang BL (2005) Why do Nogo/Nogo-66 receptor gene knockouts result in inferior regeneration compared to treatment with neutralizing agents? J Neurochem. 94: 865-874. Abstract

Walker AK (2010) Protein disulfide isomerase and the endoplasmic reticulum in amyotrophic lateral sclerosis. J Neurosci. 30: 3865-3867. Abstract

Walker AK, Farg MA, Bye CR, McLean CA, Horne MK and Atkin JD (2010) Protein disulphide isomerase protects against protein aggregation and is S-nitrosylated in amyotrophic lateral sclerosis. Brain 133: 105-116. Abstract

Yang YS, Harel NY, Strittmatter SM (2009) Reticulon-4A (Nogo-A) redistributes protein disulfide isomerase to protect mice from SOD1-dependent amyotrophic lateral sclerosis. J Neurosci. 29: 13850-13859. Abstract

View all comments by Felicia Y.T. Teng