The accumulation of autophagic vacuoles is a consistent feature of many neurodegenerative diseases (Shintani and Klionsky, 2004
). The significance of the described perturbations in the autophagic pathways in Alzheimer’s disease gained support by a report by Lee et al. (2010)
, in which the authors concluded that the absence of PSEN1
expression impaired the clearance of autophagosomes during macroautophagy due to a selective impairment in lysosomal acidification. These deficits were attributed to failed association of PS1 holoprotein with STT3B and V0a1, and defects in posttranslational glycosylation and targeting of the V0a1 to lysosomes. Moreover, human fibroblasts expressing FAD-linked PS1 variants also exhibited lysosomal/autophagy deficits similar to cells lacking PS1.
In view of the provocative outcomes reported by Lee et al. (2010)
, we reexamined several aspects of the model and now offer several insights that collectively, fail to support the conclusions drawn by the authors. First, we document that while LC3-II levels are considerably diminished in cultured PSdko-ES cells compared to WT-ES cells, autophagic flux assays reveal that this is the result of an active autophagic pathway in cells lacking PSEN
Second, we demonstrate that the mean vesicle pH in cultured PS1ko-ES cells and WT-ES cells are essentially identical, but the vesicle pH in PSdko-ES cells exhibits a shift towards more acidic pH values. The mechanism(s) underlying this observation remains to be determined, but Neely et al. (2011)
recently showed that vesicle acidification was unchanged in MEFs lacking PSEN
compared to WT MEFs. Third, we show that proteolytic processing of CatD is indistinguishable in cultured WT-ES, PS1ko-ES and PSdko-ES cells. Furthermore, we show that CatD processing in hippocampi from PScdko mice and littermate controls are identical. As the dissociation of CatD from mannose 6-phosphate receptors in lysosomes requires acidic pH and CatD processing is mediated by proteases with acidic pH optima, we would infer that the absence of PSEN
has no significant influence on lysosomal pH. Fourth, we demonstrate that N-linked glycosylation of V0a1 in MEFs or ES cells that lack PSEN
is indistinguishable from corresponding WT cells. We confirmed this result by showing that the maturation of V0a1 in the hippocampi of PScdko mice is identical to that in their littermate controls. Hence, these results are entirely consistent with our observation that lysosomal pH and function is unimpaired in the absence of PSEN
. Fifth, we failed to observe any differences in N-linked glycosylation of V0a1 and CatD processing in N2a cells or in brains of mice expressing WT or FAD-linked PS1 variants, findings that fail to support the studies reported by Lee et al. (2010)
. Sixth, we examined the association of PS1 holoprotein with V0a1 and/or STT3B. In this regard, we document that PS1 holoprotein is essentially undetectable at steady-state in both cells and brain; we estimate that the endoproteolytic derivatives of PS1 accumulate to ~0.0025% of total brain protein (Thinakaran et al., 1996
). Hence, we chose to use PEN-2 knockdown cell line (Prokop et al., 2004
), which has an elevated steady-state level of PS1 holoprotein, or stable N2a cells that overexpress WT human PS1. In neither of these cell lines did we find an association of PS1 holoprotein with either V0a1 or STT3B. Finally, we asked whether V0a1 is a substrate for N-linked glycosylation by STT3B. Indeed, we were intrigued by the model proposed by Lee et al. (2010)
because the 834–856 amino acid mouse V0a1 isoforms harbor a single lumenally exposed potential N-linked glycosylation site (asparagine 489) (Nishi and Forgac, 2000
;Leng et al., 1999
). However, we have shown that N-linked sites that serve as STT3B substrates are generally found at either amino- or carboxyl-terminal regions of the polypeptide backbone (Ruiz-Canada et al., 2009
). When tested in cells wherein STT3A or STT3B was depleted, we failed to confirm that V0a1 is uniquely glycosylated by STT3B.
In summary, we now report that cells lacking expression of PS1 either singly, or in combination with PS2 exhibit robust autophagy, and that the loss of PS function neither alters intracellular pH, N-linked glycosylation of V0a1, or processing of CatD. Finally, we fail to confirm the association of PS1 holoprotein with either V0a1 or STT3B. How can we reconcile these findings with those reported by Lee et al. (2010)
? We speculate that the PS1ko blastocysts, from which the bulk of the data were obtained by Lee and colleagues that revealed deficits in autophagy, elevated lysosomal pH, inefficient glycosylation of V0a1 and CatD processing, are, in some manner, compromised, perhaps as a consequence of issues related to clonal drift and/or compensation. In this regard, we found it curious that a series of “rescue” experiments intended to demonstrate that human PS1 can reverse the reported phenotypes in PS1ko blastocysts were not conducted in the PS1ko blastocysts, but inexplicably, were performed in PSdko blastocysts. Lee et al. (2010)
reported that human PS1 fully “rescues” the deficits in protein degradation, vesicle acidification, macroautophagy responses, V0a1 maturation and CatD processing that existed in PS1ko cells. What was conspicuously absent in this dataset was information pertaining to the aforementioned biological outcomes in naïve PSdko cells, particularly in view of the findings reported herein that failed to reveal a deficit in any of these processes in these cells.
To these latter insights, we assessed the expression of TFEB and genes involved in autophagy, and now report no differences in TFEB mRNA between cultured PSdko-ES and WT-ES cells. Moreover, transcriptome analysis of cultured PS1ko-ES and PSdko-ES cells failed to reveal any significant alteration in the expression of autophagy-specific genes. However, ATG9B
expression was elevated in both the cultured PS1ko-ES and PSdko-ES cells, but its is presently unclear as the function of this phagophore/pre-autophagosomal structure (PAS) (Suzuki et al., 2001
;Yen and Klionsky, 2007
)-associated polytopic membrane protein has not been established. Similarly, transcriptome analysis of PScdko mice failed to uncover any alterations in the expression of TFEB or genes involved in autophagy. Meanwhile, we show a significant elevation in “CLEAR” network genes in the frontal cortices (FC) and hippocampi (HC) of 6 month old PScdko mice compared to littermate controls. In contrast, we observed a small, but significant decrease in the populational expression of “CLEAR” network genes in cultured PS1ko-ES and PSdko-ES cells compared to WT-ES cells. Moreover, with the exception of elevated CTSS
transcript levels, the “CLEAR” gene expression changes between PSko-ES cells and PScdko brains do not overlap. While these latter results would seem conflicting, we would argue that the observed differences represent cell type-specific mechanisms that lead to elevated lysosomal function. ES cells are clonal, proliferative, pluripotent stem cells and the response of these cells to PSEN
-deficiency maybe significantly different than that observed in the brains of PScdko mice in which PSEN1
is selectively ablated in postmitotic, excitatory neurons. In this regard, while we observe an elevation in steady-state levels of CatD in extracts from PS1ko and PSdko ES cells, this is not due to an increase in CatD
mRNA (). In contrast, both CatD mRNA and protein are elevated in brains of PScdko mice (Mirnics et al., 2008
; and ). It is instructive that the basal, steady-state levels of the autophagy substrate, LC3-II, are lower in PS1ko and PSdko ES cells compared to WT ES cells, a finding which would suggest that lysosomal hydrolase activity is elevated, leading to enhanced turnover of this substrate in the absence of PS. Indeed, in PS1ko and PSdko cells treated with bafilomycin, the levels of LC3-II are markedly elevated, and autophagic flux is strongly enhanced. Hence, it is conceivable that in PS1ko and PSdko cells, the steady-state levels or catalytic activity of other lysosomal proteases are also elevated without corresponding increases in the mRNAs encoding these polypeptides. This hypothesis remains to be tested.
The mechanism(s) by which loss of PSEN expression elicits an increase in the expression of CLEAR genes in PScdko brains is not fully understood. However, we would offer the tentative proposal that in the absence of PS/γ-secretase activity, the membrane-tethered stubs derived from type 1 membrane proteins that would normally be processed by γ-secretase in endosomal, late Golgi compartments or the plasma membrane would now accumulate in lysosomal compartments wherein the proteases critical for lysosomal function would be “saturated”. As a result, excitatory neurons lacking PSEN expression would respond by inducing CLEAR genes as a compensatory mechanism. We should note that in addition to scoring expression in excitatory neurons, CLEAR gene expression in the PScdko brains is the summation of expression patterns in inhibitory interneurons, microglia, astrocytes, pericytes and endothelial cells. Moreover, and in view of the fact that astro-glial activation is associated with neurodegeneration in 6 month old PScdko brain, other gene expression changes will certainly contribute to the transcriptome.
In any event, the fact that all the genes in the CLEAR network contain TFEB binding site(s), and that TFEB mRNA levels are not impacted by the expression of PS, we can only conclude that in mouse brain, PS regulates pathway(s) critical for lysosomal biogenesis, but in a TFEB-independent manner. The mechanism(s) that underlies this unanticipated, but novel aspect of PS biology remains to be investigated.