Expression of the C terminus of the amyloid precursor protein alters growth factor responsiveness in stably transfected PC12 cells.

Sandhu, Faheem A.; Kim, Yong; Lapan, Kirsty A.; Salim, Mohammed T.A.; Aliuddin, Viqar; Zain, Sayeeda

doi:10.1073/pnas.93.5.2180

Cited by 15 publications

(5 citation statements)

References 51 publications

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“…To examine the Rab5 + endosomes in axons, we expressed C99 and C83 following NGF treatment. Consistent with earlier studies (73)(74)(75), expression of C99 markedly suppressed NGF-induced activation of Erk1/2 at both 5 and 30 minutes (Supplemental Figure 2, B and C). While having no effect at 5 minutes, C83 induced a small decrease in pErk1/2 at 30 minutes.…”

Section: M596vsupporting

confidence: 79%

Amyloid precursor protein–mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration

Xu¹,

Weissmiller²,

White³

et al. 2016

Journal of Clinical Investigation

153

225

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R e s e a R c h a R t i c l e1 8 1 6jci.org Volume 126Number 5 May 2016 26, 54, 55). We assayed the level of GTP-Rab5 in brains of 12-monthold Ts65Dn and 2N mice following a published protocol (56). As previously reported (28), the level of full-length APP in Ts65Dn was approximately 1.5-fold than in 2N samples ( We next tested whether the increase in App gene dose in Ts65Dn BFCNs was responsible for enlargement of Rab5 + endosomes (Figure 1). By immunoblotting, the APP siRNA caused an approximately 30% reduction in the level of full-length APP, as compared with control siRNA ( Figure 2E). Rab5 + puncta in BFCNs treated with either the APP siRNA or control siRNA were analyzed (Figure 2, F and G) as in Figure 1. Large, sometimes lobulated Rab5 + puncta were seen in cultures treated with the control siRNA, whereas these structures were typically smaller and rounded in cultures treated with the APP siRNA ( Figure 2G). Treatment with the APP siRNA significantly reduced the size of Rab5 + puncta in Ts65Dn neurons to a value equivalent to that in 2N neurons ( Figure 2F). Thus, increased App gene dose is necessary for increased Rab5 activation and for early endosome enlargement in Ts65Dn neurons.Full-length APP and β-CTF caused enlargement of early endosomes in PC12 cells. To determine how increased APP expression caused an increase in Rab5 activation, we asked which APP product(s) were responsible (Supplemental Figure 1A). We transfected PC12M cells with full-length APP-GFP, C99-GFP (β-CTF), C83-GFP (α-CTF), or AICD-GFP and examined endosomes by live cell imaging (Supplemental Figure 1B). Bright foci of GFP + intracellular structures were present in PC12M cells that overexpressed APP-GFP or C99-GFP. In contrast, cells expressing C83-GFP or AICD-GFP showed diffuse, hazy signals for GFP, with occasional foci in C83-GFP cells. In APP-GFP and C99-GFP cells, the GFP + intracellular structures were, on average, approximately 2 μm 2 (Supplemental Figure 1E). GFP signals in C83-GFP or AICD-GFP cells contained speckled small puncta within the haze, as well as a small number of larger bright puncta, as seen in cells expressing C99-GFP and APP-GFP (Supplemental Figure 1B). However, the average puncta size in C83-GFP and AICD-GFP cells was approximately 1.2 and 1.3 μm 2 , respectively. Thus, overexpressing APP and β-CTF, but not α-CTF or AICD, routinely induced formation of enlarged, bright intracellular structures. We also tested two APP mutants: APP M596V and APP SWE . APP M596V , which abolishes β-secretase cleavage, prevents production of β-CTF (57); APP SWE enhances β-secretase cleavage to increase the level of β-CTF (57). Both induced the formation of enlarged intracellular structures (Supplemental Figure 1C).We examined colocalization of APP or C99 with Rab5 in cotransfection experiments; APP-mCherry with GFP-Rab5 WT ( Figure 3B); C99-GFP with mCherry-Rab5 WT ( Figure 3C); and Rab5 + endosomes (26, 28, 37) was correlated with reduced endosomal trafficking and signaling of nerve growth factor (NGF), leading to degeneration...

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Section: M596vsupporting

confidence: 79%

Amyloid precursor protein–mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration

Xu¹,

Weissmiller²,

White³

et al. 2016

Journal of Clinical Investigation

153

225

View full text Add to dashboard Cite

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“…Thus, primary hippocampal neurons may be more vulnerable to the stresses resulting from overexpression of h-APP than are the PC12 cells. As discussed above, elevated intracellular levels of the full-length APP in the infected cells may lead to more CTF and/or A␤ protein production, which may have toxic effects on the cultured cells (Koh et al, 1990;Cotman et al, 1992;Malouf, 1992;Busciglio et al, 1993;Jarrett et al, 1993;Shearman et al, 1994;Simmons et al, 1994;Kaneko et al, 1995;Harris et al, 1996;Itoh et al, 1996;Sandhu et al, 1996;Blanc et al, 1997;Mark et al, 1997;Lambert et al, 1998). Nishimura et al (1998) reported that injection of adenovirus overexpressing wt human APP in rat hippocampus caused neuronal apoptosis.…”

Section: Discussionmentioning

confidence: 99%

Death of PC12 cells and hippocampal neurons induced by adenoviral-mediated FAD human amyloid precursor protein gene expression

et al. 1999

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We used adenoviral-mediated gene transfer of human amyloid precursor proteins (h-APPs) to evaluate the role of various h-APPs in causing neuronal cell death. We were able to infect PC12 cells with very high efficiency because approximately 90% of the cells were cytochemically positive for beta-galactosidase activity when an adenoviral vector containing LacZ cDNA was used to infect cells. Cells infected with adenovirus containing h-APP cDNA showed high-level transcription and expression of h-APP as measured by reverse transcriptase-polymerase chain reaction and Western immunoblot analyses, respectively. Intracellular and extracellular levels of h-APP were elevated approximately 17-and 24-fold in cultures infected with recombinant adenovirus containing wild-type mutant and 13- and 17-fold with V642F mutant. No elevation in h-APP was seen in cultures infected with antisense h-APP or null adenovirus. H-APP levels were maximal 3 days after infection. Overexpression of V642F mutant h-APP in PC12 cells and hippocampal neurons resulted in about a twofold increase in death compared with overexpression of wild-type h-APP. These results demonstrate the usefulness of recombinant adenoviral mediated gene transfer in cell culture studies and suggest that overexpression of a familial Alzheimer's disease mutant APP may be toxic to neuronal cells.

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“…This antibody precipitates total A␤ and p3 (and small variable amounts of APP s ) from the medium of cultured cells (18). Polyclonal antibody 1963 was raised to synthetic A␤ [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] (18). The monoclonal antibody 2G3, specific for A␤ x-40, was produced by injecting female A͞J mice intraperitoneally with…”

Section: Pulse-chase Experiments and Immunoprecipitationsmentioning

confidence: 99%

Evidence that the 42- and 40-amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities

Citron

Diehl

Gordon

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

293

216

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Cerebral deposition of the amyloid ␤ protein (A␤) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of A␤ (A␤ 40 ) accounts for Ϸ90% of all A␤ normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (A␤ 42 ), accounting for only Ϸ10% of secreted A␤, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of A␤ 42 production is a prime therapeutic goal. The same protease, ␥-secretase, is assumed to generate the C termini of both A␤ 40 and A␤ 42 . Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit ␥-secretase, on ␤-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various A␤ 40 -and A␤ 42 -specific antibodies, we demonstrate a much stronger inhibition of the ␥-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the A␤ 40 and A␤ 42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general ␤-amyloid precursor protein metabolism, including A␤ 40 production, but specifically block the generation of the pathogenic A␤ 42 peptide.The 39-to 43-amino acid amyloid ␤ protein (A␤) is deposited as amyloid in the brains of all patients with Alzheimer disease (AD). It has been shown that the primary component of deposits in the cerebral vasculature is short A␤, ending with a C terminus at residue 39 or 40 (A␤ 40 ) (1, 2), whereas long A␤, ending with a C terminus at residue 42 (A␤ 42 ), accumulates initially and predominantly in parenchymal plaques (3-7). A␤ is constitutively secreted by a wide variety of cells and exists in a soluble form in biological fluids (8, 9). During the last 2 years, evidence has accumulated suggesting that A␤ 42 plays the key role in the process of plaque formation. (i) In vitro data demonstrate that A␤ 42 accelerates the formation of A␤ fibrils by a nucleation dependent mechanism (10). (ii) While accounting for only 10% of total A␤ secreted from cells (roughly 90% is A␤ 40 ) (11, 12), A␤ 42 is the major plaque component (3, 4). Furthermore, all three early-onset familial AD genes identified to date have been shown to lead to an increase in A␤ 42 . Only the Swedish ␤-amyloid precursor protein (␤APP) missense mutation also increases A␤ 40 (11), whereas the ␤APP 717 mutations and the recently described presenilin 1 and 2 mutations do not (13,14). Thus, the long A␤ 42 peptide appears to be a prime target for therapeutic intervention. However, none of the enzymes involved in the major steps of ␤APP processing have been identified, including ␥-secretase, the protease that generates the C terminus of A␤. It has generally been assumed that the same enzyme(s) generate both A␤ 40 a...

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Expression of the C terminus of the amyloid precursor protein alters growth factor responsiveness in stably transfected PC12 cells.

Abstract: The

Cited by 15 publications

References 51 publications

Amyloid precursor protein–mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration

Amyloid precursor protein–mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration

Death of PC12 cells and hippocampal neurons induced by adenoviral-mediated FAD human amyloid precursor protein gene expression

Evidence that the 42- and 40-amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities

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