NGF induces Tyr phosphorylation of APP
To test whether NGF triggers phosphorylation of APP on Tyr residues in physiological conditions, we treated primary hippocampal neurons with 50 ng/ml of NGF for the indicated time periods (). Lysates from treated neurons were immuno-precipitated with an α–APP antibody, and precipitants were analysed by WB analysis with an α–pTyr antibody (α-pY). As shown in , we detected a protein of molecular mass similar to mature APP that is immunoprecipitated by the α–APP antibody and is phosphorylated on Tyr residue(s). This phosphorylation is detectable 10min after NGF exposure and persists for at least 24hrs (). In reciprocal experiments, we found that APP is immunoprecipitated by the α–pTyr antibody ((α-pY) from samples treated with NGF (). The evidence that the TrkA receptor is phosphorylated upon NGF treatment indicates that the NGF/TrkA signaling pathways is properly activated in this experimental setting (). In addition, the phosphorylation was very rapid and long-lasting indicating a direct and sustained NGF effect and further supporting the specificity of the NGF/TrkA signaling involvement. These results strongly suggest that NGF treatment induces APP phosphorylation on tyrosine(s) in primary neuronal cells.
NGF triggers phosphorylation of APP via the TrkA receptor
It is likely that the NGF signaling in primary hippocampal neuronal cultures () is due to the cholinergic afferent population, that is considered one of the most responsive NGF population (Siegel GJ et al., 1999
) and accounted for approximately 20-25% of the overall population in our experimental conditions (). We speculated whether such NGF dependent APP phosphorylation may occur also in hippocampal slices, where most of neuronal connections are preserved, and in septal nuclei slices (data not shown), that are considered to be the major one’s cholinergic output of the CNS (J. Hartikka and F. Hefti, 1988
). 10 min of NGF exposure induced APP phosphorylation on Tyr residues in hippocampal slices as well as in septum slices. Again, a band corresponding to mature APP is immunoprecipitated by the α-pY antibody in samples treated with NGF and this phosphorylation is synchronous to the activation of TrkA (). Activation of TrkA is necessary to mediate Tyr-phosphorylation of APP, since the TrKA inhibitors CEP-2563 and K-252a prevented phosphorylation of APP (). Thus, NGF/TrkA signaling mediate Tyr-phosphorylation of APP in two anatomical regions known to be involved in Alzheimer pathogenesis. Altogether the results indicate that activation of TrkA by NGF produces phosphorylation of APP on one or more tyrosine residues under physiological conditions. Although it is conceivable that TrkA, a receptor with tyrosine kinase activity, directly phosphorylates APP, phosphorylation of APP may also be mediated by other kinases that are activated by TrkA.
APP regulates NGF-mediated TrkA activation and the sensitivity of neurons to the trophic action of NGF
APP contains three tyrosine residues in the cytoplasmic tail (Y653
, and Y687
of the APP695
isoform). The latter two are included in the APP-Y682
sequence, which is instrumental for the association with the PTB domains of the APP interacting proteins identified to date (G. D. King and R. Scott Turner, 2004
). We, and others, have previously shown that over-expression of TrkA as well as constitutively active form of the tyrosine kinase Abl induces phosphorylation of APP on Y682
(N. Zambrano et al., 2001
; P. E. Tarr et al., 2002b
), suggesting that this tyrosine is the phosphorylation target of the NGF/TrkA signaling pathway. To test this hypothesis we have taken advantage of an APP Y682
G knock-in (KI) mouse, in which Y682
has been mutated into Glycine, to understand the physiological functions of Y682
(A. P. Barbagallo et al., 2010
). Hippocampal slices prepared from either APPYG/YG
mice or Wild Type (WT) littermates were treated with NGF. The APPYG/YG
mutant is not phosphorylated on Tyr 10 min after NGF treatment (), suggesting that Y682
is either the phosphorylation target of the NGF/TrkA signaling pathway or that it is necessary for phosphorylation of other Tyr residues of APP by TrkA. However, and surprisingly, we found that TrkA phosphorylation induced by NGF was absent in APPYG/YG
mice (, bottom panel). Lack of TrkA phosphorylation did not depend on reduced TrkA expression by APPYG/YG
mice () and suggests that TrkA signaling is impaired by the mutation at Y682
. This prediction was confirmed by the finding that NGF fails to activate down-stream signaling molecules, such as Akt, in APPYG/YG
APP and Y682 are necessary for activation of TrkA by NGF
Next we asked whether APPYG/YG
mutation abolishes NGF/TrkA signaling by either a loss or a gain of function mechanism. We answered this question by analyzing APP null mice. Of note, the Y682
G mutation acts like a null allele when the essential function of APP in development are analyzed in vivo
(H. Li et al., 2010
; A. P. Barbagallo et al., 2011
). Consistent with this, we found that NGF-dependent TrkA activation is also impaired in APP-/-
hippocampal slice, indicating that APP plays an important role in TrkA signaling and that this function requires Y682
(). Moreover, no significant differences were observed in p75 expression and processing (data not shown).
We than tested whether the role of APP in TrkA signaling had functional consequences. NGF exerts a trophic activity on DRG and CVS neurons, which express high levels of TrkA (). Notably, DRG and CVS neurons isolated from APPYG/YG mice were insensitive to this trophic function of NGF (+NGF) and an extensive neuronal loss was assessed when compared to the corresponding +NGF samples from WT mice ().
Table 1 reports neuronal nuclei assessment in APP WT and APPYG/YG mice
APP interacts with TrkA and both modulate their cellular distribution
To dissect the molecular and biochemical mechanism by which APP regulates TrkA signaling, we tested whether APP and TrkA physically interact in vivo, under physiological condition. To this end, we prepared protein samples from septum either of WT, APPYG/YG or APP-/- mice. Samples were immunoprecipitated with an α-TrkA antibody and analyzed by WB with an α-APP antibody. As shown in , a fraction of endogenous TrkA is complexed to endogenous APP in mouse brain. Interestingly, the APPYG/YG mutant does not interact with TrkA although APPYG/YG mice express normal amounts of APP, suggesting an essential role for Y682 in the APP/TrkA interaction. However the lack of a selective α–TrkA antibody fails to rule out the possibility that also TrkB may be involved in these events.
APP interacts with TrkA, regulates TrkA cellular distribution
Next, we determined whether APP regulates the sub-cellular distribution of TrkA. For this, we prepared from both WT and APPYG/YG mice primary neuronal cells derived from the DRG () and SCG (not shown). In WT mice, at low magnification, TrkA immunofluorescence was distributed on neuronal cell bodies and at high intensities along neuritis. At high magnification, TrkA immunofluorescence, localized in vesicles of small and homogeneous size, showed a preferential distribution on the cellular membrane or immediately beneath (; arrows). Of particular interest, TrkA staining was increased in proximity, and along, the neuritic domains and displayed a more grainy or beaded appearance with the tendency to organize in clusters (; arrows). These clusters filled the neuritis and sometime was possible to observe a certain number of TrkA positive vesicles organized in a row suggesting an anterograde transport through cytoskeletric structures (). The distribution pattern of APP was slight different than that of TrkA. APP immunopositive vesicles appeared less structurally defined and of more variable size (). As for TrkA, also APP appeared to increase in immunoreactivity in proximity and along the neuritic domains and showed a preferential membrane distribution (; arrows). The increase and accumulation of both APP and TrkA immunoreactivity in proximity and along neuritis () suggest the presence of an intense trafficking toward the cellular peripheral structures (axonal terminals, spines), where both proteins may exert their primary functions. Colocalization between TrkA and APP appeared to be selectively confined to the proximity and along neuritic domains (; asterisks).
In APPYG/YG mice, at low magnification, TrkA immunofluorescence resembled the distribution pattern observed in WT mice. However, at high magnification, several marked differences were evident. The TrkA prevalent membrane distribution showed in WT mice was lost in APPYG/YG mice in favor of a more intracytoplasmatic, clearly perinuclear, distribution pattern (). The loss of an organized distribution pattern was also clearly evident when analyzing the neuritic domains where a marked reduction of TrkA immunofluorescence can be observed. Very often, only few labeled grains, distantly interspaced, and organized in a row, could be followed (): suggesting the disruption of the TrkA cytoskeletric transport. As for TrkA, APP immunoreactivity was highly decreased from the cell membrane and neuritic domains, while appeared to increase in the intracellular and perinuclear regions (). Colocalization pattern between TrkA and APP appeared to be switched from the proximity and along neuritic domains (; asterisks) in WT mice to the intracellular and perinuclear regions (, asterisks) in APPYG/YG mice. Altogether these results suggest that in APPYG/YG mice both TrkA-APP interaction and cellular distribution are affected.