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The Amyloid Precursor Protein Controls PIKfyve Function.

Balklava Z, Niehage C, Currinn H, Mellor L, Guscott B, Poulin G, Hoflack B, Wassmer T - PLoS ONE (2015)

Bottom Line: Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals.Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo.This regulation is required for maintaining endosomal and neuronal function.

View Article: PubMed Central - PubMed

Affiliation: Aston University, School of Life and Health Sciences, Aston Triangle, Birmingham, B4 7ET, United Kingdom.

ABSTRACT
While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease.

No MeSH data available.


Related in: MedlinePlus

APL-1/PPK-3 interplay is required for neuronal function.(A) The C. elegans synaptic marker GFP::RAB-3 was used to visualise the central nervous system of control animals, single and double mutants. While the nervous system was intact at the light microscopic level in control animals and single mutants, in the apl-1(yn5) ppk-3(n2835) double mutant the accumulation of the synaptic vesicle marker GFP::RAB-3 was impaired leading to a more homogeneous, less well-defined staining of the ventral nerve chord. Enlargement of the respective areas is shown in the second row of panels. Bar, 50 μm. (B) Average intensity difference between GFP::RAB-3 maxima (corresponding to synapses) and GFP::RAB-3 minima (intersynaptic space) measured along the C. elegans ventral nerve chord demonstrated the reduced ability of the apl-1(yn5) ppk-3(n2835) double mutant to concentrate RAB-3 in synapses compared to the N2 control (p<0.01, two-tailed t-test, n>13). Error bars are s.e.m. (C) Thrashing analysis of single and double mutants demonstrated that the interplay between APL-1 and the PPK-3 complex is required for motor control in C. elegans. The double mutants apl-1(yn5); vacl-14(ok1877) and apl-1(yn5) ppk-3(n2835) were significantly impaired in their thrashing compared to the Bristol N2 and lon-2(e678) controls and the single mutants (p values student’s t-test <0.01, n = 20 per strain). Error bars are s.e.m.
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pone.0130485.g006: APL-1/PPK-3 interplay is required for neuronal function.(A) The C. elegans synaptic marker GFP::RAB-3 was used to visualise the central nervous system of control animals, single and double mutants. While the nervous system was intact at the light microscopic level in control animals and single mutants, in the apl-1(yn5) ppk-3(n2835) double mutant the accumulation of the synaptic vesicle marker GFP::RAB-3 was impaired leading to a more homogeneous, less well-defined staining of the ventral nerve chord. Enlargement of the respective areas is shown in the second row of panels. Bar, 50 μm. (B) Average intensity difference between GFP::RAB-3 maxima (corresponding to synapses) and GFP::RAB-3 minima (intersynaptic space) measured along the C. elegans ventral nerve chord demonstrated the reduced ability of the apl-1(yn5) ppk-3(n2835) double mutant to concentrate RAB-3 in synapses compared to the N2 control (p<0.01, two-tailed t-test, n>13). Error bars are s.e.m. (C) Thrashing analysis of single and double mutants demonstrated that the interplay between APL-1 and the PPK-3 complex is required for motor control in C. elegans. The double mutants apl-1(yn5); vacl-14(ok1877) and apl-1(yn5) ppk-3(n2835) were significantly impaired in their thrashing compared to the Bristol N2 and lon-2(e678) controls and the single mutants (p values student’s t-test <0.01, n = 20 per strain). Error bars are s.e.m.

Mentions: As defective PIKfyve function has been shown to cause profound neurodegeneration in mice and in humans [21, 25] and as APP is crucially implicated in Alzheimer's disease we wanted to test whether mutations in apl-1, ppk-3 or vacl-14 can lead to impaired neuronal function in C. elegans. We characterised the overall morphology of the neuronal system of C. elegans using the synaptic marker GFP::RAB-3 by fluorescence microscopy (Fig 6). In single apl-1(yn5) and ppk-3(n2835) mutants the neuronal system appeared to be largely unaffected. However, the apl-1(yn5) ppk-3(2835) double mutant failed to accumulate GFP::RAB-3 label in the characteristic “pearls-on-a-string” fashion to the same extent compared to control animals, leading to more homogeneous RAB-3 staining along the ventral nerve chord (Fig 6). Lack of concentrating RAB-3 vesicles in synapses along the ventral nerve chord suggests that synaptic transmission may be impaired.


The Amyloid Precursor Protein Controls PIKfyve Function.

Balklava Z, Niehage C, Currinn H, Mellor L, Guscott B, Poulin G, Hoflack B, Wassmer T - PLoS ONE (2015)

APL-1/PPK-3 interplay is required for neuronal function.(A) The C. elegans synaptic marker GFP::RAB-3 was used to visualise the central nervous system of control animals, single and double mutants. While the nervous system was intact at the light microscopic level in control animals and single mutants, in the apl-1(yn5) ppk-3(n2835) double mutant the accumulation of the synaptic vesicle marker GFP::RAB-3 was impaired leading to a more homogeneous, less well-defined staining of the ventral nerve chord. Enlargement of the respective areas is shown in the second row of panels. Bar, 50 μm. (B) Average intensity difference between GFP::RAB-3 maxima (corresponding to synapses) and GFP::RAB-3 minima (intersynaptic space) measured along the C. elegans ventral nerve chord demonstrated the reduced ability of the apl-1(yn5) ppk-3(n2835) double mutant to concentrate RAB-3 in synapses compared to the N2 control (p<0.01, two-tailed t-test, n>13). Error bars are s.e.m. (C) Thrashing analysis of single and double mutants demonstrated that the interplay between APL-1 and the PPK-3 complex is required for motor control in C. elegans. The double mutants apl-1(yn5); vacl-14(ok1877) and apl-1(yn5) ppk-3(n2835) were significantly impaired in their thrashing compared to the Bristol N2 and lon-2(e678) controls and the single mutants (p values student’s t-test <0.01, n = 20 per strain). Error bars are s.e.m.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488396&req=5

pone.0130485.g006: APL-1/PPK-3 interplay is required for neuronal function.(A) The C. elegans synaptic marker GFP::RAB-3 was used to visualise the central nervous system of control animals, single and double mutants. While the nervous system was intact at the light microscopic level in control animals and single mutants, in the apl-1(yn5) ppk-3(n2835) double mutant the accumulation of the synaptic vesicle marker GFP::RAB-3 was impaired leading to a more homogeneous, less well-defined staining of the ventral nerve chord. Enlargement of the respective areas is shown in the second row of panels. Bar, 50 μm. (B) Average intensity difference between GFP::RAB-3 maxima (corresponding to synapses) and GFP::RAB-3 minima (intersynaptic space) measured along the C. elegans ventral nerve chord demonstrated the reduced ability of the apl-1(yn5) ppk-3(n2835) double mutant to concentrate RAB-3 in synapses compared to the N2 control (p<0.01, two-tailed t-test, n>13). Error bars are s.e.m. (C) Thrashing analysis of single and double mutants demonstrated that the interplay between APL-1 and the PPK-3 complex is required for motor control in C. elegans. The double mutants apl-1(yn5); vacl-14(ok1877) and apl-1(yn5) ppk-3(n2835) were significantly impaired in their thrashing compared to the Bristol N2 and lon-2(e678) controls and the single mutants (p values student’s t-test <0.01, n = 20 per strain). Error bars are s.e.m.
Mentions: As defective PIKfyve function has been shown to cause profound neurodegeneration in mice and in humans [21, 25] and as APP is crucially implicated in Alzheimer's disease we wanted to test whether mutations in apl-1, ppk-3 or vacl-14 can lead to impaired neuronal function in C. elegans. We characterised the overall morphology of the neuronal system of C. elegans using the synaptic marker GFP::RAB-3 by fluorescence microscopy (Fig 6). In single apl-1(yn5) and ppk-3(n2835) mutants the neuronal system appeared to be largely unaffected. However, the apl-1(yn5) ppk-3(2835) double mutant failed to accumulate GFP::RAB-3 label in the characteristic “pearls-on-a-string” fashion to the same extent compared to control animals, leading to more homogeneous RAB-3 staining along the ventral nerve chord (Fig 6). Lack of concentrating RAB-3 vesicles in synapses along the ventral nerve chord suggests that synaptic transmission may be impaired.

Bottom Line: Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals.Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo.This regulation is required for maintaining endosomal and neuronal function.

View Article: PubMed Central - PubMed

Affiliation: Aston University, School of Life and Health Sciences, Aston Triangle, Birmingham, B4 7ET, United Kingdom.

ABSTRACT
While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease.

No MeSH data available.


Related in: MedlinePlus