GDE2-RECK controls ADAM10 α-secretase-mediated cleavage of amyloid precursor protein

Abstract

A disintegrin and metalloprotease 10 (ADAM10) is the α-secretase for amyloid precursor protein (APP). ADAM10 cleaves APP to generate neuroprotective soluble APPα (sAPPα), which precludes the generation of Aβ, a defining feature of Alzheimer's disease (AD) pathophysiology. Reduced ADAM10 activity is implicated in AD, but the mechanisms mediating ADAM10 modulation are unclear. We find that the plasma membrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2) stimulates ADAM10 APP cleavage by shedding and inactivating reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a glycosylphosphatidylinositol (GPI)-anchored inhibitor of ADAM10. In AD, membrane-tethered RECK is highly elevated and GDE2 is abnormally sequestered inside neurons. Genetic ablation of GDE2 phenocopies increased membrane RECK in AD, which is causal for reduced sAPPα, increased Aβ, and synaptic protein loss. RECK reduction restores the balance of APP processing and rescues synaptic protein deficits. These studies identify GDE2 control of RECK surface activity as essential for ADAM10 α-secretase function and physiological APP processing. Moreover, our results suggest the involvement of the GDE2-RECK-ADAM10 pathway in AD pathophysiology and highlight RECK as a potential target for therapeutic development.

Fig. 1.. GDE2 distribution is impaired in AD.

A-D, Immunohistochemical staining of human parietal (BA40) cortex sections of control individuals (CTL), and patients with AD and HD. Arrows highlight cells with GDE2 accumulations. B, NeuN (blue) marks neurons, GDE2 is labeled in brown. C, Representative images of CTL and AD neurons. Arrows highlight neurons with low and high GDE2 accumulation (GDE2^low, GDE2^high). Quantification of the number of GDE2^low (***p=0.0002) and GDE2^high (***p=1.49E-09) neurons in CTL (n=10) and AD (n=10). Total number of neurons counted: CTL = GDE2^low (262), GDE2^high (6) and AD = GDE2^low (670), GDE2^high (796). Mean ± SEM, two-tailed unpaired Student’s t-test. D, Arrows mark GDE2 accumulations. Autopsy numbers are at the bottom right of each panel. Ab = antibody. Scale bars: A, D, 3μm; B, 5μm; C, 10μm.

Fig. 2.. Membrane RECK is increased in brain of patients with AD.

A-D, Representative Western blots of Triton X-114 fractionated parietal (BA40) and pre-frontal (BA9) cortical extracts prepared from postmortem brain from CTL individuals, and patients with AD and HD. Detergent-rich fractions (DT-R), detergent-poor fractions (DT-P), total unfractionated extract (Tot). Na⁺/K⁺ ATPase and Actin are loading controls and confirm separation of membrane and non-membrane fractions (compare between individual total fractions; between individual DT-R fractions; between individual DT-P fractions). A, Braak V/VI parietal cortex: Mem. RECK/total RECK **p=0.0088, Mem. RECK/Na⁺/K⁺ ATPase **p=0.0096; released RECK/total RECK *p=0.0123, released RECK/Actin **p=0.0084; total RECK/Actin n.s. p=0.7508. n=4 CTL, n=5 AD. B, Braak V/VI pre-frontal cortex: Mem. RECK/total RECK *p=0.0251, Mem. RECK/Na⁺/K⁺ ATPase **p=0.0083; released RECK/total RECK *p=0.0486, released RECK/Actin **p=0.0039; total RECK/Actin n.s. p=0.5837. n=13 CTL, n=12 AD. C, Braak III/IV AD pre-frontal cortex: Mem. RECK/total RECK ***p=2.55E-05, Mem. RECK/Na⁺/K⁺ ATPase **p=0.0017; released RECK/total RECK ***p=0.0005, released RECK/Actin *p=0.0102; total RECK/Actin n.s. p=0.6641. n=10 CTL, n=10 AD. D, HD parietal cortex: Mem. RECK/total RECK n.s. p=0.9840, Mem. RECK/Na⁺/K⁺ ATPase n.s. p=0.2460; released RECK/total RECK n.s. p=0.0597, released RECK/Actin n.s. p=0.3916; total RECK/Actin n.s. p=0.2244. n=10 CTL, n=10 HD. All graphs: Mean ± SEM, two-tailed unpaired Student’s t-test.

Fig. 3.. GDE2 ablation reduces α-secretase and increases β-secretase APP cleavage.

A, Western blots and graphs quantifying sAPPα (sAPPα/APP *p=0.0122), sAPPβ (sAPPβ/APP **p=0.0071) and sAPPα:sAPPβ (*p=0.0201) in 19-month Gde2−/− cortices compared with WT. (n=3). B, Western blots and graphs quantifying sAPPα (sAPPα/APP **p=0.0097), sAPPβ (sAPPβ/APP ***p=0.0004) and sAPPα:sAPPβ (**p=0.0060) in DIV14 Gde2−/− cortical neurons compared with WT. (n=3). C, Western blots and graphs quantifying GDE2 expression (GDE2/Actin *p=0.0158), sAPPα (sAPPα/APP ***p=0.0004), sAPPβ (sAPPβ/APP *p=0.0176), and sAPPα:sAPPβ ratio (***p=6.69E-05) in 13-month RosaCreER;Gde2lox/− cortical lysates (n=3). Tamoxifen was injected on postnatal (p) days 10 and 11 in RosaCreER;Gde2+/− and RosaCreER;Gde2lox/− mice, and animals were harvested at 13-months of age. All graphs: Mean ± SEM, two-tailed unpaired Student’s t-test.

Fig. 4.. GDE2 ablation promotes amyloidogenic APP processing.

A, ELISA quantifications of TBS-soluble Aβ40 (*p=0.0252), Aβ42 (**p=0.0088) and Aβ40/42 ratios (*p=0.0461) in Gde2−/− 19-month cortical extracts (n=3); and TBS-soluble Aβ40 (*p=0.0206), Aβ42 (***p=0.0003) and Aβ40/42 ratios (***p=0.0002) in Gde2−/− DIV14 cortical neuronal cultures (n=3). B, Quantification of ELISA in APP/PS1 and APP/PS1 Gde2−/− 4-month cortical extracts for soluble Aβ peptides (TBS-soluble Aβ40 ***p=0.0006; Aβ42 ***p=4.62E-06, n=3) and Aβ aggregates (Formic acid-soluble Aβ40 ***p=7.0E-06; Aβ42 ***p=0.0001, n=3), with associated Aβ40/42 ratios (TBS-soluble Aβ40/42 ratios *p=0.0399; Formic acid-soluble Aβ40/42 ratios *p=0.0444). C, Immunostaining of 4-month APP/PS1 and APP/PS1;Gde2−/− cortical sections with corresponding quantification of amyloid load [area fraction of 6E10-positive plaque (red)], male (***p=0.0007) and female (*p=0.0208). D, Western blots and quantification of phosphorylated tau (Ser202 and Thr205 pTau/Tau, **p=0.00127) in 4-month male APP/PS1 and APP/PS1;Gde2−/− cortical extracts (n=3). All graphs: Mean ± SEM, two-tailed unpaired Student’s t-test.

Fig. 5.. Loss of GDE2 results in synaptic protein reduction.

A, Western blots and quantification of Synaptophysin/Actin (***p=0.0009), PSD95/Actin (*p=0.0455) and Homer/Actin (*p=0.0160), (n=3) in 19-month WT and Gde2−/− cortical extracts. B, Western blots and quantification of Synaptophysin/Actin (**p=0.0056), PSD95/Actin (*p=0.0135) and Homer/Actin (**p=0.0023), (n=3) in extracts of DIV14 WT and Gde2−/− cortical neuronal cultures. C, Western blots and quantification of Synaptophysin/Actin (***p=0.0008), PSD95/Actin (***p=6.68E-06), and Homer/Actin (***p=3.24E-05) in cortical extracts from13month tamoxifen-treated RosaCreER;Gde2+/− and RosaCreER;Gde2lox/− animals (n=3). D, Representative images of sections from 19-month WT and Gde2−/− mouse cortex. Circles mark synapses visualized by colocalization of Synaptophysin and PSD95. Graph quantifying synapse numbers (*p=0.0475, n=3). E, Western blots and quantification of Synaptophysin/Actin (*p=0.0272), PSD95/Actin (**p=0.0038) and Homer/Actin (*p=0.0444) (n=3) in cortical extracts from brains of 4-month old APP/PS1 and APP/PS1;Gde2−/− male mice. Actin is used as a loading control. All graphs: Mean ± SEM, two-tailed unpaired Student’s t-test.

Fig. 6.. Surface RECK inhibits ADAM10 α-secretase activity and causes reduction in synaptic proteins.

A, Western blots and quantification of sAPPα (**p=0.0056), sAPPβ (**p=0.0084), and sAPPα:sAPPβ (*p=0.0372) in GFP- and CD2RECK-transduced DIV14 cortical neuronal cultures (n=3). Asterisk marks CD2RECK expression, arrow marks endogenous RECK. B, Western blots and quantification of Synaptophysin/Actin (**p=0.0017), PSD95/Actin (**p=0.0011), and Homer/Actin (***p=5.02E-06) (n=3) in GFP- and CD2RECK-transduced DIV14 cortical neuronal cultures. Actin is used as a loading control. All graphs: Mean ± SEM, two-tailed unpaired Student’s t-test.

Fig. 7.. GDE2 promotes ADAM10 α-secretase activity by regulating RECK

A, Western blots and quantification of RECK (*p=0.0495), sAPPα (*p=0.0403), sAPPβ (p*=0.0125), and sAPPα:sAPPβ (***p=0.0002) in extracts from 19-month Gde2−/− and Gde2−/−;Reck+/− cortices (n=3). B, Western blots and quantification of Synaptophysin/Actin (*p=0.0119), PSD95/Actin (*p=0.0184), and Homer/Actin (**p=0.0027) in extracts from 19-month Gde2−/− and Gde2−/−;Reck+/− cortices (n=3). Actin is used as a loading control. C, Quantification of ELISA for TBS-soluble Aβ40 (*p=0.0435) and Aβ42 (***p=0.0009), with associated Aβ40/42 ratio (***p=4.32E-05) (n=3) in Gde2−/− DIV14 cortical neuronal cultures treated with control or Reck siRNAs. D, ELISA analysis of APP/PS1 Gde2−/− DIV14 cortical neuronal cultures treated with control or Reck siRNAs; TBS-soluble Aβ40 ***p=4.47E-05; TBS-soluble Aβ42 ***p=4.80E-06; TBS-soluble Aβ40/42 ratios *p=0.02434, (n= 3). All graphs: Mean ± SEM, two-tailed unpaired Student’s t-test.