Facilitation of glutamate, but not GABA, release in Familial Alzheimer's APP mutant Knock-in rats with increased β-cleavage of APP

Abstract

Amyloid precursor protein (APP) modulates glutamate release via cytoplasmic and intravesicular interactions with the synaptic vesicle release machinery. The intravesicular domain, called ISVAID, contains the BACE1 cleavage site of APP. We have tested the functional significance of BACE1 processing of APP using App-Swedish (App^s ) knock-in rats, which carry an App mutation that causes familial Alzheimer's disease (FAD) in humans. We show that in App^s rats, β-cleavage of APP is favored over α-cleavage. App^s rats show facilitated glutamate, but not GABA, release. Our data support the notion that APP tunes glutamate release, and that BACE1 cleavage of the ISVAID segment of APP facilitates this function. We define this phenomenon as BACE1 on APP-dependent glutamate release (BAD-Glu). Unsurprisingly, App^s rats show no evidence of AD-related pathology at 15 days and 3 months of age, indicating that alterations in BAD-Glu are not caused by pathological lesions. The evidence that a pathogenic APP mutation causes an early enhancement of BAD-Glu suggests that alterations of BACE1 processing of APP in glutamatergic synaptic vesicles could contribute to dementia.

Keywords: APP; Alzheimer's Disease; BACE1; glutamate release; synaptic transmission.

Figure 1

Characterization of App ^h, App ^s, and App ^δ7 KI rats. (a) Left panel. To verify that the humanizing and Swedish mutations were correctly inserted in the App exon‐16, we amplified by PCR the App gene exon‐16 from App ^w/w , App ^h/h, and App ^s/s rats. Sequencing of the PCR products shows that the humanizing mutations and the humanizing plus Swedish mutations were correctly inserted into the App ^h/h and App ^s/s genomes, respectively. Sequencing analysis of genomic DNA confirms the expected G to C, T to A, and GC to AT substitution in App ^h/h rats and the GA to TC, G to C, T to A, and GC to AT substitution in App ^s/s rats. Substituted nucleotides are highlighted in gray. The amino acid sequences are indicated above the DNA sequences and the amino acid substitutions introduced by the mutations are highlighted in gray (GA to TC=KM to NL; G to C=G to R; T to A=Y to F; and GC to AT=R to H). Right panel. Predicted sequences of WT and App ^δ7 cDNAs and proteins. For space reasons, only Exon 16 (black) and Exon 17 (red) are shown. The 7‐bp deletion (in white characters and boxed in black), the transmembrane domain of APP (underlined), and the novel predicted COOH‐terminal of sAPP ^δ7 (bold and italic) are indicated. (b) Levels of App mRNA were measured in 21‐day‐old App ^w/w, App ^δ7/δ7 , App ^h/h, and App ^s/s rats (2 females and 3 males for each genotype). App mRNA expression was normalized to Gapdh mRNA expression. The δ7 mutant allele was a product of aberrant homology‐directed repair but is useful because this mutation will either produce a truncated soluble APPδ7 protein (sAPPδ7) or a hypomorphic allele. Data were analyzed by ordinary one‐way ANOVA followed by post hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences and presented as average (App/Gapdh) ± SEM. (c) Schematic representation of APPWT rat, APPh, APPSw, and the metabolites derived from α‐ and β‐secretase processing. The amino acid changes that humanize the Aβ region of are in red and underlined (G>R, F>Y, R>H); the amino acids changes that introduce the Swedish mutation are in blue and underlined (K>N, M>L); the epitopes recognized by antibodies are highlighted as follows: Y188 is highlighted in gray, M3.2 is highlighted in green, 6E10 highlighted in yellow, anti‐sAPPα is highlighted in cyan, anti‐sAPPβWT is highlighted in red, and anti‐sAPPβSw is highlighted in magenta. The αCTF and βCTF derived from APPh and APPSw are identical; thus, in the paper, we will refer to both as αCTF or βCTF. The App ^δ7 allele could produce sAPPδ7: The sequence in white highlighted in black indicates the novel amino acid sequence produced by the deletion of 7 bp, which causes a frameshift. Casp. Indicates the site of cleavage of APP in the cytoplasmic region by caspases, which leads to the generation of JCasp (Gervais et al., 1999; Pellegrini, Passer, Tabaton, Ganjei & D'Adamio, 1999). (d) Summary of the expected immunoreactivities of the anti‐APP antibodies used in this study: + = positive reactivity; − = no reactivity. (e) Western blot analysis of postnuclear supernatant isolated from App ^w/w, App ^δ7/δ7, App ^h/h, and App ^s/s rats with Y188 (detects mAPP, imAPP, αCTF, and βCTF from all animals except the App ^δ7/δ7 rats). (f) M3.2 (detects only rat WT mAPP, imAPP, and βCTF). (g) 6E10 (detects only mAPP, imAPP, and βCTF carrying the humanizing mutation). (h) Western blot analysis of brain soluble fractions with two anti‐sAPPβWT antibodies (which detect sAPPβ App ^h/h rats only). (i) An anti‐sAPPβSw antibody (detects sAPPβ in App ^s/s rats only). (j) 22C11 (detects sAPPβ and sAPPα in App ^h/h and App ^s/s rats). (k) Anti‐sAPPα (detects sAPPα in App ^h/h and App ^s/s rats)

Figure 2

Increased processing by β‐secretase and decreased processing by α‐secretase of APPSw. To test whether the App ^s/s rats present the expected changes in APP metabolism, we analyzed brain samples isolated from 21‐day‐old App ^h/h and App ^s/s rats, 2 females and 3 males for each genotype. (a) Western blot (WB) of postnuclear supernatant from App ^s/s and App ^h/h rats with Y188 and 6E10 (bottom). (b) WB analysis of soluble brain fractions with an ant‐sAPPα antibody showed that levels of sAPPα are significantly lower in App ^s/s compared to App ^h/h brains. (c) Because sAPPβh and sAPPβSw cannot be detected by the same antibody (see Figures 3a,b and 4d,e), to compare sAPPβh and sAPPβSw amounts, we used 6E10 and 22C11. Signal intensity was quantified with Image Lab software (Bio‐Rad). Quantification is shown on the right. (d) Aβ is produced by γ‐cleavage of βCTF, which is increased in App ^s/s brains. ELISA measurements of endogenous Aβ40 and Aβ42 in brain homogenates of 21‐day‐old animals. Levels of steady‐state endogenous Aβ40 and Aβ42 are increased in App ^s/s brains as compared to App ^h/h brains. The Aβ42/Aβ40 ratio did not change. These ELISA kits are specific for human Aβ40‐42, as attested by the fact that they gave no signal on brain homogenates from App ^w/w (expressing rat Aβ) and App ^δ7/δ7 (expressing no Aβ) rats (not shown). Data are represented as mean ± SEM. Data were analyzed by Student's t test and shown as average ± SEM. **p < .01; ****p < .0001

Figure 3

The Swedish mutations do not alter the effect of Ex/TM on glutamate release. (a) Sequence of the Ex/TM and Ex/TM‐Sw peptides. (b) Average PPF at 50 and 200 ms ISI. Representative traces of EPSCs evoked at 50 ms ISI are shown. Ex/TM and Ex/TM‐Sw significantly decrease PPF. (c) Ex/TM and Ex/TM‐Sw significantly increase mEPSC frequency. Amplitudes and decay time of mEPSCs were not changed by these peptides. Representative recording traces of mEPSCs are shown. Data were analyzed by ordinary one‐way ANOVA followed by post hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences. Four male and four female rats were used for each group. The number of recordings analyzed for each group is indicated inside the bars. All data represent means ± SEM

Figure 4

Knock‐in App ^s/s rats show increased glutamatergic, but not GABAergic, transmission at SC–CA3>CA1 pyramidal cell synapses. (a) App ^s/s rats show significantly increased frequency of mEPSC. (b) App ^s/s rats show significantly increased amplitude of mEPSC. (c) Decay time of mEPSCs was not changed by the Swedish mutation. (d) Representative recording traces of mEPSCs. (e) Average PPF at 50 ms ISI. Representative traces are shown on the right of the panel. (f) Average PPF at 200 ms ISI. Representative traces are shown on the right of the panel. (g) AMPA/NMDA ratio is not significantly changed by APPSw. (h) The App ^s mutation does not significantly change frequency of mIPSC. (i) Amplitude of mIPSC is not altered in App ^s rats. (j) Decay time of mIPSCs was not changed by the Swedish mutation. (k) Representative recording traces of mIPSCs. (l) Average PPF at 50 ms ISI. (m) Average PPF at 200 ms ISI. Representative traces are shown on the right of the panel. Data were analyzed by ordinary one‐way ANOVA followed by post hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences. For these experiments, we used: 6 male and 6 female App ^h/h, App ^s/h, and App ^s/s rats for glutamate recordings and 6 male and 6 female App ^h/h, App ^s/h, and App ^s/s rats for GABA recordings. The number of recordings analyzed for each group is indicated inside the bars. All data represent means ± SEM

Figure 5

The App ^s/h and App ^s/s rats do not show AD‐like histopathology at 15 days and 3 months of age. Representative IHC and histology from the anterior hippocampus in a representative 15‐day‐ and 3‐month‐old subject for each genotype. Three female and three male rats per each genotype and age were tested

Figure 6

Modeling BAD‐Glu mechanisms and how the Swedish mutation may alter BAD‐Glu. (a) APP undergoes complex proteolysis. In the amyloidogenic proteolytic cascade, APP is cleaved by β‐secretase/BACE1 into sAPPβ and the COOH‐terminal fragment βCTF. Cleavage of βCTF by γ‐secretase produces Aβ peptides and the intracellular domain (AID/AICD). Alternatively, APP is processed by α‐secretase into sAPPα and the COOH‐terminal fragment αCTF. αCTF can be cleaved by γ‐secretase to produce P3 and AID/AICD (Passer et al., 2000; Sisodia & St George‐Hyslop, 2002). The Swedish mutations cause more β‐cleavage with a corresponding increase in direct and indirect metabolites (filled in black), but less α‐cleavage with a corresponding decrease in direct and indirect metabolites (filled in white). In addition, mAPP levels are reduced. The quantitative alterations in one or more of these APP metabolites can participate in BAD‐Glu dysregulation caused by the Swedish mutations. (b) The Swedish mutations cause changes in the primary sequence of several APP metabolites, including APP (APPSw), sAPPβ (sAPPβSw), and sAPPα (sAPPαSw). The qualitative alterations in one or more of these APP metabolites can participate in BAD‐Glu dysregulation caused by the Swedish mutations. (c) APP present in synaptic vesicles can interact with SV proteins and proteins regulating exocytosis via an intraluminal (ISVAID) and a cytosolic (JCasp) domain. Intraluminal and cytosolic interactions may have an opposite effect: the former tunes down glutamate release while the latter facilitates glutamate release. Cleavage of APP by BACE1 inside the ISVAID can abrogate the intravesicular interaction triggering the facilitator function of the cytosolic interaction. Increased β‐cleavage of APPSw can dysregulate BAD‐Glu dysregulation and facilitate glutamate release