Regulation of the hippocampal translatome by Apoer2-ICD release

Abstract

Background: ApoE4, the most significant genetic risk factor for late-onset Alzheimer's disease (AD), sequesters a pro-synaptogenic Reelin receptor, Apoer2, in the endosomal compartment and prevents its normal recycling. In the adult brain, Reelin potentiates excitatory synapses and thereby protects against amyloid-β toxicity. Recently, a gain-of-function mutation in Reelin that is protective against early-onset AD has been described. Alternative splicing of the Apoer2 intracellular domain (Apoer2-ICD) regulates Apoer2 signaling. Splicing of juxtamembraneous exon 16 alters the γ-secretase mediated release of the Apoer2-ICD as well as synapse number and LTP, and inclusion of exon 19 ameliorates behavioral deficits in an AD mouse model. The Apoer2-ICD has also been shown to alter transcription of synaptic genes. However, the role of Apoer2-ICD release upon transcriptional regulation and its role in AD pathogenesis is unknown.

Methods: To assess in vivo mRNA-primed ribosomes specifically in hippocampi transduced with Apoer2-ICD splice variants, we crossed wild-type, cKO, and Apoer2 cleavage-resistant mice to a Cre-inducible translating ribosome affinity purification (TRAP) model. This allowed us to perform RNA-Seq on ribosome-loaded mRNA harvested specifically from hippocampal cells transduced with Apoer2-ICDs.

Results: Across all conditions, we observed ~4,700 altered translating transcripts, several of which comprise key synaptic components such as extracellular matrix and focal adhesions with concomitant perturbation of critical signaling cascades, energy metabolism, translation, and apoptosis. We further demonstrated the ability of the Apoer2-ICD to rescue many of these altered transcripts, underscoring the importance of Apoer2 splicing in synaptic homeostasis. A variety of these altered genes have been implicated in AD, demonstrating how dysregulated Apoer2 splicing may contribute to neurodegeneration.

Conclusions: Our findings demonstrate how alternative splicing of the APOE and Reelin receptor Apoer2 and release of the Apoer2-ICD regulates numerous translating transcripts in mouse hippocampi in vivo. These transcripts comprise a wide range of functions, and alterations in these transcripts suggest a mechanistic basis for the synaptic deficits seen in Apoer2 mutant mice and AD patients. Our findings, together with the recently reported AD-protective effects of a Reelin gain-of-function mutation in the presence of an early-onset AD mutation in Presenilin-1, implicate the Reelin/Apoer2 pathway as a target for AD therapeutics.

Keywords: Alternative splicing; Alzheimer’s TRAP-Seq; ApoE; Apoer2; Reelin; Synaptic homeostasis.

Fig. 1

Apoer2-ICD regulation of translating transcripts in vivo. A Rosa^26fs-TRAP mice crossed with the Apoer2^WT, conditional KO, and cleavage-resistant Apoer2 transgenic mice (Apoer2^Δ16±19) were injected with lentiviruses expressing Cre resulting in a GFP-tagged ribosomal subunit (L10a:GFP) to allow for pull-down of ribosome-bound transcripts (intrahippocampal injections; coordinates were AP: -2.2, ML: ±1.3, DV, -1.3). Using an Internal Ribosome Entry Site (IRES), Apoer2-ICD^[±19] was co-expressed to assess the effect of overexpression of either Apoer2-ICD in Apoer2^WT or rescue of effects of lack of the Apoer2-ICD (B). C-D Heatmaps representing the proportion of genes altered in Apoer2 transgenic mice that are either rescued or not rescued with the Apoer2-ICD[±19] (C) or altered by overexpression of either Apoer2-ICD in Apoer2^WT or (D) effects of the ICD independent of the lack of ICD-release. E Enrichment analysis of the ~4700 transcripts differentially translated across all conditions, demonstrating enrichment for synaptic compartments, neuronal processes and pathways, as well as diseases of the brain. F Gene-term network of the ClueGO/Cluepedia enrichment analysis of synaptic transcripts annotated in the SynGO database. (For each mouse line: Cre-only and Cre+Apoer2-ICD[+19]: n= 4 individual and one pooled set of 4 RNA samples, Cre+Apoer2-ICD[Δ19]: n= 4 individual RNA samples)

Fig. 2

Basal translatome differences in Apoer2^cKO and cleavage-deficient hippocampi. A, B Supervenn and heatmap depicting the overlap of transcripts significantly altered in Apoer2 transgenic mice compared to Apoer2^WT. A Translatome overlap of transcripts significantly (p-value< 0.05, |log₂FC|> 0.58) altered in the Apoer2 KI/cKO genotypes compared to Apoer2^WT. B Heatmap of the 53 translating SynGO (bolded) and disease-related transcripts (symbols) significantly altered in all three Apoer2 KI/cKO compared to Apoer2^WT. C Expanded translatome overlap of the ~3,000 transcripts altered in the Apoer2 KI/cKO genotypes compared to Apoer2^WT (|log₂FC|>0.58, p-value<0.05 in at least one). D Heatmap demonstrating the log₂FC of expanded basal translatome in Apoer2^cKO and cleavage-deficient hippocampi. E Enrichment analysis of the transcripts in Panel D. F Diagram depicting the SynGO transcripts differentially-translated in the Apoer2 KI/cKO (Panel D) compared to Apoer2^WT (up, red circles; down, blue circles) in the network from Fig. 1F. AD, Alzheimer’s disease; SCZ, schizophrenia; ASD, autism spectrum disorders; ID, intellectual disability

Fig. 3

Synaptic effects of overexpressing either Apoer2-ICD in Apoer2^WT. A, B Schematic representation of the experiment. C Diagram depicting the up- (purple symbols) and down- (yellow symbols) regulated transcripts by both ICDs in the same direction (circles), ICD[+19] (plus-sign) or ICD[Δ19] (triangles). D Heatmap of the log₂FC differentially-transcribing transcripts in Apoer2^WT with either Apoer2-ICD from the network in Fig. 1F. Heatmap displaying the log₂FC expression of the synaptic transcripts not represented in the networks in Panels A and B, respectively. *p<0.05, **p<0.01

Fig. 4

Synaptic effects of Apoer2-ICD in Apoer2^cKO. A, B Diagrams depicting the transcripts differentially-translated in the Apoer2^cKO at baseline (Cre-only) compared to Apoer2^WT (up, red circles; down, blue circles) (A) or differentially-translated in Apoer2^cKO neurons expressing either ICD[+19] (plus-sign) or ICD[Δ19] (triangles) compared to the baseline Apoer2^cKO in the network from Fig. 1F (up, purple symbols; down, yellow symbols) (B). Note in Panel B, transcripts regulated by both ICDs in the same direction are depicted with circles and those differentially regulated by either the ICD[+19] or ICD[Δ19] are represented by plus-signs or triangles, respectively. C, D Heatmap displaying the log₂FC expression of the synaptic transcripts not represented in the networks in Panels A and B, respectively. *p<0.05, **p<0.01

Fig. 5

Synaptic effects of Apoer2-ICD in Apoer2^Δ16+19. A, B Diagrams depicting the transcripts differentially-translated in the Apoer2^Δ16+19 knockin at baseline (Cre-only) compared to Apoer2^WT (up, red circles; down, blue circles) (A) or differentially-translated in Apoer2^Δ16+19 neurons expressing either ICD[+19] (plus-sign) or ICD[Δ19] (triangles) compared to the baseline Apoer2^Δ16+19 in the network from Fig. 1F (up, purple symbols; down, yellow symbols) (B). Note in Panel B, transcripts regulated by both ICDs in the same direction are depicted with circles and those differentially regulated by either the ICD[+19] or ICD[Δ19] are represented by plus-signs or triangles, respectively. C, D Heatmap displaying the log₂FC expression of the synaptic transcripts not represented in the networks in Panels A and B, respectively. *p<0.05, **p<0.01, ***p<0.001

Fig. 6

Synaptic effects of Apoer2-ICD in Apoer2^Δ16Δ19. A, B Diagrams depicting the transcripts differentially-translated in the Apoer2^Δ16Δ19 knockin at baseline (Cre-only) compared to Apoer2^WT (up, red circles; down, blue circles) (A) or differentially-translated in Apoer2^Δ16Δ19 neurons expressing either ICD[+19] (plus-sign) or ICD[Δ19] (triangles) compared to the baseline Apoer2^Δ16Δ19 in the network from Fig. 1F (up, purple symbols; down, yellow symbols) (B). Note in Panel B, transcripts regulated by both ICDs in the same direction are depicted with circles and those differentially regulated by either the ICD[+19] or ICD[Δ19] are represented by plus-signs or triangles, respectively. C, D Heatmap displaying the log₂FC expression of the synaptic transcripts not represented in the networks in Panels A and B, respectively. *p<0.05, **p<0.01, ***p<0.001

Fig. 7

Apoer2-ICD regulation of AD GWAS and Reelin signaling transcripts. Heatmaps depicting the log₂FC of translating AD GWAS (A) or Reelin signaling pathway (B) transcripts between the Apoer2cKO/KI neurons expressing only Cre or Cre with either Apoer2-ICD compared to Apoer2^WT expressing only Cre (left panels of A,B) or between neurons expressing either ICD[+19] or ICD[Δ19] compared to the Cre-only translation within each genotype (right panels of A,B). B Reelin pathway transcripts are sorted into four groups: the core Reelin receptor complex and associated tyrosine kinases (1), the signaling pathway regulating cadherin trafficking (2), the signaling pathway regulating tau (MAP1B) phosphorylation (3), and the other members of the canonical Reelin pathway (4). Significance is represented by the size of the node