9  Gene Enrichment Analysis

To identify biological pathways and processes affected by Cre-mediated ApoE4→ApoE2 conversion in liver tissue, we performed complementary enrichment analyses using the high-confidence differentially expressed genes from Method 1 (combined batches, no batch adjustment).

9.1 Liver

9.1.1 Over-Representation Analysis (ORA)

Over-representation analysis using g:Profiler2 identified two significantly enriched Gene Ontology Biological Process terms among the 63 liver DEGs (Method 1, FDR < 0.01, |log₂FC| > 1). The most significant enrichment was for positive regulation of low-density lipoprotein particle receptor catabolic process (GO:0032805, p = 1.7×10⁻², 3 genes), directly implicating lipoprotein receptor regulation in the transcriptional response.

The second enriched term was sulfur compound metabolic process (GO:0006790, p = 4.5×10⁻², 323 genes), a broad metabolic category that may reflect secondary metabolic remodeling following altered lipid handling. While this enrichment is less specific, it suggests that ApoE isoform conversion triggers broader metabolic adaptations beyond lipid metabolism alone.

The limited number of significantly enriched terms in ORA likely reflects the modest size of the high-confidence DEG list (63 genes after stringent filtering), which reduces statistical power to detect over-representation in narrowly defined functional categories. This motivated the use of Gene Set Enrichment Analysis as a complementary approach that leverages information from all genes rather than only those passing significance thresholds.

9.1.2 Gene Set Enrichment Analysis (GSEA)

Gene Set Enrichment Analysis (GSEA) using the complete ranked gene list identified five significantly enriched pathways (FDR < 0.01), all related to mitochondrial oxidative metabolism:

pathway	NES	padj	size
GOBP_PROTON_MOTIVE_FORCE_DRIVEN_ATP_SYNTHESIS	2.136	0.004	70
GOBP_NADH_DEHYDROGENASE_COMPLEX_ASSEMBLY	2.111	0.004	62
GOBP_MITOCHONDRIAL_RESPIRATORY_CHAIN_COMPLEX_ASSEMBLY	2.110	0.001	101
GOBP_TRANSLATION_AT_SYNAPSE	2.106	0.006	48
GOBP_OXIDATIVE_PHOSPHORYLATION	1.893	0.007	137

Notably, all five pathways exhibited positive enrichment scores, indicating coordinated upregulation of genes involved in these processes following ApoE4→ApoE2 conversion. The enrichment plot for proton motive force-driven ATP synthesis illustrated that genes in this pathway were strongly concentrated among highly upregulated genes, with peak enrichment occurring within the top ~25% of ranked genes. This distribution pattern indicates genuine coordinated regulation rather than random association.

APOE2 Enhances Mitochondrial ATP Production

The convergent enrichment of multiple interconnected mitochondrial pathways—respiratory chain complex assembly (which builds the electron transport chain), NADH dehydrogenase complex assembly (Complex I assembly specifically), proton motive force-driven ATP synthesis (ATP synthase function), and oxidative phosphorylation (the overall process)—suggests a coherent biological program. Specifically, these findings indicate that ApoE2 expression enhances mitochondrial bioenergetic capacity compared to ApoE4 expression in hepatocytes.

The inclusion of “translation at synapse” among enriched pathways was unexpected in a liver tissue analysis, as this term typically applies to neuronal tissues. This likely represents either: (1) annotation artifacts where genes involved in general translational machinery are also annotated to synapse-specific processes, or (2) genuine enrichment of ribosomal/translational components that support increased protein synthesis for mitochondrial biogenesis

9.2 Brain

9.2.1 Over-Representation Analysis (ORA)

Over-representation analysis of the 69 brain DEGs (Method 1, FDR < 0.01, |log₂FC| > 1) identified ten significantly enriched Gene Ontology terms spanning multiple functional domains. Unlike the liver, where enrichment centered on lipoprotein metabolism, the brain showed a diverse functional landscape.

This heterogeneous enrichment pattern suggests that the transcriptional response to ApoE4→ApoE2 conversion in brain affects multiple distinct biological systems rather than a single coherent pathway. The prominence of neuroendocrine-related terms (neurohypophyseal hormone activity, vasopressin receptor binding, maternal aggressive behavior) is particularly notable and may reflect effects on hypothalamic-pituitary signaling. The enrichment of basic metabolic pathways (glycolysis, amino acid biosynthesis) alongside behavioral and neuroendocrine terms suggests broad metabolic and functional remodeling in response to ApoE isoform switching.

9.2.2 Gene Set Enrichment Analysis (GSEA)

GSEA of brain tissue revealed seventeen significantly enriched pathways (FDR < 0.05), predominantly related to chromatin regulation, neuronal structure, and gene expression control:

pathway	NES	padj	size
GOBP_REGULATION_OF_CHROMATIN_ORGANIZATION	2.239	0.019	35
GOBP_EPOXYGENASE_P450_PATHWAY	2.228	0.019	26
GOBP_ARACHIDONIC_ACID_METABOLIC_PROCESS	2.188	0.009	59
GOBP_XENOBIOTIC_METABOLIC_PROCESS	2.137	0.005	98
GOBP_SPLICEOSOMAL_COMPLEX_ASSEMBLY	2.136	0.020	49
GOBP_URONIC_ACID_METABOLIC_PROCESS	2.128	0.031	15
GOBP_DENDRITE_MORPHOGENESIS	1.891	0.009	188
GOBP_POSITIVE_REGULATION_OF_MRNA_CATABOLIC_PROCESS	1.885	0.027	91
GOBP_CELLULAR_RESPONSE_TO_XENOBIOTIC_STIMULUS	1.864	0.019	153
GOBP_ACTION_POTENTIAL	1.837	0.018	160
GOBP_POSITIVE_REGULATION_OF_MRNA_METABOLIC_PROCESS	1.828	0.019	125
GOBP_REGULATION_OF_MRNA_PROCESSING	1.818	0.019	129
GOBP_REGULATION_OF_MRNA_METABOLIC_PROCESS	1.792	0.005	274
GOBP_NEGATIVE_REGULATION_OF_GENE_EXPRESSION_EPIGENETIC	1.768	0.027	145
GOBP_NEURON_PROJECTION_ORGANIZATION	1.734	0.031	116
GOBP_DENDRITE_DEVELOPMENT	1.688	0.006	315
GOBP_SYNAPSE_ASSEMBLY	1.583	0.023	267
GOBP_POSTSYNAPSE_ORGANIZATION	1.558	0.031	283
GOBP_CHROMATIN_REMODELING	1.538	0.006	472
GOBP_MRNA_PROCESSING	1.467	0.019	453
GOBP_REGULATION_OF_SYNAPSE_STRUCTURE_OR_ACTIVITY	1.418	0.041	365

All pathways exhibited positive enrichment scores, indicating coordinated upregulation of genes in these functional categories following ApoE4→ApoE2 conversion. The enrichment pattern in brain contrasts markedly with liver, where mitochondrial oxidative metabolism dominated. Instead, brain tissue shows coordinated changes in three major functional domains: (1) chromatin organization and epigenetic regulation, suggesting remodeling of gene expression programs; (2) cytochrome P450-mediated metabolism of arachidonic acid and xenobiotics, implicating changes in lipid mediator synthesis and detoxification capacity; and (3) neuronal structural plasticity and synaptic organization, indicating potential effects on neural circuit architecture.

9.2.2.1 Visualizing Leading Edge Gene Overlaps with UpSet Plot

9.2.2.2 Reducing Redundant Pathways with collapsePathways

Original significant pathways: 21 

After collapsing: 11 

pathway	NES	padj	size
GOBP_REGULATION_OF_CHROMATIN_ORGANIZATION	2.239	0.019	35
GOBP_XENOBIOTIC_METABOLIC_PROCESS	2.137	0.005	98
GOBP_SPLICEOSOMAL_COMPLEX_ASSEMBLY	2.136	0.020	49
GOBP_URONIC_ACID_METABOLIC_PROCESS	2.128	0.031	15
GOBP_ACTION_POTENTIAL	1.837	0.018	160
GOBP_REGULATION_OF_MRNA_METABOLIC_PROCESS	1.792	0.005	274
GOBP_DENDRITE_DEVELOPMENT	1.688	0.006	315
GOBP_SYNAPSE_ASSEMBLY	1.583	0.023	267
GOBP_CHROMATIN_REMODELING	1.538	0.006	472
GOBP_MRNA_PROCESSING	1.467	0.019	453
GOBP_REGULATION_OF_SYNAPSE_STRUCTURE_OR_ACTIVITY	1.418	0.041	365