Signatures for Viral Infection and Inflammation in the Proximal Olfactory System 1 in Familial Alzheimer’s Disease 2

Objective: Alzheimer’s disease (AD) is characterized by loss of smell and olfactory 33 system pathology that precedes the diagnosis of dementia. Understanding these early 34 processes can potentially identify diagnostic and therapeutic targets to slow AD 35 progression. Here we analyzed differential gene and protein expression in the olfactory 36 bulb (OB) and olfactory tract (OT) of familial AD (FAD) individuals carrying the 37 autosomal dominant presenilin 1 E280A paisa mutation and age-matched controls. Methods: Formalin-fixed, paraffin-embedded sections containing both the OB and OT 40 from 6 FAD individuals and 6 age-matched controls were obtained. Tissue morphology 41 and composition were characterized by immunohistochemistry using antibodies against 42 the myelin marker proteolipid protein (PLP), amyloid-beta (Aβ), and 43 microglia/macrophage markers Iba1 and CD68, respectively. OB and OT were analyzed 44 separately by targeted RNA sequencing of the whole human transcriptome (BioSpyder 45 TempO-Seq); ingenuity pathway analysis and R-computational program were used to 46 identify differentially expressed genes and pathways between groups. The nanoString 47 spatial proteomics assay for 88 proteins, including markers for AD and immune 48 responses, was used to complement gene expression findings. 49 50 Results: Compared to control OT, FAD OT had significantly increased immunostaining 51 for Aβ and CD68 in the high and low myelinated regions, as well as increased 52 immunostaining for Iba1 in the high myelinated region only; both control and FAD OT 53 samples had similar total area of high and low myelinated regions. In FAD samples, 54 RNA sequencing showed a transcription profile consistent with: (1) viral infection in the 55 OB; (2) inflammation in the OT that carries information via entorhinal cortex from the OB 56 to hippocampus, a brain region essential for learning and memory; and (3) decreased 57 oligodendrocyte deconvolved transcripts, indicating dysregulation of myelination. Interestingly, spatial proteomic analysis confirmed altered myelination we Our findings reveal a transcriptomic signature for viral infection in the OB coupled with inflammation and dysregulation of myelination in the OT in FAD samples compared to control samples; results were confirmed by proteomic analysis. While our only identified human transcripts/proteins and were not designed to identify specific olfactory An the Iba1+CD68+ Iba1+CD68- d.f. , p<0.05) Iba1-CD68+ 1 p<0.05). FAD exhibit an increase in Iba1+CD68+ surface and Iba1+CD68- 483 surface in the high myelinated OT, and an increase in Iba1-CD68+ surface low 484 myelinated OT, compared to Ctrl. Quantification of Aβ surface that is overlapping 485 with Iba1+CD68+ cells. An ANOVA yields a significant difference for the genotype for 486 Aβ surface that is overlapping with Iba1+CD68+ cells (F=24.14, 1 d.f., p<0.001), 487 Iba1+CD68- cells 1 d.f. , p<0.01) and Iba1-CD68+ cells (F=12.28, 1 d.f., 488 p<0.01). An ANOVA yields a significant difference between high and low myelination areas for Aβ surface that is overlapping with Iba1+CD68- cells (F=8, 1 d.f., p<0.05) and free from microglial 1 d.f. , p<0.001). Aβ surface that is all three microglial in the high myelin OT of to Ctrl. Aβ surface that is overlapping with Iba1-CD68+ cells is in the low myelin OT. Aβ free from microglial is the myelin the Ctrl FAD.