Sequence diversity and encoded enzymatic differences of monocistronic L1 ORF2 mRNA variants in the aged normal and Alzheimer's disease brain.

Reverse transcriptase (RT) activity in the human brain has been inferred through somatic retroinsertion/retrotransposition events, however actual endogenous enzymatic activities and sources remain unclear. L1 (LINE-1) retrotransposons bicistronically express ORF2, containing RT and endonuclease (EN) domains, and RNA binding protein ORF1, together enabling L1 retrotransposition and contributing to somatic genomic mosaicism (SGM). Here, we assessed endogenous RT activities and L1 mRNA diversity from cerebral cortical samples of 31 Alzheimer's disease (AD) and non-diseased (ND) brains (both sexes) using enzymatic functional assays, targeted PacBio HiFi long-read sequencing, and quantitative spatial transcriptomics. Expected bicistronic, full-length L1 transcripts were absent from most samples, constituting <0.01% of L1 sequences, of which >80% were non-coding. Monocistronic ORF1 and ORF2 transcripts were identified across all samples, consistent with quantitative spatial transcriptomics that identified discordant ORF2 and ORF1 expression in neurons. All brains had RT activity, with AD samples showing less activity, consistent with neuronal loss of terminal AD vs. aged ND donors. Brain RT activity was higher in grey matter and correlated with increased neuronal ORF2 expression, further supporting neuronal contributions. Remarkably, >550 protein-encoding, polyA+ ORF2 sequence variants were identified, over 2x more than identified in the human reference genome (hg38). Experimental overexpression of full-length and truncated ORF2 variants revealed ∼50-fold RT and ∼1.3-fold EN activity ranges, supporting endogenous functional capacity of monocistronic ORF2 variants in the human brain. The vast sequence diversity of monocistronic ORF2 mRNAs could underlie functional differences in RT-mediated somatic gene recombination/retroinsertion and resulting genomic mosaicism in the normal and diseased brain.Significance Statement Human brain reverse transcriptase activity has been inferred through the "copy-and-paste life-cycle" of L1, which can generate genomic mosaicism via self-retrotransposition via a full-length L1 mRNA. However, their presence in aged and Alzheimer's disease neurons remains unclear. We examined aged normal and Alzheimer's brains for reverse transcriptase activity in prefrontal and medial-temporal cortices and its relationship to L1 via enzymatic activity assays and targeted PacBio sequencing. Reverse transcriptase activity was pervasive, however full-length L1 was largely absent. Instead, hundreds of different, truncated, novel L1 mRNA variants were identified, and experimental sampling revealed diverse reverse transcriptase activities. These data implicate truncated L1 variants as a source of functionally diverse and novel reverse transcriptases in the normal and Alzheimer's disease brain.