Dana Shulman

INTRODUCTION: Females with Alzheimer's disease (AD) suffer accelerated dementia and loss of cholinergic neurons compared to males, but the underlying mechanisms are unknown. Seeking causal contributors to both these phenomena, we pursued changes in transfer RNS (tRNA) fragments (tRFs) targeting cholinergic transcripts (CholinotRFs). METHODS: We analyzed small RNA-sequencing (RNA-Seq) data from the nucleus accumbens (NAc) brain region which is enriched in cholinergic neurons, compared to hypothalamic or cortical tissues from AD brains; and explored small RNA expression in neuronal cell lines undergoing cholinergic differentiation. RESULTS: NAc CholinotRFs of mitochondrial genome origin showed reduced levels that correlated with elevations in their predicted cholinergic-associated mRNA targets. Single-cell RNA seq from AD temporal cortices showed altered sex-specific levels of cholinergic transcripts in diverse cell types; inversely, human-originated neuroblastoma cells under cholinergic differentiation presented sex-specific CholinotRF elevations. DISCUSSION: Our findings support CholinotRFs contributions to cholinergic regulation, predicting their involvement in AD sex-specific cholinergic loss and dementia.

Transfer RNA fragments (tRFs) have recently been shown to be an important family of small regulatory RNAs with diverse functions. Recent reports have revealed modified tRF blood levels in a number of nervous system conditions including epilepsy, ischemic stroke, and neurodegenerative diseases, but little is known about tRF levels in the cerebrospinal fluid (CSF). To address this issue, we studied age, sex, and Parkinson's disease (PD) effects on the distributions of tRFs in the CSF and blood data of healthy controls and PD patients from the NIH and the Parkinson's Progression Markers Initiative (PPMI) small RNA-seq datasets. We discovered that long tRFs are expressed in higher levels in the CSF than in the blood. Furthermore, the CSF showed a pronounced age-associated decline in the level of tRFs cleaved from the 3'-end and anti-codon loop of the parental tRNA (3'-tRFs, i-tRFs), and more pronounced profile differences than the blood profiles between the sexes. In comparison, we observed moderate age-related elevation of blood 3'-tRF levels. In addition, distinct sets of tRFs in the CSF and in the blood segregated PD patients from controls. Finally, we found enrichment of tRFs predicted to target cholinergic mRNAs (Cholino-tRFs) among mitochondrial-originated tRFs, raising the possibility that the neurodegeneration-related mitochondrial impairment in PD patients may lead to deregulation of their cholinergic tone. Our findings demonstrate that the CSF and blood tRF profiles are distinct and that the CSF tRF profiles are modified in a sex-, age-, and disease-related manner, suggesting that they reflect the inter-individual cerebral differences and calling for incorporating this important subset of small RNA regulators into future studies.

Maternal perceived prenatal stress (PPS) is a known risk factor for diverse developmental impairments in newborns, but the underlying molecular processes are incompletely understood. Here, we report that maternal PPS altered the birth profiles of blood transfer RNA fragments (tRFs), 16-50 nt long non-random cleavage products of tRNAs, in a sex-dependent manner. Importantly, comparing stressed versus control maternal and umbilical cord blood serum presented alterations that were not limited to individual tRFs, but rather reflected selective changes in particular tRF families grouped by their mitochondrial or nuclear genome origin, parental tRNA coded amino acid, and cleavage type. Specifically, tRF families that show stress- and sex-specific effects, revealed shared length and expression patterns which were strongest in the female newborns. Several of these tRFs carry complementary motifs to particular cholinergic mRNAs, suggesting possible translational regulation similar to microRNAs. Compatible with the cholinergic regulation of stress reactions, those "CholinotRFs" achieved an AUC of 95% when classifying female newborns according to maternal PPS. Moreover, we found altered catalytic activity of serum acetylcholinesterase, which was particularly elevated in male newborns, marking a second sex-specific effect. Our findings demonstrate an association of tRF families' patterns with newborns' sex-specific stress response to PPS and may lead to better diagnosis and therapeutic tools for these and other stressors.

Introduction: Females with Alzheimer's disease (AD) suffer accelerated dementia and loss of cholinergic neurons compared to males, but the underlying mechanisms are unknown. Seeking causal contributors to both these phenomena, we pursued changes in tRNA fragments (tRFs) targeting cholinergic transcripts (CholinotRFs). Methods: (NAc) brain region which is enriched in cholinergic neurons, compared to hypothalamic or cortical tissues from AD brains; and explored small RNA expression in neuronal cell lines undergoing cholinergic differentiation. Results: NAc CholinotRFs of mitochondrial genome origin showed reduced levels that correlated with elevations in their predicted cholinergic-associated mRNA targets. Single cell RNA seq from AD temporal cortices showed altered sex-specific levels of cholinergic transcripts in diverse cell types; inversely, human-originated neuroblastoma cells under cholinergic differentiation presented sex-specific CholinotRF elevations. Discussion: Our findings support CholinotRFs contributions to cholinergic regulation, predicting their involvement in AD sex-specific cholinergic loss and dementia.

Abstract Transfer RNA fragments (tRFs) have recently been shown to be an important family of small regulatory RNAs with diverse functions. Recent reports have revealed modified tRF blood levels in a number of nervous system conditions including epilepsy, ischemic stroke and neurodegenerative diseases, but little is known about tRF levels in the cerebrospinal fluid (CSF). To address this issue, we studied age, sex and Parkinson’s disease (PD) distributions of tRFs in the CSF and blood data of PD patients and healthy controls from the NIH and the PPMI small RNA-seq datasets. The higher levels of long tRFs were found in the CSF than in the blood. Furthermore, the CSF showed pronounced age-associated declines of the level of 3’-tRFs and i-tRFs and more pronounced differences between the sexes. Blood showed moderate elevation of 3’-tFs levels with age. In addition, different distinct sets of tRFs segregated PD patients from controls in the CSF and in the blood. Finally, we found enrichment of tRFs predicted to target cholinergic mRNAs (Cholino-tRFs) in the mitochondrial originated tRFs, raising the possibility that the neurodegeneration-related mitochondrial impairment may lead to deregulation of cholinergic tone. Our findings suggest that CSF expressed tRFs are not a mirror of blood tRFs but rather potentially reflect the cerebral changes. Further, both CSF and blood present modified levels of tRFs in a sex-, age-and disease-related manner, calling for including this important subset of small RNA regulators to future studies.