Lamivudine, a reverse transcriptase inhibitor, rescues cognitive deficits in a mouse model of down syndrome

Abstract An elevated activity of retrotransposons is increasingly recognized to be implicated in a wide range of neurodegenerative and neurodevelopmental diseases. Down syndrome (DS) is the most common genetic disorder associated with intellectual disability and a genetic form of Alzheimer's disease. For this reason, we hypothesized that treatment with reverse transcriptase inhibitors could ameliorate DS phenotypes. In this proof of concept study, we treated trisomic (Ts65Dn) mice, a model of DS, with lamivudine, a reverse transcriptase inhibitor. We detected a significant improvement of neurobehavioural phenotypes, and a complete rescue of the hippocampal‐dependent recognition memory upon treatment with lamivudine. Despite clinical studies in patients with DS are warranted, this study lays the groundwork for a novel and actionable therapeutic approach.


| INTRODUC TI ON
Down syndrome (DS) is the most common known genetic disorder associated with moderate to severe intellectual disability due to a total or partial trisomy of the autosomal chromosome 21 (HSA21) and a genetic form of Alzheimer's disease (AD). 1 Overexpression of HSA21 gene products such as DYRK1A 2 , SOD1 3 or S100B 4 has been proposed to contribute to the neurological and neurobehavioural DS phenotypes. However, treatments targeted to candidate genes 5 or mechanisms have only been partially successful. 6 This is possibly explained because besides the HSA21 gene-dosage effects, triplication of HSA21 also leads to a genome-wide transcriptional deregulation. 7 Recently, single-nucleus long-read RNA sequencing has revealed thousands of unannotated RNA transcripts containing intra-exonic junctions, involving a myriad of genes, including the amyloid precursor protein (APP). 8 Notably, increased brain transcription and increased copy numbers of APP have been linked to APP somatic gene recombination associated with sporadic AD and with DS, and could contribute to their cognitive deficits. 9 Retrotransposable elements like the long interspersed nuclear element 1 (LINE1 or L1 -6 kb) are thought to participate in this process in mammals. 10 Increased retrotransposition is observed in cell senescence and with ageing 11 and has been implicated in several neurodegenerative diseases including AD, frontotemporal dementia, prion disease, but also in developmental disorders such as Rett's syndrome, autism or fragile X syndrome. 12 Nucleoside reverse transcriptase inhibitors (NRTIs) have been used to suppress retrotransposition, and thus NRTIs could theoretically improve these pathologies. Importantly, in patients with HIV-1, NRTIs treatment is associated with reduced risk of HIV-associated neurocognitive disorder, AD, Parkinson's disease and other dementias. 13 Preclinical studies have shown that the reverse transcrip- improves cognition in senescence-accelerated prone 8 (SAMP8) mice, a model for studying human ageing and age-related diseases 14 and treatment with NRTIs doubled the lifespan of progeroid Sirt6deficient mice 15

| Locomotor activity
Spontaneous locomotor activity in the home cage was measured using an automated infrared light-beam monitoring system (ActiMot2, TSE system) and the ambulatory beam breaks (X axis) were counted for 24 h. Total general activity was analysed. Mice were isolated for assessing locomotor activity during 24 h and immediately regrouped after the test.

| Novel object recognition test
The . Exploratory behaviour was defined as the animal directing its nose towards the object at a distance of <2 cm and manually registered by the experimenter. Sitting on or resting against the object was not considered as exploration. All the objects used were made of plastic and induced similar exploration levels. The arena and objects were deeply cleaned between animals to avoid olfactory cues.

| Elevated plus maze
The elevated plus maze paradigm was used to study anxiety-related

| Statistical analyses
Two-way analysis of variance (anova) repeated measures was used for testing genotype and treatment differences on the locomotor activity and the elevated plus maze. Two-way anova was used to ana-

| Lamivudine ameliorated hyperactivity in TS mice
We

| Lamivudine increased anxiety-like behaviour in WT and TS mice
We also assessed anxiety-like behaviour in the elevated plus maze paradigm to understand whether TS mice would be more sensitive to this side effect of lamivudine ( Figure 1C-E). We first analysed locomotor activity (distance travelled) ( Figure 1C). We did not detect genotype-dependent differences, but both genotypes

| Lamivudine rescues recognition memory deficits in TS mice
Recognition memory was evaluated using the novel object recognition paradigm that has been shown to be a robust and

ACK N OWLED G EM ENT
We thank Cèsar Sierra for critical reading of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.