The lncRNA LOC102549805 (U1) modulates neurotoxicity of HIV-1 Tat protein

Abstract

HIV-1 Tat is a potent neurotoxic protein that is released by HIV-1 infected cells in the brain and perturbs neuronal homeostasis, causing a broad range of neurological disorders in people living with HIV-1. Furthermore, the effects of Tat have been addressed in numerous studies to investigate the molecular events associated with neuronal cells survival and death. Here, we discovered that exposure of rat primary neurons to Tat resulted in the up-regulation of an uncharacterized long non-coding RNA (lncRNA), LOC102549805 (lncRNA-U1). Our observations showed that increased expression of lncRNA-U1 in neurons disrupts bioenergetic pathways by dysregulating homeostasis of Ca²⁺, mitigating mitochondrial oxygen reduction, and decreasing ATP production, all of which point mitochondrial impairment in neurons via the Tat-mediated lncRNA-U1 induction. These changes were associated with imbalances in autophagy and apoptosis pathways. Additionally, this study showed the ability of Tat to modulate expression of the neuropeptide B/W receptor 1 (NPBWR1) gene via up-regulation of lncRNA-U1. Collectively, our results identified Tat-mediated lncRNA-U1 upregulation resulting in disruption of neuronal homeostasis.

Fig. 1. Validation of the Tat-mediated lncRNA-U1 overexpression using RT-PCR and qRT-PCR.

a Schematic representation of genomic and cDNA structures of lncRNA-U1 isoforms. b RT-PCR was carried out using lncRNA-U1 primer pair with the total RNA prepared from Ad-Tat/Ad-Null (MOI: 2) transduced rat primary neuronal culture. Representative agarose gel shows two different bands amplified in HIV-1 Tat samples vs. control. The lower molecular weight (MW) band (U1a) corresponds to lncRNA-U1. The higher (MW) band (U1₆₆) corresponds to the minor lncRNA-U1 splice variant. c Agarose gel representing RT-PCR results for the exon-specific primers, comparison of the expression of lncRNA-U1 isoforms expression. d q-PCR analysis measuring changes in gene expression based on the fold changes in U1₆₆ isoform and the whole lncRNA-U1 expression. e The relative ratio of basal expression of U1₆₆ and total lncRNA-U1 expression. f The stability and half-life of lncRNA-U1 was determined using Actinomycin D to inhibit transcription in Ad-Null/Tat/U1 (MOI: 2) treated samples. qPCR analysis following transcriptional inhibition indicated a lncRNA-U1 half-life of 2 h and 4 h in Ad-Null and A-U1 transduced neurons, respectively. However, in Tat expressing neurons, lncRNA-U1 was increased 2–3 folds during this same period (i.e. 2–4 h), and remained elevated throughout the duration of the experiment.

Fig. 2. NPBWR1 expression changes.

a The representative bar chart for the results of the qPCR analysis showed the increased expression of NPBWR1 in rat primary neurons transduced with Ad-Tat (MOI: 2, 72 h). b Immunoblotting showed an increase in NPBWR1 protein upon Tat expression. c qPCR showed upregulation of NPBWR1 in lncRNA-U1 induced primary neurons, independent of Tat expression. qPCR analysis indicated a 2-fold increased expression of NPBWR1 when primary neurons were transduced with Adeno-U1 (MOI:2) for 72 h. d Immunoblotting confirmed the overexpression of NPBWR1 upon lncRNA-U1 up-regulation in primary neurons. e ICC confirmed increased NPBWR1 expression in primary neurons upon Tat/U1 treatment, the result of immunoblotting revealed the formation of protein aggregates in the cell borders upon manipulating lncRNA-U1 expression in neurons. f q-PCR analysis compared the NPBWR1 expression in neurons transduced with Ad-U1, Ad-U1₆₆, Ad-U1R as well as Tat and co-transduction of Tat and U1R revealing a correlation between lncRNA-U1 overexpression and changes in NPBWR1 expression. U1₆₆ splice variant did not, however, alter NPBWR1 expression. g immunoblotting analysis showed no lncRNA-U1 minor splice variant and confirmed the results from q-PCR analysis.

Fig. 3. LncRNA-U1 impaired Ca²⁺ signaling in primary neurons.

a Confocal microscopy using Fluo-4 indicated that Ad-U1 and Ad-Tat transduction increased cytosolic Ca2+ in primary neurons. b–e Mitochondrial Ca²⁺ uptake was dysregulated upon the transduction of neurons with Ad-Tat or Ad-U1. Increased Ca²⁺ clearance was observed in neurons expressing Ad-Tat and Ad-U1. f Mitochondrial membrane potential ΔΨm collapsed in neurons transduced with Ad-Tat or Ad-U1. g The Ad-U1 neurons had lower levels of ATP production compared to Ad-Null. h Immunoblotting showed alterations in the levels of MCU expression in primary neurons upon overexpression of lncRNA-U1.

Fig. 4. LncRNA-U1 modualtes neuronal respiration and mitochondrial bioenergetics.

a Confocal microscopy confirmed ROS production upon Ad-Tat and Ad-U1 transduction (red). b–g Quantification of basal oxygen consumption rate (OCR), maximal OCR, and ATP coupled respiration, spare capacity of the mitochondria and proton leak show impaired mitochondrial respiration in Ad-Tat, Ad-U1 expressing neurons. Ad-Tat and Ad-U1 dysregulated mitochondrial bioenergetics. b Measurements were conducted after oligomycin (a), FCCP (b), and rotenone/antimycin A (c) were added as indicated by dashed vertical lines. b–g Measurement of OCR in neurons expressing Ad-Tat and Ad-U1 for 48h showed decreased basal OCR, maximal OCR levels and ATP coupled respiration. h Non-mitochondiral respiration did not show significant changes in neurons transduced with either Ad-Tat or Ad-U1.

Fig. 5. Neuronal survival pathways adversly affected by lncRNA-U1 overexpression.

a SytoxGreen assay indicated increased cell death in the Ad-U1 transduced neurons. b Immunoblotting showed autophagy activation as determined by increased LC3-II and decreased p62, when neurons expressed Ad-Tat or Ad-U1, in the absence or presence of bafilomycin A1 50 nM. c Immunoblotting showed increased apoptosis in neurons expressing the Ad-Tat or Ad-U1 for 72h. Apoptosis marker cleaved-caspase3 was significantly increased, while the anti-apoptotic markers Bcl2 and BAG3 decreased significantly.

Fig. 6. lncRNA-U1 is upregulated in doxycycline-induced Tat transgenic mice.

a RT-PCR confirmed expression alterations of lncRNA-U1 in the Tat expressing mice. b q-RT PCR analysis confirmed increased expression of lncRNA-U1 in three different brain regions (hippocampus, prefrontal cortex, cerebellum) of Tat expressing transgenic mice, the result indicated more significant increases in hippocampus compared to other brain regions. No changes were observed in the brain stem.

Fig. 7. Schematic showing the hypothetical downstream effects of lncRNA-U1 overexpression in primary hippocampal neurons.

a Schematic representation of normal signaling in neurons before the increased expression of lncRNA-U1. b Increased lncRNA-U1 expression alters the neuronal environment by changing the abundance of NPBWR1 followed by increased mitochondrial Ca²⁺ due to increased MCU channel expression. Ultimately, neuronal homeostasis is altered leading to the activation of both apoptosis and autophagy pathways.