CD4+ T-cell transcription factors as early predictors of phenoconversion in idiopathic rapid eye movement sleep behaviour disorder

Ethical statement

The Ethics Committee of the Neurological Institute “C. Mondino” of Pavia approved the protocol (number 2021008499, 10/02/2021) and all the participants signed a written informed consent before enrolment.

Participants

31 patients were followed at the Sleep Center of the Neurological Institute “C. Mondino” of Pavia. The clinical evaluation included: motor symptoms, assessed by means of the Unified Parkinson’s Disease Rating Scale (UPDRS) part III⁷; cognitive function, by the Mini Mental State Examination (MMSE)^8,9; constipation and orthostatic hypotension by means of the Scale for Outcomes in Parkinson’s disease-Autonomic (SCOPA-AUT)⁷; depressive symptoms, by means of the Hamilton Rating Scale for Depression (HAM-D).¹⁰

At the original enrolment,⁵ each patient provided a venous blood sample, which was processed to isolate CD4+ T cells, according to an established procedure.⁶

Subjects with iRBD were followed prospectively with periodic in-person evaluations to diagnose phenoconversion, i.e., parkinsonism, DLB and MSA.

Isolation of CD4+ T Cells

Peripheral blood mononuclear cells were separated by Ficoll-Paque Plus density gradient centrifugation; then CD4+ T cells were obtained by immunomagnetic sorting. Cell viability was 95% ± 1% (range, 90%–98%), and purity was 98% ± 2% (range, 97%–100%). Expression of the TF genes TBX21, STAT1, STAT3, STAT4, STAT6, RORC, GATA3, FOXP3, and NR4A2 was measured by real-time polymerase chain reaction.

Real-time polymerase chain reaction assay

For real-time PCR assays of CD4+ T cells, at least 50,000 CD4+ T cells were resuspended in PerfectPure RNA lysis buffer (5 Prime GmbH, Hamburg, Germany), and total RNA was extracted by PerfectPure RNA Cell Kit™ (5 Prime GmbH, code 2302340). The amount of extracted RNA was estimated by spectrophotometry at λ = 260 nm. Total mRNA was then reverse-transcribed using a random primer and a high-capacity cDNA RT kit (Applied Biosystems, code 4368813), and the resulting amount of cDNA was estimated by spectrophotometry at λ = 260 nm. Real-Time PCR reactions were then started with 1 μM cDNA. At the beginning, we always loaded 1 μg/μl of cDNA per reaction (final reaction mix volume + sample = 20 μl). This means that for each reaction we load 2 μl of aqueous solution containing the cDNA (therefore, 2 μg of cDNA in total). The following thermal protocol was used for each sample: 20 s at 95°C (x 1, hot start); 2-step cycles as follows: 1 s at 95°C, 20 s at 60°C (x 40). All the reagents (probes and mix) were used according to the manufacturer’s instructions (www.bio-rad.com).