The importance of early biomarkers in the identification of neurodegenerative disorders

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.⁴

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 III⁵; cognitive function, by the Mini Mental State Examination (MMSE)⁶; 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).⁸

At the original enrolment,⁴ each patient provided a venous blood sample, which was processed to isolate CD4+ T cells, according to an established procedure.⁹

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).