Reprogramming astrocytes into dopaminergic neurons to restore motor dysfunction in Parkinson's disease model rats.

Abstract

Objectives: Parkinson's disease (PD) is a neurodegenerative disorder primarily caused by the loss of dopaminergic neurons (DA) in the brain. Since DA neurons are non-renewable, conventional therapies only alleviate symptoms without addressing the root cause. This study aims to reprogram astrocyte (AS) into DA neurons for transplantation into the brain to reconstruct damaged neural circuits and treat PD.

Methods: Astrocytes were isolated from neonatal rat brain tissues. A lentiviral vector carrying the transcription factors nuclear receptor-related factor 1 (UNRR1) and achaete-scute family bHLH transcription factor 1 (ASCL1), named LV-NURR1-ASCL1, was constructed and used to infect cultured rat AS in vitro. Immunofluorescence, Western blotting, and reverse transcription polymerase chain reaction (RT-PCR) were employed to detect and compare the expression levels of NURR1 and ASCL1 in lentivirus-infected AS (LV group) and AS cultured in complete medium without LV-NURR1-ASCL1 (Con group). The virus-infected AS was then cultured in neuronal induction medium for 18 days. Immunofluorescence was used to detect the expression of DA markers, including tyrosine hydroxylase (TH) and forkhead box A2 (FOXA2), as well as the neuronal marker class III beta tubulin (TUJ1). To establish the PD rat model, 6-hydroxydopamine (6-OHDA) was injected into 2 sites in the medial forebrain bundle (MFB) region of the right brain in rats. The reprogrammed cells (AS-iDA) were quantified and transplanted into the right MFB region of the PD model rats using a stereotaxic instrument. Four weeks after transplantation, immunofluorescence was used to assess the survival, differentiation, and migration of AS-iDA in the brain and the expression of TH, TUJ1, and FOXA2 in the brain tissue of PD rats. Eight weeks post-transplantation, the recovery of motor function in PD rats was evaluated using the apomorphine (APO)-induced rotation test, rotarod fatigue test, and open-field test.

Results: Immunofluorescence analysis showed positive expression of NURR1 and ASCL1 in AS after lentiviral infection. RT-PCR results demonstrated that the mRNA expression levels of NURR1 and ASCL1 in the LV group were significantly higher than those in the Con group, with increases of (7.483±0.706)-fold and (10.830±1.940)-fold, respectively. Western blotting analysis further confirmed that the protein expression levels of NURR1 and ASCL1 in the LV group were (2.403±0.511)-fold and (4.423±0.603)-fold higher, respectively, compared to the control group. After 18 days of directed induction culture lentivirus-infected AS (AS-iDA) displayed significant morphological changes, developing neuron-like long neurites. At this stage, AS-iDA highly expressed the neuronal marker TUJ1 as well as the DA markers TH and FOXA2. Four weeks post-transplantation, immunofluorescence on brain slices from PD rats revealed that AS-iDA survived in the transplant region, migrated to surrounding areas, and expressed TUJ1, TH, and FOXA2. At 8 weeks post-transplantation, compared to untreated PD rats, PD rats transplanted with AS-iDA exhibited significantly reduced rotational behavior in the APO-induced rotation test, increased mobility in the open-field test, and extended time on the rotarod in the fatigue test (all P<0.05).

Conclusions: Lentiviral overexpression of NURR1 and ASCL1 efficiently reprograms AS into DA neurons. Transplantation of reprogrammed DA neurons significantly improves motor function in PD rats, highlighting their potential as donor cells for the treatment of neurodegenerative diseases.