MANF safeguards mitochondria-associated endoplasmic reticulum membrane integrity in nucleus pulposus-derived mesenchymal stem cells to maintain homeostasis of the intervertebral disc

Intervertebral disc (IVD) degeneration (IDD) is a leading cause of low back pain, with limited treatment options. The degenerative disc’s harsh microenvironment promotes nucleus pulposus-derived mesenchymal stem cells (NP-MSCs) death and hinders self repair. Mesencephalic astrocyte-derived neurotrophic factor (MANF), an atypical neurotrophic factor, has protective effects in degenerative diseases. However, its role in IDD is unclear. Assessment of MANF expression was conducted in both human nucleus pulposus tissues and a rat IVD puncture model. An in vitro model of degeneration was established by acid treatment of NP-MSCs, and the functional role of MANF was explored through its knockdown and overexpression. RNA sequencing was employed to identify downstream targets. The therapeutic potential of MANF-overexpressing NP-MSCs was evaluated in a rat puncture model. MANF expression was markedly downregulated in degenerated IVD tissues from both human patients and rat models. Correspondingly, in vitro experiments demonstrated that MANF knockdown exacerbated, while its overexpression mitigated, acid-induced apoptosis of NP-MSCs. Mechanistically, MANF attenuated mitochondrial dysfunction and ER stress of NP-MSCs under acidic conditions by maintaining MAM integrity, as demonstrated by the complete abolition of this protection upon treatment with the MAM uncoupler FATE1. Transcriptomic analysis and subsequent validation identified receptor expression-enhancing protein 1 (REEP1) as a critical downstream effector through which MANF safeguards MAM integrity. We further elucidated that MANF upregulates REEP1 expression by directly inhibiting miR-33b-5p. In vivo, transplantation of MANF-overexpressing NP-MSCs effectively attenuated IDD in a rat model. MANF protected NP-MSCs from acidosis by sustaining MAM integrity via the MiR-33b-5p/REEP1 axis. These findings reveal MANF’s mechanism and therapeutic potential for IDD.