Mesencephalic astrocyte–derived neurotrophic factor is an ER-resident chaperone that protects against reductive stress in the heart

Abstract

We have previously demonstrated that ischemia/reperfusion (I/R) impairs endoplasmic reticulum (ER)-based protein folding in the heart and thereby activates an unfolded protein response sensor and effector, activated transcription factor 6α (ATF6). ATF6 then induces mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER-resident protein with no known structural homologs and unclear ER function. To determine MANF's function in the heart in vivo, here we developed a cardiomyocyte-specific MANF-knockdown mouse model. MANF knockdown increased cardiac damage after I/R, which was reversed by AAV9-mediated ectopic MANF expression. Mechanistically, MANF knockdown in cultured neonatal rat ventricular myocytes (NRVMs) impaired protein folding in the ER and cardiomyocyte viability during simulated I/R. However, this was not due to MANF-mediated protection from reactive oxygen species generated during reperfusion. Because I/R impairs oxygen-dependent ER protein disulfide formation and such impairment can be caused by reductive stress in the ER, we examined the effects of the reductive ER stressor DTT. MANF knockdown in NRVMs increased cell death from DTT-mediated reductive ER stress, but not from nonreductive ER stresses caused by thapsigargin-mediated ER Ca2+ depletion or tunicamycin-mediated inhibition of ER protein glycosylation. In vitro, recombinant MANF exhibited chaperone activity that depended on its conserved cysteine residues. Moreover, in cells, MANF bound to a model ER protein exhibiting improper disulfide bond formation during reductive ER stress but did not bind to this protein during nonreductive ER stress. We conclude that MANF is an ER chaperone that enhances protein folding and myocyte viability during reductive ER stress.

Protective Functions of Reactive Astrocytes Following Central Nervous System Insult

“…Both NTFs have been associated to numerous tissue-protective and anti-inflammatory functions in models of PD, ischemia and nerve injury (162-164, 168, 169). In addition, a series of recent studies demonstrated that MANF and CDNF are partially retained within the endoplasmic reticulum (ER), where they sense and respond to ER stress by negatively regulating NF-kB dependent inflammatory programs (84, 165,[169][170][171][172][173][174]. In astrocytes, upregulation of both MANF and CDNF has been observed in response to ER stress and experimental stroke, where they alleviate the secretion of pro-inflammatory cytokines IL-1b, TNF-a, and IL-6 (84-86).…”

Cerebral dopamine neurotrophic factor reduces α-synuclein aggregation and propagation and alleviates behavioral alterations in vivo

“…31,32 Moreover, MANF, the paralog of CDNF, functions as a chaperon in the ER. 33,34 Both 15 N for each amino acid of CDNF after addition of monomeric a-synuclein. The mean (black line) and standard deviation (dashed line) of all Dd (ppm) are shown.…”

Implications of Oxidative and Nitrosative Post-Translational Modifications in Therapeutic Strategies against Reperfusion Damage

“…The detrimental effects triggered by caNrf2-driven reductive stress include pathological cardiac remodeling [ 120 , 185 ] along with cytoskeletal alterations, deregulation of Ca 2+ handling, and alteration of the contractile apparatus [ 120 ]. Recently, it has been reported that the mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) resident, exerts an adaptive response to reductive stress, improving ER protein folding and cardiac myocyte viability during IR [ 186 ].…”