Abstract
BackgroundMesencephalic astrocyte-derived neurotrophic factor (MANF), a 20 kDa secreted protein, was originally derived from a rat mesencephalic type-1 astrocyte cell line. MANF belongs to a novel evolutionally conserved family of neurotrophic factors along with conserved dopamine neurotrophic factor. In recent years, ever-increasing evidence has shown that both of them play a remarkable protective role against various injuries to neurons in vivo or in vitro. However, the characteristics of MANF expression in the different types of glial cells, especially in astrocytes, remain unclear.MethodsThe model of focal cerebral ischemia was induced by rat middle cerebral artery occlusion. Double-labeled immunofluorescent staining was used to identify the types of neural cells expressing MANF. Primarily cultured glial cells were used to detect the response of glial cells to endoplasmic reticulum stress stimulation. Propidium iodide staining was used to determine dead cells. Reverse transcription PCR and western blotting were used to detect the levels of mRNA and proteins.ResultsWe found that MANF was predominantly expressed in neurons in both normal and ischemic cortex. Despite its name, MANF was poorly expressed in glial cells, including astrocytes, in normal brain tissue. However, the expression of MANF was upregulated in the glial cells under focal cerebral ischemia, including the astrocytes. This expression was also induced by several endoplasmic reticulum stress inducers and nutrient deprivation in cultured primary glial cells. The most interesting phenomenon observed in this study was the pattern of MANF expression in the microglia. The expression of MANF was closely associated with the morphology and state of microglia, accompanied by the upregulation of BIP/Grp78.ConclusionsThese results indicate that MANF expression was upregulated in the activated glial cells, which may contribute to the mechanism of ischemia-induced neural injury.
Highlights
Mesencephalic astrocyte-derived neurotrophic factor (MANF), a 20 kDa secreted protein, was originally derived from a rat mesencephalic type-1 astrocyte cell line
Ischemia-induced MANF expression in the astrocytes MANF was so named because it was originally isolated from a rat mesencephalic astrocyte line [1]
Negative controls were performed to show the specificity of anti-MANF antibody in the brain tissue by substituting the primary antibody with Phosphate-buffered saline (PBS) (Figure 1M,N, O,P)
Summary
Mesencephalic astrocyte-derived neurotrophic factor (MANF), a 20 kDa secreted protein, was originally derived from a rat mesencephalic type-1 astrocyte cell line. MANF belongs to a novel evolutionally conserved family of neurotrophic factors along with conserved dopamine neurotrophic factor. Mesencephalic astrocyte-derived neurotrophic factor (MANF), a 20 kDa secreted protein, was initially isolated and purified from culture medium of immortalized rat type-1 astrocytes ( named ventral mesencephalic cell line 1 (VMCL1)) [1]. Subsequent studies showed that MANF protein does not contain an argininerich region and has neurotrophic effects with selectivity for dopaminergic neurons [1,5]. MANF, together with conserved dopamine neurotrophic factor (CDNF), belongs to a novel and evolutionally conserved family of neurotrophic factors [5]. The cysteine bridge may be involved in catalyzing the formation of intramolecular disulfide bonds and protein folding in the endoplasmic reticulum (ER) because of its similarity to the active site motif of thiol/disulfide oxidoreductases [8,9]
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