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Ceruloplasmin (Cp) is a ferroxidase that also plays a role in iron efflux from cells. It can thus help to regulate cellular iron homeostasis. In the CNS, Cp is expressed as a membrane-anchored form by astrocytes. Here, we assessed the role of Cp in permanent middle cerebral artery occlusion (pMCAO) comparing wildtype and Cp null mice. Our studies show that the lesion size is larger and functional recovery impaired in Cp null mice compared to wildtype mice. Expression of Cp increased ninefold at 72 h after pMCAO and remained elevated about twofold at day 14. We also assessed changes in mRNA and protein expression of molecules involved in iron homeostasis. As expected there was a reduction in ferroportin in Cp null mice at 72 h. There was also a remarkable increase in DMT1 protein in both genotypes at 72 h, being much higher in wildtype mice (19.5-fold), that then remained elevated about twofold at 14 days. No difference was seen in transferrin receptor 1 (TfR1) expression, except a small reduction in wildtype mice at 72 h, suggesting that the increase in DMT1 may underlie iron uptake independent of TfR1-endosomal uptake. There was also an increase of ferritin light chain in both genotypes. Iron histochemistry showed increased iron accumulation after pMCAO, initially along the lesion border and later throughout the lesion. Immunofluorescence labeling for ferritin (a surrogate marker for iron) and GFAP or CD11b showed increased ferritin in GFAP+ astrocytes along the lesion border in Cp null mice, while CD11b+ macrophages expressed ferritin equally in both genotypes. Increased lipid peroxidation assessed by 4HNE staining was increased threefold in Cp null mice at 72 h after pMCAO; and 3-nitrotyrosine labeling showed a similar trend. Three key pro-inflammatory cytokines (IL-1β, TNFα, and IL-6) were markedly increased at 24 h after pMCAO equally in both genotypes, and remained elevated at lower levels later, indicating that the lack of Cp does not alter key inflammatory cytokine expression after pMCAO. These data indicate that Cp expression is rapidly upregulated after pMCAO, and loss of Cp results in dysregulation of iron homeostasis, increased oxidative damage, greater lesion size and impaired recovery of function.

Astrocytes are the most abundant glial cells in the central nervous system (CNS) with the capacity to sense and react to injury and inflammatory events. While it has been widely documented that astrocytes can exert tissue-degenerative functions, less is known about their protective and disease-limiting roles. Here, we report the upregulation of pleiotrophin (PTN) by mouse and human astrocytes in multiple sclerosis (MS) and its preclinical model experimental autoimmune encephalomyelitis (EAE). Using CRISPR-Cas9-based genetic perturbation systems, we demonstrate in vivo that astrocyte-derived PTN is critical for the recovery phase of EAE and limits chronic CNS inflammation. PTN reduces pro-inflammatory signaling in astrocytes and microglia and promotes neuronal survival following inflammatory challenge. Finally, we show that intranasal administration of PTN during the late phase of EAE successfully reduces disease severity, making it a potential therapeutic candidate for the treatment of progressive MS, for which existing therapies are limited.

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