BackgroundThe pathological features of the common neurodegenerative conditions, Alzheimer’s disease (AD), Parkinson’s disease and multiple sclerosis are all known to be associated with iron dysregulation in regions of the brain where the specific pathology is most highly expressed. Iron accumulates in cortical plaques and neurofibrillary tangles in AD where it participates in redox cycling and causes oxidative damage to neurons. To understand these abnormalities in the distribution of iron the expression of proteins that maintain systemic iron balance was investigated in human AD brains and in the APP-transgenic (APP-tg) mouse.ResultsProtein levels of hepcidin, the iron-homeostatic peptide, and ferroportin, the iron exporter, were significantly reduced in hippocampal lysates from AD brains. By histochemistry, hepcidin and ferroportin were widely distributed in the normal human brain and co-localised in neurons and astrocytes suggesting a role in regulating iron release. In AD brains, hepcidin expression was reduced and restricted to the neuropil, blood vessels and damaged neurons. In the APP-tg mouse immunoreactivity for ferritin light-chain, the iron storage isoform, was initially distributed throughout the brain and as the disease progressed accumulated in the core of amyloid plaques. In human and mouse tissues, extensive AD pathology with amyloid plaques and severe vascular damage with loss of pericytes and endothelial disruption was seen. In AD brains, hepcidin and ferroportin were associated with haem-positive granular deposits in the region of damaged blood vessels.ConclusionOur results suggest that the reduction in ferroportin levels are likely associated with cerebral ischaemia, inflammation, the loss of neurons due to the well-characterised protein misfolding, senile plaque formation and possibly the ageing process itself. The reasons for the reduction in hepcidin levels are less clear but future investigation could examine circulating levels of the peptide in AD and a possible reduction in the passage of hepcidin across damaged vascular endothelium. Imbalance in the levels and distribution of ferritin light-chain further indicate a failure to utilize and release iron by damaged and degenerating neurons.

Chondroitin sulfate (CS) proteoglycans in perineuronal nets (PNNs) from the central nervous system (CNS) are involved in the control of plasticity and memory. Removing PNNs reactivates plasticity and restores memory in models of Alzheimer’s disease and ageing. Their actions depend on the glycosaminoglycan (GAG) chains of CS proteoglycans, which are mainly sulfated in the 4 (C4S) or 6 (C6S) positions. While C4S is inhibitory, C6S is more permissive to axon growth, regeneration and plasticity. C6S decreases during critical period closure. We asked whether there is a late change in CS-GAG sulfation associated with memory loss in aged rats. Immunohistochemistry revealed a progressive increase in C4S and decrease in C6S from 3 to 18 months. GAGs extracted from brain PNNs showed a large reduction in C6S at 12 and 18 months, increasing the C4S/C6S ratio. There was no significant change in mRNA levels of the chondroitin sulfotransferases. PNN GAGs were more inhibitory to axon growth than those from the diffuse extracellular matrix. The 18-month PNN GAGs were more inhibitory than 3-month PNN GAGs. We suggest that the change in PNN GAG sulfation in aged brains renders the PNNs more inhibitory, which lead to a decrease in plasticity and adversely affect memory.

Brain injury induces gliosis and scar formation; its principal cell types are mainly astrocytes and some oligodendrocytes. The origin of the astrocytes and oligodendrocytes in the scar remains unclear together with the underlying mechanism of their fate choice. We examined the response of oligodendrocyte transcription factor (Olig)2(+) glial progenitors to acute brain injury. Both focal cortical (mechanical or excitotoxic) and systemic (kainic acid-induced seizure or lipopolysaccharide-induced inflammation) injury caused cytoplasmic translocation of Olig2 (Olig2(TL)) exclusively in affected brain regions as early as 2 h after injury in two-thirds of Olig2(+) cells. Many of the proliferating Olig2(+) cells reacting to injury co-expressed chondroitin sulphate proteoglycan neuron/glia antigen 2 (NG2). Using 5-bromodeoxyuridine (BrdU) tracing protocols, proliferating Olig2(TL)GFAP(+)BrdU(+) cells were observed from 2 days post-lesion (dpl). Immature oligodendrocytes were also seen from 2 dpl and all of them retained Olig2 in the nucleus (Olig2(Nuc)). From 5 dpl Olig2(TL)NG2(+)GFAP(+) cells were observed in the wound and some of them were proliferative. From 5 dpl NG2(+)RIP(+) cells were also seen, all of which were Olig2(Nuc) and some of which were also BrdU(+). Our results suggest that, in response to brain injury, NG2(+) progenitors may generate a subpopulation of astrocytes in addition to oligodendrocytes and their fate choice was associated with Olig2(TL) or Olig2(Nuc). However, the NG2(+)GFAP(+) phenotype was only seen within a limited time window (5-8 dpl) when up to 20% of glial fibrillary acidic protein (GFAP) cells co-expressed NG2. We also observed Olig2(TL)GFAP(+) cells that appeared after injury and before the NG2(+)GFAP(+) phenotype. This suggests that not all astrocytes are derived from an NG2(+) population.

We have developed a compartmentalised culture model for the purification of axonal mRNA from embryonic, neonatal and adult rat dorsal root ganglia. This mRNA was used un-amplified for RT-qPCR. We assayed for the presence of axonal mRNAs encoding molecules known to be involved in axon growth and guidance. mRNAs for beta-actin, beta-tubulin, and several molecules involved in the control of actin dynamics and signalling during axon growth were found, but mRNAs for microtubule-associated proteins, integrins and cell surface adhesion molecules were absent. Quantification of beta-actin mRNA by means of qPCR showed that the transcript is present at the same level in embryonic, newborn and adult axons. Using the photoconvertible reporter Kaede we showed that there is local translation of beta-actin in axons, the rate being increased by axotomy. Knock down of beta-actin mRNA by RNAi inhibited the regeneration of new axon growth cones after in vitro axotomy, indicating that local translation of actin-related molecules is important for successful axon regeneration.

BackgroundHepcidin is a peptide hormone belonging to the defensin family of cationic antimicrobial molecules that has an essential role in systemic iron homeostasis. The peptide is synthesised by hepatocytes and transported in the circulation to target tissues where it regulates the iron export function of the ferrous iron permease, ferroportin. In the brain hepcidin protein has been identified using immuno-histochemistry and mRNA by real-time PCR but not by in situ hybridisation raising the question of whether there is measurable transcription of the hepcidin gene in the central nervous system. Alternatively hepcidin could be transported as a hormone to the brain via the circulation.ResultsBy RT-PCR hepcidin mRNA was present at low level throughout normal rat brain while in situ hybridisation to detect low-abundant mRNA revealed that transcripts were restricted to endothelium of blood vessels and choroid plexus. In contrast, hepcidin protein analysed by immuno-histochemistry was highly expressed in blood vessels, in endothelium and in pericytes. Hepcidin was also present in glial cells and in the olfactory bulb, sub-ventricular zone and dentate gyrus, areas where neurogenesis and synaptic plasticity are maintained throughout adult life. The hepcidin species identified by Western blotting in sub-ventricular zone, cortex and hippocampus migrated as a ~2.8 kDa band, identical in size to hepcidin present in normal rat serum suggesting that hepcidin in brain was the full-length biologically active 25 amino acid peptide. Hepcidin co-localised with ferroportin in ependymal cells of the sub-ventricular zone and in the corpus callosum consistent with a regulatory role in iron metabolism at these sites.ConclusionsHepcidin protein was widely expressed in brain parenchyma while levels of hepcidin gene transcription appeared to be below the limits of detection of the in situ hybridisation probes. This disparity suggests that not all hepcidin in the brain is transcribed in situ and may originate in part outside the brain. The properties of hepcidin as a cationic peptide hormone are reflected in the finding of hepcidin in the walls of blood vessels and in pericytes and glia, cells that may be involved in transporting the peptide into brain interstitium.