High Iron and Iron Household Protein Contents in Perineuronal Net-Ensheathed Neurons Ensure Energy Metabolism with Safe Iron Handling

Abstract

A subpopulation of neurons is less vulnerable against iron-induced oxidative stress and neurodegeneration. A key feature of these neurons is a special extracellular matrix composition that forms a perineuronal net (PN). The PN has a high affinity to iron, which suggests an adapted iron sequestration and metabolism of the ensheathed neurons. Highly active, fast-firing neurons-which are often ensheathed by a PN-have a particular high metabolic demand, and therefore may have a higher need in iron. We hypothesize that PN-ensheathed neurons have a higher intracellular iron concentration and increased levels of iron proteins. Thus, analyses of cellular and regional iron and the iron proteins transferrin (Tf), Tf receptor 1 (TfR), ferritin H/L (FtH/FtL), metal transport protein 1 (MTP1 aka ferroportin), and divalent metal transporter 1 (DMT1) were performed on Wistar rats in the parietal cortex (PC), subiculum (SUB), red nucleus (RN), and substantia nigra (SNpr/SNpc). Neurons with a PN (PN⁺) have higher iron concentrations than neurons without a PN: PC 0.69 mM vs. 0.51 mM, SUB 0.84 mM vs. 0.69 mM, SN 0.71 mM vs. 0.63 mM (SNpr)/0.45 mM (SNpc). Intracellular Tf, TfR and MTP1 contents of PN⁺ neurons were consistently increased. The iron concentration of the PN itself is not increased. We also determined the percentage of PN⁺ neurons: PC 4%, SUB 5%, SNpr 45%, RN 86%. We conclude that PN⁺ neurons constitute a subpopulation of resilient pacemaker neurons characterized by a bustling iron metabolism and outstanding iron handling capabilities. These properties could contribute to the low vulnerability of PN⁺ neurons against iron-induced oxidative stress and degeneration.

Keywords: DMT1; MTP1; brain; cellular quantification; ferritin H/L; iron; iron proteins; neurodegeneration; perineuronal net; transferrin; transferrin receptor.

Figure 1

Quantitative element maps of groups of PN⁺ and PN⁻ neurons in the rat brain regions parietal cortex (PC), subiculum (SUB), and substantia nigra pars reticulata (SNpr). PNs appear in the nickel map due to Ni-DAB-enhanced WFA-staining. Intracellular iron concentrations were extracted from manually drawn ROIs.

Figure 2

Intracellular iron concentrations of neurons with (PN⁺) and without (PN⁻) a PN in the parietal cortex (PC), subiculum (SUB), and substantia nigra pars reticulata (SNr)/compacta (SNc). PN⁺ vs. PN⁻ p$<$0.01, SNc p$<$0.02 (Student’s t-test).

Figure 3

Correlative fluorescence and element microscopy. Co-localization of ferritin H and iron in the cytoplasm of an interneuron from the parietal cortex. Left panel: Cy3 signal from immunohistochemically stained ferritin H. Right panel: µPIXE quantitative element map of iron. Middle panel: Pixel scatter plot of signal correlation between ferritin H and iron. The pixels in the quadrant with the linear correlation define the area of co-localization (regions encircled in green).

Figure 4

Immunohistochemistry. Brain slices were immunohistochemically stained for iron proteins (antibody, Cy3-labeled) and perineuronal nets (WFA, Cy2). Neurons (NeuN, Cy5), or in case of TfR nuclei of cells (DAPI), were counterstained. The representative images from parietal cortex (PC), red nucleus (RN) and substantia nigra pars reticulata (SNpr) show that all iron proteins can be visualized and are distinctly visible in the cytoplasm of the cells. Ferritin L, which is supposed to be more prominent in glial cells, is also well represented in neurons. A difference in iron protein content of PN⁺ and PN⁻ neurons cannot be distinguished by naked eye, but was revealed by quantification of the immunofluorescence signal intensity using SBC. Scale bar: 50 μm.

Figure 5

Slide-based cytometry. (A) Overview images of rat brain sections with triple-labelling for neurons (NeuN, Cy2 in green), PNs (WFA, Cy3 in orange), and selected iron proteins (Cy5, not shown). The ROIs selected for analysis are highlighted. (B) Sample of high resolution images of neurons from PC. The intense Cy3-WFA signal surrounding the NeuN-area (centred) identifies the neuron as PN⁺. (C) Ratio of averaged intracellular iron protein contents PN⁺ to PN⁻ from fluorescence intensity data. The dashed line marks the reference value 100% set by the values from PN⁻ neurons. Data bars: Mean ± 95% confidence interval.

Figure 6

Percentage of PN⁺ neurons as obtained by single cell counting during slide-based cytometry. PN⁺ and PN⁻ neurons were counted in the left and right hemispheres and averaged over 14 brain slices from two rats (seven slices each). Total cell counts within the selected ROIs were 42,593 (PC), 12,301 (SUB), 2958 (NR), and 2726 (SN).

Figure 7

qRT-PCR. (A) All cDNA amplification products were verified according to their sequence lengths (data shown for SN). (B) Relative quantities of mRNA from PN-rich brain regions with respect to mRNA content in EC (PN-poor region). The dotted line marks the base level ratio of one, i.e., no difference in mRNA expression. Data given as mean ± 95% confidence interval.

Figure 8

Western blot. (A) Iron protein specific antibodies were verified by the molecular weights (kDa) of the target proteins. The DcytB specific antibody showed an insufficient signal which impeded WB analysis. (B) Relative quantities of proteins from PN-rich brain regions (PC, SUB, RN, SN) with respect to protein content in the PN-poor region (EC). The dotted line marks the base level ratio of one, i.e., no difference in protein expression. Data given as mean ± 95% confidence interval.

Figure 9

Cytochrome c oxidase enzyme histochemical staining. (A) The PN-rich regions red nucleus (RN) and substantia nigra (SN) show increased reaction (DAB-enhanced) for cytochrome c oxidase (CytOx), which is a marker for a high metabolic rate. (B) CytOx activity in the parietal cortex (PC). The strongest CytOx reaction is found in PN⁺ neurons (arrows). PN⁻ neurons (arrow head) have a weaker CytOx reaction. Scale bar: 10 μm.