Increased Plp1 gene expression leads to massive microglial cell activation and inflammation throughout the brain

Abstract

PMD (Pelizaeus-Merzbacher disease) is a rare neurodegenerative disorder that impairs motor and cognitive functions and is associated with a shortened lifespan. The cause of PMD is mutations of the PLP1 [proteolipid protein 1 gene (human)] gene. Transgenic mice with increased Plp1 [proteolipid protein 1 gene (non-human)] copy number model most aspects of PMD patients with duplications. Hypomyelination and demyelination are believed to cause the neurological abnormalities in mammals with PLP1 duplications. We show, for the first time, intense microglial reactivity throughout the grey and white matter of a transgenic mouse line with increased copy number of the native Plp1 gene. Activated microglia in the white and grey matter of transgenic mice are found as early as postnatal day 7, before myelin commences in normal cerebra. This finding indicates that degeneration of myelin does not cause the microglial response. Microglial numbers are doubled due to in situ proliferation. Compared with the jp (jimpy) mouse, which has much more oligodendrocyte death and hardly any myelin, microglia in the overexpressors show a more dramatic microglial reactivity than jp, especially in the grey matter. Predictably, many classical markers of an inflammatory response, including TNF-α (tumour necrosis factor-α) and IL-6, are significantly up-regulated manyfold. Because inflammation is believed to contribute to axonal degeneration in multiple sclerosis and other neurodegenerative diseases, inflammation in mammals with increased Plp1 gene dosage may also contribute to axonal degeneration described in patients and rodents with PLP1 increased gene dosage.

Keywords: BrdU, bromodeoxyuridine; CCL3, CC chemokine ligand 3; CCR1, CC chemokine receptor 1; CD11b, cluster of differentiation molecule 11B; CD8, cluster of differentiation 8; CNS, central nervous system; CRP, C-reactive protein; CXCL, CXC chemokine ligand; DAB, diaminobenzidine; DPN, day postnatal; EAE, experimental allergic encephalomyelitis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; IL-1β, interleukin-1β; Iba1, ionized calcium-binding adaptor molecule 1; MOG, myelin oligodendrocyte glycoprotein; PLP1, proteolipid protein 1 gene (human); PMD, Pelizaeus–Merzbacher disease; Pelizaeus–Merzbacher disease; Plp1, proteolipid protein 1 gene (non-human); QPCR, quantitative PCR; TNF-α, tumour necrosis factor-α; Ta, Tabby; iNOS, inducible nitric oxide synthase; inflammation; jp, jimpy; microglia; myelin; oligodendrocyte; proteolipid protein; qRT–PCR, quantitative reverse transcription–PCR.

Figure 1. Iba1 immunostaining of 50 μm Vibratomed sections from 36–46-day-old normal B6CBA mouse cerebra visualized with DAB alone (A) or cobalt-enhanced after DAB development (B–D)

High magnification of a typical ramified microglial cell (A) shows several long, thin processes emanating from an ellipsoid cell body. These primary processes branch into secondary processes (arrows). In areas of the cortex (B) and striatum (C), resting microglia are evenly dispersed throughout grey matter with typical ramified morphology. In the corpus callosum (D), microglia are less abundant than in grey matter, but retain their ramified morphology. The cingulum is at the top of the Figure. Scale bars: (A) 10 μm and (B–D) 50 μm.

Figure 2. Iba1 cobalt-enhanced immunostaining of 50 μm Vibratomed sections from a 50-day-old B6CBA cortex (A) and striatum (B) and Plp1tg cortex (C, E) and striatum (D, F) of 19DPN (C, D) and 30DPN (E, F) transgenics

Microglia from B6CBA mice at 50 DPN have similar morphology to microglia at younger ages. Activated microglia are often clustered together with their cell bodies having a fuzzy appearance due to numerous, short, thick processes. In the striatum, more elongated and straighter processes are visible, possibly following axonal trajectories. Microglia exhibit more processes at 30 DPN (E, F) than at earlier ages (C, D). Scale bar, 50 μm.

Figure 3. Iba1 cobalt-enhanced immunostaining of 50 μm Vibratomed sections from the corpus callosum of 19- (A), 30- (B) and 50-day-old (C) Plp1tg mice display activated microglia, where processes follow trajectories of callosal axons

At 30 and 50 DPN, microglia are more activated in the white matter compared with the 19 DPN. Scale bar, 50 μm.

Figure 4. Iba1 cobalt-enhanced immunostaining of 50 μm Vibratomed sections from 7 DPN B6CBA (A, B) and Plp1tg mice (C–E)

Microglia in the cortices (A) and striatum (B) of normal mice already display the characteristic morphology of microglia at older ages. Their long processes sometimes terminate in boutons (arrows) that are less prominent in microglia from older mice. In the cortex (C), striatum (D) and corpus callosum (E), some microglial cells in Plp1tg mice are already activated at this young age, indicated by short, numerous processes emanating from microglia with large perikarya (arrows). Note the absence of long processes on some of these activated microglia. A mix of ramified and activated microglial cells is visible in all three regions. (F) A 1 μm plastic section of a normal 7 DPN B6CBA striatum has no myelin around axons at this age. Scale bars: (A–E) 50 μm and (F) 10 μm.

Figure 5. Combined Iba1 and BrdU immunostaining of 50 μm Vibratomed sections from a 8 DPN Plp11tg mouse

Mice were injected with BrdU at 6 DPN and killed 40 h later on 8 DPN. Brown chromogen indicates Iba1 cytoplasmic staining and black chromagen indicates BrdU nuclear staining. (A) A double-labelled microglia cell with numerous processes indicates that this cell underwent cell division approx. 40 h before the mouse was killed. Two BrdU⁺/Iba1⁻ cells (arrows) are shown in the upper right. (B) An Iba1⁺ cell that is in mitosis (arrow). (C) Four BrdU⁺/Iba1⁻ cells (arrows), a BrdU⁻/Iba1⁺ cell (arrowhead) and a pair of lightly labelled Brdu⁺/Iba1⁺ cells (crossed arrows). (D) A double-labelled microglial cell with one process (arrow). Scale bar, 50 μm.

Figure 6. Quantification of microglia in Plp1tg versus control mice

(A) A 50 μm Vibratomed hemi-section from a 30 DPN mouse cerebrum at low magnification shows boxed areas used for quantification of Iba1 immunostained cells. Cells were counted at ×500 directly under the microscope at the level of the anterior commissure (arrow). Microglial cells with distinct nuclei were counted in the three areas selected: (A) dorsal cortex, (B) striatum and (C) ventral–lateral cortex. (B) Quantification of Iba1-immunostained cells in the cerebrum of 19–27 DPN Plp1tg and 23–30 DPN B6CBA mice. Values are means±S.E.M. for Iba1 cells in the three areas selected, three sections per animal were averaged, and the number of animals from both groups is shown in parentheses. The two-tailed Student's t test was used to compare Plp11tg mice with B6CBA mice; ***P<0.0001.

Figure 7. Ultrastructure of microglia in Plp1tg mice

(A–D) Electron micrographs from a 43 DPN Plp1tg mouse. (A) This typical microglial cell is characterized by dense heterochromatin lining the nuclear membrane. Contrasting light cytoplasm is typical of microglia, whereas oligodendrocytes have much more electron-dense cytoplasm. Long, thin strands of endoplasmic reticulum (arrows) with scattered ribosomes on their surface are likewise a distinguishing feature of microglia. A large inclusion (asterisk), as well as the remnants of an engulfed nucleus (arrowhead), is present in the cytoplasm. (B) A microglial cell with a long, thickened process (arrows) contains debris (asterisk). The large diameter process probably corresponds to the thickened processes seen in light micrographs. (C) An electron-dense cell with sparse cytoplasm lacks sufficient nuclear and cytoplasm features for positive identification. (D) Another electron-dense cell that is difficult to identify. This electron-dense cell abuts the perikaryon of a neuron, and these are characteristics of perineuronal oligodendrocytes. Scale bar, 1 μm.

Figure 8. Light micrographs of the brain from a 19 DPN jp mouse stained with Iba1 antibody

(A) Microglial staining is strongest in corpus callosum (CC), but weak in cerebral cortex (CeCo) and striatum (St). Microglial staining in grey matter is limited to white matter bundles including cingulum in cortex and Combs fibres in striatum. (B) The microglia in jp corpus callosum do not exhibit the intense reactivity found in Plp1tg mice manifested by short, thick processes. Scale bar, 100 μm.

Figure 9. F4/80 immunostaining of 50 μm Vibratomed cervical spinal cords from an 8–9-month-old EAE mouse (A), a Plp1tg at 43 DPN (B), and the Plp11tg without the F4/80 primary antibody (C)

(A) The EAE spinal cord shows macrophage infiltration (arrows) indicated by cytoplasmic and process staining of membranes. (B) The picture from the Plp11tg ventral funiculus illustrates the most intense staining observed in these mutants. The axoplasm is non-specifically stained in these mutants. Scale bar, 5 μm.