Proteolipid protein gene mutation induces altered ventilatory response to hypoxia in the myelin-deficient rat

Abstract

Pelizaeus Merzbacher disease is an X-linked dysmyelinating disorder of the CNS, resulting from mutations in the proteolipid protein (PLP) gene. An animal model for this disorder, the myelin-deficient (MD) rat, carries a point mutation in the PLP gene and exhibits a phenotype similar to the fatal, connatal disease, including extensive dysmyelination, tremors, ataxia, and death at approximately postnatal day 21 (P21). We postulated that early death might result from disruption of myelinated neural pathways in the caudal brainstem and altered ventilatory response to oxygen deprivation or hypercapnic stimulus. Using barometric plethysmography to measure respiratory function, we found that the MD rat develops lethal hypoxic depression of breathing at P21, but hypercapnic ventilatory response is normal. Histologic examination of the caudal brainstem in the MD rat at this age showed extensive dysmyelination and downregulation of NMDA and to a lesser extent GABA(A) receptors on neurons in the nucleus tractus solitarius, hypoglossal nucleus, and dorsal motor nucleus of the vagus. Unexpectedly, immunoreactive PLP/DM20 was detected in neurons in the caudal brainstem. Not all biosynthetic functions and structural elements were altered in these neurons, because phosphorylated and nonphosphorylated neurofilament and choline acetyltransferase expression were comparable between MD and wild-type rats. These findings suggest that PLP is expressed in neurons in the developing brainstem and that PLP gene mutation can selectively disrupt central processing of afferent neural input from peripheral chemoreceptors, leaving the central chemosensory system for hypercapnia intact.

Fig. 1.

Ventilatory response to inhalation of 8% O₂ or 10% CO₂ in the wild-type (n = 19) and MD (n = 18) rat. MD affected males (open triangles) and normal male littermates (filled squares) exhibited similar responses to inhalation of 8% O₂ for 5 min (P14,A; P18, B). At P21–P24, MD rats exhibited significant depression of ventilation in response to 8% O₂ compared with wild-type males (C) (*p < 0.05). At all ages, the ventilatory response to inhalation of 10% CO₂ for 10 min_. was not different in the MD and wild-type rats (P14, D; P18, E; P21–P24,F).

Fig. 2.

Cells stained with cresyl violet were evenly distributed at the level of the area postrema in the caudal brainstem in P21 wild-type rats (A) and MD rats (B). Scale bar, 100 μm.

Fig. 3.

NeuN expression in the MD and WT rat at P21. Neurons were stained throughout the caudal brainstem at the level of the area postrema in the wild-type rat (A) and MD rat (B). There appeared to be reduced staining for NeuN in the nucleus tractus solitarius and the hypoglossal nucleus (B, D), which was not significant when quantified (Table 1). Boxed areas in Aand B are enlarged in C andD. Scale bars: A, B, 100 μm; C, D, 50 μm.

Fig. 4.

Immunoreactivity for nonphosphorylated neurofilament in the caudal brainstem was distributed similarly in wild-type (A) and MD (B) rats at P21. Boxed areas in A andB are enlarged in C and D. Scale bars: A, B, 100 μm;C, D, 50 μm.

Fig. 5.

Expression of PLP in the wild-type and MD rat at P21. In the caudal brainstem, immunostaining for PLP was greatly diminished in the MD rat (B) compared with the wild type (A). Furthermore, neurons immunoreactive for PLP were present in the hypoglossal nucleus (B). When sections from the wild-type rat were double labeled for NeuN (red) and PLP/DM20 (green), no cells immunoreactive for both were observed (C). However, in the MD rat, cells were found that were double labeled for NeuN and PLP/DM20 (yellow) (D). Boxed areas in A and B represent comparable areas with the sites imaged in C andD. Scale bars: A, B, 100 μm; C, D, 33 μm.

Fig. 6.

In transgenic mice (P12) expressing thePLP promoter driving expression of EGFP (B), neurons (yellow) were found in the nucleus tractus solitarius that were immunoreactive for NeuN (red) and also expressed EGFP-PLP(green). Nontransgenic mice expressed no autofluorescence in neurons in the NTS (A). Scale bars: A, B, 33 μm.

Fig. 7.

NMDA NR₁-immunoreactive neurons in the WT and MD rat at P21. In the caudal medulla, at the level of the area postrema, NMDA NR₁-reactive neurons were observed in the nucleus tractus solitarius, hypoglossal nucleus, and dorsal motor nucleus of the vagus (A). In contrast, in the MD rat, neurons immunoreactive for NMDA NR₁ were strikingly diminished in these areas (B). Boxed areas in A and B are enlarged inC–F: top box, C,D; bottom box, E,F. Scale bars: A, B, 100 μm; C–F, 50 μm.

Fig. 8.

GABA_A β2 subunit immunoreactivity in the WT and MD rat at P21. In the caudal brainstem, at the level of the area postrema, GABA_A β2 subunit-immunoreactive neurons were present in the nucleus tractus solitarius, dorsal motor nucleus of the vagus, and hypoglossal nucleus (A). Immunoreactivity for this receptor was diminished in the MD rat at P21 (B). Scale bars: A,B, 100 μm.

Fig. 9.

Immunoreactivity for ChAT was similar in the WT (A) and MD (B) rat in the DMV and hypoglossal nuclei. Scale bars: A,B, 100 μm.

Fig. 10.

Expression of PLP/DM20, MBP, NMDA NR₁, and GABA_A β2 and γ subunits assayed by immunoblot in tissue from the brainstem in the wild-type and MD rat at P21. Actin is present as a loading control. In the MD rat, there was downregulation of NMDA NR₁, PLP/DM20, and MBP.