Hypoglossal neuropathology and respiratory activity in pompe mice

Abstract

Pompe disease is a lysosomal storage disorder associated with systemic deficiency of acid α-glucosidase (GAA). Respiratory-related problems in Pompe disease include hypoventilation and upper airway dysfunction. Although these problems have generally been attributed to muscular pathology, recent work has highlighted the potential role of central nervous system (CNS) neuropathology in Pompe motor deficiencies. We used a murine model of Pompe disease to test the hypothesis that systemic GAA deficiency is associated with hypoglossal (XII) motoneuron pathology and altered XII motor output during breathing. Brainstem tissue was harvested from adult Gaa(-/-) mice and the periodic acid Schiff method was used to examine neuronal glycogen accumulation. Semi-thin (2 μm) plastic sections showed widespread medullary neuropathology with extensive cytoplasmic glycogen accumulation in XII motoneuron soma. We next recorded efferent XII bursting in anesthetized and ventilated Gaa(-/-) and B6/129 mice both before and after bilateral vagotomy. The coefficient of variation of respiratory cycle duration was greater in Gaa(-/-) compared to B6/129 mice (p < 0.01). Vagotomy caused a robust increase in XII inspiratory burst amplitude in B6/129 mice (239 ± 44% baseline; p < 0.01) but had little impact on burst amplitude in Gaa(-/-) mice (130 ± 23% baseline; p > 0.05). We conclude that CNS GAA deficiency results in substantial glycogen accumulation in XII motoneuron cell bodies and altered XII motor output. Therapeutic strategies targeting the CNS may be required to fully correct respiratory-related deficits in Pompe disease.

Keywords: Pompe; acid α-glucosidase; brainstem; glycogen; hypoglossal.

Figure 1

Medullary sections showing XII motor neurons. (A–D) Show paraffin-embedded tissues cut at 10 μm and stained with the PAS method. Positive PAS staining was not observed within neuronal cell bodies of B6/129 mice (A,B). In contrast, XII motoneurons and cells in the surrounding areas showed robust PAS staining in Gaa^−/− mice (C,D). (B and D) Are higher magnification images of the areas indicated in (A and C); arrows indicate individual XII motoneurons. (E) Shows an example of CT-β labeling of the XII nuclei following tongue injection. (F) Shows a paraffin-embedded medullary section from a Gaa^−/− mouse in which XII motoneurons were retrogradely labeled with CT-β. This section was also stained with the PAS method, and labeled XII motoneurons (arrows) show PAS staining consistent with what was presented in (C,D). cc, Central canal; scale bars: (A,C,E): 100 μm; (B,D,F): 50 μm.

Figure 2

Plastic-embedded semi-thin medullary sections (2 μm) show neuronal glycogen accumulation in Gaa^−/− mice. Sections were stained with PAS and toluidine blue. (A) Provides a low power view including both hypoglossal nuclei; (B) is a higher resolution image of the area indicated by the box in (A). These images demonstrate that PAS staining within the XII nuclei is restricted to neuronal cell bodies. Ependymal cells around the central canal (CC) also showed robust PAS staining (A). (C) Shows a high magnification of PAS staining within a XII motoneuron – note the extensive accumulation of glycogen droplets in the cytoplasm with no positive nuclear staining. The image in (D) is from the ventral medulla near the pyramidal decussation. PAS positive neuroglial cells were noted (indicated by the arrows) but axons appear to be healthy with no evidence of glycogen accumulation. Similarly, glycogen accumulation was not observed in medullary axons including cranial nerve fibers (E). Finally, (F) depicts the extensive neuronal glycogen accumulation within the nucleus ambiguous in the rostral medulla. Although not the primary focus of this work, this image is included to illustrate that neuropathology in upper airway motor systems in Gaa^−/− mice is not limited to the XII motor nucleus. Scale bars: (A): 100 μm; (B,D–F): 50 μm; (C): 20 μm.

Figure 3

Examples of efferent XII nerve activity in B6/129 and Gaa^−/− mice under vagal-intact and vagotomized conditions. Both “raw” and integrated (∫) neurograms are shown. In the vagal-intact condition, the amplitude of the XII signal is similar between the two mice, but the Gaa^−/− mouse shows more variability in the timing of the respiratory cycle. Following bilateral cervical vagotomy there is a robust increase in XII burst amplitude in the B6/129 mouse whereas the Gaa^−/− mouse shows little change.

Figure 4

Examples of respiratory cycle variability in representative B6/129 and Gaa^−/− mice. The left panel shows a sequential plot of the total duration of the respiratory cycle (T_TOT) for 60 consecutive cycles. An increase in the breath-to-breath variability in T_TOT can be seen in the Gaa^−/− mouse under both vagal-intact and vagotomized conditions. The middle and right panels show histograms depicting the overall distribution of values for T_TOT during the 60 respiratory cycles depicted in the left panel.

Figure 5

The coefficient of variation (CV) of inspiratory [T_I, (A)], expiratory [T_E, (B)], and total respiratory cycle [T_TOT, (C)] duration during vagal-intact and vagotomized conditions. CV of T_I and T_TOT is significantly higher in vagotomized Gaa^−/− mice than B6/129 mice. *p < 0.05; **p < 0.01 significant different from B6/129 mice. #p < 0.05 compared with the value during vagal-intact condition.

Figure 6

Examples of Pre-I XII bursting in B6/129 and Gaa^−/− mice. These examples were taken from vagotomized, ventilated mice. We noted anecdotally that Pre-I XII bursting (i.e., bursting before the onset of the phrenic burst) was evident in B6/129 but not Gaa^−/− mice. In the examples, the solid vertical line indicates the onset of inspiratory phrenic bursting and the vertical dashed line represents the onset of Pre-I XII activity. The horizontal line indicates the end-expiratory amplitude prior to inspiration. This particular Gaa^−/− mouse example was chosen to illustrate that in cases where Gaa^−/− mice showed more robust phrenic bursting, the same animal tended to show reduced efferent XII output (see text for further description).

Figure 7

Inspiratory phrenic and XII burst amplitude B6/129 and Gaa^−/− mice. The amplitude of phrenic (A) and XII (B) neurogram inspiratory burst amplitudes were expressed as arbitrary units (a.u.), and normalized to the activity recorded under vagal-intact conditions (C). Cervical vagotomy caused robust increases in XII burst amplitude in B6/129 but not Gaa^−/− mice. See text for a commentary on the phrenic burst amplitude. *p < 0.05; **p < 0.01 significant different from B6/129 mice. ^#p < 0.05; ^##p < 0.01 compared with the value during vagal-intact condition.