The pre-Bötzinger complex has been identified as an essential part of the medullary respiratory network in mammals. Although well described in experimental animals, its localization in the human brain has remained elusive. Using serially sectioned brainstems from 19 normal individuals and patients suffering from neurodegenerative diseases (multiple system atrophy, n = 10; spinocerebellar ataxia type 3, n = 8), we have identified a circumscribed area of the ventrolateral medulla that represents the human homologue of the pre-Bötzinger complex and have mapped its longitudinal and horizontal extents. The presumed human pre-Bötzinger complex is characterized by an aggregation of loosely scattered, small and lipofuscin-rich neurons, which contain neurokinin 1 receptor as well as somatostatin, but are negative for markers of monoaminergic neurons and of motoneurons. In brains of patients suffering from multiple systems atrophy (with central respiratory deficits but without swallowing problems), pre-Bötzinger complex neurons were reduced, whereas pharyngeal motoneurons of the ambigual nucleus were not affected. In contrast, in brains of patients with spinocerebellar ataxia 3 (no reported central respiratory deficits but with dysphagia), pre-Bötzinger complex neurons were preserved, whereas ambigual motoneurons, which control swallowing, were diminished. These pathoanatomical findings support the view, that affection of the central respiratory network, including the pre-Bötzinger complex, contributes to breathing disorders in multiple system atrophy, whereas damage to ambigual motoneurons is important for pathogenesis of breathing disturbances and dysphagia in patients with spinocerebellar ataxia type 3. On the basis of these findings, the putative human homologue of the pre-Bötzinger complex can now be reliably delineated on pigment-Nissl-stained sections, making neuropathological investigations of central respiratory disturbances feasible.

Oculopharyngodistal myopathy (OPDM) is an adult-onset neuromuscular disease characterized by progressive ocular, facial, pharyngeal and distal limb muscle involvement. Trinucleotide repeat expansions in LRP12 or GIPC1 were recently reported to be associated with OPDM. However, a significant portion of OPDM patients have unknown genetic causes. In this study, long-read whole-genome sequencing and repeat-primed PCR were performed and we identified GGC repeat expansions in the NOTCH2NLC gene in 16.7% (4/24) of a cohort of Chinese OPDM patients, designated as OPDM type 3 (OPDM3). Methylation analysis indicated that methylation levels of the NOTCH2NLC gene were unaltered in OPDM3 patients, but increased significantly in asymptomatic carriers. Quantitative real-time PCR analysis indicated that NOTCH2NLC mRNA levels were increased in muscle but not in blood of OPDM3 patients. Immunofluorescence on OPDM muscle samples and expressing mutant NOTCH2NLC with (GGC)69 repeat expansions in HEK293 cells indicated that mutant NOTCH2NLC-polyglycine protein might be a major component of intranuclear inclusions, and contribute to toxicity in cultured cells. In addition, two RNA-binding proteins, hnRNP A/B and MBNL1, were both co-localized with p62 in intranuclear inclusions in OPDM muscle samples. These results indicated that a toxic protein gain-of-function mechanism and RNA gain-of-function mechanism may both play a vital role in the pathogenic processes of OPDM3. This study extended the spectrum of NOTCH2NLC repeat expansion-related diseases to a predominant myopathy phenotype presenting as OPDM, and provided evidence for possible pathogenesis of these diseases.