Our study revealed that LC noradrenergic neurons of Ndntm1ky mice showed significantly decreased spontaneous activities as well as impaired excitability, which were mediated by enhanced A-type voltage-dependent potassium currents. Ndntm1ky mice also possessed some neonatal phenotypes possibly related with LC-NE dysfunction, such as transient hypotonia and poor ventilatory responses to hypercapnia. Our data suggest that LC—the primary source of norepinephrine in CNS—is involved in PWS pathogenesis.
Previously, the hypothalamus had been the region of interest for determining PWS pathophysiology because of the endocrinological manifestations of the disease, which include hyperphagia, obesity, hypogonadism, and growth hormone deficiency (31, 32). However, hypothalamus and pituitary gland dysfunction do not correlate with central apnea and hypotonia, which are important risk factors for premature death in infants and children with PWS (33, 34). Therefore, this study focused on another important region in the brainstem—LC—using necdin-deficient mice. Most previous studies have focused on respiratory abnormalities in necdin-deficient mice because of their high postnatal lethality following respiratory instability (35–38). Other behavior-related data of these mice, however, are lacking. Our study demonstrated that, in addition to breathing, Ndntm1ky mice show defective postnatal motor development, rendering this mouse strain a suitable platform for investigating the neurobiological mechanisms underlying the neonatal symptoms of PWS.
Necdin, transcribed from the paternal Ndn allele in both mice and humans, is a neural differentiation-associated protein and is expressed exclusively in postmitotic neurons of the CNS and peripheral nervous system (39, 40). Molecular studies have demonstrated integral roles of necdin in neuronal mitotic arrest, differentiation, and survival via interactions with regulatory proteins such as the transcription factors E2F1, E2F4, and p53 and the neurotrophin receptors p75 and TrkA (18). Therefore, it is not surprising that necdin deficiency results in widespread abnormalities of the parts of the nervous system, such as reticular formation (36), forebrain GABAergic neurons (18), and medullary serotonergic neurons (19). Furthermore, our study demonstrated LC dysfunction in Ndntm1ky mice and corroborated the previous histological findings of Pagliardini et al. who used another necdin-deficient mouse model (Ndntm2stw) to reveal the abnormal distribution and appearance of noradrenergic neurons in the medulla (36). Considering the neonatal phenotypes of necdin-deficient mice, including hypotonia and blunted respiratory responses, the noradrenergic system dysfunction in both Ndntm1ky and Ndntm2stw mice may be an important etiology of neonatal PWS.
In this study, we evaluated the electrophysiological properties of LC neurons in Ndntm1ky mice. First, the SFR of LC neurons was lower in the necdin-deficient mice. LC is important for attention and behavior due to its functional switch between the phasic and tonic modes of output (41). Thus, the decreased SFR of LC neurons might affect the vital function of the LC-NE system in Ndntm1ky mice. Next, we analyzed the spontaneous AP morphology of LC neurons in Ndntm1ky mice but found no significant differences compared with WT mice. AP morphology is determined primarily by the dynamics of voltage-gated sodium and potassium channels, which are also associated with the provocation of seizures (42). This electrophysiological finding is consistent with the clinical observation that patients with PWS scarcely develop seizures (43). Finally, the latency of the first AP firing following the injection of current into LC-NE neurons of Ndntm1ky mice was significantly delayed. This delayed AP firing latency was due mostly to A-type potassium currents, which participate in neuronal dendritic calcium signaling, signal integration, and synaptic plasticity (44). In the brainstem noradrenergic neurons of the A7 catecholamine cell groups, A-type potassium currents play important roles in the regulation of the shape and firing frequency of APs as well as in synaptic integration (27). Our data indicate that necdin deficiency increases A-type potassium currents in LC-NE neurons, decreases their excitability, and possibly decreases SFR. Therefore, the baseline phasic activity of LC-NE neurons in Ndntm1ky mice may be affected.
We provide evidence that necdin deficiency—a universal presentation of PWS—leads to dysfunction of the LC-NE system. In addition to its roles in arousal and cognitive processes, the LC-NE system is the primary responder to stress in CNS (41, 45, 46). Chronic stress leads to long-lasting hyperactivity and increased sensitivity of LC neurons as well as the excessive activity of NE neurons under stress, which lead to stress-induced deficits in cognitive functions depending on the prefrontal cortex (46). LC hyper-responsiveness has been observed in patients with post-traumatic stress disorder (47), which manifests as significant arousal and emotional symptoms of angry outbursts. Such symptoms are also frequently found in patients with PWS (48). Moreover, psychiatric symptoms including temper tantrums and aggressive behaviors significantly interfere with the quality of life of patients with PWS (49).
There are some limitations to this study. First, we could not attain the causality between neonatal phenotypes and impaired LC-NE function in this observational study. Second, Ndntm1ky +m/-p mice were deficient in necdin in the whole body, and we cannot exclude the possibility that hypotonia and blunted ventilatory responses to hypercapnia were induced by myopathy or peripheral chemosensory failure. However, this is the first study to demonstrate LC-NE neuronal dysfunction in necdin-deficient mice, a widely used PWS animal model, and indicate that LC-NE dysfunction is possibly involved in the pathogenesis of some PWS symptoms. Using optogenetic or chemogenetic experiments, the cell type-specific neuronal firing activity can be modulated and the causality between neuronal activity and physiological function can be evaluated, but these approaches are difficult to use for neonatal phenotypes in the present study. Other psychiatric or behavioral phenotypes of PWS that develop in adolescence or adulthood, such as temper tantrums and aggressive behaviors, will be good targets to evaluate the causal relationship between the LC-NE dysfunction and PWS phenotypes.