Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are the only functional photoreceptive cells in the eye of newborn mice. Through postnatal day 9, in the absence of functional rods and cones, these ipRGCs mediate a robust avoidance behavior to a light source, termed negative phototaxis. To determine whether this behavior is associated with an aversive experience in neonatal mice, we characterized light-induced vocalizations and patterns of neuronal activation in regions of the brain involved in the processing of aversive and painful stimuli. Light evoked distinct melanopsin-dependent ultrasonic vocalizations identical to those emitted under stressful conditions, such as isolation from the litter. In contrast, light did not evoke the broad-spectrum calls elicited by acute mechanical pain. Using markers of neuronal activation, we found that light induced the immediate-early gene product Fos in the posterior thalamus, a brain region associated with the enhancement of responses to mechanical stimulation of the dura by light, and thought to be the basis for migrainous photophobia. Additionally, light induced the phosphorylation of extracellular-related kinase (pERK) in neurons of the central amygdala, an intracellular signal associated with the processing of the aversive aspects of pain. However, light did not activate Fos expression in the spinal trigeminal nucleus caudalis, the primary receptive field for painful stimulation to the head. We conclude that these light-evoked vocalizations and the distinct pattern of brain activation in neonatal mice are consistent with a melanopsin-dependent neural pathway involved in processing light as an aversive but not acutely painful stimulus.

The demyelinating peripheral neuropathy Charcot-Marie-Tooth type 4B (CMT4B) is characterized by axonal degeneration and myelin outfoldings. CMT4B results from mutations in either myotubularin-related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2), phosphoinositide (PI) 3-phosphatases that dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns(3,5)P2, lipids which regulate endo-lysosomal membrane traffic. The catalytically active MTMR2 and catalytically inactive MTMR13 physically associate, although the significance of this association is not well understood. Here we show that Mtmr13 loss leads to axonal degeneration in sciatic nerves of older mice. In addition, CMT4B2-like myelin outfoldings are present in Mtmr13(-/-) nerves at postnatal day 3. Thus, Mtmr13(-/-) mice show both the initial dysmyelination and later degenerative pathology of CMT4B2. Given the key role of PI 3-kinase-Akt signaling in myelination, we investigated the state of the pathway in nerves of CMT4B models. We found that Akt activation is unaltered in Mtmr13(-/-) and Mtmr2(-/-) mice. Mtmr2 and Mtmr13 are found within the Schwann cell cytoplasm, where the proteins are partially localized to punctate compartments, suggesting that Mtmr2-Mtmr13 may dephosphorylate their substrates on specific intracellular compartments. Mtmr2-Mtmr13 substrates play essential roles in endo-lysosomal membrane traffic. However, endosomes and lysosomes of Mtmr13(-/-) and Mtmr2(-/-) Schwann cells are morphologically indistinguishable from those of controls, indicating that loss of these proteins does not cause wholesale dysregulation of the endo-lysosomal system. Notably, Mtmr2 and Mtmr13 depend upon each other to achieve wild-type levels of protein expression. Mtmr2 stabilizes Mtmr13 on membranes, indicating that the Mtmr13 pseudophosphatase is regulated by its catalytically active binding partner.

The PI 3-kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3-phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34-Schwann cell knockout (Vps34SCKO) mice show severe hypomyelination in peripheral nerves. Vps34−/− Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34−/− Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34−/− Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic though endosomes. In Vps34SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to Nrg1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34SCKO mice.