In the adult human, perception of pain and temperature depends on stimulation of skin free nerve endings from nociceptive neurons in the dorsal root ganglion (DRG) that are characterized by their small size and unmyelinated peripheral and central neuronal processes. After damage to skin, uninjured nociceptive neurons sprout collateral branches that restore sensation to adjacent areas of damaged skin. In the previous project period, we found that disruption of peripherin intermediate filaments (IF) by expression of a dominant negative mutant peripherin gene inhibited the establishment of extensive neurite trees by these neurons when cultured in vitro. Similarly, disruption of IF limited the ability of these neurons to sprout collaterals to adjacent damaged skin in vivo, a finding that appears to echo the in vitro result. Further, knockout of the peripherin gene in mice results in proportionally fewer small-sized DRG neurons and a smaller number of central processes in the dorsal root. These findings indicate peripherin IF are crucial for differentiation and collateral sprouting of DRG neurons. To define the precise role of peripherin IF, the first Aim of this proposal focuses on determining whether loss of peripherin from birth in the knockout mouse results in actual death of small DRG neurons or simply an alteration in the proportion of these cells. In addition, we will determine whether loss of peripherin, like expression of mutant peripherin, critically affects branching of these neurons, this time in their central processes. Then, we will investigate one plausible reason why peripherin IF may be required for the sprouting, branching, and survival of neurons. In normal neurons, mitochondria are concentrated at growth cones and axonal branch points, where they are thought to provide local sources of energy essential for neurite outgrowth and branching. Defects in mitochondria distribution lead to neuronal degeneration. We have found that an intact IF network is essential for proper distribution of mitochondria in sensory neuronal processes. Similarly, in muscle, the IF protein desmin is critical for normal mitochondria! distribution and function. In muscle, IF are linked to mitochondria by a large protein called plectin. Based on our preliminary findings that that peripherin binds to plectin and that disruption of peripherin in sensory neurons causes defects in mitochondrial distribution, we will test the hypothesis that plectin mediates the interaction of peripherin IF with mitochondria and that peripherin has a critical role in distribution of mitochondria in sensory neurons. Defects in mitochondrial distribution could explain limitations on the length and collateralization of neuronal processes and, perhaps, death of nociceptors seen in the disrupter and knockout mice. These mice serve as models for human disease that could be induced by peripherin gene mutations.
