Regulation and function of axonal spheroids in the peripheral nervous system
Yong, Yu, Biology - Graduate School of Arts and Sciences, University of Virginia
Deppmann, Christopher, AS-Biology, University of Virginia
Axons are the primary transmission cables of the nervous system. In response to injury, a process of axonal fragmentation, or Wallerian degeneration occurs, often resulting in loss of neural function. After axon fragmentation, the swift dismantling and clearance of injured axons contributes to the restoration of nerve function. Degeneration of axons is one of the earliest events associated with degenerative disorders such as Alzheimer’s and Parkinson’s disease. Despite the importance of persevering axons as therapeutic strategies, very little is known about the mechanisms underlying axon degeneration. Beyond pathological situations, axon degeneration is also an important aspect of nervous system refinement during development. Recent studies have revealed that the absence of sufficient neurotrophic signaling permits prodestructive cues originating from tumor necrosis factor receptor (TNFR) superfamily members, such as p75 neurotrophic receptor (p75NTR) and death receptor 6 (DR6), to promote axon degeneration in peripheral nervous system (PNS). Moreover, previous work in our lab has shown that loss of DR6 delays Wallerian degeneration. However, the relative contributions of p75NTR and DR6 specifically to latent and catastrophic phases of axon degeneration in distinct etiologies have largely been overlooked. The key pathways related to developmental and Wallerian degeneration are reviewed in Chapter 1 of this thesis.
To identify the hallmarks of axon degeneration in distinct etiologies, I firstly determined the morphological changes and calcium dynamics in axons after trophic deprivation or axotomy. Using an in vitro microfluidic culture system and live imaging, I found that after trophic deprivation or axotomy, intra-axonal calcium increases before catastrophic degeneration. This is accompanied by the formation of calcium-rich spheroids that grow and then rupture, releasing their contents (≤10 kDa) to the extracellular space while allowing an influx of extracellular molecules into the intra-axonal space. Additionally, prodegenerative molecules (e.g., calcium) released into the extracellular space are capable of hastening entry of latent phase into the catastrophic phase of axon degeneration. Further, I show that in response to trophic deprivation, p75NTR promotes spheroid formation, intra-axonal calcium rise, and membrane rupture in a Rho-dependent manner. In contrast, DR6 is required for transition into the catastrophic phase in response to conditioned media from degenerating axons but not for spheroid formation or rupture. This finding places p75NTR and DR6 upstream and downstream of spheroidal rupture, respectively. Furthermore, this work supports the existence of an interaxonal degenerative signal that promotes catastrophic degeneration. These findings are described in Chapter 2 (published on J. Neuroscience) and Chapter 3 (published on Scientific Reports). The methods and materials used for these analyses are listed in Chapter 5 (partially published on Springer Protocols).
In conclusion, this work established the notion that DR6 and p75NTR play separable but interactive roles in regulating axon degeneration. It also revealed regulatory pathways for the formation of functionally important axonal spheroids. However, several open questions remain to be addressed such as, the identity of prodegenerative cues released by spheroid rupture, the in vivo effects of axonal spheroids in progression of neurodegenerative diseases, and the non-cell autonomous interaction of DR6 and p75NTR in degeneration. As the universal hallmark of degeneration, axonal spheroids and their formation mechanisms are critical for investigating the pathology of neurodegenerative disorders. The significance and future directions are discussed in Chapter 4.
PHD (Doctor of Philosophy)
Degeneration, Axonal spheroids, Death receptors