p75NTR and DR6 Regulate Distinct Phases of Axon Degeneration Demarcated by Spheroid Rupture.

The regressive events associated with trophic deprivation are critical for sculpting a functional nervous system. After nerve growth factor withdrawal, sympathetic axons derived from male and female neonatal mice maintain their structural integrity for ∼18 h (latent phase) followed by a rapid and near unison disassembly of axons over the next 3 h (catastrophic phase). Here we examine the molecular basis by which axons transition from latent to catastrophic phases of degeneration following trophic withdrawal. Before catastrophic degeneration, we observed an increase in intra-axonal calcium. This calcium flux is accompanied by p75 neurotrophic factor receptor-Rho-actin-dependent expansion of calcium-rich axonal spheroids that eventually rupture, releasing their contents to the extracellular space. Conditioned media derived from degenerating axons are capable of hastening transition into the catastrophic phase of degeneration. We also found that death receptor 6, but not p75 neurotrophic factor receptor, is required for transition into the catastrophic phase in response to conditioned media but not for the intra-axonal calcium flux, spheroid formation, or rupture that occur toward the end of latency. Our results support the existence of an interaxonal degenerative signal that promotes catastrophic degeneration among trophically deprived axons.SIGNIFICANCE STATEMENT Developmental pruning shares several morphological similarities to both disease- and injury-induced degeneration, including spheroid formation. The function and underlying mechanisms governing axonal spheroid formation, however, remain unclear. In this study, we report that axons coordinate each other's degeneration during development via axonal spheroid rupture. Before irreversible breakdown of the axon in response to trophic withdrawal, p75 neurotrophic factor receptor-RhoA signaling governs the formation and growth of spheroids. These spheroids then rupture, allowing exchange of contents ≤10 kDa between the intracellular and extracellular space to drive death receptor 6 and calpain-dependent catastrophic degeneration. This finding informs not only our understanding of regressive events during development but may also provide a rationale for designing new treatments toward myriad neurodegenerative disorders.

Transcytosis of TrkA leads to diversification of dendritic signaling endosomes.

The development of the peripheral nervous system relies on long-distance signaling from target organs back to the soma. In sympathetic neurons, this long-distance signaling is mediated by target derived Nerve Growth Factor (NGF) interacting with its axonal receptor, TrkA. This ligand receptor complex internalizes into what is commonly referred to as the signaling endosome which is transported retrogradely to the soma and dendrites to mediate survival signaling and synapse formation, respectively. The molecular identity of signaling endosomes in dendrites has not yet been determined. Here, we perform a detailed analysis of TrkA endosomal compartments and trafficking patterns. We find that signaling endosomes are not uniform but molecularly diversified into Rab7 (late endosome) and Rab11 (recycling endosome) populations in axons and dendrites in vitro and in the soma in vivo. Surprisingly, TrkA-NGF signaling endosomes in dendrites undergo dynamic trafficking events, including putative fusion and fission. Overall, we find that signaling endosomes do not remain as a singular endosomal subtype but instead exist in multiple populations that undergo dynamic endosomal trafficking events. These dynamic events might drive functional diversification of the signaling endosome.

The neurotrophin receptor signaling endosome: Where trafficking meets signaling.

Neurons are the largest cells in the body and form subcellular compartments such as axons and dendrites. During both development and adulthood building blocks must be continually trafficked long distances to maintain the different regions of the neuron. Beyond building blocks, signaling complexes are also transported, allowing for example, axons to communicate with the soma. The critical roles of signaling via ligand-receptor complexes is perhaps best illustrated in the context of development, where they are known to regulate polarization, survival, axon outgrowth, dendrite development, and synapse formation. However, knowing 'when' and 'how much' signaling is occurring does not provide the complete story. The location of signaling has a significant impact on the functional outcomes. There are therefore complex and functionally important trafficking mechanisms in place to control the precise spatial and temporal aspects of many signal transduction events. In turn, many of these signaling events affect trafficking mechanisms, setting up an intricate connection between trafficking and signaling. In this review we will use neurotrophin receptors, specifically TrkA and TrkB, to illustrate the cell biology underlying the links between trafficking and signaling. Briefly, we will discuss the concepts of how trafficking and signaling are intimately linked for functional and diverse signaling outputs, and how the same protein can play different roles for the same receptor depending on its localization. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

TrkA Bumps into Its Future Self.

In neurons, correct targeting of receptors to the axon is critical for cell survival and circuit formation. In this issue of Developmental Cell, Yamashita et al. (2017) report that the ER-resident phosphatase PTP1B is required to prime TrkA for axonal transport.

The related neuronal endosomal proteins NEEP21 (Nsg1) and P19 (Nsg2) have divergent expression profiles in vivo.

Endosomal maturation and transport constitutes a complex trafficking system present in all cell types. Neurons have adapted their endosomal system to meet their unique and complex needs. These adaptations include repurposing existing proteins to diversify endocytosis and trafficking, as well as preferential expression of certain regulators more highly in neurons than other cell types. These neuronal regulators include the family of Neuron-Specific Gene family members (Nsg), NEEP21 (Nsg1), and P19 (Nsg2). NEEP21/Nsg1 plays a role in the trafficking of multiple receptors, including the cell adhesion molecule L1/NgCAM, the neurotransmitter receptor GluA2, and ß-APP. Recently, we showed that NEEP2/Nsg1 and P19/Nsg2 are not expressed in all neuronal cell types in vitro. However, it is not known where and when NEEP21/Nsg1 and P19/Nsg2 are expressed in vivo, and whether both proteins are always coexpressed. Here, we show that NEEP21/Nsg1 and P19/Nsg2 are present in both overlapping and distinct cell populations in the hippocampus, neocortex, and cerebellum during development. NEEP21/Nsg1 and P19/Nsg2 levels are highest during embryonic development, and expression persists in the juvenile mouse brain. In particular, a subset of layer V cortical neurons retains relatively high expression of both NEEP21/Nsg1 and P19/Nsg2 at postnatal day 16 as well as in the CA1-3 regions of the hippocampus. In the cerebellum, NEEP21/Nsg1 expression becomes largely restricted to Purkinje neurons in adulthood whereas P19/Nsg2 expression strikingly disappears from the cerebellum with age. This divergent and restricted expression likely reflects differential needs for this class of trafficking regulators in different neurons during different stages of maturation.

Minor Changes in Expression of the Mismatch Repair Protein MSH2 Exert a Major Impact on Glioblastoma Response to Temozolomide.

Glioblastoma (GBM) is often treated with the cytotoxic drug temozolomide, but the disease inevitably recurs in a drug-resistant form after initial treatment. Here, we report that in GBM cells, even a modest decrease in the mismatch repair (MMR) components MSH2 and MSH6 have profound effects on temozolomide sensitivity. RNAi-mediated attenuation of MSH2 and MSH6 showed that such modest decreases provided an unexpectedly strong mechanism of temozolomide resistance. In a mouse xenograft model of human GBM, small changes in MSH2 were sufficient to suppress temozolomide-induced tumor regression. Using The Cancer Genome Atlas to analyze mRNA expression patterns in tumors from temozolomide-treated GBM patients, we found that MSH2 transcripts in primary GBM could predict patient responses to initial temozolomide therapy. In recurrent disease, the absence of microsatellite instability (the standard marker for MMR deficiency) suggests a lack of involvement of MMR in the resistant phenotype of recurrent disease. However, more recent studies reveal that decreased MMR protein levels occur often in recurrent GBM. In accordance with our findings, these reported decreases may constitute a mechanism by which GBM evades temozolomide sensitivity while maintaining microsatellite stability. Overall, our results highlight the powerful effects of MSH2 attenuation as a potent mediator of temozolomide resistance and argue that MMR activity offers a predictive marker for initial therapeutic response to temozolomide treatment.