A Proteogenomic Approach to Understanding MYC Function in Metastatic Medulloblastoma Tumors.

Brain tumors are the leading cause of cancer-related deaths in children, and medulloblastoma is the most prevalent malignant childhood/pediatric brain tumor. Providing effective treatment for these cancers, with minimal damage to the still-developing brain, remains one of the greatest challenges faced by clinicians. Understanding the diverse events driving tumor formation, maintenance, progression, and recurrence is necessary for identifying novel targeted therapeutics and improving survival of patients with this disease. Genomic copy number alteration data, together with clinical studies, identifies c-MYC amplification as an important risk factor associated with the most aggressive forms of medulloblastoma with marked metastatic potential. Yet despite this, very little is known regarding the impact of such genomic abnormalities upon the functional biology of the tumor cell. We discuss here how recent advances in quantitative proteomic techniques are now providing new insights into the functional biology of these aggressive tumors, as illustrated by the use of proteomics to bridge the gap between the genotype and phenotype in the case of c-MYC-amplified/associated medulloblastoma. These integrated proteogenomic approaches now provide a new platform for understanding cancer biology by providing a functional context to frame genomic abnormalities.

Proteomic profiling of high risk medulloblastoma reveals functional biology.

Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior.

Cytoskeletal changes during development and aging in the cortex of neurofilament light protein knockout mice.

The neurofilament light (NFL) subunit is considered as an obligate subunit polymer for neuronal intermediate filaments comprising the neurofilament (NF) triplet proteins. We examined cytoskeletal protein levels in the cerebral cortex of NFL knockout (KO) mice at postnatal day 4 (P4), 5 months, and 12 months of age compared with age-matched wild-type (WT) mice of a similar genetic background (C57BL/6). The absence of NFL protein resulted in a significant reduction of phosphorylated and dephosphorylated NFs (NF-P, NF-DP), the medium NF subunit (NFM), and the intermediate filament α-internexin (INT) at P4. At 5 months, NF-DP, NFM, and INT remained significantly lower in knockouts. At 12 months, NF-P was again significantly decreased, and INT significantly increased, in KOs compared with wild type. In addition, protein levels of class III neuron-specific ß-tubulin and microtubule-associated protein 2 were significantly increased in NFL KO mice at P4, 5 months, and 12 months, whereas ß-actin levels were significantly decreased at P4. Immunocytochemical studies demonstrated that NF-DP accumulated abnormally in the perikarya of cortical neurons by 5 months of age in NFL KO mice. Neurons that lacked NF triplet proteins, such as calretinin-immunolabeled nonpyramidal cells, showed no alterations in density or cytoarchitectural distribution in NFL KO mice at 5 months relative to WT mice, although calretinin protein levels were decreased significantly after 12 months in NFL KO mice. These findings suggest that a lack of NFL protein alters the expression of cytoskeletal proteins and disrupts other NF subunits, causing intracellular aggregation but not gross structural changes in cortical neurons or cytoarchitecture. The data also indicate that changes in expression of other cytoskeletal proteins may compensate for decreased NFs.

Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice.

BACKGROUND: Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented. METHODS: In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4-6), adolescent animals (P25-28) and mature adults (P50+). Cultures were prepared using the membrane interface method. RESULTS: Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers. CONCLUSION: In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.

Selective vulnerability of non-myelinated axons to stretch injury in an in vitro co-culture system.

Diffuse axonal injury (DAI) is an evolving axonopathy commonly characterized clinically as widespread damage to the white matter tracts. In recent electrophysiological studies, researchers have proposed that myelinated and unmyelinated axons differ in their vulnerability and functional recovery following DAI. In this study we present for the first time an in vitro stretch-injury approach that utilizes a novel myelinating co-culture system to determine the differential response between myelinated and non-myelinated axon bundles to injury. In implementing this technique we demonstrate that myelinated axon bundles are less vulnerable to stretch injury compared to caliber-matched non-myelinated bundles. Interestingly, moderate axonal strain did not induce demyelination, but instead caused an increase in the proportion of degenerated myelin basic protein over time. Additionally, there were no significant differences in the expression of axonal swellings, which is indicative of disrupted axonal transport. In summary, we present an ideal in vitro model that permits further mechanistic investigations into the role of myelin and oligodendrocyte-neuron interactions in response to DAI.

Initial calcium release from intracellular stores followed by calcium dysregulation is linked to secondary axotomy following transient axonal stretch injury.

Acute axonal shear and stretch in the brain induces an evolving form of axonopathy and is a major cause of ongoing motor, cognitive and emotional dysfunction. We have utilized an in vitro model of mild axon bundle stretch injury, in cultured primary cortical neurons, to determine potential early critical cellular alterations leading to secondary axonal degeneration. We determined that transient axonal stretch injury induced an initial acute increase in intracellular calcium, principally derived from intracellular stores, which was followed by a delayed increase in calcium over 48 h post-injury (PI). This progressive and persistent increase in intracellular calcium was also associated with increased frequency of spontaneous calcium fluxes as well as cytoskeletal abnormalities. Additionally, at 48 h post-injury, stretch-injured axon bundles demonstrated filopodia-like sprout formation that preceded secondary axotomy and degeneration. Pharmacological inhibition of the calcium-activated phosphatase, calcineurin, resulted in reduced secondary axotomy (p < 0.05) and increased filopodial sprout length. In summary, these results demonstrate that stretch injury of axons induced an initial substantial release of calcium from intracellular stores with elevated intracellular calcium persisting over 2 days. These long-lasting calcium alterations may provide new insight into the earliest neuronal abnormalities that follow traumatic brain injury as well as the key cellular changes that lead to the development of diffuse axonal injury and secondary degeneration.

Axonopathy and cytoskeletal disruption in degenerative diseases of the central nervous system.

There has been growing interest in the axon as the initial focus of pathological change in a number of neurodegenerative diseases of the central nervous system. This review concentrates on three major neurodegenerative conditions--amyotrophic lateral sclerosis, multiple sclerosis and Alzheimer's disease--with emphasis on key cellular changes that may underlie early axonal dysfunction and pathology and, potentially, the degeneration of neurons. In particular, this review will address recent data that indicate that the main pathological stimuli for these conditions, though often not definitively determined, result in an initial perturbation of the axon and its cytoskeleton, which then results in slow neuronal degeneration and loss of connectivity. The identification of a degenerative process initiated in the axon may provide new therapeutic targets for early intervention to inhibit the grim outcomes related to the progression of these diseases.

Disruption of the ubiquitin proteasome system following axonal stretch injury accelerates progression to secondary axotomy.

The ubiquitin proteasome system (UPS) plays a vital role in the regulation of protein degradation. Ubiquitination of proteins has been implicated in the pathological cascade associated with neuronal degeneration in both neurodegenerative disease and following acquired central nervous system (CNS) injury. In the present study, we have investigated the role of the UPS following mild to moderate in vitro axonal stretch injury to mature primary cortical neurons, a model of the evolving axonal pathology characteristic of diffuse axonal injury following brain trauma. Transient axonal stretch injury in this model does not involve primary axotomy. However, delayed accumulation of ubiquitin in neuritic swellings at 48 h post-injury (PI) was present in axonal bundles, followed by approximately 60% of axonal bundles progressing to secondary axotomy at 72 h PI. This delayed accumulation of ubiquitin was temporally and spatially associated with cytoskeletal damage. Pharmacological inhibition of the UPS with both MG132 and lactacystin prior to axonal injury resulted in a significant (p < 0.05) increase in the number of axonal bundles progressing to secondary axotomy at 48 and 72 h PI. These results demonstrate that, following mild to moderate transient axonal stretch injury, UPS activity may assist structural reorganization within axons, potentially impeding secondary axotomy. Protein ubiquitination in the axon may therefore have a protective role relative to the diffuse axonal changes that follow traumatic brain injury.

Acute reactive and regenerative changes in mature cortical axons following injury.

Live-imaging brain slice techniques were utilized to study the acute changes in transected adult mammalian neocortical neuronal processes. Transected distal axons, but not axon segments directly emerging from the cell body or dendrites, undergo rapid morphological changes leading to attempted sprouting within hours after injury. The stereotypical response involved an initial retraction of the severed axon segments, followed by rapid stabilization. Subsequently, the cut-end underwent extensive swelling, forming large singular or multiple bulb-like structures. Two to three hours after transection, sprout-like protuberances emanated from the swollen bulbs. These axonal sprouts were highly dynamic, with many showing increased length over time and a capacity to change direction. These results indicate that damaged mature axons have an intrinsic capacity to react adaptively and attempt regeneration.

Mild axonal stretch injury in vitro induces a progressive series of neurofilament alterations ultimately leading to delayed axotomy.

We report a new model of transient axonal stretch injury involving pressurized fluid deflection of bundles of axons, resulting in a transient 1-6% increase in original axon length to investigate the slow progression of axonal alterations that are characteristic of diffuse axonal injury (DAI). We found no discernable difference in axon bundle morphology or cytoskeletal neurofilament protein arrangement between unstretched and stretched axonal bundles at 24 h post-injury. However, by 48 h post-injury, there was a stereotypical response of stretched axons involving characteristic neurofilament alterations that bear similarities to in vivo neuronal responses associated with DAI that have been reported previously. For instance, neurofilament protein immunoreactivity (SMI-312) was increased in axons contained within 51% of all injured axon bundles at 48 h compared to surrounding unstretched axon bundles, suggestive of neurofilament compaction. Furthermore, axonal bundle derangement occurred in 25% of injured axon bundles, with individual fibres segregating from each other and becoming undulating and wavy. By 72 h post-stretch, 70% of injured axon bundles underwent secondary axotomy, becoming completely severed at the site of initial stretch injury. While these results suggest a temporal series of stereotypical responses of axons to injury, we were able to distinguish very clear differences between mildly (100-103% increase in original axonal length) injured and strongly injured (106%+) axons. For instance, mildly injured axons developed increased neurofilament immunoreactivtity (SMI-312) within 48 h, and the marked development of ring-like neurofilament immunoreactive structures within axonal bundles, which were rarely axotomized. Conversely, at more severe strain levels increased neurofilament immunoreactivity was less apparent, while axons often became distorted and disorganised within axonal bundles and eventually became completely disconnected. Almost no ring-like neurofilament structures were observed in these severely injured axonal bundles. This suggests that axons do not respond in a stereotypical manner to a transient stretch insult, and indeed that variable degrees of stretch injury activate different responses within axons, with dramatically different outcomes. Hence, it is possible that the cytoskeletal characteristics that we have used in this study may be useful parameters for discriminating between mildly and severely injured axons following TBI.