Microtubule targeting agents (MTAs) often lead to treatment limiting and life threatening side effects, including chemotherapy-induced peripheral neuropathy (CIPN). The frequency of severe CIPN varies among different MTAs. Since the microtubule binding interactions and mechanisms of action also vary among MTAs, we hypothesized that these distinct mechanisms may underlie the variability in frequency of severe CIPN. Using a two-week, maximum tolerated dose model, we morphologically and biochemically analyzed sciatic nerves from mice treated with either paclitaxel or eribulin. These drugs differ in their manner of microtubule binding and mechanisms of action and reports indicate paclitaxel also induces a higher frequency of severe CIPN than does eribulin. Morphologically, paclitaxel increased the frequency of observed signs of axon degeneration more significantly than did eribulin. Alternatively, eribulin but not paclitaxel induced occasional myelin "halo" structures. Biochemically, paclitaxel, and eribulin both induced α-tubulin expression (~1.9- and ~2.5-fold, respectively) and tubulin acetylation, a marker for microtubule stability, (~5- and ~11.7-fold, respectively). Eribulin but not paclitaxel-induced EB1 expression ~2.2-fold while paclitaxel but not eribulin mildly suppressed EB3 expression. Both EB proteins are associated with microtubule growth. Eribulin's combination of relatively mild deleterious morphological effects coupled with more potent biochemical changes promoting microtubule stability and growth in mice correlate with lower frequencies of severe CIPN in humans. We suggest that these eribulin-induced effects create a relatively stable microtubule network that compensates, in part, for the toxic anti-cancer effects of the drug, leading to fewer reported incidences of CIPN than for paclitaxel.
Despite extensive structure-function analyses, the molecular mechanisms of normal
and pathological tau action remain poorly understood. How does the C-terminal
microtubule-binding region regulate microtubule dynamics and bundling? In what biophysical
form does tau transfer trans-synaptically from one neuron to another, promoting
neurodegeneration and dementia? Previous biochemical/biophysical work led to the
hypothesis that tau can dimerize via electrostatic interactions between two N-terminal
“projection domains” aligned in an anti-parallel fashion, generating a
multivalent complex capable of interacting with multiple tubulin subunits. We sought to
test this dimerization model directly. Native gel analyses of full-length tau and deletion
constructs demonstrate that the N-terminal region leads to multiple bands, consistent with
oligomerization. Ferguson analyses of native gels indicate that an N-terminal fragment
(tau45-230) assembles into heptamers/octamers. Ferguson analyses of
denaturing gels demonstrates that tau45-230 can dimerize even in SDS. AFM
reveals multiple levels of oligomerization by both full-length tau and
tau45-230. Finally, ion-mobility mass spectroscopic analyses of
tau106-144, a small peptide containing the core of the hypothesized
dimerization region, also demonstrate oligomerization. Thus, multiple independent
strategies demonstrate that the N-terminal region of tau can mediate higher-order
oligomerization, which may have important implications for both normal and pathological
tau action.
Recombinant expression of proteins of interest in Escherichia coli is an important tool in the determination of protein structure. However, lack of expression and insolubility remain significant challenges to the expression and crystallization of these proteins. The SSGCID program uses a wheat germ cellfree expression system as a rescue pathway for proteins that are either not expressed or insoluble when produced in E. coli. Testing indicates that the system is a valuable tool for these protein targets. Further increases in solubility were obtained by the addition of the NVoy polymer reagent to the reaction mixture. These data indicate that this eukaryotic cell-free expression system has a high success rate and that the addition of specific reagents can increase the yield of soluble protein.
Chemotherapy induced peripheral neuropathy (CIPN) is a common side effect of anti-cancer treatment with microtubule targeted agents (MTAs). The frequency of severe CIPN, which can be dose limiting and even life threatening, varies widely among different MTAs. For example, paclitaxel induces a higher frequency of severe CIPN than does eribulin. Different MTAs also possess distinct mechanisms of microtubule-targeted action. Recently, we demonstrated that paclitaxel and eribulin differentially affect sciatic nerve axons, with paclitaxel inducing more pronounced neurodegenerative effects and eribulin inducing greater microtubule stabilizing biochemical effects. Here, we complement and extend these axonal studies by assessing the effects of paclitaxel and eribulin in the cell bodies of sciatic nerve axons, housed in the dorsal root ganglia (DRG). Importantly, the microtubule network in cell bodies is known to be significantly more dynamic than in axons. Paclitaxel induced ATF3 expression, a marker of neuronal stress/injury. Paclitaxel also increased expression levels of acetylated tubulin and EB1, markers of microtubule stability and growth, respectively. These effects are hypothesized to be detrimental to the dynamic microtubule network within the cell bodies. In contrast, eribulin had no significant effect on any of these parameters in the cell bodies. Taken together, DRG cell bodies and their axons, two distinct neuronal cell compartments, contain functionally distinct microtubule networks that exhibit unique biochemical responses to different MTA treatments. We hypothesize that these distinct mechanistic actions may underlie the variability seen in the initiation, progression, persistence and recovery from CIPN.
Aggregates of Aβ peptide and the microtubule-associated protein tau are key molecular hallmarks of Alzheimer’s disease (AD). However, the interaction between these two pathologies and the mechanisms underlying disease progression have remained unclear. Numerous failed clinical trials suggest the necessity for greater mechanistic understanding in order to refine strategies for therapeutic discovery and development. To this end, we have generated a transgenic Caenorhabditis elegans model expressing both human Aβ1-42 peptide and human tau protein pan-neuronally. We observed exacerbated behavioral dysfunction and age-dependent neurodegenerative changes in the Aβ;tau transgenic animals. Further, these changes occurred in the Aβ;tau transgenic animals at greater levels than worms harboring either the Aβ1-42 or tau transgene alone and interestingly without changes to the levels of tau expression, phosphorylation or aggregation. Functional changes were partially rescued with the introduction of a genetic suppressor of tau pathology. Taken together, the data herein support a synergistic role for both Aβ and tau in driving neuronal dysfunction seen in AD. Additionally, we believe that the utilization of the genetically tractable C. elegans model will provide a key resource for dissecting mechanisms driving AD molecular pathology.
Chemotherapy-induced peripheral neuropathy is a common, dose-limiting side effect of cancer treatment. This conference was the first of its kind to bring together a wide range of clinicians, researchers, and industry professionals to address the potential causes, preventions, and treatments for this drug toxicity. Intraepidermal nerve fiber loss, axonal degeneration, immune cell infiltration, alterations in tubulin protein expression and microtubule stability, axonal transport, and mitochondrial dysfunction were addressed as possible mechanisms. Problems with animal models of the disease were discussed, as well as the potential of patient-derived induced sensory neurons to serve as a novel in vitro model. Cancer Res; 75(18); 3696-8. Ó2015 AACR.
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