Developmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal ...resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.
•During synapse elimination, retreating axon branches dismantle their microtubules•Microtubules are destabilized due to branch-specific severing•Microtubule stabilization delays axon branch removal during synapse elimination•The disease-associated microtubule severing protein spastin mediates microtubule loss
Brill, Kleele, Ruschkies et al. show that microtubule destabilization mediates dismantling of retreating axon branches and excludes them from organelle delivery by axonal transport. This mechanism is branch specific and involves the neurodegeneration-associated microtubule severing protein spastin.
The relationship between cardiorespiratory fitness (CRF) and mortality risk has typically been assessed using a single measurement, though some evidence suggests the change in CRF over time ...influences risk. This evidence is predominantly based on studies using estimated CRF (CRFe). The strength of this relationship using change in directly measured CRF over time in apparently healthy men and women is not well understood.
To examine the association of change in CRF over time, measured using cardiopulmonary exercise testing (CPX), with all-cause and disease-specific mortality and to compare baseline and subsequent CRF measurements as predictors of all-cause mortality.
Participants included 833 apparently healthy men and women (42.9 ± 10.8 years) who underwent two maximal CPXs, the second CPX being ≥1 year following the baseline assessment (mean 8.6 years, range 1.0 to 40.3 years). Participants were followed for up to 17.7 (SD 11.8) years for all-cause-, cardiovascular disease- (CVD), and cancer mortality. Cox-proportional hazard models were performed to determine the association between the change in CRF, computed as visit 1 (CPX1) peak oxygen consumption (VO2peak mL·kg−1·min−1) – visit 2 (CPX2) VO2peak, and mortality outcomes. A Wald-Chi square test of equality was used to compare the strength of CPX1 to CPX2 VO2peak in predicting mortality.
During follow-up, 172 participants died. Overall, the change in CPX-CRF was inversely related to all-cause, CVD, and cancer mortality (p < 0.05). Each 1 mL·kg−1·min−1 increase was associated with a ~11, 15, and 16% (all p < 0.001) reduction in all-cause, CVD, and cancer mortality, respectively. The inverse relationship between CRF and all-cause mortality was significant (p < 0.05) when men and women were examined independently, after adjusting for years since first CPX, baseline VO2peak, and age. Further, the Wald Chi-square test of equality found CPX2 VO2peak to be a significantly stronger predictor of all-cause mortality than CPX1 VO2peak (p < 0.05).
The change in CRF over time was inversely related to mortality outcomes, and mortality was better predicted by CRF measured at subsequent test than CPX1 CRF. These findings emphasize the importance of adopting lifestyle behaviors that promote CRF, as well as support the need for routine assessment of CRF in clinical practice to better assess risk.
In many parts of the developing nervous system, the number of axonal inputs to each postsynaptic cell is dramatically reduced. This synapse elimination has been extensively studied at the ...neuromuscular junction, but how axons are lost is unknown. Here, we combine time-lapse imaging of fluorescently labeled axons and serial electron microscopy to show that axons at neuromuscular junctions are removed by an unusual cellular mechanism. As axons disappear, they shed numerous membrane bound remnants. These “axosomes” contain a high density of synaptic organelles and are formed by engulfment of axon tips by Schwann cells. After this engulfment, the axosome's contents mix with the cytoplasm of the glial cell. Axosome shedding might underlie other forms of axon loss and may provide a pathway for interactions between axons and glia.
Clearance of cellular debris is a critical feature of the developing nervous system, as evidenced by the severe neurological consequences of lysosomal storage diseases in children. An important ...developmental process, which generates considerable cellular debris, is synapse elimination, in which many axonal branches are pruned. The fate of these pruned branches is not known. Here, we investigate the role of lysosomal activity in neurons and glia in the removal of axon branches during early postnatal life. Using a probe for lysosomal activity, we observed robust staining associated with retreating motor axons. Lysosomal function was involved in axon removal because retreating axons were cleared more slowly in a mouse model of a lysosomal storage disease. In addition, we found lysosomal activity in the cerebellum at the time of, and at sites where, climbing fibers are eliminated. We propose that lysosomal activity is a central feature of synapse elimination. Moreover, staining for lysosomal activity may serve as a marker for regions of the developing nervous system undergoing axon pruning.
Staphylococcus aureus is a leading causative agent in sepsis, endocarditis, and pneumonia. An emerging concept is that prognosis worsens when the infecting S. aureus strain has the capacity to not ...only colonize tissue as an extracellular pathogen, but to invade host cells and establish intracellular bacterial populations. In previous work, we identified host CDC42 as a central regulator of endothelial cell invasion by S. aureus. In the current work, we report that ML 141, a first-in-class CDC42 inhibitor, decreases invasion and resultant pathogenesis in a dose-dependent and reversible manner. Inhibition was found to be due in part to decreased remodeling of actin that potentially drives endocytic uptake of bacteria/fibronectin/integrin complexes. ML 141 decreased binding to fibronectin at these complexes, thereby limiting a key pathogenic mechanism used by S. aureus to invade. Structural analogs of ML 141 were synthesized (designated as the RSM series) and a subset identified that inhibit invasion through non-cytotoxic and non-bactericidal mechanisms. Our results support the development of adjunctive therapeutics targeting host CDC42 for mitigating invasive infection at the level of the host.
Patients on a statin regimen have a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus ...aureus, the most common etiologic agent of sepsis. Inhibition was due in part to depletion of isoprenoid intermediates within the cholesterol biosynthesis pathway and led to the cytosolic accumulation of the small GTPases CDC42, Rac, and RhoB. Actin stress fiber disassembly required for host invasion was attenuated by simvastatin and by the inhibition of phosphoinositide 3-kinase (PI3K) activity. PI3K relies on coupling to prenylated proteins, such as this subset of small GTPases, for access to membrane-bound phosphoinositide to mediate stress fiber disassembly. Therefore, we examined whether simvastatin restricts PI3K cellular localization. In response to simvastatin, the PI3K isoform p85, coupled to these small-GTPases, was sequestered within the cytosol. From these findings, we propose a mechanism whereby simvastatin restricts p85 localization, inhibiting the actin dynamics required for bacterial endocytosis. This approach may provide the basis for protection at the level of the host in invasive infections by S. aureus.
We describe a method for in vivo confocal fluorescence imaging of synaptic terminals and subsequent electron microscopic reconstructions of the same terminals. By iontophoretically applying ...lipophilic dye to nerve terminals at a single neuromuscular junction with a sharp microelectrode in living neonatal mice, we were able to quickly label other synaptic terminals of the same motor unit. This vital labeling technique allows the same synapses to be imaged in living animals for several days. By using two dyes applied to separate junctions we could visualize competing axons converging at the same site. We also show that similar approaches can be used to study synaptic inputs to neurons. Following photoconversion, the dye labeled axons and synapses were easily identified and distinguished from unlabeled synapses of other axons ultrastructurally. This new labeling technique thus provides a useful means to study reorganization of synaptic structure at high temporal and spatial resolution.
Mouse soleus muscles were denervated by crushing the soleus nerve where it enters the muscle to determine if denervation followed by self-reinnervation can permanently alter the mix of fiber types in ...a muscle. Reinnervated and contralateral control muscles were sectioned at 2 and 7 months postdenervation and histochemically stained for myosin ATPase to determine the percentages of fast and slow twitch fibers in the muscles. It was found that, at both 2 and 7 months postdenervation, reinnervated muscles had a significantly higher percentage of slow twitch fibers than did contralateral control muscles (86.7 versus 67.8% at 2 months and 90.0 versus 69.3% at 7 months). Soleus muscles were also denervated by crushing the soleus nerve where it exits the gastrocnemius muscle (approximately 4 mm proximal to where the nerve enters the soleus muscle) to ascertain if the location of the nerve lesion plays a role in the ultimate outcome of the process of self-reinnervation. Reinnervated muscles and their contralateral muscles were sectioned at 2 months postdenervation and histochemically stained for myosin ATPase as before. It was found that, in contrast to muscles denervated at the point of nerve entry, muscles denervated 4 mm more proximal exhibited only a small increase in their percentage of slow twitch fibers which was not statistically significant (71.4 versus 68.4%). These results suggest that denervation followed by self-reinnervation can cause a permanent change in a muscle's fiber type mix and that the location of the nerve lesion strongly influences the final outcome of the reinnervation process.
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