ALS-associated TBK1 variant p.G175S is defective in phosphorylation of p62 and impacts TBK1-mediated signalling and TDP-43 autophagic degradation.

Mutations affecting SQSTM1 coding for p62 and TANK-Binding Kinase 1 (TBK1) have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TBK1 is a serine-threonine kinase that regulates p62's activity as an autophagy receptor via phosphorylation and also has roles in neuroinflammatory signalling pathways. The mechanisms underlying ALS and FTLD pathogenesis as a result of TBK1 mutations are incompletely understood, however, loss of TBK1 function can lead to dysregulated autophagy and mitophagy. Here, we report that an ALS-associated TBK1 variant affecting the kinase domain, p.G175S, is defective in phosphorylation of p62 at Ser-403, a modification critical for regulating its ubiquitin-binding function, as well as downstream phosphorylation at Ser-349. Consistent with these findings, expression of p.G175S TBK1 was associated with decreased induction of autophagy compared to wild type and reduced degradation of the ALS-linked protein TDP-43. Expression of wild type TBK1 increased NF-κB signalling ~300 fold in comparison to empty vector cells, whereas p.G175S TBK1 was unable to promote NF-κB signalling above levels observed in empty vector transfected cells. We also noted a hitherto unknown role for TBK1 as a suppressor of oxidative stress (Nrf2) signalling and show that p.G175S TBK1 expressing cells lose this inhibitory function. Our data suggest that TBK1 ALS mutations may broadly impair p62-mediated cell signalling, which ultimately may reduce neuronal survival, in addition TDP-43 was not efficiently degraded, together these effects may contribute to TBK1 mutation associated ALS and FTLD pathogenesis.

Ontogenetic allometry and architectural properties of the paravertebral and hindlimb musculature in Eastern cottontail rabbits (Sylvilagus floridanus): functional implications for developmental changes in locomotor performance.

Due to small body size, an immature musculoskeletal system, and other growth-related limits on performance, juvenile mammals frequently experience a greater risk of predation than their adult counterparts. As a result, behaviorally precocious juveniles are hypothesized to exhibit musculoskeletal advantages that permit them to accelerate rapidly and evade predation. This hypothesis was tested through detailed quantitative evaluation of muscle growth in wild Eastern cottontail rabbits (Sylvilagus floridanus). Cottontail rabbits experience high rates of mortality during the first year of life, suggesting that selection might act to improve performance in growing juveniles. Therefore, it was predicted that muscle properties associated with force and power capacity should be enhanced in juvenile rabbits to facilitate enhanced locomotor performance. We quantified muscle architecture from 24 paravertebral and hindlimb muscles across ontogeny in a sample of n = 29 rabbits and evaluated the body mass scaling of muscle mass (MM), physiological cross-sectional area (PCSA), isometric force (Fmax ), and instantaneous power (Pinst ), along with several dimensionless architectural indices. In contrast to our hypothesis, MM and PCSA for most muscles change with positive allometry during growth by scaling at Mb1.3 and Mb1.1 , respectively, whereas Fmax and Pinst generally scale indistinguishably from isometry, as do the architectural indices tested. However, scaling patterns indicate that the digital flexors and ankle extensors of juvenile S. floridanus have greater capacities for force and power, respectively, than those in adults, suggesting these muscle properties may be a part of several compensatory features that promote enhanced acceleration performance in young rabbits. Overall, our study implies that body size constraints place larger, more mature rabbits at a disadvantage during acceleration, and that adults must develop hypertrophied muscles in order to maintain mechanical similarity in force and power capacities across development. These findings challenge the accepted understanding that juvenile animals are at a performance detriment relative to adults. Instead, for prey-predator interactions necessitating short intervals of high force and power generation relative to body mass, as demonstrated by rapid acceleration of cottontail rabbits fleeing predators, it may be the adults that struggle to keep pace with juveniles.

Shorter heels are linked with greater elastic energy storage in the Achilles tendon.

Previous research suggests that the moment arm of the m. triceps surae tendon (i.e., Achilles tendon), is positively correlated with the energetic cost of running. This relationship is derived from a model which predicts that shorter ankle moment arms place larger loads on the Achilles tendon, which should result in a greater amount of elastic energy storage and return. However, previous research has not empirically tested this assumed relationship. We test this hypothesis using an inverse dynamics approach in human subjects (n = 24) at speeds ranging from walking to sprinting. The spring function of the Achilles tendon was evaluated using specific net work, a metric of mechanical energy production versus absorption at a limb joint. We also combined kinematic and morphological data to directly estimate tendon stress and elastic energy storage. We find that moment arm length significantly determines the spring-like behavior of the Achilles tendon, as well as estimates of mass-specific tendon stress and elastic energy storage at running and sprinting speeds. Our results provide support for the relationship between short Achilles tendon moment arms and increased elastic energy storage, providing an empirical mechanical rationale for previous studies demonstrating a relationship between calcaneal length and running economy. We also demonstrate that speed and kinematics moderate tendon performance, suggesting a complex relationship between lower limb geometry and foot strike pattern.

p62 overexpression induces TDP-43 cytoplasmic mislocalisation, aggregation and cleavage and neuronal death.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) that exist on a spectrum of neurodegenerative disease. A hallmark of pathology is cytoplasmic TDP-43 aggregates within neurons, observed in 97% of ALS cases and ~ 50% of FTLD cases. This mislocalisation from the nucleus into the cytoplasm and TDP-43 cleavage are associated with pathology, however, the drivers of these changes are unknown. p62 is invariably also present within these aggregates. We show that p62 overexpression causes TDP-43 mislocalisation into cytoplasmic aggregates, and aberrant TDP-43 cleavage that was dependent on both the PB1 and ubiquitin-associated (UBA) domains of p62. We further show that p62 overexpression induces neuron death. We found that stressors (proteasome inhibition and arsenic) increased p62 expression and that this shifted the nuclear:cytoplasmic TDP-43 ratio. Overall, our study suggests that environmental factors that increase p62 may thereby contribute to TDP-43 pathology in ALS and FTLD.