Kyle E. Miller

Axonal mitochondrial transport and potential are correlated

Disruption of axonal transport leads to a disorganized distribution of mitochondria and other organelles and is thought to be responsible for some types of neuronal disease. The reason for bidirectional transport of mitochondria is unknown. We have developed and applied a set of statistical methods and found that axonal mitochondria are uniformly distributed. Analysis of fast axonal transport showed that the uniform distribution arose from the clustering of the stopping events of fast axonal transport in the middle of the gaps between stationary mitochondria. To test whether transport was correlated with ATP production, we added metabolic inhibitors locally by micropipette. Whereas applying CCCP (a mitochondrial uncoupler) blocked mitochondrial transport, as has been previously reported, treatment with antimycin (an inhibitor of electron transport at complex III) caused increases in retrograde mitochondrial transport. Application of 2-deoxyglucose did not decrease transport compared with the mannitol control. To determine whether mitochondrial transport was correlated with mitochondrial potential, we stained the neurons with the mitochondrial potential-sensing dye JC-1. We found that ∼90% of mitochondria with high potential were transported towards the growth cone and ∼80% of mitochondria with low potential were transported towards the cell body. These experiments show for the first time that a uniform mitochondrial distribution is generated by local regulation of the stopping events of fast mitochondrial transport, and that the direction of mitochondrial transport is correlated with mitochondrial potential. These results have implications for axonal clogging, autophagy, apoptosis and Alzheimers disease.

Direct evidence for coherent low velocity axonal transport of mitochondria

Axonal growth depends on axonal transport. We report the first global analysis of mitochondrial transport during axonal growth and pauses. In the proximal axon, we found that docked mitochondria attached to the cytoskeletal framework that were stationary relative to the substrate and fast axonal transport fully accounted for mitochondrial transport. In the distal axon, we found both fast mitochondrial transport and a coherent slow transport of the mitochondria docked to the axonal framework (low velocity transport [LVT]). LVT was distinct from previously described transport processes; it was coupled with stretching of the axonal framework and, surprisingly, was independent of growth cone advance. Fast mitochondrial transport decreased and LVT increased in a proximodistal gradient along the axon, but together they generated a constant mitochondrial flux. These findings suggest that the viscoelastic stretching/creep of axons caused by tension exerted by the growth cone, with or without advance, is seen as LVT that is followed by compensatory intercalated addition of new mitochondria by fast axonal transport.

A physical model of axonal elongation: force, viscosity, and adhesions govern the mode of outgrowth.

Whether the axonal framework is stationary or moves is a central debate in cell biology. To better understand this problem, we developed a mathematical model that incorporates force generation at the growth cone, the viscoelastic properties of the axon, and adhesions between the axon and substrate. Using force-calibrated needles to apply and measure forces at the growth cone, we used docked mitochondria as markers to monitor movement of the axonal framework. We found coherent axonal transport that decreased away from the growth cone. Based on the velocity profiles of movement and the force applied at the growth cone, and by varying the attachment of the axonal shaft to the coverslip, we estimate values for the axial viscosity of the axon (3 x 10(6) +/- 2.4 x 10(6) Pa.s) and the friction coefficient for laminin/polyornithine-based adhesions along the axon (9.6 x 10(3) +/- 7.5 x 10(3) Pa.s). Our model suggests that whether axons elongate by tip growth or stretching depends on the level of force generation at the growth cone, the viscosity of the axon, and the level of adhesions along the axon.

Characterization of myosin V binding to brain vesicles.

Myosin II and V are important for the generation and segregation of subcellular compartments. We observed that vesicular myosin II and V were associated with the protein scaffolding of a common subset of vesicles by density sedimentation, electron microscopy, and immunofluorescence. Solubilization of either myosin II or V was caused by polyphosphates with the following efficacy at 10 mm: for myosin II ATP-Mg2+ = ATP = AMP-PNP (5′-adenylyl imidodiphosphate) > pyrophosphate = tripolyphosphate ≫ tetrapolyphosphate = ADP > cAMP = Mg2+; and for myosin V pyrophosphate = tripolyphosphate > ATP-Mg2+ = ATP = AMP-PNP ≫ ADP = tetrapolyphosphate > cAMP = Mg2+. Consequently, we suggest solubilization was not an effect of phosphorylation, hydrolysis, or disassociation of myosin from actin filaments. Scatchard analysis of myosin V binding to stripped dense vesicles showed saturable binding with a K m of 10 nm. Analysis of native vesicles indicates that these sites are fully occupied. Together, these data show there are over 100 myosin Vs/vesicle (100-nm radius). We propose that polyphosphate anions bind to myosin II and V and induce a conformational change that disrupts binding to a receptor.

Growth and elongation within and along the axon

Mechanical tension is a particularly effective stimulus for axonal elongation, but little is known about how it leads to the formation of new axon. To better understand this process, we examined the movement of axonal branch points, beads bound to the axon, and docked mitochondria while monitoring axonal width. We found these markers moved in a pattern that suggests elongation occurs by viscoelastic stretching and volume addition along the axon. To test the coupling between “lengthening” and “growth,” we measured axonal width while forcing axons to grow and then pause by controlling the tension applied to the growth cone or to the cell body. We found axons thinned during high rates of elongation and thickened when the growth cones were stationary. These findings suggest that forces cause lengthening because they stretch the axon and that growth occurs, in a loosely coupled step, by volume addition along the axon.

What is slow axonal transport

While the phenomenon of slow axonal transport is widely agreed upon, its underlying mechanism has been controversial for decades. There is now persuasive evidence that several different mechanisms could contribute to slow axonal transport. Yet proponents of different theories have been hesitant to explicitly integrate what were, at least initially, opposing models. We suggest that slow transport is a multivariate phenomenon that arises through mechanisms that minimally include: molecular motor-based transport of polymers and soluble proteins as multi-protein complexes; diffusion; and en bloc transport of the axonal framework by low velocity transport and towed growth (due to increases in body size). In addition to integrating previously described mechanisms of transport, we further suggest that only a subset of transport modes operate in a given neuron depending on the region, length, species, cell type, and developmental stage. We believe that this multivariate approach to slow axonal transport better explains its complex phenomenology: including its bi-directionality; the differing velocities of transport depending on cargo, as well differing velocities due to anatomy, cell type and developmental stage.

Impaired motoneuronal retrograde transport in two models of SBMA implicates two sites of androgen action

Spinal and bulbar muscular atrophy (SBMA) impairs motor function in men and is linked to a CAG repeat mutation in the androgen receptor (AR) gene. Defects in motoneuronal retrograde axonal transport may critically mediate motor dysfunction in SBMA, but the site(s) where AR disrupts transport is unknown. We find deficits in retrograde labeling of spinal motoneurons in both a knock-in (KI) and a myogenic transgenic (TG) mouse model of SBMA. Likewise, live imaging of endosomal trafficking in sciatic nerve axons reveals disease-induced deficits in the flux and run length of retrogradely transported endosomes in both KI and TG males, demonstrating that disease triggered in muscle can impair retrograde transport of cargo in motoneuron axons, possibly via defective retrograde signaling. Supporting the idea of impaired retrograde signaling, we find that vascular endothelial growth factor treatment of diseased muscles reverses the transport/trafficking deficit. Transport velocity is also affected in KI males, suggesting a neurogenic component. These results demonstrate that androgens could act via both cell autonomous and non-cell autonomous mechanisms to disrupt axonal transport in motoneurons affected by SBMA.

Cytoplasmic dynein pushes the cytoskeletal meshwork forward during axonal elongation

ABSTRACT During development, neurons send out axonal processes that can reach lengths hundreds of times longer than the diameter of their cell bodies. Recent studies indicate that en masse microtubule translocation is a significant mechanism underlying axonal elongation, but how cellular forces drive this process is unknown. Cytoplasmic dynein generates forces on microtubules in axons to power their movement through ‘stop-and-go’ transport, but whether these forces influence the bulk translocation of long microtubules embedded in the cytoskeletal meshwork has not been tested. Here, we use both function-blocking antibodies targeted to the dynein intermediate chain and the pharmacological dynein inhibitor ciliobrevin D to ask whether dynein forces contribute to en bloc cytoskeleton translocation. By tracking docked mitochondria as fiducial markers for bulk cytoskeleton movements, we find that translocation is reduced after dynein disruption. We then directly measure net force generation after dynein disruption and find a dramatic increase in axonal tension. Taken together, these data indicate that dynein generates forces that push the cytoskeletal meshwork forward en masse during axonal elongation.

Emerging Brain Morphologies from Axonal Elongation

Understanding the characteristic morphology of our brain remains a challenging, yet important task in human evolution, developmental biology, and neurosciences. Mathematical modeling shapes our understanding of cortical folding and provides functional relations between cortical wavelength, thickness, and stiffness. Yet, current mathematical models are phenomenologically isotropic and typically predict non-physiological, periodic folding patterns. Here we establish a mechanistic model for cortical folding, in which macroscopic changes in white matter volume are a natural consequence of microscopic axonal growth. To calibrate our model, we consult axon elongation experiments in chick sensory neurons. We demonstrate that a single parameter, the axonal growth rate, explains a wide variety of in vitro conditions including immediate axonal thinning and gradual thickness restoration. We embed our axonal growth model into a continuum model for brain development using axonal orientation distributions motivated by diffusion spectrum imaging. Our simulations suggest that white matter anisotropy—as an emergent property from directional axonal growth—intrinsically induces symmetry breaking, and predicts more physiological, less regular morphologies with regionally varying gyral wavelengths and sulcal depths. Mechanistic modeling of brain development could establish valuable relationships between brain connectivity, brain anatomy, and brain function.

Organ donation terminology: are we communicating life or death?

The continued functioning of organ transplantation depends on obtaining the permission of the next of kin. This communication process between medical or transplant professionals and a donor family hinges heavily on the understanding of certain critical terms like brain dead, life support, and transplantation. Communication issues in obtaining organ donation consent were examined, with particular focus on what are literally life-and-death decisions. Using an experimental simulation methodology, data are offered in support of the claim that much miscommunication occurs in such situations. Directions for improving such communication by allaying latent fears and more carefully defining crucial terminology are offered.