Opposite rheological properties of neuronal microcompartments predict axonal vulnerability in brain injury

OPEN SUBJECT AREAS: BIOPHYSICS BIOLOGICAL PHYSICS Opposite rheological properties of neuronal microcompartments predict axonal vulnerability in brain injury Thomas Grevesse1, Borna E. Dabiri2, Kevin Kit Parker2 & Sylvain Gabriele1 Received 30 November 2014 Accepted 5 March 2015 Published 30 March 2015 Correspondence and requests for materials should be addressed to S.G. (sylvain. . be) 1 Mechanobiology & Soft Matter Group, Interfaces and Complex Fluids Laboratory, Research Institute for Biosciences, CIRMAP, University of Mons, 20 Place du Parc B-7000 Mons, Belgium, 2Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02138, USA. Although pathological changes in axonal morphology have emerged as important features of traumatic brain injury (TBI), the mechanical vulnerability of the axonal microcompartment relative to the cell body is not well understood. We hypothesized that soma and neurite microcompartments exhibit distinct mechanical behaviors, rendering axons more sensitive to a mechanical injury. In order to test this assumption, we combined protein micropatterns with magnetic tweezer rheology to probe the viscoelastic properties of neuronal microcompartments. Creep experiments revealed two opposite rheological behaviors within cortical neurons: the cell body was soft and characterized by a solid-like response, whereas the neurite compartment was stiffer and viscous-like. By using pharmacological agents, we demonstrated that the nucleus is responsible for the solid-like behavior and the stress-stiffening response of the soma, whereas neurofilaments have a predominant contribution in the viscous behavior of the neurite. Furthermore, we found that the neurite is a mechanosensitive compartment that becomes softer and adopts a pronounced viscous state on soft matrices. Together, these findings highlight the importance of the regionalization of mechanical and rigidity-sensing properties within neuron microcompartments in the preferential damage of axons during traumatic brain injury and into potential mechanisms of axonal outgrowth after injury. M icrocompartments are an essential design feature in mammalian cells. For instance, motile cells use filopodia and lamellipodia to probe their mechanochemical environment and to orient their movement1,2, while cilia at the tip of ciliated cells are essential for sweeping the mucus and foreign particles out of the lung and trachea3. Compartmentalization is also prominent in neuronal function: neurons possess cablelike microcompartments (dendrites and axons) that propagate information in the form of action potentials, whereas the neuronal body microcompartment (soma) houses most of the genetic content and is the site of a large part of the protein synthesis. This compartmentalization is especially relevant in understanding the cellular manifestations of traumatic brain injury (TBI). Currently, it is proposed that the initial event in TBI is the pathological strain of axons as the result of an inertial loading4. This mechanical deformation is thought to damage the internal structure of axons causing diffuse axonal injury (DAI), which is one of the most common and important pathological features of TBI5,6. To date, a unifying model of axonal degeneration considers that nerve insults lead to impaired expression of a local axonal survival factor, which results in increased intra-axonal calcium levels and calcium-dependent cytoskeletal breakdown7. Damage to neurofilaments and microtubules typical of axonal focal swellings can arise from stress-induced cell membrane poration, leading to Ca21 ion entry and subsequent activation of calpains that degrade proteins non-specifically8. Alternatively, integrins, which are transmembrane proteins that physically couple the neuronal cytoskeleton to the extracellular matrix9 (ECM), have been shown to be an important contributor to DAI by propagating mechanical forces through the cytoskeleton10. In contrast, the soma is seemingly unaffected by mechanical insult. Although several reports have indicated shrunken somas11 with pycnotic nuclei (i.e. condensation of chromatin leading to a shrunken nucleus) or DNA damage12 after brain injury, important differences in the rate of degeneration between soma and cell processes must be taken into account. Indeed, prominent axonal pathology often precedes cell body loss that arises from the gradual degeneration of axons toward the cell body. Central to understanding the induction of axonal pathology is deciphering the mechanical vulnerability of the axonal microcompartment over the cellular body. SCIENTIFIC REPORTS | 5 : 9475 | DOI: 10.1038/srep09475 1 www.nature.com/scientificreports Figure 1 | Rheological characterization of cortical neuronal microcompartments. (A) Immunofluorescence image of microtubules (red) and nucleus (blue) of a bipolar neuron adhering to 10 mm wide laminin lines (green). The scale bar is 20 mm. (B) Schematic description of the experimental setup for probing rheological properties of neuronal microcompartments with magnetic tweezers. Individual cortical neurons were grown on LM stripes (green) to impose a reproducible bipolar morphology. The inset DIC image depicts a bipolar neuron with magnetic beads attached to the neurite and the soma (white arrowheads). The scale bar is 15 mm. (C) Temporal evolution of a typical bead displacement curve (blue data) in response to a single force protocol (red line). (D) Logarithmic representation of the creep function J(t) obtained from the bead displacement d(t). Gray dashed lines represent solidlike (b 5 0) and fluid-like (b 5 1) behaviors. We hypothesized that specific cytoskeletal organization within neuronal microcompartments may lead to distinct rheological properties that potentiate a greater vulnerability of axons to injury. To test this, we combined micropatterning with magnetic tweezers to apply local stresses to individual microcompartments of bipolar neurons. We found that the rheological behaviors of soma and neurite were dominated by elastic and viscous properties, respectively. Mechanical testing of neuronal microcompartments treated with pharmacological agents causing specific cytoskeletal disruption further indicated that neurofilaments and microtubules were the principal mechanical load bearing elements of the neurite, whereas the rheology of the soma was dominated by the nucleus. Furthermore, we assessed whether the rheological properties of both neuronal microcompartments can be affected by stiffness changes of their microenvironment, as observed in many injury-related pathological responses. We found that the neurite compartment tuned its internal stiffness to match the compliance of the substrate and adopted a pronounced viscous state on soft microenvironments. In contrast, the cell body was insensitive to matrix stiffness changes and remained (...truncated)