Alejandra Kun

RNA Trafficking in Axons

A substantial number of studies over a period of four decades have indicated that axons contain mRNAs and ribosomes, and are metabolically active in synthesizing proteins locally. For the most part, little attention has been paid to these findings until recently when the concept of targeting of specific mRNAs and translation in subcellular domains in polarized cells emerged to contribute to the likelihood and acceptance of mRNA targeting to axons as well. Trans‐acting factor proteins bind to cis‐acting sequences in the untranslated region of mRNAs integrated in ribonucleoprotein (RNPs) complexes determine its targeting in neurons. In vitro studies in immature axons have shown that molecular motors proteins (kinesins and myosins) associate to RNPs suggesting they would participate in its transport to growth cones. Tau and actin mRNAs are transported as RNPs, and targeted to axons as well as ribosomes. Periaxoplasmic ribosomal plaques (PARPs), which are systematically distributed discrete peripheral ribosome‐containing, actin‐rich formations in myelinated axons, also are enriched with actin and myosin Va mRNAs and additional regulatory proteins. The localization of mRNAs in PARPs probably means that PARPs are local centers of translational activity, and that these domains are the final destination in the axon compartment for targeted macromolecular traffic originating in the cell body. The role of glial cells as a potentially complementary source of axonal mRNAs and ribosomes is discussed in light of early reports and recent ultrastructural observations related to the possibility of glial‐axon trans‐endocytosis.

Neurofilament mRNAs are present and translated in the normal and severed sciatic nerve.

Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT‐PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT‐PCR demonstrated the presence of NF‐L, NF‐M and NF‐H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF‐L and NF‐M, but not NF‐H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF‐L and NF‐M mRNA in injured nerve, however, indicated that there was an up‐regulation in response to nerve injury. In situ hybridization showed that NF‐L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon. J. Neurosci. Res. 62:65–74, 2000.

Ribosomal distributions in axons of mammalian myelinated fibers

The distribution of ribosomes and polysomes in uninjured myelinated axons of rat sciatic nerve was analyzed. Ribosomes were identified by immunocytochemistry at the light and electron microscopic levels. A polyclonal antibody developed against ribosomes recognized both rRNA and ribosomal proteins. The distribution of the immunoreaction product was similar to that obtained with human anti‐ribosomal P protein. The immunoreaction product distributions were of two types in axons: 1) periodic localization in the cortical region of axoplasm that appeared as a compact structural aggregate, consistent with that described as a periaxoplasmic ribosomal plaques (PARP) domain (Koenig et al. [ 2000 ] J. Neurosci. 20:8390–8400), and 2) scattered small immuno‐reactive clusters of varying sizes (RNP) within the central core of the axon. The latter observation suggested the possibility that RNP‐like particles could be associated with the axonal transport system and in transit. Immunoreaction product was also associated with a novel structural inclusion, possibly multi‐vesicular in makeup that was located in the axon and at the myelin‐axon interface, and visible at the light and EM levels. The potential significance of this structural peculiarity is considered.

Myosin-Va-Dependent Cell-To-Cell Transfer of RNA from Schwann Cells to Axons

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

Glia to axon RNA transfer.

The existence of RNA in axons has been a matter of dispute for decades. Evidence for RNA and ribosomes has now accumulated to a point at which it is difficult to question, much of the disputes turned to the origin of these axonal RNAs. In this review, we focus on studies addressing the origin of axonal RNAs and ribosomes. The neuronal soma as the source of most axonal RNAs has been demonstrated and is indisputable. However, the surrounding glial cells may be a supplemental source of axonal RNAs, a matter scarcely investigated in the literature. Here, we review the few papers that have demonstrated that glial‐to‐axon RNA transfer is not only feasible, but likely. We describe this process in both invertebrate axons and vertebrate axons. Schwann cell to axon ribosomes transfer was conclusively demonstrated (Court et al. [2008]: J. Neurosci 28:11024–11029; Court et al. [2011]: Glia 59:1529–1539). However, mRNA transfer still remains to be demonstrated in a conclusive way. The intercellular transport of mRNA has interesting implications, particularly with respect to the integration of glial and axonal function. This evolving field is likely to impact our understanding of the cell biology of the axon in both normal and pathological conditions. Most importantly, if the synthesis of proteins in the axon can be controlled by interacting glia, the possibilities for clinical interventions in injury and neurodegeneration are greatly increased.

Myelinating and demyelinating phenotype of Trembler-J mouse (a model of Charcot-Marie-Tooth human disease) analyzed by atomic force microscopy and confocal microscopy.

The accumulation of misfolded proteins is associated with various neurodegenerative conditions. Mutations in PMP‐22 are associated with the human peripheral neuropathy, Charcot–Marie–Tooth Type 1A (CMT1A). PMP‐22 is a short‐lived 22 kDa glycoprotein, which plays a key role in the maintenance of myelin structure and compaction, highly expressed by Schwann cells. It forms aggregates when the proteasome is inhibited or the protein is mutated. This study reports the application of atomic force microscopy (AFM) as a detector of profound topographical and mechanical changes in Trembler‐J mouse (CMT1A animal model). AFM images showed topographical differences in the extracellular matrix and basal lamina organization of Tr‐J/+ nerve fibers. The immunocytochemical analysis indicated that PMP‐22 protein is associated with type IV collagen (a basal lamina ubiquitous component) in the Tr‐J/+ Schwann cell perinuclear region. Changes in mechanical properties of single myelinating Tr‐J/+ nerve fibers were investigated, and alterations in cellular stiffness were found. These results might be associated with F‐actin cytoskeleton organization in Tr‐J/+ nerve fibers. AFM nanoscale imaging focused on topography and mechanical properties of peripheral nerve fibers might provide new insights into the study of peripheral nervous system diseases. Copyright

Unravelling crucial biomechanical resilience of myelinated peripheral nerve fibres provided by the Schwann cell basal lamina and PMP22

There is an urgent need for the research of the close and enigmatic relationship between nerve biomechanics and the development of neuropathies. Here we present a research strategy based on the application atomic force and confocal microscopy for simultaneous nerve biomechanics and integrity investigations. Using wild-type and hereditary neuropathy mouse models, we reveal surprising mechanical protection of peripheral nerves. Myelinated peripheral wild-type fibres promptly and fully recover from acute enormous local mechanical compression while maintaining functional and structural integrity. The basal lamina which enwraps each myelinated fibre separately is identified as the major contributor to the striking fibres resilience and integrity. In contrast, neuropathic fibres lacking the peripheral myelin protein 22 (PMP22), which is closely connected with several hereditary human neuropathies, fail to recover from light compression. Interestingly, the structural arrangement of the basal lamina of Pmp22−/− fibres is significantly altered compared to wild-type fibres. In conclusion, the basal lamina and PMP22 act in concert to contribute to a resilience and integrity of peripheral nerves at the single fibre level. Our findings and the presented technology set the stage for a comprehensive research of the links between nerve biomechanics and neuropathies.

F-actin distribution at nodes of Ranvier and Schmidt-Lanterman incisures in mammalian sciatic nerves†

Very little is known about the function of the F‐actin cytoskeleton in the regeneration and pathology of peripheral nerve fibers. The actin cytoskeleton has been associated with maintenance of tissue structure, transmission of traction and contraction forces, and an involvement in cell motility. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and intracellular transport therein. In this work, we analyze the distribution of F‐actin at Schmidt‐Lanterman Incisures (SLI) and nodes of Ranvier (NR) domains in normal, regenerating and pathologic Trembler J (TrJ/+) sciatic nerve fibers, of rats and mice. F‐actin was quantified and it was found increased in TrJ/+, both in SLI and NR. However, SLI and NR of regenerating rat sciatic nerve did not show significant differences in F‐actin, as compared with normal nerves. Cytochalasin‐D and Latrunculin‐A were used to disrupt the F‐actin network in normal and regenerating rat sciatic nerve fibers. Both drugs disrupt F‐actin, but in different ways. Cytochalasin‐D did not disrupt Schwann cell (SC) F‐actin at the NR. Latrunculin‐A did not disrupt F‐actin at the boundary region between SC and axon at the NR domain. We surmise that the rearrangement of F‐actin in neurological disorders, as presented here, is an important feature of TrJ/+ pathology as a Charcot‐Marie‐Tooth (CMT) model.

Association of Myosin Va and Schwann cells-derived RNA in mammal myelinated axons, analyzed by immunocytochemistry and confocal FRET microscopy.

Evidence from multiple sources supports the hypothesis that Schwann cells in the peripheral nervous system transfer messenger RNA and ribosomes to the axons they ensheath. Several technical and methodological difficulties exist for investigators to unravel this process in myelinated axons - a complex two-cell unit. We present an experimental design to demonstrate that newly synthesized RNA is transferred from Schwann cells to axons in association with Myosin Va. The use of quantitative confocal FRET microscopy to track newly-synthesized RNA and determine the molecular association with Myosin Va, is described in detail.

Localization of mRNA in Vertebrate Axonal Compartments by In Situ Hybridization

The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demonstrate the importance of axon micro-dissection to obtain a whole mount perspective of mRNA distribution in the axonal territory. We describe a protocol to perform fluorescent ISH in isolated axons and guidelines for the preservation of structural and molecular integrity of cortical RNA-containing domains (e.g., Periaxoplasmic Ribosomal Plaques, or PARPs) in isolated axoplasm.