Delaminating myelin membranes help seal the cut ends of severed earthworm giant axons

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5–60 min post-transection in physiological salines and Ca²⁺-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20–100 μm from the cut end at 5–60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15–30 min post-transection. At 30–60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca²⁺-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca²⁺-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 945–960, 1997     

Depression-like behavior in the forced swimming test in PACAP-deficient mice: amelioration by the atypical antipsychotic risperidone

Axonal degeneration is a key component of many neurodegenerative diseases. Injured axons undergo a program of self-destruction termed Wallerian degeneration that is an active, well-regulated process. The pathways leading to axon fragmentation are uncharacterized, but experiments with wld ^s mutant mice led to the discovery that over-expression of NMN adenylyltransferase 1 or treatment with NAD⁺ can inhibit axonal degeneration. In this study, we show that the purine nucleosides adenosine and guanosine, but not inosine, inhibit injury-induced axonal degeneration in cultured dorsal root ganglia neurons. Axons can be preserved by adding adenosine within 6 h of the axonal injury. The presence of adenosine was required continuously after the injury to maintain axonal protection. Together these results suggest that adenosine does not alter the neuronal response to injury, but instead inhibits a local axonal pathway necessary for the commitment and/or execution of the axon destructive program.     

Calcium-activated proteolysis of neurofilament proteins in goldfish Mauthner axons

We have examined the proteolytic breakdown of neurofilament proteins (NFPs) in isolated Mauthner axoplasm (M-axoplasm). Documentation of proteolytic breakdown of NFPs in M-axoplasm is important because NFPs are not degraded in distal segments of severed Mauthner axons (M-axons) maintained in vivo for up to 62 days at 20°C. By incubating M-axoplasm with 2 mM calcium in vitro, we have demonstrated that M-axoplasm contains an endogenous calcium-activated neutral protease that degrades NFPs. This calcium-activated proteolysis of M-axoplasm NFPs produced novel bands on silver-stained gels. These novel bands were presumed to be NFP breakdown products because they reacted with antibodies to the α-intermediate filament antigen (anti-IFA) on immunoblots from these gels. Incubations of M-axoplasm with 2 mM calcium plus exogenous calpain produced novel bands similar to those observed for M-axoplasm incubated with 2 mM calcium. Incubations of M-axoplasm with 2m M calcium plus calpain inhibitors did not produce these novel bands. These in vitro data indicate that M-axoplasm contains calpain that degrades NFPs and produces novel bands similar to those observed from distal segments of severed M-axons maintained in vivo longer than 62 days postseverance. Factors that affect the activity of calpain or affect the ability of calpain to degrade NFPs could account for the delayed degradation of NFPs in distal segments of severed M-axons maintained in vivo. © 1995 John Wiley & Sons, Inc.     

Oligodendroglial modulation of fast axonal transport in a mouse model of hereditary spastic paraplegia

Oligodendrocytes are critical for the development of the plasma membrane and cytoskeleton of the axon. In this paper, we show that fast axonal transport is also dependent on the oligodendrocyte. Using a mouse model of hereditary spastic paraplegia type 2 due to a null mutation of the myelin Plp gene, we find a progressive impairment in fast retrograde and anterograde transport. Increased levels of retrograde motor protein subunits are associated with accumulation of membranous organelles distal to nodal complexes. Using cell transplantation, we show categorically that the axonal phenotype is related to the presence of the overlying Plp null myelin. Our data demonstrate a novel role for oligodendrocytes in the local regulation of axonal function and have implications for the axonal loss associated with secondary progressive multiple sclerosis.     

Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.     

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.     

Injury-Induced Inhibition of Bystander Neurons Requires dSarm and Signaling from Glia

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Heterophilic binding of L1 on unmyelinated sensory axons mediates Schwann cell adhesion and is required for axonal survival.

This study investigated the function of the adhesion molecule L1 in unmyelinated fibers of the peripheral nervous system (PNS) by analysis of L1- deficient mice. We demonstrate that L1 is present on axons and Schwann cells of sensory unmyelinated fibers, but only on Schwann cells of sympathetic unmyelinated fibers. In L1-deficient sensory nerves, Schwann cells formed but failed to retain normal axonal ensheathment. L1-deficient mice had reduced sensory function and loss of unmyelinated axons, while sympathetic unmyelinated axons appeared normal. In nerve transplant studies, loss of axonal-L1, but not Schwann cell-L1, reproduced the L1-deficient phenotype. These data establish that heterophilic axonal-L1 interactions mediate adhesion between unmyelinated sensory axons and Schwann cells, stabilize the polarization of Schwann cell surface membranes, and mediate a trophic effect that assures axonal survival.     

Broadening the therapeutic scope for rapamycin treatment

The role of autophagy in the degradation of aggregate-prone proteins has been well established. As a result, autophagy upregulation has become an attractive therapeutic strategy for the treatment of proteinopathies, a group of diseases caused by the accumulation of mutant misfolded proteins. We have previously shown that rapamycin attenuates the phenotype in a mouse model of Huntington disease when administered pre-symptomatically and have recently extended this to demonstrate the effectiveness of rapamycin in a transgenic mouse model of spinocerebellar ataxia type 3, a polyglutamine disorder caused by mutations in the ataxin-3 gene. Rapamycin, administered from the initial onset of disease signs, improves motor coordination and results in a decrease in the levels of soluble mutant ataxin-3 and protein aggregates in the brain.     

Axonal degeneration and regeneration: a mechanistic tug-of-war

Axonal degeneration is a common hallmark of both nerve injury and many neurodegenerative conditions, including motor neuron disease, glaucoma, and Parkinson's, Alzheimer's, and Huntington's diseases. Degeneration of the axonal compartment is distinct from neuronal cell death, and often precedes or is associated with the appearance of the symptoms of the disease. A complementary process is the regeneration of the axon, which is commonly observed following nerve injury in many invertebrate neurons and in a number of vertebrate neurons of the PNS. Important discoveries, together with innovative imaging techniques, are now paving the way towards a better understanding of the dynamics and molecular mechanisms underlying these two processes. In this study, I will discuss these recent findings, focusing on the balance between axonal degeneration and regeneration.     

ROCK2 is a major regulator of axonal degeneration,neuronal death and axonal regeneration in the CNS

The Rho/ROCK/LIMK pathway is central for the mediation of repulsive environmental signals in the central nervous system. Several studies using pharmacological Rho-associated protein kinase (ROCK) inhibitors have shown positive effects on neurite regeneration and suggest additional pro-survival effects in neurons. However, as none of these drugs is completely target specific, it remains unclear how these effects are mediated and whether ROCK is really the most relevant target of the pathway. To answer these questions, we generated adeno-associated viral vectors to specifically downregulate ROCK2 and LIM domain kinase (LIMK)-1 in rat retinal ganglion cells (RGCs) in vitro and in vivo. We show here that specific knockdown of ROCK2 and LIMK1 equally enhanced neurite outgrowth of RGCs on inhibitory substrates and both induced substantial neuronal regeneration over distances of more than 5 mm after rat optic nerve crush (ONC) in vivo. However, only knockdown of ROCK2 but not LIMK1 increased survival of RGCs after optic nerve axotomy. Moreover, knockdown of ROCK2 attenuated axonal degeneration of the proximal axon after ONC assessed by in vivo live imaging. Mechanistically, we demonstrate here that knockdown of ROCK2 resulted in decreased intraneuronal activity of calpain and caspase 3, whereas levels of pAkt and collapsin response mediator protein 2 and autophagic flux were increased. Taken together, our data characterize ROCK2 as a specific therapeutic target in neurodegenerative diseases and demonstrate new downstream effects of ROCK2 including axonal degeneration, apoptosis and autophagy.     

Amyloid fibrils of mammalian prion protein induce axonal degeneration in NTERA2-derived terminally differentiated neurons

Defects in axonal transport and synaptic dysfunctions are associated with early stages of several neurodegenerative diseases including Alzheimer's, Huntington's, Parkinson's, and prion diseases. Here, we tested the effect of full-length mammalian prion protein (rPrP) converted into three conformationally different isoforms to induce pathological changes regarded as early subcellular hallmarks of prion disease. We employed human embryonal teratocarcinoma NTERA2 cells (NT2) that were terminally differentiated into neuronal and glial cells and co-cultured together. We found that rPrP fibrils but not alpha-rPrP or soluble beta-sheet rich oligomers caused degeneration of neuronal processes. Degeneration of processes was accompanied by a collapse of microtubules and aggregation of cytoskeletal proteins, formation of neuritic beads, and a dramatic change in localization of synaptophysin. Our studies demonstrated the utility of NT2 cells as valuable human model system for elucidating subcellular events of prion pathogenesis, and supported the emerging hypothesis that defects in neuronal transport and synaptic abnormalities are early pathological hallmarks associated with prion diseases.