Neuropathology of ALS: Spheroids

I briefly review spheroids observed in the anterior horns of the spinal cord in amyotrophic lateral sclerosis (ALS). Spheroids are argentophilic bodies more than 20 μm in diameter. Recently, some connections between the proximal axonal swellings including spheroids and the perikarya have been reported in some ALS patients with a short clinical course or mild depletion of anterior horn neurons. Most of the cell bodies directly connected with the axonal swellings appear normal, and spheroids are considered to be one of the hallmarks of the early histological changes in this disorder. Spheroids are strongly positive with anti-phosphorylated neurofilament antibody, and are also positive with calcitonin gene-related peptide and anti-peripherin antibody. Some spheroids are immunostained with anti-synaptophysin antibody and anti-ubiquitin antibody. Spheroids are not immunostained with anti-phosphorylated tau antibody, or high molecular weight microtubule associated proteins. Electron microscopically, spheroids are usually composed of densely packed accumulation of 10 nm neurofilaments with a variety of orientations, plus vesicles, dense bodies and mitochondria. When the swellings of the initial segment is relatively pronounced, the undercoating is obscured and the neurofilaments become interwoven in some parts. In the first internode of the myelinated axons, as the swellings become larger, the neurofilaments lose their parallel orientation and become intermingled. Large accumulation of neurofilaments resembling spheroids in the perikarya of large anterior horn cells suggests that spheroids could be derived not only from the axon including the proximal portion, but also from the perikarya. Structures apparently identical to axonal spheroids are observed at the light and electron microscopic levels in the proximal portion of axons of anterior horn cells in animal models intoxicated with β, β'-iminodipropionitrile (IDPN), or with aluminum, in hereditary canine spinal muscular atrophy (HCSMA). The pathogenetic mechanism is probably associated with an impairment in slow axonal transport which particularly affects the neurofilaments in IDPN and aluminum intoxication. Impairment of slow axonal transport of neurofilaments also plays an important role in the pathogenesis of ALS. The average diameter of even normalappearing initial segment is larger in ALS than in the controls. The perikarya connected with the swollen proximal axons and their dendrites almost always appear normal. These findings suggest that the slow axonal transport of neurofilaments is probably impaired in this portion of the axon at an early stage in ALS as well as animal models for human ALS. However, techniques to analyze slow axonal transport in humans still remain tobe developed. Recently, overexpression of neurofilament subunits in transgenic mice produces a condition resembling ALS. The transgenic model may offer an interesting perspective not only for testing therapeutic strategies but also for investigating in a systematic way the various genetic and environment factors controlling the onset and progression of the disease and might yield new insights on the etiology of ALS.     

Changes in glial fibrillary acidic protein mRNA expression after corticospinal axotomy in the adult hamster

We examined changes in the expression of glial fibrillary acidic protein (GFAP) mRNA during Wallerian degeneration in the corticospinal system of the adult Golden hamster following axotomy. GFAP is the product of a type III intermediate filament (IF) gene that is expressed specifically in mature astrocytes. A well-studied component of a complex response termed reactive astrogliosis that occurs after various types of CNS injury is the increased production of astrocytic processes filled with GFAP-containing IFs. While increased expression of GFAP during reactive astrogliosis has been well established at the protein level, little is known about whether or not changes in GFAP mRNA levels occur after CNS injury. In the present study we used in situ hybridization methods to examine this issue. A 35S-labeled mouse GFAP cDNA probe was used for in situ hybridizations of sections of the brain stem obtained 2, 7, and 14 days after unilateral transections of the corticospinal tract in the caudal medulla. Film as well as emulsion autoradiography showed a dramatic increase in GFAP mRNA labeling associated with the degenerating corticospinal tract. GFAP mRNA levels were already dramatically increased in the injured corticospinal tract by 2 days post axotomy and remained elevated at 14 days. Interestingly, in addition to the robust increase in GFAP mRNA levels specifically associated with the degenerating tract, a diffuse increase in GFAP mRNA labeling was observed throughout the grey matter of the brain stem at 2 days post-axotomy, but not after this time. Immunoblotting and immunocytochemical experiments verified that the increased GFAP mRNA levels in the degenerating corticospinal system were accompanied by an increased expression of the protein. These results demonstrate that an increase in GFAP mRNA levels occurs during Wallerian degeneration in the CNS and suggest that increased expression of the GFAP gene is a major contributor to CNS scarring that results after direct traumatic injury.     

Involvement of ADAM10 in axonal outgrowth and myelination of the peripheral nerve

The disintegrin and metalloproteinase 10 (ADAM10) is a membrane‐anchored metalloproteinase with both proteolytic and disintegrin characteristics. Here, we investigate the expression, regulation, and functional role of ADAM10 in axonal outgrowth and myelination of the peripheral nerve. Expression pattern analysis of 11 ADAM family members in co‐cultures of rat dorsal root ganglia (DRG) neurons and Schwann cells (SCs) demonstrated the most pronounced mRNA expression for ADAM10. In further studies, ADAM10 was found to be consistently upregulated in DRG‐SC co‐cultures before the induction of myelination. Neurons as well as SCs widely expressed ADAM10 at the protein level. In neurons, the expression of ADAM10 was exclusively limited to the axons before the induction of myelination. Inhibition of ADAM10 activity by the hydroxamate‐based inhibitors GI254023X and GW280264X resulted in a significant decrease in the mean axonal length. These data suggest that ADAM10 represents a prerequisite for myelination, although its activity is not required during the process of myelination itself as demonstrated by expression analysis of myelin protein zero (P0) and Sudan black staining. Hence, during the process of myelin formation, ADAM10 is highly upregulated and appears to be critically involved in axonal outgrowth that is a requirement for myelination in the peripheral nerve. © 2009 Wiley‐Liss, Inc.     

Growth cones and axon trajectories of a sensory pathway in the amphibian spinal cord

Central axons of sensory ganglion (SG) neurons of the Xenopus tail enter the spinal cord via the ventral roots and travel dorsally and rostrally following a diagonal course within the lateral marginal zone (LMZ) to reach the dorsolateral fasciculus (DLF) (Nordlander et al.: Brain Res., 440:391-395, 1988). Axons are dispersed as they cross the cord. At the DLF they turn and travel together rostrally, sharing the fascicle with axons of primary sensory neurons (Rohon-Beard cells) already present in the tract. In this paper we analyze the growth patterns of the central projections of SG axons in the tail by using HRP applied to proximal branches of tail spinal nerves. Growth cones of the diagonal route are variable in configuration, often bearing processes that spread within the LMZ. Once the DLF, growth cones change shape, becoming distinctly linear. While growth cones navigating the diagonal part of the route never contact or fasciculate with other diagonal SG axons, SG growth cones and axons of the DLF are more closely associated with their fellows. Measurements of the slopes of SG axons in the diagonal route indicated a limited range with a mean of 23 degrees with respect to the cord axis. On the basis of these observations, we conclude that 1) navigational patterns for growth cones of this pathway differ for the diagonal versus the DLF part of its course, and 2) fasciculation is not a mechanism used by SG axons to reach the DLF, but that instead, each axon is able to find its way independently.     

Automatic algorithm for correcting motion artifacts in time-resolved two-dimensional magnetic resonance angiography using convex projections.

Time-resolved contrast enhanced magnetic resonance angiography (MRA) may suffer from involuntary patient motion. It is noted that while MR signal change associated with motion is large in magnitude and has smooth phase variation in k-phase, signal change associated with vascular enhancement is small in magnitude and has rapid phase variation in k-space. Based upon this observation, a novel projection onto convex sets (POCS) algorithm is developed as an automatic iterative method to remove motion artifacts. The presented POCS algorithm consists of high-pass phase filtering and convex projections in both k-space and image space. Without input of detailed motion knowledge, motion effects are filtered out, while vasculature information is preserved. The proposed method can be effective for a large class of nonrigid motions, including through-plane motion. The algorithm is stable and converges quickly, usually within five iterations. A double-blind evaluation on a set of clinical MRA cases shows that a completely unsupervised version of the algorithm produces significantly better rank scores (P=0.038) when compared to angiograms produced manually by an experienced radiologist.     

轴突分叉点位置计算法适用性的分析

某些中枢神经元通过分叉轴突向两个以上核团投射。轴突分叉点的位置通常由计算轴突主干传导时间予以估测。但是,本工作用该法的4个公式计算细胞内或细胞外记录到的数据未能得到一致和可信的数值。结果提示,计算法的原理是不合理的,并至少对在细胞内或细胞外记录条件下估测轴突分叉点是不适用的。     

Localization of proopiomelanocortin mRNA in functional subsets of neurons defined by their axonal projections

In situ cDNA:mRNA hybridization is a technique that has been developed for the visualization of cDNA:mRNA hybrids in individual cells. To use this technique to answer questions of regulation in heterogeneous populations of cells in the brain, it must be combined with other procedures allowing for the identification of functional subgroups of neurons. We report here a procedure by which in situ cDNA:mRNA hybridization may be combined with retrograde axonal tracing using the fluorescent tracer fast blue. Using this technique, it now becomes possible to measure mRNA regulation in functional subsets of cells defined by their axonal projections.     

Novel C59T leader peptide mutation in the MPZ gene associated with late-onset, axonal, sensorimotor polyneuropathy

The objective of this study was to report a novel exon-1 mutation in the myelin protein zero (MPZ) gene, resulting in axonal Charcot–Marie–Tooth neuropathy with recurrent hyper-CK-emia. In a 64-year-old woman slowly progressive distal lower limb weakness, muscle cramps in the lower limb muscles, and stocking-type numbness had developed from the age of 61. Neurologic examination revealed discrete hip flexor weakness, weakness for foot extension, diffuse wasting of the distal lower limb muscles, reduced patella tendon reflexes, and absent Achilles tendon reflexes. There was recurrently elevated creatine kinase with a maximum of 607 U/l ( n , <145 U/l). Stimulation of the peroneal and tibial nerves did not evoke a muscular response. Electromyography was neurogenic. Biopsy of the right sural nerve showed diffuse axonal degeneration and loss of axons of all diameters. Muscle biopsy showed increased fiber-size variability, angulated fibers, internalized nuclei, accumulations of nuclei, grouped atrophic muscle fibers, and fiber splitting. Molecular genetic analysis by PCR and direct nucleotide sequencing revealed the heterozygous C59T exon-1 MPZ gene mutation, resulting in the amino acid exchange S20F of the MPZ signal protein domain (leader peptide). The novel C59T mutation in the leader peptide of the MPZ gene is pathogenic and manifests as severe, late-onset, axonal, symmetric sensorimotor polyneuropathy (CMT2) and hyper-CK-emia.