Abnormal expression of neurofilament proteins in dysmyelinating axons located in the central nervous system of jimpy mutant mice

Myelination in the peripheral nervous system is considered to increase the phosphorylation level of neurofilament proteins in the axon, resulting in an increase in axonal calibre. To understand the relationship between myelination and neurofilament proteins in axons, we examined jimpy mutant mice with a point mutation in the proteolipid protein gene and dysmyelination in the central nervous system. The jimpy mice exhibited a characteristic similarity in neurofilament nature to the myelin-deficient mice in the peripheral nervous system reported previously. The following novel results were obtained in the jimpy mice: dysmyelinated axons, in which the amount of non-phosphorylated neurofilament-H was drastically increased without a significant reduction of the phosphorylated form, compared with the control myelinated axons, did not suffer any decrease in their diameters. Expression levels of all neurofilament subunit proteins and their mRNAs were enhanced in the central nervous system tissue. Because the above biochemical data were obtained from the cytoskeletal fraction, at least some of the increased neurofilament-H and -M proteins appeared to be coassembled into neurofilaments but remained non-phosphorylated. Axonal neurofilaments of the jimpy were, probably due to this abnormal stoichiometry and phosphorylation state in neurofilaments, more compact and random in alignment with less prominent cross-bridges than those of the control, providing possible evidence for disturbing the axonal transport of other organelles. These results suggest that myelination regulates both the expression and phosphorylation of neurofilament proteins, and is essential for the cytoplasmic organization of myelinated axons.     

Ammoniacal silver staining of degenerating axons

Summary Degenerating axons in the central nervous system can be selectively stained by ammoniacal silver nitrate and citric acid-formalin reducer (after Nauta and Gygax, 1954) alone. This selective staining can be obtained without any type of pretreatment. It has been found that if the tissue sections are treated first with various lipid solvents (e.g. acetone, carbon tetrachloride, chloroform-methanol, etc.) and then stained by ammoniacal silver nitrate and citric acid-formalin reducer, degenerating axons are still selectively stained. These results are discussed in relation to earlier histochemical work on the Nauta and Gygax (1954) and Nauta (1957) methods.The author wishes to acknowledge the critical advice and help of Aaron S. Abramovitz and to thank Mrs. Elaine Langer for her help in preparing the photographic plates.     

Myelination by transplanted fetal and neonatal oligodendrocytes in a dysmyelinating mutant

Demyelination is a major feature of CNS injury and disease, including multiple sclerosis. To examine the potential for myelination by transplanted oligodendrocytes, initially described by Gumpel et al., we have transplanted neonatal cortex of mice with normal myelin into a dysmyelinating mutant, the shiverer mouse. We have found that oligodendrocyte precursors mature and synthesize myelin following transplantation. Immunostaining with antibodies to myelin basic protein (MBP), neurofilament protein and glial fibrillary acidic protein, demonstrates myelination both within the graft and extending out into the host, axonal sprouting from the graft which parallels the MBP-reactivity, and minimal astrocytic proliferation in response to the transplant.     

Axonal degeneration of ascending sensory neurons in gracile axonal dystrophy mutant mouse

Summary The distribution of axonal spheroids was examined in the central nervous system of gracile axonal dystrophy (GAD) mutant mice. Only few spheroids are observed in the gracile nucleus of the medulla in normal mice throughout the period examined, while they are first noted in GAD mice as early as 40 days after birth. The incidence of spheroids shifts from the gracile nucleus to the gracile fasciculus of the spinal cord with the progress of disease, suggesting that the degenerating axonal terminals of the dorsal ganglion cells back from the distal presynaptic parts in the gracile nucleus, along the tract of the gracile fasciculus, toward the cell bodies in the dorsal root ganglion. This phenomenon indicates that the distribution of spheroids is age dependent and reflects a dying-back process in degenerating axons. In addition to the gracile nucleus and the gracile fasciculus, which is one of the main ascending tracts of primary sensory neurons, it was noted that the other primary sensory neurons joined with some of the second-order neurons at the dorsal horn and neurons at all levels of the dorsal nucleus (Clarke's column) are also severely affected in this mutant. The incidence of the dystrophic axons are further extended to the spinocerebellar tract and to particular parts of the white matter of the cerebellum, such as the inferior cerebellar peduncle and the lobules of I–III and VIII in the vermis. These results indicate that this mutant mouse is a potential animal model for human degenerative disease of the nervous system, such as neuroaxonal dystrophy and the spinocerebellar ataxia.Supported by a grant (62-11-02 63-1-03) from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan and in part by a grant from Japan Health Science Foundation     

Microglial nodules in early multiple sclerosis white matter are associated with degenerating axons

Microglial nodules in the normal-appearing white matter have been suggested as the earliest stage(s) of multiple sclerosis (MS) lesion formation. Such nodules are characterized by an absence of leukocyte infiltration, astrogliosis or demyelination, and may develop into active demyelinating MS lesions. Although the etiology of MS is still not known, inflammation and autoimmunity are considered to be the central components of this disease. Previous studies provide evidence that Wallerian degeneration, occurring as a consequence of structural damage in MS lesions, might be responsible for observed pathological abnormalities in connected normal-appearing white matter. As innate immune cells, microglia/macrophages are the first to react to even minor pathological changes in the CNS. Biopsy tissue from 27 MS patients and autopsy and biopsy tissue from 22 normal and pathological controls were analyzed to determine the incidence of microglial nodules. We assessed MS periplaque white matter tissue from early disease stages to determine whether microglial nodules are associated with altered axons. With immunohistochemical methods, the spatial relation of the two phenomena was visualized using HLA-DR antibody for MHC II expression by activated microglia/macrophages and by applying antibodies against damaged axons, i.e., SMI32 (non-phosphorylated neurofilaments) and amyloid precursor protein as well as neuropeptide Y receptor Y1, which marks axons undergoing Wallerian degeneration. Our data demonstrate that the occurrence of microglial nodules is not specific to MS and is associated with degenerating as well as damaged axons in early MS. In addition, we show that early MS microglial nodules exhibit both pro- and antiinflammatory phenotypes.     

The toppler mouse: a novel mutant exhibiting loss of Purkinje cells

We describe the genetic and neurological features of toppler, a spontaneous autosomal mutation that appeared in a colony of FVB/N mice and that manifests as severe ataxia appearing at around 12 days of age, worsening with age. The lifespan of affected mice is 8-12 months, with occasional mice living longer. Both homozygous males and females are fertile, and females are able to nurture litters. Histological examination of brain revealed no striking abnormalities other than the loss of cerebellar Purkinje cells. The toppler mutation was mapped to mouse chromosome 8, and to assess whether it was novel or a recurrence of a previously described chromosome 8 mouse mutant, toppler mice were crossed with the nervous and tottering mouse mutants. These studies demonstrate that toppler is a unique mouse mutation. Purkinje cell abnormalities in toppler mice were obvious around postnatal day (P) 14, i.e., toppler Purkinje cells already exhibited abnormal morphology. Staining for calbindin, a calcium binding protein enriched in Purkinje cells, showed altered dendritic morphology. Between P14 and P30, dramatic Purkinje cell loss occurred, although there were differences in the degree of Purkinje cell loss in each lobule. At P30, the surviving Purkinje cells expressed zebrin II. From P30 through 6 months, many of the remaining Purkinje cells gradually degenerated. Purkinje cell loss was analyzed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL), and Purkinje cells were TUNEL-positive most abundantly at P21. In addition, Bergmann glia were TUNEL positive at P21, and they expressed activated caspase-3 at earlier time points. Interestingly, despite the apparent death of some Bergmann glia, there was up-regulation of glial fibrillary acidic protein, expressed in astrocytes as well as Bergmann glia. Given the changes in both Purkinje cells and glia in toppler cerebellum, this may be a very useful model in which to investigate the developmental interaction of Purkinje cells and Bergmann glia.     

Response of olfactory axons to loss of synaptic targets in the adult mouse

Glomerular convergence has been proposed to rely on interactions between like olfactory axons, however topographic targeting is influenced by guidance molecules encountered in the olfactory bulb. Disruption of these cues during development misdirects sensory axons, however little is known about the role of bulb-derived signals in later life, as new axons arise during turnover of the olfactory sensory neuron (OSN) population. To evaluate the contribution of bulb neurons in maintaining topographic projections in adults, we ablated them with N-methyl-d-aspartate (NMDA) in P2-IRES-tauLacZ mice and examined how sensory axons responded to loss of their postsynaptic partners. NMDA lesion eliminated bulb neurons without damage to sensory axons or olfactory ensheathing glia. P2 axons contained within glomeruli at the time of lesion maintained convergence at these locations; there was no evidence of compensatory growth into the remnant tissue. Delayed apoptosis of OSNs in the target-deprived epithelium led to declines in P2 neuron number as well as the gradual atrophy, and in some cases complete loss, of P2 glomeruli in lesioned bulbs by 3 weeks. Increased cell proliferation in the epithelium partially restored the OSN population, and by 8 weeks, new P2 axons distributed within diverse locations in the bulb remnant and within the anterior olfactory nucleus. Prior studies have suggested that initial development of olfactory topography does not rely on synapse formation with target neurons, however the present data demonstrate that continued maintenance of the sensory map requires the presence of sufficient numbers and/or types of available bulbar synaptic targets.     

Neuroprotective effect of the immune system in a mouse model of severe dysmyelinating hereditary neuropathy: enhanced axonal degeneration following disruption of the RAG-1 gene

In mouse models of later onset forms of human hereditary demyelinating neuropathies, the immune system plays a crucial pathogenic role. Here, we investigated the influence of immune cells on early onset dysmyelination in mice homozygously deficient of the myelin component P0. In peripheral nerves of P0(-/-) mice, CD8+ T-lymphocytes increased with age. Macrophages peaked at 3 months followed by a substantial decline. They were mainly of hematogenous origin. To evaluate the functional role of immune cells, we cross-bred P0(-/-) mutants with RAG-1-deficient mice. At 3 months, the number of endoneurial macrophages did not differ from the macrophage number of immunocompetent myelin mutants, but the later decline of macrophages was not observed. Quantitative electron microscopy revealed that in plantar nerves of 6-month-old double mutants, significantly more axons had degenerated than in immunocompetent littermates. These data suggest a neuroprotective net effect of T-lymphocytes on axon survival in inherited, early onset dysmyelination.     

Abnormal Ca regulation in oligodendrocytes from the dysmyelinating jimpy mouse

Jimpy (jp) is a point mutation in the gene on the X chromosome which codes for the major myelin proteolipid protein. Most oligodendrocytes (OLs) in the jp mouse undergo cell death at the time when they should be actively myelinating. Loss of mature OLs results in severe CNS dysmyelination. Dying jp OLs have the morphology of apoptotic cells but it is not clear how the mutation activates biochemical pathways which lead to programmed death of OLs in jp CNS. There is compelling evidence from a number of systems that high levels of intracellular Ca²⁺ ([Ca²⁺]_i) can activate downstream processes which result in both apoptotic and necrotic cell death. To determine whether [Ca²⁺]_i dysregulation might be involved in the death of jp OLs, we used ratiometric imaging to determine levels of [Ca²⁺]_i in OLs cultured from jp and normal CNS and in immortalized cell lines derived from jp and normal OLs. Immortalized jp OLs and OLs isolated directly from jp brain both showed a similar elevation in [Ca²⁺]_i ranging from 60% to 150% over control values. A higher baseline [Ca²⁺]_i in jp OLs might increase their vulnerability to other insults due to abnormal protein processing or changes in signaling pathways which act as a final trigger for cell death.     

DNA repair in the degenerating mouse retina

In light of different recent results suggesting that the adult mammalian central nervous system can produce new neurons, possibly as an endogenous repair mechanism, we investigated whether neurogenesis occurs in response to photoreceptor degeneration in the rd1 mouse, a model of human-inherited retinal dystrophy. Bromodeoxy-Uridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression experiments detected cell proliferation in the extreme peripheral retina, in both wt and rd1 retina, independent of degeneration. BrdU incorporation and PCNA expression also occurred in rd1 photoreceptors. Our results strongly suggest that these photoreceptors undergo DNA repair: p53, PCNA, and DNA ligase IV are expressed before photoreceptor death, consistent with a model where photoreceptors expressing the rd1 mutation activate a process of DNA repair but which is overwhelmed by the disease mutation leading to apoptotic death. The existence of such a balance offers potential new targets for neuroprotective approaches.     

Growth of injured rabbit optic axons within their degenerating optic nerve

Spontaneous growth of axons after injury is extremely limited in the mammalian central nervous system (CNS). It is now clear, however, that injured CNS axons can be induced to elongate when provided with a suitable environment. Thus injured CNS axons can elongate, but they do not do so unless their environment is altered. We now show apparent regenerative growth of injured optic axons. This growth is achieved in the adult rabbit optic nerve by the use of a combined treatment consisting of: (1) supplying soluble substances originating from growing axons to be injured rabbit optic nerves (Schwartz et al., Science, 228:600-603, 1985), and (2) application of low energy He-Ne laser irradiation, which appears to delay degenerative changes in the injured axons (Schwartz et al., Lasers Surg. Med., 7:51-55, 1985; Assia et al., Brain Res., 476:205-212, 1988). Two to 8 weeks after this treatment, unmyelinated and thinly myelinated axons are found at the lesion site and distal to it. Morphological and immunocytochemical evidence indicate that these thinly myelinated and unmyelinated axons are growing in close association with glial cells. Only these axons are identified as being growing axons. These newly growing axons transverse the site of injury and extend into the distal stump of the nerve, which contains degenerating axons. Axons of this type could be detected distal to the lesion only in nerves subjected to the combined treatment. No unmyelinated or thinly myelinated axons in association with glial cells were seen at 6 or 8 weeks postoperatively in nerves that were not treated, or in nerves in which the two stumps were completely disconnected. Two millimeters distal to the site of injury, the growing axons are confined to a compartment comprising 5%-30% of the cross section of the nerve. A temporal analysis indicates that axons have grown as far as 6 mm distal to the site of injury, by 8 weeks postoperatively. Anterograde labeling with horseradish peroxidase, injected intraocularly, indicates that some of these newly growing axons arise from retinal ganglion cells.     

Cloning of M-calpain 80 kD subunit from the axonal degeneration-resistant WLDs mouse mutant

Calpains are calcium-activated cysteine proteases that are involved in cellular degradation in models of neurodegeneration. Calpains are the effectors of cytoskeletal disruption during axonal degeneration, a pathological feature of many neurological disorders. The WLD^S mouse mutant is resistant to axonal degeneration and demonstrates prolonged survival of the cytoskeleton after nerve injury. To investigate the possibility that a mutation in calpain or abnormalities in calpain protein expression is responsible for the resistance to axonal degeneration seen in the WLD^Smouse mutant, we 1) cloned and sequenced the large subunit of the high calcium-requiring form of calpain (m-calpain) from nervous system tissues of WLD^S and from wild-type C57BL/6 mice, and 2) generated polyclonal m-calpain antibodies for comparison of relative protein levels by Western blot. We found our sequence for mouse m-calpain to be almost identical to another recently published mouse sequence, and the wild-type and WLD^S sequences to be identical. Our fusion protein and peptide polyclonal antibodies were specific for the 80 kD subunit and recognized appropriate protein bands from pure m-calpain, fusion protein, and in tissue. There was no apparent difference in m-calpain expression in nerve or spinal cord in noninjured adult animals. These data suggest that a defect in m-calpain 80 kD subunit does not likely underlie the WLD^S phenotype, but raise questions about other subunits of calpain and possibly other proteases. J. Neurosci. Res. 52:653–660, 1998. © 1998 Wiley-Liss, Inc.     

The astrocyte as a locus of carbonic anhydrase in the brains of normal and dysmyelinating mutant mice

There is some controversy in the literature whether carbonic anhydrase occurs in astrocytes, as well as in oligodendrocytes and myelin, in the mammalian brain. In the present study this issue was addressed by double immunostaining for carbonic anhydrase and two astrocytic "markers" in the brains of normal mice and two dysmyelinating mutants, jimpy and shiverer. In the brains of young mice, carbonic anhydrase and glutamine synthetase were colocalized in astrocytes in the cortical gray matter. In gray matter of the adult mouse brain, it was possible to immunostain both carbonic anhydrase and glial fibrillary acidic protein (GFAP) in the same cells. However, in contrast to the findings in gray matter, in and near subcortical white matter carbonic anhydrase could be detected only in oligodendrocytes and myelinated fibers. In the brains of jimpy mice, virtually all the carbonic-anhydrase-positive cells were also GFAP positive, even in regions normally occupied by white matter. In the brains of young and adult shiverer mice, carbonic anhydrase was localized in astrocytes in the gray matter, but in and near the tracts normally occupied by white matter carbonic anhydrase could be detected only in oligodendrocytes and their abundant processes. The findings confirmed the oligodendrocyte-myelin unit to be the primary locus of carbonic anhydrase in the normal mouse brain and showed the astrocytes in gray matter normally to be a secondary locus of carbonic anhydrase. The immunostaining in the jimpy mouse brain suggested further that reactive astrocytes, in particular, might be rich in carbonic anhydrase.     

Successful silver impregnation of degenerating axons after long survivals in the human brain

Degenerating axons in the human brain were successfully impregnated with reduced silver methods. The appearance of degenerating fibers did not differ markedly with survival times of three weeks and two, six, and twelve years following cerebral infarction or contusion of the brain. Impregnated fibers were found only along the appropriate corticofugal pathways. Electron microscopic examination of Vibratome-cut, silver-stained sections demonstrated silver deposition almost exclusively within axon fragments. Previous studies using anterograde degeneration methods in the human brain have limited their choice of cases to those with short periods of survival. Relaxing the restriction of short survival times extends the range of cases which can be used to study neural connections which may be unique to, or different in, the human brain.     

Dying back type axonal degeneration of sensory nerve terminals in muscle spindles of the gracile axonal dystrophy (GAD) mutant mouse

A disorder of the gracile axonal dystrophy (GAD) mutant mouse is characterized by a neuromuscular disease with sensory ataxia (detectable 30 days after birth) and paresis of the hindlimbs (detectable at 80 days). In the sensory ataxia stage, histological study of the primary sensory system shows that, in addition to the lesions in the central nervous system, peripherally projecting axons have also started to degenerate at their distal ends in muscle spindles. Although the structure of Ia fibre endings appear normal until 15 days after birth, initial changes in the annulo-spiral structure are detected around the 20th day by a degeneration of the terminal axons. Degeneration then progressed proximally and the secondary endings also start to degenerate. Neuron cell bodies located in the dorsal root ganglia are morphologically intact until the later stages. Chronological studies indicate that, although axonal degeneration progresses throughout life, it is accentuated during the rapid somatic growth period. Around 50 days of age, transient regeneration takes place at axonal endings when somatic growth has attained a plateau. Such primary sensory endings tend to be restored by fine, multiple axons which gain access to the intrafusal fibres through the original endoneurial tubes. Ultrastructural observations at the fully affected stage show intrafusal muscle fibres lying scattered within spindles due to loss of the fine network of inner capsule layers and an almost complete loss of sensory endings from the surface of intrafusal muscle fibres. These results indicate that this mutant mouse is a useful model for naturally occurring 'dying back' type axonal degeneration or 'central and peripheral distal axonopathies', and would provide significant information about the complete evolution of the pathological processes involved.     

Measurement of axonal conduction velocity in single mammalian motor axons

In deeply anesthetized cats, determinations of motor-axonal conduction velocity (CV) were made using extracellular potentials recorded from single, functionally isolated motor axons innervating the muscle tibialis posterior. Axons were activated by suprathreshold electrical stimulation at the ventral-root level. Action potentials were recorded with 3 bipolar electrodes located on the muscle nerve at the level of the popliteal fossa. The most proximal and distal of the bipolar muscle-nerve electrodes were 16.4-22.0 mm apart. Estimates were made of CV from ventral root to muscle nerve (conventional CV) and between the proximal and the distal pairs of muscle-nerve electrodes (muscle-nerve CV). An evaluation was based on comparison of these CVs, estimates of uncertainties in time and distance measurements and simulations of the effects of recording conditions on CV estimates. The analysis indicated that the uncertainty in the conventional CV measurement of mammalian motor axons is at least +/- 2%. However, variability may be as great as 20% between muscle-nerve CV measurements from different experiments, probably due to such factors as regional variation in CV and differences in recording configuration.