Impaired Axonal Transport Research Articles

Neuropharmacology and neurotoxicity of 3,4-methylenedioxymethamphetamine.

The existing data indicate that MDMA produces long-term deficits in markers of 5-HT axon terminals in the rodent brain. Increased cleavage of the cytoskeletal protein tau, impairment of axonal transport, and functional consequences associated with a 5-HT depleting regimen of MDMA support the view that MDMA induces structural brain damage, that is, axonal degeneration. A confluence of oxidative stress and bioenergetic stress induced by MDMA is hypothesized to underlie the process of MDMA neurotoxicity (Fig. 3). The actions of MDMA on the 5-HT transporter to promote free radical formation and/or intracellular calcium may synergize with MDMA-induced disturbances in cellular energetics and hyperthermia to effect selective toxicity to 5-HT axon terminals.

The morphological and neurochemical effects of diffuse brain injury on rat central noradrenergic system

The central noradrenergic system is widely distributed throughout the brain and is closely related to spontaneous motility and level of consciousness. The study presented here evaluated the morphological as well as neurochemical effects of diffuse brain injury on the central noradrenergic system in rat. Adult male Sprague–Dawley rats were subjected to impact–acceleration brain injury produced with a weight-drop device. Morphological changes in locus coeruleus (LC) neurons were examined by using immuno-histochemistry for dopamine-β-hydroxylase, and norepinephrine (NE) turnover in the cerebral cortex was measured by high performance liquid chromatography with electrochemical detection. The size of LC neurons increased by 11% 24 h after injury but had decreased by 27% seven days after injury. Axons of noradrenergic neurons were swollen 24 h and 48 h after injury but the swelling had dwindled in seven days. NE turnover was significantly reduced seven days after injury and remained at a low level until eight weeks after injury. These results suggest that focal impairment of axonal transport due to diffuse brain injury causes cellular changes in LC and that the neurochemical effect of injury on the central noradrenargic system lasts over an extended period of time. Chronic suppression of NE turnover may explain the sustained behavioral and psychological abnormalites observed in a clinical situation.

Impaired axonal transport of cortical neurons in Alzheimer’s disease is associated with neuropathological changes

Using a novel in vitro post mortem tracing method, we demonstrate a decrease of axonal transport in the temporal cortex neurons as compared to axonal transport in the prefrontal cortex neurons in AD patients, but not in non-demented controls. The decrease of axonal transport is related to the degree of neuropathological changes, as the temporal cortex undergoes more severe neuropathological changes in AD. The present study provides, for the first time, direct evidence of the presence of impaired axonal transport in AD brains.

Synaptic pathology and cell death in the cerebellum in Creutzfeldt-Jakob disease.

Prion protein (PrP(c)) is a cell membrane glycoprotein particularly abundant in the synapses. Prion diseases are characterized by the replacement of the normal PrPc by a protease-resistant, sheet-containing isoform (PrP(CJD), PrP(Sc), PrP(BSE)) that is pathogenic. Creutzfeldt-Jakob disease (CJD) in humans, scrapie (Sc) in sheep and goats, and bovine spongiform encephalopathy (BSE) in cattle are typical prion diseases. Classical CJD can be presented as sporadic, infectious or familial, whereas the new variant of CJD (nvCJD) is considered a BSE-derived human disease. Spongiform degeneration, glial proliferation, involving astrocytes and microglia, neuron loss and abnormal PrP deposition are the main neuopathological findings in most human and animal prion diseases. Yet recent data point to synapses as principal targets of abnormal PrP deposition. Loss of synapses is an early abnormality in experimental scrapie. Decreased expression of crucial proteins linked to exocytosis and neurotransmission, covering synaptophysin, synaptosomal-associated protein of 25,000 mol wt (SNAP-25), synapsins, syntaxins and Rab3a occurs in the cerebral cortex and cerebellum in sporadic CJD. Moreover, impairment of glomerular synapses and attenuation of parallel fiber pre-synaptic terminals on Purkinje cell dendrites is a cardinal consequence of abnormal PrP metabolism in CJD. Accumulation of synaptic proteins in the soma and axonal torpedoes of Purkinje cells suggests additional impairment of axonal transport. Increase in nuclear DNA vulnerability leading to augmented numbers of cells bearing nuclear DNA fragments is a common feature in the brains of humans affected by prion diseases examined at post-mortem, but also in archival biopsy samples processed with the method of in situ end-labeling of nuclear DNA fragmentation. This form of cell death is reminiscent of apoptosis found in experimental scrapie in rodents. It is not clear that all forms of cell death in human and animal prion diseases are due to apoptosis. Yet new observations have shown cleaved (active) caspase-3 (17 kDa), a main executioner of apoptosis, expressed in scattered cells in the brains of mice with experimental scrapie and in the cerebellum of patients with sporadic CJD. Together, these data suggest activation of the caspase pathway of apoptosis in human and animal prion diseases.

Optic nerve degeneration and mitochondrial dysfunction: genetic and acquired optic neuropathies

Selective degeneration of the smallest fibers (papillo-macular bundle) of the human optic nerve occurs in a large number of optic neuropathies characterized primarily by loss of central vision. The pathophysiology that underlies this peculiar pattern of cell involvement probably reflects different forms of genetic and acquired mitochondrial dysfunction. Maternally inherited Leber’s hereditary optic neuropathy (LHON), dominant optic atrophy (Kjer disease), the optic atrophy of Leigh’s syndrome, Friedreich ataxia and a variety of other conditions are examples of inherited mitochondrial disorders with different etiologies. Tobacco–alcohol amblyopia (TAA), the Cuban epidemic of optic neuropathy (CEON) and other dietary (Vitamins B, folate deficiencies) optic neuropathies, as well as toxic optic neuropathies such as due to chloramphenicol, ethambutol, or more rarely to carbon monoxide, methanol and cyanide are probably all related forms of acquired mitochondrial dysfunction. Biochemical and cellular studies in LHON point to a partial defect of respiratory chain function that may generate either an ATP synthesis defect and/or a chronic increase of oxidative stress. Histopathological studies in LHON cases and a rat model mimicking CEON revealed a selective loss of retinal ganglion cells (RGCs) and the corresponding axons, particularly in the temporal-central part of the optic nerve. Anatomical peculiarities of optic nerve axons, such as the asymmetric pattern of myelination, may have functional implications on energy dependence and distribution of mitochondrial populations in the different sections of the nerve. Histological evidence suggests impaired axonal transport of mitochondria in LHON and in the CEON-like rat model, indicating a possible common pathophysiology for this category of optic neuropathies. Histological evidence of myelin pathology in LHON also suggests a role for oxidative stress, possibly affecting the oligodendrocytes of the optic nerves.

Intra-axonal Neurofilament Compaction Does Not Evoke Local Axonal Swelling in all Traumatically Injured Axons

Traumatic axonal injury (TAI) contributes to morbidity and mortality following traumatic brain injury (TBI). Single-label immunocytochemical studies employing antibodies to neurofilament compaction (NFC), RM014, and antibodies to APP, a marker of impaired axonal transport (AxT), have shown that TAI involves both NFC and disruption of AxT. Although it may be hypothesized that both events occur within the same injured axon, this has not been confirmed. To determine the relationship between NFC and impaired AxT, dual-label immunofluorescence was employed. To compare and contrast specific changes associated with these two markers of TAI, single-label electron microscopy was also used. Rats were subjected to an impact acceleration injury (30 min–6 h survival), and their brains were prepared for dual-label immunofluorescence and single-label electron microscopy. APP and RM014 were consistently found in two distinct classes of TAI. One, which showed only RM014 immunoreactivity, was thin and elongate, was sometimes vacuolated, and revealed little progressive change over time. The second was distinguished by focal axonal swellings containing APP immunoreactivity alone in small-caliber axons or in combination with RM014 immunoreactivity in large-caliber axons. These swellings were localized to either nodal or internodal loci and underwent progressive swelling over time, ultimately leading to secondary axotomy. Ultrastructural examination of these two classes of TAI revealed NFC together with mitochondrial dilation without organelle pooling in the RM014 single-labeled axons. However, the APP single-labeled small-caliber axons and APP/RM014 dual-labeled large-caliber axons revealed a progressive accumulation of organelles associated with increased axonal swelling over time. In contrast to previous thought, it now appears that NFC may occur independent of impaired AxT in TAI. This finding underscores the complexity of TAI, suggesting the need for multiple immunocytochemical approaches to fully assess the overall axonal response to TBI.

Impaired retrograde axonal transport of adenovirus-mediated E. coli LacZ gene in the mice carrying mutant SOD1 gene

A replication-defective recombinant adenoviral vector containing E. coli lacZ gene was injected into the gastrocnemius muscles of transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) gene and non-transgenic wild-type mice at 40 weeks of age. After 60 and 90 h of the injection, lacZ staining was observed at the distal ends of the sciatic nerves in both mice groups, with the number and the distances greatly reduced in the transgenic mice. Mean velocities of retrograde transport for lacZ was estimated to be 2.1 and 0.05 mm/24 h in non-transgenic and transgenic mice, respectively. These results indicate that the retrograde axonal transport of foreign gene product is impaired in the mice model for familial amyotrophic lateral sclerosis.

Long-term impairment of anterograde axonal transport along fiber projections originating in the rostral raphe nuclei after treatment with fenfluramine or methylenedioxymethamphetamine.

To further evaluate the serotonin (5-HT) neurotoxic potential of substituted amphetamines, we used tritiated proline to examine anterograde transport along ascending axonal projections originating in the rostral raphe nuclei of animals treated 3 weeks previously with (+/-)fenfluramine (FEN, 10 mg/kg, every 2 h x 4 injections; i.p.) or (+/-)3,4-methylenedioxymethamphetamine (MDMA, 20 mg/kg, twice daily for 4 days; s.c.). The documented 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT, 75 microg; ICV; 30 min after pretreatment with pargyline, 50 mg/kg; i.p., and desipramine 25 mg/kg; i.p.), served as a positive control. Along with anterograde axonal transport, we measured two 5-HT axonal markers, 5-HT and 5-hydroxyindoleacetic acid (5-HIAA). Prior treatment with FEN or MDMA led to marked reductions in anterograde transport of labeled material to various forebrain regions known to receive 5-HT innervation. These reductions were associated with lasting decrements in 5-HT axonal markers. In general, decreases in axonal transport were less pronounced than those in 5-HT and 5-HIAA. However, identical changes were observed after 5,7-DHT. These results further indicate that FEN and MDMA, like 5,7-DHT, are 5-HT neurotoxins.

Altered time course of mRNA expression of alpha tubulin in the central nervous system of hens treated with diisopropyl phosphorofluoridate (DFP).

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus-ester induced delayed neurotoxicity (OPIDN) in the hen, human and other sensitive species. We studied the effect of single dose of DFP (1.7 mg/kg/s.c.) on the expression of alpha tubulin which is one of the major sub-unit of tubulin polymers that constitute an important constituent of cellular architecture. The hens were sacrificed at different time points i.e. 1, 2, 5, 10, and 20 days. Total RNA was extracted from the following brain regions: cerebrum, cerebellum, and brainstem as well as spinal cord. Northern blots prepared using standard protocols were hybridized with alpha tubulin as well as with beta-actin and 28S RNA cDNA (controls) probes. The results indicate a differential/spatial/temporal regulation of alpha tubulin levels which may be the result of perturbed microtubule dynamics not only in the axons but also in perikarya of neurons in the CNS of DFP treated hens. In the highly susceptible tissues like brainstem and spinal cord the initial down-regulation of mRNA levels could be attributed to DFP induced stress response resulting in inhibited cell metabolism and or cell injury/cell death. Increase in levels of mRNA at 5 days and thereafter coincided with increased tubulin transport which may be due to increased phosphorylation of tubulins in both axons and perikarya and other intraaxonal changes resulting in impaired axonal transport. DFP induced decreased rate of tubulin polymerization resulting in increased levels of free tubulin monomers may be involved in the altered alpha tubulin mRNA expression at different time points by autoregulatory circuits. Cerebellum being the less susceptible tissue showed only a moderate decline at day 2, while the alpha tubulin remained at near control levels at day 1. Delayed down-regulation may be due to the co-ordinated up or down-regulation of different sub-types of alpha and beta tubulins as well as the differential response of specialised cell types in cerebellum. Continuous overexpression of alpha tubulin in cerebrum from the beginning may be its effective protective strategy to safeguard itself from neurotoxicity. Differential expression pattern observed could be due to the differential susceptibility and variability in the rate of axonal transport of different regions besides the tubulin heterogenity of CNS. Hence our results indicte differential expression of alpha tubulin is either one of the reasons for the development of OPIDN or the result of progressive changes taking place during OPIDN.

Prominent Axonopathy and Disruption of Axonal Transport in Transgenic Mice Expressing Human Apolipoprotein E4 in Neurons of Brain and Spinal Cord

The epsilon 4 allele of the human apolipoprotein E gene (ApoE4) constitutes an important genetic risk factor for Alzheimer's disease. Recent experimental evidence suggests that human ApoE is expressed in neurons, in addition to being synthesized in glial cells. Moreover, brain regions in which neurons express ApoE seem to be most vulnerable to neurofibrillary pathology. The hypothesis that the expression pattern of human ApoE might be important for the pathogenesis of Alzheimer's disease was tested by generating transgenic mice that express human ApoE4 in neurons or in astrocytes of the central nervous system. Transgenic mice expressing human ApoE4 in neurons developed axonal degeneration and gliosis in brain and in spinal cord, resulting in reduced sensorimotor capacities. In these mice, axonal dilatations with accumulation of synaptophysin, neurofilaments, mitochondria, and vesicles were documented, suggesting impairment of axonal transport. In contrast, transgenic mice expressing human ApoE4 in astrocytes remained normal throughout life. These results suggest that expression of human ApoE in neurons of the central nervous system could contribute to impaired axonal transport and axonal degeneration. The possible contribution of hyperphosphorylation of protein Tau to the resulting phenotype is discussed.

Autonomic nervous function assessment using thermal reactivity of microcirculation

There are only a few reliable objective methods of diagnosing peripheral neuronal damage suitable for routine use; the most important is based on measurement of nerve conduction velocity, which only shows changes when severe disturbances are already present. However, it is precisely at this stage that the possibilities of therapy are no longer satisfactory. As small fibres are affected earlier in the course of most forms of PNP than the large ones, assessment of afferent as well as efferent C-fibre function gains importance in the management of this widespread disease. In assessment of autonomic dysfunction, variability of the heartbeat with deep breathing or the Valsalva manoeuvre is a good and generally accepted test, although not strongly associated with other PNP test abnormalities. However, axonal degeneration starts in the most distal parts of the axon due to impaired axonal transport. Therefore, the longest C-fibres, i.e. in the lower extremities, are affected first, and incipient changes are most prominent there. For this reason HLDF, a reflex response of the skin blood flow stimulated by heat, has advantages in assessment of early C-fibre dysfunction. Considering the fact that the afferent and efferent sympathetic C-fibres are involved in regulation of microcirculation, the skin blood flow regulation is investigated by means of laser Doppler flowmetry. The microcirculation is stimulated by heat and the reaction of microcirculation is assessed as a value for the function of afferent and efferent (sympathetic) C-fibres. The results of this method are in close correlation with electrophysiologic tests, which is not achieved with sudomotor function.

Damage to the cytoskeleton of large diameter sensory neurons and myelinated axons in vincristine‐induced painful peripheral neuropathy in the rat

Vincristine, along with other antimitotic chemotherapeutic drugs, produces a peripheral neuropathy in humans that is accompanied by painful paresthesias, dysesthesias, and occasionally hypoesthesia, and by hyporeflexia (Holland et al. [1973] Cancer Res. 33:1258–1264; McLeod and Penny [1969] J Neurol Neurosurg Psychiatry 32:297–304; Postma et al. [1993] J Neurooncol. 15:23–27; Sandler et al. [1969] Neurology 19:367–374). Systemic administration of vincristine causes swelling of unmyelinated axons and disorientation of axonal microtubules (Tanner et al. [1998a1998a] J Comp Neurol. 395:481–492) at a time when it also produces allodynia and mechanical hyperalgesia (Aley et al. [1996] Neuroscience 73:259–265; Authier et al. [1999] Neuroreport 10:965–968) and enhanced responsiveness in C-fibers in the rat (Tanner et al. [1998b] J Neurosci. 18:6480–6491). Because slowing of A-fiber conduction velocities had also been demonstrated (Tanner et al. [1998b] J Neurosci. 18:6480–6491), and mechanical hyperalgesia can occur secondary to damage to large diameter sensory afferents (Basbaum et al. [1991] Can J Physiol Pharmacol. 69:647–651; Coggeshall et al. [1993] Pain 52:233–242; Woolf and Mannion [1999] Lancet 353:1959–1964), we sought to determine whether vincristine also induced ultrastructural changes in myelinated A-fibers. Moreover, since systemic treatment with vincristine did not cause profound microtubule depolymerization in the unmyelinated axons of the peripheral nerve, we hypothesized that the drug's effects may be more extensive in the cell body, because in the spinal ganglion, the blood-nerve barrier is less restrictive. We used quantitative ultrastructural methods to analyze the microtubule cytoskeleton in myelinated axons in the mid-shaft of the saphenous nerve and in the sensory ganglion cells. Vincristine induced swelling of the whole nerve and an increase in the cross-sectional areas of myelinated axons but no loss of myelinated axons. There was a significant decrease in axonal microtubules, as well as microtubule disorganization, in myelinated fibers from vincristine-treated rats. In the spinal ganglion, vincristine induced swelling of large diameter sensory neurons and a build-up of neurofilaments in the cell bodies and proximal axons, suggestive of impaired anterograde axonal transport. J. Comp. Neurol. 424:563–576, 2000. © 2000 Wiley-Liss, Inc.

Axonal damage revealed by accumulation of β-amyloid precursor protein in HTLV-I-associated myelopathy

We investigated the localization and extent of β-amyloid precursor protein (APP) immunoreactivity as a sensitive marker for impairment of fast axonal transport in the spinal cords of patients with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). The results from this study show that APP, used as a marker of early axonal damage in HAM/TSP lesions, is more intensively expressed in areas of active-inflammatory lesions than those of inactive-chronic lesions. The close localization to the areas containing inflammation (activation of macrophage/microglia) is striking and suggests that axonal damage is closely associated with inflammation in active-chronic lesions. Although inflammatory cell infiltration in the central nervous system (CNS) is rarely found in inactive-chronic lesions, a few clusters of APP+ axons are found in the spinal cord white matter in some cases. The presence of APP+ axons without relation to inflammatory cells in inactive-chronic lesions, suggest that soluble neurotoxic factors might induce axonal changes in the CNS of HAM/TSP. The occasional myelinated fibers in the anterior and posterior spinal roots in lower thoracic to lumbar levels had APP+ axons, suggesting that spinal nerve roots can be affected in HAM/TSP, especially in lower thoracic to lumbar levels. Impairment of fast axonal transport may contribute to the development of disability in patients with HAM/TSP.

Retrograde axonal transport impairment of large- and medium-sized retinal ganglion cells in diabetic rat

Retrograde axonal transport impairment of large- and medium-sized retinal ganglion cells in diabetic rat

Retrograde axonal transport impairment of large- and medium-sized retinal ganglion cells in diabetic rat

Purpose. Several abnormalities in visual pathway functions in diabetic humans and animals have been reported. We demonstrated retrograde axonal transport impairment in retinal ganglion cells of streptozotocin-diabetic rats. Methods. Diabetes was induced in male Wistar albino rats by intraperitoneal injection of streptozotocin. Three months after the induction of diabetes, fluoro-gold was injected into the dorsal lateral geniculate nucleus. Percentages of fluoro-gold-labeled large-, medium- and small-sized retinal ganglion cells per total population were calculated in wholemount retinas of diabetic and control rats. The same sections were stained with cresyl violet and each retinal ganglion cell type evaluated by light microscopy. Results. Although a quantitative decrease in the population of each retinal ganglion cell type was not observed, mean percentages of fluoro-gold-labeled large- and medium-sized retinal ganglion cells per total population were significantly decreased in diabetic rats compared with controls. Conclusions. Our results suggest that diabetes affects the retrograde axonal transport in large- and medium-sized retinal ganglion cells despite the absence of morphological changes in the perikaryon and decrease in total cell population. Diabetes-induced impairment of retrograde axonal transport in large- and medium-sized retinal ganglion cells precede optic nerve involvement. However, this may merely be a consequence of metabolic changes in diabetic states.

Damage to the cytoskeleton of large diameter sensory neurons and myelinated axons in vincristine-induced painful peripheral neuropathy in the rat

Vincristine, along with other antimitotic chemotherapeutic drugs, produces a peripheral neuropathy in humans that is accompanied by painful paresthesias, dysesthesias, and occasionally hypoesthesia, and by hyporeflexia (Holland et al. [1973] Cancer Res. 33:1258-1264; McLeod and Penny [1969] J Neurol Neurosurg Psychiatry 32:297-304; Postma et al. [1993] J Neurooncol. 15:23-27; Sandler et al. [1969] Neurology 19:367-374). Systemic administration of vincristine causes swelling of unmyelinated axons and disorientation of axonal microtubules (Tanner et al. [1998a1998a] J Comp Neurol. 395:481-492) at a time when it also produces allodynia and mechanical hyperalgesia (Aley et al. [1996] Neuroscience 73:259-265; Authier et al. [1999] Neuroreport 10:965-968) and enhanced responsiveness in C-fibers in the rat (Tanner et al. [1998b] J Neurosci. 18:6480-6491). Because slowing of A-fiber conduction velocities had also been demonstrated (Tanner et al. [1998b] J Neurosci. 18:6480-6491), and mechanical hyperalgesia can occur secondary to damage to large diameter sensory afferents (Basbaum et al. [1991] Can J Physiol Pharmacol. 69:647-651; Coggeshall et al. [1993] Pain 52:233-242; Woolf and Mannion [1999] Lancet 353:1959-1964), we sought to determine whether vincristine also induced ultrastructural changes in myelinated A-fibers. Moreover, since systemic treatment with vincristine did not cause profound microtubule depolymerization in the unmyelinated axons of the peripheral nerve, we hypothesized that the drug's effects may be more extensive in the cell body, because in the spinal ganglion, the blood-nerve barrier is less restrictive. We used quantitative ultrastructural methods to analyze the microtubule cytoskeleton in myelinated axons in the mid-shaft of the saphenous nerve and in the sensory ganglion cells. Vincristine induced swelling of the whole nerve and an increase in the cross-sectional areas of myelinated axons but no loss of myelinated axons. There was a significant decrease in axonal microtubules, as well as microtubule disorganization, in myelinated fibers from vincristine-treated rats. In the spinal ganglion, vincristine induced swelling of large diameter sensory neurons and a build-up of neurofilaments in the cell bodies and proximal axons, suggestive of impaired anterograde axonal transport.

Immunoreactivity of beta-amyloid precursor protein in amyotrophic lateral sclerosis.

We investigated the localization and extent of beta-amyloid precursor protein (beta-APP695) immunoreactivity as a sensitive marker for impairment of fast axonal transport in the spinal cords of 21 patients with amyotrophic lateral sclerosis (ALS), paying special attention to anterior horn neurons. Specimens from 18 patients without neurological disease served as controls. Increased beta-APP immunoreactivity was frequently recognized in the anterior horns of the ALS patients with short clinical courses or with mild depletion of anterior horn cells, while no beta-APP immunoreactivity was demonstrated in those with severe depletion of anterior horn neurons or with long-standing clinical courses. Increased beta-APP immunoreactivity in the anterior horn neurons was mainly confined to the perikarya and no immunoreactivity was recognized in the dendrites or proximal axons directly emanating from the somata, except some spheroids (proximal axonal swellings) which showed increased immunoreactivity of beta-APP. Increased beta-APP immunoreactivity was spotted or focally aggregated in the perikarya of normal-looking large anterior horn neurons, while it was frequently diffuse in that of degenerative neurons such as central chromatolytic cells and or those with simple atrophy. On the other hand, the controls showed no immunostaining with beta-APP in the spinal cord. These findings suggest that increased immunoreactivity of beta-APP in neuronal perikarya of the anterior horn cells and in some proximal axonal swellings is an early change of ALS, and may be a response of the increased synthesis of beta-APP resulting from neuronal damage, or the impairment of fast axonal transport.

Neuroaxonal pathology in Creutzfeldt-Jakob disease.

Neuroaxonal pathology has met little attention in transmissible spongiform encephalopathies. In brains of a series of 39 consecutive Creutzfeldt-Jakob disease (CJD) cases, we detected numerous abnormal neurites that labeled for neurofilament proteins (NFP) by immunocytochemistry. Three types of abnormally NFP-accumulating structures were more prominently observed in CJD brains than in age-matched control brains: 1. Neurons with NFP in their somata were seen in 29 CJD cases (74%), some appearing distended with eccentrically placed nuclei and homogenously stained cytoplasm. 2. Neuritic distensions (neuritic swellings, dystrophic neurites) were observed mainly in the white matter but also at the junction between gray and white matter. In some axons many such swellings could be traced along the visible part of the axonal segment. 3. Axonal spheroids were observed mainly in the medulla, predominantly but not exclusively in posterior nuclei, and were more numerous than in age-matched control brains. Several neurons, axons and spheroids demonstrated immunoreactivity for amyloid precursor protein. Their number was much smaller, however, than that of NFP-immunoreactive structures and varied from one or two immunopositive neurites per section to small clusters of beaded or spiral axons. Focal expression of apolipoprotein E in cells of microglial and astrocytic morphology was observed in areas with most pronounced axonal damage. We conclude that neuroaxonal pathology is a frequent and important part of brain lesioning in CJD, probably reflecting profound impairment of axonal transport.

Selective impairment of fast anterograde axonal transport in the peripheral nerves of asymptomatic transgenic mice with a G93A mutant SOD1 gene

Transgenic mice that express a mutant Cu/Zn superoxide dismutase (SOD1) gene have been provided a valuable model for human amyotrophic lateral sclerosis (ALS). We studied a possible impairment of fast axonal transport in transgenic mice carrying a Gly 93→Ala (G93A) mutant SOD1 gene found in human familial ALS (FALS). Left sciatic nerve was ligated for 6 h in transgenic (Tg) and age-matched wild-type (WT) mice. Immunohistochemical analyses were performed for accumulations of kinesin and cytoplasmic dynein on both sides of the ligation site. Clinical function and histology in the spinal cords, sciatic nerves and gastrocnemius muscles were also assessed. The mice were examined at an early asymptomatic stage (aged 19 weeks) and a late stage (30 weeks) just before the development of the symptoms. WT mice showed an apparent increase in immunoreactivities for kinesin and cytoplasmic dynein at proximal and distal of the ligation, respectively. In contrast, the young Tg mice showed a selective decrease of kinesin accumulation in the proximal of the ligation. The mice were asymptomatic with a mild histological change only in muscles. The old Tg mice showed a marked reduction of the immunoreactivity for kinesin and cytoplasmic dynein on both sides of the ligation. They had a significant loss of spinal motor neurons, relatively small myelinated fiber densities of sciatic nerves, and severe muscular changes. These results provide direct evidence that the SOD1 mutation leads to impaired fast axonal transport, particularly in the anterograde direction at an early, asymptomatic stage preceding loss of spinal motor neurons and peripheral axons. This impairment may contribute to subsequent selective motor neuron death in the present model implicated for human FALS.

Axonal swellings and degeneration in mice lacking the major proteolipid of myelin.

Glial cells produce myelin and contribute to axonal morphology in the nervous system. Two myelin membrane proteolipids, PLP and DM20, were shown to be essential for the integrity of myelinated axons. In the absence of PLP-DM20, mice assembled compact myelin sheaths but subsequently developed widespread axonal swellings and degeneration, associated predominantly with small-caliber nerve fibers. Similar swellings were absent in dysmyelinated shiverer mice, which lack myelin basic protein (MBP), but recurred in MBP*PLP double mutants. Thus, fiber degeneration, which was probably secondary to impaired axonal transport, could indicate that myelinated axons require local oligodendroglial support.