Distal effects in a model of proximal axonopathy: 3,3'-iminodipropionitrile causes specific loss of neurofilaments in rat vestibular afferent endings

3,3'-Iminodipropionitrile (IDPN) is a neurotoxic compound that causes both a proximal neurofilamentous axonopathy and loss of the vestibular sensory hair cells. We used immunocytochemistry to examine changes in the expression of heavy, medium and light neurofilament (NF-H, NF-M, NF-L) proteins in the afferent terminals of vestibular sensory epithelia after IDPN exposure in rats. Acute, repeated and subchronic IDPN exposure induced a marked loss of NFs in the nerve terminals. The effect of subchronic IDPN was specific, as demonstrated by comparison with the synaptic membrane protein SNAP-25. In addition, Western blot analysis indicated specific loss of NFs in the vestibular receptors. Ultrastructural analysis revealed that afferent endings in the vestibular receptors were significantly preserved in animals exposed to subchronic IDPN, but that these endings showed NF segregation from microtubules followed by NF loss. These effects were closely paralleled by ultrastructural changes in the nerve terminals, particularly in the afferent contacts with the hair cells, and preceded hair cell loss. Thus, distal NF loss and nerve terminal pathology occur in the IDPN model of proximal neurofilamentous axonopathy. Similar distal pathology could also occur in human diseases characterized by proximal axonal swellings, particularly in amyotrophic lateral sclerosis.     

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.     

Cerebrospinal fluid and serum antibodies against neurofilaments in patients with amyotrophic lateral sclerosis

Background: The aim of the study was to assess autoimmune involvement in amyotrophic lateral sclerosis (ALS). Methods: We measured IgG antibodies against light (NFL) and medium (NFM) subunits of neurofilaments using ELISA in paired cerebrospinal fluid (CSF) and serum samples from 38 ALS patients and 20 controls. Results: Serum levels of anti‐NFL were higher in ALS patients than in controls (P < 0.005). Serum anti‐NFL antibodies and intrathecal anti‐NFM antibodies were related to patient disability (serum anti‐NFL: P < 0.05; intrathecal anti‐NFM: P < 0.05). Anti‐NFL levels were significantly correlated with anti‐NFM levels in ALS (P < 0.001) and the control group (P < 0.0001) in the CSF, but not in serum. Anti‐NFL and anti‐NFM antibodies significantly correlated between serum and CSF in the ALS group (anti‐NFL: P < 0.0001; anti‐NFM: P < 0.001) and in the control group (anti‐NFL: P < 0.05; anti‐NFM: P < 0.05). Conclusions: Autoimmune humoral response to neurocytoskeletal proteins is associated with ALS.     

Review: The role of mitochondria in the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of upper and lower motor neurones leading to muscle weakness and paralysis. Despite recent advances in the genetics of ALS, the mechanisms underlying motor neurone degeneration are not fully understood. Mitochondria are known to be involved in the pathogenesis of ALS, principally through mitochondrial dysfunction, the generation of free radicals, and impaired calcium handling in ALS patients and models of disease. However, recent studies have highlighted the potential importance of altered mitochondrial morphology and defective axonal transport of mitochondria in ALS. Here, we review the evidence for mitochondrial involvement in ALS and discuss potential therapeutic strategies targeting mitochondria.     

RNS与SFEMG在检测ALS患者神经肌肉接头功能紊乱的比较研究

目的探讨重复神经电刺激(RNS)与单纤维肌电图(SFEMG)在检测肌萎缩侧索硬化(ALS)患者神经肌肉接头功能紊乱中的吻合率及RNS低频递减阳性率与SFEMG指标纤维密度(FD)、颤抖(jitter)、阻滞(block)的关系。方法收集2008-5—2009-4在北京协和医院神经科门诊或病房确诊或拟诊的ALS患者43例,同时行RNS及SFEMG检查。比较RNS与SFEMG在判断ALS患者神经肌肉接头紊乱的敏感性和特异性,并分析RNS低频递减与SFEMG参数指标jitter、block、FD的相关性。结果(1)43例患者中26例RNS(+),占60.5%,17例RNS(-),占39.5%。SFEMG(+)34例,占79.1%,SFEMG(-)9例,占20.9%。其中SFEMG(+)+RNS(+)者共25例,SFEMG(-)+RNS(-)者8例。RNS在判断ALS存在神经肌肉接头受累方面与SFEMG比较有一定的吻合性(Kappa=0.47,P0.01)。(2)RNS阴性和阳性组FD间比较无统计学差异(t=-0.1405,P0.05)。RNS阳性组Block程度明显高于RNS阴性组(χ~2=11.432,P0.01),jitter值也明显高于RNS阴性组(t=2.906,P0.01)。桡神经RNS波幅递减程度与jitter值呈正相关(r=0.626,P0.05)。结论 RNS与SFEMG比较有一定的吻合率。RNS检查灵敏度较高,具有操作简单,费用低,耗时短,无创,不需患者特殊配合,近远端肌肉均可操作,易于推广的特点,对ALS患者的辅助诊断具有意义。     

Calbindin‐D28K is increased in the ventral horn of spinal cord by neuroprotective factors for motor neurons

Slow glutamate‐mediated neuronal degeneration is implicated in the pathophysiology of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The calcium‐binding proteins calbindin‐D_28K and parvalbumin have been reported to protect neurons against excitotoxic insults. Expression of calbindin‐D_28K is low in adult human motor neurons, and vulnerable motor neurons additionally may lack parvalbumin. Thus, it has been speculated that the lack of calcium‐binding proteins may, in part, be responsible for early degeneration of the population of motor neurons most vulnerable in ALS. Using a rat organotypic spinal cord slice system, we examined whether the most potent neuroprotective factors for motor neurons can increase the expression of calbindin‐D_28K or parvalbumin proteins in the postnatal spinal cord. After 4 weeks of incubation of spinal cord slices with 1) glial cell line‐derived neurotrophic factor (GDNF), 2) neurturin, 3) insulin‐like growth factor I (IGF‐I), or 4) pigment epithelium‐derived factor (PEDF), the number of calbindin‐D_28K‐immunopositive large neurons (>20 μm) in the ventral horn was higher under the first three conditions, but not after PEDF, compared with untreated controls. Under the same conditions, parvalbumin was not upregulated by any neuroprotective factor. The same calbindin increase was true of IGF‐I and GDNF in a parallel glutamate toxicity model of motor neuron degeneration. Taken together with our previous reports from the same model, which showed that all these neurotrophic factors can potently protect motor neurons from slow glutamate injury, the data here suggest that upregulation of calbindin‐D_28K by some of these factors may be one mechanism by which motor neurons can be protected from glutamate‐induced, calcium‐mediated excitotoxicity. © 2015 Wiley Periodicals, Inc.     

Impaired hypoxic sensor Siah‐1, PHD3, and FIH system in spinal motor neurons of an amyotrophic lateral sclerosis mouse model

We recently reported spinal blood flow–metabolism uncoupling in the Cu/Zn‐superoxide dismutase 1 (SOD1)‐transgenic (Tg) mouse model of amyotrophic lateral sclerosis (ALS), suggesting relative hypoxia in the spinal cord. However, the hypoxic stress sensor pathway in ALS has not been well studied. In the present work, we examined the temporal and spatial changes of hypoxic stress sensor proteins (Siah‐1, PHD3, and FIH) following motor neuron (MN) degeneration in the spinal cord of normoxic ALS mice. The expression of Siah‐1 and PHD3 proteins progressively increased in the surrounding glial cells of presymptomatic Tg mice (10 weeks, 10 weeks) compared with the large MN of the anterior horn. In contrast, a significant reduction in Siah‐1 and PHD3 protein expression was evident in end‐stage ALS mice (18 weeks, 18 weeks). Double‐immunofluorescence analysis revealed PHD3 plus Siah‐1 double‐positive cells in the surrounding glia of symptomatic Tg mice (14–18 weeks), with no change in the large MNs. In contrast, FIH protein expression decreased in the surrounding glial cells of Tg mice at end‐stage ALS (18 weeks). The present study suggests a partial loss in the neuroprotective response of spinal MNs in ALS results from a relative hypoxia through the Siah‐1, PHD3, and FIH system under normoxic conditions. This response could be an important mechanism of neurodegeneration in ALS. © 2012 Wiley Periodicals, Inc.     

Binding of IDPN (β,β′-iminodipropionitrile) to rat spinal cord: Possible implication in the mechanism of spheroid formation in amyotrophic lateral sclerosis

β,β′-iminodipropionitrile (IDPN) produces ‘spheroids’ similar to those in certain cases of amyotrophic lateral sclerosis (ALS). Therefore, the target molecule of IDPN could be important to the understanding of the molecular mechanism of spheroid formation in ALS. Wistar rats were injected ip with ¹⁴C-labeled IDPN (¹⁴C-IDPN) and killed at 0.5, 1, 3, 6, 12 and 24 h thereafter. The radioactivity in each organ increased rapidly and reached the maximum at 0.5–1 h after ¹⁴C-IDPN injection. Thereafter, a rapid decrease occurred until 6 h, followed by a gradual decline until 24 h. The radioactivity in the cerebral cortex, diencephalon and cerebellum was higher than in the pons, medulla oblongata and spinal cord. Although high in the visceral organs and skeletal muscles, no or little radioactivity was detected in fat tissue. Autoradiography also confirmed these results. In three rats, ¹⁴C-IDPN was injected to the lumbar enlargement of the spinal cord. Six hours after injection, the segment was removed and homogenized with physiological saline (PS). After centrifugation, the supernatant was obtained (PS fraction). The pellet was resuspended with 4 mol/L urea and the supernatant was obtained (urea fraction). Each fraction was analysed by gel filtration. A peak of radioactivity was observed at the elution fraction Nos 19 and 20 (consistent with free ¹⁴C-IDPN) when PS fraction was applied. On application of urea fraction, another peak was obtained at the elution fractions Nos 8 and 9 (MW 60～80 kDa). The present study demonstrates that ¹⁴C-IDPN does not selectively accumulate to the spinal cord and suggests that an IDPN-binding molecule with an MW of 60–80 kDa is present in the spinal cord. The molecule may be related to the pathological process of spheroid formation in ALS.     

Quantitative immunocytochemical analysis of the spinal cord in G86R superoxide dismutase transgenic mice: Neurochemical correlates of selective vulnerability

Transgenic mice with a G86R mutation in the mouse superoxide dismutase (SOD-1) gene, which corresponds to a mutation that has been observed in familial amyotrophic lateral sclerosis (ALS), display progressive loss of motor function and provide a valuable model of ALS. The pathology in the spinal cords of these mice was evaluated to determine whether there are chemically identified populations of neurons that are either highly vulnerable or resistant to degeneration. Qualitatively, there were phosphorylated neurofilament protein (NFP)-immunoreactive inclusions and a pronounced loss of motoneurons in the ventral horn of the spinal cord without the presence of vacuoles that has been reported in other SOD-1 transgenic mice. Neuron counts from SOD-1 and control spinal cords revealed that the percentage loss of NFP-, choline acetyltransferase (ChAT)-, and calretinin (CR)-immunoreactive neurons was greater than the percentage loss of total neurons, suggesting that these neuronal groups are particularly vulnerable in SOD-1 transgenic mice. In contrast, calbindin-containing neurons did not degenerate significantly and represent a protected population of neurons. Quantitative double-labeling experiments suggested that the vulnerability of ChAT- and CR-immunoreactive neurons was due primarily to the presence of NFP within a subset of these neurons, which degenerated preferentially to ChAT- and CR-immunoreactive neurons that did not colocalize with NFP. Our findings suggest that NFP, which has been demonstrated previously to be involved mechanistically in motoneuron degeneration, may also be important in the mechanism of degeneration that is initiated by the SOD-1 mutation. © 1996 Wiley-Liss, Inc.     

On the early diagnosis of IVIg-responsive chronic multifocal acquired motor axonopathy

Abstract. Multifocal acquired motor axonopathy (MAMA) is a treatable, immune mediated motor neuropathy with purely axonal electrophysiological features. Distinction from degenerative neuronopathies such as progressive muscular atrophy (PMA) or early motor neuron disease (MND) can be difficult because of the similar clinical and electrophysiological findings. Here, we report the clinical, electrophysiological and laboratory findings in 6 patients with MAMA. Electrophysiological testing showed purely axonal findings with evidence of pathological spontaneous activity and chronic neurogenic changes. Of particular note, pathological spontaneous activity in paraspinal myotoms was not detectable in any of the patients even though it had been documented in peripheral muscles of the corresponding myotome(s). Elevated serum ganglioside antibody levels,most frequently anti-GD1a antibodies, were present in all 6 patients. IV Ig treatment led to clinical improvement in all but one patient, who showed an allergic response when exposed to IVIg. Our findings indicate that paraspinal EMG and anti-GD1a antibodies can facilitate the early identification of treatable, IVIg responsive, patients with MAMA.     

Loss of hnRNPA1 in ALS spinal cord motor neurons with TDP‐43‐positive inclusions

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons and appearance of skein‐like inclusions. The inclusions are composed of trans‐activation response (TAR) DNA‐binding protein 43 (TDP‐43), a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. hnRNPA1 and hnRNPA2/B1 are hnRNPs that interact with the C‐terminus of TDP‐43. Using immunohistochemistry, we investigated the association between TDP‐43 and hnRNPA1 in ALS spinal motor neurons. We examined spinal cords of seven ALS cases and six muscular dystrophy cases (used as controls) for the presence of TDP‐43 and hnRNPA1 protein. In the control cases, hnRNPA1 immunoreactivity in motor neurons was intense in the nucleus and weak in the cytoplasm where it showed a fine granular appearance. In the ALS cases, hnRNPA1 immunoreactivity in motor neurons was reduced in the nuclei of neurons with skein‐like inclusions but was not detected in the skein‐like inclusions. The marked loss of hnRNPA1 in motor neurons with concomitant cytoplasmic aggregation of TDP‐43 may represent a severe disturbance of mRNA processing, suggesting a key role in progressive neuronal death in ALS.     

Motor unit number estimation in the rat tail using a modified multipoint stimulation technique

Motor unit number estimation (MUNE) of the rodent hindlimb has been used mainly for following the progression of motor neuron disorders. By performing MUNE in the tail, however, progression of axonal neuropathy could also be assessed, as both proximal and distal regions would be available for study. In this investigation, three raters performed a modified multipoint stimulation MUNE technique in the tails of 14 healthy adult rats. The technique was straightforward to perform, with a relatively narrow range of motor unit number estimates of 40 ± 16 (standard deviation) for the proximal tail and 21 ± 11 for the distal tail. Intrarater reliability coefficients were 0.31 (P = 0.033) and 0.32 (P = 0.028) for the proximal and distal tail, respectively. Interrater reliability coefficients were 0.22 (P = 0.086) and 0.44 (P = 0.004). These reliability assessments, along with the relatively low motor unit estimates and narrow range of values, support the idea that rat tail MUNE may have utility in the evaluation of rodent models of neuromuscular disease, including length‐dependent neuropathy. Muscle Nerve 40: 115–121, 2009     

Development of a rat model of amyotrophic lateral sclerosis expressing a human SOD1 transgene

Mutations in copper–zinc superoxide dismutase gene (SOD1) have been linked to some familial cases of ALS. We report here that rats that express a human SOD1 transgene with two different ALS‐associated mutations (G93A and H46R) develop striking motor neuron degeneration and paralysis. By comparing the two transgenic rats with different SOD1 mutations, we demonstrate that the time course in these rats was similar to human SOD1‐mediated familial ALS. As in the human disease and transgenic ALS mice, pathological analysis shows selective loss of motor neurons in the spinal cords of these transgenic rats. In addition, typical neuronal Lewy body‐like hyaline inclusions as well as astrocytic hyaline inclusions identical to those in human familial ALS are observed in the spinal cords. The larger size of this rat model as compared with the ALS mice will facilitate studies involving manipulations of spinal fluid (implantation of intrathecal catheters for chronic therapeutic studies; CSF sampling) and spinal cord (e.g., direct administration of viral‐ and cell‐mediated therapies).     

Motor unit number estimation predicts disease onset and survival in a transgenic mouse model of amyotrophic lateral sclerosis

Motor unit number estimation (MUNE) has proved useful in predicting rate of progression and survival in patients with amyotrophic lateral sclerosis (ALS). In animal models, it has demonstrated physiological effects of experimental medications that were not evident behaviorally. We sought to determine more specifically what aspects of function and survival that MUNE could predict in the G93A transgenic mouse model of ALS. Transgenic mice were examined in two distinct treatment studies, neither of which showed an effect of drug on survival, behavioral measures, or MUNE. MUNE was performed using a modification of the incremental stimulation method by stimulating the sciatic nerve at the sciatic notch, and recording with a circumferential surface electrode around the ipsilateral distal hindlimb. Both limbs were studied and the results averaged. MUNE was performed longitudinally on all animals from near onset to premorbid state. Each study was evaluated separately. For both studies, MUNE at initial study correlated significantly with behavioral determination of disease onset, and MUNE slope from initial to final study correlated significantly with disease duration, as measured from onset to time of death. However, the final MUNE value did not correlate with survival. Thus, in two studies involving animals with quite different disease courses, initial MUNE effectively predicted symptom onset and MUNE slope predicted survival. This suggests that MUNE has potential efficacy as a useful functional outcome measure in both animal and human studies of ALS.     

Early and selective pathology of light chain neurofilament in the spinal cord and sciatic nerve of G86R mutant superoxide dismutase transgenic mice

Pathologic accumulation of neurofilament protein (NF), both within spheroids of the proximal axon and within inclusions of motor neuron somata, is a hallmark of neurodegeneration in amyotrophic lateral sclerosis (ALS). Transgenic mice that express mutations in superoxide dismutase (SOD-1), which were genetically linked to familial ALS, develop symptomatology and pathology that strongly resemble ALS and therefore provide a useful model for studying the disease. Examining NF in the G86R mutant SOD-1 transgenic mice, we previously demonstrated that phosphorylated NF accumulates in motor neuron somata of symptomatic transgenic mice. In the present study, we expand these results by examining the immunocytochemical distribution of the three subunits of NF (i.e., light, medium, and heavy chains) as well as tubulin in presymptomatic and symptomatic SOD-1 transgenic mice. Although all NF subunits, but not tubulin, accumulate along with phosphorylated NF in the spinal cord inclusions of symptomatic mice, numerous inclusions containing only light chain NF are found in the spinal cord of presymptomatic SOD-1 transgenic mice. In addition to these results in the spinal cord, intensely immunoreactive aggregates of NF-L, but not the other NF subunits or tubulin, were observed in the sciatic nerve of both symptomatic and presymptomatic mutant SOD-1 transgenic mice. These results suggest that the mechanism of NF alteration in SOD-1 transgenic mice, and also perhaps in ALS patients, originates with the disruption of NF-L, only later involving the other subunits.     

ROCK inhibition improves axonal regeneration in a preclinical model of amyotrophic lateral sclerosis

Alteration of the RhoA/ROCK (Rho kinase) pathway has been shown to be neuroprotective in SOD1^G93A mice, the most commonly used animal model of ALS. Since previous studies indicate that, apart from neuroprotection, ROCK inhibitor Y-27632 can also accelerate regeneration of motor axons, we here assessed the regenerative capability of axons in SOD1^G93A mice with and without treatment with Y-27632. Regeneration of axons was examined after sciatic nerve crush in pre- and symptomatic SOD1^G93A mice. Proregenerative effects of Y-27632 were studied during the disease course in the SOD1^G93A mouse model. In symptomatic SOD1^G93A mice, axonal regeneration was markedly reduced compared to presymptomatic SOD1^G93A mice and wild types. Treatment with Y-27632 improved functional and morphological measures of motor axons after sciatic crush in all tested conditions. Y-27632 treatment did not increase the lifespan of symptomatic SOD1^G93A mice, but did improve axonal (re)innervation of neuromuscular junctions. Our study provides proof of concept that axonal regeneration of motor neurons harboring SOD1^G93A is impaired, but amenable for pharmacological interventions aiming to accelerate axonal regeneration. Given the lack of treatments for ALS, approaches to improve axonal regeneration, including by inhibiting ROCK, should be further explored.