Overexpression of ErbB2 and ErbB3 receptors in Schwann cells of patients with Charcot-Marie-tooth disease type 1A

Axon-derived neuregulins (NRGs) are a family of growth factors whose binding to ErbB tyrosine kinase receptors promotes the maturation, proliferation and survival of Schwann cells (SCs). Correct NRG/ErbB signaling is essential for the homeostasis of axonal-glial complexes and seems to play a role in peripheral nerve repair. The potential involvement of ErbB receptors in human peripheral neuropathies has not been clarified. Therefore, we assessed the immunoreactivity for EGFR (ErbB1), ErbB2, and ErbB3 in nerve biopsies from patients with different forms of Charcot-Marie-Tooth disease, type 1, (CMT1), as compared to others with inflammatory neuropathies and controls. The most notable changes consisted in the overexpression of ErbB2 and ErbB3 by SCs of nerves from CMT1A patients. These findings are consistent with an impairment of SC differentiation and expand the molecular phenotype of CMT1A. The upregulation of these receptors may play a role in the inhibition of myelination or in the promotion of recurrent demyelination and axonal damage.     

NCAM1与致痫大鼠认知功能障碍发病机制之间相关性的研究

目的 研究神经细胞黏附分子1(NCAMl)与癫痫大鼠认知功能障碍发病机制之间的相关性,探讨其在癫痫认知功能障碍中发挥的作用. 方法 采用随机数字表法将120只Wistar大鼠分为实验组和对照组.实验组又分为致痫组、卡马西平治疗组、奥卡西平治疗组、茴拉西坦治疗组、盐酸多奈哌齐治疗组,每组20只;采用匹罗卡品诱导癫痫模型,后4组并灌服相应药物.对照组(n=20)不造模,灌服生理盐水造模.通过Morris水迷宫实验测试大鼠的学习记忆能力,并通过RT-PCR法检测大鼠海马组织中NCAM1 mRNA表达,免疫组化法检测大鼠海马组织中NCAM1蛋白表达. 结果 各组大鼠水迷宫实验逃逸潜伏期差异有统计学意义(F=91.920,P=0.000),按长短排序为:卡马西平组＞奥卡西平组＞茴拉西坦组＞致痫组＞盐酸多奈哌齐组＞对照组.各组大鼠免疫组化及RT-PCR结果差异亦有统计学意义(F=324.510,P=0.000; F=81.160,P=0.000),按表达量多少排序为:盐酸多奈哌齐组＞茴拉西坦组＞致痫组＞奥卡西平组＞卡马西平组＞对照组. 结论 癫痫发作30d后海马的NCAM1表达水平升高,参与了癫痫认知功能障碍的发病;抗癫痫药物能加重癫痫认知功能障碍的发生,促智药物可明显改善癫痫的认知功能.     

Accumulation of peripheral myelin protein 22 in onion bulbs and Schwann cells of biopsied nerves from patients with Charcot-Marie-Tooth disease type 1A

Peripheral myelin protein 22 (PMP-22) is a glycoprotein expressed in the myelin sheath of myelinated Schwann cells. Duplication of the PMP-22 gene and its gene dosage effect have been postulated to be involved in the pathogenesis in the majority of individuals with Charcot-Marie-Tooth disease type 1A (CMT1A). Northern blot analysis has demonstrated that the mean relative ratio of PMP-22 mRNA/β-actin mRNA in biopsied nerves of patients with CMT1A is significantly higher than that in disease controls. To investigate whether the elevated expression of PMP-22 mRNA is reflected in the amount and the localization of PMP-22, we analyzed PMP-22, myelin basic protein (MBP), protein zero (P0), and S-100 immunoreactivities in biopsied nerves from six patients with CMT1A, five patients with other types of CMT, five patients with acquired demyelinating neuropathies, and two normal subjects. In all patients with CMT other than CMT1A and acquired demyelinating neuropathy, as well as in normal subjects, the myelin sheath was immunoreactive for PMP-22, MBP, and P0, while the Schwann cell cytoplasm was immunoreactive only for S-100. In five out of six patients with CMT1A, however, the PMP-22 immunoreactivity was present not only on the myelin sheath but also in the Schwann cell cytoplasm and onion bulbs (OBs). Although OBs are nonspecific and also seen in other inherited or acquired demyelinating neuropathies, the PMP-22-positive OBs were seen exclusively in CMT1A.The finding suggested that the expression of PMP-22 was abnormal for its localization and probably for the amount in patients with CMT1A carrying duplication of the PMP-22 gene. Received: 5 February 1996 / Revised, accepted: 20 May 1996     

c-Kit receptor expression in normal human Schwann cells and Schwann cell lines derived from neurofibromatosis type 1 tumors

The growth factor receptor c-Kit has several well-characterized functions during the development of numerous cell types, including red blood cells, mast cells, and melanocytes. Its role in Schwann cells has been described in transformed cells derived from malignant peripheral nerve sheath tumors from patients with neurofibromatosis type 1 (NF1 MPNST; Badache et al. [1998] Oncogene 17:795-800). However, c-Kit functions have not been investigated in normal Schwann cells. We report here that neonatal rat Schwann cells express low c-Kit levels, whereas expression levels for c-Kit are high for Schwann cells derived from MPNST of NF1 patients. In addition, c-Kit expression is not detectable in normal adult human Schwann cells. Although the c-Kit ligand stem cell factor (SCF) induces the phosphorylation of protein kinase B (or Akt) and prevents apoptosis in Schwann cells, SCF has no effect on the proliferation or differentiation of Schwann cells.     

Myasthenia gravis and Charcot-Marie-Tooth disease type 1A: An unusual combination of diseases

Concurrence of myasthenia gravis (MG) and Charcot-Marie-Tooth type 1 (CMT1A) neuropathy is rare. We describe a 60-year-old woman with MG and genetically proved CMT1A. The fluctuating ocular symptoms and proximal limb weakness were markedly improved by pyridostigmine treatment. Recognition of the possible association of MG and CMT1A in the same patient is important because the therapeutic result is rewarding. © 1997 John Wiley & Sons, Inc. Muscle Nerve 20: 1457–1459, 1997     

Early abnormalities in sciatic nerve function and structure in a rat model of Charcot-Marie-Tooth type 1A disease

We investigated early peripheral nervous system impairment in PMP22-transgenic rats ("CMT rat"), an established animal model for Charcot-Marie-Tooth disease 1A, at postnatal day 30 (P30), when the clinical phenotype is not yet apparent. Hemizygous CMT1A rats and wildtype littermates were studied by means of behavioral examination, electrophysiology, molecular biology, and light microscopy analysis. Behavioral studies only showed, a mild, but significant, decrease in toe spread 1-5, suggesting a weakness of distal foot muscles in CMT1A rats compared with normal littermates. Nerve conduction studies disclosed a severe slowing in motor conduction velocity, a temporal dispersion and a dramatic decrease of amplitude of motor waves in P30 transgenic animals. Coherently with a demyelinating process, affected nerves showed a significant thinning of myelin. Interestingly, axonal diameter and area were unchanged, but expression of non-phosphorylated neurofilaments was increased in CMT1A rats compared with normal controls. Our results confirm the fidelity of this animal model to human disease. Similarly, in young CMT1A patients, the MCV is significantly reduced and the muscle weakness is confined to distal segments, whereas morphological and morphometrical signs of axonal atrophy are absent. However, the presence of a molecular and functional damage of the axons suggests that this may be the correct moment to start neuroprotective therapies.     

Neuropeptides and neural cell adhesion molecule (NCAM) in CSF from patients with ALS

In 10 patients with amyotrophic lateral sclerosis (ALS), the CSF content of the neuropeptides vasoactive intestinal polypeptide (VIP) and cholecystokinin (CCK) as well as neural cell adhesion molecule (NCAM) was investigated. Compared with normal controls, no deviations were found in CCK or NCAM, while the values of VIP were significantly lower in ALS patients. This finding may reflect a loss of motor neurons.     

Charcot-Marie-Tooth disease type 1A and hereditary neuropathy with liability to pressure palsies: a SacI polymorphism in the proximal CMT1A-REP elements may lead to genetic misdiagnosis

A male patient with clinical signs and symptoms of a demyelinating neuropathy was shown to have a duplication of the 1.5-Mb region on chromosome 17p11.2 by means of two-color fluorescence in situ hybridization (FISH). This duplication is typical for the vast majority of Charcot-Marie-Tooth type 1A (CMT1A) cases. Analysis of DNA extracted from peripheral blood used to detect an EcoRI/SacI 3.2-kb junction fragment with probe pLR7.8 confirmed the CMT1A duplication, but also revealed a 7.8-kb fragment usually observed in patients with a hereditary neuropathy with liability to pressure palsies (HNPP). Both fragments observed in one patient canot result from one unequal crossover. In EcoRI/SacI Southern hybridization experiments with probe pLR7.8 DNA of his healthy parents also revealed a 7.8-kB restriction fragment. A subsequent two-color FISH analysis, however, indicated a normal status for interphase nuclei of the parents. Hence we hypothesize that the 7.8-kb fragment observed in our patient and his parents is not the product of unequal crossover during meiosis but due to a polymorphism of the SacI site in a proximal CMT1A-REP element. Received: May 28, 1998 / Accepted July 27, 1998 / Published online: December 9, 1998     

Selected items from the Charcot-Marie-Tooth (CMT) Neuropathy Score and secondary clinical outcome measures serve as sensitive clinical markers of disease severity in CMT1A patients

This study evaluates primary and secondary clinical outcome measures in Charcot-Marie-Tooth disease type 1A (CMT1A) with regard to their contribution towards discrimination of disease severity. The nine components of the composite Charcot-Marie-Tooth disease Neuropathy Score and six additional secondary clinical outcome measures were assessed in 479 adult patients with genetically proven CMT1A and 126 healthy controls. Using hierarchical clustering, we identified four significant clusters of patients according to clinical severity. We then tested the impact of each of the CMTNS components and of the secondary clinical parameters with regard to their power to differentiate these four clusters. The CMTNS components ulnar sensory nerve action potential (SNAP), pin sensibility, vibration and strength of arms did not increase the discriminant value of the remaining five CMTNS components (Ulnar compound motor action potential [CMAP], leg motor symptoms, arm motor symptoms, leg strength and sensory symptoms). However, three of the six additional clinical outcome measures – the 10 m-timed walking test (T10MW), 9 hole-peg test (9HPT), and foot dorsal flexion dynamometry – further improved discrimination between severely and mildly affected patients. From these findings, we identified three different composite measures as score hypotheses and compared their discriminant power with that of the CMTNS. A composite of eight components CMAP, Motor symptoms legs, Motor symptoms arms, Strength of Legs, Sensory symptoms), displayed the strongest power to discriminate between the clusters. As a conclusion, five items from the CMTNS and three secondary clinical outcome measures improve the clinical assessment of patients with CMT1A significantly and are beneficial for upcoming clinical and therapeutic trials.     

Central nervous system impairment detected by somatosensory evoked potentials in patients with Charcot-Marie-Tooth disease type 1A

Diseases related to peripheral myelin protein 22 (PMP22) have been implicated to involve the central nervous system (CNS). This study aimed to detect central nerve impairment using somatosensory evoked potentials (SSEPs) in patients with Charcot-Marie-Tooth disease (CMT) 1A. A total of 30 CMT1A patients and 26 healthy volunteers were included. Baseline characteristics, brain MRI and segmental SSEPs were collected from the participants. The peak latencies of N9, N13 and N20 were recorded, and central conduction velocity (CCT) was calculated and compared between groups. Significant differences were found in the peak latencies and amplitudes of N9, N13 and N20 between the two groups. CCT was significantly prolonged in the CMT group (7.05 ± 2.09 ms) compared to the control group (5.40 ± 1.79 ms) (p = 0.003). Six of 30 CMT patients had abnormal MRI signals, but no correlation with CCT was found. The central somatosensory pathway that carries SSEPs was impaired in CMT1A patients, which implies an important underlying role of PMP22 in the CNS.     

Degeneration of anterior horn cell in neuronal type of Charcot-Marie-Tooth disease (hereditary motor and sensory neuropathy type II): A Golgi study

Summary A morphological study using the Golgi impregnation method was carried out on the anterior horn cells at cervical (C), thoracic (Th), and lumbar (L) levels of the spinal cord in a patient with neuronal type of Charcot-Maire-Tooth disease (hereditary motor and sensory neuropathy type II) and an age-matched control. The present study demonstrated an uneven cell body surface, loss of cells (particularly large cells), loss of dendrites, reduced dendritic extent and an irregular surface and shape of dendrites at the C and L levels. In contrast, hematoxylin and eosin and Klüver-Barrera staining showed only simple atrophy or no change. The Th level of the patient showed none of these changes. Our results suggest that the degeneration or loss of dendrites of anterior horn cells by the Golgi staining method, which is most severe at the L level, is closely related to clinical findings such as muscle atrophy and weakness in neuronal type of Charcot-Marie-Tooth disease.     

Recovery of emotional behaviour in neural cell adhesion molecule (NCAM) null mutant mice through transgenic expression of NCAM180

In the present study we further investigate functions of the neural cell adhesion molecule (NCAM) in the mature central nervous system and its implications for animal behaviour. To this end we generated transgenic mice expressing the major NCAM isoform with the largest cytoplasmic domain, NCAM180, under control of a promoter for the small form neurofilament gene. Transgenic mice were also bred with mice deficient in endogenous NCAM (Ncam-/- mice) so that effects of NCAM180 could be analysed in the presence and absence of endogenous NCAM. While overexpression of transgenic NCAM180 was without apparent behavioural or morphological effect, its expression in Ncam-/- mice counteracted NCAM ablation-induced aggressive, anxiety-like and antidepressant-like behaviour. It furthermore prevented a hypersensitivity of Ncam-/- mice to the anxiolytic serotonin1A (5-HT1A) receptor agonist buspirone. Such recovery of emotional behaviour and behavioural 5-HT1A response occurred in spite of misdevelopment of the olfactory bulb and hippocampus that is characteristic of Ncam-/- mice, and without an apparent change in the expression of 5-HT1A binding sites in the brain. Hippocampus- and amygdala-dependent learning, though disturbed in Ncam-/- mice, remained unaffected by the transgenic NCAM180. We suggest an involvement of NCAM180-mediated cell recognition processes in the serotonergic modulation of emotional behaviour in adult mice.     

Establishment and characterization of immortalized Schwann cells from murine model of Niemann-Pick disease type C (spm/spm)

Niemann-Pick disease type C (NPC) is characterized by an accumulation of unesterified cholesterol in the endosomal/lysosomal (E/L) system, resulting in progressive neurodegeneration and death during early childhood. To investigate the cellular pathomechanism of nervous system involvement in NPC, continuous neural cell lines are desirable. In this study, we obtained neuronal and Schwann cell cultures and established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of NPC model mouse (spm/spm). One of the cell lines, designated SPMS9, had distinct Schwann cell phenotypes and was maintained over 10 months without phenotypic alterations. The level of Npc1 mRNA was markedly decreased, and NPC1 protein was not detectable in SPMS9 cells. These cells contained intracytoplasmic granules positive for filipin cholesterol staining and immunoreactive for GM2 ganglioside. Electron-microscopically, intracytoplasmic polymorphous membranous inclusions and vacuoles were demonstrated in SPMS9 cells. The treatment with an inhibitor of ceramide-specific glucosyltransferase, N-butyldeoxynojirimysin (NB-DNJ) markedly reduced the intracytoplasmic granular immunofluorescence for GM2 ganglioside in SPMS9 cells, whereas the amount of filipin-positive granules remained unchanged. The SPMS9 cells retained vesicular fluorescence of cationic dye acriflavine 16-24 hours after loading, indicating the defect of transmembrane efflux pump activity of NPC1 in the E/L compartment in these cells. These immortalized Schwann cells can be useful in studies on the nervous system lesions in NPC.     

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) expression by human brain microvessel endothelial cells in primary culture

PECAM-1 expression was investigated in primary cultures of human brain microvessel endothelial cells (HBMEC). HBMEC constitutively express PECAM-1 along their apical cell surface, advancing processes and on the basal surface at points of contact with the extracellular matrix. Surface expression is not altered by cytokine or lipopolysaccharide treatment. This distribution may mediate cell-cell contact and migration during angiogenesis and HBMEC-leukocyte interactions in CNS inflammation.     

Cation binding at the node of Ranvier in biopsied peripheral nerves of patients with Charcot-Marie-Tooth disease type 1A and hereditary neuropathy with liability to pressure palsies

The node of Ranvier in myelinated fibers is known to have an affinity to bind cations. Demyelination and remyelination due to abnormal expression of a myelin protein may affect cation binding or vice versa under pathological conditions. To study the cation binding at the node of Ranvier in inherited demyelinating neuropathies associated with over- and under-expression of the peripheral myelin protein 22 (PMP-22), the reaction with ferric ion and ferrocyanide was used to visualize the cation binding sites in biopsied nerves of four patiens with Charcot-Marie-Tooth disease type 1A (CMT1A) and two patients with hereditary neuropathy with liability to pressure palsies (HNPP), and the results were compared with those of four patients having acquired neuropathies with normal PMP-22 expression. In CMT1A, nodal widening or paranodal demyelination was associated with dense precipitates focally on both sides of the widened node. Although fainter precipitates were present at the node between remyelinated internodes, the percentage of nodes exhibiting the reaction product between normal and remyelinated internodes was not statistically different from that between normal internodes in CMT1A. In acquired neuropathies, on the other hand, the difference was significant between the two (P < 0.05), with reduction between normal and remyelinated internodes. At the nodes neighboring demylinated internodes, the percentage of nodes exhibiting the reaction product was reduced significantly in both CMT1A and acquired neuropathies, but to a lesser degree in CMT1A. Precipitates were clearly seen at the nodes neighboring a tomaculum in HNPP. The results suggest that preserved cation binding at the node may allow nerves to keep the electrical excitability in CMT1A and HNPP where myelin remodeling takes place at high frequency. Received: 6 June 1995 / Revised: 28 September 1995 / Revised, accepted: 29 November 1995     

Differential expression of intercellular adhesion molecule-1 (ICAM-1) in the A‚-containing lesions in brains of patients with dementia of the Alzheimer type

Inflammatory processes have been implicated in the formation of senile plaques in the cerebral cortex of patients with dementia of the Alzheimer type (DAT), since several inflammation-induced proteins are present within these plaques. The relation between inflammatory components and other amyloid β protein (Aβ)-containing lesions of the DAT brain [cerebrovascular amyloidosis (CA) and cerebellar senile plaques] is unclear. We studied the distribution of the inflammation-inducible protein intercellular adhesion molecule-1 (ICAM-1) in CA and in senile plaques of the cerebellum, using an immunohistochemical approach. We observed striking differences in ICAM-1 reactivity between the different types of Aβ-containing lesions. ICAM-1 was only expressed in classic senile plaques in the granular and Purkinje cell layer of the cerebellum, and not in diffuse senile plaques of the molecular layer. Also, ICAM-1 was not associated with CA; only when the vascular amyloid extended into the neuropil (dyshoric angiopathy) was perivascular ICAM-1 reactivity observed. This is in contrast to the putative primary involvement of inflammation in the formation of cerebrocortical classic and diffuse senile plaques. Our findings indicate that ICAM-1 expression, which may be an indicator of an inflammatory reaction, is induced in the neuropil depending on the specific site of Aβ production. Received: 2 October 1995 / Revised, accepted: 4 December 1995     

The α-chemokine CXCL14 is up-regulated in the sciatic nerve of a mouse model of Charcot–Marie–Tooth disease type 1A and alters myelin gene expression in cultured Schwann cells

At present the pathogenesis of CMT1A neuropathy, caused by the overexpression of PMP22, has not yet been entirely understood. The PMP22-overexpressing C61 mutant mouse is a suitable animal model, which mimics the human CMT1A disorder. We observed that myelin gene expression in the sciatic nerve of the C61 mouse was up-regulated at postnatal day 4 to 7 (P4–P7). When investigating the morphology of peripheral nerves in C61 and wildtype mice at early stages of postnatal development, hypermyelination could be detected in the femoral quadriceps and sciatic nerve of transgenic animals at postnatal day 7 (P7). In order to identify genes, other than Pmp22, that are modulated in sciatic nerve of P7 transgenic mice, we applied microarray technology. Amongst the regulated genes, the gene encoding the α-chemokine CXCL14 was most prominently up-regulated. We report that Cxcl14 was expressed exclusively by Schwann cells of the sciatic nerve, as well as by cultured Schwann cells triggered to differentiate. Furthermore, in cultured Schwann cells CXCL14 modulated the expression of myelin genes and altered cell proliferation. Our findings demonstrate that early overexpression of PMP22, in a mouse model of CMT1A, results in a strong up-regulation of CXCL14, which seems to play a novel regulatory role in Schwann cell differentiation.     

Quantitative assessment of muscle echogenicity in Charcot-Marie-Tooth disease type 1A by automatic thresholding methods

ObjectiveTo investigate the utility of automatic thresholding methods for quantitative muscle echogenicity assessment as a marker of disease severity in Charcot-Marie-Tooth disease type 1A (CMT1A).MethodsMuscle ultrasound was performed in 15 CMT1A patients and 7 healthy controls. Muscle echogenicity of six limb muscles in each subject was assessed by 16 automatic thresholding methods and conventional grey-scale analysis. Echogenicity of each method in CMT1A patients was compared with that in controls. A correlation between the echogenicity and CMT neuropathy score (CMTNS) was also analysed in CMT1A patients.ResultsSignificant differences in mean echogenicity of the 6 muscles between CMT1A patients and controls were found both in grey-scale analysis (p < 0.01) and 11 of the 16 automatic thresholding methods (p < 0.05 in each method). In CMT1A patients, mean echogenicity of the 6 muscles was positively correlated with CMTNS in 8 of the 16 automatic thresholding methods, but not in grey-scale analysis.ConclusionAutomatic thresholding methods can be used to detect the difference in muscle echogenicity between CMT1A patients and controls. Echogenicity parameters correlate with the disease severity.SignificanceQuantitative muscle echogenicity assessment by automatic thresholding methods shows potential as a surrogate marker of disease progression in CMT1A.     

Influence of DRD2 and ANKK1 genotypes on apomorphine-induced growth hormone (GH) response in alcohol-dependent patients

Background

D₂ receptor function can be assessed by growth hormone (GH) response to apomorphine. Several association studies between dopamine receptor polymorphisms and results of the apomorphine challenge test with normal and alcohol-dependent subjects yielded inconsistent results. In this pilot study, we tested polymorphisms from the DRD2 region for GH response to apomorphine challenge in more detail.

Methods

Apomorphine challenge tests measuring GH responses on 5 time points were performed on day 1 of alcohol detoxification in 43 patients with alcohol dependence; patients were genotyped for 11 polymorphisms including DRD2, ANKK1, NCAM1 and TTC12.

Results

Associations (p < 0.05) were found for ANKK1 (rs11604671, rs1800497) and DRD2 (rs6276, rs1076560), which are located on adjacent chromosomal positions. Consistent with PET studies suggesting a reduced D₂ receptor availability in patients carrying the ANKK1 rs1800497 T polymorphism (formerly known as DRD2 TaqI A1) we found a reduced GH response to apomorphine in those subjects.

Conclusion

This has been the first study showing significant associations between apomorphine-induced GH response and SNPs in DRD2 and ANKK1 in alcohol-dependent patients. In this respect, our preliminary results are in line with other reports which suggested that DRD2 and ANKK1 polymorphisms influence D₂ receptor availability and signal transduction in the dopaminergic pathways. Small sample size in our study limits the generalizability of our results.