Gisela Stoltenburg-Didinger

Fetal alcohol syndrome and mental retardation: spine distribution of pyramidal cells in prenatal alcohol-exposed rat cerebral cortex; a Golgi study.

Among the various possible effects of the fetal alcohol-syndrome (FAS), mental retardation can be considered the most deleterious. In animal studies, prenatal alcohol exposure has been shown to result in increased neonatal mortality, retarded cerebellar development and a significant decrease in neonatal brain weight. In a Golgi study on Wistar rats that were prenatally exposed to alcohol the spine distribution in proximal apical dendrites of layer V pyramidal cells of the parietal cortex was examined. As compared with controls, a distinct spine abnormality could be demonstrated at 12 days and at 40 days of postnatal age: a persistent predominance of long, thin and entangled spines, and a decreased number of normal stubby and mushroom-shaped spines. These abnormal dendritic patterns show a striking resemblance to those described by Purpura in mentally retarded children of normal karyotype.

Charcot-Marie-Tooth neuropathy type 2 and P0 point mutations: two novel amino acid substitutions (Asp61Gly; Tyr119Cys) and a possible "hotspot" on Thr124Met.

Mutations in the gene for the major protein component of peripheral nerve myelin, myelin protein zero (MPZ, P0), cause hereditary disorders of Schwann cell myelin such as Charcot‐Marie‐Tooth neuropathy type 1B (CMT1B), Dejerine‐Sottas syndrome (DSS), and congenital hypomyelinating neu‐ropathy (CHN). More recently, P0 mutations were identified in the axonal type of CMT neuropathy, CMT2, which is different from the demyelinating variants with respect to electroneurography and nerve pathology. We screened 49 patients with a clinical and histopathological diagnosis of CMT2 for mutations in the P0 gene. Three heterozygous single nucleotide changes were detected: two novel mis‐sense mutations, Asp61Gly and Tyr119Cys, and the known Thr124Met substitution, that has already been reported in several CMT patients from different European countries. Haplotype analysis for the P0 locus proved that our patients with the 124Met allele were not related to a cohort of patients with the same mutation, all of Belgian descent and all found to share a common ancestor (7).Our data suggest that P0 mutations account for a detectable proportion of CMT2 cases with virtually every patient harbouring a different mutation but recurrence of the Thr124Met amino acid substitution.The high frequency of this peculiar genotype in the European CMT population is presumably not only due to a founder effect but Thr124Met might constitute a mutation hotspot in the P0 gene as well.

Heterozygous myogenic factor 6 mutation associated with myopathy and severe course of Becker muscular dystrophy

Myogenic factors (MYF) belong to the basic helix-loop-helix (bHLH) transcription factor family and regulate myogenesis and muscle regeneration. The physiological importance of both functions was demonstrated in homozygous Myf knockout mice and mdx mice. Myf5 and Myod are predominantly expressed in proliferating myoblasts while Myf4 and Myf6 are involved in differentiation of myotubes. In a boy with myopathy and an increase of muscle fibres with central nuclei we detected a heterozygous 387G-->T nucleotide transversion in the MYF6 gene (MIM*159991). Protein-protein interaction of mutant MYF6 was reduced, and DNA-binding potential and transactivation capacity were abolished, thus demonstrating MYF6 haploinsufficiency. The boys father carried the identical mutation and, in addition, an in-frame deletion of exons 45-47 in his dystrophin gene. This mutation is normally associated with a mild to moderate course of Becker muscular dystrophy but the father suffered from a severe course of Becker muscular dystrophy suggesting MYF6 as a modifier.

Normal and Abnormal Development of the Human Cerebral Cortex

The functional organization of the developing human brain does not only differ substantially from that of the mature brain, but it also undergoes continuous changes. During fetal brain development, transient neuronal circuitries are formed which are essential for the subsequent development of mature projections. Transitory connections are linked to transient structures which are particularly prominent in the human fetal brain and are susceptible to damage under pathological circumstances. The First International Symposium on Normal and Abnormal Development of the Human Fetal Brain aimed to gather basic information on these transitory organizations and the major developmental events that occur in the fetal brain. This paper summarizes a roundtable discussion at the symposium on transient characteristics and injuries in the developing human cerebral cortex. An overview of cortical development is first presented and then the development of several fetal structures, including the subventricular zone, ganglionic eminence, marginal zone, subplate and cortical plate, is discussed with regard to their important contribution to the formation of the correct fiber projections in the adult. The last section focuses on the anomalies that are commonly found in the premature fetal brain on the one hand and on the other hand are related to the transitory characteristics of the developing brain.

Increased mRNA expression of tissue inhibitors of metalloproteinase-1 and -2 in Duchenne muscular dystrophy

In dystrophinopathies, disease severity is generally related to the extent of muscle fibrosis. To determine whether a decrease in matrix degradation contributes to the severe fibrosis seen in Duchenne muscular dystrophy (DMD), we quantified RNA transcript numbers for the fibrolytic matrix metalloproteinases (MMP)-1 and −2 and their natural tissue inhibitors (TIMP)-1 and −2 in DMD muscle as well as in pathological and normal controls. In addition, we investigated gelatinase (MMP-2) enzyme activity by zymography. We found an up-regulation of TIMP-1, TIMP-2 and MMP-2 RNA in DMD muscle. Zymography revealed an increase in MMP-2 activity in DMD muscle homogenates, which was absent in pathological and normal controls. Therefore, besides enhanced fibrogenesis, a disturbance of matrix degradation may play a significant role in muscle fibrosis in DMD. TIMP-1 should be investigated further as a promising target for pharmacological intervention to prevent muscle fibrosis in DMD.

Glial fibrillary acidic protein and RNA expression in adult rat hippocampus following low-level lead exposure during development

The astroglial cytoskeletal element, glial fibrillary acidie protein (GFAP), is a generally accepted sensitive indicator for neurotoxic effects in the mature brain. We used GFAP as a marker for structural changes in rat hippocampus related to chronic low level lead exposure during different developmental periods. Four groups of rats were investigated: a control group, a perinatal group, which was exposed during brain development (EO-P16), a permanent group, exposed during and after brain development (E0-P100), and a postweaning group, exposed after brain development (P16–P100). Sections were processed for light microscopy (hematoxylin-eosin, Nissl, periodic acid Schiff (PAS) and GFAP-specific immunohistology), for electron microscopy, and for in-situ hybridization (GFAP). Sections were prepared from animals tested for active avoidance learning (AAL) and long-term potentiation (LTP). Chronic lead exposure did not affect glial and neuronal functions, as assessed by LTP and AAL, when lead exposure started after brain development (postweaning group). In this group, astrocytes displayed increased GFAP and GFAP gene transcript levels. However, lead exposure affected neuronal and glial function when the intoxication fell into the developmental period of the brain (perinatal and permanent groups). In these groups, LTP and AAL were impaired, and astrocytes failed to react to the toxic exposure with an adequate increase of GFAP and GFAP gene transcripts. Although GFAP is an accepted marker for neurotoxicity, our data suggest the marker function of GFAP to be restricted to postnatal toxic insult.

Analysis of TP53 and PTEN in gliomatosis cerebri.

Gliomatosis cerebri (GC) is a rare glial neoplasm with extensive diffuse brain infiltration but relative preservation of the underlying architecture. Previous molecular studies, mostly analyzing biopsy samples, have suggested an astrocytic origin of GC, but a larger collective of autopsy tissue has not been investigated so far. Furthermore, whether the widespread neoplastic infiltration is based on a monoclonal process is still a matter of debate. In the present study, we screened paraffin-embedded brain tissue from different areas of 18 cases (8 autopsy cases and 10 biopsies) for alterations in the TP53 and PTEN genes. Nuclear accumulation of p53 protein was detected in 9 cases (50%). Somatic TP53 mutations occurred in two autopsy cases (11% of all cases). In the first case, a C→T transition in codon 273 (Arg→Cys) was detected in all tumor samples. In the second case, in tumor samples from one hemisphere, nuclear accumulation of p53 was caused by a G→A transition in codon 244 (Gly→Asp). In the present series, no mutations within the coding region of PTEN were found. Pten expression was observed in two autopsy cases (25%) and seven biopsy samples (70%). These data suggest that TP53 is affected in some cases, but other yet-unidentified genetic alterations might contribute to tumorigenesis in GC. Furthermore, although GC might be a monoclonal process, the presence of different tumor clones cannot be ruled out.

Effects of lipoic acid in hexacarbon-induced neuropathy

The effects of lipoic acid on hexacarbon neurotoxicity in rats were investigated. Rats were exposed by inhalation to n-hexane for 24 hours/day, 7 days/week, up to a total period of 9 weeks. Eight animals were exposed to 700 ppm n-hexane only, and eight animals were exposed to 700 ppm n-hexane and additionally received 100 mumol/kg lipoic acid PO daily. Clinical status of the animals was evaluated by examination of general condition, motor performance tests and neurophysiological measurements of caudal nerve motor conduction velocity. Results showed that animals exposed to 700 ppm n-hexane developed severe motor neuropathy leading to paralysis by the 6th week. Motor distal latencies of these animals were severely prolonged. In contrast, in animals treated with lipoic acid the onset of motor neuropathy was delayed for approximately 3 weeks as could be demonstrated by motor performance tests and measurements of motor distal latencies.

Alterations of cell cycle regulators in gliomatosis cerebri.

SummaryGliomatosis cerebri (GC) is regarded as a rare glial neoplasm of unknown origin, and a detailed analysis of molecular alterations underlying this disease has started only recently. However, because GC characteristically affects large parts of the brain and spinal cord, the distribution of genetic alterations may be highly variable between different tumor areas. Additionally, tumor areas with varying degrees of differentiation may be present, raising the possibility to model the genetic events associated with astrocytoma progression. Here we analyzed various tumor regions with features of low-grade and high-grade astrocytomas from 9 autopsy-proven GC cases for the immunoexpression of the cell cycle-controlling proteins mdm2, p21, p27/kip1, p16, and Rb. The samples were also screened for EGFR expression, and for amplification of the EGFR and MDM2 genes. Furthermore, allelic losses of the CDKN2A gene and of a PTEN flanking region of chromosome 10 were determined. We detected tumor regions with immunoexpression of p21 only rarely in our series, without association to the tumor grade. No MDM2 gene amplification was detected. In contrast, three cases demonstrated maintained Rb expression. The expression of p27kip1 showed a clear reduction with increasing astrocytoma malignancy in 7 cases. Allelic loss of the CDKN2A gene occurred in 5 patients but was not related to the tumor grading, nor to the intensity of p16 immunoexpression. No homozygous CDKN2Adeletions were detected. EGFR amplification was also absent in our series, but one case demonstrated EGFR expression only in the high-grade tumor area. Allelic losses on chromosome 10 were found in one out of six informative cases. However, marked differences in the immunoexpression, as well as in the distribution of genetic aberrations were seen between different tumor samples within a given case. The distribution of the alterations suggests that these molecular genetic changes represent secondary events, which may develop within tumor clones derived from a common founder tumor clone characterized by extraordinary spreading through the brain. Moreover, the detected aberrations in gliomatosis cerebri can reflect the tumor progression associated with secondary malignant astrocytoma formation even within a single case.

Prenatal methylmercury exposure results in dendritic spine dysgenesis in rats

To test the prenatal neurotoxic effect of low doses of methylmercury chloride (MM), the toxic agent was given by gavage to pregnant Wistar rats on days 6 to 9 after conception in doses of 0.025, 0.05, 0.5 and 5.0 mg/kg/day. The offspring of these animals were subjected to a routine developmental and behavioral testing battery. In essence, functional changes including impaired swimming behavior, increased auditory startle amplitude, increased passiveness and increased locomotor stereotypy compared to controls were the result of prenatal MM exposure, tested from the second to the seventh month postnatally. Further, in each group, ten prenatally treated rats were investigated histopathologically at the age of twelve months. The most striking effect was a distinct difference in the morphology of the dendritic spines of the pyramidal neurons in the somatosensory cortex demonstrated by Golgi impregnation. The spine abnormalities in the experimental animals consisted of a reduction of stubby and mushroom-shaped spines and a predominance of long and tortuous spines. Dendritic spine dysgenesis implies defective development and might be the pathological feature of the impaired behavior and learning of the MM-exposed animals.