Christoph Münch

Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS

The authors report mutation screening of the p150 subunit of dynactin (DCTN1) and the cytoplasmic dynein heavy chain (DNCHC1) genes in 250 patients with ALS and 150 unrelated control subjects. Heterozygous missense mutations of the DCTN1 gene were detected in one apparently sporadic case of ALS (T1249I), one individual with familial ALS (M571T), two patients with familial ALS, and two unaffected relatives in the same kindred (R785W). The allelic variants of the DCTN1 gene may represent a previously unknown genomic risk factor for ALS.

Heterozygous R1101K mutation of the DCTN1 gene in a family with ALS and FTD.

A heterozygous R1101K mutation of the p150 subunit of dynactin (DCTN1) is reported in a family with amyotrophic lateral sclerosis (ALS) and co‐occurrence of frontotemporal dementia (FTD). Two members of our kindred were affected with motor neuron disease and two with dementia in an autosomal dominant pattern of inheritance. We excluded the involvement of the ALS and FTD‐linked genes for copper/zinc superoxide dismutase (SOD1) and tau. The R1101K sequence alteration of the DCTN1 gene may predispose subjects to ALS and FTD. Ann Neurol 2005;58:777–780

The RNA of the glutamate transporter EAAT2 is variably spliced in amyotrophic lateral sclerosis and normal individuals

Impaired re-uptake of synaptic glutamate, and a reduced expression of the glutamate transporter EAAT2 have been found in the motor cortex of patients with amyotrophic lateral sclerosis (ALS). Two splice forms of the EAAT2 RNA resulting from retention of intronic sequences (EAAT2/Int) and deletion of one protein coding exon (EAAT2/C1) have been reported to account for the EAAT2 protein loss in ALS. In this study we investigated the presence of two known (EAAT2/C1; EAAT2/Int) and three novel (EAAT2/C2-4) EAAT2 RNA in motor cortex of 17 ALS cases and 11 controls. Reverse transcription and PCR were carried out to amplify the complementary DNA of the complete and variably spliced EAAT2 transcripts. Nested PCR was followed to generate amplicons specific for EAAT2/C1-4 and EAAT2/Int. EAAT2/Int was detected in 59% of ALS specimens as compared to 36% of controls showing a trend but no statistical significance of a more frequent expression in ALS (Type I error 24.6%). EAAT2/C1-4 were found to be equally expressed in ALS patients and controls. Our results indicate that the involvement of EAAT2 transcripts in ALS is unlikely to be primary, and more complex than previously recognized. Alterations of quantitative expression of distinct EAAT2 splice forms in ALS cannot be excluded from this study and remain to be investigated.

Alternative splicing of the glutamate transporter EAAT2 (GLT-1).

The human glutamate transporter EAAT2 (GLT-1) is of major importance for synaptic glutamate reuptake, and reportedly, a candidate gene for neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimers disease and epilepsy. Here we report the polymerase chain reaction (PCR) cloning of two novel EAAT2 transcripts, named EAAT2-C1 and EAAT2-C2, which originate from alternative splicing of the human EAAT2 gene. EAAT2-C1 results from skipping of the protein coding exon eight. In contrast, EAAT2-C2 is characterized by usage of internal splice sites in the exons five and six. The splicing events lead to a deletion of 45 and 107 amino acids, respectively, located in the C-terminal and central part of the putative protein.

Alternative splicing of the 5′‐sequences of the mouse EAAT2 glutamate transporter and expression in a transgenic model for amyotrophic lateral sclerosis

Glutamate‐mediated neurotoxicity and a reduced expression of the excitatory amino acid transporter 2 (EAAT2) have been described in the pathogenesis of several acute and chronic neurological conditions. EAAT2 is the major carrier of glutamate in the mammalian brain. However, the principles of EAAT2 expression regulation are not fully understood. For the human brain, extensive alternative splicing of the EAAT2 RNA has been shown. To delineate the complex RNA regulation of EAAT2 we investigated whether the murine species is a suitable model for the study of EAAT2 splicing events. We identified five splice variants (mEAAT2/5UT1–5) encoding different 5′‐untranslated sequences and two distinct N‐termini of the putative EAAT2 polypeptide. In the murine CNS we found a region‐specific expression pattern of the novel 5′‐variants of EAAT2 as shown by in situ hybridization, dot blotting and competitive reverse transcription polymerase chain reaction. Furthermore, we performed an expression analysis of the EAAT2 splice variants in the spinal cord of a transgenic model (SOD1G93A) of amyotrophic lateral sclerosis, a motor neurone disease for which altered splicing of EAAT2 has been discussed. We found an increased expression of mEAAT2/5UT4 and a reduction of mEAAT2/5UT5 in the early course of the disease. We conclude that alternative splicing of 5′‐sequences may contribute to the regional expression of the EAAT2 RNA and was altered in the pre‐symptomatic stage of the SOD1G93A‐mouse model for amyotrophic lateral sclerosis.

The EAAT2 (GLT-1) gene in motor neuron disease: absence of mutations in amyotrophic lateral sclerosis and a point mutation in patients with hereditary spastic paraplegia

OBJECTIVES To investigate if sequence alterations of the excitatory amino acid transporter gene EAAT2 (GLT-1) may be a contributory factor to the pathogenesis of motor system degeneration. EAAT2 serves as a candidate gene as its reduced expression was reported in patients with amyotrophic lateral sclerosis (ALS). Furthermore, neurolathyrism, a motor neuron disease clinically related to hereditary spastic paraplegia (HSP), has been associated with an exogenous excitotoxin. METHODS Sequence alterations were screened for in the coding region of EAAT2 in 55 patients with ALS and one family with autosomal dominant HSP (AD-HSP). RESULTS In ALS, no sequence alteration in the EAAT2 gene have been found. Interestingly, a heterozygous A79G mutation of the EAAT2 gene was detected in two of seven affected patients with AD-HSP in the same kindred. The absence of cosegregation with the familial disease showed that the detected variant was not the cause of disease. The A79G sequence variant was not found in 55 patients with ALS or in 50 non-neurological controls. CONCLUSION The allelic variant of the EAAT2 gene in conjunction with the primary gene defect may be a modifying factor for the highly variable AD-HSP phenotype.

Rituximab in chronic inflammatory demyelinating polyneuropathy associated with diabetes mellitus

The authors report a 57-year-old patient with chronic inflammatory demyelinating polyneuropathy (CIDP) associated with diabetes mellitus (DM) who was treated successfully with rituximab. The B lymphocyte suppression using rituximab was followed 4 weeks later by neurological improvement and a stable disease course of over 10 months. We suggest that rituximab may be a treatment option in CIDP increasingly less responsive to intravenous immunoglobulin, particularly in patients with concurrent DM.

Impaired RNA splicing of 5′-regulatory sequences of the astroglial glutamate transporter EAAT2 in human astrocytoma

A loss of the glutamate transporter EAAT2 has been reported in the neoplastic transformation of astrocytic cells and astrocytoma. The RNA expression of EAAT2 and five 5′-regulatory splice variants was investigated to identify alterations of the post-transcriptional EAAT2 gene regulation in human astrocytic tumours. Three known (EAAT2, HBGTII, and HBGTIIC) and two novel (EAAT2/3 and EAAT2/31) EAAT2 transcripts originating from alternative splicing of 5′-regulatory sequences were subject to an RNA expression analysis using reverse transcription and competitive PCR. Specimens of astrocytoma World Health Organisation (WHO) grade I-IV in 14 patients and control brain tissue obtained from three normal persons were studied. The main EAAT2 RNA was found to be equally expressed in normal human brain and astrocytic tumour samples. By contrast, the expression pattern of four 5′-variants of the transporter transcript was altered in the investigated series of astrocytoma compared with normal brain. HBGTII, HBGTIIC, and EAAT2/3 were amplified from seven and four tumours and one sample, respectively. EAAT2/31 was expressed in none of the tumour specimens studied. In conclusion,in astrocytic tumours of different histopathological grades there was a substantial reduction of RNA splicing events in EAAT2. The impairment of EAAT2 splicing indicates an altered expression which is not primarily involved in the tumorigenesis but may contribute to some biological properties of astrocytoma such as oedema, necrosis, and tumour related seizures.

Differential Regulation of 5 Splice Variants of the Glutamate Transporter EAAT2 in an In Vivo Model of Chemical Hypoxia Induced by 3-Nitropropionic Acid

Defective glutamate uptake has been implicated as a pathogenic event of neuronal damage related to cerebral ischemia and hypoxia. In several models of ischemia‐hypoxia, a reduced immunoreactivity and altered RNA expression of excitatory amino acid transporter 2 (EAAT2), the major excitatory amino acid transporter, have been reported. However, the gene regulation of EAAT2 under these conditions is incompletely understood. In this study, we investigated alternative splicing of EAAT2 in an in vivo mouse model of chemical hypoxia as induced by 3‐nitropropionic acid (3‐NP). The neurotoxin 3‐NP is an inhibitor of mitochondrial energy production. Furthermore, it is known to inhibit glutamate reuptake directly, representing at least one of the mechanisms responsible for 3‐NP‐induced neurodegeneration. Here we report an expression analysis of five known (mEAAT2/5UT1–5) and two novel (mEAAT2/5UT6, ‐7) 5′ splice variants of EAAT2 using semiquantitative PCR. The RNA expression was studied at 2, 12, 24, 48, and 72 hr and 7 days after 3‐NP administration. mEAAT2/5UT4 and mEAAT2/5UT5 were up‐regulated in the frontal cortex and down‐regulated in the hippocampus 12–72 hr after chemical hypoxia. In the cerebellum, there was an increased expression of mEAAT2/5UT4 and a down‐regulation of mEAAT2/5UT5. mEAAT2/5UT3 show a different regional expression pattern, being regulated in the cerebellum only. mEAAT2/5UT1–7 encoded distinct 5′ regulatory sequences, including conserved elements of translational control. It is easily conceivable that expression alterations of 5′ splice variants of EAAT2 are related to glutamate transporter malfunction after chemical hypoxia. Our findings contribute to the hypothesis that RNA splicing events can serve as a molecular mechanism of posthypoxic gene regulation.

5'-heterogeneity of the human excitatory amino acid transporter cDNA EAAT2 (GLT-1).

TWO novel transcripts of the human excitatory amino acid transporter EAAT2 (GLT-1) were cloned using PCR cloning techniques. Comparative sequence analysis of the 5′-untranslated region (UTR) of five EAAT2 transcripts led to the definition of five putative 5′-untranslated exons that are combined in a variable manner. Alternative splicing is a likely mechanism for the 5′ complexity of the EAAT2 cDNA. The potential functional meaning of this finding such as differential expression of the EAAT2 transcripts in the central nervous system remains to be demonstrated.