RESUMO
We cloned four novel transcripts of the excitatory amino acid transporter 2, named EAAT2/3UT1-4, resulting from differential cleavage and polyadenylation. Tandem poly (A) sites were found to be functional at 72, 654, 973 nucleotides and more than 2 kb downstream of the stop codon. A tissue-specific expression was identified for 3'-variants of the EAAT2 RNA, most prominently for EAAT2/3UT4 (hippocampus>cortex>>cerebellum>thalamus) as demonstrated by Northern blot analysis and quantitative PCR. We conclude, that alternative poly (A) selection may contribute to the reported differential EAAT2 protein expression under normal and diseased conditions.
Assuntos
Química Encefálica/genética , RNA Mensageiro/genética , Receptores de Neurotransmissores/genética , Sequência de Bases , Northern Blotting , Clonagem Molecular , Primers do DNA , Transportador 2 de Aminoácido Excitatório , Expressão Gênica/genética , Humanos , Dados de Sequência Molecular , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/análise , Análise de Sequência de DNARESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Transcrição Gênica , Transportadores de Cassetes de Ligação de ATP/genética , Processamento Alternativo , Sistema X-AG de Transporte de Aminoácidos , Transporte Biológico , Clonagem Molecular , DNA Complementar/metabolismo , Éxons , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossínteseRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/genética , Variação Genética , Splicing de RNA , RNA/genética , Receptores de Neurotransmissores/genética , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Cadáver , Transportador 2 de Aminoácido Excitatório , Humanos , Córtex Motor/metabolismo , RNA/metabolismo , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Stem and progenitor cells provide a promising therapeutic strategy for amyotrophic lateral sclerosis (ALS). To comparatively evaluate the therapeutic potentials of human bone marrow-derived mesodermal stromal cells (hMSCs) and umbilical cord blood cells (hUBCs) in ALS, we transplanted hMSCs and hUBCs and their neuroectodermal derivatives (hMSC-NSCs and hUBC-NSCs) into the ALS mouse model over-expressing the G93A mutant of the human SOD1 gene. We used a standardized protocol similar to clinical studies by performing a power calculation to estimate sample size prior to transplantation, matching the treatment groups for gender and hSOD-G93A gene content, and applying a novel method for directly injecting 100,000 cells into the CSF (the cisterna magna). Ten days after transplantation we found many cells within the subarachnoidal space ranging from frontal basal cisterns back to the cisterna magna, but only a few cells around the spinal cord. hMSCs and hMSC-NSCs were also located within the Purkinje cell layer. Intrathecal cell application did not affect survival times of mice compared to controls. Consistently, time of disease onset and first pareses, death weight, and motor neuron count in lumbar spinal cord did not vary between treatment groups. Interestingly, transplantation of hMSCs led to an increase of pre-symptomatic motor performance compared to controls in female animals. The negative outcome of the present study is most likely due to insufficient cell numbers within the affected brain regions (mainly the spinal cord). Further experiments defining the optimal cell dose, time point and route of application and particularly strategies to improve the homing of transplanted cells towards the CNS region of interest are warranted to define the therapeutic potential of mesodermal stem cells for the treatment of ALS.
Assuntos
Esclerose Lateral Amiotrófica/terapia , Movimento Celular/fisiologia , Coluna Vertebral/fisiologia , Transplante de Células-Tronco , Envelhecimento/fisiologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Contagem de Células , Sobrevivência Celular , Cisterna Magna/fisiologia , Progressão da Doença , Humanos , Camundongos , Camundongos Transgênicos , Desempenho Psicomotor/fisiologia , Tamanho da Amostra , Espaço Subaracnóideo/fisiologia , Superóxido Dismutase/genéticaRESUMO
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.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Astrócitos/metabolismo , Astrocitoma , Neoplasias Encefálicas , Transportador 2 de Aminoácido Excitatório/genética , Genes Reguladores/genética , Splicing de RNA/genética , Processamento Alternativo/genética , Astrócitos/patologia , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Expressão Gênica/genética , Variação Genética , Humanos , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.