New alternative splicing variants of the ATXN2 transcript.

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells and selective loss of neurons in the brainstem. This neurodegenerative disorder is caused by the expansion of a polyglutamine domain in ataxin-2. Ataxin-2 is composed of 1312 amino acids, has a predicted molecular weight of 150-kDa and is widely expressed in neuronal and non-neuronal tissues. To date, the putative functions of ataxin-2 on mRNA translation and endocytosis remain ill-defined. Differential splicing with a lack of exons 10 and 21 was described in humans, and additional splicing of exon 11 in mice. In this study, we observed that the molecular size of transfected full-length wild-type ataxin-2 (22 glutamines) is different from endogenous ataxin-2 and that this variation could not be explained by the previously published splice variants alone. METHODS: Quantitative immunoblots and qualitative reverse-transcriptase polymerase-chain-reaction (RT-PCR) were used to characterize isoform variants, before sequencing was employed for validation. RESULTS: We report the characterization of further splice variants of ataxin-2 in different human cell lines and in mouse and human brain. Using RT-PCR from cell lines HeLa, HEK293 and COS-7 throughout the open reading frame of ataxin-2 together with PCR-sequencing, we found novel splice variants lacking exon 12 and exon 24. These findings were corroborated in murine and human brain. The splice variants were also found in human skin fibroblasts from SCA2 patients and controls, indicating that the polyglutamine expansion does not abolish the splicing. CONCLUSIONS: Given that Ataxin-2 interacts with crucial splice modulators such as TDP-43 and modulates the risk of Amyotrophic Lateral Sclerosis, its own splice isoforms may become relevant in brain tissue to monitor the RNA processing during disease progression and neuroprotective therapy.

Ataxin-2 modulates the levels of Grb2 and SRC but not ras signaling.

Ataxin-2 (ATXN2) is implicated mainly in mRNA processing. Some ATXN2 associates with receptor tyrosine kinases (RTK), inhibiting their endocytic internalization through interaction of proline-rich domains (PRD) in ATXN2 with SH3 motifs in Src. Gain of function of ATXN2 leads to neuronal atrophy in the diseases spinocerebellar ataxia type 2 (SCA2) and amyotrophic lateral sclerosis (ALS). Conversely, ATXN2 knockout (KO) mice show hypertrophy and insulin resistance. To elucidate the influence of ATXN2 on trophic regulation, we surveyed interactions of ATXN2 with SH3 motifs from numerous proteins and observed a novel interaction with Grb2. Direct binding in glutathione S-transferase (GST) pull-down assays and coimmunoprecipitation of the endogenous proteins indicated a physiologically relevant association. In SCA2 patient fibroblasts, Grb2 more than Src protein levels were diminished, with an upregulation of both transcripts suggesting enhanced protein turnover. In KO mouse embryonal fibroblasts (MEF), the protein levels of Grb2 and Src were decreased. ATXN2 absence by itself was insufficient to significantly change Grb2-dependent signaling for endogenous Ras levels, Ras-GTP levels, and kinetics as well as MEK1 phosphorylation, suggesting that other factors compensate for proliferation control. In KO tissue with postmitotic neurons, a significant decrease of Src protein levels is prominent rather than Grb2. ATXN2 mutations modulate the levels of several components of the RTK endocytosis complex and may thus contribute to alter cell proliferation as well as translation and growth.

ATXN2-CAG42 sequesters PABPC1 into insolubility and induces FBXW8 in cerebellum of old ataxic knock-in mice.

Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)(31). This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissue-specific effects, which may be partially alleviated by the induction of FBXW8.

Ataxin-2 associates with rough endoplasmic reticulum.

Ataxin-2 is a novel protein, normally with a domain of 22 consecutive glutamine (Q) residues, which may expand beyond a threshold of (Q)(32), causing a neurodegenerative disease named Spinocerebellar ataxia type 2 (SCA2). To obtain clues about the functions of ataxin-2, we used fluorescence microscopy and centrifugation fractionation analyses. Immunocytochemical detection in non-neuronal and neuronal cells showed endogenous and transfected ataxin-2 distributed throughout the cytoplasm, with perinuclear preference and a granular appearance. Triple-labelling and confocal microscopy demonstrated co-localisation with the endoplasmic reticulum (ER) markers calreticulin, calnexin and CFP-ER. The pathogenic form of ataxin-2 with an expanded polyQ domain showed the same distribution pattern. Subcellular fractionation of mouse brain homogenates showed endogenous ataxin-2 associated with rough ER (rER) membranes, in a manner dependent on RNA, salt and phosphorylation. Our data are in agreement with recent findings that ataxin-2 directly interacts with poly(A)-binding protein (PABP), thus associating with polyribosomes under normal conditions and being recruited to stress granules under environmental stress. These data, in conjunction with the presence of Lsm domains within ataxin-2, suggest that ataxin-2 is involved in the processing of mRNA and/or the regulation of translation.

Ataxin-2 associates with the endocytosis complex and affects EGF receptor trafficking.

Ataxin-2 is a novel protein, where the unstable expansion of an internal polyglutamine domain can cause the neurodegenerative disease Spinocerebellar Ataxia type 2 (SCA2). To elucidate its cellular function, we have used full-length ataxin-2 as bait in a yeast two-hybrid screen of human adult brain cDNA. As binding partners we found endophilin A1 and A3, two brain-expressed members of the endophilin A family involved in synaptic vesicle endocytosis. Co-immunoprecipitation studies confirmed the binding of these proteins as an endogenous complex in mouse brain. In vitro binding experiments narrowed the binding interfaces down to two proline-rich domains on ataxin-2, which interacted with the SH3 domain of endophilin A1/A3. Ataxin-2 and endophilin associated at the endoplasmic reticulum as well as at the plasma membrane as determined by immunofluorescence microscopy of transfected cell lines, and by centrifugation fractionation studies of mouse brain. Importantly, the pattern observed in transfected cells was conserved in rat hippocampal neurons. In the mouse brain, an association of ataxin-2 with endocytic proteins such as the adaptor CIN85 and the ubiquitin ligase c-Cbl was also demonstrated. GST pull-down assays showed ataxin-2 to directly interact with the SH3 domains A and C of CIN85 and with the SH3 domain of Src, a kinase activated after receptor stimulation. Functional studies demonstrated that ataxin-2 affects endocytic trafficking of the epidermal growth factor receptor (EGFR). Taken together, these data implicate ataxin-2 to play a role in endocytic receptor cycling.

Insulin receptor and lipid metabolism pathology in ataxin-2 knock-out mice.

Ataxin-2 is a cytoplasmic protein, product of the SCA2 gene. Expansion of the normal polyglutamine tract in the protein leads to the neurodegenerative disorder Spino-Cerebellar Ataxia type 2 (SCA2). Although ataxin-2 has been related to polyribosomes, endocytosis and actin-cytoskeleton organization, its biological function remains unknown. In the present study, an ataxin-2 deficient mouse (Sca2(-/-)) was generated to investigate the functional role of this protein. Homozygous mice exhibited reduced fertility and locomotor hyperactivity. In analyses up to the age of 6 months, the absence of ataxin-2 led to abdominal obesity and hepatosteatosis. This was associated with reduced insulin receptor expression in liver and cerebellum, although the mRNA levels were increased indicating a post-transcriptional effect of ataxin-2 on the insulin receptor status. As in insulin resistance syndromes, insulin levels were increased in pancreas and blood serum. In the cerebellum, increased levels of gangliosides and sulfatides, as well as decreased cholesterol dynamics, may be relevant for cellular membrane functions, and alterations in the sphingomyelin cycle may affect second messengers. Thus, the data suggest altered signaling in ataxin-2 deficient organisms.

Ataxin-2 promotes apoptosis of human neuroblastoma cells.

Neuroblastoma is a highly heterogeneous tumor of young children. Although many advances have been made towards understanding the molecular mechanisms dictating the phenotypic heterogeneity, the prognosis of children with neuroblastoma, particularly of progressively growing variants, has remained dire. About 10% of neuroblastomas regress spontaneously, probably by apoptosis, while another 20% have amplified the MYCN gene resulting in a poor prognosis. In pursuit of identifying cell death-associated genes in neuroblastoma, we encountered the SCA2 gene, coding for ataxin-2, as an important player. Here, we report that enforced expression of wild-type ataxin-2, but not of mutant ataxin-2, sensitizes neuroblastoma cells for apoptosis. In line with this, higher levels of ataxin-2 were detected in apoptotic cells compared to nonapoptotic cells. Neuroblastoma tumors with amplified MYCN contain significantly less ataxin-2 protein than tumors without amplified MYCN. Collectively, our data suggest that ataxin-2 has an important role in regulating the susceptibility of neuroblastoma cells to apoptotic stimuli in vitro and in vivo.

[The effect of farnesyl methyl ether on molting rhythm and testes development inEphestia kühniella. Studies in vivo and in vitro]. / Der Einflu\ von FarnesylmethylÄther auf die HÄutungsfolge und die Hodenentwicklung beiEphestia kühniella. Untersuchungen in vivo und in vitro.

Oral application of farnesyl methyl ether (FME), which allows for a continuous supply, inhibited the pupal differentiations of the integument. Animals fed during the course of the last larval instar reached the stage of grown-up feeding larvae, but did not enter the pharate pupal phase. By this way pupation could practically be delayed for any length of time. When the FME was administered from the first day of this instar a supernumerary larval molt occurred in some animals. A further molt, however, did not take place. An earlier treatment increased the number of superlarvae. Beginning the exposure to FME at the end of the last larval instar, some larval-pupal intermediates could be obtained with predominantly larval characteristics.When the continuous application was discontinued and the animals were transferred to FME-free food, a normal pupation took place.In the testes the metamorphosis changes of fusion and torsion were similarly inhibited by the juvenile hormone analogue.Furthermore, FME influenced testis growth. A dilution series was tested on two age groups (1- or 7-day-old last instar larvae). On transforming the data into the logarithms, two negative linear dose-response relationships were obtained. The regression analysis showed that both sets of data fitted simple straight-line models. The regression curves could be regarded as parallel whereby the older animals had higher testis size values at a given dose. However, compared with the size at the beginning of the FME-application, in the older animals a real decrease in volume took place.When the animals were transferred to untreated food, their testes became normal in size, fused and twisted.The dose-response relationship could be verified in vitro. Here too, a negative linear regresion of the log (testis size) on the log (FME-dose) was found. The regression coefficient did not differ significantly from that calculated from the in vivo data. Hence, FME acts directly on the testis sheath and does not control the organ size by alterations of the hemolymph.Testes will fuse autonomously if they have passed the critical period of this process. Culturing organs after this period on a FME-containing medium, they fused after one day despite the presence of the juvenile hormone analogue. However, with further incubation these fused partners separated. Testes which were already fused at the beginning of the incubation remained together. This indicates that there is a period of modifiable surface properties of the testes during which a fusion can be reversed by FME.The process of torsion does not show such a reversibility.

[The humoral control of testis development ofEphestia kühniella during metamorphosis]. / Die humorale Kontrolle der Hodenentwicklung vonEphestia kühniella in der Metamorphose.

1. The testes ofEphestia kühniella go through two morphogenetic processes during metamorphosis: fusion of both organs in the prepupa (stage A3) and torsion in the early pupal phase (day 1-5; 23° C). 2. By ligating behind the thorax the critical periods of these processes have been determined: for fusion the transition to prepupal phase, for torsion the second half (A3-A4) of this developmental stage. 3. The humoral conditions during prepupal phase do not enable testis to make an autonomous torsion. This process is connected to a pupal milieu. 4. When testes from donors, which have not yet passed the critical periods of fusion and torsion, are directly transplanted into pupae differentiation will only occur, if they are exposed to the influence of secretions of endocrine organs during this stage. The effectiveness of the morphogenetic factors decreases with aging of this stage and is no more detectable towards its end. 5. Testes of various ages show different responsiveness to an effective humoral milieu: the younger the grafts the later they respond. Investigations with progressively younger larvae, however, did not yield a threshold of competence until the 3. instar (younger larvae withdrew from operation because of their small size). 6. On the base of these results a conception of the control of testis differentiation during metamorphosis is traced. It is suggested that beside the stimulus of prothoracic gland hormone during the prepupal stage a second, pupal factor is necessary for torsion.