Eberhard Scherzinger

Formation of Neuronal Intranuclear Inclusions Underlies the Neurological Dysfunction in Mice Transgenic for the HD Mutation

Huntingtons disease (HD) is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. We have observed that mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients.

Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.

The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntingtons disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimers disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.

Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010

We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.

SH3GL3 Associates with the Huntingtin Exon 1 Protein and Promotes the Formation of Polygln-Containing Protein Aggregates

The mechanism by which aggregated polygins cause the selective neurodegeneration in Huntingtons disease (HD) is unknown. Here, we show that the SH3GL3 protein, which is preferentially expressed in brain and testis, selectively interacts with the HD exon 1 protein (HDex1p) containing a glutamine repeat in the pathological range and promotes the formation of insoluble polyglutamine-containing aggregates in vivo. The C-terminal SH3 domain in SH3GL3 and the proline-rich region in HDex1p are essential for the interaction. Coimmunoprecipitations and immunofluorescence studies revealed that SH3GL3 and HDex1p colocalize in transfected COS cells. Additionally, an anti-SH3GL3 antibody was also able to coimmunoprecipitate the full-length huntingtin from an HD human brain extract. The characteristics of the interaction between SH3GL3 and huntingtin and the colocalization of the two proteins suggest that SH3GL3 could be involved in the selective neuronal cell death in HD.

Aggregation of proteins with expanded glutamine and alanine repeats of the glutamine-rich and asparagine-rich domains of Sup35 and of the amyloid β-peptide of amyloid plaques

The exon-1 peptide of huntingtin has 51 Gln repeats and produces the symptoms of Huntingtons disease in transgenic mice. Aggregation of the yeast Sup35 protein into prions has been attributed to its glutamine-rich and asparagine-rich domain. Here, we show that poly-l-asparagine forms polar zippers similar to those of poly-l-glutamine. In solution at acid pH, the glutamine-rich and asparagine-rich 18-residue Sup35 peptide, rendered soluble by the addition of two aspartates at the amino end and two lysines at the carboxyl end, gives a β-sheet CD spectrum; it aggregates at neutral pH. A poly-alanine peptide D2A10K2 gives an α-helical CD spectrum at all pHs and does not aggregate; a peptide with the sequence of the C-terminal helix of the α-chain of human hemoglobin, preceded by two aspartates and followed by two lysines, exhibits a random coil spectrum and does not aggregate either. Alignment of several β-strands with the sequence of the 42-residue Alzheimers amyloid β-peptide shows that they can be linked together by a network of salt bridges. We also asked why single amino acid replacements can so destabilize the native structures of proteins that they unfold and form amyloids. The difference in free energy of a protein molecule between its native, fully ordered structure and an amorphous mixture of randomly coiled chains is only of the order of 10 kcal/mol. Theory shows that destabilization of the native structure by no more than 2 kcal/mol can increase the probability of nucleation of disordered aggregates from which amyloids could grow 130,000-fold.

Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates.

Publisher Summary The accumulation of polyglutamine-containing protein aggregates in neuronal intranuclear inclusions (NIIs) has been demonstrated for several progressive neurodegenerative diseases such as Huntingtons disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), and spinocerebellar ataxia (SCA) types 1, 3, and 7. Furthermore, it has been shown in vitro that the proteolytic cleavage of fusion proteins of glutathione S-transferase (GST) and the polyglutamine-containing huntingtin peptide coded for by the first exon of the HD gene s leads to the formation of insoluble high molecular weight protein aggregates with a fibrillar or ribbonlike morphology reminiscent of β -amyloid fibrils in Alzheimers disease and scrapie prion rods. This chapter demonstrates that the cellulose acetate filter retardation assay can be a useful tool for the identification, structural characterization, and quantification of SDS-insoluble polyglutamine-containing protein aggregates formed in vitro and in vivo. In addition to the histochemical identification of amyloids, it useful in detecting insoluble protein aggregates in all types of human and animal amyloidoses, including the polyglutamine diseases, and also in screening compound libraries for potential aggregation inhibitors. Currently, attempts to develop a microtiter plate-based high-throughput filter retardation assay to identify chemical compounds that slow down the rate of formation of polyglutamine-containing fibrils in vitro are in progress. The amyloid-binding agents arising from this screen then will be tested in a HD cell culture model system and in the HD animal model for their therapeutic potential.

Stimulation of T7 DNA polymerase by a new phage-coded protein.

SummaryA bacteriophage-induced DNA-binding protein was purified from T7 infected E. coli. The protein has a molecular weight of about 25000, as judged by SDS-polyacrylamide gel electrophoresis. The purified protein binds to single-stranded but not to native T7 DNA. Like the T4 gene-32 protein and the 22000-dalton “unwinding protein” of E. coli, the T7 25000 protein lowers the melting temperature of poly d(A-T). Using partially single-stranded T7 DNA as template-primer, the protein stimulates in vitro DNA synthesis by T7 DNA polymerase about five-fold. It was also found that the DNA-unwinding protein of E. coli stimulates T7 DNA polymerase to approximately the same extent. However, neither of the unwinding proteins stimulate DNA polymerase I of E. coli.

Detection of polyglutamine aggregation in mouse models

Publisher Summary The first steps in the molecular pathogenesis of HD are beginning to be unraveled. The huntingtin protein is subjected to an initial cleavage step that releases an N-terminal fragment. The precise nature of this fragment and the cleavage mechanism are unknown, although caspase 3 has been proposed to fill this role. The N-terminal fragment can move into the nucleus, but once again, the mechanism by which this occurs is unknown. Nuclear inclusions have been described in juvenile onset HD with a frequency of 38–50% in cortical neurons, whereas dystrophic neurites (axonal inclusions) are reported to predominate in the adult form of the disease. Inclusions are observed in brain regions not traditionally associated with neuro-degeneration. Therefore, the presence of an inclusion does not necessarily lead to cell death and symptoms may be caused by dysfunction in brains regions not previously associated with HD. Dystrophic neurites were also identified in layer VI of the cortex of an individual who was presymptomatic for HD and had died of other causes. This one case suggests that, as in mice, inclusions may form prior to the onset of symptoms. Although the evidence is currently circumstantial, the early detection of inclusions suggests that they may be causative of the disease symptoms. A major application of the R6 transgenic lines is to test drugs that slow down or prevent poly(Q) aggregation. In order to demonstrate causality, it will be necessary to show that by slowing down or preventing the formation of inclusions, the onset of symptoms is correspondingly prevented or delayed. This chapter also provides protocols by which the appearance of aggregates can be monitored.

Replication Determinants of the Broad Host-Range Plasmid RSF1010

RSF1010 is a small (8.7 kb), multicopy plasmid of the incompatibility group IncQ that confers resistances to streptomycin and sulfonamide to its host cells (1). It is very similar or identical with the 2 other representatives of the IncQ group, R300 (2) and R1162 (3). One of the most striking properties of this plasmid is its extraordinary broad host-range among gram-negative bacteria. This feature has made it attractive as a replicon for construction of vectors for gene cloning outside of Escherichia coli species. Both general- and special-purpose cloning vectors, derived from RSF1010, have proved very useful for gene manipulation in different species of soil bacteria (for review, see Ref. 4,5).

Replication of the colicin E1 plasmid in extracts of Escherichia coli: Uncoupling of leading strand from lagging strand synthesis

SummaryThe replication of the ColE1 plasmid was studied in extracts from E. coli dnaG mutants. It was found that the synthesis of the complementary strands of ColE1 DNA can be carried out in these extracts in two consecutive steps: (1) synthesis of the leading L strand independent of the dnaG function, and (2) synthesis of the lagging H strand depending upon addition of wild-type dnaG protein. In contrast to L strand synthesis, the latter reaction is insensitive to rifampicin and novobiocin. Both synthetic pathways are however blocked by antiserum directed against dnaB protein. This indicates an additional role of the dnaB protein in duplex DNA replication besides assisting the dnaG protein in the priming of lagging strand synthesis. The T7 gene-4 protein acting in conjunction with T7 DNA polymerase can substitute for both the function of the dnaB and dnaG protein. It is concluded that plasmid replication proceeds by a semi-discontinuous mechanism.