'Tissue' transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease.

By crossing Huntington's disease (HD) R6/1 transgenic mice with 'tissue' transglutaminase (TG2) knock-out mice, we have demonstrated that this multifunctional enzyme plays an important role in the neuronal death characterising this disorder in vivo. In fact, a large reduction in cell death is observed in R6/1, TG2(-/-) compared with R6/1 transgenic mice. In addition, we have shown that the formation of neuronal intranuclear inclusions (NII) is potentiated in absence of the 'tissue' transglutaminase. These phenomena are paralleled by a significant improvement both in motor performances and survival of R6/1, TG2(-/-) versus R6/1 mice. Taken together these findings suggest an important role for tissue transglutaminase in the regulation of neuronal cell death occurring in Huntington's disease.

Early alterations in gene expression and cell morphology in a mouse model of Huntington's disease.

Several mouse models for Huntington's disease (HD) have been produced to date. Based on differences in strain, promoter, construct, and number of glutamines, these models have provided a broad spectrum of neurological symptoms, ranging from simple increases in aggressiveness with no signs of neuropathology, to tremors and seizures in absence of degeneration, to neurological symptoms in the presence of gliosis and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) positivity, and finally to selective striatal damage associated with electrophysiological and behavioral abnormalities. We decided to analyze the morphology of striatum and hippocampus from a mouse transgenic line obtained by microinjection of exon 1 from the HD gene after introduction of a very high number of CAG repeat units. We found a massive darkening and compacting of striatal and hippocampal neurons in affected mice, associated with a lower degree of more classical apoptotic cell condensation. We then explored whether this morphology could be explained with alterations in gene expression by hybridizing normal and affected total brain RNA to a panel of 588 known mouse cDNAs. We show that some genes are significantly and consistently up-regulated and that others are down-regulated in the affected brains. Here we discuss the possible significance of these alterations in neuronal morphology and gene expression.

A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice.

Huntington's disease (HD) is a dominant disorder characterized by premature and progressive neurodegeneration. In order to generate an accurate model of the disease, we introduced an HD-like mutation (an extended stretch of 72-80 CAG repeats) into the endogenous mouse Hdh gene. Analysis of the mutation in vivo reveals significant levels of germline instability, with expansions, contractions and sex-of-origin effects in evidence. Mice expressing full-length mutant protein display abnormal social behaviour in the absence of acute neurodegeneration. Given that psychiatric changes, including irritability and aggression, are common findings in HD patients, our data are consistent with the hypothesis that some clinical features of HD may be caused by pathological processes that precede gross neuronal cell death. This implies that effective treatment of HD may require an understanding and amelioration of these dysfunctional processes, rather than simply preventing the premature death of neurons in the brain. These mice should facilitate the investigation of the molecular mechanisms that underpin the pathway from genotype to phenotype in HD.

Mutation of the prion protein gene at codon 208 in familial Creutzfeldt-Jakob disease.

Four point mutations and one insertion within the prion protein (PrP) gene have been tightly linked to the development of inherited prion disease. We developed a denaturing gradient gel electrophoresis system that allowed us to screen the entire open reading frame of the PrP gene. Using this system, we found a new mutation of the PrP gene in a patient with pathologically confirmed Creutzfeldt-Jakob disease and a negative family history for dementia. DNA sequencing revealed an adenine substitution for guanine at the second position of codon 208, which results in the nonconservative substitution of histidine for arginine. The same PrP mutation was identified in another younger member of the pedigree but was not present in more than 200 alleles tested. Such findings suggest that the frequency of inherited prion disease might be higher than ascertained by clinical history alone.

NGF retards apoptosis in chick embryo bursal cell in vitro.

Recent studies have demonstrated that the action of nerve growth factor (NGF) is not restricted to neuronal cells but also affects cells of the immune system. In a previous work on the effect of NGF on the chick embryo bursa of Fabricius both in vivo and in vitro, we observed that NGF prolongs bursal cell survival in vitro. In the present study we report that the increase of viable cells in NGF-treated cultures is not due to a proliferative effect of NGF on bursal cells but to a reduction of cell mortality. The morphological analysis revealed that bursal cells in cultures die by apoptosis, which was also shown by the typical pattern of DNA fragmentation, a hallmark of this cell death process. It is concluded that NGF, with an action similar to that described in sympathetic neurons and PC12, could retard bursal cell death by influencing apoptosis.

An explanation for the neutral effect of DR2 on IDDM susceptibility in central Italy.

Susceptibility to IDDM is strongly associated with HLA. Some HLA allelic combinations (haplotypes) can be found in most patients, whereas other haplotypes are encountered only rarely. It has been proposed that this difference in susceptibility depends on the absence (in the DR3 and DR4 haplotypes) or the presence (in the DR2 haplotype) of Asp57 in the DQ beta-chain. Data on southern European populations challenge this hypothesis because the DR2 haplotype has not been associated negatively with IDDM, as reported in northern European populations. This study on a selected panel of DR2-positive Italian IDDM patients shows that 19 of 21 (90.5%) DR2 haplotypes possess a non-Asp57 DQB allele. Moreover, the same non-Asp57 subtype has a comparatively high frequency (9/28, or 32.1%, DR2 haplotypes) also in the DR2-positive healthy Italian population. The difference between patients and control subjects is significant (P less than 0.0001). This is the largest series of DR2-positive patients analyzed so far. Comparison with cumulated data in various white populations shows a distinct northern European-to-southern European gradient. Toward southern Europe, the relative frequency of the non-Asp57 DR2 subtype increases. Concomitantly, the apparent protective effect of the DR2 haplotype disappears. Therefore, the observed differences in DR2-IDDM association in white populations can be explained adequately by the Asp57 hypothesis, which this study's data strongly support.

Detection of complex alleles by direct analysis of DNA heteroduplexes.

DNA molecules derived from three alleles of the HLA-DRB3 locus and differing from each other at several nucleotide sites were denatured and cross-hybridized. Each allelic combination was found to generate a pair of heteroduplexes of different mobility. Their retardation as compared to homoduplexes was proportional to the number of mismatches. In each heteroduplexes pair the component possessing the highest number of Pyr-Pyr oppositions was the most retarded. The results are those predicted by a theoretical model implying a correlation between base-pair opening and bending of the DNA double helix. These observations introduce a new HLA typing method at the genomic level and indicate an experimental approach to the analysis of the superhelical DNA conformation as related to different types of base oppositions.

Arg74 in HLA-DRB1 and DRB3 controls a DR3-related epitope.

TR81 is a specificity closely related to or identical with DR3. In Caucasoids two amino acids, Tyr at position 26 and Arg at position 74 of HLA class II DR beta chains, have been found to be associated with the presence of TR81. Recently, a variant of DRB1*03 identified in American Blacks has been shown to possess Arg at position 74 but Phe at position 26. This codon combination is found to be present in four other cell lines where it still specifies the TR81 determinant. This suggests that the TR81 specificity is uniquely dependent on the presence of Arg at position 74.

DNA typing of an HLA-DR bilocus specificity by gene amplification and oligonucleotide hybridization.

The structure of the DRB1*03 gene has been interpreted as the product of a gene conversion event involving a DRB3 gene as donor and resulting in the introduction of two short segments of the DRB3 sequence into the DRB1 locus. The serological counterpart of this double insertion is the TR81 specificity. Consequently, the TR81-specifying sequences can reside on either DRB1 or DRB3, or on both loci. Within each of the two sequence stretches a single nucleotide may be responsible for the generation of the TR81 alloantigen. Oligonucleotide probes corresponding to these stretches and to their allelic variants were constructed. They were used, under stringent hybridization conditions, to detect TR81-specifying sequences in the DNA of HLA-homozygous cell lines carrying different haplotypes of the DRw52 family. Prior to hybridization the DNA was amplified with either DRB1-specific or DRB3-specific primers. Using this approach it was possible to perform a "DNA typing" of the TR81-specifying sites separately on both the DRB1 locus and the DRB3 locus.