Quantitative immunocytochemical analysis of the spinal cord in G86R superoxide dismutase transgenic mice: neurochemical correlates of selective vulnerability.

Transgenic mice with a G86R mutation in the mouse superoxide dismutase (SOD-1) gene, which corresponds to a mutation that has been observed in familial amyotrophic lateral sclerosis (ALS), display progressive loss of motor function and provide a valuable model of ALS. The pathology in the spinal cords of these mice was evaluated to determine whether there are chemically identified populations of neurons that are either highly vulnerable or resistant to degeneration. Qualitatively, there were phosphorylated neurofilament protein (NFP)-immunoreactive inclusions and a pronounced loss of motoneurons in the ventral horn of the spinal cord without the presence of vacuoles that has been reported in other SOD-1 transgenic mice. Neuron counts from SOD-1 and control spinal cords revealed that the percentage loss of NFP-, choline acetyltransferase (ChAT)-, and calretinin (CR)-immunoreactive neurons was greater than the percentage loss of total neurons, suggesting that these neuronal groups are particularly vulnerable in SOD-1 transgenic mice. In contrast, calbindin-containing neurons did not degenerate significantly and represent a protected population of neurons. Quantitative double-labeling experiments suggested that the vulnerability of ChAT- and CR-immunoreactive neurons was due primarily to the presence of NFP within a subset of these neurons, which degenerated preferentially to ChAT- and CR-immunoreactive neurons that did not colocalize with NFP. Our findings suggest that NFP, which has been demonstrated previously to be involved mechanistically in motoneuron degeneration, may also be important in the mechanism of degeneration that is initiated by the SOD-1 mutation.

Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder primarily involving motoneurons. A subset of individuals with familial autosomal dominant forms of the disease have mutations of the copper/zinc superoxide dismutase (Cu/Zn SOD, SOD-1) gene, which encodes a ubiquitously expressed enzyme that plays a key role in oxygen free radical scavenging. This observation suggests that altered or reduced SOD-1 activity may play a role in the neurodegenerative process. To explore this possibility further, we have introduced a mutation into the mouse SOD-1 gene that corresponds to one of the changes found in the human gene in familial amyotrophic lateral sclerosis. Integration and expression of this mouse gene in transgenic mice was identified by the presence of a unique restriction enzyme site in the transgene coding sequence generated by introduction of the mutation. We report here that high expression of this altered gene in the central nervous systems of transgenic mice is associated with an age-related rapidly progressive decline of motor function accompanied by degenerative changes of motoneurons within the spinal cord, brain stem, and neocortex. These findings indicate a causative relationship between altered SOD activity and motoneuron degeneration. Moreover, biochemical studies indicate normal levels of total SOD activity in transgenic mouse tissues, results that indicate that the neurodegenerative disorder does not result from a diminution of activity and, as such, represents a dominant "gain of function" mutation.

Molecular basis of five apolipoprotein B gene polymorphisms in noncoding regions.

Restriction fragment length polymorphisms (RFLPs) are useful in linkage and clinical association studies of human diseases. In this report, we characterize the molecular basis and frequencies of two new RFLPs, AvaII and BalI, two previously reported RFLPs, HincII and PvuII, and one new sequence polymorphism in the human apolipoprotein B gene. For the AvaII RFLP, the two alleles yield either a 1 kb fragment or 0.7 and 0.3 kb fragments, and have frequencies of 20% and 80%, respectively. The polymorphic site is about 4 kb upstream of exon 1. For the BalI RFLP, the two alleles yield either a 4.9 or 6.2 kb fragment, and have about equal frequencies. The polymorphic site is within an Alu sequence in intron 20, 146 bp 5' to exon 21. The BalI recognition sequence TGGCCA is replaced by TAGCCA. For the HincII RFLP, the two alleles yield either a 1.7 or 1.3 kb fragment and have frequencies of 80% and 20%, respectively. The polymorphic site is in intron 4, 171 bp 3' to exon 4. The HincII recognition sequence GTTAAC, present in the minor allele, is replaced by GTTACC. HincII fragments of 7.4 and 7.0 kb, previously reported for this polymorphism, are the result of partial digestion at the invariant HincII site in intron 3, 334 bp 3' to exon 3. For the PvuII RFLP, the two alleles yield either a 7.5 or 5.5 kb fragment and have frequencies of 96% and 4%, respectively. The polymorphic site is within an Alu sequence in intron 4, 523 bp 5' to exon 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypobetalipoproteinemia due to an apolipoprotein B gene exon 21 deletion derived by Alu-Alu recombination.

We report the molecular defect in an individual with homozygous hypobetalipoproteinemia. A unique TaqI restriction fragment length polymorphism was found in the midportion of the apolipoprotein B (apoB) gene using the genomic probe, pB51. The probe, which identifies TaqI fragments of 8.4 and 2.8 kilobases (kb) in normal individuals, hybridized to a single 11-kb fragment in the proband. The parents of the proband showed all three TaqI fragments, implying that they are heterozygotes for the mutant apoB allele. In this family, the mutant allele cosegregated with low total cholesterol levels and formal linkage analysis gave a decimal logarithm of the ratio score of 3.3 at a recombination frequency of 0. The polymorphic TaqI site was localized to an EcoRI fragment of 4 kb in normal individuals. The corresponding fragment in the proband was 3.4 kb, suggesting a 0.6-kb deletion in the mutant allele. Both the normal 4-kb EcoRI fragment and the mutant 3.4-kb EcoRI fragment were cloned and sequenced. In the normal allele, the 4-kb EcoRI fragment extends from intron 20 to 23. Exon 21 is flanked by Alu sequences that are in the same orientation. The mutant allele had a 694-bp deletion in this region which included a small part of the Alu sequence in intron 20, the entire exon 21, and most of the Alu sequence in intron 21. The polymorphic TaqI site, which lies within the Alu sequence in intron 21, was absent in the proband as a result of the deletion. The deletion of exon 21 results in a frame shift mutation and the introduction of a stop codon. Translation of the encoded mRNA would yield a prematurely terminated protein. This mutant apoB protein would be 1085 amino acids long with the 73 carboxyl-terminal residues out of frame. We postulate that the deletion of exon 21 is the consequence of a crossover event between the Alu sequences in introns 20 and 21 resulting in nonreciprocal exchange between two chromosomes.