Genetic analysis of the mouse brain proteome

Proteome analysis is a fundamental step in systematic functional genomics. Here we have resolved 8,767 proteins from the mouse brain proteome by large-gel two-dimensional electrophoresis. We detected 1,324 polymorphic proteins from the European collaborative interspecific backcross. Of these, we mapped 665 proteins genetically and identified 466 proteins by mass spectrometry. Qualitatively polymorphic proteins, to 96%, reflect changes in conformation and/or mass. Quantitatively polymorphic proteins show a high frequency (73%) of allele-specific transmission in codominant heterozygotes. Variations in protein isoforms and protein quantity often mapped to chromosomal positions different from that of the structural gene, indicating that single proteins may act as polygenic traits. Genetic analysis of proteomes may detect the types of polymorphism that are most relevant in disease-association studies.     

A critical role for the protein tyrosine phosphatase receptor type Z in functional recovery from demyelinating lesions

Several lines of evidence suggest that tyrosine phosphorylation is a key element in myelin formation, differentiation of oligodendrocytes and Schwann cells, and recovery from demyelinating lesions. Multiple sclerosis is a demyelinating disease of the human central nervous system, and studies of experimental demyelination indicate that remyelination in vivo requires the local generation, migration or maturation of new oligodendrocytes, or some combination of these. Failure of remyelination in multiple sclerosis could result from the failure of any of these processes or from the death of oligodendrocytes. Ptprz encodes protein tyrosine phosphatase receptor type Z (Ptpz, also designated Rptpbeta), which is expressed primarily in the nervous system but also in oligodendrocytes, astrocytes and neurons. Here we examine the susceptibility of mice deficient in Ptprz to experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We observe that mice deficient in Ptprz show impaired recovery from EAE induced by myelin oligodendrocyte glycoprotein (MOG) peptide. This sustained paralysis is associated with increased apoptosis of mature oligodendrocytes in the spinal cords of mutant mice at the peak of inflammation. We further demonstrate that expression of PTPRZ1, the human homolog of Ptprz, is induced in multiple sclerosis lesions and that the gene is specifically expressed in remyelinating oligodendrocytes in these lesions. These results support a role for Ptprz in oligodendrocyte survival and in recovery from demyelinating disease.     

Mutations in Rab3a alter circadian period and homeostatic response to sleep loss in the mouse

Rab3a is the most abundant Rab (ras-associated binding) protein in the brain and has a regulatory role in synaptic vesicle trafficking. Mice with a targeted loss-of-function mutation in Rab3a have defects in Ca(2+)-dependent synaptic transmission: the number of vesicles released in response to an action potential is greater than in wildtype mice, resulting in greater synaptic depression and the abolishment of CA3 mossy-fiber long term potentiation. The effect of these changes on behavior is unknown. In a screen for mouse mutants with abnormal rest-activity and sleep patterns, we identified a semidominant mutation, called earlybird, that shortens the circadian period of locomotor activity. Sequence analysis of Rab3a identified a point mutation in the conserved amino acid (Asp77Gly) within the GTP-binding domain of this protein in earlybird mutants, resulting in significantly reduced levels of Rab3a protein. Phenotypic assessment of earlybird mice and a null allele of Rab3a revealed anomalies in circadian period and sleep homeostasis, providing evidence that Rab3a-mediated synaptic transmission is involved in these behaviors.     

Mutant deoxynucleotide carrier is associated with congenital microcephaly

The disorder Amish microcephaly (MCPHA) is characterized by severe congenital microcephaly, elevated levels of alpha-ketoglutarate in the urine and premature death. The disorder is inherited in an autosomal recessive pattern and has been observed only in Old Order Amish families whose ancestors lived in Lancaster County, Pennsylvania. Here we show, by using a genealogy database and automated pedigree software, that 23 nuclear families affected with MCPHA are connected to a single ancestral couple. Through a whole-genome scan, fine mapping and haplotype analysis, we localized the gene affected in MCPHA to a region of 3 cM, or 2 Mb, on chromosome 17q25. We constructed a map of contiguous genomic clones spanning this region. One of the genes in this region, SLC25A19, which encodes a nuclear mitochondrial deoxynucleotide carrier (DNC), contains a substitution that segregates with the disease in affected individuals and alters an amino acid that is highly conserved in similar proteins. Functional analysis shows that the mutant DNC protein lacks the normal transport activity, implying that failed deoxynucleotide transport across the inner mitochondrial membrane causes MCPHA. Our data indicate that mitochondrial deoxynucleotide transport may be essential for prenatal brain growth.     

Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells

The mechanism of disease-associated trinucleotide repeat instability involves cis-acting factors (cis-elements) in the vicinity of the repeat, but the nature of these elements is unknown. One cis-element may be the location of the replication origin relative to the repeat. We have used an SV40 DNA replication system to investigate the effect of the location of replication initiation on (CTG)(n)*(CAG)(n) stability in primate cells. Depending on the distance between the SV40 replication origin and the repeat tract, templates with 79 repeats yield predominantly expansions or predominantly deletions or remain intact. All templates with 17 repeats are stable. Thus, cis-elements that affect the sites of Okazaki fragment initiation relative to the repeat are crucial determinants of instability. This model system recapitulates the bias for expansions observed in many of the diseases associated with trinucleotide repeats. Our results might explain the variable amounts of CTG/CAG instability that are observed in different chromosomal contexts.