Noise-Induced Dysregulation of Quaking RNA Binding Proteins Contributes to Auditory Nerve Demyelination and Hearing Loss.

Noise exposure causes auditory nerve (AN) degeneration and hearing deficiency, though the proximal biological consequences are not entirely understood. Most AN fibers and spiral ganglion neurons are ensheathed by myelinating glia that provide insulation and ensure rapid transmission of nerve impulses from the cochlea to the brain. Here we show that noise exposure administered to mice of either sex rapidly affects myelinating glial cells, causing molecular and cellular consequences that precede nerve degeneration. This response is characterized by demyelination, inflammation, and widespread expression changes in myelin-related genes, including the RNA splicing regulator Quaking (QKI) and numerous QKI target genes. Analysis of mice deficient in QKI revealed that QKI production in cochlear glial cells is essential for proper myelination of spiral ganglion neurons and AN fibers, and for normal hearing. Our findings implicate QKI dysregulation as a critical early component in the noise response, influencing cochlear glia function that leads to AN demyelination and, ultimately, to hearing deficiency.SIGNIFICANCE STATEMENT Auditory glia cells ensheath a majority of spiral ganglion neurons with myelin, protect auditory neurons, and allow for fast conduction of electrical impulses along the auditory nerve. Here we show that noise exposure causes glial dysfunction leading to myelin abnormality and altered expression of numerous genes in the auditory nerve, including QKI, a gene implicated in regulating myelination. Study of a conditional mouse model that specifically depleted QKI in glia showed that QKI deficiency alone was sufficient to elicit myelin-related abnormality and auditory functional declines. These results establish QKI as a key molecular target in the noise response and a causative agent in hearing loss.

Reproductive and neurological Quaking(viable) phenotypes in a severe combined immune deficient mouse background.

The quaking(viable) (qkv) mutation, a spontaneous deletion of a multigenic region encompassing roughly 1 Mb at 5.9 cM on the proximal end of mouse chromosome 17, causes severe trembling in all homozygous animals and infertility in all homozygous males. Physiologically, quaking mice exhibit dysmyelination and postmeiotic spermatogenic arrest. Molecular defects in Qkv mice occur in the affected tissues, indicating the primary causes of these pathologies are cell autonomous. However, because both the reproductive and neurological defects are in immune-privileged sites and because some similar pathologies at both sites have been shown to be immune mediated, we tested whether the immune system participates secondarily in manifestation of Qkv phenotypes. The qkv mutation was bred into a severe combined immune-deficient mouse line (SCID; devoid of mature B and T cells) and penetrance of the neurological and the male sterile phenotypes was measured. Results showed that neither defect was ameliorated in the immune-deficient background. We conclude that the Qkv pathologies do not likely involve a B- or T-cell-dependent response against these immune-privileged sites.

The neurological mutant quaking(viable) is Parkin deficient.

The mouse mutant quaking(viable) ( qk(v)) has been studied for almost four decades as a model for dysmyelination of the central nervous system (CNS). The genetic lesion associated with the qk(v) phenotype is a large deletion of approximately 1 Megabase on mouse Chromosome (Chr) 17. This deficiency alters the expression of transcripts from the qkI locus in oligodendrocytes, resulting in improper myelination of the CNS in animals homozygous for the deletion. To determine whether other genes within the deletion contribute to the quaking(viable) phenotype, we physically mapped and sequenced the deleted interval. We determined that the mouse Parkin gene, as well as the Parkin co-regulated gene ( Pacrg), lies within the qk(v) deletion. We determined that qk(v) mutants completely lack the expression of the Parkin gene product. Loss-of-function mutations in the human PARKIN gene cause autosomal juvenile Parkinson's disease (AR-JP). Our studies show that the deletion of Parkin in qk(v) brains does not result in the loss of dopaminergic neurons typical of AR-JP patients. Also, alpha-synuclein, a target of Parkin-dependent ubiquitination, does not accumulate in qk(v) mutant brains. Despite the lack of AR-JP-like neuropathology in qk(v) mice, this mutant may constitute a readily available model for the study of the cellular function of Parkin. This is the first report of a gene distinct from qkI affected by the qk(v) deletion. The discovery of the multigenic nature of this classical mouse mutation calls for the re-evaluation of its phenotypic characterization.

It's a double knock-out! The quaking mouse is a spontaneous deletion of parkin and parkin co-regulated gene (PACRG).

Mutations in the parkin gene (PRKN) are the commonest cause of juvenile and early-onset parkinsonism. However, the pathogenic mechanism by which loss of parkin protein results in degeneration of dopaminergic neurons remains elusive. Animal models provide a useful tool for the study of development and disease, and the recent production of transgenic fly and mouse parkin deficient models allows investigation of the molecular role of parkin in dopamine regulation and nigrostriatal function. We have identified the mouse mutant Quaking as a spontaneously occurring PRKN knockout. The quaking mutation is a deletion of approximately 1.17 Mb of mouse chromosome 17, resulting in the deletion of the entire promoter and first five coding exons of PRKN In addition, the recently described Parkin Co-Regulated Gene (PACRG) is completely deleted. Homozygous Quaking mice show a complete loss of PRKN and PACRG mRNA and protein. These mice will constitute a useful additional model for studies of the molecular role of parkin and PACRG in neurodegeneration.

Quaking*shiverer double-mutant mice survive for at least 100 days with no CNS myelin.

Mice expressing mutations that produce CNS hypomyelination often die prematurely: the more severe the hypomyelination, the shorter the life span. However, we have previously described jimpy-msd mice that survive twice as long as usual; although they acquire significantly increased amounts of myelin, they still succumb long before their unaffected littermates. This result contradicts any postulated causal relationship between extent of CNS hypomyelination and premature death of the animal. Here we have addressed this question in another way, by using an animal model that does not involve a proteolipid protein (Plp) gene mutation. We demonstrate that quaking*shiverer double-mutant mice can survive for at least 100 days without any CNS myelin whatsoever. Therefore, at least for a mouse, absence of CNS myelin is not lethal per se.

Molecular defects in the dysmyelinating mutant quaking.

The quaking [or quakingviable (qk[v])] mutant mouse, which exhibits severe dysmyelination of the central nervous system (CNS), has been studied extensively over the last 30 years. The genetic defect responsible for the dysmyelinating phenotype had remained elusive, however, until the recent cloning of a candidate gene, qkI (Ebersole et al.: Nature Genet 12:260-265, 1996). qkI encodes three proteins, QKI-5, QKI-6, and QKI-7, which are abundant in myelin-forming cells in wild-type mice but whose levels are severely reduced in myelin-forming cells of qk(v) mice, consistent with the notion that abnormalities of qkI expression underlie the qk(v) phenotype. This review discusses some of the known molecular defects in qk(v) in the context of this new information and the potential role of QKI proteins in myelinogenesis.

A 1.2-Mb YAC contig spans the quaking region.

We describe here a 1.2-Mb yeast artificial chromosome (YAC) contig within the region of mouse chromosome 17 between Brachyury (T) and D17Rp17e, and spanning the quaking (qk) region. We describe six new probes distributed across 1.2 Mb: D17Leh502, D17Leh503, D17Leh504, D17Leh505, D17Leh506, and D17Leh507. Probes D17Leh502 and D17Leh507 are at the extreme ends of the YAC contig. With the exception of D17Leh507, all of these probes are within a deletion associated with the quaking(viable) (qkv) allele of quaking. We have positioned these probes on a detailed YAC physical map together with two previously published probes, D17Leh508 and D17Aus119. We show here that D17Leh508 is also within the qkv deletion. Genetic mapping of D17Leh504 and D17Leh507 on two high-resolution genetic crosses carrying qkv and quaking(lethal-1) (qkl-1) alleles shows that these probes do not recombine with quaking and are therefore within 0.04 cM of qkv and 0.05 cM of qkl-1 mutations. The deletion breakpoint contained within the YAC contig has been positioned to within 90 kb by restriction mapping of wildtype and mutant DNA. This contig will form the basis for identification and mapping of expressed sequences and for an investigation of genome organization.

Oligotriche and quaking gene mutations. Phenotypic effects on mouse spermatogenesis and testicular steroidogenesis.

The phenotypic actions of the oligotriche gene mutation on testicular function have not been elucidated, although it is known that male mice homozygous for the mutation are infertile. In the present study, the effect of the oligotriche gene mutation on mouse testicular function was analyzed by comparing normal and mutant mice. Spermatogenesis was analyzed by enumerating germ cells in seminiferous tubules at specific stages of spermatogenesis and by electron microscopy. Steroidogenic potential was estimated by radioimmunometric determination of testosterone secreted by testes perfused in vitro. Parallel studies were completed for male mice homozygous for the quaking gene mutation, a mutation known to cause male mouse sterility by disrupting sperm tail development. The experimental results suggest that the oligotriche and quaking gene mutations interfere with sperm tail formation by different mechanisms. Testicular steroidogenesis was not affected by either gene mutation. The results provide the first evidence that the oligotriche gene mutation induces male mouse sterility by effecting the complete absence of a sperm tail. This phenotypic action is different from that of the quaking gene mutation.

Quaking phenotype influences brain lipid-related mRNA levels.

Although lipids compose almost 80% of myelin, the influence of quaking on mRNAs encoding lipid biosynthetic enzymes and transport proteins has not been previously reported. Understanding the influence of quaking on myelin-specific and lipid-related mRNAs will be useful in determining the mechanism of the quaking defect. Stearoyl CoA desaturase (SCD) catalyzes a key step in the biosynthesis of oleic acid (C18:1, n-9), a major fatty acid in myelin. SCD, LDL receptor (LDLR) and apolipoprotein E (Apo E) mRNA levels are all reduced in neonatal quaking brains. In contrast to brain, quaking hepatic LDLR and Apo E mRNA levels are normal. These results indicate that lipid-related mRNAs are reduced in neonatal quaking brain, but the quaking liver is unaffected. The quaking defect influences gene expression in multiple cell types of glial lineage in the developing CNS.

The proximal end of mouse chromosome 17: new molecular markers identify a deletion associated with quakingviable.

Five randomly identified cosmids have been mapped proximal to the Leh66D locus on mouse chromosome 17. Two of these cosmids, Au10 and Au119, map near the neurological mutation quaking. Au119 is deleted in qkviable/qkviable DNA, whereas Au10 is not. Au76 maps to a gene-rich region near the Time locus. The Au76 locus encodes a member of a low copy gene family expressed in embryos, the adult central nervous system and testis. A second member of this family has been mapped to chromosome 15 near c-sis (PDGF-B). At the centromeric end of chromosome 17, Au116 maps near the Tu1 locus, and along with Au217rs identifies a region of unusually high recombinational activity between t-haplotypes and wild-type chromosomes. Au217I and II map to the large inverted repeats found at the proximal end of the wild-type chromosome. In addition, the Au217I and/or II loci encode testis transcripts not expressed from t-haplotypes.

Developmental expression of glial fibrillary acidic protein and actin-encoding messages in quaking and control mice.

Quaking is a neurological mutation leading to pleiotropic phenotypic expression, the most prominent being disturbed myelin formation in the central nervous system (CNS) with minor abnormalities in the peripheral nervous system. Previous immunochemical measurements of glial fibrillary acidic protein (GFAP) revealed a marked increase in the protein in several areas of the CNS. To further characterize the regulation parameters of GFAP synthesis, we analyzed the levels of GFAP mRNA in 5 regions of the CNS, some with elevated levels of GFAP and some without. This was compared to the developmental expression of GFAP transcripts in the same regions in normal mice. To establish the specificity of the variations observed with this astroglial specific message, we conducted a similar investigation with actin RNA which is expressed by several cell types in the CNS. Both the actin and the GFAP message were found to be increased in the adult mutant throughout the CNS. In 2-year-old normal mice the messengers for both cytoskeleton proteins were expressed in a higher amount than in young adults.

Spatial distribution of myelin basic protein mRNA and polypeptide in quaking oligodendrocytes in culture.

In the CNS, myelin is formed from the expansion of oligodendrocyte processes. In order to study myelin assembly in the hypomyelinating mutant mouse quaking (qk), cultures of oligodendrocytes were established from affected and control animals. The cytoarchitecture of the oligodendrocytes was analyzed by performing morphometric measurements after immunostaining with antitubulin. The results indicate that the gross morphology of the processes is similar in control and mutant cells. The localization of the message for the myelin structural component, myelin basic protein (MBP), was examined by in situ hybridization. In control oligodendrocytes, 80% of MBP mRNA is found in the processes. In contrast, only 23% of MBP mRNA is localized to these structures in the mutant; the majority of MBP mRNA remains in the cell body. The mutant cells are capable of distributing mRNAs to the periphery as shown by the presence of tubulin mRNA in their processes. MBP polypeptide was visualized by immunofluorescence and found in the perikaryon, processes and membranous expansions of the control cells. In the mutant, it is largely confined to the perikaryon, reflecting the distribution of the mRNA. These results suggest that the localization of MBP polypeptide is achieved by restricting the distribution of its mRNA, and that MBP assembly into the myelin membrane occurs in the processes. This step appears to be blocked in qk oligodendrocytes in culture.

Pleiotropic action of the murine quaking locus: structure of the qkv allele.

The spontaneous allele quakingviable (qkv) exerts effects on myelination and spermiogenesis. The defects generated by qkv were not separated in a multilocus mapping cross that provided a mapping resolution of 0.1 centiMorgans (cM). Furthermore, no distortions suggestive of a large chromosomal anomaly associated with qkv were apparent. One plausible interpretation is that the quaking locus contains more than one functional domain, either organized into overlapping genes or expressed by alternative splicing mechanisms. The cloning needed to analyze this locus will be enhanced by the very high resolution of the meiotic mapping cross reported here. The recombinational distances on this qkv map were compressed compared with those previously reported in a high-resolution map for qkl-1, an embryonic lethal allele of quaking induced by ethylnitrosourea. Additional crosses confirmed prior reports that the sex and the genetic background of the heterozygous parent can affect recombinational distances. These joint effects on recombination are strong enough to account for the discrepancy between the two maps. This variability of two-factor map values leads to the preferred multilocus map-building protocol discussed in the accompanying paper.

Quaking shiverer double mutant mice: morphological phenotypes support possible dual actions of the shiverer locus.

Mice doubly homozygous for the two different hypomyelination mutations, quaking (qk) and shiverer (shi) or shiverer myelin-deficient (shimld) (abbreviations: qk*shi and qk*shimld), both have much less myelin than either single mutant ancestor, myelin morphology resembling shi or shimld rather than qk, and abundant shi-type oligodendrocytic microprocesses. The qk*shimld double mutant differs from qk*shi only in having small amounts of normal or abnormal major dense line, in keeping with the morphologic difference between the shi and shimld single mutants. By contrast, shi*jp and shimld*jp have clearly different morphological phenotypes; unexpectedly the major dense line is present in the CNS myelin of shi*jp but not shimld*jp. When shi and shimld act alone, their different DNA abnormalities produce similar protein abnormalities. We speculate that the two mutations interact with qk at a different, later step of DNA expression than they interact with jp. In the interaction with qk, the similar proteins produce similar morphologies. In the interaction with jp, the different DNAs are somehow caused to produce protein differences that are reflected in different morphologies. In this study we have observed for the first time a morphological effect of these mutant genes in heterozygous animals. Of particular importance, animals whose genomes combine shi/+ or shimld/+ with qk/qk produce qk-type, compacted myelin but abundant shi-type oligodendrocyte microprocesses. We consider this as evidence that both shi and shimld have two effects: non-production of a normal structural protein, myelin basic protein, and production of an abnormal protein which perturbs the cytogogic function we postulate to be normally exercised by the myelin basic protein gene.

Expression of cellular protooncogenes in the mouse male germ line: a distinctive 2.4-kilobase pim-1 transcript is expressed in haploid postmeiotic cells.

We report that a 2.4-kilobase (kb) pim-1 transcript is expressed in the germ cells of mouse testis. Analysis of purified populations of spermatogenic cell types indicates that the 2.4-kb transcript is selectively expressed in haploid postmeiotic early spermatids. The evidence for a developmentally regulated expression of pim-1 in haploid spermatids suggests a possible developmental role for this protooncogene product. The 2.4-kb pim-1 transcript present in postmeiotic cells differs in size from the 2.8-kb transcript usually detected in somatic tissues. Similar testis-specific transcripts have been seen for mos and abl genes. These data suggest specificity in transcription or processing of certain genes in haploid male germ cells. We have also analyzed other representative protooncogenes, including examples of protein kinases, the ras family, and the "nuclear" protooncogenes. The results indicate that additional protooncogenes are preferentially expressed in either meiotic pachytene cells or postmeiotic early spermatids. These findings suggest a differential regulation of gene expression in these two developmental stages of germ cells. In particular, analysis of expression of the three members of the ras gene family indicates a distinct temporal differential regulation in the expression of the Harvey, Kirsten, and N-ras genes in these germ cells.

Saturation germ line mutagenesis of the murine t region including a lethal allele at the quaking locus.

The proximal region of mouse chromosome 17 contains many genes affecting embryonic development, germ cell differentiation, and the immune system. Although the study of natural variation, including t haplotypes, has yielded some information about the function of these genes, spontaneous variants often exhibit manifold genetic effects and are generally not carried on inbred backgrounds. To clearly connect phenotypes with the actions of individual genes, mutants in which genes are altered singly are needed. Therefore, we used a highly efficient point mutagen, N-ethyl-N-nitrosourea, in combination with classical breeding schemes to induce and identify recessive lethal mutations in the t region. Of 350 mutagenized gametes examined, at least 10 independent recessive embryonic lethal mutations have been identified; an additional two are perinatal lethals. A spontaneous brachyury mutation, TWis, arose on a genetic background that permits high-resolution mapping of the induced recessive mutations against cloned DNA sequences from the t region. One lethal mutation is an allele at the quaking locus. The multiple alleles of quaking and the feasibility of high-resolution mapping permit investigation of the pleiotropic action of this locus in mammalian development.

Expression of a myelin basic protein gene in transgenic shiverer mice: correction of the dysmyelinating phenotype.

Mice homozygous for the autosomal recessive mutation shiverer (shi) lack myelin basic protein (MBP) and exhibit a distinct behavioral pattern including tremors (shivering), convulsions, and early death. We have previously demonstrated that shiverer mice have a partial deletion in the gene encoding MBP. We now have introduced the wild-type MBP gene into the germ line of shiverer mice by microinjection into fertilized eggs. Transgenic shiverer mice homozygous for the introduced gene have MBP mRNA and protein levels that are approximately 25% of normal, and produce compacted myelin with major dense lines. Correct temporal and spatial expression of the MBP gene is achieved with a genomic MBP cosmid clone containing 4 kb of 5' flanking sequence and 1 kb of 3' flanking sequence. Moreover, the four different forms of MBP produced by alternative patterns of RNA splicing are present. These homozygous transgenic shiverer mice no longer shiver nor die prematurely.

Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin.

Mice homozygous for the mutation myelin deficient (mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mld mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.

Myelin basic protein gene expression in quaking, jimpy, and myelin synthesis-deficient mice.

Jimpy (jp), myelin synthesis-deficient (jpmsd), and quaking (qk) are mutations which affect myelination to different degrees in the mouse central nervous system (CNS). Total messenger RNA (mRNA) and myelin basic protein (MBP)-specific mRNA from brains of these three mutants have been analyzed by in vitro translation and immunoprecipitation with antibody to MBP. The results indicate that the three mutations do not affect the level of total MBP-specific mRNA in the CNS but do affect the relative proportions of the various MBP-related translation products encoded in vitro. In each case the proportions of 14K and 12K Mr MBP-related translation products are reduced and the proportions of 21.5K, 18.5K, and 17K Mr MBP-related translation products are increased relative to wild type. This effect is most pronounced in jp, less so in jpmsd, and least pronounced in qk animals. The MBP-related polypeptides that accumulate in vivo have also been analyzed in the three mutants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with antibody to MBP. The levels of all the major MBP-related polypeptides that accumulate in vivo are reduced in all three mutations. The reduction is most pronounced in jp, less in jpmsd, and least pronounced in qk animals. These results indicate that the jp, jpmsd, and qk mutations exhibit qualitatively similar phenotypic effects on MBP gene expression but the magnitude of the effect is proportional to the extent of hypomyelination in each mutant.

Failure of 5-hydroxytryptophan to modulate brain stem auditory evoked responses in myelin deficient mutant qk mice.

The effect of 5-hydroxytryptophan (5-HTP) on brain stem auditory evoked response (BAER) amplitude in Quaking (qk) and normal littermate mice was examined. Administration of 5-hydroxytryptophan (5-HTP) (75 mg/kg, i.p.) to normal mice increased the amplitude of BAER peaks I, II, and III but had no effect on peak IV. In qk mice, however, 5-HTP did not affect the amplitude of any BAER peaks. Our data indicate that although 5-HTP increased BAER amplitude in normal mice, it failed to modulate BAER in qk mice. These findings are consistent with the possibility that 5-HTP receptor sites associated with myelin basic protein may be reduced in the myelin-deficient mutant qk mice.