Progesterone Antagonist Therapy in a Pelizaeus-Merzbacher Mouse Model

Pelizaeus-Merzbacher disease (PMD) is a severe hypomyelinating disease, characterized by ataxia, intellectual disability, epilepsy, and premature death. In the majority of cases, PMD is caused by duplication of PLP1 that is expressed in myelinating oligodendrocytes. Despite detailed knowledge of PLP1, there is presently no curative therapy for PMD. We used a Plp1 transgenic PMD mouse model to test the therapeutic effect of Lonaprisan, an antagonist of the nuclear progesterone receptor, in lowering Plp1 mRNA overexpression. We applied placebo-controlled Lonaprisan therapy to PMD mice for 10 weeks and performed the grid slip analysis to assess the clinical phenotype. Additionally, mRNA expression and protein accumulation as well as histological analysis of the central nervous system were performed. Although Plp1 mRNA levels are increased 1.8-fold in PMD mice compared to wild-type controls, daily Lonaprisan treatment reduced overexpression at the RNA level to about 1.5-fold, which was sufficient to significantly improve the poor motor phenotype. Electron microscopy confirmed a 25% increase in the number of myelinated axons in the corticospinal tract when compared to untreated PMD mice. Microarray analysis revealed the upregulation of proapoptotic genes in PMD mice that could be partially rescued by Lonaprisan treatment, which also reduced microgliosis, astrogliosis, and lymphocyte infiltration.     

Therapy of Pelizaeus-Merzbacher disease in mice by feeding a cholesterol-enriched diet

Duplication of PLP1 (proteolipid protein gene 1) and the subsequent overexpression of the myelin protein PLP (also known as DM20) in oligodendrocytes is the most frequent cause of Pelizaeus-Merzbacher disease (PMD), a fatal leukodystrophy without therapeutic options. PLP binds cholesterol and is contained within membrane lipid raft microdomains. Cholesterol availability is the rate-limiting factor of central nervous system myelin synthesis. Transgenic mice with extra copies of the Plp1 gene are accurate models of PMD. Dysmyelination followed by demyelination, secondary inflammation and axon damage contribute to the severe motor impairment in these mice. The finding that in Plp1-transgenic oligodendrocytes, PLP and cholesterol accumulate in late endosomes and lysosomes (endo/lysosomes), prompted us to further investigate the role of cholesterol in PMD. Here we show that cholesterol itself promotes normal PLP trafficking and that dietary cholesterol influences PMD pathology. In a preclinical trial, PMD mice were fed a cholesterol-enriched diet. This restored oligodendrocyte numbers and ameliorated intracellular PLP accumulation. Moreover, myelin content increased, inflammation and gliosis were reduced and motor defects improved. Even after onset of clinical symptoms, cholesterol treatment prevented disease progression. Dietary cholesterol did not reduce Plp1 overexpression but facilitated incorporation of PLP into myelin membranes. These findings may have implications for therapeutic interventions in patients with PMD.     

Minimal role for caspase 12 in the unfolded protein response in oligodendrocytes in vivo

The unfolded protein response (UPR) is implicated in many neurodegenerative disorders including Alzheimer, Parkinson and prion diseases, and the leukodystrophy, Pelizaeus-Merzbacher disease (PMD). Critical features of degeneration in several of these diseases involve activation of cell death pathways in various neural cell populations, and the initiator caspase 12 has been proposed to play a central role. Accordingly, pharmacological strategies to inhibit caspase 12 activity have received remarkable attention in anticipation of effecting disease amelioration. Our investigation in animal models of PMD demonstrates that caspase 12 is activated following accumulation of mutant proteins in oligodendrocytes; however, eliminating caspase 12 activity does not alter pathophysiology with respect to levels of apoptosis, oligodendrocyte function, disease severity or life span. We conclude that caspase 12 activation by UPR signaling is an epiphenomenon that plays little discernable role in the loss of oligodendrocytes in vivo and may portend the inconsequence of caspase 12 to the pathophysiology of other protein conformational diseases.     

Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

Cholesterol availability is rate-limiting for myelination, and prior studies have established the importance of cholesterol synthesis by oligodendrocytes for normal CNS myelination. However, the contribution of cholesterol uptake through the endocytic pathway has not been fully explored. To address this question, we used mice with a conditional null allele of the Npc1 gene, which encodes a transmembrane protein critical for mobilizing cholesterol from the endolysosomal system. Loss of function mutations in the human NPC1 gene cause Niemann-Pick type C disease, a childhood-onset neurodegenerative disorder in which intracellular lipid accumulation, abnormally swollen axons, and neuron loss underlie the occurrence of early death. Both NPC patients and Npc1 null mice exhibit myelin defects indicative of dysmyelination, although the mechanisms underlying this defect are incompletely understood. Here we use temporal and cell-type-specific gene deletion in order to define effects on CNS myelination. Our results unexpectedly show that deletion of Npc1 in neurons alone leads to an arrest of oligodendrocyte maturation and to subsequent failure of myelin formation. This defect is associated with decreased activation of Fyn kinase, an integrator of axon-glial signals that normally promotes myelination. Furthermore, we show that deletion of Npc1 in oligodendrocytes results in delayed myelination at early postnatal days. Aged, oligodendocyte-specific null mutants also exhibit late stage loss of myelin proteins, followed by secondary Purkinje neuron degeneration. These data demonstrate that lipid uptake and intracellular transport by neurons and oligodendrocytes through an Npc1-dependent pathway is required for both the formation and maintenance of CNS myelin.     

In vitro insertion of the myelin proteolipid apoprotein into oligodendrocyte plasma membranes

Uncoated vesicles (UCV) loaded with the myelin proteolipid apoprotein covalently tagged with fluorescein (PLP_F) were found to interact with isolated oligodendrocytes from bovine brain at 4°C as well as at 37°C. After 1.5 hours of incubation, the labeled protein was localized in the cell membranes. After 2.5 hours the fluorescence intensity associated with the oligodendrocytes decreased and completely disappeared at t=3.5 hours. Addition of KCl or EDTA in the incubation medium significantly hindered the interaction with cells. In contrast, the elimination of membrane proteins from UCV did not perturb cell labeling. A specific role of PLP was suggested since UCV loaded with a soluble protein (BSA_F) led to a weak cell labeling.Abbreviations IAF 5-iodacetamidofluorescein - BSA bovine serum albumin - BSA BSA labelled with IAF - PLP proteolipid apoprotein - PLP_F aqueous form of PLP tagged with IAF - CV coated vesicles - UCV uncoated vesicles - UCV^*PLP_F UCV loaded with PLP_F - MV model vesicles This work was suported by Cnrs and INSERM.     

Hypomyelinating leukodystrophy-associated mutation of RARS leads it to the lysosome,inhibiting oligodendroglial morphological differentiation

Pelizaeus-Merzbacher disease (PMD) is a central nervous system (CNS) demyelinating disease in human, currently known as prototypic hypomyelinating leukodystrophy 1 (HLD1). The gene responsible for HLD1 encodes proteolipid protein 1 (PLP1), which is the major myelin protein produced by oligodendrocytes. HLD9 is an autosomal recessive disorder responsible for the gene differing from the plp1 gene. The hld9 gene encodes arginyl-tRNA synthetase (RARS), which belongs to a family of cytoplasmic aminoacyl-tRNA synthetases. Herein we show that HLD9-associated missense mutation of Ser456-to-Leu (S456L) localizes RARS proteins as aggregates into the lysosome but not into the endoplasmic reticulum (ER) and the Golgi body. In contrast, wild-type proteins indeed distribute throughout the cytoplasm. Expression of S456L mutant constructs in cells decreases lysosome-related signaling through ribosomal S6 protein phosphorylation, which is known to be required for myelin formation. Cells harboring the S456L mutant constructs fail to exhibit phenotypes with myelin web-like structures following differentiation in FBD-102b cells, as part of the mammalian oligodendroglial cell model, whereas parental cells exhibit them. Collectively, HLD9-associated RARS mutant proteins are specifically localized in the lysosome with downregulation of S6 phosphorylation involved in myelin formation, inhibiting differentiation in FBD-102b cells. These results present some of the molecular and cellular pathological mechanisms for defect in myelin formation underlying HLD9.     

Differential expression of apoptotic markers in jimpy and in Plp overexpressors: evidence for different apoptotic pathways

Point mutations and duplications of proteolipid protein (PLP) gene in mammals cause dysmyelination and oligodendrocyte cell death. The jimpy mouse, which has a lethal Plp point mutation, is the best characterized of the mutants; transgenic mice, which have additional copies of Plp gene, are less characterized. While oligodendrocyte death is a prominent feature in jimpy, the pathways leading to death have not been investigated in jimpy and Plp overexpressors. Using immunohistochemistry and immunobloting, we examined expression of cleaved caspase-3, Poly (ADP-ribose) polymerase (PARP), caspase-12, and mitochondrial apoptotic markers in spinal cord in jimpy males and Plp overexpressors. Compared to controls, cleaved caspase-3 is increased 10× in jimpy white matter spinal cord, and 3× in Plp overexpressor. In jimpy, the number of cleaved caspase-3 cells far exceeds the number of TUNEL⁺ cells. The majority of cleaved caspase-3⁺ cells were not TUNEL⁺ and these cells exhibited staining in perikarya and in processes. Only 30% of the cleaved caspase-3⁺ cells were TUNEL⁺ and exhibited both nuclear and perinuclear staining. This observation suggests that activation of caspase-3 begins earlier and overlaps for a period of time with DNA fragmentation. In both Plp mutants, quantitative immunobloting of PARP showed a 45% increase in total as well as cleaved form, indicating that oligodendrocytes die via apoptosis. Most interestingly, cleavage of caspase-12, a caspase associated with unfolded protein response, is dramatically increased in jimpy but not at all in Plp overexpressors. Mitochondrial markers cytochrome c and Bcl-X_L are upregulated in both Plp mutants but levels of expression are different between mutants, suggesting that apoptosis in these two Plp mutants follows different pathways. In jimpy, mitochondrial apoptotic markers may play a role in amplifying the apoptotic signal. Our data shows for the first time, in vivo, that mutations in Plp gene increase oligodendrocyte death by activating the caspase cascade but the trigger to upregulate this cascade follows different pathways.     

Depletion of Molecular Chaperones from the Endoplasmic Reticulum and Fragmentation of the Golgi Apparatus Associated with Pathogenesis in Pelizaeus-Merzbacher Disease

Missense mutations in the proteolipid protein 1 (PLP1) gene cause a wide spectrum of hypomyelinating disorders, from mild spastic paraplegia type 2 to severe Pelizaeus-Merzbacher disease (PMD). Mutant PLP1 accumulates in the endoplasmic reticulum (ER) and induces ER stress. However, the link between the clinical severity of PMD and the cellular response induced by mutant PLP1 remains largely unknown. Accumulation of misfolded proteins in the ER generally leads to up-regulation of ER chaperones to alleviate ER stress. Here, we found that expression of the PLP1-A243V mutant, which causes severe disease, depletes some ER chaperones with a KDEL (Lys-Asp-Glu-Leu) motif, in HeLa cells, MO3.13 oligodendrocytic cells, and primary oligodendrocytes. The same PLP1 mutant also induces fragmentation of the Golgi apparatus (GA). These organelle changes are less prominent in cells with milder disease-associated PLP1 mutants. Similar changes are also observed in cells expressing another disease-causing gene that triggers ER stress, as well as in cells treated with brefeldin A, which induces ER stress and GA fragmentation by inhibiting GA to ER trafficking. We also found that mutant PLP1 disturbs localization of the KDEL receptor, which transports the chaperones with the KDEL motif from the GA to the ER. These data show that PLP1 mutants inhibit GA to ER trafficking, which reduces the supply of ER chaperones and induces GA fragmentation. We propose that depletion of ER chaperones and GA fragmentation induced by mutant misfolded proteins contribute to the pathogenesis of inherited ER stress-related diseases and affect the disease severity.     

Histone Deacetylase Inhibitor Upregulates Peroxisomal Fatty Acid Oxidation and Inhibits Apoptotic Cell Death in Abcd1-Deficient Glial Cells

In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL''s in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.     

Increased Plp1 gene expression leads to massive microglial cell activation and inflammation throughout the brain

PMD (Pelizaeus–Merzbacher disease) is a rare neurodegenerative disorder that impairs motor and cognitive functions and is associated with a shortened lifespan. The cause of PMD is mutations of the PLP1 [proteolipid protein 1 gene (human)] gene. Transgenic mice with increased Plp1 [proteolipid protein 1 gene (non-human)] copy number model most aspects of PMD patients with duplications. Hypomyelination and demyelination are believed to cause the neurological abnormalities in mammals with PLP1 duplications. We show, for the first time, intense microglial reactivity throughout the grey and white matter of a transgenic mouse line with increased copy number of the native Plp1 gene. Activated microglia in the white and grey matter of transgenic mice are found as early as postnatal day 7, before myelin commences in normal cerebra. This finding indicates that degeneration of myelin does not cause the microglial response. Microglial numbers are doubled due to in situ proliferation. Compared with the jp (jimpy) mouse, which has much more oligodendrocyte death and hardly any myelin, microglia in the overexpressors show a more dramatic microglial reactivity than jp, especially in the grey matter. Predictably, many classical markers of an inflammatory response, including TNF-α (tumour necrosis factor-α) and IL-6, are significantly up-regulated manyfold. Because inflammation is believed to contribute to axonal degeneration in multiple sclerosis and other neurodegenerative diseases, inflammation in mammals with increased Plp1 gene dosage may also contribute to axonal degeneration described in patients and rodents with PLP1 increased gene dosage.     

Elevated CSF N-acetylaspartylglutamate suggests specific molecular diagnostic abnormalities in patients with white matter diseases

Background: In order to identify biomarkers useful for the diagnosis of genetic white matter disorders we compared the metabolic profile of patients with leukodystrophies with a hypomyelinating or a non-hypomyelinating MRI pattern. Methods: We used a non-a priori method of in vitro ¹H-NMR spectroscopy on CSF samples of 74 patients with leukodystrophies. Results: We found an elevation of CSF N-acetylaspartylglutamate (NAAG) in patients with Pelizaeus–Merzbacher disease (PMD)—PLP1 gene, Pelizaeus–Merzbacher-like disease—GJC2 gene and Canavan disease—ASPA gene. In the PMD group, NAAG was significantly elevated in the CSF of all patients with PLP1 duplication (19/19) but was strictly normal in 6 out of 7 patients with PLP1 point mutations. Additionally, we previously reported increased CSF NAAG in patients with SLC17A5 mutations. Conclusions: Elevated CSF NAAG is a biomarker that suggests specific molecular diagnostic abnormalities in patients with white matter diseases. Our findings also point to unique pathological functions of the overexpressed PLP in PMD patients with duplication of this gene.     

综述与专论: 少突胶质细胞病——佩梅病的临床特点及致病机制

佩梅病（Pelizaeus-Merzbacher disease,PMD）是髓鞘形成低下性脑白质营养不良疾病中最常见的一种,其临床特点主要表现为发育落后尤其是大运动落后、眼震、肌张力低下等。其致病机制主要为脑白质髓鞘形成细胞-少突胶质细胞发生病理性改变从而导致髓鞘形成不良,相应理论基础包括以往研究中PLP1点突变通过影响PLP1/DM20寡聚体的形成,进而影响少突胶质细胞的存活,髓鞘分子结构的形成等;而PLP1重复突变则使少突胶质细胞及髓鞘脂的发育停止。近年来对细胞器互作网络（organelle interaction network,OIN）的研究进一步揭示了PLP1突变的致病机制：PLP1点突变通过影响PLP1蛋白上膜进而影响少突胶质细胞髓鞘化。PLP1重复突变则改变内质网线粒体间的连接,继而影响线粒体的形态功能等产生致病作用。目前已有相关研究表明,一些小分子化合物或药物例如胆固醇、吡拉西坦等以及基因疗法在动物体内对PMD临床症状有改善作用,其在PMD 患者体内的疗效有待进一步证实。     

Cell-type-specific expression pattern of ceramide synthase 2 protein in mouse tissues

Ceramide synthase 2 (CerS2) catalyzes the synthesis of dihydroceramides from dihydrosphingosine and very long fatty acyl (C22–C24)-CoAs. CerS2-deficient (gene trap) mice were reported to exhibit myelin and behavioral abnormalities, associated with the expression of CerS2 in oligodendrocytes and neurons based on expression of lacZ reporter cDNA instead of the cers2 gene in these mice. In order to clarify the cell-type-specific expression of CerS2 protein, we have raised antibodies that specifically recognize the glycosylated and non-glycosylated CerS2 protein in wild-type but not in CerS2-deficient mouse tissues. In early postnatal, juvenile and adult mouse brain, the new antibodies detect CerS2 protein only in oligodendrocytes but not in neurons, suggesting that the gene trap vector in CerS2-deficient mice led to ectopic expression of the lacZ reporter gene in neurons. In liver, the CerS2 protein is expressed in hepatocytes but not in Ito cells or Kupffer cells. We conclude that the behavioral abnormalities observed in CerS2-deficient mice originate primarily in oligodendrocytes and not in neurons. The identification of specific cell types in which CerS2 protein is expressed is prerequisite to further mechanistic characterization of phenotypic abnormalities exhibited by CerS2-deficient mice. The amount of CerS2 protein detected in different tissues by immunoblot analyses does not strictly correspond to the activity of the CerS2 enzyme. Disproportional results are likely due to post-translational regulation of the CerS2 protein.     

Structure-Function Analysis of Barley NLR Immune Receptor MLA10 Reveals Its Cell Compartment Specific Activity in Cell Death and Disease Resistance

Plant intracellular immune receptors comprise a large number of multi-domain proteins resembling animal NOD-like receptors (NLRs). Plant NLRs typically recognize isolate-specific pathogen-derived effectors, encoded by avirulence (AVR) genes, and trigger defense responses often associated with localized host cell death. The barley MLA gene is polymorphic in nature and encodes NLRs of the coiled-coil (CC)-NB-LRR type that each detects a cognate isolate-specific effector of the barley powdery mildew fungus. We report the systematic analyses of MLA10 activity in disease resistance and cell death signaling in barley and Nicotiana benthamiana. MLA10 CC domain-triggered cell death is regulated by highly conserved motifs in the CC and the NB-ARC domains and by the C-terminal LRR of the receptor. Enforced MLA10 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that MLA10 activity in cell death signaling is suppressed in the nucleus but enhanced in the cytoplasm. By contrast, nuclear localized MLA10 is sufficient to mediate disease resistance against powdery mildew fungus. MLA10 retention in the cytoplasm was achieved through attachment of a glucocorticoid receptor hormone-binding domain (GR), by which we reinforced the role of cytoplasmic MLA10 in cell death signaling. Together with our data showing an essential and sufficient nuclear MLA10 activity in disease resistance, this suggests a bifurcation of MLA10-triggered cell death and disease resistance signaling in a compartment-dependent manner.     

Glucose Deprivation Induces G2/M Transition-Arrest and Cell Death in N-GlcNAc2-Modified Protein-Producing Renal Carcinoma Cells

Some cancer cells can survive under glucose deprivation within the microenvironment of a tumor. Recently, we reported that N-linked (β-N-acetylglucosamine)₂ [N-GlcNAc₂]-modified proteins induce G2/M arrest and cell death under glucose deprivation. Here, we investigated whether such a response to glucose deprivation contributes to the survival of renal cell carcinomas, which are sensitive to nutritional stress. Specifically, we analyzed seven renal carcinoma cell lines. Four of these cell lines produced N-GlcNAc₂-modified proteins and led G2/M-phase arrest under glucose deprivation, leading to cell death. The remaining three cell lines did not produce N-GlcNAc₂-modified proteins and undergo G1/S-phase arrest under glucose deprivation, leading to survival. The four dead cell lines displayed significant up-regulation in the UDP-GlcNAc biosynthesis pathway as well as increased phosphorylation of p53, which was not observed in the surviving three cell lines. In addition, the four dead cell lines showed prolonged up-regulated expression of ATF3, which is related to unfolded protein response (UPR), while the surviving three cell lines showed only transient up-regulation of ATF3. In this study, we demonstrated that the renal carcinoma cells which accumulate N-GlcNAc₂-modified proteins under glucose deprivation do not survive with abnormaly prolonged UPR pathway. By contrast, renal carcinoma cells that do not accumulate N-GlcNAc₂-modified proteins under these conditions survive. Morover, we demonstrated that buformin, a UPR inhibitor, efficiently reduced cell survival under conditions of glucose deprivation for both sensitive and resistant phenotypes. Further studies to clarify these findings will lead to the development of novel chemotherapeutic treatments for renal cancer.     

Cerebellar Hypoplasia in Mice Lacking Selenoprotein Biosynthesis in Neurons

Selenium exerts many, if not most, of its physiological functions as a selenocysteine moiety in proteins. Selenoproteins are involved in many biochemical processes including regulation of cellular redox state, calcium homeostasis, protein biosynthesis, and degradation. A neurodevelopmental syndrome called progressive cerebello-cortical atrophy (PCCA) is caused by mutations in the selenocysteine synthase gene, SEPSECS, demonstrating that selenoproteins are essential for human brain development. While we have shown that selenoproteins are required for correct hippocampal and cortical interneuron development, little is known about the functions of selenoproteins in the cerebellum. Therefore, we have abrogated neuronal selenoprotein biosynthesis by conditional deletion of the gene encoding selenocysteyl tRNA^[Ser]Sec (gene symbol Trsp). Enzymatic activity of cellular glutathione peroxidase and cytosolic thioredoxin reductase is reduced in cerebellar extracts from Trsp-mutant mice. These mice grow slowly and fail to gain postural control or to coordinate their movements. Histological analysis reveals marked cerebellar hypoplasia, associated with Purkinje cell death and decreased granule cell proliferation. Purkinje cell death occurs along parasagittal stripes as observed in other models of Purkinje cell loss. Neuron-specific inactivation of glutathione peroxidase 4 (Gpx4) used the same Cre driver phenocopies tRNA^[Ser]Sec mutants in several aspects: cerebellar hypoplasia, stripe-like Purkinje cell loss, and reduced granule cell proliferation. Parvalbumin-expressing GABAergic interneurons (stellate and/or basket cells) are virtually absent in tRNA^[Ser]Sec-mutant mice, while some remained in Gpx4-mutant mice. Our data show that selenoproteins are specifically required in postmitotic neurons of the developing cerebellum, thus providing a rational explanation for cerebellar hypoplasia as occurring in PCCA patients.     

Brain DNA metabolism in myelin deficient mutant jp,jpmsd and qk mice

We studied metabolism of brain DNA in three myelin deficient mutants qk, jp and jp^msd mice. The DNA content, the in vivo incorporation of [¹⁴C]thymidine in DNA and the activity of acid DNase in tissues (cerebellum and cerebrum) from normal littermates and affected mice were compared. The results showed that neither the DNA content, the incorporation of [¹⁴C]thymidine in DNA nor the activity of acid DNase in brain were altered in qk affected mice. In jp^msd mice, however, the DNA content as well as the incorpation of thymidine in DNA were reduced in both cerebellum and cerebrum, but the activity of acid DNase was reduced in cerebrum only. In jp mice, although the DNA content was reduced in both cerebellum and cerebrum, the incorporation of thymidine in DNA and the activity of acid DNase were reduced in cerebrum only. The data suggest a) that in qk mutants DNA metabolism and hence cell (glial) proliferation is not affected; b) that in jp^msd mutants DNA synthesis, and thus the cell proliferation is reduced in cerebellum as well as in cerebrum of the affected mice and c) that in jp mutants the synthesis of DNA and the cell proliferation is reduced in cerebrum but not in cerebellum.     

The QKI-6 RNA Binding Protein Regulates Actin-interacting Protein-1 mRNA Stability during Oligodendrocyte Differentiation

The quaking viable (qk^v) mice represent an animal model of dysmyelination. The absence of expression of the QKI-6 and QKI-7 cytoplasmic isoforms in oligodendrocytes (OLs) during CNS myelination causes the qk^v mouse phenotype. The QKI RNA-binding proteins are known to regulate RNA metabolism of cell cycle proteins and myelin components in OLs; however, little is known of their role in reorganizing the cytoskeleton or process outgrowth during OL maturation and differentiation. Here, we identify the actin-interacting protein (AIP)-1 mRNA as a target of QKI-6 by using two-dimensional differential gel electrophoresis. The AIP-1 mRNA contains a consensus QKI response element within its 3′-untranslated region that, when bound by QKI-6, decreases the half-life of the AIP-1 mRNA. Although the expression of QKI-6 is known to increase during OL differentiation and CNS myelination, we show that this increase is paralleled with a corresponding decrease in AIP-1 expression in rat brains. Furthermore, qk^v/qk^v mice that lack QKI-6 and QKI-7 within its OLs had an increased level of AIP-1 in OLs. Moreover, primary rat OL precursors harboring an AIP-1 small interfering RNA display defects in OL process outgrowth. Our findings suggest that the QKI RNA-binding proteins regulate OL differentiation by modulating the expression of AIP-1.