PLP overexpression perturbs myelin protein composition and myelination in a mouse model of Pelizaeus-Merzbacher disease

Duplication of PLP1, an X‐linked gene encoding the major myelin membrane protein of the human CNS, is the most frequent cause of Pelizaeus‐Merzbacher disease (PMD). Transgenic mice with extra copies of the wild type Plp1 gene, a valid model of PMD, also develop a dysmyelinating phenotype dependant on gene dosage. In this study we have examined the effect of increasing Plp1 gene dosage on levels of PLP/DM20 and on other representative myelin proteins. In cultured oligodendrocytes and early myelinating oligodendrocytes in vivo, increased gene dosage leads to elevated levels of PLP/DM20 in the cell body. During myelination, small increases in Plp1 gene dosage (mice hemizygous for the transgene) elevate the level of PLP/DM20 in oligodendrocyte soma but cause only minimal and transient effects on the protein composition and structure of myelin suggesting that cells can regulate the incorporation of proteins into myelin. However, larger increases in dosage (mice homozygous for the transgene) are not well tolerated, leading to hypomyelination and alteration in the cellular distribution of PLP/DM20. A disproportionate amount of PLP/DM20 is retained in the cell soma, probably in autophagic vacuoles and lysosomes whereas the level in myelin is reduced. Increased Plp1 gene dosage affects other myelin proteins, particularly MBP, which is transitorily reduced in hemizygous mice but consistently and markedly lower in homozygotes in both myelin and naïve or early myelinating oligodendrocytes. Whether the reduced MBP is implicated in the pathogenesis of dysmyelination is yet to be established. © 2006 Wiley‐Liss, Inc.     

Characterization of a PLP-overexpressing transgenic rat, a model for the connatal form of Pelizaeus-Merzbacher disease

Pelizaeus-Merzbacher disease (PMD) most frequently results from duplication of the Plp1 gene with a correlation between disease severity and increasing copy number of the gene. Animal models of PMD, in particular those overexpressing the Plp1 gene, have been sought in attempts to provide systems in which potential therapies can be tested. Here we describe a rat model of the severe connatal form of PMD and provide a detailed characterization of its pathology and molecular biology, prior to testing therapeutic approaches. We determined the exact copy number of Plp1, and the resulting effects on RNA and protein expression. Distinct differences in myelin and disparate distributions of myelin protein markers in comparison to wild-type controls were observed. Altered expression of Plp1 also caused an increase in the apoptotic cell death of oligodendrocytes. These results provide the platform from which to test the effectiveness of in vivo therapies.     

Evidence that CNS hypomyelination does not cause death of jimpy-msd mutant mice

Mice expressing three of the proteolipid protein (Plp) mutations in the mouse (jimpy, jimpy-msd, and jimpy-4J) all have a severe deficiency of CNS myelin and oligodendrocytes (OLs), and die sometime in their 4th postnatal week. The prevailing view has been that the animals' shortened life span and lack of myelin are causally related. Here we describe the survival of jimpy-msd males for as long as postnatal day (P) 210. Although these spontaneously occurring longer-lived jimpy-msd males show a 2- to 8-fold increase in numbers of myelinated axons in many CNS regions, this does not protect them from a later but still premature death. Investigating the cause of premature death may reveal previously undiscovered properties of the myelin genes or the cells that express them, or perhaps additional unsuspected cellular responses that contribute to the disease. This study identifies small accumulations of inflammatory cells in the brain parenchyma of jimpy-msd mice as young as P14 and as old as P60, suggesting that the pathology of the disease produced by at least this Plp mutation may be far more complex than has been previously recognized.     

Analysis of the human tyrosine hydroxylase promoter-chloramphenicol acetyltransferase chimeric gene expression in transgenic mice.

To investigate cis-elements responsible for catecholaminergic (CAnergic) neuron-specific expression of the tyrosine hydroxylase (TH) gene, we produced lines of transgenic mice carrying 5.0-kb, 2.5-kb and 0.2-kb fragments from the 5'-flanking region of the human TH gene fused to a reporter gene, chloramphenicol acetyltransferase (CAT), and designated them as TC 50, TC 25, and TC 02, respectively, and reporter gene expression in transgenic mice was analyzed by CAT assay by immunocytochemistry with anti-CAT antibody. High-level CAT expression was observed in the brain and adrenal gland using the 5.0-kb promoter of the TC 50 mice, but ectopic expression was consistently observed in several somatic tissues, e.g. thymus, colon, and testis. In brain, expression was achieved in CAnergic neurons with the largest construct (5.0 kb), but not with 2.5 kb or 0.2 kb of 5' flanking sequence. However, TC 50 mice also expressed CAT immunoreactivity in non-CAnergic neurons. In the TC 25 line CAT immunoreactivity was detected only in some non-CAnergic neurons. In the TC 02 line no CAT immunoreactivity was detected in any of the tissues examined. These results indicate that the 5.0-kb DNA fragment of the TH gene upstream region contains activity to express CAT in CAnergic neurons and surprisingly, lacks some regulatory elements attenuating ectopic expression, and that the 2.5-kb and 0.2-kb fragment are not sufficient for the proper expression. We discuss the presence of the tissue-specific regulatory elements in the structure portion of the TH gene and/or 3'-flanking region.     

Transgenic mice expressing yellow fluorescent protein under control of the human tyrosine hydroxylase promoter

Pathogenesis of Parkinson's disease and related catecholaminergic neurological disorders is closely associated with changes in the levels of tyrosine hydroxylase (TH). Therefore, investigation of the regulation of the TH gene system should assist in understanding the pathomechanisms involved in these neurological disorders. To identify regulatory domains that direct human TH expression in the central nervous system (CNS), we generated two transgenic mouse lines in which enhanced yellow fluorescent protein (EYFP) is expressed under the control of either 3.2‐kb (hTHP‐EYFP construct) human TH promoter or 3.2‐kb promoter with 2‐kb 3′‐flanking regions (hTHP‐ex3‐EYFP construct) of the TH gene. In the adult transgenic mouse brain, the hTHP‐EYFP construct directs neuron‐specific EYFP expression in various CNS areas, such as olfactory bulb, striatum, interpeduncular nucleus, cerebral cortex, hippocampus, and particularly dentate gyrus. Although these EYFP‐positive cells were identified as mature neurons, few EYFP‐positive cells were TH‐positive neurons. On the other hand, we could detect the EYFP mRNA expression in a subset of neurons in the olfactory bulb, midbrain, and cerebellum, in which expression of endogenous TH is enriched, with hTHP‐ex3‐EYFP transgenic mice. These results indicate that the 3.2‐kb sequence upstream of the TH gene is not sufficient for proper expression and that the 2‐kb sequence from the translation start site to exon 3 is necessary for expression of EYFP in a subset of catecholaminergic neurons. © 2012 Wiley Periodicals, Inc.     

Four-kilobase sequence of the mouse CNP gene directs spatial and temporal expression of lacZ in transgenic mice

The gene encoding 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) is one of the earliest myelin genes to be expressed in the brain. It is expressed at basal levels in some non-neural tissues but at much higher levels in the nervous system, and its relevance and mechanism are unknown. Using transgenic mice, we examined the expression pattern conferred by a 4-kilobase (-kb) 5′-flanking sequence of the mouse CNP gene coupled to the bacterial lacZ reporter gene. Here we report that this 4-kb fragment contains sufficient information to direct expression of the transgene to the tissue and/or cell type, in which CNP is normally expressed. In the central nervous system (CNS), CNP-lacZ expression was regulated in a temporal manner, consistent with endogenous CNP expression. Transgene expression was detected in embryonic brain and spinal cord in immature oligodendrocytes, and it significantly increased with age. In adult mice, β-galactosidase activity (which appeared to be oligodendrocyte specific) was found essentially in white matter areas of the CNS. Moreover, the transgene was expressed in peripheral nervous system, testis, and thymus—tissues that normally express CNP. Taken together, our results provide strong evidence that cis-acting regulatory elements, necessary to direct spatial and temporal expression of the transgene in oligodendrocytes, are located within the 4-kb 5′-flanking sequence of the mouse CNP gene. This promoter could be a valuable tool to target specific expression of other transgenes to oligodendrocytes, and may provide important new insights into myelination or dysmyelination. J. Neurosci. Res. 53:393–404, 1998. © 1998 Wiley-Liss, Inc.     

Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-mediated excitotoxic axonal damage is attenuated in the absence of myelin proteolipid protein

In vivo and in vitro studies have shown that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor-mediated excitotoxicity causes cytoskeletal damage to axons. AMPA/kainate receptors are present on oligodendrocytes and myelin, but currently there is no evidence to suggest that axon cylinders contain AMPA receptors. Proteolipid protein (PLP) and DM20 are integral membrane proteins expressed by CNS oligodendrocytes and located in compact myelin. Humans and mice lacking normal PLP/DM20 develop axonal swellings and degeneration, suggesting that local interactions between axons and the oligodendrocyte/myelin unit are important for the normal functioning of axons and that PLP/DM20 is involved in this process. To determine whether perturbed glial-axonal interaction affects AMPA-receptor-mediated axonal damage, AMPA (1.5 nmol) was injected into the caudate nucleus of anesthetized Plp knockout and wild-type male mice (n = 13). Twenty-four hours later, axonal damage was detected by using neurofilament 200 (NF 200) immunohistochemistry and neuronal damage detected via histology. AMPA-induced axonal damage, assessed with NF 200 immunohistochemistry, was significantly reduced in Plp knockout mice compared with wild-type mice (P = 0.015). There was no significant difference in the levels of neuronal perikaryal damage between the Plp knockout and wild-type mice. In addition, there was no significant difference in the levels of glutamate receptor subunits GluR1-4 or KA2 in Plp knockout compared with wild-type littermates. The present study suggests that PLP-mediated interactions among oligodendrocytes, myelin, and axons may be involved in AMPA-mediated axonal damage.     

The wmN1 enhancer region in intron 1 is required for expression of human PLP1

The myelin proteolipid protein gene (PLP1) encodes the most abundant protein present in myelin from the central nervous system (CNS). Its expression must be tightly controlled as evidenced by mutations that alter PLP1 dosage; both overexpression (elevated PLP1 copy number) and lack thereof (PLP1 deletion) result in X‐linked genetic disorders in man. However, not much is known about the mechanisms that govern expression of the human gene. To address this, transgenic mice were generated which utilize human PLP1 (hPLP1) sequences (proximal 6.2 kb of 5′‐flanking DNA to the first 38 bp of exon 2) to drive expression of a lacZ reporter cassette. LoxP sites were incorporated around a 1.5‐kb section of hPLP1 intron 1 since it contains sequence orthologous to the wmN1 region from mouse which, previously, was shown to augment expression of a minimally‐promoted transgene coincident with the active myelination period of CNS development. Eight transgenic lines were generated with the parental, 6.2hPLP(+)Z/FL, transgene. All lines expressed the transgene appropriately in brain as evidenced by staining with X‐gal in white matter regions and olfactory bulb. Removal of the “wmN1” region from 6.2hPLP(+)Z/FL with a ubiquitously expressed Cre‐driver caused a dramatic reduction in transgene activity. These results demonstrate for the first time that the wmN1 enhancer region: (1) is functional in hPLP1; (2) works in collaboration with its native promoter—not just a basal heterologous promoter; (3) is required for high levels of hPLP1 gene activity; (4) has a broader effect, both spatially and temporally, than originally projected with mPlp1.     

PLP/DM20 Expression and turnover in a transgenic mouse model of pelizaeus‐merzbacher disease

The most common cause of Pelizaeus‐Merzbacher (PMD) is due to duplication of the PLP1 gene but it is unclear how increased gene dosage affects PLP turnover and causes dysmyelination. We have studied the dynamics of PLP/DM20 in a transgenic mouse model of PMD with increased gene dosage of the proteolipid protein gene (Plp1). The turnover of PLP/DM20 were investigated using an ex‐vivo brain slice system and cultured oligodendrocytes. Homozygous mice have reduced PLP translation, markedly enhanced PLP degradation, and markedly reduced incorporation of PLP into myelin. Proteasome inhibition (MG132) prevented the enhanced degradation. Numerous autophagic vesicles are present in homozygous transgenic mice that may influence protein dynamics. Surprisingly, promoting autophagy with rapamycin decreases the degradation of nascent PLP suggesting autophagic vacuoles serve as a cellular storage compartment. We suggest that there are multiple subcellular fates of PLP/DM20 when overexpressed: the vast majority being degraded by the proteasome, a proportion sequestered into autophagic vacuoles, probably fused with endolysosomes, and only a small proportion entering the myelin sheath, where its association with lipid rafts is perturbed. Transgenic oligodendrocytes have fewer membrane sheets and this phenotype is improved with siRNA‐mediated knockdown of PLP expression that promotes the formation of MBP+ myelin‐like sheets. This finding suggests that RNAi technology is in principle applicable to improve CNS myelination when compromised by PLP/DM20 overexpression. © 2010 Wiley‐Liss, Inc.