Identification of the regulatory region of the peripheral myelin protein 22 (PMP22) gene that directs temporal and spatial expression in development and regeneration of peripheral nerves

Minor changes in PMP22 gene dosage have profound effects on the development and maintenance of peripheral nerves. This is evident from the genetic disease mechanisms in Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) as well as transgenic animals with altered PMP22 gene dosage. Thus, regulation of PMP22 is a crucial aspect in understanding the function of this protein in health and disease. In this study, we have generated transgenic mice containing 10 kb of the 5'-flanking region of the PMP22 gene, including the two previously identified alternative promoters, fused to a lacZ reporter gene. We show that this part of the PMP22 gene contains the necessary information to mirror the endogenous expression pattern in peripheral nerves during development and regeneration and in mouse models of demyelination due to genetic lesions. Transgene expression is strongly regulated during myelination, demyelination, and remyelination in Schwann cells, demonstrating the crucial influence of neuron-Schwann cell interactions in the regulation of PMP22. In addition, the region of the PMP22 gene present on this transgene confers also neuronal expression in sensory and motor neurons. These results provide the crucial basis for further dissection of the elements that direct the temporal and spatial regulation of the PMP22 gene and to elucidate the molecular basis of the master program regulating peripheral nerve myelination.     

A novel P0 glycoprotein transgene activates expression of lacZ in myelin-forming Schwann cells

P0 glycoprotein, the most abundant protein in peripheral nerve, is expressed specifically in the Schwann cell lineage. Upstream of the rat P0 gene 1.1 kb of DNA can activate expression of cDNAs specifically in Schwann cells in transgenic mice. However, the expression of P0 promoter-based transgenes has been inconsistent. As much as 9 kb of 5' flanking sequence fused to lacZ never yielded detectable levels of beta-galactosidase in multiple lines of mice. We describe transgenic mice that express lacZ in peripheral nerve, using the complete mouse P0 gene, including 6 kb of 5' flanking sequence, all exons and introns, and the natural polyadenylation signal. This vector activated lacZ expression specifically in cultured Schwann cells, and myelin-forming Schwann cells in four out of six transgenic lines. Transgene expression paralleled that of the endogenous P0 gene, both during development and after Wallerian degeneration. lacZ expression was lower than endogenous P0 expression, and was not detected in neural crest or Schwann cell precursors, where low levels of P0 mRNA are present. However, when the same vector contained a small myc tag instead of the 3.2-kb lacZ insert, the resulting transgenic mRNA was expressed at levels comparable to endogenous P0 mRNA. These data suggest that intragenic or 3' flanking sequences are necessary to generate the remarkable levels of endogenous P0 gene expression.     

Developmental profile of kainate receptor subunit KA1 revealed by Cre expression in YAC transgenic mice

To determine the spatio-temporal expression in brain of the high-affinity kainate receptor subunit KA1, we generated transgenic mice expressing Cre recombinase from the KA1 gene on a chromosomally integrated 550 kb yeast artificial chromosome (YAC). Activity of the KA1 gene promoter during brain development was visualized by Cre immunohistochemistry, and by X-gal staining of beta-galactosidase induced by Cre recombinase in double transgenic KA1-Cre/lacZ indicator mice. During early brain development, expression from the YAC-carried KA1-Cre transgene was observed in all major brain areas, predicting a function for KA1 in the developing central nervous system. In the adult brain, KA1-Cre transgene expression was restricted mainly to hippocampal CA3 pyramidal and dentate gyrus granule cells, an adult expression pattern characteristic for the endogenous KA1 alleles. KA1-Cre transgenic mice may help in elucidating the role of floxed genes ablated in vivo in KA1 expressing neurons.     

Cellular expression of an HMGCR promoter-CAT fusion gene in transgenic mouse brain: Evidence for a developmental regulation in oligodendrocytes

The HMGCR gene encodes the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the key enzyme for cholesterol synthesis. Mice transgenic for the prokaryotic chloramphenicol acetyl transferase (CAT) reporter gene fused with a 5′ Bam H1 fragment including the promoter sequence for murine HMGCR gene have been obtained. Homozygote transgenic mice were derived from a particular line selected for similar regulation of endogenous HMGCR and the transgene expression by nutritional conditions in different tissue. In addition, high expression of the transgene was evidenced in the brain. Cellular expression of the CAT gene in the central nervous system (CNS) was investigated by immunohistochemistry (IHC). This study was performed on frozen sections of the developing and adult brain, using a rabbit anti-CAT antiserum especially raised for that purpose. CAT expression was observed in some rare individuals in different neural cell types including Purkinje cells and astrocytes. But the most outstanding observation was the high level of CAT expression correlated with differentiated pattern of oligodendrocyte (01) distribution observed in white-matter tracts. Double and triple labeling for CAT and stage-specific antigens were performed on transgenic Ol-enriched preparations and cultures. This study showed a normal sequence of differentiation in the transgenic oligodendroglial cell lineage and demonstrated a strict correlation between late differentiation and activation of the CAT gene in these cells: CAT expression started in transgenic Ols between galactocerebroside (GC)-positive and myelin basic protein (MBP)-positive stages and was detected in MBP-positive cells during the myelination period. After myelination, the number of CAT-positive Ols decreased in the adult brain. These observations demonstrate a developmental regulation of the CAT transgene in Ols during myelination in CNS and reinforce the hypothesis of endogenous synthesis as major source of cholesterol during myelination. © 1994 Wiley-Liss, Inc.     

Proximal regions of the olfactory marker protein gene promoter direct olfactory neuron-specific expression in transgenic mice

Olfactory marker protein (OMP) expression is highly restricted to mature olfactory neurons (ON). Less than 0.3 kb of upstream 5′ flanking sequence of the OMP gene directs lacZ expression preferentially to ON in several independently derived lines of transgenic mice. A larger transgene with 0.8 kb of upstream flanking sequence also gave lacZ expression in ON and in a few ectopic sites in the central nervous system (CNS). In addition to the main olfactory epithelium, endogenous OMP is also expressed in chemosensory neurons of the vomeronasal and septal organs, and lacZ expression was detected in neurons of these sites as well. This confirmed the presence of regulatory sequences in the proximal portion of the OMP gene. Endogenous OMP expression in ON was normal in all transgenic lines. Strikingly, in several transgenic lines lacZ expression was restricted to subsets of ON. In one such line, ON axons were intensely stained for lacZ and projected to a subset of olfactory bulb glomeruli. Although identifiable subsets of ON and their termination fields have been described previously, this is the first demonstration of this phenomenon in transgenic mice. These lines of transgenic mice thus provide in vivo models for characterization of genetic elements regulating developmental and functional organization of the olfactory neuroepithelium. © 1996 Wiley-Liss, Inc.     

Distinct disease mechanisms in peripheral neuropathies due to altered peripheral myelin protein 22 gene dosage or a Pmp22 point mutation

Point mutations affecting PMP22 can cause hereditary demyelinating and dysmyelinating peripheral neuropathies. In addition, duplication and deletion of PMP22 are associated with Charcot-Marie-Tooth disease Type 1A (CMT1A) and Hereditary Neuropathy with Liability to Pressure Palsy (HNPP), respectively. This study was designed to elucidate disease processes caused by misexpression of Pmp22 and, at the same time, to gain further information on the controversial molecular function of PMP22. To this end, we took advantage of the unique resource of a set of various Pmp22 mutant mice to carry out comparative expression profiling of mutant and wild-type sciatic nerves. Tissues derived from Pmp22-/- ("knockout"), Pmp22tg (increased Pmp22 copy number), and Trembler (Tr; point mutation in Pmp22) mutant mice were analyzed at two developmental stages: (i) at postnatal day (P)4, when normal myelination has just started and primary causative defects of the mutations are expected to be apparent, and (ii) at P60, with the goal of obtaining information on secondary disease effects. Interestingly, the three Pmp22 mutants exhibited distinct profiles of gene expression, suggesting different disease mechanisms. Increased expression of genes involved in cell cycle regulation and DNA replication is characteristic and specific for the early stage in Pmp22-/- mice, supporting a primary function of PMP22 in the regulation of Schwann cell proliferation. In the Tr mutant, a distinguishing feature is the high expression of stress response genes. Both Tr and Pmp22tg mice show strongly reduced expression of genes important for cholesterol synthesis at P4, a characteristic that is common to all three mutants at P60. Finally, we have identified a number of candidate genes that may play important roles in the disease process or in myelination per se.     

Behavioral and Molecular Exploration of the AR-CMT2A Mouse Model Lmna R298C/R298C

In 2002, we identified LMNA as the first gene responsible for an autosomal recessive axonal form of Charcot-Marie-Tooth disease, AR-CMT2A. All patients were found to be homozygous for the same mutation in the LMNA gene, p.Arg298Cys. In order to investigate the physiopathological mechanisms underlying AR-CMT2A, we have generated a knock-in mouse model for the Lmna p.Arg298Cys mutation. We have explored these mice through an exhaustive series of behavioral tests and histopathological analyses, but were not able to find any peripheral nerve phenotype, even at 18 months of age. Interestingly at the molecular level, however, we detect a downregulation of the Lmna gene in all tissues tested from the homozygous knock-in mouse Lmna (R298C/R298C) (skeletal muscle, heart, peripheral nerve, spinal cord and cerebral trunk). Importantly, we further reveal a significant upregulation of Pmp22, specifically in the sciatic nerves of Lmna (R298C/R298C) mice. These results indicate that, despite the absence of a perceptible phenotype, abnormalities exist in the peripheral nerves of Lmna (R298C/R298C) mice that are absent from other tissues. Although the mechanisms leading to deregulation of Pmp22 in Lmna (R298C/R298C) mice are still unclear, our results support a relation between Lmna and Pmp22 and constitute a first step toward understanding AR-CMT2A physiopathology.     

Antiprogesterone therapy uncouples axonal loss from demyelination in a transgenic rat model of CMT1A neuropathy

OBJECTIVE: Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy, and a duplication of the Pmp22 gene causes the most frequent subform CMT1A. Using a transgenic rat model of CMT1A, we tested the hypothesis that long-term treatment with anti-progesterone (Onapristone) reduces Pmp22 overexpression and improves CMT disease phenotype of older animals, thereby extending a previous proof-of-concept observation in a more clinically relevant setting. METHODS: We applied placebo-controlled progesterone-antagonist therapy to CMT rats for 5 months and performed grip-strength analysis to assess the motor phenotype. Quantitative Pmp22 RT-PCR and complete histological analysis of peripheral nerves and skin biopsies were performed. RESULTS: Anti-progesterone therapy significantly increased muscle strength and muscle mass of CMT rats and reduced the performance difference to wildtype rats by about 50%. Physical improvements can be explained by the prevention of axon loss. Surprisingly, the effects of anti-progesterone were not reflected by improved myelin sheath thickness. Electrophysiology confirmed unaltered NCV, but less reduced CMAP recordings in the treatment group. Moreover, the reduction of Pmp22 mRNA, as quantified in cutaneous nerves, correlated with the clinical phenotype at later stages. INTERPRETATION: Progesterone-antagonist long-term therapy reduces [corrected] Pmp22 overexpression to a degree at which the axonal support function of Schwann cells is better maintained than myelination. This suggests that axonal loss in CMT1A is not caused by demyelination, but rather by a Schwann cell defect that has been partially uncoupled by anti-progesterone treatment. Pmp22 expression analysis in skin may provide a prognostic marker for disease severity and for monitoring future clinical trials.     

Analysis of cis-regulatory elements in the 5' flanking region of the Drosophila melanogaster choline acetyltransferase gene.

We have analyzed the cis-regulatory elements in the 5' flanking region of the Drosophila choline acetyltransferase gene (ChAT, E.C.2.3.1.6). DNA fragments were fused to the Escherichia coli lacZ reporter gene and introduced into the Drosophila germ line by P-element-mediated transformation. A 7.4 kb 5' flanking sequence directed beta-galactosidase expression in the adult optic lobes and other well-defined CNS structures with a pattern very similar to the distribution of endogenous ChAT protein. In contrast, the proximal 3.3 kb and 1.2 kb of 5' flanking DNA directed lacZ expression in only selected subsets of the structures seen with the 7.4 kb lacZ construct. Our results indicate that both qualitative and quantitative regulatory elements are present in the 5' flanking DNA and that these elements distinguish various subsets of cholinergic neurons. We have also fused the same 5' flanking DNA sequences to wild-type ChAT cDNA and used these constructs to transform Chatsl mutant flies. Not only the 7.4 kb cDNA construct, but also the 3.3 and 1.2 kb constructs, rescued Chatsl from temperature-dependent paralysis and adult lethality, indicating that the regulatory information in any of these genomic fragments can drive sufficient wild-type ChAT expression to overcome these mutant phenotypes.     

Retroviral labeling of Schwann cells: in vitro characterization and in vivo transplantation to improve peripheral nerve regeneration

Transplantation of Schwann cells (SCs) is a promising treatment modality to improve neuronal regeneration. Identification of the transplanted cells is an important step when studying the development of this method. Genetic labeling is the most stable and reliable method of cell identification, but it is still unclear whether it has deleterious effect on SC characteristics. Our aim was to achieve a stable population of SCs transduced with the lacZ gene at a high frequency using a retroviral vector in vitro, and to follow the labeled SC in vitro to assess their viability and phenotypic marker expression. Furthermore, we transplanted lacZ-labeled SCs in a conduit to repair peripheral nerve to investigate their effect on nerve regeneration in vivo. Rat and human SCs were cultured and transduced with an MFG lacZ nls marker gene, achieving a transduction rate of 80% and 70%, respectively. Rat SCs were kept in culture for 27 weeks and examined every 4 weeks for expression of lacZ, viability, and phenotypic marker expression of GFAP, p75, MHC I and II. Throughout this period, transduced rat SCs remained viable and continued to proliferate. The proportion of cells expressing lacZ dropped only by 10% and the expression of phenotypic markers remained stable. Transduced human SCs were followed up for 4 weeks in culture. They proliferated and continued to express the lacZ gene and phenotypic marker expression of GFAP and p75 was preserved. Primary culture of transduced rat SCs were transplanted, syngeneically, in a conduit to bridge a 10 mm gap in sciatic nerve and the grafts were examined after 3 weeks for the presence and participation of labeled SCs and for axonal regeneration distance. Transplanted transduced rat SCs were clearly identified, taking part in the regeneration process and enhancing the axonal regeneration rate by 100% (at the optimal concentration) compared to conduits without SCs. Thus, retroviral introduction of lacZ gene has no deleterious effect on SCs in vitro and these SCs take part and enhance nerve regeneration in vivo.     

5' flanking sequences of the rat tyrosine hydroxylase gene target accurate tissue-specific, developmental, and transsynaptic expression in transgenic mice.

Transgenic mice were generated in which sequences that flank the rat tyrosine hydroxylase (TH) gene were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Mice bearing 4.8 kilobases (kb) of 5' flanking DNA exhibited correct tissue-specific expression in the CNS and periphery. Expression was more robust in the CNS than in the periphery, although CAT activity was clearly detected in sympathetic ganglia (superior cervical ganglia) and the adrenal, the two peripheral tissues that contain TH-positive cells. Within the brain, CAT expression was seen in all the expected areas containing TH-positive cell bodies, with little or no expression in other regions. In the olfactory bulb, which contains the majority of the CNS TH cells, developmental expression of CAT was quantifiable and was found to parallel the postnatal rise in endogenous TH, with both TH and CAT reaching adult levels by postnatal day 21. Since TH activity in the olfactory bulb requires afferent input, the dependence of CAT activity on transsynaptic input was also assayed in transgenic mice. Like the endogenous TH activity, CAT levels were also reduced by deafferentation, in parallel with loss in endogenous dopamine levels. While previous experiments demonstrated that shorter 5' flanking regions (2.5 kb and 3.5 kb of 5' upstream sequences of the human and mouse TH gene, respectively) failed to direct accurate tissue-specific expression, our data demonstrate that 4.8 kb of 5' flanking sequence of the rat TH gene contains sufficient regulatory information to mediate appropriate tissue-specific expression in all CNS and PNS tissues, as well as to mediate developmental and transsynaptic expression in the olfactory bulb.     

Desmin-lacZ transgene, a marker of regenerating skeletal muscle

Transgenic C57 mice bearing a transgene of the desmin gene linked to the lacZ reporter gene which encoded for the enzyme β-galactosidase were used. In the muscle cell, a blue nuclear product appearing in the presence of the X-gal substrate for the enzyme provided evidence of the expression of the desmin gene. However, no transgene expression was observed 2 weeks postnatal in skeletal muscles, even though endogenous desmin was present. In order to investigate the regulatory mechanisms of the desmin gene during regeneration, adult pectoralis fragments (without expression of the desmin transgene) from transgenic mice were implanted into the tibialis anterior of 4 day or 6 week old Swiss mice. Adult pectoralis transplants re-expressed the transgene from day 4 to 10 after implantation. In addition, lesions were performed in adult transgenic pectoralis and transgenic expression in injured muscles was observed 2 days later. This new transgenic mouse is a powerful tool for the study of the various steps of skeletal muscle regeneration.     

Generation of a transgenic model to address regulation and function of the human neurokinin 1 receptor (NK1R)

We have generated mouse transgenic lines using yeast artificial chromosome (YAC) technology which demonstrate expression from the human NK1 receptor (NK1R) locus. We introduced a 380 kb fragment encompassing the human NK1R gene and flanking regions which we hoped would recapitulate the expected endogenous expression of the human gene. To visualise this expression the NK1 locus co-expresses the green fluorescence protein gene (GFP) under the control of an internal ribosome entry site (IRES) sequence. We have generated five mouse lines that express the human NK1 receptor gene with and without the marker gene. All the lines incorporating the marker gene appear to exhibit the same expression pattern in analysis of selected anatomical regions throughout the mouse. The lack of a human specific NK1R antibody determined that we could not distinguish between expression of the transgene and endogenous NK1R. Our analysis has shown transgene expression in brain areas known to express NK1R in human such as the hippocampus and caudate putamen. The majority of these cells were also positive for GFP fluorescence. These transgenic lines may prove a good pre-clinical model as drugs can be addressed against both the human receptor and modulators of its expression in vivo.     

Distinct elements of the peripheral myelin protein 22 (PMP22) promoter regulate expression in Schwann cells and sensory neurons

Genetic disease mechanisms in the demyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1A (CMTA) and hereditary neuropathy with liability to pressure palsies (HNPP) as well as transgenic animals with altered PMP22 gene dosage revealed that alterations in PMP22 gene expression have profound effects on the development and maintenance of peripheral nerves. Consequently, the regulation of PMP22 is a crucial aspect in understanding the function of this protein in health and disease. In this study, we dissected and analyzed different cis-acting elements in the 5'-flanking region of the Pmp22 gene in vivo. We found two separate elements that contribute to different aspects of Pmp22 expression. The first is located 5' distally to promoter 1 and is involved in gene regulation during late phases of myelination in development ["late myelination Schwann cell-specific element" (LMSE)] and in remyelination after injury. The second element was identified upstream of promoter 2 and guides Pmp22 expression in sensory neurons. These results suggest that multiple distinct signaling pathways regulating Pmp22 expression in myelination as well as in neurons converge on distinct segments of the PMP22 promoter region. The underlying molecular mechanisms are likely to be crucially involved in the maintenance of the integrity of myelinated peripheral nerves.     

Identification of a new Pmp22 mouse mutant and trafficking analysis of a Pmp22 allelic series suggesting that protein aggregates may be protective in Pmp22-associated peripheral neuropathy

We have identified and characterized a new peripheral myelin protein 22 (Pmp22) mouse mutant. The mutation results in a serine to threonine amino acid substitution at residue 72, which is a hot spot for mutation in human PMP22, leading to the peripheral neuropathy Dejerine-Sottas syndrome. We have previously described two other Pmp22 mutants, providing an allelic series for gene function analysis. Pmp22 mutations generally lead to abnormal intracellular trafficking of Pmp22, and we show that each mutant protein in the allelic series has a unique pattern of intracellular localization in transfected cell lines. The mutant protein from the less severely affected mutants occurs in large aggregates, while the mutant protein from the most severely affected mutant occurs in a diffuse perinuclear pattern that largely colocalizes with wild-type protein. This suggests that large Pmp22 aggregates may be protective in this form of peripheral neuropathy.