Huiyuan Jiang

Heterozygous P0 Knockout Mice Develop a Peripheral Neuropathy that Resembles Chronic Inflammatory Demyelinating Polyneuropathy (CIDP)

Demyelinating peripheral neuropathies are clinically divided into inherited and acquired types. Inherited demyelinating neuropathies are caused by mutations in genes expressed by myelinating Schwann cells, whereas acquired ones, including chronic inflammatory demyelinating polyneuropathy (CIDP), are probably caused by autoimmune mechanisms. We find that heterozygous P0 knockout (P0+/-) mice develop a neuropathy that resembles CIDP. By one year of age, P0+/- mice develop severe, asymmetric slowing of motor nerves, with temporal dispersion or conduction block, which are features of acquired demyelinating neuropathies including CIDP. Moreover, morphological analysis of affected nerves reveals severe and selective demyelination of motor fibers, focal regions of demyelination, and inflammatory cells. These data suggest that immune-mediated mechanisms may contribute to the pathogenesis of the neuropathy in P0+/- mice.

Regulation of Oleoyl‐CoA Synthesis in the Peripheral Nervous System: Demonstration of a Link with Myelin Synthesis

Abstract: We studied the regulation of oleic acid synthesis in the PNS. During mouse postnatal development, the proportion of 18:1 rises in the sciatic nerve from 17% at 5 days of age to 33% at 25 days. However, this rise does not occur in the dysmyelinating mutant mouse trembler. In normal mouse development, the total stearoyl‐CoA desaturase (SCD) activity measured in sciatic nerve homogenates is high during the first 3 weeks. Yet in trembler nerves, this SCD activity represents only 15% of normal values. Using the RT‐PCR technique, we demonstrate that the SCD2 isoform is predominantly expressed in the PNS. Northern blot analysis showed that the mRNA levels for SCD2 parallel those of other specific myelin proteins in both normal mouse and trembler mutant development. Similar experiments in a rat demyelination‐remyelination model confirmed that SCD2 mRNA levels are regulated in the PNS in a similar manner to myelin‐specific proteins.

Absence of P0 leads to the dysregulation of myelin gene expression and myelin morphogenesis.

P0, the major peripheral nervous system (PNS) myelin protein, is a member of the immunoglobulin supergene family of membrane proteins and can mediate homotypic adhesion. P0 is an essential structural component of PNS myelin; mice in which P0 expression has been eliminated by homologous recombination (P0−/−) develop a severe dysmyelinating neuropathy with predominantly uncompacted myelin. Although P0 is thought to play a role in myelin compaction by promoting adhesion between adjacent extracellular myelin wraps, as an adhesion molecule it could also have a regulatory function. Consistent with this hypothesis, Schwann cells in adult P0−/− mice display a novel molecular phenotype: PMP22 expression is down‐regulated, MAG and PLP expression are up‐regulated, and MBP expression is unchanged. As in quaking viable mutant mice (qkv), which have uncompacted myelin morphologically similar to that found in P0−/− mice, neither the qKI‐6 or qKI‐7 proteins are expressed in P0−/− peripheral nerve. In addition to these changes in gene expression in the P0 knockout, PLP/DM‐20 accumulates in the endoplasmic reticulum of P0−/− Schwann cells, whereas MAG accumulates in redundant loops of uncompacted myelin, not at nodes of Ranvier or Schmidt‐Lantermann incisures. Taken together, these results demonstrate that P0 is involved, either directly or indirectly, in the regulation of both myelin gene expression and myelin morphogenesis. J. Neurosci. Res. 60:714–724, 2000.

Proteolipid Protein mRNA Stability Is Regulated by Axonal Contact in the Rodent Peripheral Nervous System

Proteolipid protein (PLP) and its alternatively spliced isoform, DM20, are the main intrinsic membrane proteins of compact myelin in the CNS. PLP and DM20 are also expressed by Schwann cells, the myelin-forming cells in the PNS, and are necessary for normal PNS function in humans. We have investigated the expression of PLP in the PNS by examining transgenic mice expressing a LacZ transgene under the control of the PLP promoter. In these animals, myelinating Schwann cells expressed beta-galactosidase more prominently than nonmyelinating Schwann cells. PLP/DM20 mRNA levels, but not those of LacZ mRNA, increased during sciatic nerve development and decreased after axotomy, with resultant Wallerian degeneration. PLP/DM20 transcription rates, in nuclear run off experiments, however, did not increase in developing rat sciatic nerve despite robust increases in PLP/DM20 mRNA levels during the same period. In RNAse protection studies, PLP mRNA levels fell to undetectable levels following nerve transection whereas levels of DM20 were essentially unchanged despite both being transcribed from the same promoter. Finally, cotransfection studies demonstrated that PLP-GFP, but not DM20-GFP mRNA is down-regulated in Schwann cells cultured in the absence of forskolin. Taken together these data demonstrate that steady state levels of PLP mRNA are regulated at a posttranscriptional level in Schwann cells, and that this regulation is mediated by Schwann cell-axonal contact. Since the difference between these two mRNAs is a 105-bp sequence in PLP and not in DM20, this sequence is likely to play a role in the regulation of PLP mRNA.

Regulation of Myelin‐Specific Gene Expression: Relevance to CMT1

ABSTRACT: Schwann cells, the myelinating cells of the peripheral nervous system, are derived from the neural crest. Once neural crest cells are committed to the Schwann cell fate, they can take on one of two phenotypes to become myelinating or nonmyelinating Schwann cells, a decision that is determined by interactions with axons. The critical step in the differentiation of myelinating Schwann cells is the establishment of a one‐to‐one relationship with axons, the so‐called “promyelinating” stage of Schwann cell development. The transition from the promyelinating to the myelinating stage of development is then accompanied by a number of significant changes in the pattern of gene expression, including the activation of a set of genes encoding myelin structural proteins and lipid biosynthetic enzymes, and the inactivation of a set of genes expressed only in immature or nonmyelinating Schwann cells. These changes are regulated mainly at the transcriptional level and also require continuous interaction between Schwann cells and their axons.

Fatty acid synthase expression during peripheral nervous system myelination

The expression of fatty acid synthase (FAS) in rat and mouse sciatic nerves during postnatal development was investigated. FAS activity was not sensitive to the nutritional status of the animals. During development, the specific activity of FAS was low in rat and mouse nerves immediately after birth. Then, there was a steady increase in the activity (8- to 10-fold) which reached a maximal level around postnatal day 11, plateaued till day 32, and decreased to reach 30% of the maximum at day 80. A similar developmental profile was obtained when the amount of FAS protein was quantified, thus suggesting that the variations in activity observed during sciatic nerve development are mainly due to variations in FAS protein content. Northern blot analysis showed that the mRNA levels for FAS parallels those of the ceramide galactosyl transferase (CGT) during mouse sciatic nerve development and in a rat demyelination-nerve regeneration model. In addition, we measured FAS expression in the sciatic nerves of the trembler mutant, which is a mouse model of PNS dysmyelination. In 20-day-old trembler nerves, FAS specific activity, protein amount and mRNA levels represented only 25% of the normal values. Altogether, our data indicate that FAS expression is linked to the PNS myelination process, and that the main regulation occurs at the level of the gene expression.

Modulation of cell-mediated immunity prolongs adenovirus-mediated transgene expression in sciatic nerve.

In a previous report, we demonstrated that a first-generation (E1- and E3-deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenovirus-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral transgene expression over time. In our current work we have optimized adenoviral vector-mediated transgene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2-week-old rats, decreases thereafter, and there is minimal associated tissue injury. In contrast, few lacZ-expressing Schwann cells are found in nerve of adult animals 10 days after injection, probably owing to immune clearance of virus-infected cells. Consistent with this notion, high levels of LacZ are found in sciatic nerve 30 days after injection of adult SCID mice, which have a genetic defect in both cellular and humoral immunity, of adult beta2-microglobulin-deficient mice (beta2M4-/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenovirus-infected Schwann cells cocultured with axons in vitro, in the absence of a host immune response, ensheathe axons and express lacZ for at least 8 weeks. These data thus demonstrate that lacZ transgene expression of first-generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell-mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

Minimal role for activating transcription factor 3 in the oligodendrocyte unfolded protein response in vivo.

To further our goal of identifying and characterizing the functions of major components of the unfolded protein response (UPR) in oligodendrocytes, the gene encoding the activator of transcription factor 3 protein (ATF3) has been ablated in mice expressing mutant forms of the Proteolipid protein 1 (Plp1) gene and the phenotype of double mutants characterized at several levels. Mature oligodendrocytes in Plp1 mutant mice undergo UPR‐induced cell stress, induce ATF3 expression and exhibit a greater propensity to die by apoptosis, which is consistent with pro‐death function of ATF3 proposed from in vitro studies. However, we find that the absence of ATF3 has no effect on the levels of apoptosis in Plp1 mutants. Furthermore, we find that oligodendrocyte function appears normal in Atf3−/− mice and that motor coordination and neural communication are similarly unaffected. Accordingly, we conclude that ATF3, at best, plays a minor role in UPR signaling and its expression is more likely induced by the UPR as a secondary event in oligodendrocytes that is unrelated to cell death.

Overcoming Cellular Immunity to Prolong Adenoviral-Mediated Gene Expression in Sciatic Nerve

ABSTRACT: In a previous report, we demonstrated that a first generation (E1‐ and E3‐deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenoviral‐mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral gene expression over time. In our current work we have optimized adenoviral‐mediated gene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2‐week‐old animals, decreases thereafter, and there is minimal associated tissue injury. In contrast, very few adenoviral‐infected Schwann cells are found in nerves of adult animals 10 days after injection, probably due to immune clearance of viral‐infected cells. Consistent with this notion, high levels of lacZ are found in sciatic nerve 30 days after injection of adult SCID mice, which have a genetic defect in both cellular and humoral immunity, of adult β2 microglobulin‐deficient mice (β2 M −/−), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenoviral‐infected Schwann cells co‐cultured with axons in vitro, in the absence of a host immune response, ensheath axons and express lacZ for at least 8 weeks. These data thus demonstrate that expression of first generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell‐mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

The absence of myelin P0 protein produces a novel molecular phenotype in Schwann cells.

ABSTRACT: In order to better understand the pathogenesis of demyelination in P0 knockout (P0−/−) mice, we analyzed the myelin gene expression and the localization of myelin proteins in P0 null mouse sciatic nerve. We have demonstrated that the severe demyelinating neuropathy of P0‐knockout mouse is associated with changes in the program of myelin gene expression. Some changes in myelin gene expression occur early, others occur during adulthood. We also provide evidence that the absence of P0 is associated with changes in the localization of specific paranodal proteins in the peripheral nerve. These data suggest that P0 plays an important role, either directly or indirectly, in the program of Schwann cell gene expression and in the specific distribution of peripheral myelin proteins. Furthermore, myelin gene dysregulation and improper localization of paranodal proteins may account, in part, for the pathogenesis of demyelination in P0‐knockout mice, as well as in human demyelinating peripheral neuropathy associated with mutations in the P0 gene.