Effects of Altered Thyroid States on Myelinogenesis

Abstract: Myelinogenesis was studied in controls and in rats treated since birth with Methimazole (hypothyroid) or thyroxine (hyperthyroid). The amount of myelin in forebrain and its protein composition were determined between 13 and 40 days of age, the period of most rapid myelin accumulation. Hypothyroid rats had reduced body and brain weights relative to controls and the yield of myelin was reduced on both a per brain and a per milligram brain protein basis. Developmental changes in the protein composition of isolated myelin followed the pattern of control animals (the percentage of total myelin protein present as proteolipid protein, large basic protein, and small basic protein increased, as did the ratio of proteolipid/large basic protein) but were delayed temporally by 1–2 days. Hyperthyroid rats also had reduced body and brain weights. At 13 days myelin accumulation was greater than that of controls, corresponding to an earlier initiation of myelination. At later ages myelin yield was reduced on a per brain basis but not on a per milligram brain protein basis. The developmental pattern of myelin protein composition was accelerated temporally by 1–2 days. Myelination in optic nerve, assayed by proteolipid protein content, also was slightly delayed in hypothyroid animals and somewhat accelerated in hyperthyroid animals. The relative synthesis of myelin proteins (determined as incorporation of intracranially injected [³H]glycine into myelin protein relative to incorporation into whole brain protein), as well as distribution of radioactivity among individual myelin proteins, was determined. The results supported the conclusion of the myelin protein accumulation study; hypothyroidism retards the developmental program for myelinogenesis, whereas in the hyperthyroid state myelin synthesis is initiated earlier but is also terminated earlier.     

Regulation of Myelination: Schwann Cell Transition from a Myelin-Maintaining State to a Quiescent State After Permanent Nerve Transection

Permanent nerve transection of the adult rat sciatic nerve forces Schwann cells in the distal nerve segment from a myelin-maintaining to a quiescent state. This transition was followed by serial morphometric evaluation of the percentage fascicular area having myelin (myelin percent of area) in transverse sections of the distal nerve segment and revealed a rapid decline from a normal value of 36.6% to 3.2% by 14 days for the sciatic nerve to less than 1.0% throughout the remaining time course (up to 105 days). No evidence of axonal reentry into the distal nerve segment or new myelin formation was observed at times under 70 days. In some of the distal nerve segments at 70, 90, and 105 days, new myelinated fibers were observed that usually consisted of only a few myelinated fibers at the periphery and in the worst case amounted to 1.6% (myelin percent of area). Radioactive precursor incorporation of [3H]mannose into endoneurial slices at 4 and 7 days after transection revealed two species of the major myelin glycoprotein, P0, with Mr of 28,500 and 27,700. By 14 days after nerve transection, only the 27,700 Mr species remained. Incorporation of [3H]mannose into the 27,700 Mr species increased progressively to 35 days after transection and then began to decline at 70 and 105 days. Alterations in the oligosaccharide structure of this down-regulated myelin glycoprotein accounted for the progressive increase in mannose incorporation. Lectin affinity chromatography of pronase-digested P0 glycopeptides on concanavalin A-Sepharose revealed that the 28,500 Mr species of P0 had the complex-type oligosaccharide as the predominant oligosaccharide structure (92%). In contrast, the high mannose-type oligosaccharide was the predominate structure for the 27,700 Mr form, which increased to 70% of the total radioactivity by 35 days after nerve transection. Since the biosynthesis of the complex-type oligosaccharide chains on glycoproteins involves high mannose-type intermediates, the mechanism of down-regulation in the biosynthesis of this major myelin glycoprotein, therefore, results in a biosynthetic switch from the complex-type oligosaccharide structure as an end product to the predominantly high mannose-type oligosaccharide structure as a biosynthetic intermediate. This biosynthetic switch occurs gradually between 7 and 14 days after nerve transection and likely reflects a decreased rate of processing through the Golgi apparatus. It remains to be determined if the high mannose-type oligosaccharide chain on P0 can undergo additional processing steps in this permanent nerve transection model.     

The myelination of the cerebellar cortex in the cat

Summary The myelination of the cerebellar cortex of the cat was investigated in 61 cats aged from 3 hrs post partum to two and a half years. The first myelinated fibers appear at the time of birth in the central medullary ray. Before the onset of myelination, all fibers reach a critical diameter of about 1 m. About the 14th day of life the number of oligodendrocytes in the prospective white matter increases markedly. Thereafter, the oligodendrocytes invade the inner granular layer. It therefore seems that the myelination of the cerebellar cortex proceeds from the central medullary ray towards the granular layer. At the 60th day of postnatal life, most of the afferent and efferent fiber systems are myelinated. These findings are discussed in relation to the development of function and the maturation of the electrical activity of the cerebellar circuit.Dedicated to Prof. Dr. H. Leonhardt in honour of his 60th birthdaySupported by the Deutsche Forschungsgemeinschaft (La 184/3)     

Characterization of the MAL2-positive compartment in oligodendrocytes

Oligodendrocytes (OLs), the myelin-producing cells of the central nervous system, segregate different surface subdomains at the plasma membrane as do other differentiated cells such as polarized epithelia and neurons. To generate the complex membrane system that characterizes myelinating OLs, large amounts of membrane proteins and lipids need to be synthesized and correctly targeted. In polarized epithelia, a considerable fraction of apical proteins are transported by an indirect pathway involving a detour to the basolateral membrane before being internalized and transported across the cell to the apical membrane by a process known as transcytosis. The apical recycling endosome (ARE) or its equivalent, the subapical compartment (SAC), of hepatocytes is an intracellular trafficking station involved in the transcytotic pathway. MAL2, an essential component of the machinery for basolateral-to-apical transcytosis, is an ARE/SAC resident protein. Here, we show that, after differentiation, murine oligodendrocyte precursor and human oligodendroglioma derived cell lines, Oli-neu and HOG, respectively, up-regulate the expression of MAL2 and accumulate it in an intracellular compartment, exhibiting a peri-centrosomal localization. In these oligodendrocytic cell lines, this compartment shares some of the main features of the ARE/SAC, such as colocalization with Rab11a, sensitivity to disruption of the microtubule cytoskeleton with nocodazole, and lack of internalized transferrin. Therefore, we suggest that the MAL2-positive compartment in oligodendrocytic cells could be a structure analogous to the ARE/SAC and might have an important role in the sorting of proteins and lipids for myelin assembly during oligodendrocyte differentiation.     

Brain-derived neurotrophic factor (BDNF) induces polarized signaling of small GTPase (Rac1) protein at the onset of Schwann cell myelination through partitioning-defective 3 (Par3) protein

Brain-derived neurotrophic factor (BDNF) was shown to play a role in Schwann cell myelination by recruiting Par3 to the axon-glial interface, but the underlying mechanism has remained unclear. Here we report that Par3 regulates Rac1 activation by BDNF but not by NRG1-Type III in Schwann cells, although both ligands activate Rac1 in vivo. During development, active Rac1 signaling is localized to the axon-glial interface in Schwann cells by a Par3-dependent polarization mechanism. Knockdown of p75 and Par3 individually inhibits Rac1 activation, whereas constitutive activation of Rac1 disturbs the polarized activation of Rac1 in vivo. Polarized Rac1 activation is necessary for myelination as Par3 knockdown attenuates myelination in mouse sciatic nerves as well as in zebrafish. Specifically, Par3 knockdown in zebrafish disrupts proper alignment between the axon and Schwann cells without perturbing Schwann cell migration, suggesting that localized Rac1 activation at the axon-glial interface helps identify the initial wrapping sites. We therefore conclude that polarization of Rac1 activation is critical for myelination.     

缺氧缺血诱导对原代新生大鼠前脑混合细胞髓鞘形成和细胞骨架的影响

目的:该研究探讨了缺氧缺血诱导对新生大鼠前脑混合细胞髓鞘形成和细胞骨架的影响。方法:在原代培养的新生大鼠前脑混合细胞中,应用免疫组化染色髓鞘碱性蛋白(MBP)和劳克坚牢蓝(LFB)染色检测髓鞘和轴突的发育情况;Western印迹分析NF155蛋白的表达情况;免疫荧光分析参与细胞骨架调节的ERM-Rho GTP酶家族相关蛋白表达。结果:缺氧缺血对混合细胞的进一步分化和成熟起到抑制作用,MBP染色阳性率降低57%,髓鞘和轴突染色阳性率降低74%;NF155蛋白表达降低51%;Rho GTP酶家族成员Rac1、Cdc42分别降低81%和75%。结论:缺氧缺血使细胞突起的形成和骨架重组受到阻碍,继而影响髓鞘的发育和成熟,这一过程与ERM-Rho GTP酶细胞骨架通路有关。     

Interactions between oligodendrocyte precursors control the onset of CNS myelination

The formation of CNS myelin is dependent on the differentiation of oligodendrocyte precursor cells (OPCs) and oligodendrocyte maturation. How the initiation of myelination is regulated is unclear, but it is likely to depend on the development of competence by oligodendrocytes and receptivity by target axons. Here we identify an additional level of control of oligodendrocyte maturation mediated by interactions between the different cellular components of the oligodendrocyte lineage. During development oligodendrocyte precursors mature through a series of stages defined by labeling with monoclonal antibodies A2B5 and O4. Newly differentiated oligodendrocytes begin to express galactocerebroside recognized by O1 antibodies and subsequently mature to myelin basic protein (MBP)-positive cells prior to formation of compact myelin. Using an in vitro brain slice culture system that supports robust myelination, the consequences of ablating cells at different stages of the oligodendrocyte lineage on myelination have been assayed. Elimination of all OPC lineage cells through A2B5+, O4+, and O1+ complement-mediated cell lysis resulted in a delay in development of MBP cells and myelination. Selective elimination of early OPCs (A2B5+) also unexpectedly resulted in delayed MBP expression compared to controls suggesting that early OPCs contribute to the timing of myelination onset. By contrast, elimination of differentiated (O1+) immature oligodendrocytes permanently inhibited the appearance of MBP+ cells suggesting that oligodendrocytes are critical to facilitate the maturation of OPCs. These data illuminate that the presence of intra-lineage feed-forward and feedback cues are important for timely myelination by oligodendrocytes.     

Cleavage of neuregulin-1 by BACE1 or ADAM10 protein produces differential effects on myelination

Neuregulin-1 (Nrg1) is encoded by a single gene and exists in naturally secreted and transmembrane isoforms. Nrg1 exerts its signaling activity through interaction with its cognate ErbB receptors. Multiple membrane-anchored Nrg1 isoforms, present in six different membrane topologies, must be processed by a protease to initiate a signaling cascade. Here, we demonstrate that BACE1 and ADAM10 can process type I and III Nrg1 at two adjacent sites. Our cleavage site mapping experiments showed that the BACE1 cleavage site is located eight amino acids downstream of the ADAM10 cleavage site, and this order of cleavage is the opposite of amyloid precursor protein cleavage by these two enzymes. Cleavages were further confirmed via optimized electrophoresis. Cleavage of type I or III Nrg1 by ADAM10 and BACE1 released a signaling-capable N-terminal fragment (ntf), either Nrg1-ntfα or Nrg1-ntfβ, which could similarly activate an ErbB receptor as evidenced by increased phosphorylation of Akt and ERK, two downstream signaling molecules. Although both Nrg1-ntfα and Nrg1-ntfβ could initiate a common signaling cascade, inhibition or down-regulation of ADAM10 alone in a co-culture system did not affect normal myelination, whereas specific inhibition of BACE1 impaired normal myelination. Thus, processing of Nrg1 by BACE1 appears to be more critical for regulating myelination. Our results imply that a significant inhibition of BACE1 could potentially impair Nrg1 signaling activity in vivo.     

The Effects of Prenatal Iron Deficiency and Risperidone Treatment on the Rat Frontal Cortex: A Proteomic Analysis

Prenatal iron deficiency (pID) has been described to increase the risk for neurodevelopmental disorders such as autism and schizophrenia; however, the precise molecular mechanisms are still unknown. Here, we utilized high‐throughput MS to examine the proteomic effects of pID in adulthood on the rat frontal cortex area (FCA). In addition, the FCA proteome was examined in adulthood following risperidone treatment in adolescence to see if these effects could be prevented. We identified 1501 proteins of which 100 were significantly differentially expressed in the FCA at postnatal day 90. Pathway analysis of proteins affected by pID revealed changes in metabolic processes, including the tricyclic acid cycle, mitochondrial dysfunction, and P13K/Akt signaling. Interestingly, most of these protein changes were not present in the adult pID offspring who received risperidone in adolescence. Considering the link between pID and several neurodevelopmental disorders such as autism and schizophrenia these presented results bring new perspectives to understand the role of iron in metabolic pathways and provide novel biomarkers for future studies of pID.