Hypomyelinated vps16 Mutant Zebrafish Exhibit Systemic and Neurodevelopmental Pathologies.

Homotypic Fusion and Protein Sorting (HOPS) and Class C-core Vacuole/Endosome Tethering (CORVET) complexes regulate the correct fusion of endolysosomal bodies. Mutations in core proteins (VPS11, VPS16, VPS18, and VPS33) have been linked with multiple neurological disorders, including mucopolysaccharidosis (MPS), genetic leukoencephalopathy (gLE), and dystonia. Mutations in human Vacuolar Protein Sorting 16 (VPS16) have been associated with MPS and dystonia. In this study, we generated and characterized a zebrafish vps16(-/-) mutant line using immunohistochemical and behavioral approaches. The loss of Vps16 function caused multiple systemic defects, hypomyelination, and increased neuronal cell death. Behavioral analysis showed a progressive loss of visuomotor response and reduced motor response and habituation to acoustic/tap stimuli in mutants. Finally, using a novel multiple-round acoustic/tap stimuli test, mutants showed intermediate memory deficits. Together, these data demonstrate that zebrafish vps16(-/-) mutants show systemic defects, neurological and motor system pathologies, and cognitive impairment. This is the first study to report behavior abnormalities and memory deficiencies in a zebrafish vps16(-/-) mutant line. Finally, we conclude that the deficits observed in vps16(-/-) zebrafish mutants do not mimic pathologies associated with dystonia, but more align to abnormalities associated with MPS and gLE.

NAD supplementation improves mitochondrial performance of cardiolipin mutants.

Cardiolipin (CL) deficiency causes mitochondrial dysfunction and aberrant metabolism that are associated in humans with the severe disease Barth syndrome (BTHS). Several metabolic abnormalities are observed in BTHS patients and model systems, including decreased oxidative phosphorylation, reduced tricarboxylic acid (TCA) cycle flux, and accumulated lactate and D-ß-hydroxybutyrate, which strongly suggests that nicotinamide adenine dinucleotide (NAD) redox metabolism may be altered in CL-deficient cells. In this study, we identified abnormal NAD+ metabolism in multiple BTHS model systems and demonstrate that supplementation of NAD+ precursors such as nicotinamide mononucleotide (NMN) improves mitochondrial function. Improved mitochondrial function in the Drosophila model was associated with restored exercise endurance, which suggests a potential therapeutic benefit of NAD+ precursor supplementation in the management of BTHS patients.

Prophylactic Knockdown of the miR-183/96/182 Cluster Ameliorates Pseudomonas aeruginosa-Induced Keratitis.

Purpose: Previously, we demonstrated that miR-183/96/182 cluster (miR-183C) knockout mice exhibit decreased severity of Pseudomonas aeruginosa (PA)-induced keratitis. This study tests the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis indicative of a therapeutic potential. Methods: Eight-week-old miR-183C wild-type and C57BL/6J inbred mice were used. Locked nucleic acid-modified anti-miR-183C or negative control oligoribonucleotides with scrambled sequences (NC ORNs) were injected subconjunctivally 1 day before and then topically applied once daily for 5 days post-infection (dpi) (strain 19660). Corneal disease was graded at 1, 3, and 5 dpi. Corneas were harvested for RT-PCR, ELISA, immunofluorescence (IF), myeloperoxidase and plate count assays, and flow cytometry. Corneal nerve density was evaluated in flatmounted corneas by IF staining with anti-ß-III tubulin antibody. Results: Anti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1ß at 1 dpi, which was decreased at 5 dpi; fewer polymorphonuclear leukocytes (PMNs) at 5 dpi; lower viable bacterial plate count at both 1 and 5 dpi; increased percentages of MHCII+ macrophages (Mϕ) and dendritic cells (DCs), consistent with enhanced activation/maturation; and decreased severity of PA keratitis. Anti-miR-183C treatment in the cornea of naïve mice resulted in a transient reduction of corneal nerve density, which was fully recovered one week after the last anti-miR application. miR-183C targets repulsive axon-guidance receptor molecule Neuropilin 1, which may mediate the effect of anti-miR-183C on corneal nerve regression. Conclusions: Prophylactic miR-183C knockdown is protective against PA keratitis through its regulation of innate immunity, corneal innervation, and neuroimmune interactions.

Targeting Inflammation Driven by HMGB1 in Bacterial Keratitis-A Review.

Pseudomonas (P.) aeruginosa is a Gram-negative bacteria that causes human infectionsinfections. It can cause keratitis, a severe eye infection, that develops quickly and is a major cause of ulceration of the cornea and ocular complications globally. Contact lens wear is the greatest causative reason in developed countries, but in other countries, trauma and predominates. Use of non-human models of the disease are critical and may provide promising alternative argets for therapy to bolster a lack of new antibiotics and increasing antibiotic resistance. In this regard, we have shown promising data after inhibiting high mobility group box 1 (HMGB1), using small interfering RNA (siRNA). Success has also been obtained after other means to inhinit HMGB1 and include: use of HMGB1 Box A (one of three HMGB1 domains), anti-HMGB1 antibody blockage of HMGB1 and/or its receptors, Toll like receptor (TLR) 4, treatment with thrombomodulin (TM) or vasoactive intestinal peptide (VIP) and glycyrrhizin (GLY, a triterpenoid saponin) that directly binds to HMGB1. ReducingHMGB1 levels in P. aeruginosa keratitis appears a viable treatment alternative.

Development and characterization of a chronic photoreceptor degeneration model in adult zebrafish that does not trigger a regenerative response.

Zebrafish (Danio rerio) have become a highly-utilized model system in the field of regenerative biology because of their endogenous ability to regenerate many tissues and organs, including the retina. The vast majority of previous research on retinal regeneration in adult zebrafish utilizes acute methodologies for retinal damage. Acute retinal cell death triggers a reactive gliosis response of Müller glia (MG), the resident macroglia of the retina. In addition, each activated MG undergoes asymmetric cell division to produce a neuronal progenitor, which continues to divide and ultimately gives rise to new retinal neurons. Studies using these approaches have uncovered many crucial mechanisms by which MG respond to acute damage. However, they may not adequately mimic the chronic neuronal degeneration observed in many human retinal degenerative diseases. The current study aimed to develop a new long-term, chronic photoreceptor damage and degeneration model in adult zebrafish. Comparing the subsequent cellular responses to that of the commonly-used acute high-intensity model, we found that low, continuous light exposure damaged the outer segments of both rod and cone photoreceptors, but did not result in significant apoptotic cell death, MG gliosis, or MG cell-cycle re-entry. Instead, chronic light nearly completely truncated photoreceptor outer segments and resulted in a recruitment of microglia to the area. Together, these studies present a chronic photoreceptor model that can be performed in a relatively short time frame (21 days), that may lend insight into the cellular events underlying non-regenerative photoreceptor degeneration observed in other model systems.

Characterization of the Expression of Vacuolar Protein Sorting 11 (Vps11) in Mammalian Oligodendrocytes.

A founder mutation in human VPS11 (Vacuolar Protein Sorting 11) was recently linked to a genetic leukoencephalopathy in Ashkenazi Jews that presents with the classical features of white matter disorders of the central nervous system (CNS). The neurological deficits include hypomyelination, hypotonia, gradual loss of vision, and seizures. However, the cells expressing the mutation were not identified. Here we describe, using immunocytochemistry, the strong expression of Vps11 in mouse oligodendrocytes and, specifically, its localization with Myelin Associated Glycoprotein (MAG) in the inner tongue of myelin. In longitudinal sections of myelin, it forms a bead-like structure, alternating with Myelin Basic Protein (MBP). Immunofluorescent staining with Vps11 and neurofilament proteins indicates the absence of Vps11 in axons in vivo. Finally, changes in Vps11 expression are associated with altered proteolipid protein (PLP) levels based upon mice with duplications or deletions of the Plp1 gene. To determine potential functional contributions of Vps11, we combined Vps11 with Platelet Derived Growth Factor Receptor-α (PDGFRα) in vitro and in vivo: in both conditions, co-localization of the two proteins was frequently found in round vesicles of OPCs/oligodendrocytes, suggesting retrograde transport for degradation by the endolysosomal system. Neuron-to-glial communication has been invoked to explain degenerative changes in myelin followed by degenerative changes in axons, and vice versa; but to our knowledge, no specific proteins in retrograde transport from the myelin inner tongue to oligodendrocyte perikarya have been identified. The identification of mutations in VPS11 and its localization at the axon-myelin interface should open new avenues of research.

Ocular Effects of Glycyrrhizin at Acidic and Neutral pH.

PURPOSE: To test the effects of acidic vs. neutral pH glycyrrhizin (GLY) on the unwounded and wounded normal mouse cornea and after infection with Pseudomonas aeruginosa isolates KEI 1025 and multidrug-resistant MDR9. METHODS: Acidic or neutral GLY vs. phosphate-buffered saline (PBS) was topically applied to normal or wounded corneas of C57BL/6 mice. In unwounded corneas, goblet cells and corneal nerves were stained and quantitated. After wounding, corneas were fluorescein stained and photographed using a slit lamp. Mice also were infected with KEI 1025 or MDR9 and the protective effects of GLY pH evaluated comparatively. RESULTS: In the unwounded cornea, application of acidic or neutral GLY vs. PBS reduced the number of bulbar conjunctival goblet cells but did not alter corneal nerve density. Similar application of GLY to scarified corneas delayed wound closure. After KEI 1025 infection, none of the GLY vs. PBS-treated corneas perforated; GLY treatment also decreased plate count (neutral pH more effective) and reduced MPO and several cytokines. Similarly, for MDR9, GLY at either pH was protective and also enhanced the effects of moxifloxacin to which MDR9 is resistant. CONCLUSION: Acidic or neutral pH GLY decreased goblet cell number but had no effect on nerve density. After corneal wounding, GLY at either pH (1) delayed wound closure and, (2) after infection, decreased keratitis when used alone or in combination with moxifloxacin. Neutral pH did not alter the therapeutic effect of GLY and would be preferred if used clinically.

Effects of Glycyrrhizin Treatment on Diabetic Cornea.

Purpose: To test how glycyrrhizin (GLY) affects mouse corneal epithelial cells (MCEC) and the diabetic murine cornea. Methods: Viability of MCEC grown under normal or high glucose (HG) with/without GLY was tested by an MTT assay. In addition, C57BL/6 mice were injected with streptozotocin and a subset of control and diabetic mice received GLY in their drinking water. mRNA and protein levels of proinflammatory and oxidative stress molecules were tested by reverse transcription-polymerase chain reaction (RT-PCR) in both models. Ex vivo studies using human diabetic versus control corneas analyzed proinflammatory and oxidative stress markers using RT-PCR and enzyme-linked immunosorbent assay. Results: GLY protected against loss of cell viability induced by HG and significantly reduced HMGB1, IL-1ß, TLR2, TLR4, NLRP3, COX2, SOD2, HO-1, GPX2, and GR1. In vivo, corneas of GLY-treated diabetic mice showed significantly decreased mRNA expression for CXCL2, iNOS, and all molecules listed above; GLY also lowered HMGB1 and IL-1ß proteins (in vitro and in vivo). Ex vivo studies using diabetic human corneas revealed elevated mRNA levels of inflammatory and oxidative stress molecules (as listed above for in vivo) versus normal age-matched controls. Protein levels for HMGB1 and IL-1ß also were elevated in diabetic human versus control corneas. Conclusions: The data provide evidence that GLY treatment attenuates inflammation and oxidative stress in vitro in MCEC and in vivo in the cornea of diabetic mice. Ex vivo data support the similarities of proinflammatory and oxidative stress data in mouse compared to human, suggesting that GLY treatment would have relevancy to patient care.

Effects of Glycyrrhizin on Multi-Drug Resistant Pseudomonas aeruginosa.

The effects of glycyrrhizin (GLY) on multi-drug resistant (MDR) systemic (MDR9) vs. ocular (B1045) Pseudomonas aeruginosa clinical isolates were determined. Proteomes of each isolate with/without GLY treatment were profiled using liquid chromatography mass spectrometry (LC-MS/MS). The effect of GLY on adherence of MDR isolates to immortalized human (HCET) and mouse (MCEC) corneal epithelial cells, and biofilm and dispersal was tested. Both isolates were treated with GLY (0.25 minimum inhibitory concentration (MIC), 10 mg/mL for MDR9 and 3.75 mg/mL for B1045) and subjected to proteomic analysis. MDR9 had a greater response to GLY (51% of identified proteins affected vs. <1% in B1045). In MDR9 vs. controls, GLY decreased the abundance of proteins for: antibiotic resistance, biofilm formation, and type III secretion. Further, antibiotic resistance and type III secretion proteins had higher control abundances in MDR9 vs. B1045. GLY (5 and 10 mg/mL) significantly reduced binding of both isolates to MCEC, and B1045 to HCET. MDR9 binding to HCET was only reduced at 10 mg/mL GLY. GLY (5 and 10 mg/mL) enhanced dispersal for both isolates, at early (6.5 h) but not later times (24-72 h). This study provides evidence that GLY has a greater effect on the proteome of MDR9 vs. B1045, yet it was equally effective at disrupting adherence and early biofilm dispersal.

Airborne Particulates Affect Corneal Homeostasis and Immunity.

Purpose: To determine the effects of airborne particulate matter (PM) <2.5 µm in vitro and on the normal and Pseudomonas aeruginosa (PA)-infected cornea. Methods: An MTT viability assay tested the effects of PM2.5 on mouse corneal epithelial cells (MCEC) and human corneal epithelial cells (HCET). MCEC were tested for reactive oxygen species using a 2',7'-dichlorodihydrofluorescein assay; RT-PCR determined mRNA levels of inflammatory and oxidative stress markers in MCEC (HMGB1, toll-like receptor 2, IL-1ß, CXCL2, GPX1, GPX2, GR1, superoxide dismutase 2, and heme oxygenase 1) and HCET (high mobility group box 1, CXCL2, and IL-1ß). C57BL/6 mice also were infected and after 6 hours, the PM2.5 was topically applied. Disease was graded by clinical score and evaluated by histology, plate count, myeloperoxidase assay, RT-PCR, ELISA, and Western blot. Results: After PM2.5 (25-200 µg/mL), 80% to 90% of MCEC and HCET were viable and PM exposure increased reactive oxygen species in MCEC and mRNA expression levels for inflammatory and oxidative stress markers in mouse and human cells. In vivo, the cornea of PA+PM2.5 exposed mice exhibited earlier perforation over PA alone (confirmed histologically). In cornea, plate counts were increased after PA+PM2.5, whereas myeloperoxidase activity was significantly increased after PA+PM2.5 over other groups. The mRNA levels for several proinflammatory and oxidative stress markers were increased in the cornea in the PA+PM2.5 over other groups; protein levels were elevated for high mobility group box 1, but not toll-like receptor 4 or glutathione reductase 1. Uninfected corneas treated with PM2.5 did not differ from normal. Conclusions: PM2.5 triggers reactive oxygen species, upregulates mRNA levels of oxidative stress, inflammatory markers, and high mobility group box 1 protein, contributing to perforation in PA-infected corneas.

Insertion of proteolipid protein into mitochondria but not DM20 regulates metabolism of cells.

Proteolipid protein (PLP), besides its adhesive role in myelin, has been postulated to have multiple cellular functions. One well-documented function of PLP is regulation of oligodendrocyte (Olg) apoptosis. In contrast, DM20, an alternatively spliced product of the PLP1/Plp1 gene, has been proposed to have functions that are unique from PLP but these functions have never been elucidated. Here, we compare metabolism of PLP and DM20, and show that oxidative phosphorylation (OxPhos) was significantly decreased in Plp1 but not DM20 or EGFP expressing cells. The reserve OxPhos capacity of Plp1 expressing cells was half of control cells, suggesting that they are very vulnerable to stress. ATP in media of Plp1 expressing cells is significantly increased more than two-fold compared to controls; markers of apoptosis are increased in cells over-expressing Plp1, indicating that abnormal metabolism of PLP is most likely the direct cause leading to Olg apoptosis. We hypothesize that abnormal metabolism, mediated by increased insertion of PLP into mitochondria, underlies demyelination in Pelizaeus-Merzbacher Disease (PMD) and in models of PMD. To understand why PLP and DM20 function differently, we mutated or deleted amino acids located in the PLP-specific region. All these mutations and deletions of the PLP-specific region prevented insertion of PLP into mitochondria. These findings demonstrate that the PLP-specific region is essential for PLP's import into mitochondria, and now offer an explanation for deciphering unique functions of PLP and DM20.

Novel pathologic findings in patients with Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is an X-linked inherited hypomyelinating disorder caused by mutations in the gene encoding proteolipid protein (PLP), the major structural protein in central nervous system (CNS) myelin. Prior to our study, whether hypomyelination in PMD was caused by demyelination, abnormally thin sheaths or failure to form myelin was unknown. In this study, we compared the microscopic pathology of myelin from brain tissue of 3 PMD patients with PLP1 duplications to that of a patient with a complete PLP1 deletion. Autopsy tissue procured from PMD patients was embedded in paraffin for immunocytochemistry and plastic for electron microscopy to obtain highresolution fiber pathology of cerebrum and corpus callosum. Through histological stains, immunocytochemistry and electron microscopy, our study illustrates unique pathologic findings between the two different types of mutations. Characteristic of the patient with a PLP1 deletion, myelin sheaths showed splitting and decompaction of myelin, confirming for the first time that myelin in PLP1 deletion patients is similar to that of rodent models with gene deletions. Myelin thickness and g-ratios of some fibers, in relation to axon diameter was abnormally thin, suggesting that oligodendrocytes remain metabolically functional and/or are attempting to make myelin. Many fibers showed swollen, progressive degenerative changes to axons in addition to the dissolution of myelin. All three duplication cases shared remarkable fiber pathology including swellings, constriction and/or transection and involution of myelin. Characteristic of PLP1 duplication patients, many axons showed segmental demyelination along their length. Still other axons had abnormally thick myelin sheaths, suggestive of continued myelination. Thus, each type of mutation exhibited unique pathology even though commonality to both mutations included involution of myelin, myelin balls and degeneration of axons. This pathology study describes findings unique to each mutation that suggests the mechanism causing fiber pathology is likewise heterogeneous.

Insertion of proteolipid protein into oligodendrocyte mitochondria regulates extracellular pH and adenosine triphosphate.

Proteolipid protein (PLP) and DM20, the most abundant myelin proteins, are coded by the human PLP1 and non-human Plp1 PLP gene. Mutations in the PLP1 gene cause Pelizaeus-Merzbacher disease (PMD) with duplications of the native PLP1 gene accounting for 70% of PLP1 mutations. Humans with PLP1 duplications and mice with extra Plp1 copies have extensive neuronal degeneration. The mechanism that causes neuronal degeneration is unknown. We show that native PLP traffics to mitochondria when the gene is duplicated in mice and in humans. This report is the first demonstration of a specific cellular defect in brains of PMD patients; it validates rodent models as ideal models to study PMD. Insertion of nuclear-encoded mitochondrial proteins requires specific import pathways; we show that specific cysteine motifs, part of the Mia40/Erv1 mitochondrial import pathway, are present in PLP and are required for its insertion into mitochondria. Insertion of native PLP into mitochondria of transfected cells acidifies media, partially due to increased lactate; it also increases adenosine triphosphate (ATP) in the media. The same abnormalities are found in the extracellular space of mouse brains with extra copies of Plp1. These physiological abnormalities are preventable by mutations in PLP cysteine motifs, a hallmark of the Mia40/Erv1 pathway. Increased extracellular ATP and acidosis lead to neuronal degeneration. Our findings may be the mechanism by which microglia are activated and proinflammatory molecules are upregulated in Plp1 transgenic mice (Tatar et al. (2010) ASN Neuro 2:art:e00043). Manipulation of this metabolic pathway may restore normal metabolism and provide therapy for PMD patients.

Gait abnormalities and progressive myelin degeneration in a new murine model of Pelizaeus-Merzbacher disease with tandem genomic duplication.

Pelizaeus-Merzbacher disease (PMD) is a hypomyelinating leukodystrophy caused by mutations of the proteolipid protein 1 gene (PLP1), which is located on the X chromosome and encodes the most abundant protein of myelin in the central nervous sytem. Approximately 60% of PMD cases result from genomic duplications of a region of the X chromosome that includes the entire PLP1 gene. The duplications are typically in a head-to-tail arrangement, and they vary in size and gene content. Although rodent models with extra copies of Plp1 have been developed, none contains an actual genomic rearrangement that resembles those found in PMD patients. We used mutagenic insertion chromosome engineering resources to generate the Plp1dup mouse model by introducing an X chromosome duplication in the mouse genome that contains Plp1 and five neighboring genes that are also commonly duplicated in PMD patients. The Plp1dup mice display progressive gait abnormalities compared with wild-type littermates. The single duplication leads to increased transcript levels of Plp1 and four of the five other duplicated genes over wild-type levels in the brain beginning the second postnatal week. The Plp1dup mice also display altered transcript levels of other important myelin proteins leading to a progressive degeneration of myelin. Our results show that a single duplication of the Plp1 gene leads to a phenotype similar to the pattern seen in human PMD patients with duplications.

Determinants of central nervous system adult neurogenesis are sex, hormones, mouse strain, age, and brain region.

Multiple sclerosis is a sexually dimorphic (SD) disease that causes oligodendrocyte death, but SD of glial cells is poorly studied. Here, we analyze SD of neural progenitors in 6-8 weeks and 6-8 months normal C57BL/6, SJL/J, and BALB/c mice in the subventricular zone (SVZ), dorsolateral horn (DLC), corpus callosum (CC), and parenchyma. With a short 2-h bromodeoxyuridine (BrdU) pulse, no gender and strain differences are present at 6-8 weeks. At 6-8 months, the number of BrdU(+) cells decreases twofold in each sex, strain, and region, indicating that a common aging mechanism regulates BrdU incorporation. Strikingly, 2× more BrdU(+) cells are found in all brain regions in 6-8 months C57BL/6 females versus males, no gender differences in 6-8 months SJL/J, and fewer BrdU(+) cells in females versus males in BALB/cs. The number of BrdU(+) cells modestly fluctuates throughout the estrous cycle in C57BL/6 and SJLs. Castration causes a dramatic increase in BrdU(+) cells in SVZ and DLC. These findings indicate that testosterone is a major regulator of adult neural proliferation. At 6-8 months, the ratio of PDGFRα(+) cells in the CC to BrdU(+) cells in the DLC of both strains, sexes, estrous cycle, and castrated mice was essentially the same, suggesting that BrdU(+) cells in the DLC differentiate into CC oligodendrocytes. The ratio of TUNEL(+) to BrdU(+) cells does not match proliferation, indicating that these events are differentially regulated. Differential regulation of these two processes leads to the variation in glial numbers between gender and strain. Explanations of neural proliferation based upon data from one sex or strain may be very misleading.

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.

Different proteolipid protein mutants exhibit unique metabolic defects.

PMD (Pelizaeus-Merzbacher disease), a CNS (central nervous system) disease characterized by shortened lifespan and severe neural dysfunction, is caused by mutations of the PLP1 (X-linked myelin proteolipid protein) gene. The majority of human PLP1 mutations are caused by duplications; almost all others are caused by missense mutations. The cellular events leading to the phenotype are unknown. The same mutations in non-humans make them ideal models to study the mechanisms that cause neurological sequelae. In the present study we show that mice with Plp1 duplications (Plp1tg) have major mitochondrial deficits with a 50% reduction in ATP, a drastically reduced mitochondrial membrane potential and increased numbers of mitochondria. In contrast, the jp (jimpy) mouse with a Plp1 missense mutation exhibits normal mitochondrial function. We show that PLP in the Plp1tg mice and in Plp1-transfected cells is targeted to mitochondria. PLP has motifs permissive for insertion into mitochondria and deletions near its N-terminus prevent its co-localization to mitochondria. These novel data show that Plp1 missense mutations and duplications of the native Plp1 gene initiate uniquely different cellular responses.

Sexual dimorphism in the white matter of rodents.

Sexual dimorphism of astrocytes and neurons is well documented in many brain and spinal cord structures. Sexual dimorphism of oligodendrocytes (Olgs) and myelin has received less attention. We recently showed that density of Olgs in corpus callosum, fornix, and spinal cord of wild-type male rodents is more densely packed than in females; myelin proteins and myelin gene expression are likewise greater in males than in female rodents. However, glial cell proliferation and cell death were two times greater in female corpus callosum. Endogenous sex hormones, specifically lack of androgens, produce an Olg female phenotype in castrated male mouse. In vitro studies using Olgs culture also showed differences between males and females Olg survival and signaling pathways in response to sexual hormones. Sexual dimorphism of white matter tracts and glia in rodents indicates the necessity for controlling gender in the experimental studies of neurodegenerative disorders. Most importantly, our studies suggest that hormones may contribute to sexual dimorphism observed in certain human diseases including multiple sclerosis.

Sexual dimorphism of oligodendrocytes is mediated by differential regulation of signaling pathways.

Sexual dimorphism of white matter has not been considered important, the assumption being that sex hormones are not essential for glial development. We recently showed exogenous hormones in vivo differentially regulate in male and female rodents the life span of oligodendrocytes (Olgs) and amount of myelin (Cerghet et al. [2006] J. Neurosci. 26:1439-1447). To determine which hormones regulate male and female Olg development, we prepared enriched Olg cultures grown in serum-free medium with estrogen (E2), progesterone (P2), and dihydrotestosterone (DHT) or their combinations. P2 significantly increased the number of Olgs in both sexes, but more so in females; E2 had minor effects on Olg numbers; and DHT reduced Olgs numbers in both sexes, but more so in females. Combinations of hormones affected Olg numbers differently from single hormones. The change in Olg numbers was due to changes not in proliferation but rather in survival. P2 increased pAKT by many-fold, but MAPK levels were unchanged, indicating that activation of the Akt pathway by P2 is sufficient to regulate Olg differentiation. DHT reduced pAkt in both sexes but differentially increased pMAPK in males and decreased it in females. Stressing Olgs reveals that both sexes are protected by P2, but females are slightly better protected than males. Females always showed greater differences than males regarding changes in Olg numbers and in signaling molecules. Given the greater fluctuation of neurosteroids in women than in men and the higher incidence of multiple sclerosis (MS) in women, these sexually dimorphic differences may contribute to differences in male and female MS lesions.

Plasticity of neurons and glia following neonatal hypoxic-ischemic brain injury in rats.

Periventricular white matter injury in premature infants is linked to chronic neurological dysfunction. Periventricular white matter injury is caused by many mechanisms including hypoxia-ischemia (HI). Animal models of HI in the neonatal rodent brain can replicate some important features of periventricular white matter injury. Most rodent studies have focused upon early cellular and tissue events following unilateral neonatal HI that is elicited by unilateral carotid artery ligation and followed by timed exposure to moderate hypoxia. Milder hypoxic-ischemic insults elicit preferential white matter injury. Little information is available about long-term cellular effects of unilateral HI. One month after unilateral neonatal hypoxia ischemia, we show that all the components for structural reorganization of the brain are present in moderately injured rats. These components in the injured side include extensive influx of neurites, axonal and dendritic growth cones, abundant immature synapses, and myelination of many small axons. Surprisingly, this neural recovery is often found in and adjacent to cysts that have the ultrastructural features of bone extracellular matrix. In contrast, brains with severe hypoxia ischemia one month after injury still undergo massive neuronal degeneration. While massive destruction of neurons and glia are striking events shortly after brain HI, neural cells re-express their intrinsic properties and attempt an anatomical recovery long after injury.