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.     

Insulin-like growth factor I stimulates oligodendrocyte development and myelination in rat brain aggregate cultures.

Insulin-like growth factor I (IGF-I) and high concentrations of insulin have been shown to stimulate an increase in the number of oligodendrocytes that appear in developing monolayer cultures of rat brain cells (McMorris et al., Proc Natl Acad Sci USA 83: 822-826, 1986; McMorris et al., Ann NY Acad Sci 605:101-109, 1990; McMorris and Dubois-Dalcq, J Neurosci Res 21:199-209, 1988). In the present study, we investigated whether IGF-I or insulin treatment induces a corresponding increase in the synthesis and accumulation of myelin. Aggregate cultures, established from 16-day-old fetal rat brains, were treated with either 100 ng/ml IGF-I or 5,000 ng/ml insulin and analyzed for the number of oligodendrocytes, activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), total amount of myelin, and synthesis rate of myelin proteins. Cultures treated with IGF-I beginning on day 2 after explantation contained 35-80% more oligodendrocytes and had 60-160% higher CNP activity than controls when tested on day 13, 20, or 27. By day 27, treated cultures had 35-90% more myelin than controls. Similar results were observed in response to 5,000 ng/ml insulin, a concentration at which insulin binds to IGF receptors and acts as an analogue of IGF-I. The synthesis rate of myelin proteins was measured in experiments using 5,000 ng/ml insulin. When treatment was begun at day 20 rather than day 2, cultures did not exhibit an increased number of oligodendrocytes over control during the following 4-6 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Transplantation of bulk-separated oligodendrocytes into the brains of shiverer mutant mice: Immunohistochemical and electron microscopic studies on the myelination

Normal oligodendrocytes were separated from 7-day-old mouse (BALB/c) brains by the Percoll gradient method. Immunohistochemical staining with an anti-galactocerebroside serum revealed that about 85% of the separated cells were oligodendrocytes. The oligodendrocytes were transplanted into the corpus striatum of 4-week-old shiverer mutant mice which are characterized by the lack of myelin basic protein (MBP). Myelination by the implanted oligodendrocytes was investigated immunohistochemically and electron microscopically 6 weeks after operation. Certain areas in the corpus striatum were intensely stained with antiserum to MBP. Electron microscopic examination showed that some axons were surrounded by normal type myelin sheaths with major dense lines. These results clearly indicate that matured oligodendrocytes are able to survive and myelinate the host axons even in the adult brain.     

The impact of erythropoietin and iron status on brain myelination in the newborn rat

Erythropoietin (Epo) drives iron (Fe) utilization for erythropoiesis, but the potentially resultant tissue iron deficiency (ID) can also impede brain development. Conversely, Epo binds to Epo receptors (EpoR) on immature brain oligodendrocytes and neurons, promoting growth and differentiation. The objective of the study was to examine the interaction between Epo and Fe on myelination in brain development during daily Epo treatment. Male and female Sprague‐Dawley rats from postnatal day (P) P4‐P12 modeled premature newborns. Dam‐fed Fe‐sufficient (IS) or postnatal ID groups were given daily subcutaneous sham or erythropoietic Epo injections (425 U^.kg^?1.d^?1), ± oral Fe (6 mg^.kg^?1.d^?1). Tissues and blood were collected and studied at P12. Epo in the ID groups, in the absence of oral Fe, stimulated microcytic ID anemia along with raising inflammatory markers. Both the microcytic anemia and inflammation improved in the ID + Epo + Fe group. Fe treatment positively impacted erythropoiesis and body Fe (µg/g) in all groups. Relative brain Fe (µg/g rat) was improved in the IS + Epo + Fe group. Brain Fe was not worsened in +Epo groups. Brain weight and brain Fe were related to plasma Epo levels. Amount of myelination was impacted by feeding type, but was not inhibited by Epo. Expression of a protein in myelin, mylein basic protein, was greater in all +Fe groups than –Fe groups. With therapeutic Epo, available body Fe was prioritized for erythropoiesis instead of brain, but Epo did not worsen brain Fe and potentially Epo improved myelination and maturation in the brain.     

Proteolytic processing of the Alzheimer's disease amyloid precursor protein in brain and platelets

Proteolytic processing of the amyloid precursor protein by beta -and gamma-secretases results in the production of Alzheimer's disease (AD) Abeta amyloid peptides. Modulation of secretase activity is being investigated as a potential therapeutic approach. Recent studies with human brain have revealed that the beta-secretase protein, BACE, is increased in cortex of AD patients. Analysis of betaCTF (or C99), the amyloid precursor protein (APP) product of BACE cleavage that is the direct precursor to Abeta, shows it is also elevated in AD, underlying the importance of beta-secretase cleavage in AD pathogenesis. The C-terminal product of gamma-secretase cleavage of APP, epsilonCTF (or AICD), is enriched in human brain cortical nuclear fractions, a subcellular distribution appropriate for a putative involvement of APP cytosolic domain in signal transduction. Analysis of AD cortex samples, particularly that of a carrier of a familial APP mutation, suggests that processing of APP transmembrane domain generates an alternative CTF product. All these particularities observed in the AD brain demonstrate that APP processing is altered in AD. The transgenic mouse model Tg2576 seems to be a promising laboratory tool to test potential modulators of Abeta formation. Indeed, C-terminal products of alpha-, beta-, and gamma-secretase cleavage are readily detectable in the brain of these transgenic mice. Finally, the finding of the same secretase products in platelets and neurons make platelets a potentially useful and easily accessible clinical tool to monitor effects of novel therapies based on inhibition of beta- or gamma-secretase.     

Quantitation of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and central nervous system and its relationship to the clinicopathologic features of autoimmune encephalomyelitis

There is controversy whether the amount of autoantigens expressed in the thymus regulates negative selection of autoreactive T cells and determine susceptibility or resistance to experimental autoimmune encephalomyelitis (EAE). In the present study, we have addressed this issue by quantifying neuroantigens in the thymus of two EAE-susceptible (LEW and LEW.1AV1) and one EAE-resistant (BN) rat strains. We further examined whether amounts of neuroantigens in various parts of the central nervous system (CNS) affect the clinical course and lesion distribution of acute and chronic EAE. Real-time PCR and histologic analyses showed that there was no significant difference in the amount and distribution of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and CNS among the three strains and that both acute and chronic EAE lesions in the CNS were preferentially distributed in the area where neuroantigens were abundantly present. These findings suggest that susceptibility or resistance to EAE is not regulated by the amount of the neuroantigens expressed in the thymus. Furthermore, the lesion distribution, but not the clinical course, of EAE is related to the neuroantigen expression in the CNS.     

A novel GLP‐1/GIP/Gcg triagonist reduces cognitive deficits and pathology in the 3xTg mouse model of Alzheimer's disease

Type 2 diabetes mellitus (T2DM) is an important risk factor for Alzheimer's disease (AD). Glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP) have been identified to be effective in T2DM treatment and neuroprotection. In this study, we further explored the effects of a novel unimolecular GLP‐1/GIP/Gcg triagonist on the cognitive behavior and cerebral pathology in the 7‐month‐old triple transgenic mouse model of AD (3xTg‐AD), and investigated its possible electrophysiological and molecular mechanisms. After chronic administration of the GLP‐1/GIP/Gcg triagonist (10 nmol/kg bodyweight, once daily, i.p.) for 30 days, open field, Y maze and Morris water maze tests were performed, followed by in vivo electrophysiological recording, immunofluorescence and Western blotting experiments. We found that the chronic treatment with the triagonist could improve long‐term spatial memory of 3xTg‐AD mice in Morris water maze, as well as the working memory in Y maze task. The triagonist also alleviated the suppression of long‐term potentiation (LTP) in the CA1 region of hippocampus. In addition, the triagonist significantly reduced hippocampal pathological damages, including amyloid‐β (Aβ) and phosphorylated tau aggregates, and upregulated the expression levels of ^S133p‐CREB, ^T286p‐CAMKII and ^S9p‐GSK3β in the hippocampus of the 3xTg‐AD mice. These results demonstrate for the first time that the novel GLP‐1/GIP/Gcg triagonist is efficacious in ameliorating cognitive deficits and pathological damages of 3xTg‐AD mice, suggesting that the triagonist might be potentially beneficial in the treatment of AD.     

Spatial patterns of atrophy,hypometabolism, and amyloid deposition in Alzheimer's disease correspond to dissociable functional brain networks

Recent neuroimaging studies of Alzheimer's disease (AD) have emphasized topographical similarities between AD‐related brain changes and a prominent cortical association network called the default‐mode network (DMN). However, the specificity of distinct imaging abnormalities for the DMN compared to other intrinsic connectivity networks (ICNs) of the limbic and heteromodal association cortex has not yet been examined systematically. We assessed regional amyloid load using AV45‐PET, neuronal metabolism using FDG‐PET, and gray matter volume using structural MRI in 473 participants from the Alzheimer's Disease Neuroimaging Initiative, including preclinical, predementia, and clinically manifest AD stages. Complementary region‐of‐interest and voxel‐based analyses were used to assess disease stage‐ and modality‐specific changes within seven principle ICNs of the human brain as defined by a standardized functional connectivity atlas. Amyloid deposition in AD dementia showed a preference for the DMN, but high effect sizes were also observed for other neocortical ICNs, most notably the frontoparietal‐control network. Atrophic changes were most specific for an anterior limbic network, followed by the DMN, whereas other neocortical networks were relatively spared. Hypometabolism appeared to be a mixture of both amyloid‐ and atrophy‐related profiles. Similar patterns of modality‐dependent network specificity were also observed in the predementia and, for amyloid deposition, in the preclinical stage. These quantitative data confirm a high vulnerability of the DMN for multimodal imaging abnormalities in AD. However, rather than being selective for the DMN, imaging abnormalities more generally affect higher order cognitive networks and, importantly, the vulnerability profiles of these networks markedly differ for distinct aspects of AD pathology. Hum Brain Mapp 37:35–53, 2016. © 2015 Wiley Periodicals, Inc.     

Accumulation of caspase cleaved amyloid precursor protein represents an early neurodegenerative event in aging and in Alzheimer's disease

The activation of caspase-3 and possibly other caspases during apoptosis may lead to the cleavage of the amyloid precursor protein (APP) and subsequent accumulation of APP cleavage products (cAPP). We examined the association between activated caspase-3 and cAPP in human brain by qualitative and quantitative analysis of in situ immunohistochemistry and Western blots. Frontal cortex and hippocampal tissue from age-matched control and Alzheimer's brains (AD) was used. Both activated caspase-3 and cAPP are increased in AD [Braak and Braak (BB) stage IV-VI] compared to aged control (BB stage 0-1) and transitional (BB stage II-III) cases in the hippocampal and frontal cortex. Caspase-3 activation and the accumulation of APP cleavage fragments appear to either parallel or precede neurofibrillary tangle formation. These findings raise the possibility that the activation of caspase-3 and cleavage of APP may be involved with neuronal degeneration and that pathways characteristic of apoptosis are activated in AD.     

Co-clustering of galactosylceramide and membrane proteins in oligodendrocyte membranes on interaction with polyvalent carbohydrate and prevention by an intact cytoskeleton

We have shown previously that addition of liposomes containing the two major glycosphingolipids of myelin, galactosylceramide (GalC) and cerebroside sulfate (CBS), to cultured oligodendrocytes (OLs) caused clustering of GalC on the extracellular surface and myelin basic protein (MBP) on the cytosolic surface to the same membrane domains. It also caused depolymerization of actin microfilaments and microtubules, indicating that interaction of the liposomes with the OL surface induces transmembrane signal transmission. We show that a multivalent form of galactose conjugated to bovine serum albumin has a similar effect as the multivalent GalC/CBS-containing liposomes. Because GalC and CBS can interact with each other across apposed membranes and because anti-GalC and anti-CBS antibodies also cause redistribution of GalC/CBS and depolymerization of microtubules, we believe that the multivalent carbohydrate interacts with GalC and CBS in the OL membrane. Several myelin-specific transmembrane proteins could be involved in this transmembrane signal transmission from GalC/CBS. We looked at co-clustering of several myelin constituents by confocal microscopy to determine if they are located in or redistribute to GalC/MBP-containing domains. Myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), MAPK, and some phosphotyrosine-containing proteins were found to co-cluster with GalC and MBP, but myelin-associated glycoprotein (MAG) and phosphatidylinositol-4,5-bisphosphate (PIP(2)) did not. These results suggest that MOG and PLP, but probably not MAG, are possible candidates for transmembrane transmission of the signal received by GalC/CBS. To determine if depolymerization of actin microfilaments was required for co-clustering, or was secondary to clustering, we stabilized F-actin with jasplakinolide. This also prevented depolymerization of the microtubules and prevented clustering of all constituents, including GalC. The prevention of clustering or redistribution of these glycolipids and proteins by an intact cytoskeleton is consistent with the picket fence model.     

Chronic Theiler's virus infection in mice: appearance of myelin basic protein in the cerebrospinal fluid and serum antibody directed against MBP

Myelin basic protein (MBP) appears frequently in the cerebrospinal fluid (CSF) of mice with chronic demyelination following intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV); antibody to MBP can frequently be found in the sera. The peaks of the immune responses to both MBP and TMEV coincide with the time course of the clinical signs of disease. Adsorption of mouse sera with TMEV or MBP indicate the non-identity of the antigens and the specificity of the antisera as measured by ELISA. Immunoblot analysis of sera confirmed the ELISA findings. The mechanism of induction of antibody directed against MBP and its role in TMEV-associated demyelination remain to be determined.     

Decreased levels of PDI and P5 in oligodendrocytes in Alzheimer's disease

Protein disulfide isomerase (PDI) is a chaperone protein located in the endoplasmic reticulum (ER). Nitric oxide‐induced S‐nitrosylation of PDI inhibits its enzymatic activity, leading to protein accumulation and activation of the unfolded protein response. Protein disulfide isomerase P5 (P5) is a member of the PDI family that mostly localizes to the ER lumen. Both S‐nitrosylated PDI and S‐nitrosylated P5 are found in Alzheimer's disease (AD) brain. Previously, we showed that expression of the ER stress marker, growth arrest, and DNA damage protein (GADD34) was significantly increased in neurons and oligodendrocytes in AD brain. In the present study, we showed that PDI and P5 levels were significantly decreased in oligodendrocytes in the brains of AD patients and an AD mouse model. Interestingly, these decreases were evident before the animals displayed typical AD pathology. Because we previously showed that small short interfering RNA knockdown of PDI or P5 could affect the viability of neuronal cells under ER stress, dysfunction of PDI and P5 under ER stress could cause apoptosis of neuronal cells. In summary, we showed that the levels of PDI and P5 were significantly decreased in the oligodendrocytes of AD patients. This phenomenon was also found in an AD mouse model before the animals displayed AD pathology. The overall findings suggest that oligodendrocytes may play important roles in AD pathogenesis.     

Axonopathy, tau abnormalities, and dyskinesia, but no neurofibrillary tangles in p25-transgenic mice

Neurofibrillary tangles, one of the pathologic hallmarks of Alzheimer's disease (AD), are composed of abnormally polymerized tau protein. The hyperphosphorylation of tau alters its normal cellular function and is thought to promote the formation of neurofibrillary tangles. Growing evidence suggests that cyclin-dependent kinase 5 (cdk5) plays a role in tau phosphorylation, but the function of the enzyme in tangle formation remains uncertain. In AD, cdk5 is constitutively activated by p25, a highly stable, 25kD protein thought to be increased in the AD brain. To test the hypothesis that p25/cdk5 interactions promote neurofibrillary pathology, we created transgenic mouse lines that overexpress the human p25 protein specifically in neurons. Mice with high transgenic p25 expression have augmented cdk5 activity and develop severe hindlimb semiparalysis and mild forelimb dyskinesia beginning at approximately 3 months of age. Immunohistochemical and ultrastructural analyses showed widespread axonal degeneration with focal accumulation of tau in various regions of the brain and, to a lesser extent, the spinal cord. However, there was no evidence of neurofibrillary tangles in neuronal somata or axons, nor were paired helical filaments evident ultrastructurally. These studies confirm that p25 overexpression can lead to tau abnormalities and axonal degeneration in vivo but do not support the hypothesis that p25-related induction of cdk5 is a primary event in the genesis of neurofibrillary tangles.     

Altered static and dynamic functional network connectivity in Alzheimer's disease and subcortical ischemic vascular disease: shared and specific brain connectivity abnormalities

Subcortical ischemic vascular disease (SIVD) is a major subtype of vascular dementia with features that overlap clinically with Alzheimer's disease (AD), confounding diagnosis. Neuroimaging is a more specific and biologically based approach for detecting brain changes and thus may help to distinguish these diseases. There is still a lack of knowledge regarding the shared and specific functional brain abnormalities, especially functional connectivity changes in relation to AD and SIVD. In this study, we investigated both static functional network connectivity (sFNC) and dynamic FNC (dFNC) between 54 intrinsic connectivity networks in 19 AD patients, 19 SIVD patients, and 38 age‐matched healthy controls. The results show that both patient groups have increased sFNC between the visual and cerebellar (CB) domains but decreased sFNC between the cognitive‐control and CB domains. SIVD has specifically decreased sFNC within the sensorimotor domain while AD has specifically altered sFNC between the default‐mode and CB domains. In addition, SIVD has more occurrences and a longer dwell time in the weakly connected dFNC states, but with fewer occurrences and a shorter dwell time in the strongly connected dFNC states. AD has both similar and opposite changes in certain dynamic features. More importantly, the dynamic features are found to be associated with cognitive performance. Our findings highlight similar and distinct functional connectivity alterations in AD and SIVD from both static and dynamic perspectives and indicate dFNC to be a more important biomarker for dementia since its progressively altered patterns can better track cognitive impairment in AD and SIVD.     

Preparation of a monoclonal antibody to citrullinated epitopes: its characterization and some applications to immunohistochemistry in human brain

Using hybridoma technology, an IgM monoclonal antibody (mAb), designated as F95, was developed against a deca-citrullinated peptide (DCP) consisting of 10 citrulline residues and a carboxyl Gly-Gly-Cys through which DCP was covalently linked to an activated carrier protein, keyhole limpet hemocyanin (KLH). Clones were selected on the basis of not reacting with human unmodified and noncitrullinated myelin basic protein (MBP), MBP-C1, but reacting well with human citrullinated MBP (MBP-C8). When tested by ELISA, this mAb demonstrated minimal reactivity with human MBP-C1, varying reactivity with the C2-C5 isomers of human MBP, moderate binding with guinea pig MBP-C8, and strong reactivity with human MBP-C8. By ELISA, mAb F95 was directed predominantly against citrulline, not MBP, as revealed by its binding to DCP linked with activated KLH, bovine serum albumin (BSA), or ovalbumin (OA), but not with KLH, BSA, or OA alone. Immunohistochemistry of normal human brain demonstrated that F95 stained central nervous system myelin and a subset of astrocytes. Given the citrulline-directed features of mAb F95, this immunohistochemical pattern suggests that certain astroglial filaments expressing glial fibrillary acidic protein also contain citrulline-bearing components. These potentially implicate citrullinated proteins, notably in astroglial filaments, in a variety of normal and pathological neurobiological processes.     

Chemical and morphological alterations of spines within the hippocampus and entorhinal cortex precede the onset of Alzheimer's disease pathology in double knock-in mice

Mice with knock-in of two mutations that affect beta amyloid processing and levels (2xKI) exhibit impaired spatial memory by 9-12 months of age, together with synaptic plasticity dysfunction in the hippocampus. The goal of this study was to identify changes in the molecular and structural characteristics of synapses that precede and thus could exert constraints upon cellular mechanisms underlying synaptic plasticity. Drebrin A is one protein reported to modulate spine sizes and trafficking of proteins to and from excitatory synapses. Thus, we examined levels of drebrin A within postsynaptic spines in the hippocampus and entorhinal cortex. Our electron microscopic immunocytochemical analyses reveal that, by 6 months, the proportion of hippocampal spines containing drebrin A is reduced and this change is accompanied by an increase in the mean size of spines and decreased density of spines. In the entorhinal cortex of 2xKI brains, we detected no decrement in the proportion of spines labeled for drebrin A and no significant change in spine density at 6 months, but rather a highly significant reduction in the level of drebrin A immunoreactivity within each spine. These changes are unlike those observed for the somatosensory cortex of 2xKI mice, in which synapse density and drebrin A immunoreactivity levels remain unchanged at 6 months and older. These results indicate that brains of 2xKI mice, like those of humans, exhibit regional differences of vulnerability, with the hippocampus exhibiting the first signatures of structural changes that, in turn, may underlie the emergent inability to update spatial memory in later months.