The p38α MAPK Deletion in Oligodendroglia does not Attenuate Myelination Defects in a Mouse Model of Periventricular Leukomalacia.

Periventricular leukomalacia (PVL) is a severe type of white matter damage in premature infants and the most common cause of cerebral palsy. It is generally known to be caused by hypoxia and inflammation. Currently there is no effective treatment available, in part due to that the pathogenesis of the disease has not been well understood. The p38α mitogen-activated protein kinase (MAPK) is the serine/threonine kinase and several in vitro studies demonstrated that p38 MAPK is essential for oligodendroglial differentiation and myelination. Indeed, our nerve/glial antigen 2 (NG2)-specific oligodendroglial p38α MAPK conditional knockout (CKO) mice revealed its complex roles in myelination and remyelination. To identify the specific in vivo roles of oligodendroglial p38α MAPK in PVL, we generated a mouse PVL model by combination of LPS-mediated inflammation and hypoxia-ischemia in NG2-p38α MAPK CKO mice. Our results demonstrate that a selective deletion of p38α MAPK in oligodendrocyte did not attenuate myelination defects in the mouse model of PVL. Myelination phenotype revealed by MBP immunostaining was not significantly affected in the p38α MAPK CKO mice compared to the wildtype after PVL induction. The electron microscopic images demonstrated that the microstructure of myelin structures was not significantly different between the wild-type and p38α MAPK CKO mice. In addition, oligodendrocyte degeneration in the corpus callosum white matter area was unaffected in the p38α MAPK CKO during and after the PVL induction. These data indicate that p38α MAPK in oligodendrocyte has minimal effect on myelination and oligodendrocyte survival in the mouse PVL model.

Class IIa histone deacetylases link cAMP signaling to the myelin transcriptional program of Schwann cells.

Schwann cells respond to cyclic adenosine monophosphate (cAMP) halting proliferation and expressing myelin proteins. Here we show that cAMP signaling induces the nuclear shuttling of the class IIa histone deacetylase (HDAC)-4 in these cells, where it binds to the promoter and blocks the expression of c-Jun, a negative regulator of myelination. To do it, HDAC4 does not interfere with the transcriptional activity of MEF2. Instead, by interacting with NCoR1, it recruits HDAC3 and deacetylates histone 3 in the promoter of c-Jun, blocking gene expression. Importantly, this is enough to up-regulate Krox20 and start Schwann cell differentiation program-inducing myelin gene expression. Using conditional knockout mice, we also show that HDAC4 together with HDAC5 redundantly contribute to activate the myelin transcriptional program and the development of myelin sheath in vivo. We propose a model in which cAMP signaling shuttles class IIa HDACs into the nucleus of Schwann cells to regulate the initial steps of myelination in the peripheral nervous system.

Increased BACE1 activity inhibits peripheral nerve regeneration after injury.

Axons of the peripheral nervous system possess the capacity to regenerate following injury. Previously, we showed that genetically knocking out Beta-Site APP-Cleaving Enzyme 1 (BACE1) leads to increased nerve regeneration. Two cellular components, macrophages and neurons, contribute to enhanced nerve regeneration in BACE1 knockout mice. Here, we utilized a transgenic mouse model that overexpresses BACE1 in its neurons to investigate whether neuronal BACE1 has an inverse effect on regeneration following nerve injury. We performed a sciatic nerve crush in BACE1 transgenic mice and control wild-type littermates, and evaluated the extent of both morphological and physiological improvements over time. At the earliest time point of 3days, we observed a significant decrease in the length of axonal sprouts growing out from the crush site in BACE1 transgenic mice. At later times (10 and 15days post-crush), there were significant reductions in the number of myelinated axons in the sciatic nerve and the percentage of re-innervated neuromuscular junctions in the gastrocnemius muscle. Transgenic mice had a functional electrophysiological delay in the recovery up to 8weeks post-crush compared to controls. These results indicate that BACE1 activity levels have an inverse effect on peripheral nerve repair after injury. The results obtained in this study provide evidence that neuronal BACE1 activity levels impact peripheral nerve regeneration. This data has clinical relevance by highlighting a novel drug target to enhance peripheral nerve repair, an area which currently does not have any approved therapeutics.

Src and phospho-FAK kinases are activated by allopregnanolone promoting Schwann cell motility, morphology and myelination.

Schwann cells' (SCs) development and maturation require coordinate and complementary activation of several signals and intracellular pathways. Among factors controlling these processes, the signalling intermediates Src tyrosine kinase and focal adhesion kinase (FAK) are relevant for SCs', participating in regulation of their adhesion, motility and migration. Recently, the progesterone metabolite allopregnanolone (ALLO) was proved to be synthesized by SCs, whereas it acts autocrinally on SCs motility and proliferation, which are crucial processes for nerve development, maturation and regeneration. Herein, we investigate the hypothesis that the molecular mechanisms behind the ALLO's action on SCs involve the signalling intermediates Src and FAK. We first demonstrated that ALLO 10-6  M regulates SCs morphology, motility and myelination, also increasing the internode distance in the in vitro myelination model of neuron/SCs co-culture. ALLO's actions were mediated by the modulation of Src/FAK pathway, since they were counteracted by PP2 10-5  M, a selective inhibitor of Src kinase. Then, we proved that Src/FAK activation in SCs involves GABA-A dependent mechanisms and actin re-arrangements. In conclusion, our findings are the first to corroborate the importance of the neuroactive steroid ALLO in regulating SCs development and maturation via the Src and phospho-FAK signalling activation. Cover Image for this issue: doi: 10.1111/jnc.13795.

The role of p38alpha in Schwann cells in regulating peripheral nerve myelination and repair.

Myelination in the peripheral nervous system (PNS) is controlled by both positive and negative regulators within Schwann cells to ensure timely onset and correct myelin thickness for saltatory conduction by neurons. Transcription factors such as Sox10, octamer-binding transcription factor 6 (Oct6) and Krox20 form a positive regulatory network, whereas negative regulators such as cJun and Sox2 oppose myelination in Schwann cells. The role of the p38 MAPK pathway has been studied in PNS myelination, but its precise function remains unclear, with both positive and negative effects of p38 activity reported upon both myelination and processes of nerve repair. To clarify the role of p38 MAPK in the PNS, we have analysed mice with a Schwann cell-specific ablation of the major p38 isoform, p38alpha. In line with previous findings of an inhibitory role for p38 MAPK, we observe acceleration of post-natal myelination in p38alpha null nerves, a delay in myelin down-regulation following injury, together with a small increase in levels of re-myelination following injury. Finally we explored roles for p38alpha in controlling axonal regeneration and functional repair following PNS injury and observe that loss of p38alpha function in Schwann cells does not appear to affect these processes as previously reported. These studies therefore provide further proof for a role of p38 MAPK signalling in the control of myelination by Schwann cells in the PNS, but do not show an apparent role for signalling by this MAP kinase in Schwann cells controlling other elements of Wallerian degeneration and functional repair following injury. Cover Image for this issue: doi: 10.1111/jnc.13793.

The COMT Val158Met polymorphism moderates the association between cognitive functions and white matter microstructure in schizophrenia.

OBJECTIVES: Impaired cognitive functioning is a core feature of schizophrenia. Cognitive impairment in schizophrenia has been associated with white-matter (WM) abnormalities and degenerative changes of cortical myelin in the cerebral cortex. Furthermore, findings suggested a role of the COMT gene in affecting both WM and neuropsychological performances.We thus hypothesized that the COMT ValMet genotype would affect the association between cognitive functions and WM microstructure in a sample of schizophrenic patients. MATERIALS AND METHODS: Seventy-eight schizophrenic patients performed the brief assessment of cognition in schizophrenia for assessment of cognitive performances. Sixty-nine patients provided a venous blood sample for genotypic analysis. WM integrity was evaluated using tract-based spatial statistics with threshold-free cluster enhancement (P<0.05). RESULTS: Analysis indicated an association between cognitive functions and WM microstructure in the Val/Val group, but not in the Met carriers group. WM tracts include the corpus callosum, thalamic radiations, corona radiata, forceps major and minor, superior and inferior longitudinal fasciculus, inferior fronto-occipital fasciculus, corticospinal tract, and cingulum. CONCLUSION: Results suggested a moderating effect of the COMT ValMet polymorphism on the association between cognitive functioning and WM microstructure. Our findings support the importance of myelination in cognition, identifying measures of WM microstructure as important neurobiological features of cognitive performances.

The protein tyrosine phosphatase Shp2 is required for the generation of oligodendrocyte progenitor cells and myelination in the mouse telencephalon.

The protein tyrosine phosphatase Shp2 (PTPN11) is crucial for normal brain development and has been implicated in dorsal telencephalic neuronal and astroglia cell fate decisions. However, its roles in the ventral telencephalon and during oligodendrogenesis in the telencephalon remain largely unknown. Shp2 gain-of-function (GOF) mutations are observed in Noonan syndrome, a type of RASopathy associated with multiple phenotypes, including cardiovascular, craniofacial, and neurocognitive abnormalities. To gain insight into requirements for Shp2 (LOF) and the impact of abnormal Shp2 GOF mutations, we used a Shp2 conditional mutant allele (LOF) and a cre inducible Shp2-Q79R GOF transgenic mouse in combination with Olig2(cre/+) mice to target embryonic ventral telencephalic progenitors and the oligodendrocyte lineage. In the absence of Shp2 (LOF), neuronal cell types originating from progenitors in the ventral telencephalon were generated, but oligodendrocyte progenitor cell (OPC) generation was severely impaired. Late embryonic and postnatal Shp2 cKOs showed defects in the generation of OPCs throughout the telencephalon and subsequent reductions in white matter myelination. Conversely, transgenic expression of the Shp2 GOF Noonan syndrome mutation resulted in elevated OPC numbers in the embryo and postnatal brain. Interestingly, expression of this mutation negatively influenced myelination as mice displayed abnormal myelination and fewer myelinated axons in the white matter despite elevated OPC numbers. Increased proliferating OPCs and elevated MAPK activity were also observed during oligodendrogenesis after expression of Shp2 GOF mutation. These results support the notion that appropriate Shp2 activity levels control the number as well as the differentiation of oligodendrocytes during development.

Catechol-O-methyltransferase Val158Met polymorphism on the relationship between white matter hyperintensity and cognition in healthy people.

BACKGROUND: White matter lesions can be easily observed on T2-weighted MR images, and are termed white matter hyperintensities (WMH). Their presence may be correlated with cognitive impairment; however, the relationship between regional WMH volume and catechol-O-methyltransferase (COMT) Val158Met polymorphism in healthy populations remains unclear. METHODS: We recruited 315 ethnic Chinese adults with a mean age of 54.9 ± 21.8 years (range: 21-89 y) to examine the genetic effect of COMT on regional WMH and the manner in which they interact to affect cognitive function in a healthy adult population. Cognitive tests, structural MRI scans, and genotyping of COMT were conducted for each participant. RESULTS: Negative correlations between the Digit Span Forward (DSF) score and frontal WMH volumes (r = -.123, P = .032, uncorrected) were noted. For the genetic effect of COMT, no significant difference in cognitive performance was observed among 3 genotypic groups. However, differences in WMH volumes over the subcortical region (P = .016, uncorrected), whole brain (P = .047, uncorrected), and a trend over the frontal region (P = .050, uncorrected) were observed among 3 COMT genotypic groups. Met homozygotes and Met/Val heterozygotes exhibited larger WMH volumes in these brain regions than the Val homozygotes. Furthermore, a correlation between the DSF and regional WMH volume was observed only in Met homozygotes. The effect size (cohen's f) revealed a small effect. CONCLUSIONS: The results indicate that COMT might modulate WMH volumes and the effects of WMH on cognition.

Allelic specificity of Ube3a expression in the mouse brain during postnatal development.

Genetic alterations of the maternal UBE3A allele result in Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, lack of speech, and difficulty with movement and balance. The combined effects of maternal UBE3A mutation and cell type-specific epigenetic silencing of paternal UBE3A are hypothesized to result in a complete loss of functional UBE3A protein in neurons. However, the allelic specificity of UBE3A expression in neurons and other cell types in the brain has yet to be characterized throughout development, including the early postnatal period when AS phenotypes emerge. Here we define maternal and paternal allele-specific Ube3a protein expression throughout postnatal brain development in the mouse, a species that exhibits orthologous epigenetic silencing of paternal Ube3a in neurons and AS-like behavioral phenotypes subsequent to maternal Ube3a deletion. We find that neurons downregulate paternal Ube3a protein expression as they mature and, with the exception of neurons born from postnatal stem cell niches, do not express detectable paternal Ube3a beyond the first postnatal week. By contrast, neurons express maternal Ube3a throughout postnatal development, during which time localization of the protein becomes increasingly nuclear. Unlike neurons, astrocytes and oligodendrotyes biallelically express Ube3a. Notably, mature oligodendrocytes emerge as the predominant Ube3a-expressing glial cell type in the cortex and white matter tracts during postnatal development. These findings demonstrate the spatiotemporal characteristics of allele-specific Ube3a expression in key brain cell types, thereby improving our understanding of the developmental parameters of paternal Ube3a silencing and the cellular basis of AS.

A novel adenoviral vector-mediated mouse model of Charcot-Marie-Tooth type 2D (CMT2D).

Charcot-Marie-Tooth disease type 2D is a hereditary axonal and glycyl-tRNA synthetase (GARS)-associated neuropathy that is caused by a mutation in GARS. Here, we report a novel GARS-associated mouse neuropathy model using an adenoviral vector system that contains a neuronal-specific promoter. In this model, we found that wild-type GARS is distributed to peripheral axons, dorsal root ganglion (DRG) cell bodies, central axon terminals, and motor neuron cell bodies. In contrast, GARS containing a G240R mutation was localized in DRG and motor neuron cell bodies, but not axonal regions, in vivo. Thus, our data suggest that the disease-causing G240R mutation may result in a distribution defect of GARS in peripheral nerves in vivo. Furthermore, a distributional defect may be associated with axonal degradation in GARS-associated neuropathies.

Lithium and GSK3-ß promoter gene variants influence white matter microstructure in bipolar disorder.

Lithium is the mainstay for the treatment of bipolar disorder (BD) and inhibits glycogen synthase kinase 3-ß (GSK3-ß). The less active GSK3-ß promoter gene variants have been associated with less detrimental clinical features of BD. GSK3-ß gene variants and lithium can influence brain gray matter structure in psychiatric conditions. Diffusion tensor imaging (DTI) measures of white matter (WM) integrity showed widespred disruption of WM structure in BD. In a sample of 70 patients affected by a major depressive episode in course of BD, we investigated the effect of ongoing long-term lithium treatment and GSK3-ß promoter rs334558 polymorphism on WM microstructure, using DTI and tract-based spatial statistics with threshold-free cluster enhancement. We report that the less active GSK3-ß rs334558*C gene-promoter variants, and the long-term administration of the GSK3-ß inhibitor lithium, were associated with increases of DTI measures of axial diffusivity (AD) in several WM fiber tracts, including corpus callosum, forceps major, anterior and posterior cingulum bundle (bilaterally including its hippocampal part), left superior and inferior longitudinal fasciculus, left inferior fronto-occipital fasciculus, left posterior thalamic radiation, bilateral superior and posterior corona radiata, and bilateral corticospinal tract. AD reflects the integrity of axons and myelin sheaths. We suggest that GSK3-ß inhibition and lithium could counteract the detrimental influences of BD on WM structure, with specific benefits resulting from effects on specific WM tracts contributing to the functional integrity of the brain and involving interhemispheric, limbic, and large frontal, parietal, and fronto-occipital connections.

JNK signaling is the shared pathway linking neuroinflammation, blood-brain barrier disruption, and oligodendroglial apoptosis in the white matter injury of the immature brain.

BACKGROUND: White matter injury is the major form of brain damage in very preterm infants. Selective white matter injury in the immature brain can be induced by lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI) in the postpartum (P) day 2 rat pups whose brain maturation status is equivalent to that in preterm infants less than 30 weeks of gestation. Neuroinflammation, blood-brain barrier (BBB) damage and oligodendrocyte progenitor apoptosis may affect the susceptibility of LPS-sensitized HI in white matter injury. c-Jun N-terminal kinases (JNK) are important stress-responsive kinases in various forms of insults. We hypothesized that LPS-sensitized HI causes white matter injury through JNK activation-mediated neuroinflammation, BBB leakage and oligodendroglial apoptosis in the white matter of P2 rat pups. METHODS: P2 pups received LPS (0.05 mg/kg) or normal saline injection followed by 90-min HI. Immunohistochemistry and immunoblotting were used to determine microglia activation, TNF-α, BBB damage, cleaved caspase-3, JNK and phospho-JNK (p-JNK), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP) expression. Immunofluorescence was performed to determine the cellular distribution of p-JNK. Pharmacological and genetic approaches were used to inhibit JNK activity. RESULTS: P2 pups had selective white matter injury associated with upregulation of activated microglia, TNF-α, IgG extravasation and oligodendroglial progenitor apoptosis after LPS-sensitized HI. Immunohistochemical analyses showed early and sustained JNK activation in the white matter at 6 and 24 h post-insult. Immunofluorescence demonstrated upregulation of p-JNK in activated microglia, vascular endothelial cells and oligodendrocyte progenitors, and also showed perivascular aggregation of p-JNK-positive cells around the vessels 24 h post-insult. JNK inhibition by AS601245 or by antisense oligodeoxynucleotides (ODN) significantly reduced microglial activation, TNF-α immunoreactivity, IgG extravasation, and cleaved caspase-3 in the endothelial cells and oligodendrocyte progenitors, and also attenuated perivascular aggregation of p-JNK-positive cells 24 h post-insult. The AS601245 or JNK antisense ODN group had significantly increased MBP and decreased GFAP expression in the white matter on P11 than the vehicle or scrambled ODN group. CONCLUSIONS: LPS-sensitized HI causes white matter injury through JNK activation-mediated upregulation of neuroinflammation, BBB leakage and oligodendrocyte progenitor apoptosis in the immature brain.

The nucleosome remodeling and deacetylase chromatin remodeling (NuRD) complex is required for peripheral nerve myelination.

Several key transcription factors and coregulators important to peripheral nerve myelination have been identified, but the contributions of specific chromatin remodeling complexes to peripheral nerve myelination have not been analyzed. Chromodomain helicase DNA-binding protein 4 (Chd4) is the core catalytic subunit of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex. Previous studies have shown Chd4 interacts with Nab (NGFI-A/Egr-binding) corepressors, which are required for early growth response 2 (Egr2/Krox20), to direct peripheral nerve myelination by Schwann cells. In this study, we examined the developmental importance of the NuRD complex in peripheral nerve myelination through the generation of conditional Chd4 knock-out mice in Schwann cells (Chd4(loxP/loxP); P0-cre). Chd4 conditional null mice were found to have delayed myelination, radial sorting defects, hypomyelination, and the persistence of promyelinating Schwann cells. Loss of Chd4 leads to elevated expression of immature Schwann cell genes (Id2, c-Jun, and p75), and sustained expression of the promyelinating Schwann cell gene, Oct6/Scip, without affecting the levels of Egr2/Krox20. Furthermore, Schwann cell proliferation is upregulated in Chd4-null sciatic nerve. In vivo chromatin immunoprecipitation studies reveal recruitment of Chd4 and another NuRD component, Mta2, to genes that are positively and negatively regulated by Egr2 during myelination. Together, these results underscore the necessity of Chd4 function to guide proper terminal differentiation of Schwann cells and implicate the NuRD chromatin remodeling complex as a requisite factor in timely and stable peripheral nerve myelination.

Angiotensin-converting enzyme and progression of white matter lesions and brain atrophy--the SMART-MR study.

High levels of angiotensin-converting-enzyme (ACE) may increase the risk of dementia through blood pressure elevation and subsequent development of cerebral small-vessel disease. However, high ACE levels may also decrease this risk through amyloid degradation which prevents brain atrophy. Within the SMART-MR study, a prospective cohort study among patients with symptomatic atherosclerotic disease, serum ACE levels were measured at baseline and a 1.5 Tesla brain MRI was performed at baseline and after on average (range) 3.9 (3.0-5.8) years of follow-up in 682 persons (mean age 58 ± 10 years). Brain segmentation was used to quantify total, deep, and periventricular white matter lesion (WML) volume, and total brain, cortical gray matter and ventricular volume (%ICV). Lacunar infarcts were rated visually. Regression analyses were used to examine the prospective associations between serum ACE and brain measures. Patients with the highest serum ACE levels (>43.3 U/L) had borderline significantly more progression of deep WML volumes than patients with the lowest ACE levels (<21.8 U/L); mean difference (95% CI) in change was 0.20 (-0.02; 0.43) %ICV. On the contrary, patients with the highest serum ACE levels had significantly less progression of cortical brain atrophy than patients with the lowest ACE levels; mean difference (95% CI) in change was 0.78 (0.21; 1.36) %ICV. Serum ACE was not associated with subcortical atrophy, periventricular WML, or lacunar infarcts. Our results show that higher ACE activity is associated with somewhat more progression of deep WML volume, but with less progression of cortical brain atrophy. This suggests both detrimental and beneficial effects of high ACE levels on the brain.

Changed histone acetylation patterns in normal-appearing white matter and early multiple sclerosis lesions.

The epigenetic identity of oligodendrocytes is modulated by posttranslational modifications of histones. Acetylation of histone H3 results from the balance between the activity of histone acetyltransferases (HATs) and histone deacetylases and modulates transcriptional activation. We have previously shown that, in rodents, histone deacetylation favors oligodendrocyte differentiation, whereas acetylation is associated with increased levels of transcriptional inhibitors of oligodendrocyte differentiation. Here, we report, in humans brains, a shift toward histone acetylation in the white matter of the frontal lobes of aged subjects and in patients with chronic multiple sclerosis (MS). Increased immunoreactivity for acetylated histone H3 was observed in the nuclei of NogoA+ oligodendrocytes in a subset of MS samples. These changes were associated with high levels of transcriptional inhibitors of oligodendrocyte differentiation (i.e., TCF7L2, ID2, and SOX2) and higher HAT transcript levels (i.e., CBP, P300) in female MS patients compared with non-neurological controls and correlated with disease duration. Chromatin immunoprecipitation from samples of MS patients revealed enrichment of acetyl-histone H3 at the promoter of the increased target genes (i.e., TCF7L2). The data in chronic lesions contrasted with findings in early MS lesions, where a marked oligodendroglial histone deacetylation was observed. Together, these data suggest that histone deacetylation is a process that occurs at the early stages of the disease and whose efficiency decreases with disease duration.

HDAC1 and HDAC2 control the transcriptional program of myelination and the survival of Schwann cells.

Histone deacetylases (HDACs) are major epigenetic regulators. We show that HDAC1 and HDAC2 functions are critical for myelination of the peripheral nervous system. Using mouse genetics, we have ablated Hdac1 and Hdac2 specifically in Schwann cells, resulting in massive Schwann cell loss and virtual absence of myelin in mutant sciatic nerves. Expression of Sox10 and Krox20, the main transcriptional regulators of Schwann cell myelination, was greatly reduced. We demonstrate that in Schwann cells, HDAC1 and HDAC2 exert specific primary functions: HDAC2 activates the transcriptional program of myelination in synergy with Sox10, whereas HDAC1 controls Schwann cell survival by regulating the levels of active ß-catenin.

HDAC-mediated deacetylation of NF-κB is critical for Schwann cell myelination.

Schwann cell myelination is tightly regulated by timely expression of key transcriptional regulators that respond to specific environmental cues, but the molecular mechanisms underlying such a process are poorly understood. We found that the acetylation state of NF-κB, which is regulated by histone deacetylases (HDACs) 1 and 2, is critical for orchestrating the myelination program. Mice lacking both HDACs 1 and 2 (HDAC1/2) exhibited severe myelin deficiency with Schwann cell development arrested at the immature stage. NF-κB p65 became heavily acetylated in HDAC1/2 mutants, inhibiting the expression of positive regulators of myelination and inducing the expression of differentiation inhibitors. We observed that the NF-κB protein complex switched from associating with p300 to associating with HDAC1/2 as Schwann cells differentiated. NF-κB and HDAC1/2 acted in a coordinated fashion to regulate the transcriptionally linked chromatin state for Schwann cell myelination. Thus, our results reveal an HDAC-mediated developmental switch for controlling myelination in the peripheral nervous system.

Decreased activity of the 20S proteasome in the brain white matter and gray matter of patients with multiple sclerosis.

Carbonylated (oxidized) proteins are known to accumulate in the cerebral white matter (WM) and gray matter (GM) of patients with multiple sclerosis (MS). Although oxidative stress is necessary for carbonyl generation, it is the failure of the degradation systems that ultimately leads to the build-up of carbonylated proteins within tissues. In this study, we measured the activity of the 20S proteasome and other proteolytic systems in the cerebral WM and GM of 13 MS patients and 13 controls. We report that the activities of the three peptidases of the 20S proteasome (i.e. chymotrypsin-like, caspase-like and trypsin-like) in both MS-WM and MS-GM are greatly reduced. Interestingly, neither the amount of proteasome nor the levels of the catalytic subunits (ß1, ß2, and ß5) are diminished in this disease. Proteins containing Lys-48 poly-ubiquitin also accumulate in MS tissues, indicating failure of the 26S proteasome as well. Levels of the regulatory caps 11S α and 19S are also lower in MS than in controls, suggesting that the activity of the more complex proteasomes may be reduced further. Finally, the activities of other proteases that might also remove oxidized proteins (calpain, cathepsin B, mitochondrial LonP) are not lessened in MS. Together, these studies suggest that direct inactivation of proteolytic centers in the 20S particle and/or the presence of specific inhibitors is the underlying cause of proteasomal dysfunction in MS.

White matter damage and the effect of matrix metalloproteinases in type 2 diabetic mice after stroke.

BACKGROUND AND PURPOSE: Diabetes mellitus leads to a higher risk of ischemic stroke and worse outcome compared to the general population. However, there have been few studies on white matter (WM) damage after stroke in diabetes mellitus. We therefore investigated WM damage after stroke in mice with diabetes mellitus. METHODS: BKS.Cg-m(+/+)Lepr(db)/J (db/db) type 2 diabetes mellitus mice and db(+) non-diabetes mellitus mice were subjected to middle cerebral artery occlusion. Functional outcome, immunostaining, zymography, Western blot, and polymerase chain reaction were used. RESULTS: After stroke, mice with diabetes mellitus exhibited significantly increased lesion volume and brain hemorrhagic and neurological deficits compared to mice without diabetes mellitus. Bielshowsky silver, luxol fast blue, amyloid precursor protein, and NG2 expression were significantly decreased, indicating WM damage, and matrix metalloproteinase (MMP)-9 activity was significantly increased in the ischemic brain of mice with diabetes mellitus. Subanalysis of similar lesions in mice with and without diabetes mellitus demonstrated mice with diabetes mellitus had significantly increased WM damage than in mice without diabetes mellitus (P<0.05). To investigate the mechanism underlying diabetes mellitus-induced WM damage, oxygen-glucose deprivation-stressed premature oligodendrocyte and primary cortical neuron cultures were used. High glucose increased MMP-2, MMP-9, cleaved caspase-3 levels, and apoptosis, as well as decreased cell survival and dendrite outgrowth in cultured primary cortical neuron. High glucose increased MMP-9, cleaved caspase-3 level, and apoptosis, and decreased cell proliferation and cell survival in cultured oligodendrocytes. Inhibition of MMP by GM6001 treatment significantly decreased high glucose-induced cell death and apoptosis in cultured primary cortical neuron and oligodendrocytes but did not alter dendrite outgrowth in primary cortical neuron. CONCLUSIONS: Mice with diabetes mellitus have increased brain hemorrhage and show more severely injured WM than mice without diabetes mellitus after stroke. MMP-9 upregulated in mice with diabetes mellitus may exacerbate WM damage after stroke in mice with diabetes mellitus.