a-versatile-murine-model-subcortical-white-matter-stroke-for-study

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

University of California, Los Angeles

Ultrastructural disruption of myelin sheaths and a loss of myelin with age are well-documented phenomena in both the human and rhesus monkey. Age-dependent activation of calpain-1 (EC 3.4.22.52) has been suggested as a plausible mechanism for increased proteolysis in the white matter of the rhesus monkey. The present study documents activation of calpain-1 throughout brain white matter in aged animals, evidenced by immunodetection of the activated enzyme as well as a calpain-derived spectrin fragment in both tissue section and Triton X-100-soluble homogenate of subcortical white matter from the frontal, temporal, and parietal lobes. Separation of myelin fractions from brain stem tissue into intact and floating myelin confirmed previous reports of an age-related increase in activated calpain-1 in the floating fraction. Measurements of calpain-1 activity using a fluorescent substrate revealed an age-related increase in calpain-1 proteolytic activity in the floating myelin fraction consistent with immunodetection of the activated enzyme in this fraction. Double-immunofluorescence demonstrated co-localization of activated calpain-1 with human leukocyte antigen-DR (HLA-DR), a marker for activated microglia, suggesting that these cells represent the major source of the increase in activated calpain-1 in the aging brain. These data solidify the role of calpain-1 in myelin protein metabolism and further implicate activated microglia in the pathology of the aging brain.

Activation of calpain-1 in myelin and microglia in the white matter of the aged rhesus monkey.

The amyloid precursor protein (APP) must fulfill important roles based on its sequence conservation from fly to human. Although multiple functions for APP have been proposed, the best-known role for this protein is as the precursor of Abeta peptide, a neurotoxic 39-43-amino acid peptide crucial to the pathogenesis of Alzheimer's disease. To investigate additional roles for APP with an eye toward understanding the molecular basis of the pleiotropic effects ascribed to APP, we isolated proteins that interacted with the plasma membrane isoform of APP. We employed a membrane-impermeable crosslinker to immobilize proteins binding to transmembrane APP in human embryonic kidney (HEK)293 cells expressing APP751 (HEK275) or rat embryonic day 18 primary neurons infected with a virus expressing APP. Notch2 was identified as a potential APP binding partner based on mass spectrometry analysis of APP complexes immunopurified from neurons. To confirm the interaction between Notch2 and APP, we carried out immunoprecipitation studies in HEK275 cells transiently expressing full-length Notch2 using Notch2 antibodies. The results indicated that APP and Notch2 interact in mammalian cells, and confirmed our initial findings. Interestingly, Notch1 also coimmunoprecipitated with APP, suggesting that APP and Notch family members may engage in intermolecular cross talk to modulate cell function. Finally, cotransfection of APP/CFP and Notch2/YFP into COS cells revealed that these two proteins colocalize on the plasma membrane. Intracellularly, however, although some APP and Notch molecules colocalize, others reside in distinct locations. The discovery of proteins that interact with APP may aid in the identification of new functions for APP.

Amyloid precursor protein interacts with notch receptors.

In myelinated axons, action potential conduction is dependent on the discrete clustering of ion channels at specialized regions of the axon, termed nodes of Ranvier. This organization is controlled, at least in part, by the adherence of myelin sheaths to the axolemma in the adjacent region of the paranode. Age-related disruption in the integrity of internodal myelin sheaths is well described and includes splitting of myelin sheaths, redundant myelin, and fluctuations in biochemical constituents of myelin. These changes have been proposed to contribute to age-related cognitive decline; in previous studies of monkeys, myelin changes correlate with cognitive performance. In the present study, we hypothesize that age-dependent myelin breakdown results in concomitant disruption at sites of axoglial contact, in particular at the paranode, and that this disruption alters the molecular organization in this region. In aged monkey and rat optic nerves, immunolabeling for voltage-dependent potassium channels of the Shaker family (Kv1.2), normally localizing in the adjacent juxtaparanode, were mislocalized to the paranode. Similarly, immunolabeling for the paranodal marker caspr reveals irregular caspr-labeled paranodal profiles, suggesting that there may be age-related changes in paranodal structure. Ultrastructural analysis of paranodal segments from optic nerve of aged monkeys shows that, in a subset of myelinated axons with thick sheaths, some paranodal loops fail to contact the axolemma. Thus, age-dependent myelin alterations affect axonal protein localization and may be detrimental to maintenance of axonal conduction.

Age-related molecular reorganization at the node of Ranvier.

We previously demonstrated that the amyloid precursor protein (APP) interacts with Notch receptors. Here, we confirmed the APP/Notch1 endogenous interaction in embryonic day 17 rat brain tissue, suggesting the interaction was not as a result of over-expression artifacts. To investigate potential homodimeric and heterodimeric interactions of APP and Notch2 (N2), we have visualized the subcellular localization of the APP/N2 complexes formed in living cells using bimolecular fluorescence complementation (BiFC) analysis. BiFC was accomplished by fusing the N-terminal fragment or the C-terminal fragment of yellow fluorescent protein (YFP) to APP, N2, and a C-terminally truncated form of N2. When expressed in COS-7 cells, these tagged proteins alone did not produce a fluorescent signal. The tagged APP homodimer produced a weak fluorescent signal, while neither full-length N2, nor a truncated N2 alone, produced a visible signal, suggesting that N2 receptors do not form homodimers. The strongest fluorescent signal was obtained with co-expression of the C-terminal fragment of YFP fused to APP and the N-terminal fragment of YFP fused to the truncated form of N2. This heterodimer localized to plasma membrane, endoplasmic reticulum (ER), Golgi and other compartments. The results were confirmed and quantified by flow cytometry. The BiFC method of specifically visualizing APP/Notch interactions can be applied to study APP and Notch signaling during development, aging and neurodegeneration.

Visualization of APP dimerization and APP-Notch2 heterodimerization in living cells using bimolecular fluorescence complementation.

The specific molecular events that underlie the age-related loss of cognitive function are poorly understood. Although not experimentally substantiated, age-dependent neuronal loss has long been considered central to age-related cognitive decline. More recently, age-related changes in brain white matter have taken precedence in explaining the steady decline in cognitive domains seen in non-diseased elderly. Characteristic alterations in the ultrastructure of myelin coupled with evidence of inflammatory processes present in the white matter of several different species suggest that specific molecular events within brain white matter may better explain observed pathological changes and cognitive deficits. This review focuses on recent evidence highlighting the importance of white matter in deciphering the course of "normal" brain aging.

What's behind the decline? The role of white matter in brain aging.

Changes in brain white matter are prominent features of the aging brain and include glial cell activation, disruption of myelin membranes with resultant reorganization of the molecular components of the node of Ranvier, and loss of myelinated fibers associated with inflammation and oxidative stress. In previous studies, overexpression of CNP, a key myelin protein, was implicated in age-related changes in myelin and axons. Here we examine the extent of CNP accumulation in brain white matter and isolated myelin of aged rhesus monkeys and its relationship to CNP degradation and partitioning in myelin. With age, excess CNP is found in myelin and throughout brain white matter accompanied by proteolytic fragments of CNP. These increases occur in the absence of changes in CNP mRNA levels. Using a combination of 2D electrophoresis, immunoprecipitation, and mass spectrometry analysis, ubiquitinated CNP was demonstrable in the Triton X-100 insoluble lipid raft associated fractions of myelin isolated from rhesus monkeys. Further, using ubiquitin-mediated fluorescence complementation (UbFC), ubiquitinated CNP was visualized by microscopy in both COS-7 and MO3.13 cells and by immunoblot in MO3.13 cells and appears to at least partially localize within lipid rafts. The findings suggest that incomplete degradation of CNP due to failure of the proteasomal system and aberrant degradation by calpain-1 leads to age-related CNP accumulation and proteolysis. In sum, we suspect these phenomena result in age-related dysfunction of CNP in the lipid raft, which may lead to myelin and axonal pathology.

Age-dependent accumulation of ubiquitinated 2',3'-cyclic nucleotide 3'-phosphodiesterase in myelin lipid rafts.

Stroke is a devastating neurological disease with limited functional recovery. Stroke affects all cellular elements of the brain and impacts areas traditionally classified as both gray matter and white matter. In fact, stroke in subcortical white matter regions of the brain accounts for approximately 30% of all stroke subtypes, and white matter injury is a component of most classes of stroke damage. However, most basic scientific information in stroke cell death and neural repair relates principally to neuronal cell death and repair. Despite an emerging biological understanding of white matter development, adult function, and reorganization in inflammatory diseases, such as multiple sclerosis, little is known of the specific molecular and cellular events in white matter ischemia. This limitation stems in part from the difficulty in generating animal models of white matter stroke. This review will discuss recent progress in studies of animal models of white matter stroke, and the emerging principles of cell death and repair in oligodendrocytes, axons, and astrocytes in white matter ischemic injury.

Models that matter: white matter stroke models.

Recovery from stroke requires neuroplasticity within surviving adjacent cortex. The axon initial segment (AIS) is the site of action potential initiation and a focal point for tuning of neuronal excitability. Remodeling of the AIS may be important to neuroplasticity after stroke.

Remodeling of the axon initial segment after focal cortical and white matter stroke.

&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x4EE5;&#x524D;&#x6240;&#x793A;&#x9AD3;&#x9798;&#x5F02;&#x5E38;&#x63CF;&#x7ED8;&#x5927;&#x8111;&#x7684;&#x6B63;&#x5E38;&#x8001;&#x5316;&#x8FC7;&#x7A0B;&#x548C;&#x4E0E;&#x5E74;&#x9F84;&#x76F8;&#x5173;&#x7684;&#x51CF;&#x5C11;&#x5728; Klotho &#x5B88;&#x6052;&#x8DE8;&#x7269;&#x79CD;&#x3002;Klotho &#x8868;&#x8FBE;&#x4E3B;&#x8981;&#x5730;&#x751F;&#x6210;&#x8111;&#x3001; &#x80BE;&#x3001; &#x5EF6;&#x4F38;&#x7684;&#x5BFF;&#x547D;&#xFF0C;&#x800C;&#x635F;&#x5931;&#x7684; Klotho &#x52A0;&#x901F;&#x8001;&#x5316;&#x8C61;&#x8868;&#x578B;&#x7684;&#x53D1;&#x5C55;&#x3002;Klotho &#x5728;&#x8111;&#x4E2D;&#x7684;&#x4F5C;&#x7528;&#x662F;&#x672A;&#x77E5;&#x7684;&#x867D;&#x7136; Klotho &#x8868;&#x8FBE;&#x7684;&#x635F;&#x5931;&#x4F1A;&#x5BFC;&#x81F4;&#x8BA4;&#x77E5;&#x529F;&#x80FD;&#x969C;&#x788D;&#x3002;&#x6211;&#x4EEC;&#x5728;&#x5C11;&#x7A81;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x529F;&#x80FD;&#xFF0C;&#x5305;&#x62EC;&#x8BF1;&#x5BFC;&#x7684;&#x6210;&#x719F;&#x7684;&#x5927;&#x9F20;&#x521D;&#x7EA7; oligodendrocytic &#x7956;&#x7EC6;&#x80DE; (OPCs) &#x548C;&#x9AD3;&#x9798;&#x4E0A;&#x53D1;&#x73B0; Klotho &#x7684;&#x91CD;&#x5927;&#x4F5C;&#x7528;&#x3002;&#x78F7;&#x86CB;&#x767D;&#x5206;&#x6790;&#x8868;&#x793A; Klotho &#x7684;&#x4E0B;&#x6E38;&#x5F71;&#x54CD;&#x6D89;&#x53CA; Akt &#x548C; ERK &#x4FE1;&#x53F7;&#x901A;&#x8DEF;&#x3002;Klotho &#x589E;&#x52A0; OPC &#x6210;&#x719F;&#x548C; Akt &#x6216; ERK &#x529F;&#x80FD;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x963B;&#x6B62;&#x8FD9;&#x79CD;&#x5BF9; OPCs &#x5F71;&#x54CD;&#x3002;Klotho &#x6572;&#x9664;&#x5C0F;&#x9F20;&#x548C;&#x63A7;&#x5236; littermates &#x7684;&#x4F53;&#x5185;&#x7814;&#x7A76;&#x8868;&#x660E;&#x6572;&#x9664;&#x5C0F;&#x9F20;&#x5728;&#x4E3B;&#x8981;&#x9AD3;&#x9798;&#x86CB;&#x767D;&#x548C;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x6709;&#x663E;&#x8457;&#x51CF;&#x5C11;&#x3002;&#x7531;&#x514D;&#x75AB;&#x7EC4;&#x5316;&#xFF0C;&#x603B;&#x6570;&#x548C;&#x6210;&#x719F;&#x5C11;&#x7A81;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x7684;&#x6570;&#x91CF;&#x662F;&#x5728; Klotho &#x6572;&#x9664;&#x5C0F;&#x9F20;&#x663E;&#x8457;&#x8F83;&#x4F4E;&#x3002;&#x5F15;&#x4EBA;&#x6CE8;&#x76EE;&#x7684;&#x662F;&#xFF0C;&#x5728;&#x8D85;&#x5FAE;&#x7ED3;&#x6784;&#x4E00;&#x7EA7;&#xFF0C;Klotho &#x6572;&#x9664;&#x5C0F;&#x9F20;&#x8868;&#x73B0;&#x51FA;&#x663E;&#x8457;&#x53D7;&#x635F;&#x7684;&#x9AD3;&#x9798;&#x7684;&#x80FC;&#x80DD;&#x4F53;&#x548C;&#x89C6;&#x795E;&#x7ECF;&#x3002;&#x8FD9;&#x4E9B;&#x5C0F;&#x9F20;&#x4E5F;&#x663E;&#x793A;&#x5728;&#x8282;&#x70B9;&#x7684;&#x90CE;&#x7684;&#x4E25;&#x91CD;&#x5F02;&#x5E38;&#x3002;&#x7834;&#x8BD1; Klotho &#x5F71;&#x54CD;&#x5C11;&#x7A81;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x7684;&#x673A;&#x5236;&#xFF0C;&#x6211;&#x4EEC;&#x7528;&#x8367;&#x5149;&#x7D20;&#x9176;&#x901A;&#x8DEF;&#x8BB0;&#x8005;&#x6765;&#x6807;&#x8BC6;&#x6240;&#x6D89;&#x53CA;&#x7684;&#x8F6C;&#x5F55;&#x56E0;&#x5B50;&#x3002;&#x5728;&#x4E00;&#x8D77;&#xFF0C;&#x8FD9;&#x4E9B;&#x7814;&#x7A76;&#x65B0;&#x578B;&#x4E3A;&#x63D0;&#x4F9B;&#x8BC1;&#x636E; Klotho &#x4F5C;&#x4E3A;&#x9AD3;&#x9798;&#x751F;&#x7269;&#x5B66;&#xFF0C;&#x56E0;&#x800C;&#x53EF;&#x80FD;&#x662F;&#x6709;&#x7528;&#x7684;&#x6CBB;&#x7597;&#x76EE;&#x6807;&#xFF0C;&#x5728;&#x52AA;&#x529B;&#x4FDD;&#x62A4;&#x8111;&#x9AD3;&#x9798;&#x5BF9;&#x5E74;&#x9F84;&#x76F8;&#x5173;&#x53D8;&#x5316;&#x548C;&#x4FC3;&#x8FDB;&#x4FEE;&#x590D;&#x7684;&#x591A;&#x53D1;&#x6027;&#x786C;&#x5316;&#x75C7;&#x4E2D;&#x7684;&#x5173;&#x952E;&#x4EBA;&#x7269;&#x3002;

&#x5C11;&#x7A81;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x6210;&#x719F;&#x548C;&#x9AD3;&#x9798;&#x7684;&#x4E2D;&#x67A2;&#x795E;&#x7ECF;&#x7CFB;&#x7EDF;&#xFF0C;&#x589E;&#x5F3A;&#x4E86;&#x6297;&#x8870;&#x8001;&#x86CB;&#x767D; Klotho&#x3002;

We have previously shown that myelin abnormalities characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. Although the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. We found significant effects of Klotho on oligodendrocyte functions, including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein analysis indicated that Klotho's downstream effects involve Akt and ERK signal pathways. Klotho increased OPC maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knock-out mice and control littermates revealed that knock-out mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knock-out mice. Strikingly, at the ultrastructural level, Klotho knock-out mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. To decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes and promote repair in multiple sclerosis.

The antiaging protein Klotho enhances oligodendrocyte maturation and myelination of the CNS.

Nous avons d&eacute;j&agrave; montr&eacute; que les anomalies de la my&eacute;line caract&eacute;risent le processus normal de vieillissement du cerveau et qu&#39;une r&eacute;duction &acirc;ge-associ&eacute;es Klotho est conserv&eacute;e selon les esp&egrave;ces. Principalement produite dans le cerveau et les reins, Klotho surexpression s&#39;&eacute;tend la dur&eacute;e de vie, alors que la perte de Klotho acc&eacute;l&egrave;re le d&eacute;veloppement de vieillissement comme ph&eacute;notypes. M&ecirc;me si on ne conna&icirc;t pas la fonction de Klotho dans cerveau, perte d&#39;expression Klotho conduit &agrave; des d&eacute;ficits cognitifs. Nous avons trouv&eacute; des effets significatifs du Klotho sur les fonctions de l&#39;oligodendrocyte, y compris induite la maturation de cellules prog&eacute;nitrices oligodendrocytic primaire rat (OPCs) in vitro et la my&eacute;linisation. Phosphoprot&eacute;ine analyse a montr&eacute; que les effets en aval de Klotho impliquent les voies de signaux Akt et ERK. Klotho a augment&eacute; de maturation de l&#39;OPC et l&#39;inhibition de la fonction Akt ou ERK bloqu&eacute; &agrave; cet effet sur l&#39;OPCs. Des &eacute;tudes in vivo des souris knock-out Klotho et contr&ocirc;le de m&ecirc;me port&eacute;e a r&eacute;v&eacute;l&eacute; que les souris knock-out ont une r&eacute;duction significative dans l&#39;expression de prot&eacute;ines et de g&egrave;nes majeurs de la my&eacute;line. Par immunohistochimie, le nombre des oligodendrocytes matures et totales &eacute;tait significativement plus faible chez les souris knock-out Klotho. &Eacute;tonnamment, au niveau ultrastructural, souris knock-out Klotho pr&eacute;sentaient significativement alt&eacute;r&eacute;e my&eacute;linisation du corps calleux et nerf optique. Ces souris ont &eacute;galement affichent des anomalies graves aux n&oelig;uds de Ranvier. Pour d&eacute;crypter les m&eacute;canismes par lesquels Klotho influence des oligodendrocytes, nous avons utilis&eacute; des journalistes voie lucif&eacute;rase pour identifier les facteurs de transcription impliqu&eacute;s. Ensemble, ces &eacute;tudes fournissent des &eacute;l&eacute;ments de preuve nouveaux pour Klotho comme un acteur cl&eacute; dans la biologie de la my&eacute;line, qui peut ainsi &ecirc;tre une cible th&eacute;rapeutique utile aux efforts visant &agrave; prot&eacute;ger la my&eacute;line cerveau contre les variations de la fonction de l&#39;&acirc;ge et favoriser la r&eacute;paration dans la scl&eacute;rose en plaques.

La prot&eacute;ine anti-&acirc;ge Klotho am&eacute;liore la maturation des oligodendrocytes et my&eacute;linisation du SNC.

Abbiamo precedentemente dimostrato che anomalie mielina caratterizzano il normale processo di invecchiamento del cervello e che una riduzione et&agrave;-associate Klotho &egrave; conservata tra specie. Prevalentemente generate nel cervello e reni, Klotho sovraespressione si estende life span, considerando che la perdita di Klotho accelera lo sviluppo di invecchiamento-come fenotipi. Anche se la funzione di Klotho nel cervello &egrave; sconosciuta, perdita di espressione di Klotho porta a deficit cognitivi. Abbiamo trovato significativi effetti di Klotho sulle funzioni oligodendrocyte, tra cui maturazione indotta delle cellule del progenitor oligodendrocytic primario ratto (PocketPC) in vitro e mielinizzazione. Fosfoproteina analisi indicano che gli effetti a valle di Klotho implicano percorsi di segnale Akt ed ERK. Klotho aumentata maturazione OPC e inibizione della funzione di Akt o ERK bloccato questo effetto su PocketPC. Studi in vivo su topi knock-out Klotho e controllo littermates ha rivelato che topi knock-out hanno una significativa riduzione nell&#39;espressione di gene e proteina mielinica principali. Immunoistochimica, il numero degli oligodendrociti maturi e totali era significativamente pi&ugrave; basso nei topi knock-out Klotho. Sorprendentemente, a livello ultrastrutturale, topi knock-out Klotho esposte significativamente ridotta mielinizzazione del nervo ottico e del corpo calloso. Questi topi visualizzati anche gravi anomalie presso i nodi di Ranvier. Per decifrare i meccanismi con cui Klotho colpisce gli oligodendrociti, abbiamo usato i reporter di luciferase pathway per identificare i fattori di trascrizione coinvolti. Insieme, questi studi forniscono prove romanzo Klotho come un giocatore chiave nella biologia della mielina, che cos&igrave; pu&ograve; essere un obiettivo terapeutico utile negli sforzi per proteggere la guaina mielinica cervello contro cambiamenti et&agrave;-dipendenti e promuovere la riparazione nella sclerosi multipla.

La proteina antiaging Klotho migliora la maturazione oligodendrocyte e la mielinizzazione del SNC.

&#x6211;&#x3005; &#x306F;&#x4EE5;&#x524D;&#x9AC4;&#x9798;&#x7570;&#x5E38;&#x6B63;&#x5E38;&#x306A;&#x8001;&#x5316;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x8133;&#x306E;&#x7279;&#x6027;&#x3092;&#x793A;&#x3059;&#x3053;&#x3068;&#x3068;&#x3001;&#x52A0;&#x9F62;&#x306B;&#x4F34;&#x3046;&#x524A;&#x6E1B; Klotho &#x3067;&#x7A2E;&#x3092;&#x6E21;&#x3063;&#x3066;&#x7BC0;&#x7D04;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x4E3B;&#x306B;&#x8133;&#x3068;&#x814E;&#x81D3;&#x306E;&#x751F;&#x6210;&#x3001;Klotho &#x306E;&#x640D;&#x5931;&#x9AD8;&#x9F62;&#x5316;&#x306E;&#x3088;&#x3046;&#x306A;&#x8868;&#x73FE;&#x578B;&#x306E;&#x958B;&#x767A;&#x3092;&#x52A0;&#x901F;&#x3059;&#x308B;&#x306E;&#x306B;&#x5BFE;&#x3057; Klotho &#x904E;&#x5270;&#x767A;&#x73FE;&#x306F;&#x3001;&#x5BFF;&#x547D;&#x3092;&#x62E1;&#x5F35;&#x3057;&#x307E;&#x3059;&#x3002;Klotho &#x8133;&#x306E;&#x6A5F;&#x80FD;&#x304C;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x640D;&#x5931; Klotho &#x8868;&#x73FE;&#x306E;&#x8A8D;&#x77E5;&#x306E;&#x8D64;&#x5B57;&#x306B; &#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x8A98;&#x5C0E;&#x6210;&#x719F;&#x30E9;&#x30C3;&#x30C8;&#x4E00;&#x6B21; oligodendrocytic &#x524D;&#x99C6;&#x7D30;&#x80DE; (Opc) in vitro &#x3067;&#x306E;&#x8133;&#x9AC4;&#x9798;&#x5F62;&#x6210;&#x3092;&#x542B;&#x3080;&#x3001;&#x30AA;&#x30EA;&#x30B4;&#x30C7;&#x30F3;&#x30C9;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306E;&#x6A5F;&#x80FD;&#x306B; Klotho &#x306E;&#x91CD;&#x8981;&#x306A;&#x52B9;&#x679C;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30EA;&#x30F3;&#x86CB;&#x767D;&#x8CEA;&#x5206;&#x6790; Klotho &#x306E;&#x4E0B;&#x6D41;&#x52B9;&#x679C; Akt &#x3068; ERK &#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x7D4C;&#x8DEF;&#x3092;&#x542B;&#x3080;&#x3053;&#x3068;&#x304C;&#x793A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;Klotho OPC &#x6210;&#x719F;&#x3057;&#x3053;&#x306E; Opc &#x306B;&#x53CA;&#x307C;&#x3059; Akt &#x307E;&#x305F;&#x306F; ERK &#x95A2;&#x6570;&#x306E;&#x6291;&#x5236;&#x3092;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3057;&#x307E;&#x3059;&#x3002;&#x751F;&#x4F53;&#x5185;&#x7814;&#x7A76; Klotho &#x30CE;&#x30C3;&#x30AF;&#x30A2;&#x30A6;&#x30C8; &#x30DE;&#x30A6;&#x30B9;&#x3068;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x306E;&#x540C;&#x8179;&#x4ED4;&#x306E;&#x30CE;&#x30C3;&#x30AF;&#x30A2;&#x30A6;&#x30C8; &#x30DE;&#x30A6;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B;&#x4E3B;&#x8981;&#x306A;&#x9AC4;&#x9798;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3068;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x306E;&#x5927;&#x5E45;&#x306A;&#x524A;&#x6E1B;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x514D;&#x75AB;&#x7D44;&#x7E54;&#x5316;&#x5B66;&#x306B;&#x3088;&#x308B;&#x5408;&#x8A08;&#x3068;&#x6210;&#x719F;&#x306E;&#x30AA;&#x30EA;&#x30B4;&#x30C7;&#x30F3;&#x30C9;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x6570; Klotho &#x30CE;&#x30C3;&#x30AF;&#x30A2;&#x30A6;&#x30C8; &#x30DE;&#x30A6;&#x30B9;&#x3067;&#x6709;&#x610F;&#x306B;&#x4F4E;&#x304B;&#x3063;&#x305F;&#x3002;&#x9A5A;&#x304F;&#x307B;&#x3069;&#x3001;&#x5FAE;&#x7D30;&#x69CB;&#x9020;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x3067;&#x306F;&#x3001;&#x5927;&#x5E45;&#x306B;&#x969C;&#x5BB3;&#x306E;&#x8133;&#x9AC4;&#x9798;&#x5F62;&#x6210;&#x306E;&#x8996;&#x795E;&#x7D4C;&#x3068;&#x8133;&#x6881; Klotho &#x30CE;&#x30C3;&#x30AF;&#x30A2;&#x30A6;&#x30C8; &#x30DE;&#x30A6;&#x30B9;&#x3092;&#x5C55;&#x793A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x306B;&#x3082;&#x6DF1;&#x523B;&#x306A;&#x7570;&#x5E38;&#x3067;&#x30E9;&#x30F3;&#x30F4;&#x30A3;&#x30A8;&#x306E;&#x8868;&#x793A;&#x3002;Klotho &#x30AA;&#x30EA;&#x30B4;&#x30C7;&#x30F3;&#x30C9;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x89E3;&#x8AAD;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x6211;&#x3005; &#x30EB;&#x30B7;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;&#x7D4C;&#x8DEF;&#x8A18;&#x8005;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x8EE2;&#x5199;&#x56E0;&#x5B50;&#x3092;&#x7279;&#x5B9A;&#x3059;&#x308B;&#x306E;&#x306B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3002;&#x4E00;&#x7DD2;&#x306B;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x7814;&#x7A76; Klotho &#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x5E74;&#x9F62;&#x4F9D;&#x5B58;&#x7684;&#x5909;&#x5316;&#x306B;&#x5BFE;&#x3059;&#x308B;&#x8133;&#x30DF;&#x30A8;&#x30EA;&#x30F3;&#x3068;&#x591A;&#x767A;&#x6027;&#x786C;&#x5316;&#x75C7;&#x306E;&#x4FEE;&#x5FA9;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x53D6;&#x308A;&#x7D44;&#x307F;&#x3067;&#x6709;&#x7528;&#x306A;&#x6CBB;&#x7642;&#x4E0A;&#x306E;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x30DF;&#x30A8;&#x30EA;&#x30F3;&#x751F;&#x7269;&#x5B66;&#x3067;&#x91CD;&#x8981;&#x306A;&#x9078;&#x624B;&#x3068;&#x3057;&#x3066;&#x65B0;&#x3057;&#x3044;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30A2;&#x30F3;&#x30C1;&#x30A8;&#x30A4;&#x30B8;&#x30F3;&#x30B0; &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; Klotho &#x30AA;&#x30EA;&#x30B4;&#x30C7;&#x30F3;&#x30C9;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306E;&#x6210;&#x719F;&#x3068;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;&#x306E;&#x8133;&#x9AC4;&#x9798;&#x5F62;&#x6210;&#x3092;&#x9AD8;&#x3081;&#x307E;&#x3059;&#x3002;

&#xC6B0;&#xB9AC;&#xB294; &#xC774;&#xC804; &#xC218; &#xCD08; &#xC774;&#xC0C1;&#xC774; &#xB1CC;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xB178;&#xD654; &#xACFC;&#xC815;&#xC744; &#xD2B9;&#xC131;&#xD654; &#xD558; &#xACE0; Klotho&#xC5D0;&#xC11C; &#xB098;&#xC774; &#xAD00;&#xB828; &#xB41C; &#xAC10;&#xC18C; &#xC885;&#xC5D0; &#xAC78;&#xCCD0; &#xBCF4;&#xC874;&#xC740; &#xB098;&#xD0C0;&#xB0AC;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC8FC;&#xB85C; &#xB1CC;&#xC640; &#xC2E0;&#xC7A5;&#xC5D0;&#xC11C; &#xC0DD;&#xC131; &#xB41C;, Klotho overexpression Klotho&#xC758; &#xC190;&#xC2E4;&#xC744; &#xAC00;&#xC18D; &#xB178;&#xD654; &#xAC19;&#xC740; &#xACE0;&#xAE30; &#xAC1C;&#xBC1C; &#xBC18;&#xBA74; &#xC218;&#xBA85;, &#xD655;&#xC7A5; &#xD569;&#xB2C8;&#xB2E4;. &#xB450;&#xB1CC;&#xC5D0; &#xC788;&#xB294; Klotho&#xC758; &#xAE30;&#xB2A5;&#xC740; &#xC54C;&#xB824;&#xC9C4;, &#xD558;&#xC9C0;&#xB9CC; Klotho &#xC2DD;&#xC758; &#xC190;&#xC2E4; &#xC778;&#xC9C0; &#xC801;&#xC790;&#xB97C; &#xB9AC;&#xB4DC;. &#xC6B0;&#xB9AC;&#xB294; &#xC720;&#xB3C4; &#xC131;&#xC219; &#xC950; &#xC8FC; oligodendrocytic &#xC870;&#xC0C1; &#xC138;&#xD3EC; (OPCs) &#xCCB4; &#xC678; &#xBC0F; myelination&#xC744; &#xD3EC;&#xD568; &#xD558; &#xC5EC; oligodendrocyte &#xD568;&#xC218;&#xC5D0; Klotho&#xC758; &#xC911;&#xC694; &#xD55C; &#xD6A8;&#xACFC; &#xBC1C;&#xACAC;. Phosphoprotein &#xBD84;&#xC11D; Klotho&#xC758; &#xB2E4;&#xC6B4;&#xC2A4;&#xD2B8;&#xB9BC; &#xD6A8;&#xACFC; ERK &#xBC0F; Akt &#xC2E0;&#xD638; &#xACBD;&#xB85C; &#xD3EC;&#xD568; &#xD45C;&#xC2DC; &#xD569;&#xB2C8;&#xB2E4;. Klotho &#xC99D;&#xAC00; OPC &#xC131;&#xC219; &#xBC0F; Akt &#xB610;&#xB294; ERK &#xAE30;&#xB2A5;&#xC758; &#xC800;&#xD574; &#xCC28;&#xB2E8; Opcs&#xC5D0;&#xC774; &#xD6A8;&#xACFC;. Klotho &#xB179;&#xC544;&#xC6C3; &#xC0DD;&#xC950;&#xC640; &#xC81C;&#xC5B4; littermates in vivo &#xC5F0;&#xAD6C; &#xB179;&#xC544;&#xC6C3; &#xC950; &#xC8FC;&#xC694; myelin &#xB2E8;&#xBC31;&#xC9C8; &#xBC0F; &#xC720;&#xC804;&#xC790; &#xD45C;&#xD604;&#xC5D0; &#xC0C1;&#xB2F9;&#xD55C; &#xAC10;&#xC18C;&#xB97C;&#xAC00;&#xC9C0;&#xACE0; &#xBC1D;&#xD614;&#xB2E4;. Immunohistochemistry, &#xCD1D; &#xBC0F; &#xC131;&#xC219; oligodendrocytes &#xC218; Klotho &#xB179;&#xC544;&#xC6C3; &#xC0DD;&#xC950;&#xC5D0;&#xC11C; &#xC0C1;&#xB2F9;&#xD788; &#xB0AE;&#xC740; &#xD588;&#xB2E4;. &#xAE30; &#xB9C9;&#xD788;&#xAC8C;, ultrastructural &#xC218;&#xC900;&#xC5D0;&#xC11C; Klotho &#xB179;&#xC544;&#xC6C3; &#xC950; &#xC2DC; &#xC2E0;&#xACBD; &#xBC0F; &#xC2E0;&#xCCB4; callosum&#xC758; &#xD06C;&#xAC8C; &#xC7A5;&#xC560;&#xC778;&#xB41C; myelination &#xC804;&#xC2DC;. &#xC774;&#xB7EC;&#xD55C; &#xB9C8;&#xC6B0;&#xC2A4;&#xB294; &#xB610;&#xD55C; &#xB178;&#xB4DC;&#xC758; Ranvier&#xC5D0;&#xC11C; &#xC2EC;&#xAC01;&#xD55C; &#xC774;&#xC0C1;&#xC774; &#xD45C;&#xC2DC; &#xB429;&#xB2C8;&#xB2E4;. Klotho oligodendrocytes&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xD574;&#xB3C5;, &#xC6B0;&#xB9AC; &#xAD00;&#xB828; &#xB41C; &#xC804;&#xC0AC; &#xC778;&#xC790;&#xB97C; &#xC2DD;&#xBCC4; &#xD558;&#xAE30; &#xC704;&#xD574; luciferase &#xD1B5;&#xB85C; &#xAE30;&#xC790; &#xC0AC;&#xC6A9;. &#xD568;&#xAED8;, &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xB530;&#xB77C;&#xC11C; &#xB1CC; myelin &#xC5F0;&#xB839;&#xC5D0; &#xB530;&#xB77C; &#xBCC0;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xBCF4;&#xD638; &#xBC0F; &#xB2E4; &#xBC1C;&#xC131; &#xACBD;&#xD654; &#xC99D;&#xC5D0;&#xC11C; &#xC218;&#xB9AC;&#xB97C; &#xD64D;&#xBCF4; &#xD558;&#xB294; &#xB178;&#xB825;&#xC5D0;&#xC11C; &#xC720;&#xC6A9;&#xD55C; &#xCE58;&#xB8CC; &#xB300;&#xC0C1; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; myelin &#xC0DD;&#xBB3C;&#xD559;&#xC758; &#xD575;&#xC2EC; &#xC120;&#xC218;&#xB85C; &#xC11C; Klotho&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xC99D;&#xAC70;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

Antiaging &#xB2E8;&#xBC31;&#xC9C8; Klotho oligodendrocyte &#xC131;&#xC219; &#xBC0F; CNS myelination&#xC744; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0B5;&#xB2C8;&#xB2E4;.

Hemos demostrado previamente que anormalidades de mielina caracterizan el proceso de envejecimiento normal del cerebro y que se conserva una reducci&oacute;n Klotho asociados con la edad entre especies. Predominante generados en el cerebro y los ri&ntilde;ones, sobreexpresi&oacute;n de Klotho extiende de vida, mientras que p&eacute;rdida de Klotho acelera el desarrollo de envejecimiento como fenotipos. Aunque se desconoce la funci&oacute;n de Klotho en cerebro, p&eacute;rdida de expresi&oacute;n de Klotho conduce a d&eacute;ficits cognoscitivos. Se encontraron efectos significativos de Klotho sobre funciones oligodendrocyte, incluyendo la maduraci&oacute;n inducida de c&eacute;lulas progenitoras oligodendrocytic primaria de rata (OPCs) in vitro y mielinizaci&oacute;n. Fosfoprote&iacute;na an&aacute;lisis indic&oacute; que los efectos aguas abajo de Klotho implican Akt y ERK v&iacute;as de se&ntilde;al. Klotho creciente maduraci&oacute;n de OPC e inhibici&oacute;n de Akt o ERK funci&oacute;n bloquea este efecto en OPC. Estudios in vivo de ratones knock-out de Klotho y hermanos de camada de control revelaron que los ratones knock-out tienen una reducci&oacute;n significativa en la expresi&oacute;n de prote&iacute;nas y genes principales de la mielina. Por inmunohistoqu&iacute;mica, el n&uacute;mero de oligodendrocitos totales y maduros fue significativamente menor en ratones knock-out de Klotho. Sorprendentemente, en el nivel ultraestructural, ratones knock-out de Klotho exhibieron significativamente deteriorado mielinizaci&oacute;n del nervio &oacute;ptico y el cuerpo calloso. Estos ratones tambi&eacute;n muestran anormalidades severas en los nodos de Ranvier. Para descifrar los mecanismos por los cuales Klotho afecta a oligodendrocitos, utilizamos reporteros camino de luciferasa para identificar los factores de transcripci&oacute;n implicados. Juntos, estos estudios proporcionan evidencia novela de Klotho como un actor clave en la biolog&iacute;a de la mielina, que as&iacute; puede ser una diana terap&eacute;utica &uacute;til en esfuerzos para proteger la mielina del cerebro contra cambios dependientes de la edad y promover la reparaci&oacute;n en la esclerosis m&uacute;ltiple.

La prote&iacute;na antienvejecimiento Klotho mejora oligodendrocyte maduraci&oacute;n y mielinizaci&oacute;n del SNC.

Tandem acute thrombotic emboli in the cervical and intracranial arteries are an unusual cause of stroke presenting unique management challenges. In regional systems of acute stroke care anchored by Comprehensive Stroke Centers (CSC), combined fibrinolytic, endovascular, and open surgical intervention is a new therapeutic option.

Drip, Ship, and Grip, then Slice and Dice: Comprehensive Stroke Center Management of Cervical and Intracranial Emboli.

In the 10 years since the FDA first cleared the use of endovascular devices for the treatment of acute stroke, definitive evidence that such therapy improves outcomes remains lacking. The decision to intubate patients undergoing stroke thrombectomy impacts multiple variables that may influence outcomes after stroke. Three main areas where intubation may deleteriously affect acute stroke management include the introduction of delays in revascularization, fluctuations in peri-procedural blood pressure, and hypocapnia, resulting in cerebral vasoconstriction. In this mini-review, we discuss the evidence supporting these limitations of intubation during stroke thrombectomy and encourage neurohospitalists, neurocritical care specialists, and neurointerventionalists to carefully consider the decision to intubate during thrombectomy and provide strategies to avoid potential complications associated with its use in acute stroke.

To Tube or Not to Tube? The Role of Intubation during Stroke Thrombectomy.

The axon plays a central role in both the injury and repair phases after stroke. This review highlights emerging principles in the study of axonal injury in stroke and the role of the axon in neural repair after stroke.

The back and forth of axonal injury and repair after stroke.

Cerebral microvascular disease predominantly affects brain white matter and deep grey matter, resulting in ischaemic damage that ranges from lacunar infarcts to white matter hyperintensities seen on magnetic resonance imaging. These lesions are common and result in both clinical stroke syndromes and accumulate over time, resulting in cognitive deficits and dementia. Magnetic resonance imaging studies suggest that these lesions progress over time, accumulate adjacent to prior lesions and have a penumbral region susceptible to further injury. The pathological correlates of this adjacent injury in surviving myelinated axons have not been previously defined. In this study, we sought to determine the molecular organization of axons in tissue adjacent to lacunar infarcts and in the regions surrounding microinfarcts, by determining critical elements in axonal function: the morphology and length of node of Ranvier segments and adjacent paranodal segments. We examined post-mortem brain tissue from six patients with lacunar infarcts and tissue from two patients with autosomal dominant retinal vasculopathy and cerebral leukoencephalopathy (previously known as hereditary endotheliopathy with retinopathy, nephropathy and stroke) who accumulate progressive white matter ischaemic lesions in the form of lacunar and microinfarcts. In axons adjacent to lacunar infarcts yet extending up to 150% of the infarct diameter away, both nodal and paranodal length increase by ∼20% and 80%, respectively, reflecting a loss of normal cell-cell adhesion and signalling between axons and oligodendrocytes. Using premorbid magnetic resonance images, brain regions from patients with retinal vasculopathy and cerebral leukoencephalopathy that harboured periventricular white matter hyperintensities were selected and the molecular organization of axons was determined within these regions. As in regions adjacent to lacunar infarcts, nodal and paranodal length in white matter of these patients is increased. Myelin basic protein and neurofilament immunolabelling demonstrates that axons in these adjacent regions have preserved axonal cytoskeleton organization and are generally myelinated. This indicates that the loss of normal axonal microdomain architecture results from disrupted axoglial signalling in white matter adjacent to lacunar and microinfarcts. The loss of the normal molecular organization of nodes and paranodes is associated with axonal degeneration and may lead to impaired conduction velocity across surviving axons after stroke. These findings demonstrate that the degree of white matter injury associated with cerebral microvascular disease extends well beyond what can be identified using imaging techniques and that an improved understanding of the neurobiology in these regions can drive new therapeutic strategies for this disease entity.

Molecular disorganization of axons adjacent to human lacunar infarcts.

Recent successful endovascular stroke trials have provided unequivocal support for these therapies in selected patients with large-vessel occlusive acute ischemic stroke. In this piece, we briefly review these trials and their utilization of advanced neuroimaging techniques that played a pivotal role in their success through targeted patient selection. In this context, the unique challenges and opportunity for advancement in current stroke networks' routine delivery of care created by these trials are discussed and recommendations to change current national stroke system guidelines are proposed.

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Lesion patterns may predict prognosis after acute ischemic stroke within the middle cerebral artery (MCA) territory; yet it remains unclear whether such imaging prognostic factors are related to patient outcome after intravenous thrombolysis.

Jason D. Hinman

Department of Neurology,
David Geffen School of Medicine,
Department of Neurology, David Geffen School of Medicine

University of California, Los Angeles

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Jason D. Hinman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neurology,David Geffen School of Medicine,Department of Neurology, David Geffen School of Medicine

University of California, Los Angeles

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Department of Neurology,
David Geffen School of Medicine,
Department of Neurology, David Geffen School of Medicine