in-vitro-modeling-for-neurological-diseases-using-direct-conversion

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

The Research Institute at Nationwide Children’s Hospital

College of Medicine, The Ohio State University

&#x808C;&#x840E;&#x7F29;&#x4FA7;&#x7D22;&#x786C;&#x5316; (ALS) &#x662F;&#x4E00;&#x79CD;&#x81F4;&#x547D;&#x7684;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x75BE;&#x75C5;&#xFF0C;&#x4E0E;&#x661F;&#x5F62;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x53D7;&#x7275;&#x8FDE;&#x4F5C;&#x4E3A;&#x5BB6;&#x65CF; (F) ALS &#x4E2D;&#x7684;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x6B7B;&#x4EA1;&#x4F5C;&#x51FA;&#x5DE8;&#x5927;&#x7684;&#x8D21;&#x732E;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x63D0;&#x8BAE;&#x7684; ALS &#x75C5;&#x7406;&#x661F;&#x5F62;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x7684;&#x4F5C;&#x7528;&#x90E8;&#x5206;&#x6765;&#x81EA;&#x4E8E;&#x556E;&#x9F7F;&#x52A8;&#x7269;&#x6A21;&#x578B;&#x7684;&#x57FA;&#x4E8E;&#x5185;&#x8D85;&#x6C27;&#x5316;&#x7269;&#x6B67;&#x5316;&#x9176;&#x7684;&#x663E;&#x6027;&#x7A81;&#x53D8;&#x7684;&#x5931;&#x771F; 1 (SOD1) &#x57FA;&#x56E0;&#xFF0C;&#x54EA;&#x4E2A;&#x5E10;&#x6237;&#x4E3A; &lt; 2%&#x7684;&#x6240;&#x6709; ALS &#x4F8B;&#x3002; &#x96F6;&#x661F; (S) ALS&#xFF0C;&#x8FD9;&#x4F1A;&#x5F71;&#x54CD;&#x5176;&#x4F5C;&#x7528; &gt; 90%&#x7684; ALS &#x60A3;&#x8005;&#x4ECD;&#x5C06;&#x8BBE;&#x7ACB;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x661F;&#x5F62;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x4EA7;&#x751F;&#x7684;&#x5931;&#x771F;&#x548C;&#x9500;&#x552E;&#x75C5;&#x4EBA;&#x6B7B;&#x540E;&#x7EC4;&#x7EC7;&#xFF0C;&#x663E;&#x793A;&#x6765;&#x81EA;&#x8FD9;&#x4E24;&#x4E2A;&#x75C5;&#x4EBA;&#x7FA4;&#x4F53;&#x7684;&#x661F;&#x5F62;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x5BF9;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x6709;&#x540C;&#x6837;&#x6BD2;&#x6027;&#x3002;&#x6211;&#x4EEC;&#x8FD8;&#x8868;&#x660E; SOD1 &#x662F;&#x9500;&#x552E;&#xFF0C;&#x4E00;&#x4E2A;&#x53EF;&#x884C;&#x7684;&#x76EE;&#x6807;&#x4F5C;&#x4E3A;&#x5176;&#x7EC4;&#x5408;&#x5F0F;&#x5927;&#x5927;&#x51CF;&#x8F7B;&#x5BF9;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x4ECB;&#x5BFC;&#x661F;&#x5F62;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x6BD2;&#x6027;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x6570;&#x636E;&#x4F5C;&#x4E3A;&#x975E;&#x7EC6;&#x80DE;&#x4E2D;&#x9500;&#x552E;&#x7684;&#x81EA;&#x4E3B;&#x7EC4;&#x4EF6;&#x7A81;&#x51FA;&#x661F;&#x5F62;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x548C;&#x63D0;&#x4F9B;&#x4F53;&#x5916;&#x6A21;&#x578B;&#x7CFB;&#x7EDF;&#x8C03;&#x67E5;&#x5E38;&#x89C1;&#x75BE;&#x75C5;&#x53D1;&#x75C5;&#x673A;&#x5236;&#x548C;&#x8BC4;&#x4F30;&#x6F5C;&#x5728;&#x7597;&#x6CD5;&#x9500;&#x552E;&#x548C;&#x5931;&#x771F;&#x3002;

&#x5BB6;&#x65CF;&#x6027;&#x4E0E;&#x6563;&#x53D1;&#x6027; ALS &#x60A3;&#x8005;&#x4ECE;&#x661F;&#x5F62;&#x80F6;&#x8D28;&#x7EC6;&#x80DE;&#x662F;&#x5BF9;&#x8FD0;&#x52A8;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x6BD2;&#x6027;&#x3002;

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.

Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.

Scl&eacute;rose lat&eacute;rale amyotrophique (SLA) est une maladie fatale des motoneurones, astrocytes impliqu&eacute;s comme contribuant consid&eacute;rablement &agrave; la mort des motoneurones en genetique ALS (F). Cependant, le r&ocirc;le des astrocytes dans la pathologie de la SLA d&eacute;coule en partie des mod&egrave;les de rongeurs de FALS issu des mutations dominantes au sein de la superoxyde dismutase 1 (SOD1) g&egrave;ne, qui repr&eacute;sentent &lt; 2 % de tous les cas d&#39;ALS. Leur r&ocirc;le dans sporadiques ALS (S), qui affecte &gt; 90 % des patients d&#39;ALS, reste &agrave; &eacute;tablir. &Agrave; l&#39;aide d&#39;astrocytes g&eacute;n&eacute;r&eacute;s &agrave; partir des tissus post-mortem de patients FALS tant SALS, nous montrons que les astrocytes provenant de deux groupes de patients sont de m&ecirc;me toxiques pour les neurones moteurs. Nous d&eacute;montrons &eacute;galement que SOD1 est une cible viable SALS, comme sa pr&eacute;cipitation att&eacute;nue consid&eacute;rablement la toxicit&eacute; induite par l&#39;astrocyte vers les neurones moteurs. Nos donn&eacute;es soulignent les astrocytes comme un composant autonome non-cellules dans SALS et fournissent un syst&egrave;me mod&egrave;le in vitro pour &eacute;tudier des m&eacute;canismes communs de la maladie et l&#39;&eacute;valuation des th&eacute;rapies potentielles de SALS et FALS.

Des patients d&#39;ALS familiales et sporadiques, les astrocytes sont toxiques pour les neurones moteurs.

Amyotrophe Lateralsklerose (ALS) ist ein schwerwiegender Motoneuron-Krankheit, mit Astrozyten verwickelt als tr&auml;gt wesentlich zur Motoneuron Tod famili&auml;re (F) ALS. Jedoch die vorgeschlagene Rolle der Astrozyten in der Pathologie des ALS teilweise von abgeleitet Nagetier Modelle der FALS basierend auf dominante Mutationen innerhalb der Superoxid-Dismutase 1 (SOD1)-Gen, die entfallen &lt; 2 % der f&auml;lle f&uuml;r alle ALS. ihre rolle bei der sporadischen (S) ALS, das betrifft &gt; 90 % der ALS-Patienten, muss noch eingerichtet werden. Anhand von Post-mortem Gewebe aus FALS und SALS Patienten generiert Astrozyten zeigen wir dass Astrozyten abgeleitet aus beiden Patientengruppen zu Motoneuronen &auml;hnlich giftig sind. Wir zeigen auch, dass die SOD1 eine tragf&auml;hige Ziel f&uuml;r SALS, ist, wie seinen Niederschlag Gliazelle-vermittelte Toxizit&auml;t gegen&uuml;ber Motoneuronen erheblich d&auml;mpft. Unsere Daten Astrozyten als autonome Komponente in SALS nicht-Zelle markieren und eine in-vitro-Modellsystem zum gemeinsamen Krankheitsmechanismen zu untersuchen und bewerten m&ouml;gliche Therapien f&uuml;r SALS und FALS sorgen.

Astrozyten aus famili&auml;ren und sporadischen ALS-Patienten sind giftig f&uuml;r Motoneuronen.

Sclerosi laterale amiotrofica (SLA) &egrave; una malattia del motoneurone fatale, con astrociti implicato come contribuire sostanzialmente alla morte del motoneurone in familial ALS (F). Tuttavia, il ruolo proposto degli astrociti nella patologia di ALS deriva in parte da roditore modelli di FALS basato su mutazioni dominanti all&#39;interno della superossido dismutasi 1 (SOD1) gene, che rappresentano &lt; 2% di tutti i casi di ALS. loro ruolo in sporadici ALS (S), che colpisce &amp;gt; 90% dei pazienti di ALS, resta da stabilire. Utilizzando gli astrociti generati da post-mortem del tessuto di pazienti sia FALS e SALS, mostriamo che gli astrociti derivati da entrambi i gruppi di pazienti sono analogamente tossici per i motoneuroni. Abbiamo anche dimostrare che la SOD1 &egrave; una destinazione valida per SALS, come suo atterramento attenua significativamente Astrocita-mediata tossicit&agrave; verso i motoneuroni. I nostri dati evidenziano gli astrociti come componente autonoma non cellule in SALS e forniscono un sistema di modello in vitro per indagare i meccanismi comuni di malattia e valutare potenziali terapie per SALS e FALS.

Astrociti da pazienti di ALS familiari e sporadici sono tossici per i neuroni motori.

&#x840E;&#x7E2E;&#x6027;&#x5074;&#x7D22;&#x786C;&#x5316;&#x75C7; (ALS) &#x3068;&#x5927;&#x5E45;&#x306B;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x6B7B; &#xFF08;F&#xFF09; &#x306E;&#x5BB6;&#x65CF;&#x6027; ALS &#x306B;&#x8CA2;&#x732E;&#x3068;&#x3057;&#x3066;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x308B;&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306E;&#x81F4;&#x547D;&#x7684;&#x306A;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x75BE;&#x60A3;&#x3067;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;ALS &#x306E;&#x75C5;&#x614B;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306E;&#x63D0;&#x6848;&#x306E;&#x5F79;&#x5272;&#x306E;&#x4E00;&#x90E8;&#x30B9;&#x30FC;&#x30D1;&#x30FC;&#x30AA;&#x30AD;&#x30B7;&#x30C9;&#x306E;&#x30C7;&#x30A3;&#x30B9;&#x30E0;&#x30BF;&#x30FC;&#x30BC;&#x5185;&#x306E;&#x652F;&#x914D;&#x7684;&#x306A;&#x5909;&#x7570;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066; FALS &#x306E;&#x9F67;&#x6B6F;&#x985E;&#x52D5;&#x7269;&#x30E2;&#x30C7;&#x30EB;&#x304B;&#x3089; 1 &#x306E; (SOD1) &#x907A;&#x4F1D;&#x5B50;&#x3092;&#x8003;&#x616E;&#x6D3E;&#x751F; &lt; 2 &#xFF05; &#x306E;&#x3059;&#x3079;&#x3066;&#x306E; ALS &#x306E;&#x30B1;&#x30FC;&#x30B9;&#x3002; &#x81EA;&#x5206;&#x306E;&#x5F79;&#x5272;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x3001;&#x6563;&#x767A;&#x7684;&#x306A;&#x306E; (S) als &#x76EE; ALS &#x60A3;&#x8005;&#x306E; 90 &#xFF05; &#x306E;&#x307E;&#x307E;&#x78BA;&#x7ACB;&#x3057;&#x307E;&#x3059;&#x3002;&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8; FALS SALS &#x60A3;&#x8005;&#x304B;&#x3089;&#x6B7B;&#x5F8C;&#x306E;&#x7D44;&#x7E54;&#x304B;&#x3089;&#x751F;&#x6210;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306E;&#x4E21;&#x65B9;&#x306E;&#x60A3;&#x8005;&#x56E3;&#x4F53;&#x304B;&#x3089;&#x6D3E;&#x751F;&#x3057;&#x305F;&#x304C;&#x540C;&#x69D8;&#x306B;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306B;&#x6709;&#x6BD2;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3; &#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x6BD2;&#x6027;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306B;&#x5411;&#x3051;&#x3066;&#x5927;&#x5E45;&#x306B;&#x6E1B;&#x8870;&#x3055;&#x305B;&#x308B;&#x3053;&#x3068;&#x307E;&#x305F; SOD1 SALS&#x3001;&#x6709;&#x671B;&#x306A;&#x6A19;&#x7684;&#x3068;&#x306A;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8; SALS &#x306E;&#x975E;&#x7D30;&#x80DE;&#x306E;&#x81EA;&#x5F8B;&#x7684;&#x306A;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x3068;&#x3057;&#x3066;&#x5F37;&#x8ABF;&#x8868;&#x793A;&#x3057;&#x3001;&#x5171;&#x901A;&#x306E;&#x75BE;&#x60A3;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x8ABF;&#x67FB;&#x3057;&#x3001;SALS FALS &#x306E;&#x6F5C;&#x5728;&#x7684;&#x7642;&#x6CD5;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E; in vitro &#x30E2;&#x30C7;&#x30EB; &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x5BB6;&#x65CF;&#x6027;&#x304A;&#x3088;&#x3073;&#x6563;&#x767A;&#x7684;&#x306A; ALS &#x60A3;&#x8005;&#x304B;&#x3089;&#x306F;&#x904B;&#x52D5;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306B;&#x6709;&#x6BD2;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#xB8E8; &#xAC8C; &#xB9AD; &#xBCD1; &#xCE21;&#xBA74; &#xACBD;&#xD654; &#xC99D; (ALS)&#xC740; &#xCE58;&#xBA85;&#xC801;&#xC778; &#xBAA8;&#xD130; &#xC2E0;&#xACBD; &#xC9C8;&#xBCD1;, &#xC5F0;&#xB8E8; &#xBAA8;&#xD130; &#xC2E0;&#xACBD; &#xC8FD;&#xC74C;&#xC73C;&#xB85C; &#xAC00;&#xC871; (F) ALS&#xC5D0; &#xC2E4;&#xC9C8;&#xC801;&#xC73C;&#xB85C; &#xCC38;&#xC5EC; &#xD558;&#xB294; &#xAC83; &#xC774;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xB8E8; &#xAC8C; &#xB9AD; &#xBCD1;&#xC758; &#xBCD1; &#xB9AC;&#xC5D0; &#xC774;&#xB2E4;&#xC758; &#xC81C;&#xC548; &#xB41C; &#xC5ED;&#xD560; &#xC77C;&#xBD80;&#xC5D0;&#xC11C; &#xD30C;&#xC0DD; superoxide dismutase &#xB0B4;&#xC758; &#xC9C0;&#xBC30;&#xC801;&#xC778; &#xBCC0;&#xC774; &#xB530;&#xB77C; FALS&#xC758; &#xC124;&#xCE58;&#xB958; &#xBAA8;&#xB378; 1 (SOD1) &#xC720;&#xC804;&#xC790;&#xB294; &#xB2F4;&#xB2F9; &lt;&#xC758; &#xBAA8;&#xB4E0; &#xB8E8; &#xAC8C; &#xB9AD; &#xBCD1;&#xC758; &#xACBD;&#xC6B0;. 2% &#xADF8;&#xB4E4;&#xC758; &#xC5ED;&#xD560;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xBBF8;&#xCE58;&#xB294; &#xC0B0;&#xBC1C;&#xC801; (S) ALS&#xC5D0; &amp;gt; &#xB8E8; &#xAC8C; &#xB9AD; &#xBCD1; &#xD658;&#xC790;&#xC758; 90%&#xB97C; &#xC124;&#xC815;&#xD560; &#xC218; &#xC788;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC774;&#xB2E4; &#xB450; &#xD658;&#xC790; &#xADF8;&#xB8F9;&#xC5D0;&#xC11C; &#xD30C;&#xC0DD; &#xB41C;&#xC740; &#xB9C8;&#xCC2C;&#xAC00;&#xC9C0;&#xB85C; &#xBAA8;&#xD130; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0; &#xB3C5;&#xC131;&#xC774; &#xD45C;&#xC2DC; &#xC774;&#xB2E4; FALS&#xC640; SALS &#xD658;&#xC790;&#xC5D0;&#xC11C; &#xC0AC;&#xD6C4; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; &#xC0DD;&#xC131; &#xB41C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;. &#xCD5C;&#xC800;&#xC758; &#xD06C;&#xAC8C; &#xC57D;&#xD654; &#xBAA8;&#xD130; &#xB274;&#xB7F0;&#xC73C;&#xB85C; &#xC0AC;&#xC774;&#xD1A0; &#xC911;&#xC7AC; &#xB3C5;&#xC131;&#xC73C;&#xB85C; &#xB610;&#xD55C; SOD1 SALS&#xC5D0; &#xB300; &#xD55C; &#xC720;&#xB825;&#xD55C; &#xB300;&#xC0C1; &#xC774;&#xB2E4; &#xC2DC;&#xC5F0; &#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xB370;&#xC774;&#xD130; SALS&#xC5D0; &#xC140;&#xC774; &#xC544;&#xB2CC; &#xC790;&#xCE58; &#xCEF4;&#xD3EC;&#xB10C;&#xD2B8;&#xB85C; &#xC774;&#xB2E4;&#xB97C; &#xAC15;&#xC870; &#xD45C;&#xC2DC; &#xD558; &#xACE0; &#xC77C;&#xBC18;&#xC801;&#xC778; &#xC9C8;&#xBCD1; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC870;&#xC0AC; &#xD558; &#xACE0; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xCE58;&#xB8CC; SALS FALS&#xC5D0; &#xB300; &#xD55C; &#xD3C9;&#xAC00;&#xB97C; &#xD558;&#xB294; &#xCCB4; &#xC678; &#xBAA8;&#xB378; &#xC2DC;&#xC2A4;&#xD15C;&#xC744; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

&#xAC00;&#xC871; &#xD558; &#xACE0; &#xC0B0;&#xBC1C;&#xC801; &#xC778; &#xB8E8; &#xAC8C; &#xB9AD; &#xBCD1; &#xD658;&#xC790;&#xC5D0; &#xAC8C; &#xC11C; &#xC774;&#xB2E4; &#xBAA8;&#xD130; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0; &#xB3C5;&#xC131;&#xC774; &#xC788;&#xB2E4;.

Esclerosis lateral amiotr&oacute;fica (ELA) es una enfermedad de la motoneurona fatal, con astrocitos implicado como contribuir substancialmente a la muerte de la neurona motora en ALS familiar de (F). Sin embargo, el papel propuesto de astrocitos en la patolog&iacute;a de ALS deriva en parte de modelos de roedores de FALS basado en mutaciones dominantes dentro de la super&oacute;xido dismutasa 1 gene (SOD1), que representan &lt; 2% de todos los casos de ALS. Su papel en ALS espor&aacute;dico de la (S), que afecta a &gt; 90% de los pacientes de ALS, queda por establecerse. Usando astrocitos generados a partir de tejido post mortem de pacientes tanto FALS y SALS, demostramos que los astrocitos derivados de ambos grupos de pacientes son igualmente t&oacute;xicos para las neuronas motoras. Tambi&eacute;n demostramos que la SOD1 es un objetivo viable para SALS, como su ca&iacute;da aten&uacute;a significativamente la toxicidad mediada por Astrocito hacia las neuronas motoras. Nuestros datos destacan astrocitos como un componente de c&eacute;lulas no aut&oacute;nomo en SALS y proporcionan un sistema modelo in vitro para investigar los mecanismos comunes de la enfermedad y evaluar posibles terapias para COPIARNOS y FALS.

Los astrocitos de pacientes de ALS familiares o espor&aacute;dicos son t&oacute;xicos para las neuronas motoras.

Transplantation of gene-corrected motor neurons as a therapeutic strategy for spinal muscular atrophy.

Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials.

Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS.

Making sense of pain: are pluripotent stem cell-derived sensory neurons a new tool for studying pain mechanisms?

Spinal Muscular Atrophy (SMA) is the most frequent lethal genetic neurodegenerative disorder in infants. The disease is caused by low abundance of the survival of motor neuron (SMN) protein leading to motor neuron degeneration and progressive paralysis. We previously demonstrated that a single intravenous injection (IV) of self-complementary adeno-associated virus 9 carrying the human SMN cDNA (scAAV9-SMN) resulted in widespread transgene expression in spinal cord motor neurons in SMA mice as well as nonhuman primates and complete rescue of the disease phenotype in mice. Here, we evaluated the dosing and efficacy of scAAV9-SMN delivered directly to the cerebral spinal fluid (CSF) via single injection. We found widespread transgene expression throughout the spinal cord in mice and nonhuman primates when using a ten times lower dose compared to the IV application. Interestingly, in nonhuman primates, lower doses than in mice can be used for similar motor neuron targeting efficiency. Moreover, the transduction efficacy is further improved when subjects are kept in the Trendelenburg position to facilitate spreading of the vector. We present a detailed analysis of transduction levels throughout the brain, brainstem and spinal cord of nonhuman primates, providing new guidance for translation towards therapy for a wide range of neurodegenerative disorders.Molecular Therapy (2014); doi:10.1038/mt.2014.210.

Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA - a dose response study in mice and nonhuman primates.

For the past century, research on neurological disorders has largely focused on the most prominently affected cell types - the neurons. However, with increasing knowledge of the diverse physiological functions of glial cells, their impact on these diseases has become more evident. Thus, many conditions appear to have more complex origins than initially thought. Since neurological pathologies are often sporadic with unknown etiology, animal models are difficult to create and might only reflect a small portion of patients in which a mutation in a gene has been identified. Therefore, reliable in vitro systems to studying these disorders are urgently needed. They might be a pre-requisite for improving our understanding of the disease mechanisms as well as for the development of potential new therapies. In this review, we will briefly summarize the function of different glial cell types in the healthy central nervous system (CNS) and outline their implication in the development or progression of neurological conditions. We will then describe different types of culture systems to model non-cell autonomous interactions in vitro and evaluate advantages and disadvantages. This article is part of a Special Issue entitled SI: Exploiting human neurons.

Glia-neuron interactions in neurological diseases: Testing non-cell autonomy in a dish.

Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic to motor neurons (MNs) and play a non-cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of MNs to cell death remain unclear. Here we report that astrocytes derived from either mice bearing mutations in genes associated with ALS or human subjects with ALS reduce the expression of major histocompatibility complex class I (MHCI) molecules on MNs; reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MNs against astrocyte toxicity. Overexpression of a single MHCI molecule, HLA-F, protects human MNs from ALS astrocyte-mediated toxicity, whereas knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, on human astrocytes results in enhanced MN death. Thus, our data indicate that, in ALS, loss of MHCI expression on MNs renders them more vulnerable to astrocyte-mediated toxicity.

Major histocompatibility complex class I molecules protect motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis.

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9ALS/FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9ALS/FTD, its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc-51-like kinase 1 (ULK1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a-dependent trafficking of the ULK1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62-positive puncta reminiscent of the p62 pathology observed in C9ALS/FTD patients. Finally, basal levels of autophagy were markedly reduced in C9ALS/FTD patient-derived iNeurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9ALS/FTD-associated p62 pathology.

The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant or digenic disorder linked to derepression of the toxic DUX4 gene in muscle. There is currently no pharmacological treatment. The emergence of DUX4 enabled development of cell and animal models that could be used for basic and translational research. Since DUX4 is toxic, animal model development has been challenging, but progress has been made, revealing that tight regulation of DUX4 expression is critical for creating viable animals that develop myopathy. Here, we report such a model - the tamoxifen-inducible FSHD mouse model called TIC-DUX4. Uninduced animals are viable, born in Mendelian ratios, and overtly indistinguishable from WT animals. Induced animals display significant DUX4-dependent myopathic phenotypes at the molecular, histological, and functional levels. To demonstrate the utility of TIC-DUX4 mice for therapeutic development, we tested a gene therapy approach aimed at improving muscle strength in DUX4-expressing muscles using adeno-associated virus serotype 1.Follistatin (AAV1.Follistatin), a natural myostatin antagonist. This strategy was not designed to modulate DUX4 but could offer a mechanism to improve muscle weakness caused by DUX4-induced damage. AAV1.Follistatin significantly increased TIC-DUX4 muscle mass and strength even in the presence of DUX4 expression, suggesting that myostatin inhibition may be a promising approach to treat FSHD-associated weakness. We conclude that TIC-DUX4 mice are a relevant model to study DUX4 toxicity and, importantly, are useful in therapeutic development studies for FSHD.

AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD.

CLN6-Batten disease, a form of neuronal ceroid lipofuscinosis is a rare lysosomal storage disorder presenting with gradual declines in motor, visual, and cognitive abilities and early death by 12-15 years of age. We developed a self-complementary adeno-associated virus serotype 9 (scAAV9) vector expressing the human CLN6 gene under the control of a chicken β-actin (CB) hybrid promoter. Intrathecal delivery of scAAV9.CB.hCLN6 into the cerebrospinal fluid (CSF) of the lumbar spinal cord of 4-year-old non-human primates was safe, well tolerated, and led to efficient targeting throughout the brain and spinal cord. A single intracerebroventricular (i.c.v.) injection at post-natal day 1 in Cln6 mutant mice delivered scAAV9.CB.CLN6 directly into the CSF, and it prevented or drastically reduced all of the pathological hallmarks of Batten disease. Moreover, there were significant improvements in motor performance, learning and memory deficits, and survival in treated Cln6 mutant mice, extending survival from 15 months of age (untreated) to beyond 21 months of age (treated). Additionally, many parameters were similar to wild-type counterparts throughout the lifespan of the treated mice.

Gene Therapy Corrects Brain and Behavioral Pathologies in CLN6-Batten Disease.

CLN8 disease is a rare form of neuronal ceroid lipofuscinosis caused by biallelic mutations in the CLN8 gene, which encodes a transmembrane endoplasmic reticulum protein involved in trafficking of lysosomal enzymes. CLN8 disease patients present with myoclonus, tonic-clonic seizures, and progressive declines in cognitive and motor function, with many cases resulting in premature death early in life. There are currently no treatments that can cure the disease or substantially slow disease progression. Using a mouse model of CLN8 disease, we tested the safety and efficacy of an intracerebroventricularly (i.c.v.) delivered self-complementary adeno-associated virus serotype 9 (scAAV9) gene therapy vector driving expression of human CLN8. A single neonatal injection was safe and well tolerated, resulting in robust transgene expression throughout the CNS from 4 to 24 months, reducing histopathological and behavioral hallmarks of the disease and restoring lifespan from 10 months in untreated animals to beyond 24 months of age in treated animals. While it is unclear whether some of these behavioral improvements relate to preserved visual function, improvements in learning/memory, or other central or peripheral benefits, these results demonstrate, by far, the most successful degree of rescue reported in an animal model of CLN8 disease, and they support further development of gene therapy for this disorder.

AAV9 Gene Therapy Increases Lifespan and Treats Pathological and Behavioral Abnormalities in a Mouse Model of CLN8-Batten Disease.

Batten disease is a family of rare, fatal, neuropediatric diseases presenting with memory/learning decline, blindness, and loss of motor function. Recently, we reported the use of an AAV9-mediated gene therapy that prevents disease progression in a mouse model of CLN6-Batten disease ( ), restoring lifespans in treated animals. Despite the success of our viral-mediated gene therapy, the dosing strategy was optimized for delivery to the brain parenchyma and may limit the therapeutic potential to other disease-relevant tissues, such as the eye. Here, we examine whether cerebrospinal fluid (CSF) delivery of scAAV9.CB.CLN6 is sufficient to ameliorate visual deficits in  mice. We show that intracerebroventricular (i.c.v.) delivery of scAAV9.CB.CLN6 completely prevents hallmark Batten disease pathology in the visual processing centers of the brain, preserving neurons of the superior colliculus, thalamus, and cerebral cortex. Importantly, i.c.v.-delivered scAAV9.CB.CLN6 also expresses in many cells throughout the central retina, preserving many photoreceptors typically lost in  mice. Lastly, scAAV9.CB.CLN6 treatment partially preserved visual acuity in  mice as measured by optokinetic response. Taken together, we report the first instance of CSF-delivered viral gene reaching and rescuing pathology in both the brain parenchyma and retinal neurons, thereby partially slowing visual deterioration.

Article	Year
Astrocytes from familial and sporadic ALS patients are toxic to motor neurons. Nature biotechnology\| PubMed ID: 21832997	2011
Transplantation of gene-corrected motor neurons as a therapeutic strategy for spinal muscular atrophy. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 23449105	2013
Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 24008656	2013
Making sense of pain: are pluripotent stem cell-derived sensory neurons a new tool for studying pain mechanisms? Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 25082088	2014
Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA - a dose response study in mice and nonhuman primates. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 25358252	2014
Glia-neuron interactions in neurological diseases: Testing non-cell autonomy in a dish. Brain research\| PubMed ID: 26778174	2017
Major histocompatibility complex class I molecules protect motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis. Nature medicine\| PubMed ID: 26928464	2016
The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy. The EMBO journal\| PubMed ID: 27334615	2016
AAV-mediated follistatin gene therapy improves functional outcomes in the TIC-DUX4 mouse model of FSHD. JCI insight\| PubMed ID: 30429376	2018
Gene Therapy Corrects Brain and Behavioral Pathologies in CLN6-Batten Disease. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 31331814	2019
AAV9 Gene Therapy Increases Lifespan and Treats Pathological and Behavioral Abnormalities in a Mouse Model of CLN8-Batten Disease. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 33010819	2021
Intracranial delivery of AAV9 gene therapy partially prevents retinal degeneration and visual deficits in CLN6-Batten disease mice. Molecular therapy. Methods & clinical development\| PubMed ID: 33665223	2021

Kathrin Meyer

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