Brain inflammation is accompanied by peripheral inflammation in Cstb(-/-) mice, a model for progressive myoclonus epilepsy

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited childhood-onset neurodegenerative disorder, characterized by myoclonus, seizures, and ataxia. Mutations in the cystatin B gene (CSTB) underlie EPM1. The CSTB-deficient (Cstb(-/-)) mouse model recapitulates key features of EPM1, including myoclonic seizures. The mice show early microglial activation that precedes seizure onset and neuronal loss and leads to neuroinflammation. We here characterized the inflammatory phenotype of Cstb(-/-) mice in more detail. We found higher concentrations of chemokines and pro-inflammatory cytokines in the serum of Cstb(-/-) mice and higher CXCL13 expression in activated microglia in Cstb(-/-) compared to control mouse brains. The elevated chemokine levels were not accompanied by blood-brain barrier disruption, despite increased brain vascularization. Macrophages in the spleen and brain of Cstb(-/-) mice were predominantly pro-inflammatory. Taken together, these data show that CXCL13 expression is a hallmark of microglial activation in Cstb(-/-)mice and that the brain inflammation is linked to peripheral inflammatory changes, which might contribute to the disease pathology of EPM1.Peer reviewe

Gene-Expression Profiling Suggests Impaired Signaling via the Interferon Pathway in Cstb(-/-) Microglia

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM254800) is an autosomal recessive neurodegenerative disorder characterized by stimulus-sensitive and action-activated myoclonus, tonic-clonic epileptic seizures, and ataxia. Loss-of-function mutations in the gene encoding the cysteine protease inhibitor cystatin B (CSTB) underlie EPM1. The deficiency of CSTB in mice (Cstb(-/-) mice) generates a phenotype resembling the symptoms of EPM1 patients and is accompanied by microglial activation at two weeks of age and an upregulation of immune system-associated genes in the cerebellum at one month of age. To shed light on molecular pathways and processes linked to CSTB deficiency in microglia we characterized the transcriptome of cultured Cstb(-/-) mouse microglia using microarray hybridization and RNA sequencing (RNA-seq). The gene expression profiles obtained with these two techniques were in good accordance and not polarized to either pro- or anti-inflammatory status. In Cstb(-/-) microglia, altogether 184 genes were differentially expressed. Of these, 33 genes were identified by both methods. Several interferon-regulated genes were weaker expressed in Cstb(-/-) microglia compared to control. This was confirmed by quantitative real-time PCR of the transcripts Irf7 and Stat1. Subsequently, we explored the biological context of CSTB deficiency in microglia more deeply by functional enrichment and canonical pathway analysis. This uncovered a potential role for CSTB in chemotaxis, antigen-presentation, and in immune-and defense response-associated processes by altering JAK-STAT pathway signaling. These data support and expand the previously suggested involvement of inflammatory processes to the disease pathogenesis of EPM1 and connect CSTB deficiency in microglia to altered expression of interferon-regulated genes.Peer reviewe

Microglial dysfunction in Cstb-/- mice, a model for the neurodegenerative disorder progressive myoclonus epilepsy of Unverricht-Lundborg type, EPM1

The autosomal recessively inherited progressive myoclonus epilepsy of Unverricht- Lundborg type (EPM1, OMIM 254800) is a neurodegenerative disease severely affecting patients motor coordination. Its onset lies around 6 to 16 years of age and the presenting symptoms are severely incapacitating, stimulus-sensitive myoclonus and/or tonic-clonic seizures. Later during disease course, the patients develop ataxia. EPM1 is caused by mutations in the cystatin B (CSTB, OMIM 601145) gene, encoding the cysteine protease inhibitor CSTB. The CSTB-deficient mouse (Cstb-/- mice) is characterized by neuronal hyperexcitability and brain atrophy. In addition, histological studies revealed that the neuronal pathology is accompanied by early microglial activation. Aberrant activation of microglia and neuroinflammation has previously been linked to neuropathology and neurodegenerative diseases. Therefore, we aimed to characterize microglia of Cstb-/- mice in more detail. Our results identified impaired interferon signaling as a potential molecular pathway underlying the phenotype of Cstb-/- mice. In addition, functional properties of activated primary Cstb-/- microglia are altered in vitro. For example, their chemokine release is enhanced, but their phagocytic activity is reduced. In pre-symptomatic Cstb-/- mouse brains at postnatal day 14, the activation of Cstb-/- microglia is shifted towards an anti-inflammatory activation, and it switches towards a pro-inflammatory activation in early symptomatic Cstb-/- mice at postnatal day 30. In line with this, inflammatory markers are upregulated, and T lymphocytes and granulocytes exist in the brain of Cstb-/- mice, which suggests an infiltration of peripheral immune cells. Pro-inflammatory activated macrophages in the spleen imply a widespread immune system activation in Cstb-/- mice. In conclusion, we show that microglia in Cstb-/- mice are dysfunctional and that their activation is aberrant. We link CSTB deficiency in young mice to inflammatory processes and suggest that microglial dysfunction might contribute to the pathology of EPM1.Tutkimuksemme kohteena on hermorappeumasairaus EPM1 eli Unverricht-Lundborgin tauti, jonka pääoireita ovat tahdottomat lihasnykäykset eli myokloniat ja/tai epileptiset kohtaukset, sekä näitä seuraava ataksia eli haparointi. Tauti ilmenee yleensä 6-16 vuoden iässä ja sen taustalla ovat perinnölliset muutokset kystatiini B -geenissä. Geeni tuottaa proteaasinestäjää, jonka puute tai muuttunut toiminta johtaa EPM1-sairauteen. Tutkimuksemme hyödyntää hiirimallia, josta kystatiini B-geeni on poistettu (kystatiini B -puutteinen hiiri). Näille hiirille kehittyy myokloniaa ja ataksiaa, ja ne muistuttavat suuresti ihmisen EPM1-sairautta. Aikaisemmat tutkimuksemme ovat osoittaneet, että aivojen immuunisolut, mikrogliasolut, ovat aktivoituneet jo nuorten kystatiin B -puutteisten hiirten aivoissa. Poikkeava mikrogliasolujen aktivaatio ja aivojen tulehdusreaktiot on aiemmin liitetty aivojen patologisiin tiloihin ja hermorappeumasairauksiin. Tässä tutkimustyössä olemmekin tutkineen yksityiskohtaisemmin mikrogliasolujen toimintaa kystatiini B -puutteisten hiirten aivoissa. Tuloksemme viittaavat interferoni-signalointireittien puutteellisen toiminnan voivan olla kystatiini B -puutteisen hiiren ilmiasun taustalla. Havaitsimme myös aktivoitujen mikrogliasolujen toiminnan muuttuneen. Kemokiinien vapauttaminen on tehostunut ja tulehdusmerkkiaineiden kuten tulehdusreaktioita lisäävän iNOS:n ja tulehduksia estävän ARG1:n tuotto kasvanut, mutta solujen fagosyyttinen aktiivisuus laskenut. Lisäksi kystatiini B -puutteisten hiirten aivoista löytyy T-lymfosyyttejä ja granulosyyttejä, mikä viittaa näiden immuunisolujen siirtyneen periferiasta aivoihin. Yleiseen immuunijärjestelmän aktivaatioon kystatiini B -puutteisella hiirellä viittaa myös pernan makrofagisolujen aktivoituminen. Olemme tässä tutkimuksessa osoittaneet, että kystatiini B -hiiren mikrogliasolujen toiminta ja aktivaatio on poikkeavaa. Tuloksemme kytkevät kystatiini B -puutteen immuunijärjestelmän toiminnan muutoksiin nuorilla hiirillä. Tällaiset muutokset liitetään usein aivojen tulehdustiloihin, mikä viittaa siihen, että mikrogliasolujen poikkeava toiminta on ainakin osittain vastuussa EPM1:n patologiasta

Gene expression values identified by microarray- and sequencing-based transcriptome profiling of <i>Cstb</i>^<i>-/-</i> microglia.

<p>(A) Venn diagram illustrating the overlap in the number of genes identified by both methods (blue), only by microarray (green), or only by RNA-seq (red). (B–E) Distribution of microarray and RNA-seq gene expression values in control and <i>Cstb</i>^<i>-/-</i> microglia. The expression values of genes identified also in the other method (blue bars) are higher than the expression values identified only by the microarray or the RNA-seq (grey bars). The number of genes with a specific gene expression value is depicted for (B) control and (C) <i>Cstb</i>^<i>-/-</i> microglia in the microarray and for (D) control and (E) <i>Cstb</i>^<i>-/-</i> microglia in the RNA-seq data. Scatter plot of the mean expression values for each gene of (F) control and (G) <i>Cstb</i>^<i>-/-</i> samples identified by both methods.</p

Upstream regulators enriched in <i>Cstb</i>^<i>-/-</i> microglia identified by microarray and RNA-seq analyses.

<p>The 12 upstream regulators most highly ranked based on their p-value in the microarray (black color) and the RNA-seq approach (grey color) are depicted. The z-scores and the fold changes of all genes potentially controlled by this regulator are illustrated for both methods.</p

DEGs identified by microarray- and sequencing-based expression profiling of <i>Cstb</i>^<i>-/-</i> microglia.

<p>(A) Scatter plot of the FCs of the differentially expressed genes (DEGs) identified by microarray and RNA-seq. DEGs common to both methods are depicted in orange, RNA-seq-specific DEGs in light blue, and microarray-specific DEGs in purple. (B) Venn diagram showing the overlap in the number of DEGs. The colors correspond to the colors in A. (C) Protein-protein interaction network of the 33 genes differentially expressed in microarray and RNA-seq. The size of each node is proportional to the number of its connections to other nodes and the color of each node illustrates the fold change of the gene it represents.</p

Canonical pathways enriched in <i>Cstb</i>^<i>-/-</i> microglia identified by microarray and RNA-seq analyses.

<p>The 14 canonical pathways most highly ranked based on their p-value in the microarray (black color) and the RNA-seq approach (grey color) are depicted. The p-values and the fold changes of the microglia genes linked to each pathway are illustrated for both methods results.</p

JAK-STAT pathway-associated transcripts differentially expressed in <i>Cstb</i>^<i>-/-</i> microglia.

<p>JAK-STAT pathway-associated transcripts differentially expressed in <i>Cstb</i>^<i>-/-</i> microglia.</p