Neuronaalisten seroidilipofuskinoosien CLN8 ja CLN10 tautimekanismien tutkimus hiiri- ja Drosophilamalleissa

The neuronal ceroid lipofuscinoses (NCLs) are a group of pediatric neurodegenerative syndromes. They are characterized by epilepsy, mental and motor regression, loss of vision and early death. Pathological findings include accumulation of lysosomal storage material together with neuron loss and glial activation in affected brains. Mutations in at least ten genes are reported to cause NCL disease. Apart from human disease, NCL phenotypes are widely present in the animal kingdom. However, the events leading to NCL disease onset and progression remain elusive and without a possibility to intervene. In this study these events were assessed in two forms of NCLs, the CLN8 and CLN10 diseases. Late-infantile onset CLN8 disease and the more protracted progressive epilepsy with mental retardation are caused by mutations in the CLN8 gene. CLN8 is a resident of the endoplasmic reticulum membrane and linked to a lipid metabolism-related protein family. Mutations in the cathepsin D (CTSD) gene cause the most severe form of NCL, CLN10, which typically leads to death shortly after birth. The lysosomal aspartic protease CTSD is involved in cellular protein degradation and apoptosis. Two animal models were used in this study, the Cln8mnd mouse carrying a spontaneous mutation in the mouse Cln8 gene, and the previously generated cathepsin D deficient Drosophila, cathD1. Both model the respective human NCL diseases with intracellular storage and neuronal death. The Drosophila cathD1 was characterized with degenerative changes in the retina. This phenotype could be utilized to study the genetic pathways of cathD in a hypothesis-based modifier screen using Drosophila genetics and histological techniques. Seven candidate modifiers involved in lipid metabolism regulation, endocytosis and oxidative stress were identified as enhancers of the retinal degeneration of cathD1. Similar processes have been described affected in other Drosophila models for NCLs, suggesting that Drosophila NCL proteins may act in overlapping genetic pathways. While the importance of these pathways needs to be assessed in the human disease, their overlap suggests a possibility for a common mechanism of neurodegeneration. Similarly to previously characterized NCL models, the CLN8 disease model Cln8mnd showed neuron loss and glial activation in sensory thalamocortical pathways. By using stereological methods and histology, neuron loss in the visual thalamocortical pathway was shown to appear relatively late compared to the previously described retinal degeneration in Cln8mnd. This suggests that the visual areas of the brain are spared even with diminished or absent input from the retina. The somatosensory thalamocortical pathway was affected first. While the cerebral cortical sensory areas were relatively spared, neuron loss progressed rapidly in the thalamic component of these pathways. Thalamic sensory nuclei have shown particular vulnerability in the majority of NCL models, indicating a common pathological pathway proceeding from thalamus to the cortex. In the future, knowledge of the differentially affected components of distinct pathways may be utilized in therapies, which should be targeted to the primary sites of pathology. In CLN8 disease these appear to be the retina and somatotosensory thalamus. Glial cells were observed to contribute to the Cln8mnd neuropathology. Especially microglial activation preceded neuron loss in Cln8mnd brain. Decreased axonal myelination, studied by stereology, light and electron microscopy, was observed even before microglial activation, yet myelination reached normal levels by the time of major brain pathology. By protein and gene expression profiling we were able to show a delayed maturation of oligodendrocytes in the Cln8mnd mouse brain and in vitro. These results suggest that perturbations in the glia-neuron-glia signaling occur well in advance of the neurodegeneration in Cln8mnd. With increasing information on the mechanisms of these interactions it may be possible to find a specific target for therapies for CLN8 or NCLs in general. The myelination defect in Cln8mnd was observed through large-scale brain lipid analysis, where decreased amounts of myelin-specific galactolipids were found in Cln8mnd cortex, especially in the early stages of Cln8mnd disease. Subsequent analyses showed a persistent defect in the galactolipid synthesis by the UDP-galactose:ceramide galactosyltransferase enzyme. The connection of CLN8 to lipid synthesis regulation is in agreement with the sequence-based hypothesis of a role for CLN8 in lipid metabolism regulation, while further studies are required to indicate a specific function. In conclusion, this study resulted in increased knowledge on the CLN8 and CLN10 disease pathogenesis and on the cellular functions of the affected proteins. Results from both cathD1 Drosophila and Cln8mnd mouse suggest both similarities and differences to other NCL models. In the future, increased knowledge on the molecular changes may take us towards targeting these diseases with therapies.Neuronaaliset seroidilipofuskinoosit (NCL-taudit) muodostavat yleisimmän syyn lapsuusiän hermorappeumaan. Vaikka tautien taustalla olevat geenivirheet pääosin tunnetaankin, molekyylitason tapahtumat, jotka johtavat taudin syntyyn, ovat edelleen pitkälti selvittämättä. Taudin etenemistä ei myöskään kyetä pysäyttämään tai tautia parantamaan. Tässä väitöskirjassa tutkittiin NCL-tauteihin lukeutuvien CLN8- ja CLN10-tautien taustalla olevien proteiinien toimintaa. Synnynnäisen NCL-taudin, CLN10:n taustalla ovat katepsiini D (CTSD) geenin virheet. Työssä käytimme CLN10-tautia mallintavaa banaanikärpästä ja saimme lisävahvistusta sille, että häiriöt solujen rasva-aineenvaihdunnassa, endosytoosissa ja solujen suojautumisessa happiradikaaleilta vaikuttavat NCL-taudeissa. Lisätutkimukset solu- ja eläinmalleissa voivat tulevaisuudessa tuoda tietoa siitä, voidaanko näitä reittejä käyttää hoitomuotojen kohdistamiseen. CLN8-geenin mutaatiot aiheuttavat paitsi lapsuusiän NCL-taudin, myös nuoruusiällä puhkeavan, suomalaisen tautiperimän tauteihin kuuluvan Pohjoisen epilepsian. CLN8-tutkimuksissa käytimme hiirikantaa, jossa on luonnollisesti syntynyt Cln8-geenin mutaatio. Tutkimme hermosolukuoleman laajuutta ja kohdentumista eri aivoalueille. Näyttääkin siltä, että aiemmin kuvattujen NCL-tautimallien lisäksi myös Cln8-hiiren aivopatologia keskittyy isoaivokuoren ja talamuksen välisiin yhteyksiin ja alkaa tunto- ja kipuaistimuksia välittävältä somatosensoriselta aivoradalta. Lisäksi kuvasimme hermotukisolujen muutoksia taudin edetessä. Havaitsimme immuunipuolustuksen aktivaatioon viittaavia muutoksia mikrogliassa jo ennen varsinaista hermosolukuolemaa. Näin ollen tulehdusreaktio näyttää edeltävän hermosolujen kuolemaa. Jatkotutkimukset ovat tarpeen, kun selvitetään, onko tulehdusreaktion aktivoituminen haitallista vai hyödyllistä. Jo ennen mikroglian aktivaatiota muutoksia nähtiin oligodendrosyyteissä, jotka muodostavat hermosolujen viejähaarakkeita ympäröivän myeliinitupen. Myeliiniä, joka on välttämätöntä nopealle signaalinvälitykselle, muodostui Cln8-hiirellä normaalia hitaammin. Tämän havaittiin liittyvän myeliiniin rikastuneiden rasva-aineiden vähentyneeseen valmistukseen. Tulokset viittaavat siihen, että CLN8:n toiminta liittyisi rasva-aineiden valmistukseen, mutta tarkan biologisen mekanismin selvittämiseksi vaaditaan lisätöitä. Yhteenvetona, väitöskirjatyön tuloksena saatiin lisää tietoa NCL-tautien aiheuttamasta aivopatologiasta ja siihen vaikuttavista tekijöistä. Lisätutkimukset tuovat valaistusta sille, miten rasva-aineenvaihdunnan häiriöt, tulehdusreaktio ja myeliinin kehityshäiriö vaikuttavat NCL-taudin puhkeamiseen

The neuronal ceroid lipofuscinosis Cln8 gene expression is developmentally regulated in mouse brain and up-regulated in the hippocampal kindling model of epilepsy

BACKGROUND: The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by accumulation of autofluorescent material in many tissues, especially in neurons. Mutations in the CLN8 gene, encoding an endoplasmic reticulum (ER) transmembrane protein of unknown function, underlie NCL phenotypes in humans and mice. The human phenotype is characterized by epilepsy, progressive psychomotor deterioration and visual loss, while motor neuron degeneration (mnd) mice with a Cln8 mutation show progressive motor neuron dysfunction and retinal degeneration. RESULTS: We investigated spatial and temporal expression of Cln8 messenger ribonucleic acid (mRNA) using in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and northern blotting. Cln8 is ubiquitously expressed at low levels in embryonic and adult tissues. In prenatal embryos Cln8 is most prominently expressed in the developing gastrointestinal tract, dorsal root ganglia (DRG) and brain. In postnatal brain the highest expression is in the cortex and hippocampus. Expression of Cln8 mRNA in the central nervous system (CNS) was also analyzed in the hippocampal electrical kindling model of epilepsy, in which Cln8 expression was rapidly up-regulated in hippocampal pyramidal and granular neurons. CONCLUSION: Expression of Cln8 in the developing and mature brain suggests roles for Cln8 in maturation, differentiation and supporting the survival of different neuronal populations. The relevance of Cln8 up-regulation in hippocampal neurons of kindled mice should be further explored

Cystatin B: mutation detection, alternative splicing and expression in progressive myclonus epilepsy of Unverricht-Lundborg type (EPM1) patients

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Fibroblast Growth Factor 21 Drives Dynamics of Local and Systemic Stress Responses in Mitochondrial Myopathy with mtDNA Deletions

AbstractMitochondrial dysfunction elicits stress responses that safeguard cellular homeostasis against metabolic insults. Mitochondrial integrated stress response (ISRmt) is a major response to mitochondrial (mt)DNA expression stress (mtDNA maintenance, translation defects), but the knowledge of dynamics or interdependence of components is lacking. We report that in mitochondrial myopathy, ISRmt progresses in temporal stages and development from early to chronic and is regulated by autocrine and endocrine effects of FGF21, a metabolic hormone with pleiotropic effects. Initial disease signs induce transcriptional ISRmt (ATF5, mitochondrial one-carbon cycle, FGF21, and GDF15). The local progression to 2nd metabolic ISRmt stage (ATF3, ATF4, glucose uptake, serine biosynthesis, and transsulfuration) is FGF21 dependent. Mitochondrial unfolded protein response marks the 3rd ISRmt stage of failing tissue. Systemically, FGF21 drives weight loss and glucose preference, and modifies metabolism and respiratory chain deficiency in a specific hippocampal brain region. Our evidence indicates that FGF21 is a local and systemic messenger of mtDNA stress in mice and humans with mitochondrial disease.</div

Outcomes of progranulin gene therapy in the retina are dependent on time and route of delivery.

Neuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin, which regulates neuronal lysosomal function. Absence of progranulin causes cerebellar atrophy, seizures, dementia, and vision loss. As progranulin gene therapies targeting the brain are developed, it is advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development: the retina is easily visible through direct imaging, can be assessed through quantitative methods in&nbsp;vivo, and requires smaller amounts of adeno-associated virus (AAV). In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. Mice heterologously expressing progranulin showed a reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV92YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with AAV2.7m8-scCAG-PGRN exhibit no improvement, and mice injected at 12&nbsp;months of age have thinner retinas than do their controls. Thus, delivery of progranulin proves to be time sensitive and dependent on route of administration, requiring early delivery for optimal therapeutic benefit

Cln5-deficiency in mice leads to microglial activation, defective myelination and changes in lipid metabolism

CLN5 disease, late infantile variant phenotype neuronal ceroid lipofuscinosis, is a severe neurodegenerative disease caused by mutations in the CLN5 gene, which encodes a lysosomal protein of unknown function. Cln5-deficiency in mice leads to loss of thalamocortical neurons, and glial activation, but the underlying mechanisms are poorly understood. We have now studied the gene expression of Cln5 in the mouse brain and show that it increases gradually with age and differs between neurons and glia, with the highest expression in microglia. In Cln5(-/-) mice, we documented early and significant microglial activation that was already evident at 3 months of age. Loss of Cln5 also leads to defective myelination in vitro and in the developing mouse brain. This was accompanied by early alterations in serum lipid profiles, dysfunctional cellular metabolism and lipid transport in Cln5(-/-) mice. Taken together, these data provide significant new information about events associated with Cln5-deficiency, revealing altered myelination and disturbances in lipid metabolism, together with an early neuroimmune response. (C) 2011 Elsevier Inc. All rights reserved