Characterization of astrocytes derived from fibroblasts of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular degenerative disease, targeting the motor neurons of the brain stem, motor cortex and spinal cord. Various lines of evidence have shown that glial cells contribute to disease development and progression. We derived astrocytes from fibroblasts of patients with familial ALS associated with SOD1 mutation and compared them to astrocytes derived from fibroblasts of age- and sex-matched controls. In order to assess astrocytic maturity, we compared the mRNA expression of astrocytic markers in astrocytes derived from three different protocols to that of human fetal astrocytes (HFA). Protocol 1 incorporated 10 weeks of astrogenesis. Protocol 2 incorporated 3 weeks of neurogenesis and 10 weeks of astrogenesis, and protocol 3 incorporated 2 weeks of astrogenesis. Our data show that astrocytes from all three protocols are similar in mRNA expression of astrocytic markers to HFA. However, morphologically, astrocytes from protocol 1 and 2 had astrocyte-like projections and therefore looked more like astrocytes compared to astrocytes from protocol 3. Next, we derived astrocytes from ALS patients, using protocol 2 and compared them to their age- and sex-matched controls. We found that the mRNA levels of EAAT2 and NESTIN were lower and the mRNA levels of glutamine synthetase, connexin43 and TGFB1 were higher in ALS astrocytes compared to their controls. Since the phenotypes observed in this study corroborate with previous literature exploring the role of astrocytes in ALS, these astrocytes may offer a system in which ALS can be studied in human cells.La sclérose latérale amyotrophique (SLA) est une maladie neuromusculaire et neurodégénérative qui a pour cible les neurones moteurs du tronc cérébral, du cortex moteur et de la moelle épinière. Diverses sources de données ont démontré que les cellules gliales contribuent au développement et à la progression de la maladie. Notre but était de dériver des astrocytes matures à partir de fibroblastes de patients atteints de la SLA et de les comparer à des astrocytes dérivés de fibroblastes provenant de témoins appariés selon l'âge et le sexe. Afin de nous assurer de la maturité astrocytaire, nous avons comparé les niveaux d'expression de l'ARNm de marqueurs astrocytaires dans des astrocytes obtenus par 3 protocoles différents aux niveaux de ces marqueurs dans des astrocytes fœtaux humains (AFH). Le protocole 1 incorporait 10 semaines d'astrogenèse. Le protocole 2 incorporait 3 semaines de neurogenèse suivies de 10 semaines d'astrogenèse, et le protocole 3 incorporait 2 semaines d'astrogenèse. Nos données montrent que les astrocytes issus de chacun des trois protocoles ont des niveaux d'expression d'ARNm de marqueurs d'astrocytes similaires aux niveaux d'expression dans les AFH. Cependant, morphologiquement, les astrocytes des protocoles 1 et 2 ressemblaient plus à des astrocytes. Par la suite, nous avons dérivé des astrocytes issus de patients atteints de la SLA en utilisant le protocole 2. Nous les avons comparés à leurs témoins appariés selon l'âge et le sexe. Nous avons découvert que les niveaux d'ARNm de EEAT2 et de NESTIN étaient plus bas et que les niveaux d'ARNm de glutamine synthéase, connexine 43 et TGFB1 étaient plus élevés dans les astrocytes de la SLA comparés aux témoins. Etant donné que les phénotypes observés dans cette études sont en accord avec la littérature, ces astrocytes pourraient constituer un système dans lequel al SLA serait étudiée dans des cellules humaines

Crosstalk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-β aggregates

Background Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aβ) and alpha-synuclein (αSYN), respectively. In order to develop effective therapies, it is crucial to understand how the Aβ/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear. Methods The aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aβ pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aβ and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized “close-culture” chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a &lt;1 mm space. Results Our data show that intracellular deposits of αSYN and Aβ are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the “close-culture” chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte. Conclusions Taken together, our data demonstrate the importance of astrocyte and microglia interactions in Aβ/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.Title in thesis list of papers: Cross-talk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-β aggregates</p

Development of a Chimeric Model to Study and Manipulate Human Microglia In&nbsp;Vivo.

iPSC-derived microglia offer a powerful tool to study microglial homeostasis and disease-associated inflammatory responses. Yet, microglia are highly sensitive to their environment, exhibiting transcriptomic deficiencies when kept in isolation from the brain. Furthermore, species-specific genetic variations demonstrate that rodent microglia fail to fully recapitulate the human condition. To address this, we developed an approach to study human microglia within a surrogate brain environment. Transplantation of iPSC-derived hematopoietic-progenitors into the postnatal brain of humanized, immune-deficient mice results in context-dependent differentiation into microglia and other CNS macrophages, acquisition of an ex&nbsp;vivo human microglial gene signature, and responsiveness to both acute and chronic insults. Most notably, transplanted microglia exhibit robust transcriptional responses to Aβ-plaques that&nbsp;only partially overlap with that of murine microglia, revealing new, human-specific Aβ-responsive genes. We therefore have demonstrated that this chimeric model provides a powerful new system to examine the in&nbsp;vivo function of patient-derived and genetically modified microglia