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. 2011 Aug 10;29(9):824-8.
doi: 10.1038/nbt.1957.

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

Amanda M Haidet-Phillips  1 , Mark E Hester, Carlos J Miranda, Kathrin Meyer, Lyndsey Braun, Ashley Frakes, SungWon Song, Shibi Likhite, Matthew J Murtha, Kevin D Foust, Meghan Rao, Amy Eagle, Anja Kammesheidt, Ashley Christensen, Jerry R Mendell, Arthur H M Burghes, Brian K Kaspar
Affiliations

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

Amanda M Haidet-Phillips et al. Nat Biotechnol. .

Abstract

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.

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Figures

Fig. 1
Fig. 1
NPCs can be differentiated into highly enriched astrocyte cultures which show a similar gene profile to spinal cord primary astrocytes. (A) Marker analysis of human NPC-derived astrocytes showing specific immunoreactivity against human nuclear antigen (hNA); high expression of the astrocyte markers CD44, Vimentin, and GFAP; absence of the glial progenitor marker NG2; and absence of the microglia marker CD11b. Scale bars, 50 μm. (B) Comparison of CD11b expression levels by quantitative RT-PCR in NPC-derived astrocyte cultures from control and ALS patients. Mesenchymal stem cells and human microglia were used as negative and positive controls, respectively. Error bars represent SEM. (C) Gene expression profile analysis of NPC-derived astrocytes. Comparisons were made to a human sample of mesenchymal stem cells, undifferentiated NPCs, and primary human astrocytes derived from spinal cord.
Fig. 2
Fig. 2
Astrocytes derived from sALS and fALS patients cause MN death in co-culture. (A) Representative microscopic field showing the morphology of Hb9-GFP+ MNs in co-culture with human astrocytes at 24 and 120 hours. Scale bars, 100 μm. (B) Soma size and neurite length of Hb9-GFP+ MNs at 48 and 96 hours when co-cultured with astrocytes from fALS, sALS and non-ALS controls. (C) Counts of Hb9-GFP+ MNs per well after 120 hours of co-culture with astrocytes from fALS, sALS and non-ALS controls. *p<0.05, **p<0.01 and ***p<0.001 compared to 63358 (non-ALS control). Error bars represent SEM. (D) Representative microscopic field showing the morphology of Hb9-GFP+ MNs treated with astrocyte-conditioned media for 24 or 120 hrs. Scale bars, 100 μm. (E) Counts of Hb9-GFP+ MNs per well after 120 hours of treatment with astrocyte conditioned media. *p<0.05, **p<0.01 and ***p<0.001 compared to 63358 (non-ALS control). Error bars represent SEM.
Fig. 3
Fig. 3
Inflammatory gene activation in fALS and sALS astrocytes. (A) Hierarchical clustering analysis of inflammatory cytokine and receptor genes differentially expressed in ALS astrocytes compared to non-ALS control line (63358). (B) Cluster of inflammatory genes most upregulated in ALS astrocytes compared to non-ALS control. Shades of red correspond to the magnitude of expression increase, the intensity of green corresponds to the magnitude of the reduction in gene transcript. Display range was set at maximum of +/− 15 fold.
Fig. 4
Fig. 4
Suppression of SOD1 in both fALS and sALS astrocytes is MN protective. (A) Representative microscopic fields showing the morphology of MNs stained with choline acetyl transferase (ChAT) after 120 hours of co-culture with human astrocytes transduced with either missense or SOD1 shRNA lentivirus. Scale bars, 100 μm. (B) Counts of MNs per well after 120 hours in co-culture with astrocytes expressing the missense shRNA (white bars) or the SOD1 shRNA (black bars). (C) Quantification of SOD1 levels in astrocyte cell lysate measured by ELISA. *p<0.05 and **p<0.01 compared to 63358 (non-ALS control). Error bars represent SEMs.
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