Page 63 - Biennial Report 2018-20 Jun 2021

INTERFERON MEDIATED GENE EXPRESSION IN NEURODEGENERATIVE DISEASES

Polyglutamine (polyQ) expansion in proteins causes neurodegenerative diseases. Such a
mutation in TATA binding protein leads to cerebellar neuronal death and ataxia (SCA17). It was
found that polyQ-TBP causes dysregulation of genes dynamically. At various time points post
transfection of expanded polyQ-TBP construct, signal transducer and activator of transcription1
(Stat1) and its four dependent genes, ubiquitin specific proteases18 (Usp18), guanylate binding
protein3 (Gbp3), interferon stimulated gene15 (Isg15) and C-XC motif chemokine10 (Cxcl10)
were significantly upregulated in Neuro2A cells. In the microarray analysis done previously,
upregulated genes showed significant enrichment for interferon and immunity related
processes, while down-regulated genes remained unclassified with no significant enrichment for
any biological process. Notably, IFN-γ induced protease, cytokines and ubiquitin-like proteins
were increased in TBP-59Q. The ectopic expression of STAT1 resulted in a 21% increase in
cytochrome-c release, a reporter of apoptotic cell death. the downregulation of miR-29a/b and
upregulation of miR-322 in cells expressing polyglutamine-TBP was previously reported. Similar
effects were found following IFN-γ treatment. Thus, neuronal cells expressing toxic
polyglutamine-TBP release interferons into the surrounding milieu, triggering the induction of
STAT1 and subsequent dysregulation of miR29a/b and miR-322.
The previous study from Beena Pillai’s laboratory showing interferon pathway upregulation was
done using SCA17 transfection model, tetracycline-inducible stable SCA17 cell lines were
generated. These were generated using lentiviral integration of the 16Q-TBP and 59Q-TBP genes
fused with eGFP in Neuro2A cells. This resulted in 16Q-TBP and 59Q-TBP cell lines that could be
induced to express the polyQ protein. Increased apoptosis in 59Q-TBP expressing cells was
observed as compared to 16Q-TBP expressing cells. In this new improved model, the mRNA
expression of the interferon pathway genes, also known as interferon stimulated genes (ISGs),
in 59Q-TBP and 16Q-TBP stable lines at multiple timepoints was rechecked. This showed a clear
upregulation of the interferon pathway.

It was hypothesized that this phenomenon of interferon activation might be present in other
polyQ diseases as well. Meta-analysis of publicly available datasets in Gene Expression Omnibus
(GEO) was performed and the expression of interferon pathway genes was checked. Indeed, it
was observed that Stat1 was upregulated in almost all datasets and genes under the control of
Stat1 were also induced in some datasets. To confirm the results of our meta-analysis,
Huntington’s disease (HD) mice model was used. Using the cortex and cerebellum from HD mice
and wild type mice, no significant upregulation of interferon gamma gene at the mRNA level was
observed. However, STAT1 protein levels were upregulated in HD mice samples albeit not
statistically significant, perhaps due to paucity of samples. Further, the mRNA levels of the
interferon pathway genes were checked and in two of the five genes checked, a significant
upregulation was observed. However, the heterogeneity of the brain tissue may have masked
changes in mRNA level in a small fraction of cells. In summary, preliminary results indicate that
the interferon pathway was upregulated in HD mice brain samples, although further validation
is needed. The 16CAG region and the 59CAG region of the 16Q-TBP and 59Q-TBP gene were
cloned in a separate vector (pcDNA3.1) to express these CAG repeat regions at the RNA level.
The mRNA levels of three known dsRNA binding RNA sensors in 16Q-TBP and 59Q-TBP expressing
cells were checked. Two RNA sensors, Rig1 and Mda5, were found to be upregulated at the
mRNA level. HD mice brain samples were then checked for Rig1 and Mda5 mRNA levels, which

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