DNMT1 regulates expression of MHC class I in post-mitotic neurons.

Major Histocompability Complex I (MHC-I) molecules present cellularly derived peptides to the adaptive immune system. Generally MHC-I is not expressed on healthy post-mitotic neurons in the central nervous system, but it is known to increase upon immune activation such as viral infections and also during neurodegenerative processes. MHC-I expression is known to be regulated by the DNA methyltransferase DNMT1 in non-neuronal cells. Interestingly DNMT1 expression is high in neurons despite these being non-dividing. This suggests a role for DNMT1 in neurons beyond the classical re-methylation of DNA after cell division. We thus investigated whether DNMT1 regulates MHC-I in post-mitotic neurons. For this we used primary cultures of mouse cerebellar granule neurons (CGNs). Our results showed that knockdown of DNMT1 in CGNs caused upregulation of some, but not all subtypes of MHC-I genes. This effect was synergistically enhanced by subsequent IFNγ treatment. Overall MHC-I protein level was not affected by knockdown of DNMT1 in CGNs. Instead our results show that the relative MHC-I expression levels among the different MHC subtypes is regulated by DNMT1 activity. In conclusion, we show that while the mouse H2-D1/L alleles are suppressed in neurons by DNMT1 activity under normal circumstances, the H2-K1 allele is not. This finding is particularly important in two instances. One: in the context of CNS autoimmunity with epitope presentation by specific MHC-I subtypes where this allele specific regulation might become important; and two: in amyotropic lateral sclerosis (ALS) where H2-K but not H2-D protects motor neurons from ALS astrocyte-induced toxicity in a mouse model of ALS.

Neurobasal media facilitates increased specificity of siRNA-mediated knockdown in primary cerebellar cultures.

BACKGROUND: Efficient and specific knockdown of proteins in post-mitotic cells such as differentiated neurons can be difficult to achieve. Further, special care must be taken to maintain the health of neurons in vitro. We wanted to achieve knockdown in primary cerebellar granule neurons, which can be effectively grown in Neurobasal™ media. NEW METHOD: We tested the efficiency of siRNA from the Accell range from Dharmacon™ when delivered in Neurobasal™ media in contrast to the recommended Accell Delivery media provided by the manufacturer. RESULTS: We observed a more specific knockdown of target in Neurobasal™ media, than in Accell Delivery media when using cerebellar granule neurons. Transfection efficiency and cell viability was comparable between the two media. COMPARISON WITH EXISTING METHODS: Delivery of siRNA in Neurobasal™ media facilitates increased specificity of the knockdown compared to delivery in Accell Delivery media. The off-target effect observed in Accell Delivery media was not a secondary biological response to downregulation of target, but rather a mixture of specific and non-specific off-target effects. CONCLUSIONS: Specific knockdown of target can be achieved in primary cerebellar granule cells using Accell siRNAs in Neurobasal™ media. This method ensures specific knockdown in post-mitotic neurons without the need for biosafety level 2 laboratories, additional reagents, or instruments needed by other transfection.