Neurodegenerative diseases are often associated with accumulation of misfolded proteins. For instance, amyotrophic lateral sclerosis (ALS) affects 5 out of 100000 people worldwide. The most notable ALS cases are Stephen Hawkin and Lou Gehrig, hence the disease is known as Lou Gehrig disease. In 20% of the ALS cases, it is due to a mutation in SOD1, while other cases without Sod1 mutation are associated with TDP-43 protein accumulation in the cytoplasm of spinal cord neurons. This study has shed light on treating ALS by accumulating RNA introns or simply delivering oligonucleotides.
Before you read on: this is a decent paper published in Nature; easy to understand; straight-forward protocols
In this study, the researchers performed a genome wide screen to see which genes are responsible for suppressing TDP-43 toxicity. They narrowed down on dbr1, which suppresses the TDP-43 and TDP-43 mutant toxicity. This suppression is, however, not due to a lowered expression of TDP-43. When human neuronal cell line is transfected with siRNA against dbr1, the toxicity caused by TDP-43 is relieved. The author also proved that Dbr1 knockdown reduces TDP-43 toxicity in primary rat neurons. Using DBR1 mutants that do not have lariat debranching enzymatic activity in yeast spotting assay (look at the figure below if you don’t know what DBR1 does: responsible for debranching the lariat, and subsequently degrading the introns to avoid accumulation of the junk DNA) , TDP-43 toxicity is reduced. As Dbr1 is responsible for debranching lariats following splicing, the knockdown of Dbr1 should increase the amount of introns in the cell. This group incorporated MS2 RNA binding protein into the intron and GFP-tagged MS2-CP protein to visualize the localization of intron. In Dbr1 null cell, intron is colocalized with TDP-43.
The accumulation of introns alleviated the TDP-43 toxicity, which suggest that the accumulation may be a way to treat ALS cases. There are currently no direct therapies against TDP43. So how can we go around this problem? Delivering oligonucleotides into ALS models may be a possible therapy in the near future. In fact, researchers had already used antisense oligonucleotide against SOD1 to treat animal models of ALS which showed slowed disease progression.
Take home message: Without Dbr1, a lariat debranching enzyme, introns accumulate in the cytoplasm which is correlated with lowered TDP-43 toxicity and TDP-43 colocalization with DBR1.
Nat Genet. 2012 Oct 28. doi: 10.1038/ng.2434. [Epub ahead of print]
Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models.
Armakola M, Higgins MJ, Figley MD, Barmada SJ, Scarborough EA, Diaz Z, Fang X, Shorter J, Krogan NJ, Finkbeiner S, Farese RV Jr, Gitler AD.
Ohara group previously published a paper with a mutant virus carrying a FLAG-tag L*. However, the location of FLAG-tag disrupts the zinc binding motif of L. The zinc binding motif is not required for suppressing anti-IFN activity, but it is shown to bind to zinc and this function is conserved in all members of cardiovirus.
Related article: new type of drug for treating HIV
Finding: the group made a series of FLAG-tag L* mutants, that do not disrupt the zinc binding motif of the L protein and mutate the start codon downstream of the start codon of L*. They found that these mutants do not change the clinical outcome at 180 days post infection compared to the wildtype virus. Though, the demyelinating lesion was not seen at 21 dpi and 45dpi in mice infected with mutant virus. In vitro studies showed similar growth rate in BHK21 cells but not in L929 cells.
Significance: If the FLAG-tag L* is functional, we can potentially use this mutant virus to study the function and the localization of L* protein. If we need to make GFP tag L* or GFP tag L to visualize their localizations within the cell under fluorescent microscopy, it is possible to incorporate the tag using the same strategy/location.
Expresison of L* protein of Theiler’s murine encephalomyelitis virus in the chronic phase of infection. Asakura K, Harunobu M, Toshiki H, Ohara, Y. 2007. PMID: 17622631
Skowronski group discovered the reason why HIV-1 can’t infect macrophage. It is due to the fact that HIV-1 lacks Vpx accessory protein that targets SAMHD1 protein to degradation. SAMHD1 has a role in regulating the host’s immune response in sensing the endogenous nucleic acid.
The first figure composed of a key experiment with coninfecting the macrophage with pseudotyped viruses. One of the viruses is vesicular stomatitis virus that carries HIV-1-IRES driven GFP. The other virus is a viral like particle derived from SIV. This viral like particle only contains the accessory protein Vpx from HIV-2 (which HIV-2 can infect macrophage). Under co-infection, the flow cytometry showed that there is an increase in cells expressing GFP, which means the co-infection, ie. Vpx protein, increases HIV-GFP expression.
Because Vpx interacts with a E3 ubiquitin ligase to target substrate to proteasomal degradation. They then ask what is targeted by this complex in figure 2. They pulled down the substrates and sent for mass spec analysis. They found SAMHD1 as one of the candidate. They later confirmed that SAMHD1 interacts with all the components.
In figure 3, they found that there is an inverse dose-dependent relationship between Vpx expression and SAMHD1 expression. In other words, more Vpx is expressed, the less SAMHD1. When MG132 proteasomes inhibitor is used, SAMHD1 degradation is inhibited, indicating that SAMHD1 degradation is carried out by proteasomes.
In figure 4, they knockdowned SAMHD1 and increased Vpx expression. They realized that combination increases the viral load of HIV-1, further confirming that Vpx can rescue the inhibition of HIV-1 replication in macrophage.
This paper is a good read, due to (1) thorough experimental designs to confirm the conclusion, (2) found SAMHD1 as a restriction factor to inhibit HIV-1 from replicating in macrophage.
2011 Jun 29;474(7353):658-61. doi: 10.1038/nature10195.
Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.