Mao X, Ou MT, Karuppagounder SS, Kam TI, Yin X, Xiong Y, Ge P, Umanah GE, Brahmachari S, Shin JH, Kang HC, Zhang J, Xu J, Chen R, Park H, Andrabi SA, Kang SU, Gonçalves RA, Liang Y, Zhang S, Qi C, Lam S, Keiler JA, Tyson J, Kim D, Panicker N, Yun SP, Workman CJ, Vignali DA, Dawson VL, Ko HS, Dawson TM. Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3. Science. 2016 Sep 30;353(6307) PubMed.

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  1. These data look quite interesting and the binding rather compelling. But I would caution that LAG3 is an immune checkpoint molecule whose expression in mice is thought to be largely restricted to T-cells and, in the brain, largely microglial cells. In humans, LAG3 expression data from RNAseq is less clear on individual cell types, but our own AMP-AD data suggest that again LAG3 in the AD and control brain is present at very low levels (<1 CPM). So, I think the in vivo data may be reflective of a more complex mechanism than what is proposed here. Indeed, given the relative binding between LAG3 and APLP1 (~fivefold difference in affinity for α-synuclein) and the fact that APLP1 is present at ~500-fold higher levels in the brain than LAG3, one would think further investigations into APLP1 are warranted. It is almost certain that both of these type 1 membrane proteins are shed into the media, and would act like decoy receptors. Again, this would seem to complicate the straightforward interpretation of this provocative data.

    View all comments by Todd E. Golde

  2. The study by Mao and collaborators is extremely interesting. It addresses the issue of whether specific mechanisms govern neuronal uptake of α-synuclein fibrils from the extracellular space. The demonstrations that lymphocyte-activation gene 3 (LAG3) protein is a surface protein that binds α-synuclein fibrils, specifically in neurons, and that it is involved in the endocytosis of the fibrils are very exciting. The authors then used a variety of approaches to show that prion-like spread of α-synuclein aggregates is mitigated when LAG3 is depleted or blocked, and show this also applies when tested in vivo in animals. The study has identified a new potential therapeutic target for Parkinson's disease and related synucleinopathies, and the future will tell if it is possible to develop strategies to slow disease progression in patients by interfering with LAG3.

    View all comments by Patrik Brundin

  3. Todd Golde suggests that LAG3 expression is largely restricted to T-cells and, in the brain, microglia. It is true that LAG3 is expressed in both T-cells and the brain. Indeed, one of the first papers characterizing LAG3 showed that it is enriched not only in the thymus and spleen, but present at high levels in the brain as well (Workman et al., 2002). However, to suggest that it is largely in microglia is not supported by empirical evidence. For instance, the Allen Brain Atlas shows that LAG3 mRNA is localized primarily to neurons, including in the substantia nigra pars compacta. In addition, LAG3 has high mRNA levels in the brain consistent with the Workman et al., paper (see Allen Brain Atlas). Moreover, the expression of mouse LAG3 is high in the hippocampus and cortex, with raw expression levels of 9.90 and 6.23, respectively. Unfortunately, the immunological reagents that are available for LAG3 are not of sufficient quality for immunolocalization in neuronal tissue, in that there is a high degree of staining in LAG3 knockout brains and cell cultures. As such, in lieu of immunohistochemistry, we showed, using western blots, that LAG3 is primarily expressed in neurons with undetectable levels in astrocytes and microglia (see Figure S5B of the paper). However, we cannot exclude the possibility that there may be very low or undetectable levels of LAG3 in astrocytes and microglia, or that LAG3 levels increase with activation of astrocytes or microglia. Future studies will be required to determine if LAG3 is expressed at detectable levels in activated astrocytes or microglia. For now, the best evidence indicates that LAG3 is predominantly expressed in neurons.

    Golde also suggests that APLP1 levels in the brain are 500-fold greater than LAG3. It is very likely that APLP1 is expressed at higher levels than LAG3, but the fold expression over LAG3 is probably orders of magnitude less. According to Allen Brain Atlas, the raw expression levels of APLP1 in the hippocampus and cortex are 37.34 and 32.85 respectively, which are only modestly higher than LAG3. Although one certainly cannot directly compare mRNA levels of APLP1 and LAG3 to make definitive conclusions, the Allen Brain Atlas mRNA data, and our western blot findings in both mouse and human neurons, indicate that APLP1 is likely to be modestly more abundant than LAG3, but certainly not over 500-fold.

    It is true, as Golde suggests, that things may be more complicated than they appear since both the extracellular domain of APLP1 and LAG3 are shed into the media. Moreover, as we report and suggest, LAG3 may not be the only way in which pathologic α-synuclein gets into cells. These and other questions await further experimentation.

    We agree with Patrik Brundin that our study identified a new potential therapeutic target and we are looking forward to the day when we test whether interfering with LAG3 is a disease-modifying therapy for Parkinson’s disease.

    References:

    Workman CJ, Rice DS, Dugger KJ, Kurschner C, Vignali DA. Phenotypic analysis of the murine CD4-related glycoprotein, CD223 (LAG-3). Eur J Immunol. 2002 Aug;32(8):2255-63. PubMed.

    View all comments by Ted Dawson

  4. Our consortium RNAseq data is available at synapse.org, along with other groups’ data.

    This data and RNAseq data from Ben Barres and colleagues at Stanford is consistent and pretty unequivocal. In humans, Lag3 RNA levels are ~500- to 1000-fold lower in the brain than APLP1. In the single study by Barres and colleagues, LAG3 RNA is almost undetectable. In mice it’s about 100-fold lower. In mice Lag3 is clearly a fairly selective microglial transcript (per the Barres study) and our data is consistent with that study. LAG3 is elevated in old APP mice (~threefold) and it falls into the microglial co-expression network. It has also previously been implicated as part of the microglial sensome.

    I think one has to be cautious in using ISH data from the Allen Brain Atlas, especially when there are data that simply don't jibe. APLP1, like APP, is very abundant in the brain. LAG3 is not. So the Allen data is almost certainly flawed.

    View all comments by Todd E. Golde

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