Lu S, Hu J, Arogundade OA, Goginashvili A, Vazquez-Sanchez S, Diedrich JK, Gu J, Blum J, Oung S, Ye Q, Yu H, Ravits J, Liu C, Yates JR 3rd, Cleveland DW. Heat-shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition. Nat Cell Biol. 2022 Sep;24(9):1378-1393. Epub 2022 Sep 8 PubMed.

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  1. This exciting study by the research group of Dr. Cleveland reports that in cultured cells the small heat shock protein HSPB1 regulates the phase separation behavior of cytoplasmic TDP-43. Interestingly, TDP-43 phase separates independently from stress granules without the requirement to bind RNA. The fact that the formation of these TDP-43 droplets leads to the depletion of nuclear TDP-43 indicates that what is observed in a cell line reflects what happens in patients. Another indication is that the expression level of HSPB1 in postmortem tissue of ALS patients is decreased in those spinal motor neurons with TDP-43 pathology. Remarkably, HSPB1 does not only interact with TDP-43 (and FUS) but also with SOD1 which inspired us several years ago to overexpress HSPB1 in the mutant SOD1 mouse model. Unfortunately without any benefit (Krishnan et al., 2008). Crossbreeding a TDP-43 mouse model with these HSPB1 overexpressing mice seems to be a logical next step. This brings us to the important question whether these insights can be translated into a therapeutic strategy beneficial for ALS patients. Upregulating the heat-shock response was the rationale to test arimoclomol in ALS (Kieran et al., 2004), which unfortunately failed in a Phase 3 trial (NCT03491462). Overall, this interesting study renews the interest into chaperones in general and HSPB1 in particular. In addition, the interaction of TDP-43 with tubulins and the requirement for intact microtubules to get TDP-43 droplet disassembly further increases the excitement.

    References:

    Krishnan J, Vannuvel K, Andries M, Waelkens E, Robberecht W, Van Den Bosch L. Over-expression of Hsp27 does not influence disease in the mutant SOD1(G93A) mouse model of amyotrophic lateral sclerosis. J Neurochem. 2008 Sep;106(5):2170-83. PubMed.

    Kieran D, Kalmar B, Dick JR, Riddoch-Contreras J, Burnstock G, Greensmith L. Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice. Nat Med. 2004 Apr;10(4):402-5. PubMed.

    View all comments by Ludo Van Den Bosch

  2. This is supremely well-designed work from the Cleveland lab. As mentioned in the article, many proteins linked to proteostasis exhibit genetic linkage to ALS. In addition, chaperones and other proteins linked to proteostasis frequently show up in molecular studies of TDP-43 protein interaction networks, as well as interaction networks of other RNA-binding proteins. Thus, the observed role of HSPB1 is not surprising because biomolecular condensates and subsequent fibrillar aggregates are associated with lots of these proteins in chaperones and other proteins regulating proteostasis.

    However, just because something is unsurprising does not mean that it is not important. The Cleveland group has nicely shown that HSPB1 regulates TDP-43 phase separation and aggregate formation. Importantly they show that modulating levels of HSPB1 is capable of regulating phase separation of TDP-43; this finding is important because chaperones able to modulate TDP-43 phase separation become potential targets for disease modification, i.e., therapy.

    The challenge is to find those interacting proteins that exhibit the strongest effects on TDP-43 phase separation. HSPB1 clearly has strong effects, particularly when one examines resolution of TDP-43 biomolecular condensates at 12 hours, where siHSPB1 prevents removal of the phase separated TDP-43 droplets (Fig. 7g). 

    Three other points:

    1) The article shows a nice correlation between cytoplasmic TDP-43 phase separation and loss of nuclear TDP-43. Such nuclear loss presumably lies behind the extensive cryptic splicing observed by Phil Wong, Len Petrucelli, Aaron Gitler, Michael Ward and others.

    2) I note the absence of co-localization between G3BP1 and TDP-43. The Cleveland group has reported this previously and suggest that TDP-43 granules are something different than the classic stress granule. There are many types of RNA granules, which might respond to differing signaling pathways. The absence of G3BP1–TDP-43 co-localization speaks to this heterogeneity.

    3) The work is overwhelmingly done in non-neuronal cells that are easy to use, but the Cleveland group does a nice job of showing that these findings translate to motor neurons, which is always a key thing to look for in such studies.

    View all comments by Benjamin Wolozin

  3. In general, this is a beautiful study of TDP43 variant dynamics in vitro and in cultured cells, focusing on the changes in cytoplasmic TDP43 mobility upon cellular stress. One of the most important findings is that HSPB1 acts as a chaperone for TDP43, regulating its biophysical state as well as its localization in cells. Consistent with this function, HSPB1 is lost from cells that exhibit TDP43 pathology (nuclear exclusion, cytoplasmic deposition) in ALS. Loss of HSPB1 is also associated with a more pervasive interruption of nucleocytoplasmic transport, with several typically nuclear proteins becoming mislocalized, not just TDP43.

    This includes RanGAP, a factor critical for regulating Ran GDP/GTP balance required for transport. Notably, HSPB1 is highly expressed in motor neurons, and mutations in the HSPB1 gene have been associated with ALS and other conditions that affect motor neurons, including subtypes of Charcot-Marie-Tooth disease. These observations support a fundamental connection between HSPB1, TDP43 and motor neuron health.

    Keeping all this in mind, it is unclear how relevant the cellular stress paradigms utilized in this work (MG132, NaAsO2, heat) are for reproducing the ALS disease state. In fact, more recent work suggests that, if anything, heat stress may be the most similar on a molecular basis, even if not an intuitive basis. Likewise, many of the manipulations utilized here could be unrelated to disease. These include transient overexpression of dNLS TDP43, and/or dNLS TDP43 carrying mutations that mimic acetylation. Most experiments were performed in U2OS cells, a transformed cell line that may exhibit unique responses to disease-related stimuli such as TDP43 accumulation.

    On a different and thought-provoking note, prior evidence suggests that disease-associated (CMT) mutations in the HSPB1 gene increase chaperone-like activity. It will be really interesting to determine if the same holds true for ALS-associated mutations, rather than a loss of function as suggested by the authors (a "probable" enrichment for missense or frame-shift variants in ALS, p=0.068). If correct, this would argue against a toxic contribution by stable (i.e. gel or fibril-like) inclusions in ALS.

    View all comments by Sami Barmada

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