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DISC1 Aggregates Stall Intracellular Transport

9 February 2012. Proteins encoded by disrupted-in-schizophrenia 1 (DISC1) can clump together and slow the transport systems within a cell, according to a study published online on January 30 in Human Molecular Genetics. Led by Josef Kittler of University College London, U.K., the study also finds that aggregations of DISC1 protein can attract other, free-floating DISC1 proteins, presumably interfering with their function. The findings highlight the idea that problems with how DISC1 moves around in a cell may contribute to chronic mental illness.

Discovered in a Scottish family beset by schizophrenia and other mental illnesses over a decade ago, DISC1 has been linked to many biological processes, including neural differentiation and migration during brain development, and synapse formation and function (see SRF related news story). While risk-associated alleles for DISC1 could alter any one of these functions to increase susceptibility to mental illness, yet another idea has emerged based on the ability of DISC1 proteins to bind to each other: “DISCopathy,” in which the aggregation of DISC1 is pathogenic, similar to the more famous protein pathologies involved in neurodegenerative diseases like Alzheimer’s (Korth, 2009).

Evidence for this stems largely from postmortem work, which has found DISC1 to be enriched in the insoluble protein fraction—which contains aggregated, non-functional protein—recovered from brains of individuals with schizophrenia, depression, and bipolar disorder compared to controls (Leliveld et al., 2008). Though corresponding DISC1 aggregates were not detected by microscope, the study found that aggregation could inhibit binding between DISC1 and one of its binding partners, NDEL1. Along with genetic studies showing that variants in HSP70, a chaperone that helps guide protein folding, are associated with schizophrenia (Kim et al., 2008) and bipolar disorder (Pae et al., 2009), these findings suggest that faulty DISC1 trafficking within the cell could contribute to mental illness.

Aggregates in aggresomes
The new study delves deeply into the cell biology to get at just what DISC1 aggregates are doing in the cell: are they inert bystanders, or do they get in the way of things? First author Talia Atkin and colleagues overexpressed GFP-tagged DISC1 protein in COS7 cells, and found large DISC1 aggregates over 5 microns in diameter formed in about 30 percent of cells. Though these types of aggregates have been found before, Atkin and colleagues further localized these clumps to the aggresome, a membrane-bound compartment that houses proteins and organelles destined for destruction. Similar results were obtained in cultured neurons.

Consistent with this aggresomal location, the DISC1 aggregates colocalized with a marker for the autophagy pathway, which disposes of the cell’s garbage. Furthermore, compounds that inhibit autophagy increased the number of cells with DISC1-containing aggresomes. Finally, playing with the different garbage disposal pathways in the cell could shift the prevalence of DISC1 aggregates. For example, inhibiting the proteasomal pathway, which uses a multiprotein proteasome complex to degrade defunct or unneeded proteins one at a time, led to more cells with DISC1-containing aggresomes. Conversely, treating cells with enhancers of the proteasomal pathway decreased the number of cells with large DISC1-containing aggregates.

Turning to rat cortical neurons, the researchers used the same methods employed in the postmortem studies to recover the insoluble proteins from cultured neurons. These contained a low level of DISC1, including a 72 kD isoform linked to aggregate formation in the postmortem study, but could be prodded to incorporate more DISC1 under free radical-enriched conditions. Applying the same technique to COS7 cells containing aggresomal GFP-DISC1, the researchers found much of this DISC1 in the insoluble fraction, thus drawing a link between aggresomes in these cells and the insoluble fraction obtained in postmortem studies.

Dead end
These DISC1 aggregates had effects beyond the aggresome. First, the DISC1 aggregates seemed able to sequester soluble, free-floating DISC1 proteins to the aggresome: overexpressing DISC1 in COS7 cells increased the amount of endogenous DISC1 in the insoluble fraction by a factor of three, and reduced the endogenous DISC1 in the soluble fraction by nearly half. Fluorescent recovery after photobleaching experiments combined with live cell imaging revealed that, once recruited to the aggresome, DISC1 did not return to the cytosol, where it is needed to carry out its functions. DISC1 aggregates were also immobile, unlike smaller clusters that moved at a velocity typical for other cargo. These findings suggest that the DISC1 aggregates are a dead end, containing DISC1 that is of no use to the cell.

To see if depletion of soluble DISC1 has consequences, the researchers monitored intracellular transport, something that DISC1 regulates. With live cell imaging, the researchers found that cells containing DISC1 aggresomes did not transport mitochondria within the axons of hippocampal neurons as readily as control neurons. While only 13 percent of mitochondria moved in neurons transfected with GFP-DISC1, 24.4 percent of mitochondria moved in control neurons transfected only with GFP. This suggests that DISC1 in aggresomes somehow interferes with cargo transport within the cell.

If perturbing non-mutant DISC1 trafficking can disrupt intracellular transport, what could risk-associated forms of DISC1 do? A chimeric protein resembling something that might result from the original Scottish translocation aggregates (Zhou et al., 2010) and other risk variants also results in DISC1 aggregates (Narayanan et al., 2011; Leliveld et al., 2009). Further experiments will have to examine the full spectrum of consequences in neurons containing these kinds of DISC1 aggregates to fully grasp the implications for mental illness.—Michele Solis.

Reference:
Atkin TA, Brandon NJ, Kittler JT. Disrupted in Schizophrenia 1 forms pathological aggresomes that disrupt its function in intracellular transport. Hum Mol Genet. 2012 Jan 30. Abstract

Comments on Related Papers


Related Paper: Convergence of Two Independent Mental Disease Genes on the Protein Level: Recruitment of Dysbindin to Cell-Invasive Disrupted-In-Schizophrenia 1 Aggresomes.

Comment by:  T.A. AtkinJosef Kittler
Submitted 13 June 2011
Posted 13 June 2011

Protein aggregation is a well-characterized disease mechanism in multiple neurodegenerative disorders. Formation of Lewy bodies is a hallmark feature of Parkinson’s disease; tau and amyloid-β aggregation precedes symptoms in Alzheimer’s disease; and mutant huntingtin aggregation occurs in Huntington’s disease (Ross and Poirier, 2004). Interestingly, protein aggregation has recently emerged as a disease mechanism in schizophrenia (Leliveld et al., 2008; Leliveld et al., 2009; Atkin TA, 2009; Zhou et al., 2010), but the function of these protein aggregates remains unknown. In a recent paper by Ottis et al., a new pathological feature of DISC1 aggregates has been revealed, in addition to a novel binding partner. First the authors find that DISC1 aggregates are cell invasive, suggesting a new pathological feature of these interesting structures. Secondly, they find that DISC1 aggregates can co-recruit soluble dysbindin, and they further demonstrate a direct interaction between soluble DISC1 and dysbindin. Moreover, they demonstrate co-precipitation of dysbindin with insoluble-DISC1 in postmortem brain tissue of patients with chronic mental illness.

These interesting findings suggest DISC1 aggregate formation leads to cytosolic depletion of both functional DISC1 and dysbindin in a dominant-negative manner. As there appears to be convergence in the roles of DISC1 and dysbindin (Camargo et al., 2007; Hayashi-Takagi et al., 2010; Ito et al., 2010), combined depletion could have compound effects on neuronal function. These findings also provide the first evidence that heterologous proteins can be co-recruited into DISC1 aggregates. As DISC1 binds multiple proteins, which of these proteins may be co-recruited to the DISC1 aggregates must now be explored.

While these are interesting findings, several key questions now emerge. Ottis et al. find that purified DISC1 aggregates invade other cells leading to aggregate formation in the invaded cell in addition to co-recruitment of soluble DISC1. Cell-invasive properties have been previously demonstrated in progressive neurodegenerative disorders (Moreno-Gonzalez and Soto, 2011), but how this can be reconciled with the chronic and often non-progressive illness of schizophrenia must now be explored. Another question this work poses is whether this spreading of DISC1 aggregates occurs in vivo. Whereas exocytosis and extracellular localization of other cell-invasive proteins occurs, such as Parkinson’s associated α-synuclein, DISC1 does not seem to be secreted, nor are DISC1 aggregates found by Ottis et al. to be released upon conditions of neurotoxicity. Therefore, whether DISC1 aggregates are found extracellularly and whether this feature of DISC1 aggregates occurs in vivo must be investigated.

These promising findings by Ottis et al. therefore open two avenues of research within the ever-growing field of DISC1 literature. They support the emerging idea of DISC1 aggregation as a pathogenic agent and suggest a mechanism by which it may also act as an infectious agent. Secondly, they reveal an interaction of DISC1 with another schizophrenia-associated protein, dysbindin, whose function seems likely to converge with that of DISC1.

References:

Atkin TA, M.A., Brandon N, Kittler JT, 2009. DISC1 forms intracellular aggregates causing disruption of intracellular trafficking. Society for Neuroscience Annual Meeting, Chicago, USA, 249.241/N217.

Camargo, L.M., Collura, V., Rain, J.C., Mizuguchi, K., Hermjakob, H., Kerrien, S., Bonnert, T.P., Whiting, P.J., Brandon, N.J., 2007. Disrupted in Schizophrenia 1 Interactome: evidence for the close connectivity of risk genes and a potential synaptic basis for schizophrenia. Mol Psychiatry 12, 74-86. Abstract

Hayashi-Takagi, A., Takaki, M., Graziane, N., Seshadri, S., Murdoch, H., Dunlop, A.J., Makino, Y., Seshadri, A.J., Ishizuka, K., Srivastava, D.P., Xie, Z., Baraban, J.M., Houslay, M.D., Tomoda, T., Brandon, N.J., Kamiya, A., Yan, Z., Penzes, P., Sawa, A., 2010. Disrupted-in-Schizophrenia 1 (DISC1) regulates spines of the glutamate synapse via Rac1. Nat Neurosci 13, 327-332. Abstract

Ito, H., Morishita, R., Shinoda, T., Iwamoto, I., Sudo, K., Okamoto, K., Nagata, K., 2010. Dysbindin-1, WAVE2 and Abi-1 form a complex that regulates dendritic spine formation. Mol Psychiatry 15, 976-986. Abstract

Leliveld, S.R., Bader, V., Hendriks, P., Prikulis, I., Sajnani, G., Requena, J.R., Korth, C., 2008. Insolubility of disrupted-in-schizophrenia 1 disrupts oligomer-dependent interactions with nuclear distribution element 1 and is associated with sporadic mental disease. J Neurosci 28, 3839-3845. Abstract

Leliveld, S.R., Hendriks, P., Michel, M., Sajnani, G., Bader, V., Trossbach, S., Prikulis, I., Hartmann, R., Jonas, E., Willbold, D., Requena, J.R., Korth, C., 2009. Oligomer assembly of the C-terminal DISC1 domain (640-854) is controlled by self-association motifs and disease-associated polymorphism S704C. Biochemistry 48, 7746-7755. Abstract

Moreno-Gonzalez, I., Soto, C., 2011. Misfolded protein aggregates: Mechanisms, structures and potential for disease transmission. Semin Cell Dev Biol. Abstract

Ross, C.A., Poirier, M.A., 2004. Protein aggregation and neurodegenerative disease. Nat. Med. 10, S10-S17. Abstract

Zhou, X., Chen, Q., Schaukowitch, K., Kelsoe, J.R., Geyer, M.A., 2010. Insoluble DISC1-Boymaw fusion proteins generated by DISC1 translocation. Mol Psychiatry 15, 669-672. Abstract

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View all comments by Josef Kittler

Related Paper: Convergence of Two Independent Mental Disease Genes on the Protein Level: Recruitment of Dysbindin to Cell-Invasive Disrupted-In-Schizophrenia 1 Aggresomes.

Comment by:  Carsten Korth, SRF Advisor
Submitted 14 June 2011
Posted 14 June 2011

These are valuable comments, and I would agree in seeing our report as the beginning rather than the end of a story. I would like to specifically comment on the cell-invasiveness of DISC1 aggresomes.

Cell invasiveness of protein aggregates may be less rare than initially thought. In the last two years, this has been established for a range of proteins associated with protein conformational diseases. The initial paper on seeded nucleation of Aβ (Meyer-Luehmann et al., 2006) was recently joined by papers demonstrating invasiveness of cytosolic proteins α-synuclein (Desplats et al., 2009), polyglutamine protein (Ren et al., 2009), tau (Clavaguera et al., 2009), and SOD1 aggregates (Münch et al., 2011), making cell-invasiveness a hallmark for progressive brain diseases like the presence of misfolded proteins in these diseases itself. A recent paper has nicely demonstrated specific mechanisms of secretion and uptake for ALS-associated SOD1 aggregates (Münch et al., 2011).

But care should be taken when using the term "infectious" for cell invasiveness, since the mere secretion and/or uptake of protein aggregates does not necessarily trigger a replicative process, whereby the invasive aggregate has to 1) be taken up, 2) convert/recruit fresh material, and 3) somehow break the aggregated material up into novel seeds before 4) releasing it in an efficient manner. To my knowledge, a complete and efficient cycle of events has not been demonstrated for any of the proteins mentioned above. But that does not mean that incomplete cycles could not have disease relevance through recruiting other proteins in a more or less specific manner (Olzscha et al., 2011). This is exactly, at this stage, what we have proposed for DISC1.

The disease relevance of the phenomenon of cell invasiveness is still unclear for the majority of diseases that the proteins named above are associated with. The only instances where a clear relation to human disease has been shown are the case reports of Parkinson's patients that received grafts that, upon autopsy many years later, were found to have incorporated α-synuclein-positive inclusions like the surrounding host tissue (Li et al., 2008).

Thus, it remains to be shown whether there is transmission of these aggregates in vivo in a significant manner, and whether transmission is related to the pathomechanisms of these diseases.

The other point we have been attempting to make is the current underappreciation of protein pathology in the world of molecular psychiatry. While it is beyond doubt that genetics has made the major breakthroughs in the discovery of candidate genes, including DISC1 and dysbindin, some major disease-relevant phenomena may not be intelligible through pure genetic analyses.

References:

Clavaguera, F., Bolmont, T., Crowther, R.A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A.K., Beibel, M., Staufenbiel, M., et al. (2009). Transmission and spreading of tauopathy in transgenic mouse brain. Nat Cell Biol 11, 909-913. Abstract

Desplats, P., Lee, H.J., Bae, E.J., Patrick, C., Rockenstein, E., Crews, L., Spencer, B., Masliah, E., and Lee, S.J. (2009). Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein. Proc Natl Acad Sci U S A 106, 13010-13015. Abstract

Li, J.Y., Englund, E., Holton, J.L., Soulet, D., Hagell, P., Lees, A.J., Lashley, T., Quinn, N.P., Rehncrona, S., Bjorklund, A., et al. (2008). Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation. Nat Med 14, 501-503. Abstract

Meyer-Luehmann, M., Coomaraswamy, J., Bolmont, T., Kaeser, S., Schaefer, C., Kilger, E., Neuenschwander, A., Abramowski, D., Frey, P., Jaton, A.L., et al. (2006). Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science 313, 1781-1784. Abstract

Munch, C., O'Brien, J., and Bertolotti, A. (2011). Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells. Proc Natl Acad Sci U S A 108, 3548-3553. Abstract

Olzscha, H., Schermann, S.M., Woerner, A.C., Pinkert, S., Hecht, M.H., Tartaglia, G.G., Vendruscolo, M., Hayer-Hartl, M., Hartl, F.U., and Vabulas, R.M. (2011). Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions. Cell 144, 67-78. Abstract

Ren, P.H., Lauckner, J.E., Kachirskaia, I., Heuser, J.E., Melki, R., and Kopito, R.R. (2009). Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates. Nat Cell Biol 11, 219-225. Abstract

View all comments by Carsten Korth