Research Departments & Organizations
We are focused on misfolding caused by mutant forms of the cytosolic enzyme superoxide dismutase (SOD1), that produces an inherited form of ALS (Lou Gehrig’s Disease), with progressive, fatal motor neuron dysfunction. We are using mice overexpressing a mutant G85R SOD1-YFP fusion protein to study the mechanism of disease causation. Notably, other forms of ALS, including both inherited and non-inherited forms in humans, are indistinguishable at a clinical level. The mouse model studied affords one of the most powerful approaches to following the development of this non-treatable neurodegenerative condition. The hope is that basic understanding may lead to directed therapy.
Specialized Terms: Chaperones in protein folding; ALS (Lou Gehrig's Disease)
Extensive Research Description
Studies of the past decade have shown that many diseases of neurodegeneration are the result of protein misfolding, and we have begun to seek an understanding of the mechanism of such degeneration. We have focused on misfolding caused by mutant forms of theanti-oxidant cytosolic enzyme SOD1 (superoxide dismutase), that producean inherited form of ALS (Lou Gehrig’s disease), with progressive,fatal motor neuron dysfunction. We are using mice expressing mutant G85R SOD1-YFP, containing a mutant version of SOD1 unable to reach the native state, studying the transgenic animals with a variety of approaches, to investigate how the mutant SOD1 produces motor neuron dysfunction.
We have identified that there is a progression of misbehavior of mutant SOD1 protein itself in the spinal cord of transgenic animals. Initially it is soluble and associated to a significant extent with the abundant cytosolic chaperone Hsc70 (by contrast the wild-type protein does not form such association, presumably because it occupies the native state). Subsequently the mutant SOD1 protein begins to form both soluble oligomers (observable by gel filtration chromatography) and insoluble aggregates and at this point an additional molecular chaperone, Hsp110 becomes associated with the soluble form. At this point the animal exhibits mild lower extremity symptoms (weakness or pulling in when held by the tail) which progress over the next two to three months to lethal paralysis, usually commencing in the lower extremities. We are currently seeking to understand ultrastructural correlates of this progression using EM, analyzing motor neuron cell bodies in the spinal cord, sciatic nerve axons, and neuromuscular junctions. Where do abnormalities first present? What is the progression? We have used laser capture microdissection of motor neurons and RNA-seq from animals at mid-course in their progression to inspect for changes in transcriptional expression, but find only a small number of differences with wild-type SOD1-YFP motor neurons. Notably, there is no heat shock response, no unfolded protein (ER) response, but there are effects on several calcium binding proteins, and the level of Hsp110 mRNA is increased. We surmise that most of the toxicity of mutant SOD1 is exerted post-transcriptionally, contrasting with pathogenesis by other proteins implicated in ALS, such as TDP43 and FUS. At the post-translational level, using affinity capture and mass spectrometry, we find a variety of associations of the mutant protein in spinal cord, the most prominent being with the molecular chaperone, Hsc70. It remains to be seen whether other lower affinity interactions, that have gone undetected by affinity capture and MS, are crucial to pathogenesis.
In the mouse system we have also recently been able, in collaboration with David McCormick's group to carry out electrophysiologic measurements of MNs by patch clamping them in spinal cord of wild-type and mutant SOD1 mice in slice preparations prepared by MSTP student Muhamed Hadzipasic in our lab. This has identified a vulnerable motor neuron that dies by the time animals are symptomatic. We are further characterizing this neuronal type as well as conducting additional circuit-related experiments to analyze how the motor sytem adapts to loss of a motor neuron firing type.
We are also studying a heterologous system that reports on mutant SOD1-YFP toxicity.
Our ability to produce and purify the mutant SOD1-YFP protein from E.coli (as well as the wild-type protein) has enabled testing in other systems. One such system involves the axoplasm from the giant axon of the squid Loligo pealei, a system in which the transport of vesicles can be examined using videomicroscopy. We observe that adding mutant SOD1-YFP (but not wild-type) produces marked slowing of anterograde but not retrograde vesicular traffic. This occurs when monomeric forms of the mutant protein are added, but also when oligomeric species, first crosslinked and then purified by gel filtration, are added. At the same time as the mutant protein produces slowing of anterograde fast axonal transport, we observe that the MAP kinase cascade is activated by the mutant protein, involving ASK1 (MAPKKK) and p38 (MAPK). Remarkably, addition of molecular chaperones, and most potently, Hsp110, restores anterograde transport to normal and abrogates activation of the kinase cascade. In affinity capture studies, we observe that Hsp110 physically associates with the mutant protein. Thus it appears that there is a pathway in which the misfolded protein, if not counteracted by molecular chaperones, is able to activate a kinase cascade that inhibits anterograde transport, most likely via the phosphorylation of kinesin. We are now seeking to understand how the misfolded protein “links” to the kinase cascade. For example, does it directly interact with ASK1, or with another component that does so?
In further tests we are studying neurotransmission in the squid giant synapse, an axonal-axonal connection, injecting mutant SOD1 protein presynaptically and testing effects on neurotransmission.
SOD1-linked ALS mice and primary cultures derived from them (at E14). Projects are designed to address early toxic alterations produced by the mutant protein, using morphologic, electrophysiologic, and ultimately molecular biochemical techniques. A range of techniques including EM, mass spectrometry, fluorescent imaging procedures, and electrophysiology measurements (the latter in collaboration with the McCormick group), are being employed.
Unfolded DapA forms aggregates when diluted into free solution, confounding comparison with folding by the GroEL/GroES chaperonin system.
Ambrose AJ, Fenton W, Mason DJ, Chapman E, Horwich AL. Unfolded DapA forms aggregates when diluted into free solution, confounding comparison with folding by the GroEL/GroES chaperonin system. FEBS Letters 2015, 589:497-499. 2015
Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS.
Hadzipasic M, Tahvildari B, Nagy M, Bian M, Horwich AL, McCormick DA. Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS. Proceedings Of The National Academy Of Sciences Of The United States Of America 2014, 111:16883-8. 2014
Absence of lipofuscin in motor neurons of SOD1-linked ALS mice.
Bandyopadhyay U, Nagy M, Fenton WA, Horwich AL. Absence of lipofuscin in motor neurons of SOD1-linked ALS mice. Proceedings Of The National Academy Of Sciences Of The United States Of America 2014, 111:11055-60. 2014
Molecular chaperones in cellular protein folding: the birth of a field.
Horwich AL. Molecular chaperones in cellular protein folding: the birth of a field. Cell 2014, 157:285-288. 2014
Production of RNA for transcriptomic analysis from mouse spinal cord motor neuron cell bodies by laser capture microdissection.
Bandyopadhyay U, Fenton WA, Horwich AL, Nagy M. Production of RNA for transcriptomic analysis from mouse spinal cord motor neuron cell bodies by laser capture microdissection. Journal Of Visualized Experiments : JoVE 2014, e51168. 2014
RNA-Seq profiling of spinal cord motor neurons from a presymptomatic SOD1 ALS mouse.
Bandyopadhyay U, Cotney J, Nagy M, Oh S, Leng J, Mahajan M, Mane S, Fenton WA, Noonan JP, Horwich AL. RNA-Seq profiling of spinal cord motor neurons from a presymptomatic SOD1 ALS mouse. PloS One 2013, 8:e53575. 2013
A biochemical screen for GroEL/GroES inhibitors.
Johnson SM, Sharif O, Mak PA, Wang HT, Engels IH, Brinker A, Schultz PG, Horwich AL, Chapman E. A biochemical screen for GroEL/GroES inhibitors. Bioorganic & Medicinal Chemistry Letters 2014, 24:786-9. 2014
Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm.
Song Y, Nagy M, Ni W, Tyagi NK, Fenton WA, López-Giráldez F, Overton JD, Horwich AL, Brady ST. Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm. Proceedings Of The National Academy Of Sciences Of The United States Of America 2013, 110:5428-33. 2013
Chaperonin-mediated protein folding.
Horwich AL. Chaperonin-mediated protein folding. The Journal Of Biological Chemistry 2013, 288:23622-32. 2013
Protein folding in the cell: an inside story.
Horwich AL. Protein folding in the cell: an inside story. Nature Medicine 2011, 17:1211-6. 2011
Hydrogen-deuterium exchange in vivo to measure turnover of an ALS-associated mutant SOD1 protein in spinal cord of mice.
Farr GW, Ying Z, Fenton WA, Horwich AL. Hydrogen-deuterium exchange in vivo to measure turnover of an ALS-associated mutant SOD1 protein in spinal cord of mice. Protein Science : A Publication Of The Protein Society 2011, 20:1692-6. 2011
Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS.
Wang J, Farr GW, Zeiss CJ, Rodriguez-Gil DJ, Wilson JH, Furtak K, Rutkowski DT, Kaufman RJ, Ruse CI, Yates JR, Perrin S, Feany MB, Horwich AL. Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS. Proceedings Of The National Academy Of Sciences Of The United States Of America 2009, 106:1392-7. 2009
Chaperonin chamber accelerates protein folding through passive action of preventing aggregation.
Apetri AC, Horwich AL. Chaperonin chamber accelerates protein folding through passive action of preventing aggregation. Proceedings Of The National Academy Of Sciences Of The United States Of America 2008, 105:17351-5. 2008
Topologies of a substrate protein bound to the chaperonin GroEL.
Elad N, Farr GW, Clare DK, Orlova EV, Horwich AL, Saibil HR. Topologies of a substrate protein bound to the chaperonin GroEL. Molecular Cell 2007, 26:415-26. 2007