2007 — 2010 |
Blackstone, Craig |
Z01Activity Code Description: Undocumented code - click on the grant title for more information. ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Molecular Pathogenesis of Hereditary Spastic Paraplegias @ Neurological Disorders and Stroke
Research in the Cellular Neurology Unit focuses on the molecular mechanisms underlying a number of neurodegenerative disorders, including mitochondrial disorders, dystonia, and the hereditary spastic paraplegias (HSPs). These disorders, which together afflict millions of Americans, worsen insidiously over a number of years, and treatment options are limited for many of them. Our laboratory is investigating inherited forms of these disorders, using molecular and cell biology approaches to study how mutations in disease genes ultimately result in cellular dysfunction. In this project, we are focusing on the HSPs. One major research theme involves the characterization and functional analysis of the hereditary spastic paraplegia type 3A (SPG3A) protein, atlastin-1. We have recently reported in the journal Cell that atlastin-1 is a member of a ubiquitous family of GTPases that interact with two families of ER shaping proteins to generate the tubular endoplasmic reticulum (ER) network. Interestingly, atlastin-1 interacts with the SPG31 protein REEP1, which is an ER shaping protein, as well as the SPG4 protein spastin, a microtubule-severing ATPase. We have very recently completed a study demonstrating that these three proteins interact with one another to organize the tubular ER network in conjunction with the microtubule cytoskeleton. Since SPG3A, SPG4, and SGP31 account for over 50% of all HSP cases, we suggest ER network defects as the predominant neuropathologic mechanism for the HSPs. In another study, we have been studying the complicated HSP known as Troyer syndrome (SPG20), which is cause by a mutation in the spartin protein. Interestingly, we have found that the spartin protein is involved in the degradation of the EGF receptor, prefiguring a critical role for spartin in endocytosis, as well as in cytokinesis through interaction with a novel ESCRT-III protein. We are currently investigating the function of spartin in the nervous system by analyzing spartin-null mice that we have generated as a murine model of Troyer syndrome. In a final project related to the HSPs, we have been investigating the complicated HSP known as MAST syndrome (SPG21), which is caused by a large deletion in the acid cluster protein maspardin. We have generated maspardin-null mice as a murine model for this disorder and are investigating these animals using behavioral techniques in addition to using neurons and other cells derived form these animals for cellular trafficking studies. Lastly, we have recently completed a study characterizing the interaction of the maspardin protein with a specific isoform of aldehyde dehydrogenase, ALDH16A1. Taken together, we expect that our studies will advance our understanding of the molecular pathogenesis of the HSPs. Such an understanding at the molecular and cellular levels will hopefully lead to novel treatments to prevent the progression of these disorders.
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0.903 |
2007 — 2018 |
Blackstone, Craig |
Z01Activity Code Description: Undocumented code - click on the grant title for more information. ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Regulation of Mitochondrial Fission and Fusion @ Neurological Disorders and Stroke
Research in the Cellular Neurology Unit focuses on the molecular mechanisms underlying a number of neurodegenerative disorders, including mitochondrial disorders, dystonia, and the hereditary spastic paraplegias (HSPs). These disorders, which together afflict millions of Americans, worsen insidiously over a number of years, and treatment options are limited for many of them. Our laboratory is investigating inherited forms of these disorders, using molecular and cell biology approaches to study how mutations in disease genes ultimately result in cellular dysfunction. In this project, we are emphasizing investigations into the regulation of mitochondrial morphology within cells. Indeed, fusion and fission events that regulate mitochondrial morphology are essential for proper mitochondrial function, and their regulation is increasingly recognized in diverse cellular functions. Mitochondrial fission events in mammals are orchestrated by at least two proteins; the dynamin-related protein Drp1 and the integral membrane protein Fis1. The reciprocal process of mitochondrial fusion also requires large GTPases of the dynamin superfamily: OPA1 and the mitofusins Mfn1 and Mfn2. Since mutations in Drp1, Mfn2, and OPA1 have been identified in patients with inherited neurological disorders, and there is prominent fragmentation of mitochondria during programmed cell death, insights into the regulation of these processes is highly relevant clinically. We have recently published a study of the Drp1 A395D mutation that caused a neonatally fatal mitochondrial disorder due to markedly diminished mitochondrial fission. In this study, we were able to show that this mutation resulted in loss of higher-order multimeric interactions of the Drp1 protein. In complementary studies, we have now identified mutation in Drp1 that dramatically stabilizes higher-order Drp1 structures. Lastly, in ongoing studies we have identified a number of Drp1-interacting proteins that may be involved in the proper distribution of mitochondria within cells as well as novel proteins that regulate the mitochondrial fission/fusion balance thorugh unknown mechanisms. Together, these studies are continuing to provide critical insights into the regulation of mitochondrial morphology within a cell, an area of increasing clinical relevance and importance.
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0.903 |
2011 — 2018 |
Blackstone, Craig |
ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Endocytic Mechanisms in the Hereditary Spastic Paraplegias @ Neurological Disorders and Stroke
Research in the Cell Biology Section, Neurogenetics Branch focuses on the molecular mechanisms underlying a number of neurodegenerative disorders, including mitochondrial disorders, dystonia, and the hereditary spastic paraplegias (HSPs). These disorders, which together afflict millions of Americans, worsen insidiously over a number of years, and treatment options are limited for many of them. Our laboratory is investigating inherited forms of these disorders, using molecular and cell biology approaches to study how mutations in disease genes ultimately result in cellular dysfunction. Over the past several years, we have studied the interplay of the proteins that are mutated in SPG11 and SPG15, the two most common autosomal recessive HSPs. These proteins interact with one another as well as with a new adaptor protein complex -- AP5, one component of which, AP5Z1, is mutated in SPG48. Importantly, we have identified a fundamental role for the SPG15 and SPG11 proteins in lysosomal biogenesis and autophagic lysosomal reformation. Studies in these areas were published in the Journal of Clinical Investigation in 2014. Studies of the SPG48 protein AP5Z1 were published in Human Molecular Genetics in 2015 and Neurology: Genetics in 2016. In 2018, we published a study in Human Molecular Genetics, in collaboration with Dr. Xue-Jun Li, investigating patient derived induced pluripotent stem cells for SPG15 and SPG48; we found abnormalities in mitochondrial structure and function within axons. Lastly, we are investigating the functions of the SPG8 protein strumpellin, which is part of the WASH protein complex implicated in the shaping of endosomes through alterations of the actin cytoskeleton; we published a mechanistic study of the SPG8 protein in Nature Communications in early 2016 and another study has just been submitted for publication. Taken together, we expect that our studies will advance our understanding of the molecular pathogenesis of the HSPs. Such an understanding at the molecular and cellular levels will hopefully lead to novel treatments to prevent the progression of these disorders.
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0.903 |
2011 — 2018 |
Blackstone, Craig |
ZIAActivity Code Description: Undocumented code - click on the grant title for more information. |
Er Network Shaping Mechanisms in the Hereditary Spastic Paraplegias @ Neurological Disorders and Stroke
Research in the Cell Biology Section, Neurogenetics Branch focuses on the molecular mechanisms underlying a number of neurodegenerative disorders, most notable those affecting long axons such as peripheral neuropathies and the hereditary spastic paraplegias (HSPs). These disorders worsen insidiously over a number of years, and treatment options are limited for many of them. Our laboratory is investigating inherited forms of these disorders, using molecular and cell biology approaches to study how mutations in disease genes ultimately result in cellular dysfunction. In this project, we are focusing on the HSPs. One major research theme involves the characterization and functional analysis of the hereditary spastic paraplegia type 3A (SPG3A) protein, atlastin-1. In 2009, we reported in the journal Cell that atlastin-1 is a member of a ubiquitous family of GTPases that interact with two families of ER shaping proteins to generate the tubular endoplasmic reticulum (ER) network. Interestingly, atlastin-1 interacts with the SPG31 protein REEP1, which is an ER shaping protein, as well as with other ER-shaping proteins and the SPG4 protein spastin, a microtubule-severing ATPase. In 2010, we published a study in the Journal of Clinical Investigation demonstrating that these three proteins interact with one another to organize the tubular ER network in conjunction with the microtubule cytoskeleton. Since SPG3A, SPG4, and SGP31 account for well over 50% of all HSP cases, we suggest ER network defects as the predominant neuropathologic mechanism for the HSPs. This is supported by the recent identification of numerous other HSP proteins that regulate ER morphology, including the CPT1C protein (in collaboration with Dr. Kurt Fischbeck). Over the past year, we have continued to develop animal models for SPG31 (knock out) and SPG3A (knockout and knock in), as well as double mutant mice, to evaluate the extent of ER morphology changes using both in vivo and ex vivo studies. We are employing both high-throughput FIB-SEM electron microscopy and super-resolution confocal microscopy to examine the changes in tubular ER within neuronal axons in response to these genetic manipulations. In addition, we have identified interactions of these proteins with several other proteins mutated in the HSPs, expanding the number of HSP cases related to defects in ER network formation. Furthermore, we are actively generating in situ models for many of the HSPs through the production of patient-derived, induced pluripotent stem cells that are then differentiated into telecephalic neurons, in collaboration with Dr. Xue-Jun Li. Several of these studies were published in 2014 in the journals Stem Cells and Human Molecular Genetics. ER morphology and dynamics in these and other cells are being evaluated using a number of emerging super-resolution microscopy techniques in collaboration with Drs. Jennifer Lippincott-Schwartz, Eric Betzig, and Harald Hess; some of this work was published in Science in 2016, and additional studies in this exciting area will be published shortly. Finally, we are working with Dr. Niamh O'Sullivan on fly models of these HSPs. A key aspect of ER function possibly related to disease pathogenesis is the formation of lipid droplets, and this is an area of emphasis for our cellular and organismal studies. We are in the process of completing several studies tying changes in ER morphology to alterations in lipid droplet biogenesis. As part of these studies, we have used CRISPR technologies to knock out all three atlastin isoforms from cell lines such as HeLa and NIH-3T3. We have also utilized advanced imaging techniques such as CT scans and MR spectroscopy to study changes in fat tissue in HSP mouse models non-invasively; one such study was published in Hum Mol Genet in 2016. Currently, we are collaborating with Dr. Lippincott-Schwartz and colleagues assessing the role of the SPG4 protein spastin in lipid droplet biogenesis and interactions. Collectively, these studies were also the inspiration for the planning of a large clinical natural history trial in patients with the three most common forms of autosomal dominant HSP (SPG4, SPG3A, and SPG31), which has been recruiting subjects for the past two years. Taken together, we expect that our studies will advance our understanding of the molecular pathogenesis of the HSPs. Such an understanding at the molecular and cellular levels will hopefully lead to novel treatments to prevent the progression of these disorders.
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0.903 |