2010 — 2014 |
Morfini, Gerardo Andres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Axonal Transport Deficits During Hereditary Spastic Paraplegia @ University of Illinois At Chicago
DESCRIPTION (provided by applicant): The hereditary spastic paraplegias (HSP) comprise a genetically heterogeneous group of disorders characterized by progressive bilateral weakness and spasticity of the lower limbs. This hallmark clinical symptom of HSP results from progressive dysfunction and degeneration of upper motor neurons in corticospinal tracts and dorsal column fibers. Over fifteen mutations in structurally and functionally unrelated genes have been identified as causative of HSP. Among these, mutations in the SPG4 locus coding for the microtubule-severing protein spastin represent the most common cause of HSP. Despite these major breakthroughs, pathogenic mechanisms underlying HSP pathogenesis remain unknown. Pathological observations from HSP patients and HSP animal models indicate that neurons affected in HSP degenerate following a "dying back" pattern, which is characterized by early abnormalities in synapses and distal axons and progressive degeneration of axons. Significantly, recent genetic data demonstrated that reductions in fast axonal transport (FAT) result in such pattern of neuronal degeneration. Moreover, loss of function mutations in the SPG10 locus coding for a specific subunit of the molecular motor protein conventional kinesin lead to HSP, suggesting that abnormalities in FAT might indeed represent a critical event in HSP pathogenesis. Our recent studies demonstrated that nanomolar levels of specific mutant spastin isoforms inhibit FAT in an axon-autonomous manner. Consistent with a role of kinases in the regulation of FAT, pharmacological studies presented in this application indicate that this effect of pathogenic spastin was mediated by the activity of casein kinase 2 (CK2). Complementing these observations, active CK2 was found to inhibit FAT. Moreover, CK2 was found to directly phosphorylate and inhibit the functionality of the molecular motor protein conventional kinesin. Based on these and additional findings herein, we propose that activation of axonal CK2 and inhibition of FAT induced by pathogenic spastin represent critical pathogenic events in HSP. Experiments in this application will characterize effects of pathogenic spastin on FAT. Biochemical, immunochemical, pharmacological and cell biological methods will be used to identify specific axonal cargoes and molecular motors associated with pathogenic spastin. Based on our previous work, lentiviral approaches will evaluate isoform-specific effects of pathogenic spastin in vivo. Finally, biochemical, and cell biological approaches will identify and characterize CK2 targets associated with pathogenic spastin including molecular motors and cytoskeletal proteins. These studies will help identifying molecular components and mechanisms mediating the inhibition of FAT induced by pathogenic spastin. Studies proposed here will characterize pathogenic mechanisms underlying the axonal defects characteristic of HSP. The ultimate goal of this project is to identify novel therapeutic targets in HSP that help prevent distal axonopathy and degeneration of motor neurons. PUBLIC HEALTH RELEVANCE: Hereditary spastic paraplegias (HSPs) comprise a heterogeneous group of genetic diseases that lead to lower limb spasticity in affected patients. This clinical symptom hallmark results from dysfunction and dying back degeneration of upper motor neurons. Although HSP pathogenesis involves early alterations in axonal and synaptic function, specific mechanisms and molecular components involved in this pathogenic event remain elusive. Mutations in the SPG4 locus coding for the microtubule-severing protein spastin represent the most common cause of HSP. The mammalian spastin gene has two start codons, resulting in the production of two alternatively spliced spastin isoforms. Consistent with genetic data linking alterations in axonal transport to HSP, our recently published studies and data presented here indicate that pathogenic forms of spastin protein inhibit axonal transport through a mechanism involving the activity of casein kinase 2 (CK2). Moreover, the effects of pathogenic spastin are isoform-specific and occur independently of changes in gene transcription. Consistent with these observations, active CK2 dramatically inhibited axonal transport. Together, our observations suggest that HSP pathogenesis might involve reductions in the delivery of axonal and synaptic proteins essential for neuronal function and survival. Experiments in this application will characterize alterations in axonal transport induced by pathogenic spastin, and evaluate underlying molecular mechanisms. The ultimate goal of this project is to define disease mechanisms and identify novel therapeutic targets in HSP.
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0.958 |
2016 — 2017 |
Morfini, Gerardo Andres |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Addressing the Contribution of Jnk3 to Axonal Pathology in Huntington's Disease @ University of Illinois At Chicago
Huntington's disease (HD) is an autosomal-dominant, progressive neurodegenerative disorder featuring devastating clinical symptoms that include motor deficits, cognitive decline, and behavioral impairments. To date, most research efforts towards the development of therapeutic strategies in HD have been largely focused on inhibition of pathways leading to the loss of neuronal somata, failing to address or even consider the progressive loss of neuritic connectivity that takes place much earlier in the disease process. Building on a solid body of published findings and strong preliminary data, this application aims to illuminate specific therapeutic targets and mechanisms underlying axonal pathology in HD. Experiments proposed under Aim 1 will directly evaluate the contribution of JNK3, a potentially druggable protein kinase, to the axonal pathology induced by mutant huntingtin (mhtt) expression in vivo. Extending these studies, experiments under Aim 2 will identify JNK3-dependent alterations in the phosphorylation of axonal proteins induced by mhtt. Together, these studies will help illuminate a molecular basis linking JNK3 activation to mhtt-induced axonal degeneration.
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0.958 |
2020 |
Bosco, Daryl Angela (co-PI) [⬀] Morfini, Gerardo Andres |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Role of P38alpha Signaling in Mutant Sod1-Linked Motor Neuron Disease @ University of Illinois At Chicago
Axonal pathology represents an early, critical pre-symptomatic event in the disease course of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder that primarily targets motor neurons. The mechanism(s) by which axons degenerate in ALS are largely unknown. Collaborative efforts by the PIs and from others showed that pathogenic forms of the superoxide dismutase (SOD1) protein associated with familial forms of ALS (fALS) inhibit fast axonal transport (FAT). The potential disease relevance of these findings was highlighted by the discovery of fALS-related mutant genes encoding motor proteins, suggesting that FAT deficits suffice to cause axonopathy and degeneration of motor neurons. Our published and preliminary data demonstrated that the toxic effect of mutant SOD1 and misfolded forms of SOD1 on FAT was mediated through aberrant activation of a mitogen-activated protein kinase (MAPK) pathway. Importantly, our work established p38alpha (p38?), the major p38 MAPK isoform expressed in the CNS, as a specific MAPK component responsible for FAT inhibition and the motor protein kinesin-1 as a novel p38? substrate relevant to this toxic effect. Although multiple independent studies have demonstrated enhanced phosphorylation (and hence activation) of p38 MAPKs in ALS mouse models and in post-mortem human CNS tissues, none have addressed the contribution(s) of specific p38 MAPKs isoforms to the progressive degeneration on MNs triggered by mutant SOD1 in vivo. While pleiotropic therapeutic compounds that only mildly inhibit p38 MAPKs still showed a beneficial effect on the survival of fALS-SOD1 mice, findings from our work led us to posit that the therapeutic effects of p38 MAPK inhibition will be more substantial when the p38? isoform is specifically targeted. To define the contribution of aberrant p38? signaling to ALS pathogenesis, two PIs with a long history of collaboration and strong track-records in both ALS and aberrant kinase signaling have assembled a synergistic MPI application that will shed light on this important issue. Taking advantage of the SOD1G93A mouse model of ALS and of p38?AF/+ knock-in mice, which feature a mutant MAPK14 allele encoding non-activatable p38?, we will use genetic approaches to directly measure the contribution of aberrant p38? signaling to mutant SOD1-mediated motor neuron degeneration in vivo (Aim 1). It is also important to define mechanism(s) by which aberrant p38? activation causes axonal degeneration. To identify p38?-dependent alterations in the phosphorylation of axonal proteins induced by mutant SOD1, an unbiased phosphoproteomics approach will be applied to both the isolated squid axoplasm preparation, a unique and powerful ex vivo system with which to study axonal-specific events, and to cultured neurons prepared from SOD1G93A mice with attenuated p38? signaling (Aim 2). The outcomes of this Aim will move the field forward by providing novel mechanistic insights linking aberrant p38? activation to axonopathy and by revealing novel biomarkers to evaluate axonopathy in ALS and related neurodegenerative disorders. Collectively, this proposal aims to gain novel mechanistic insights into the pathogenic processes underlying ALS, and to establish in vivo contribution(s) of the specific molecular target p38? to the degeneration of motor neurons triggered by mutant SOD1.
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0.958 |
2021 |
Baas, Peter W [⬀] Morfini, Gerardo Andres |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Spg4 Hereditary Spastic Paraplegia
PROJECT SUMMARY / ABSTRACT Hereditary Spastic Paraplegias (HSP) are heritable neurodegenerative diseases in which progressive degeneration of corticospinal axonal tracts results in limb weakness, spasticity and gait deficiencies. These symptoms result from a dying back pattern of degeneration of corticospinal axons, which also display prominent swellings of unclear pathological significance. The commonest form of HSP, termed SPG4-HSP, is caused by mutations in the SPAST gene, which codes for a microtubule-severing protein called spastin. To date, the prevailing mechanistic hypothesis for the etiology of SPG4-HSP is haploinsufficiency, meaning that the corticospinal tracts degenerate because of insufficient functional spastin. However, several major disease features are not readily explained by this etiology, and it is not clear how reduced microtubule severing would promote corticospinal axonal degeneration. Providing novel information that may fill a major gap in our knowledge of SPG4-HSP pathogenesis, recent work of the Principal Investigators revealed toxic properties of mutant spastin proteins, suggesting that a gain-of-function mechanism operates in SPG4-HSP. Curiously, both mechanisms negatively affect fast axonal transport (FAT), a cellular process fueled by molecular motor proteins that allows bidirectional movement of vesicular cargoes along axons. Based on a strong experimental premise, it is hypothesized in this multi-PI grant proposal that abnormalities in microtubule organization associated with reduced spastin levels cause FAT deficits and axonal swellings (loss-of-function). On the other hand, toxic effects of mutant spastin protein cause different FAT deficits that are mediated by casein kinase 2 (CK2), and these deficits promote corticospinal axon degeneration (gain-of-function). The former makes the axon more vulnerable, but it is the latter that suffices for corticospinal axon degeneration. The proposed work seeks to test these hypotheses by directly comparing a mouse model with a single SPAST allele (SPAST +/-) with a transgenic mouse model with both endogenous mouse SPAST alleles intact that additionally expresses human spastin bearing a pathogenic mutation associated with SPG4-HSP (spastin-C448Y mice). In Aim 1, these models will be individually crossed with mice that selectively express eGFP in corticospinal motor neurons (CSMN), so that loss-of and gain-of-function contributions to the disease can be investigated. In Aim 2, FAT deficits will be studied in neurons cultured from these animals, and specific hypotheses for the etiology of the deficits will be tested. In Aim 3, studies are proposed using transgenic spastin-C448Y mice in which autophagy is experimentally enhanced or CK2 levels are experimentally reduced, to test the hypothesis that these manipulations will prevent or reduce corticospinal axon degeneration and associated behavioral deficits. The overall significance of this project is to establish mechanisms underlying SPG4-HSP and forge a path toward effective therapies for patients.
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0.919 |