Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases

© The Author(s), 2017. This is the author's version of the work and is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Neurobiology of Disease 105 (2017): 273-282, doi:10.1016/j.nbd.2017.04.010.Neurons affected in a wide variety of unrelated adult-onset neurodegenerative diseases (AONDs) typically exhibit a “dying back” pattern of degeneration, which is characterized by early deficits in synaptic function and neuritic pathology long before neuronal cell death. Consistent with this observation, multiple unrelated AONDs including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and several motor neuron diseases feature early alterations in kinase-based signaling pathways associated with deficits in axonal transport (AT), a complex cellular process involving multiple intracellular trafficking events powered by microtubule-based motor proteins. These pathogenic events have important therapeutic implications, suggesting that a focus on preservation of neuronal connections may be more effective to treat AONDs than addressing neuronal cell death. While the molecular mechanisms underlying AT abnormalities in AONDs are still being analyzed, evidence has accumulated linking those to a well-established pathological hallmark of multiple AONDs: altered patterns of neuronal protein phosphorylation. Here, we present a short overview on the biochemical heterogeneity of major motor proteins for AT, their regulation by protein kinases, and evidence revealing cell type-specific AT specializations. When considered together, these findings may help explain how independent pathogenic pathways can affect AT differentially in the context of each AOND.The authors would like to acknowledge funding from CHDI and NIH grants RO1 NS066942A, R21 NS096642 (to G.M) and R01-NS023868 and R01-NS041170 and a Zenith Award from the Alzheimer’s Association (to STB).2018-04-1

ALS-linked FUS exerts a gain of toxic function involving aberrant p38 MAPK activation

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 7 (2017): 115, doi:10.1038/s41598-017-00091-1.Mutations in Fused in Sarcoma/Translocated in Liposarcoma (FUS) cause familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by progressive axonal degeneration mainly affecting motor neurons. Evidence from transgenic mouse models suggests mutant forms of FUS exert an unknown gain-of-toxic function in motor neurons, but mechanisms underlying this effect remain unknown. Towards this end, we studied the effect of wild type FUS (FUS WT) and three ALS-linked variants (G230C, R521G and R495X) on fast axonal transport (FAT), a cellular process critical for appropriate maintenance of axonal connectivity. All ALS-FUS variants impaired anterograde and retrograde FAT in squid axoplasm, whereas FUS WT had no effect. Misfolding of mutant FUS is implicated in this process, as the molecular chaperone Hsp110 mitigated these toxic effects. Interestingly, mutant FUS-induced impairment of FAT in squid axoplasm and of axonal outgrowth in mammalian primary motor neurons involved aberrant activation of the p38 MAPK pathway, as also reported for ALS-linked forms of Cu, Zn superoxide dismutase (SOD1). Accordingly, increased levels of active p38 MAPK were detected in post-mortem human ALS-FUS brain tissues. These data provide evidence for a novel gain-of-toxic function for ALS-linked FUS involving p38 MAPK activation.We are grateful for funding from NIH/NINDS (R01 NS078145, R01 NS090352, and R21 NS091860 to D.A.B., R01 NS066942A and R21 NS096642 to G.M., R01NS023868 and R01NS041170 to S.T.B.), the ALS Therapy Alliance/CVS Pharmacy (to D.A.B. and G.M.) and the ALS Association (to C.F. and J.M.)

Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS.

Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS

Engagement of Neurotropic Viruses in Fast Axonal Transport: Mechanisms, Potential Role of Host Kinases and Implications for Neuronal Dysfunction

Much remains unknown about mechanisms sustaining the various stages in the life cycle of neurotropic viruses. An understanding of those mechanisms operating before their replication and propagation could advance the development of effective anti-viral strategies. Here, we review our current knowledge of strategies used by neurotropic viruses to undergo bidirectional movement along axons. We discuss how the invasion strategies used by specific viruses might influence their mode of interaction with selected components of the host’s fast axonal transport (FAT) machinery, including specialized membrane-bounded organelles and microtubule-based motor proteins. As part of this discussion, we provide a critical evaluation of various reported interactions among viral and motor proteins and highlight limitations of some in vitro approaches that led to their identification. Based on a large body of evidence documenting activation of host kinases by neurotropic viruses, and on recent work revealing regulation of FAT through phosphorylation-based mechanisms, we posit a potential role of host kinases on the engagement of viruses in retrograde FAT. Finally, we briefly describe recent evidence linking aberrant activation of kinase pathways to deficits in FAT and neuronal degeneration in the context of human neurodegenerative diseases. Based on these findings, we speculate that neurotoxicity elicited by viral infection may involve deregulation of host kinases involved in the regulation of FAT and other cellular processes sustaining neuronal function and survival

Inhibition of fast axonal transport by pathogenic SOD1 involves activation of p38 MAP kinase

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS

Tau: a signaling hub protein

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Mueller, R. L., Combs, B., Alhadidy, M. M., Brady, S. T., Morfini, G. A., & Kanaan, N. M. Tau: a signaling hub protein. Frontiers in Molecular Neuroscience, 14, (2021): 647054, https://doi.org/10.3389/fnmol.2021.647054.Over four decades ago, in vitro experiments showed that tau protein interacts with and stabilizes microtubules in a phosphorylation-dependent manner. This observation fueled the widespread hypotheses that these properties extend to living neurons and that reduced stability of microtubules represents a major disease-driving event induced by pathological forms of tau in Alzheimer’s disease and other tauopathies. Accordingly, most research efforts to date have addressed this protein as a substrate, focusing on evaluating how specific mutations, phosphorylation, and other post-translational modifications impact its microtubule-binding and stabilizing properties. In contrast, fewer efforts were made to illuminate potential mechanisms linking physiological and disease-related forms of tau to the normal and pathological regulation of kinases and phosphatases. Here, we discuss published work indicating that, through interactions with various kinases and phosphatases, tau may normally act as a scaffolding protein to regulate phosphorylation-based signaling pathways. Expanding on this concept, we also review experimental evidence linking disease-related tau species to the misregulation of these pathways. Collectively, the available evidence supports the participation of tau in multiple cellular processes sustaining neuronal and glial function through various mechanisms involving the scaffolding and regulation of selected kinases and phosphatases at discrete subcellular compartments. The notion that the repertoire of tau functions includes a role as a signaling hub should widen our interpretation of experimental results and increase our understanding of tau biology in normal and disease conditions.This work was supported by NIH grants (R01AG067762 and R01AG044372 to NK, R01NS082730 to NK and SB, R01NS118177 and R21NS120126 to GM, R01NS023868 and R01NS041170 to SB), a gift from Neurodegenerative Research Inc. (GM), a Zenith Award from the Alzheimer’s Association (SB), a grant from the Secchia Family Foundation (NK), NIH/National Institute on Aging (NIA) funded Michigan Alzheimer’s Disease Research Center 5P30AG053760 (BC), the Office of the Assistant Secretary of Defense for Health Affairs through the Peer-Reviewed Alzheimer’s Research Program (Award No. W81XWH-20-1-0174 to BC), and an Alzheimer’s Association Research Grant 20-682085 (BC)

Inhibition of fast axonal transport by pathogenic SOD1 involves activation of p38 MAP kinase

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 8 (2013): e65235, doi:10.1371/journal.pone.0065235.Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.Support was provided by 2007/2008 Marine Biological Laboratory summer fellowships and NIH (NS066942A) grants to GM; Howard Hughes Medical Institute-USE Grant #52006287 to Hunter College of CUNY (LM); Muscular Dystrophy Association (MDA) and NIH (R01NS44170) grants to LJH; MDA and NIH (NS23868, NS23320, NS41170) grants to STB; NIH grant MH066179 to GB; NIH grants R01AG031311 and R01NS055951 to DMW; NIH (U01NS05225, R01NS050557, 1RC1NS068391, 1RC2NS070342) grants to RHB; R01NS067206 to DAB; ALS Association grants to GM, AT, RHB, and STB; and ALS/CVS Therapy Alliance grants to RHB, GM, AT, LJH, and DAB. RHB and AT received support from the Angel Fund. RHB also received support from the DeBourgknecht Fund for ALS Research, P2ALS and Project ALS

Defined tau phosphospecies differentially inhibit fast axonal transport through activation of two independent signaling pathways

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Morris, S. L., Tsai, M., Aloe, S., Bechberger, K., Konig, S., Morfini, G., & Brady, S. T. Defined tau phosphospecies differentially inhibit fast axonal transport through activation of two independent signaling pathways. Frontiers in Molecular Neuroscience, 13, (2021): 610037, https://doi.org/10.3389/fnmol.2020.610037.Tau protein is subject to phosphorylation by multiple kinases at more than 80 different sites. Some of these sites are associated with tau pathology and neurodegeneration, but other sites are modified in normal tau as well as in pathological tau. Although phosphorylation of tau at residues in the microtubule-binding repeats is thought to reduce tau association with microtubules, the functional consequences of other sites are poorly understood. The AT8 antibody recognizes a complex phosphoepitope site on tau that is detectable in a healthy brain but significantly increased in Alzheimer’s disease (AD) and other tauopathies. Previous studies showed that phosphorylation of tau at the AT8 site leads to exposure of an N-terminal sequence that promotes activation of a protein phosphatase 1 (PP1)/glycogen synthase 3 (GSK3) signaling pathway, which inhibits kinesin-1-based anterograde fast axonal transport (FAT). This finding suggests that phosphorylation may control tau conformation and function. However, the AT8 includes three distinct phosphorylated amino acids that may be differentially phosphorylated in normal and disease conditions. To evaluate the effects of specific phosphorylation sites in the AT8 epitope, recombinant, pseudophosphorylated tau proteins were perfused into the isolated squid axoplasm preparation to determine their effects on axonal signaling pathways and FAT. Results from these studies suggest a mechanism where specific phosphorylation events differentially impact tau conformation, promoting activation of independent signaling pathways that differentially affect FAT. Implications of findings here to our understanding of tau function in health and disease conditions are discussed.This research was funded by NIH grants R21NS096642 (GM); 1R01NS118177-01A1 (GM), R01 NS082730 (SB), a Zenith Award from the Alzheimer’s Association (SB), and a grant from the Tau Consortium/Rainwater Foundation (SB)

The NF2 tumor suppressor regulates microtubule-based vesicle trafficking via a novel Rac, MLK and p38SAPK pathway

© Macmillan Publishers, 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Oncogene 32 (2013): 1135–1143, doi:10.1038/onc.2012.135.Neurofibromatosis type 2 patients develop schwannomas, meningiomas and ependymomas resulting from mutations in the tumor suppressor gene, NF2, encoding a membrane-cytoskeleton adapter protein called merlin. Merlin regulates contact inhibition of growth and controls the availability of growth factor receptors at the cell surface. We tested if microtubule-based vesicular trafficking might be a mechanism by which merlin acts. We found that schwannoma cells, containing merlin mutations and constitutive activation of the Rho/Rac family of GTPases, had decreased intracellular vesicular trafficking relative to normal human Schwann cells. In Nf2−/− mouse Schwann (SC4) cells, re-expression of merlin as well as inhibition of Rac or its effector kinases, MLK and p38SAPK, each increased the velocity of Rab6 positive exocytic vesicles. Conversely, an activated Rac mutant decreased Rab6 vesicle velocity. Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38SAPK kinase. Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.This work was supported by NIH R01 CA118032 (to NR), and MBL research fellowships (to NR and GM), NIH R01 NS23868 (to STB)

Frontotemporal lobar dementia mutant tau impairs axonal transport through a protein phosphatase 1γ-dependent mechanism

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Combs, B., Christensen, K. R., Richards, C., Kneynsberg, A., Mueller, R. L., Morris, S. L., Morfini, G., Brady, S. T., & Kanaan, N. M. Frontotemporal lobar dementia mutant tau impairs axonal transport through a protein phosphatase 1γ-dependent mechanism. Journal of Neuroscience, 41(45), (2021): 9431-9451, https://doi.org/10.1523/JNEUROSCI.1914-20.2021.Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms (α, β, and γ) to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1β, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons.This work was supported by National Institutes of Health (NIH) Grants R01 NS082730 (N.M.K. and S.T.B.), R01 AG044372 (N.M.K.), and R01 AG067762 (N.M.K.); NIH/National Institute on Aging, Michigan Alzheimer's Disease Research Center Grant 5P30AG053760 (B.C.); Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Alzheimer's Research Program Award W81XWH-20-1-0174 (B.C.); Alzheimer's Association Research Grants 20-682085 (B.C.), R01 NS118177 (G.A.M.), and R21NS120126 (G.A.M.); Zenith Award from the Alzheimer's Association (S.T.B.); Tau Consortium/Rainwater Foundation (S.T.B.); Neurodegenerative Research (G.A.M.); and the Secchia Family Foundation (N.M.K.)