Pathogenic forms of tau inhibit kinesin-dependent axonal transport through a mechanism involving activation of axonal phosphotransferases

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Neuroscience 31 (2011): 9858-9868, doi:10.1523/JNEUROSCI.0560-11.2011.Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2–18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau.This work was supported by NIH Grants T32 AG020506-07 (N.M.K.); AG09466 (L.I.B.); and NS23868, NS23320, and NS41170 (S.T.B.); as well as 2007/2008 MBL Summer Research Fellowships and an ALS/CVS Therapy Alliance grant (G.M.)

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

Prion protein inhibits fast axonal transport through a mechanism involving casein kinase 2

Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrPc) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrPc that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrPc. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrPc on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases

Data from: Prion protein inhibits fast axonal transport through a mechanism involving casein kinase 2

Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrPc) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrPc that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrPc. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrPc on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases

Supplementary Figure. 3 The CK2 pharmacological inhibitor DMAT does not interfere with FAT.

Supplementary Figure. 3 The CK2 pharmacological inhibitor DMAT does not interfere with FAT

PrP induces CK2-mediated phosphorylation of kinesin light chain subunits and detachment of conventional kinesin from membrane cargoes.

<p>(<b>A-B</b>) Quantitative immunoblot analysis of kinesin-1 from two pair of sister axoplasms incubated either with control PrP-Scram or PrP_{<b>106-126</b>} peptides and (<b>C-D</b>) primary embryonic mouse cortical neurons cultured for 3 days in vitro treated for one hour. Antibody 63–90 preferentially recognizes kinesin-1 light chains (KLCs) when they are not phosphorylated by CK2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188340#pone.0188340.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188340#pone.0188340.ref035" target="_blank">35</a>]. (<b>B and D</b>) Quantitation graph bars show that PrP_{<b>106-126</b>} decreased the immunoreactivity for 63–90 versus control PrP-Scram treated axoplasms and neurons respectively. Note a significant reduction (<b>B</b>) of immunoreactivity when neurons were treated with PrP_{<b>106-126</b>} compared to PrP-Scram; (n = 5, number of independent experiments. p = 0.0313, significance was assessed at P < 0.05). (<b>D</b>) Significant reduction of 63–90 immunoreactivity in axoplasms incubated with PrP_{<b>106-126</b>} compared to control PrP-Scram, (n = 3; number of independent experiments. p = 0.0355, significance was assessed at P < 0.05). (<b>E</b>) Vesicles purified from sister axoplasms by vesicle flotation assays perfused with control PrP-Scram and PrP_{<b>106-126</b>} synthetic peptide were assayed by Western blot for KHC and TrkB. TrkB was used as membrane protein marker and for loading control. (<b>F</b>) Quantitation graph bars shows a significant reduction of kinesin-1 association to purified vesicles in PrP_{<b>106-126</b>} incubated extruded axoplasms compared to control PrP-Scram treated axoplasms, (n = 3, number of independent experiments; significance was assessed at P < 0.05). Taken together, these experiments suggest that PrP_{<b>106-126</b>} increases the intracellular activity of CK2, which in turn results in KLCs phosphorylation and kinesin-1 release from its cargo vesicles.</p

Figure 2. Prion inhibits fast axonal transport of mitochondria in primary hippocampal cultured neurons.

Figure 2. Prion inhibits fast axonal transport of mitochondria in primary hippocampal cultured neurons

Supplementary Figure 2. PrP Full length (PrP-FL) inhibits fast axonal transport.

Supplementary Figure 2. PrP Full length (PrP-FL) inhibits fast axonal transport