Gustavo Pigino

Axonal Transport Defects in Neurodegenerative Diseases

Adult-onset neurodegenerative diseases (AONDs) comprise a heterogeneous group of neurological disorders characterized by a progressive, age-dependent decline in neuronal function and loss of selected neuronal populations. Alterations in synaptic function and axonal connectivity represent early and critical pathogenic events in AONDs, but molecular mechanisms underlying these defects remain elusive. The large size and complex subcellular architecture of neurons render them uniquely vulnerable to alterations in axonal transport (AT). Accordingly, deficits in AT have been documented in most AONDs, suggesting a common defect acquired through different pathogenic pathways. These observations suggest that many AONDs can be categorized as dysferopathies, diseases in which alterations in AT represent a critical component in pathogenesis. Topics here address various molecular mechanisms underlying alterations in AT in several AONDs. Illumination of such mechanisms provides a framework for the development of novel therapeutic strategies aimed to prevent axonal and synaptic dysfunction in several major AONDs.

Altered Metabolism of the Amyloid β Precursor Protein Is Associated with Mitochondrial Dysfunction in Down's Syndrome

Most Downs syndrome (DS) patients develop Alzheimers disease (AD) neuropathology. Astrocyte and neuronal cultures derived from fetal DS brain show alterations in the processing of amyloid beta precursor protein (AbetaPP), including increased levels of AbetaPP and C99, reduced levels of secreted AbetaPP (AbetaPPs) and C83, and intracellular accumulation of insoluble Abeta42. This pattern of AbetaPP processing is recapitulated in normal astrocytes by inhibition of mitochondrial metabolism, consistent with impaired mitochondrial function in DS astrocytes. Intracellular Abeta42 and reduced AbetaPPs are also detected in DS and AD brains. The survival of DS neurons is markedly increased by recombinant or astrocyte-produced AbetaPPs, suggesting that AbetaPPs may be a neuronal survival factor. Thus, mitochondrial dysfunction in DS may lead to intracellular deposition of Abeta42, reduced levels of AbetaPPs, and a chronic state of increased neuronal vulnerability.

Alzheimer's Presenilin 1 Mutations Impair Kinesin-Based Axonal Transport

Several lines of evidence indicate that alterations in axonal transport play a critical role in Alzheimers disease (AD) neuropathology, but the molecular mechanisms that control this process are not understood fully. Recent work indicates that presenilin 1 (PS1) interacts with glycogen synthase kinase 3β (GSK3β). In vivo, GSK3β phosphorylates kinesin light chains (KLC) and causes the release of kinesin-I from membrane-bound organelles (MBOs), leading to a reduction in kinesin-I driven motility (Morfini et al., 2002b). To characterize a potential role for PS1 in the regulation of kinesin-based axonal transport, we used PS1-/- and PS1 knock-inM146V (KIM146V) mice and cultured cells. We show that relative levels of GSK3β activity were increased in cells either in the presence of mutant PS1 or in the absence of PS1 (PS1-/-). Concomitant with increased GSK3β activity, relative levels of KLC phosphorylation were increased, and the amount of kinesin-I bound to MBOs was reduced. Consistent with a deficit in kinesin-I-mediated fast axonal transport, densities of synaptophysin- and syntaxin-I-containing vesicles and mitochondria were reduced in neuritic processes of KIM146V hippocampal neurons. Similarly, we found reduced levels of PS1, amyloid precursor protein, and synaptophysin in sciatic nerves of KIM146V mice. Thus PS1 appears to modulate GSK3β activity and the release of kinesin-I from MBOs at sites of vesicle delivery and membrane insertion. These findings suggest that mutations in PS1 may compromise neuronal function by affecting GSK-3 activity and kinesin-I-based motility.

A novel CDK5‐dependent pathway for regulating GSK3 activity and kinesin‐driven motility in neurons

Neuronal transmission of information requires polarized distribution of membrane proteins within axonal compartments. Membrane proteins are synthesized and packaged in membrane‐bounded organelles (MBOs) in neuronal cell bodies and later transported to axons by microtubule‐dependent motor proteins. Molecular mechanisms underlying targeted delivery of MBOs to discrete axonal subdomains (i.e. nodes of Ranvier or presynaptic terminals) are poorly understood, but regulatory pathways for microtubule motors may be an essential step. In this work, pharmacological, biochemical and in vivo experiments define a novel regulatory pathway for kinesin‐driven motility in axons. This pathway involves enzymatic activities of cyclin‐dependent kinase 5 (CDK5), protein phosphatase 1 (PP1) and glycogen synthase kinase‐3 (GSK3). Inhibition of CDK5 activity in axons leads to activation of GSK3 by PP1, phosphorylation of kinesin light chains by GSK3 and detachment of kinesin from transported cargoes. We propose that regulating the activity and localization of components in this pathway allows nerve cells to target organelle delivery to specific subcellular compartments. Implications of these findings for pathogenesis of neurodegenerative diseases such as Alzheimers disease are discussed.

Pathogenic huntingtin inhibits fast axonal transport by activating JNK3 and phosphorylating kinesin

Selected vulnerability of neurons in Huntingtons disease suggests that alterations occur in a cellular process that is particularly critical for neuronal function. Supporting this idea, pathogenic Htt (polyQ-Htt) inhibits fast axonal transport (FAT) in various cellular and animal models of Huntingtons disease (mouse and squid), but the molecular basis of this effect remains unknown. We found that polyQ-Htt inhibited FAT through a mechanism involving activation of axonal cJun N-terminal kinase (JNK). Accordingly, we observed increased activation of JNK in vivo in cellular and mouse models of Huntingtons disease. Additional experiments indicated that the effects of polyQ-Htt on FAT were mediated by neuron-specific JNK3 and not by ubiquitously expressed JNK1, providing a molecular basis for neuron-specific pathology in Huntingtons disease. Mass spectrometry identified a residue in the kinesin-1 motor domain that was phosphorylated by JNK3 and this modification reduced kinesin-1 binding to microtubules. These data identify JNK3 as a critical mediator of polyQ-Htt toxicity and provide a molecular basis for polyQ-Htt–induced inhibition of FAT.

JNK mediates pathogenic effects of polyglutamine- expanded androgen receptor on fast axonal transport

Expansion of the polyglutamine (polyQ) stretch in the androgen receptor (AR) protein leads to spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disease characterized by lower motor neuron degeneration. The pathogenic mechanisms underlying SBMA remain unknown, but recent experiments show that inhibition of fast axonal transport (FAT) by polyQ-expanded proteins, including polyQ-AR, represents a new cytoplasmic pathogenic lesion. Using pharmacological, biochemical and cell biological experiments, we found a new pathogenic pathway that is affected in SBMA and results in compromised FAT. PolyQ-AR inhibits FAT in a human cell line and in squid axoplasm through a pathway that involves activation of cJun N-terminal kinase (JNK) activity. Active JNK phosphorylated kinesin-1 heavy chains and inhibited kinesin-1 microtubule-binding activity. JNK inhibitors prevented polyQ-AR–mediated inhibition of FAT and reversed suppression of neurite formation by polyQ-AR. We propose that JNK represents a promising target for therapeutic interventions in SBMA.

The amino terminus of tau inhibits kinesin-dependent axonal transport: implications for filament toxicity.

The neuropathology of Alzheimers disease (AD) and other tauopathies is characterized by filamentous deposits of the microtubule‐associated protein tau, but the relationship between tau polymerization and neurotoxicity is unknown. Here, we examined effects of filamentous tau on fast axonal transport (FAT) using isolated squid axoplasm. Monomeric and filamentous forms of recombinant human tau were perfused in axoplasm, and their effects on kinesin‐ and dynein‐dependent FAT rates were evaluated by video microscopy. Although perfusion of monomeric tau at physiological concentrations showed no effect, tau filaments at the same concentrations selectively inhibited anterograde (kinesin‐dependent) FAT, triggering the release of conventional kinesin from axoplasmic vesicles. Pharmacological experiments indicated that the effect of tau filaments on FAT is mediated by protein phosphatase 1 (PP1) and glycogen synthase kinase‐3 (GSK‐3) activities. Moreover, deletion analysis suggested that these effects depend on a conserved 18‐amino‐acid sequence at the amino terminus of tau. Interestingly, monomeric tau isoforms lacking the C‐terminal half of the molecule (including the microtubule binding region) recapitulated the effects of full‐length filamentous tau. Our results suggest that pathological tau aggregation contributes to neurodegeneration by altering a regulatory pathway for FAT.

Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta.

The pathological mechanism by which Aβ causes neuronal dysfunction and death remains largely unknown. Deficiencies in fast axonal transport (FAT) were suggested to play a crucial role in neuronal dysfunction and loss for a diverse set of dying back neuropathologies including Alzheimers disease (AD), but the molecular basis for pathological changes in FAT were undetermined. Recent findings indicate that soluble intracellular oligomeric Aβ (oAβ) species may play a critical role in AD pathology. Real-time analysis of vesicle mobility in isolated axoplasms perfused with oAβ showed bidirectional axonal transport inhibition as a consequence of endogenous casein kinase 2 (CK2) activation. Conversely, neither unaggregated amyloid beta nor fibrillar amyloid beta affected FAT. Inhibition of FAT by oAβ was prevented by two specific pharmacological inhibitors of CK2, as well as by competition with a CK2 substrate peptide. Furthermore, perfusion of axoplasms with active CK2 mimics the inhibitory effects of oAβ on FAT. Both oAβ and CK2 treatment of axoplasm led to increased phosphorylation of kinesin-1 light chains and subsequent release of kinesin from its cargoes. Therefore pharmacological modulation of CK2 activity may represent a promising target for therapeutic intervention in AD.

Pathogenic Forms of Tau Inhibit Kinesin-Dependent Axonal Transport through a Mechanism Involving Activation of Axonal Phosphotransferases

Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimers 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.

Complex environment experience rescues impaired neurogenesis, enhances synaptic plasticity, and attenuates neuropathology in familial Alzheimer’s disease-linked APPswe/PS1ΔE9 mice

Experience in complex environments induces numerous forms of brain plasticity, improving structure and function. It has been long debated whether brain plasticity can be induced under neuropathological conditions, such as Alzheimers disease (AD), to an extent that would reduce neuropathology, rescue brain structure, and restore its function. Here we show that experience in a complex environment rescues a significant impairment of hippocampal neurogenesis in transgenic mice harboring familial AD‐linked mutant APPswe/PS1ΔE9. Proliferation of hippocampal cells is enhanced significantly after enrichment, and these proliferating cells mature to become new neurons and glia. Enhanced neurogenesis was accompanied by a significant reduction in levels of hyperphosphorylated tau and oligomeric Aβ, the precursors of AD hallmarks, in the hippocampus and cortex of enriched mice. Interestingly, enhanced expression of the neuronal anterograde motor kinesin‐1 was observed, suggesting enhanced axonal transport in hippocampal and cortical neurons after enrichment. Examination of synaptic physiology revealed that environmental experience significantly enhanced hippocampal long‐term potentiation, without notable alterations in basal synaptic transmission. This study suggests that environmental modulation can rescue the impaired phenotype of the Alzheimers brain and that induction of brain plasticity may represent therapeutic and preventive avenues in AD.—Y.‐S. Hu, P. Xu, G. Pigino, S. T. Brady, J. Larson, O. Lazarov. Complex environment experience rescues impaired neurogenesis, enhances synaptic plasticity, and attenuates neuropathology in familial Alzheimers disease‐linked APPswe/PS1ΔE9 mice. FASEB J. 24, 1667–1681 (2010). www.fasebj.org