Protein Misfolding, Signaling Abnormalities and Altered Fast Axonal Transport: Implications for Alzheimer and Prion Diseases.

Histopathological studies revealed that progressive neuropathies including Alzheimer, and Prion diseases among others, include accumulations of misfolded proteins intracellularly, extracellularly, or both. Experimental evidence suggests that among the accumulated misfolded proteins, small soluble oligomeric conformers represent the most neurotoxic species. Concomitant phenomena shared by different protein misfolding diseases includes alterations in phosphorylation-based signaling pathways synaptic dysfunction, and axonal pathology, but mechanisms linking these pathogenic features to aggregated neuropathogenic proteins remain unknown. Relevant to this issue, results from recent work revealed inhibition of fast axonal transport (AT) as a novel toxic effect elicited by oligomeric forms of amyloid beta and cellular prion protein PrPC, signature pathological proteins associated with Alzheimer and Prion diseases, respectively. Interestingly, the toxic effect of these oligomers was fully prevented by pharmacological inhibitors of casein kinase 2 (CK2), a remarkable discovery with major implications for the development of pharmacological target-driven therapeutic intervention for Alzheimer and Prion diseases.

Nrf2 stabilization prevents critical oxidative damage in Down syndrome cells.

Mounting evidence implicates chronic oxidative stress as a critical driver of the aging process. Down syndrome (DS) is characterized by a complex phenotype, including early senescence. DS cells display increased levels of reactive oxygen species (ROS) and mitochondrial structural and metabolic dysfunction, which are counterbalanced by sustained Nrf2-mediated transcription of cellular antioxidant response elements (ARE). Here, we show that caspase 3/PKCδdependent activation of the Nrf2 pathway in DS and Dp16 (a mouse model of DS) cells is necessary to protect against chronic oxidative damage and to preserve cellular functionality. Mitochondria-targeted catalase (mCAT) significantly reduced oxidative stress, restored mitochondrial structure and function, normalized replicative and wound healing capacity, and rendered the Nrf2-mediated antioxidant response dispensable. These results highlight the critical role of Nrf2/ARE in the maintenance of DS cell homeostasis and validate mitochondrial-specific interventions as a key aspect of antioxidant and antiaging therapies.

APP/Go protein Gßγ-complex signaling mediates Aß degeneration and cognitive impairment in Alzheimer's disease models.

Deposition of amyloid-ß (Aß), the proteolytic product of the amyloid precursor protein (APP), might cause neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the direct involvement of APP in the mechanism of Aß-induced degeneration in AD remains on debate. Here, we analyzed the interaction of APP with heterotrimeric Go protein in primary hippocampal cultures and found that Aß deposition dramatically enhanced APP-Go protein interaction in dystrophic neurites. APP overexpression rendered neurons vulnerable to Aß toxicity by a mechanism that required Go-Gßγ complex signaling and p38-mitogen-activated protein kinase activation. Gallein, a selective pharmacological inhibitor of Gßγ complex, inhibited Aß-induced dendritic and axonal dystrophy, abnormal tau phosphorylation, synaptic loss, and neuronal cell death in hippocampal neurons expressing endogenous protein levels. In the 3xTg-AD mice, intrahippocampal application of gallein reversed memory impairment associated with early Aß pathology. Our data provide further evidence for the involvement of APP/Go protein in Aß-induced degeneration and reveal that Gßγ complex is a signaling target potentially relevant for developing therapies for halting Aß degeneration in AD.

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.

Conventional kinesin: Biochemical heterogeneity and functional implications in health and disease.

Intracellular trafficking events powered by microtubule-based molecular motors facilitate the targeted delivery of selected molecular components to specific neuronal subdomains. Within this context, we provide a brief review of mechanisms underlying the execution of axonal transport (AT) by conventional kinesin, the most abundant kinesin-related motor protein in the mature nervous system. We emphasize the biochemical heterogeneity of this multi-subunit motor protein, further discussing its significance in light of recent discoveries revealing its regulation by various protein kinases. In addition, we raise issues relevant to the mode of conventional kinesin attachment to cargoes and examine recent evidence linking alterations in conventional kinesin phosphorylation to the pathogenesis of adult-onset neurodegenerative diseases.

The sphingolipid psychosine inhibits fast axonal transport in Krabbe disease by activation of GSK3ß and deregulation of molecular motors.

Loss of function of galactosylceramidase lysosomal activity causes demyelination and vulnerability of various neuronal populations in Krabbe disease. Psychosine, a lipid-raft-associated sphingolipid that accumulates in this disease, is thought to trigger these abnormalities. Myelin-free in vitro analyses showed that psychosine inhibited fast axonal transport through the activation of axonal PP1 and GSK3ß in the axon. Abnormal levels of activated GSK3ß and abnormally phosphorylated kinesin light chains were found in nerve samples from a mouse model of Krabbe disease. Administration of GSK3ß inhibitors significantly ameliorated transport defects in vitro and in vivo in peripheral axons of the mutant mouse. This study identifies psychosine as a pathogenic sphingolipid able to block fast axonal transport and is the first to provide a molecular mechanism underlying dying-back degeneration in this genetic leukodystrophy.

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.

Molecular mechanisms of environmental enrichment: impairments in Akt/GSK3ß, neurotrophin-3 and CREB signaling.

Experience of mice in a complex environment enhances neurogenesis and synaptic plasticity in the hippocampus of wild type and transgenic mice harboring familial Alzheimer's disease (FAD)-linked APPswe/PS1ΔE9. In FAD mice, this experience also reduces levels of tau hyperphosphorylation and oligomeric ß-amyloid. Although environmental enrichment has significant effects on brain plasticity and neuropathology, the molecular mechanisms underlying these effects are unknown. Here we show that environmental enrichment upregulates the Akt pathway, leading to the downregulation of glycogen synthase kinase 3ß (GSK3ß), in wild type but not FAD mice. Several neurotrophic signaling pathways are activated in the hippocampus of both wild type and FAD mice, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and this increase is accompanied by the upregulation of the BDNF receptor, tyrosine kinase B (TrkB). Interestingly, neurotrophin-3 (NT-3) is upregulated in the brains of wild type mice but not FAD mice, while insulin growth factor-1 (IGF-1) is upregulated exclusively in the brains of FAD mice. Upregulation of neurotrophins is accompanied by the increase of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus following environmental enrichment. Most importantly, we observed a significant increase in levels of cAMP response element- binding (CREB) transcripts in the hippocampus of wild type and FAD mice following environmental enrichment. However, CREB phosphorylation, a critical step for the initiation of learning and memory-required gene transcription, takes place in the hippocampus of wild type but not of FAD mice. These results suggest that experience of wild type mice in a complex environmental upregulates critical signaling that play a major role in learning and memory in the hippocampus. However, in FAD mice, some of these pathways are impaired and cannot be rescued by environmental enrichment.

Axonal degeneration in Alzheimer's disease: when signaling abnormalities meet the axonal transport system.

Alzheimer's disease (AD) is characterized by progressive, age-dependent degeneration of neurons in the central nervous system. A large body of evidence indicates that neurons affected in AD follow a dying-back pattern of degeneration, where abnormalities in synaptic function and axonal connectivity long precede somatic cell death. Mechanisms underlying dying-back degeneration of neurons in AD remain elusive but several have been proposed, including deficits in fast axonal transport (FAT). Accordingly, genetic evidence linked alterations in FAT to dying-back degeneration of neurons, and FAT defects have been widely documented in various AD models. In light of these findings, we discuss experimental evidence linking several AD-related pathogenic polypeptides to aberrant activation of signaling pathways involved in the phosphoregulation of microtubule-based motor proteins. While each pathway appears to affect FAT in a unique manner, in the context of AD, many of these pathways might work synergistically to compromise the delivery of molecular components critical for the maintenance and function of synapses and axons. Therapeutic approaches aimed at preventing FAT deficits by normalizing the activity of specific protein kinases may help prevent degeneration of vulnerable neurons in AD.

Phosphorylation in the amino terminus of tau prevents inhibition of anterograde axonal transport.

Alzheimer's disease (AD) and other tauopathies are characterized by fibrillar inclusions composed of the microtubule-associated protein, tau. Recently, we demonstrated that the N-terminus of tau (amino acids [aa] 2-18) in filamentous aggregates or N-terminal tau isoforms activate a signaling cascade involving protein phosphatase 1 and glycogen synthase kinase 3 that results in inhibition of anterograde fast axonal transport (FAT). We have termed the functional motif comprised of aa 2-18 in tau the phosphatase-activating domain (PAD). Here, we show that phosphorylation of tau at tyrosine 18, which is a fyn phosphorylation site within PAD, prevents inhibition of anterograde FAT induced by both filamentous tau and 6D tau. Moreover, Fyn-mediated phosphorylation of tyrosine 18 is reduced in disease-associated forms of tau (e.g., tau filaments). A novel PAD-specific monoclonal antibody revealed that exposure of PAD in tau occurs before and more frequently than tyrosine 18 phosphorylation in the evolution of tangle formation in AD. These results indicate that N-terminal phosphorylation may constitute a regulatory mechanism that controls tau-mediated inhibition of anterograde FAT 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 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.

Complex environment experience rescues impaired neurogenesis, enhances synaptic plasticity, and attenuates neuropathology in familial Alzheimer's disease-linked APPswe/PS1DeltaE9 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 Alzheimer's 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/PS1DeltaE9. 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 Abeta, 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 Alzheimer's brain and that induction of brain plasticity may represent therapeutic and preventive avenues in AD.

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.

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

Selected vulnerability of neurons in Huntington's 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 Huntington's 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 Huntington's 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 Huntington's 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.

Synaptic transmission block by presynaptic injection of oligomeric amyloid beta.

Early Alzheimer's disease (AD) pathophysiology is characterized by synaptic changes induced by degradation products of amyloid precursor protein (APP). The exact mechanisms of such modulation are unknown. Here, we report that nanomolar concentrations of intraaxonal oligomeric (o)Abeta42, but not oAbeta40 or extracellular oAbeta42, acutely inhibited synaptic transmission at the squid giant synapse. Further characterization of this phenotype demonstrated that presynaptic calcium currents were unaffected. However, electron microscopy experiments revealed diminished docked synaptic vesicles in oAbeta42-microinjected terminals, without affecting clathrin-coated vesicles. The molecular events of this modulation involved casein kinase 2 and the synaptic vesicle rapid endocytosis pathway. These findings open the possibility of a new therapeutic target aimed at ameliorating synaptic dysfunction in AD.

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

The neuropathology of Alzheimer's 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.

Conventional kinesin holoenzymes are composed of heavy and light chain homodimers.

Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.

Approaches to kinesin-1 phosphorylation.

Most mammalian proteins undergo reversible protein modification after or during synthesis. These modifications are associated, for the most part, with changes in protein functionality. Protein phosphorylation is the most common posttranslational modification in mammalian cells, regulating critical cellular processes that include cell division, differentiation, growth, and cell-cell signaling as well as fast axonal transport (FAT). Evidence has accumulated that kinesin-1 phosphorylation plays a key regulatory role in kinesin-based FAT. Multiple kinase and phosphatase activities with the ability to regulate kinesin-1 function and FAT have been identified. Moreover, additional pathways are likely to exist for regulating FAT through reversible phosphorylation/dephosphorylation of specific motor protein subunits. The present chapter describes specific biochemical assays to determine, or to perturb experimentally, the phosphorylation status of kinesin-1. These protocols provide assays for characterization of novel effectors (i.e., trophic factors, neurotransmitters, pharmacological inhibitors, pathogenic protein expression, etc.) that affect the phosphorylation status of kinesin-1. Finally, in vitro phosphorylation assays suitable for analyzing the direct effects of specific kinases on kinesin-1 are provided.

Impairments in fast axonal transport and motor neuron deficits in transgenic mice expressing familial Alzheimer's disease-linked mutant presenilin 1.

Presenilins (PS) play a central role in gamma-secretase-mediated processing of beta-amyloid precursor protein (APP) and numerous type I transmembrane proteins. Expression of mutant PS1 variants causes familial forms of Alzheimer's disease (FAD). In cultured mammalian cells that express FAD-linked PS1 variants, the intracellular trafficking of several type 1 membrane proteins is altered. We now report that the anterograde fast axonal transport (FAT) of APP and Trk receptors is impaired in the sciatic nerves of transgenic mice expressing two independent FAD-linked PS1 variants. Furthermore, FAD-linked PS1 mice exhibit a significant increase in phosphorylation of the cytoskeletal proteins tau and neurofilaments in the spinal cord. Reductions in FAT and phosphorylation abnormalities correlated with motor neuron functional deficits. Together, our data suggests that defects in anterograde FAT may underlie FAD-linked PS1-mediated neurodegeneration through a mechanism involving impairments in neurotrophin signaling and synaptic dysfunction.

1-Methyl-4-phenylpyridinium induces synaptic dysfunction through a pathway involving caspase and PKCdelta enzymatic activities.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration has been used, in various mammalian species, as an experimental model of Parkinson's disease. The pathogenesis for such pharmacologically induced Parkinson's disease involves 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. This metabolite produces rapid degeneration of nigrostriatal dopaminergic neurons, which causes the parkinsonian syndrome. In this work, we show that injection of MPP+ into the presynaptic terminal of the squid giant synapse blocks synaptic transmission without affecting the presynaptic action potential or the presynaptic calcium currents. These effects of MPP+ were mimicked by the injection of an active form of caspase-3 and prevented by inhibitors of caspase-3 and protein kinase C delta. Ultrastructurally, MPP+-injected synapses showed a dramatic reduction in the number of neurotransmitter vesicles at the presynaptic active zone, as compared with control synapses. Otherwise, normal docking and clathrin-coated vesicles were observed, albeit at much reduced numbers. These results indicate that MPP+ acutely reduces presynaptic vesicular availability, not release, and that MPP+-induced pathogenesis results from presynaptic dysfunction that leads, secondarily, to dying-back neuropathy in affected neurons.