Gorazd B. Stokin

Axonal Transport of Amyloid Precursor Protein Is Mediated by Direct Binding to the Kinesin Light Chain Subunit of Kinesin-I

We analyzed the mechanism of axonal transport of the amyloid precursor protein (APP), which plays a major role in the development of Alzheimers disease. Coimmunoprecipitation, sucrose gradient, and direct in vitro binding demonstrated that APP forms a complex with the microtubule motor, conventional kinesin (kinesin-I), by binding directly to the TPR domain of the kinesin light chain (KLC) subunit. The estimated apparent Kd for binding is 15-20 nM, with a binding stoichiometry of two APP per KLC. In addition, association of APP with microtubules and axonal transport of APP is greatly decreased in a gene-targeted mouse mutant of the neuronally enriched KLC1 gene. We propose that one of the normal functions of APP may be as a membrane cargo receptor for kinesin-I and that KLC is important for kinesin-I-driven transport of APP into axons.

Increased App Expression in a Mouse Model of Down's Syndrome Disrupts NGF Transport and Causes Cholinergic Neuron Degeneration

Degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive dysfunction in Alzheimers disease (AD) and Downs syndrome (DS). We used Ts65Dn and Ts1Cje mouse models of DS to show that the increased dose of the amyloid precursor protein gene, App, acts to markedly decrease NGF retrograde transport and cause degeneration of BFCNs. NGF transport was also decreased in mice expressing wild-type human APP or a familial AD-linked mutant APP; while significant, the decreases were less marked and there was no evident degeneration of BFCNs. Because of evidence suggesting that the NGF transport defect was intra-axonal, we explored within cholinergic axons the status of early endosomes (EEs). NGF-containing EEs were enlarged in Ts65Dn mice and their App content was increased. Our study thus provides evidence for a pathogenic mechanism for DS in which increased expression of App, in the context of trisomy, causes abnormal transport of NGF and cholinergic neurodegeneration.

Axonal stress kinase activation and tau misbehavior induced by kinesin-1 transport defects.

Many neurodegenerative diseases exhibit axonal pathology, transport defects, and aberrant phosphorylation and aggregation of the microtubule binding protein tau. While mutant tau protein in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP17) causes aberrant microtubule binding and assembly of tau into filaments, the pathways leading to tau-mediated neurotoxicity in Alzheimers disease and other neurodegenerative disorders in which tau protein is not genetically modified remain unknown. To test the hypothesis that axonal transport defects alone can cause pathological abnormalities in tau protein and neurodegeneration in the absence of mutant tau or amyloid β deposits, we induced transport defects by deletion of the kinesin light chain 1 (KLC1) subunit of the anterograde motor kinesin-1. We found that upon aging, early selective axonal transport defects in mice lacking the KLC1 protein (KLC1−/−) led to axonopathies with cytoskeletal disorganization and abnormal cargo accumulation. In addition, increased c-jun N-terminal stress kinase activation colocalized with aberrant tau in dystrophic axons. Surprisingly, swollen dystrophic axons exhibited abnormal tau hyperphosphorylation and accumulation. Thus, directly interfering with axonal transport is sufficient to activate stress kinase pathways initiating a biochemical cascade that drives normal tau protein into a pathological state found in a variety of neurodegenerative disorders including Alzheimers disease.

Examination of potential mechanisms of amyloid-induced defects in neuronal transport

Microtubule-based neuronal transport pathways are impaired during the progression of Alzheimers disease and other neurodegenerative conditions. However, mechanisms leading to defects in transport remain to be determined. We quantified morphological changes in neuronal cells following treatment with fibrils and unaggregated peptides of beta-amyloid (Abeta). Abeta fibrils induce axonal and dendritic swellings indicative of impaired transport. In contrast, Abeta peptides induce a necrotic phenotype in both neurons and non-neuronal cells. We tested several popular hypotheses by which aggregated Abeta could disrupt transport. Using fluorescent polystyrene beads, we developed experimental models of physical blockage and localized release of reactive oxygen species (ROS) that reliably induce swellings. Like the beads, Abeta fibrils localize in close proximity to swellings; however, fibril internalization is not required for disrupting transport. ROS and membrane permeability are also unlikely to be responsible for fibril-mediated toxicity. Collectively, our results indicate that multiple initiating factors converge upon pathways of defective transport.

Linking molecular motors to Alzheimer’s disease

It is currently thought that Alzheimers disease develops due to aberrant generation of amyloid-beta peptides. However, the mechanisms underlying the aberrant generation of amyloid-beta peptides remain unknown. An emerging concept suggests that impaired axonal transport may play a pivotal role in the aberrant generation of amyloid-beta peptides. Here we review and discuss advances in understanding AD with the primary focus on the possible role of molecular motors and axonal transport in its pathogenesis.

Neuropsychiatric symptoms, APOE ε4, and the risk of incident dementia: A population-based study

Objective:To investigate the population-based interaction between a biological variable (APOE &egr;4), neuropsychiatric symptoms, and the risk of incident dementia among subjects with prevalent mild cognitive impairment (MCI). Methods:We prospectively followed 332 participants with prevalent MCI (aged 70 years and older) enrolled in the Mayo Clinic Study of Aging for a median of 3 years. The diagnoses of MCI and dementia were made by an expert consensus panel based on published criteria, after reviewing neurologic, cognitive, and other pertinent data. Neuropsychiatric symptoms were determined at baseline using the Neuropsychiatric Inventory Questionnaire. We used Cox proportional hazards models, with age as a time scale, to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). Models were adjusted for sex, education, and medical comorbidity. Results:Baseline agitation, nighttime behaviors, depression, and apathy significantly increased the risk of incident dementia. We observed additive interactions between APOE &egr;4 and depression (joint effect HR = 2.21; 95% CI = 1.24–3.91; test for additive interaction, p < 0.001); and between APOE &egr;4 and apathy (joint effect HR = 1.93; 95% CI = 0.93–3.98; test for additive interaction, p = 0.031). Anxiety, irritability, and appetite/eating were not associated with increased risk of incident dementia. Conclusions:Among prevalent MCI cases, baseline agitation, nighttime behaviors, depression, and apathy elevated the risk of incident dementia. There was a synergistic interaction between depression or apathy and APOE &egr;4 in further elevating the risk of incident dementia.Objective: To investigate the population-based interaction between a biological variable (APOE ε4), neuropsychiatric symptoms, and the risk of incident dementia among subjects with prevalent mild cognitive impairment (MCI). Methods: We prospectively followed 332 participants with prevalent MCI (aged 70 years and older) enrolled in the Mayo Clinic Study of Aging for a median of 3 years. The diagnoses of MCI and dementia were made by an expert consensus panel based on published criteria, after reviewing neurologic, cognitive, and other pertinent data. Neuropsychiatric symptoms were determined at baseline using the Neuropsychiatric Inventory Questionnaire. We used Cox proportional hazards models, with age as a time scale, to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). Models were adjusted for sex, education, and medical comorbidity. Results: Baseline agitation, nighttime behaviors, depression, and apathy significantly increased the risk of incident dementia. We observed additive interactions between APOE ε4 and depression (joint effect HR = 2.21; 95% CI = 1.24–3.91; test for additive interaction, p < 0.001); and between APOE ε4 and apathy (joint effect HR = 1.93; 95% CI = 0.93–3.98; test for additive interaction, p = 0.031). Anxiety, irritability, and appetite/eating were not associated with increased risk of incident dementia. Conclusions: Among prevalent MCI cases, baseline agitation, nighttime behaviors, depression, and apathy elevated the risk of incident dementia. There was a synergistic interaction between depression or apathy and APOE ε4 in further elevating the risk of incident dementia.

αSynuclein control of mitochondrial homeostasis in human-derived neurons is disrupted by mutations associated with Parkinson’s disease

The etiology of Parkinson’s disease (PD) converges on a common pathogenic pathway of mitochondrial defects in which α-Synuclein (αSyn) is thought to play a role. However, the mechanisms by which αSyn and its disease-associated allelic variants cause mitochondrial dysfunction remain unknown. Here, we analyzed mitochondrial axonal transport and morphology in human-derived neurons overexpressing wild-type (WT) αSyn or the mutated variants A30P or A53T, which are known to have differential lipid affinities. A53T αSyn was enriched in mitochondrial fractions, inducing significant mitochondrial transport defects and fragmentation, while milder defects were elicited by WT and A30P. We found that αSyn-mediated mitochondrial fragmentation was linked to expression levels in WT and A53T variants. Targeted delivery of WT and A53T αSyn to the outer mitochondrial membrane further increased fragmentation, whereas A30P did not. Genomic editing to disrupt the N-terminal domain of αSyn, which is important for membrane association, resulted in mitochondrial elongation without changes in fusion-fission protein levels, suggesting that αSyn plays a direct physiological role in mitochondrial size maintenance. Thus, we demonstrate that the association of αSyn with the mitochondria, which is modulated by protein mutation and dosage, influences mitochondrial transport and morphology, highlighting its relevance in a common pathway impaired in PD.

Tau Pathology is Present In Vivo and Develops In Vitro in Sensory Neurons from Human P301S Tau Transgenic Mice: A System for Screening Drugs against Tauopathies

Intracellular tau aggregates are the neuropathological hallmark of several neurodegenerative diseases, including Alzheimers disease, progressive supranuclear palsy, and cases of frontotemporal dementia, but the link between these aggregates and neurodegeneration remains unclear. Neuronal models recapitulating the main features of tau pathology are necessary to investigate the molecular mechanisms of tau malfunction, but current models show little and inconsistent spontaneous tau aggregation. We show that dorsal root ganglion (DRG) neurons in transgenic mice expressing human P301S tau (P301S-htau) develop tau pathology similar to that found in brain and spinal cord and a significant reduction in mechanosensation occurs before detectable fibrillar tau formation. DRG neuronal cultures established from adult P301S-htau mice at different ages retained the pattern of aberrant tau found in vivo. Moreover, htau became progressively hyperphosphorylated over 2 months in vitro beginning with nonsymptomatic neurons, while hyperphosphorylated P301S-htau-positive neurons from 5-month-old mice cultured for 2 months died preferentially. P301S-htau-positive neurons grew aberrant axons, including spheroids, typically found in human tauopathies. Neurons cultured at advanced stages of tau pathology showed a 60% decrease in the fraction of moving mitochondria. SEG28019, a novel O-GlcNAcase inhibitor, reduced steady-state pSer396/pSer404 phosphorylation over 7 weeks in a significant proportion of DRG neurons showing for the first time the possible beneficial effect of prolonged dosing of O-GlcNAcase inhibitor in vitro. Our system is unique in that fibrillar tau forms without external manipulation and provides an important new tool for understanding the mechanisms of tau dysfunction and for screening of compounds for treatment of tauopathies.

Mild Neurocognitive Disorder: An Old Wine in a New Bottle

AbstractThe American Psychiatric Association has recently published the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). The DSM-IV category “Dementia, Delirium, Amnestic, and Other Cognitive Disorders” has undergone extensive revision. DSM-5 has renamed this category as “Neurocognitive Disorders” (NCD), which now covers three entities: delirium, major NCD, and mild NCD. The DSM-IV version of mild NCD resembles the DSM-5 version in name only. DSM-IV defined mild NCD based on a single criterion, whereas DSM-5 defines mild NCD by using several cognitive and related criteria. The main difference between mild NCD and the Key International Symposium criteria of mild cognitive impairment (MCI) is that the research work that led to the construct of MCI primarily involved elderly study participants (even though age was not part of the definition of MCI), whereas mild NCD includes acquired cognitive disorders of all age groups. DSM-5 essentially discusses the epidemiology and diagnostic markers of mild NCD by drawing congruence between MCI and mild NCD. The DSM-5 definition of mild NCD is anchored on four criteria and two specifiers. The four criteria refer to cognitive changes, functional activities, and exclusion of delirium and competing mental disorders. The two specifiers are the presumed etiologies of mild NCD and the presence or absence of behavioral problems. While the category “mild NCD” may improve reliability of diagnoses, it has yet to withstand scientific scrutiny to be considered a valid construct. This article reviews the DSM-5 criteria for mild NCD, compares them with the Key International Symposium MCI criteria, and discusses the pros and cons of the mild NCD construct.

Imaging amyloid precursor protein in vivo: an axonal transport assay.

Fusion of fluorescent probes to axonally transported proteins represents an established approach that enables live imaging of axonal transport. In this approach, in vivo examination of fluorescent particle dynamics provides information about the length, directionality, and the velocity by which axonally transported proteins travel along axons. Analysis of these parameters provides information about the distribution of axonal proteins and their dynamics in and between different subcellular compartments. Establishing the movement behavior of amyloid precursor protein within axons indicated that live imaging approaches offer the opportunity to significantly enhance our understanding of the biology as well as pathology of axonal transport. This chapter provides a fluorescence-based procedure for measuring axonal transport of APP in cultured newborn mouse hippocampal neurons.