Kinesin Regulation in the Proximal Axon is Essential for Dendrite-selective Transport.

Neurons are polarized and typically extend multiple dendrites and one axon. To maintain polarity, vesicles carrying dendritic proteins are arrested upon entering the axon. To determine whether kinesin regulation is required for terminating anterograde axonal transport, we overexpressed the dendrite-selective kinesin KIF13A.

Reactivation of minimal change disease after Pfizer vaccine against COVID-19.

This case follows a 54-year-old woman with a medical history of hypertension who experienced reactivation of minimal change disease (MCD) after receiving the Pfizer vaccine against COVID-19. She had her first episode of MCD 15 days after receiving the influenza vaccine in 2018. She remained in remission for over 3 years following treatment with steroids.

Drug-Induced Lupus with Leukocytoclastic Vasculitis Associated with Apixaban.

Drug-induced lupus is an iatrogenic-induced autoimmune disease with common offending agents well documented in the literature. To our knowledge, there are no prior case reports of drug-induced lupus associated with apixaban or any other direct oral anticoagulant. We describe a case of drug-induced lupus with leukocytoclastic vasculitis associated with apixaban started 15 days prior, after a WATCHMAN procedure for atrial fibrillation in an 86-year-old male previously anticoagulated on rivaroxaban.

A novel strategy to visualize vesicle-bound kinesins reveals the diversity of kinesin-mediated transport.

In mammals, 15 to 20 kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each kinesin or the functional significance of such diversity. To characterize the transport mediated by different kinesins, we developed a novel strategy to visualize vesicle-bound kinesins in living cells.

The Development of Neuronal Polarity: A Retrospective View.

In 1988, Carlos Dotti, Chris Sullivan, and I published a paper on the establishment of polarity by hippocampal neurons in culture, which continues to be frequently cited 30 years later (Dotti et al., 1988). By following individual neurons from the time of plating until they had formed well developed axonal and dendritic arbors, we identified the five stages of development that lead to the mature expression of neuronal polarity.

Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila.

Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. Here, we report that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion.

The cellular mechanisms that maintain neuronal polarity.

As polarized cells, neurons maintain different sets of resident plasma membrane proteins in their axons and dendrites, which is consistent with the different roles that these neurites have in electrochemical signalling. Axonal and dendritic proteins are synthesized together within the somatodendritic domain; this raises a fundamental question: what is the nature of the intracellular trafficking machinery that ensures that these proteins reach the correct domain? Recent studies have advanced our understanding of the processes underlying the selective sorting and selective transport of axonal and dendritic proteins and have created potential avenues for future progress.

Analyzing kinesin motor domain translocation in cultured hippocampal neurons.

Neuronal microtubules are subject to extensive posttranslational modifications and are bound by MAPs, tip-binding proteins, and other accessory proteins. All of these features, which are difficult to replicate in vitro, are likely to influence the translocation of kinesin motors. Here we describe assays for evaluating the translocation of a population of fluorescently labeled kinesin motor domains, based on their accumulation in regions of the cell enriched in microtubule plus ends.

A Novel Assay to Identify the Trafficking Proteins that Bind to Specific Vesicle Populations.

Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin binding domain (FRB)-tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking.

Axonal transport plays a crucial role in mediating the axon-protective effects of NmNAT.

Deficits in axonal transport are thought to contribute to the pathology of many neurodegenerative diseases. Expressing the slow Wallerian degeneration protein (Wld(S)) or related nicotinamide mononucleotide adenyltransferases (NmNATs) protects axons against damage from a broad range of insults, but the ability of these proteins to protect against inhibition of axonal transport has received little attention. We set out to determine whether these proteins can protect the axons of cultured hippocampal neurons from damage due to hydrogen peroxide or oxygen-glucose deprivation (OGD) and, in particular, whether they can reduce the damage that these agents cause to the axonal transport machinery.

Selective microtubule-based transport of dendritic membrane proteins arises in concert with axon specification.

The polarized distribution of membrane proteins to axonal or somatodendritic neuronal compartments is fundamental to nearly every aspect of neuronal function. The polarity of dendritic proteins depends on selective microtubule-based transport; the vesicles that carry these proteins are transported into dendrites but do not enter the axon. We used live-cell imaging of fluorescently tagged dendritic and axonal proteins combined with immunostaining for initial segment and cytoskeletal markers to evaluate different models of dendrite-selective transport in cultured rat hippocampal neurons.

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.

Culture of primary rat hippocampal neurons: design, analysis, and optimization of a microfluidic device for cell seeding, coherent growth, and solute delivery.

We present the design, analysis, construction, and culture results of a microfluidic device for the segregation and chemical stimulation of primary rat hippocampal neurons. Our device is designed to achieve spatio-temporal solute delivery to discrete sections of neurons with mitigated mechanical stress. We implement a geometric guidance technique to direct axonal processes of the neurons into specific areas of the device to achieve solute segregation along routed cells.

The clathrin adaptor complex responsible for somatodendritic protein sorting.

Neuronal proteins contain "address labels" that govern their localization. In this issue of Neuron, Farías et al. (2012) identify the machinery that recognizes one class of dendritic localization signals and establish its role in the polarization of dendritic proteins, including several postsynaptic receptors.

Differential neurite outgrowth is required for axon specification by cultured hippocampal neurons.

Formation of an axon is the first morphological evidence of neuronal polarization, visible as a profound outgrowth of the axon compared with sibling neurites. One unsolved question on the mechanism of axon formation is the role of axon outgrowth in axon specification. This question was difficult to assess, because neurons freely extend their neurites in a conventional culture.

A novel split kinesin assay identifies motor proteins that interact with distinct vesicle populations.

Identifying the kinesin motors that interact with different vesicle populations is a longstanding and challenging problem with implications for many aspects of cell biology. Here we introduce a new live-cell assay to assess kinesin-vesicle interactions and use it to identify kinesins that bind to vesicles undergoing dendrite-selective transport in cultured hippocampal neurons. We prepared a library of "split kinesins," comprising an axon-selective kinesin motor domain and a series of kinesin tail domains that can attach to their native vesicles; when the split kinesins were assembled by chemical dimerization, bound vesicles were misdirected into the axon.

Oxidative stress inhibits axonal transport: implications for neurodegenerative diseases.

Background: Reactive oxygen species (ROS) released by microglia and other inflammatory cells can cause axonal degeneration. A reduction in axonal transport has also been implicated as a cause of axonal dystrophies and neurodegeneration, but there is a paucity of experimental data concerning the effects of ROS on axonal transport. We used live cell imaging to examine the effects of hydrogen peroxide on the axonal transport of mitochondria and Golgi-derived vesicles in cultured rat hippocampal neurons.

Axonal transport analysis using Multitemporal Association Tracking.

Multitemporal Association Tracking (MAT) is a new graph-based method for multitarget tracking in biological applications that reduces the error rate and implementation complexity compared to approaches based on bipartite matching. The data association problem is solved over a window of future detection data using a graph-based cost function that approximates the Bayesian a posteriori association probability. MAT has been applied to hundreds of image sequences, tracking organelle and vesicles to quantify the deficiencies in axonal transport that can accompany neurodegenerative disorders such as Huntington's Disease and Multiple Sclerosis and to quantify changes in transport in response to therapeutic interventions.

Short-term high-resolution imaging of developing hippocampal neurons in culture.

Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development. The cultures are quite homogeneous and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. Because the cultures are essentially two dimensional, there is no need to acquire images at multiple focal planes.

Long-term time-lapse imaging of developing hippocampal neurons in culture.

Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development. The cultures are quite homogeneous and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. Studying developmental processes that occur over many hours or days--for example, dendritic branching--involves capturing images of a cell at regular intervals without compromising cell survival.

General considerations for live imaging of developing hippocampal neurons in culture.

Dissociated cell cultures of the rodent hippocampus have become a standard model for studying many facets of neural development, including the development of polarity, axonal and dendritic growth, and synapse formation. The cultures are quite homogeneous--∼90% of the cells are pyramidal neurons--and it is relatively easy to express green fluorescent protein (GFP)-tagged proteins by transfection. This article describes the cultures and the key features of the system used to image them.

The translocation selectivity of the kinesins that mediate neuronal organelle transport.

Polarized kinesin-driven transport is crucial for development and maintenance of neuronal polarity. Kinesins are thought to recognize biochemical differences between axonal and dendritic microtubules in order to deliver their cargoes to the appropriate domain. To identify kinesins that mediate polarized transport, we prepared constitutively active versions of all the kinesins implicated in vesicle transport and expressed them in cultured hippocampal neurons.

Automated kymograph analysis for profiling axonal transport of secretory granules.

This paper describes an automated method to profile the velocity patterns of small organelles (BDNF granules) being transported along a selected section of axon of a cultured neuron imaged by time-lapse fluorescence microscopy. Instead of directly detecting the granules as in conventional tracking, the proposed method starts by generating a two-dimensional spatio-temporal map (kymograph) of the granule traffic along an axon segment. Temporal sharpening during the kymograph creation helps to highlight granule movements while suppressing clutter due to stationary granules.

Expression of kinesin superfamily genes in cultured hippocampal neurons.

The nature of the different kinesin family members that function in a single, specific neuron type has not been systematically investigated. Here, we used quantitative real-time PCR to analyze the developmental expression patterns of kinesin family genes in cultured mouse hippocampal neurons, a highly homogeneous population of nerve cells. For purposes of comparison, we also determined the set of kinesins expressed in embryonic and adult hippocampal tissue.