Maturational conversion of dendritic early endosomes and their roles in L1-mediated axon growth.

The function of endosomes is intricately linked to cellular function in all cell types, including neurons. Intriguingly, neurons express cell type-specific proteins that localize to endosomes, but little is known about how these neuronal proteins interface with canonical endosomes and ubiquitously expressed endosomal components, such as EEA1 (Early Endosomal Antigen 1). NEEP21 (Neuronal Early Endosomal Protein 21 kDa) localizes to somatodendritic endosomes, and downregulation of NEEP21 perturbs the correct trafficking of multiple receptors, including glutamate receptors (GluA2) during LTP and amyloidogenic processing of ßAPP. Our own work implicated NEEP21 in correct trafficking of the axonal cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM). NEEP21 dynamically localizes with EEA1-positive early endosomes but is also found in EEA1-negative endosomes. Live imaging reveals that NEEP21-positive, EEA1-negative endosomes arise as a consequence of maturational conversion of EEA1/NEEP21 double-positive endosomes. Interfering with EEA1 function causes missorting of L1/NgCAM, axon outgrowth defects on the L1 substrate, and disturbance of NEEP21 localization. Last, we uncover evidence that functional interference with NEEP21 reduces axon and dendrite growth of primary rat hippocampal neurons on L1 substrate but not on N-cadherin substrate, thus implicating endosomal trafficking through somatodendritic early endosomes in L1-mediated axon growth.

A potassium leak channel silences hyperactive neurons and ameliorates status epilepticus.

OBJECTIVE: To develop a constitutively active K(+) leak channel using TREK-1 (TWIK-related potassium channel 1; TREK-M) that is resistant to compensatory down-regulation by second messenger cascades, and to validate the ability of TREK-M to silence hyperactive neurons using cultured hippocampal neurons. To test if adenoassociated viral (AAV) delivery of TREK-M could reduce the duration of status epilepticus and reduce neuronal death induced by lithium-pilocarpine administration. METHODS: Molecular cloning techniques were used to engineer novel vectors to deliver TREK-M via plasmids, lentivirus, and AAV using a cytomegalovirus (CMV)-enhanced GABRA4 promoter. Electrophysiology was used to characterize the activity and regulation of TREK-M in human embryonic kidney (HEK-293) cells, and the ability to reduce spontaneous activity in cultured hippocampal neurons. Adult male rats were injected bilaterally with self-complementary AAV particles composed of serotype 5 capsid into the hippocampus and entorhinal cortex. Lithium-pilocarpine was used to induce status epilepticus. Seizures were monitored using continuous video-electroencephalography (EEG) monitoring. Neuronal death was measured using Fluoro-Jade C staining of paraformaldehyde-fixed brain slices. RESULTS: TREK-M inhibited neuronal firing by hyperpolarizing the resting membrane potential and decreasing input resistance. AAV delivery of TREK-M decreased the duration of status epilepticus by 50%. Concomitantly it reduced neuronal death in areas targeted by the AAV injection. SIGNIFICANCE: These findings demonstrate that TREK-M can silence hyperexcitable neurons in the brain of epileptic rats and treat acute seizures. This study paves the way for an alternative gene therapy treatment of status epilepticus, and provides the rationale for studies of AAV-TREK-M's effect on spontaneous seizures in chronic models of temporal lobe epilepsy.

Patient Engagement in Neuroradiology: A Narrative Review and Case Studies.

The field of patient engagement in radiology is evolving and offers ample opportunities for neuroradiologists to become involved. The patient journey can serve as a model that inspires patient engagement initiatives. The patient journey in radiology may be viewed in 5 stages: 1) awareness that an imaging test is needed, 2) considering having a specific imaging test, 3) access to imaging, 4) imaging service delivery, and 5) ongoing care. Here, we describe patient engagement opportunities based on literature review and paired with case studies by practicing neuroradiologists.

Mechanisms of polarized membrane trafficking in neurons -- focusing in on endosomes.

Neurons are polarized cells that have a complex and unique morphology: long processes (axons and dendrites) extending far from the cell body. In addition, the somatodendritic and axonal domains are further divided into specific subdomains, such as synapses (pre- and postsynaptic specializations), proximal and distal dendrites, axon initial segments, nodes of Ranvier, and axon growth cones. The striking asymmetry and complexity of neuronal cells are necessary for their function in receiving, processing and transferring electrical signals, with each domain playing a precise function in these processes. In order to establish and maintain distinct neuronal domains, mechanisms must exist for protein delivery to specific neuronal compartments, such that each compartment has the correct functional molecular composition. How polarized membrane domains are established and maintained is a long-standing question. Transmembrane proteins, such as receptors and adhesion molecules, can be transported to their proper membrane domains by several pathways. The biosynthetic secretory system delivers newly synthesized transmembrane proteins from the ER via the Golgi and trans-Golgi-network (TGN) to the plasma membrane. In addition, the endosomal system is critically involved in many instances in ensuring proper (re)targeting of membrane components because it can internalize and degrade mislocalized proteins, or recycle proteins from one domain to another. The endosomal system is thus crucial for establishing and maintaining neuronal polarity. In this review, we focus mainly on the intracellular compartments that serve as sorting stations for polarized transport, with particular emphasis on the emerging roles of endosomes.

Neuronal early endosomes require EHD1 for L1/NgCAM trafficking.

In neurons, the endosomal system is essential for membrane receptor trafficking to dendrites and axons and thereby participates in various neuronal functions, such as neurite outgrowth and synaptic plasticity. A multitude of regulators coordinates trafficking through endosomes, but most of them have not been studied in detail in neurons. In non-neuronal cells, EHD1 (Eps15 homology-domain containing protein 1) functions in the recycling endosome and is required for endosome-to-plasma membrane transport of multiple cargos. In this study, we analyze the role of EHD1 in neurons. In particular, we investigate whether EHD1 is required for polarized trafficking of the dendritically targeted transferrin and the axonal adhesion molecule L1/NgCAM (neuron-glia cell adhesion molecule) and, if so, in what compartment it is required. We find that endosomal recycling of both L1/NgCAM and transferrin is impaired when EHD1 is downregulated. We show that EHD1 colocalizes with L1/NgCAM and transferrin mostly in EEA1 (early endosome antigen 1)-positive early endosomes and less extensively with recycling endosomes. Using live imaging, we observe that EHD1 is stably associated with endosomal membranes during their maturation into EEA1-positive compartments and often persists on them longer than EEA1. Finally, we show that downregulation of EHD1 causes a delay of L1/NgCAM in exiting EEA1-positive endosomes, resulting in impaired targeting of L1/NgCAM to the axonal membrane. We conclude that, in neurons, EHD1 functions in early endosomes rather than (or possibly in addition to) recycling endosomes. These findings point to the existence of neuronal adaptations of the endosomal system.

Alterations of EHD1/EHD4 protein levels interfere with L1/NgCAM endocytosis in neurons and disrupt axonal targeting.

Axon growth is regulated by many proteins, including adhesion molecules, which need to be trafficked correctly to axons. The adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) travels to axons via an endocytosis-dependent pathway (transcytosis), traversing somatodendritic endosomes. The Eps15 homology domain (EHD) family proteins (EHD1-EHD4) play important roles in endosomal recycling and possibly in endocytosis. We investigated whether EHD1 regulates L1/NgCAM trafficking in neurons. Both short hairpin-mediated downregulation and overexpression of EHD1 led to dendritic mistargeting of NgCAM. Downregulation of EHD1 showed increased endosomal accumulation of NgCAM, whereas, surprisingly, overexpression of EHD1 led to impairment of L1/NgCAM internalization in neurons but not in fibroblasts. Transferrin internalization, however, was unaffected. At longer overexpression times of EHD1, NgCAM endocytosis returned to normal, suggesting rapid upregulation of compensatory endocytic pathways. EHD1 is capable of hetero-oligomerization, and an endogenous complex of EHD1 and EHD4 was identified previously. We therefore tested whether short-term overexpression of other EHD family members showed a similar endocytosis defect. Expression of EHD4, but not of EHD3, also caused a defect in L1/NgCAM endocytosis. Oligomerization of EHD1 was required to cause NgCAM endocytosis defects, and simultaneous expression of EHD1 and EHD4 rescued NgCAM endocytosis. Therefore, balanced levels of EHD1-EHD4 are important for NgCAM endocytosis in neurons. Our data suggest that EHD1 plays roles in both endosomal recycling and a specialized endocytosis pathway in neurons used by NgCAM. We propose that EHD1 and EHD4 act as hetero-oligomeric complexes in this pathway.

Cytoarchitectonic Mapping of MRI Detects Rapid Changes in Alzheimer's Disease.

The clinical and pathological progression of Alzheimer's disease often proceeds rapidly, but little is understood about its structural characteristics over short intervals. This study evaluated the short temporal characteristics of the brain structure in Alzheimer's disease through the application of cytoarchitectonic probabilistic brain mapping to measurements of gray matter density, a technique which may provide advantages over standard volumetric MRI techniques. Gray matter density was calculated using voxel-based morphometry of T1-weighted MRI obtained from Alzheimer's disease patients and healthy controls evaluated at intervals of 0.5, 1.5, 3.5, 6.5, 9.5, 12, 18, and 24 months by the MIRIAD study. The Alzheimer's disease patients had 19.1% less gray matter at 1st MRI, and this declined 81.6% faster than in healthy controls. Atrophy in the hippocampus, amygdala, and basal forebrain distinguished the Alzheimer's disease patients. Notably, the CA2 of the hippocampus was found to have atrophied significantly within 1 month. Gray matter density measurements were reliable, with intraclass correlation coefficients exceeding 0.8. Comparative atrophy in the Alzheimer's disease group agreed with manual tracing MRI studies of Alzheimer's disease while identifying atrophy on a shorter time scale than has previously been reported. Cytoarchitectonic mapping of gray matter density is reliable and sensitive to small-scale neurodegeneration, indicating its use in the future study of Alzheimer's disease.

Neuronal lysosomal dysfunction releases exosomes harboring APP C-terminal fragments and unique lipid signatures.

Defects in endolysosomal and autophagic functions are increasingly viewed as key pathological features of neurodegenerative disorders. A master regulator of these functions is phosphatidylinositol-3-phosphate (PI3P), a phospholipid synthesized primarily by class III PI 3-kinase Vps34. Here we report that disruption of neuronal Vps34 function in vitro and in vivo impairs autophagy, lysosomal degradation as well as lipid metabolism, causing endolysosomal membrane damage. PI3P deficiency also promotes secretion of unique exosomes enriched for undigested lysosomal substrates, including amyloid precursor protein C-terminal fragments (APP-CTFs), specific sphingolipids, and the phospholipid bis(monoacylglycero)phosphate (BMP), which normally resides in the internal vesicles of endolysosomes. Secretion of these exosomes requires neutral sphingomyelinase 2 and sphingolipid synthesis. Our results reveal a homeostatic response counteracting lysosomal dysfunction via secretion of atypical exosomes eliminating lysosomal waste and define exosomal APP-CTFs and BMP as candidate biomarkers for endolysosomal dysfunction associated with neurodegenerative disorders.

Studying endosomes in cultured neurons by live-cell imaging.

Endosomes play critical roles on regulating surface receptor levels as well as signaling cascades in all cell types, including neurons. Endocytosis and endosomal trafficking is routinely studied after fixation, but live imaging is increasingly being used to capture the dynamic nature of endosomes and is allowing increasingly sophisticated glimpses into trafficking processes in live neurons. In this chapter, we describe the basics of neuronal primary cultures, methods for expressing fluorescent proteins, and live imaging of cargos and endosomal regulators.

Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system.

Defects in endosomal sorting have been implicated in Alzheimer's disease. Endosomal traffic is largely controlled by phosphatidylinositol-3-phosphate, a phosphoinositide synthesized primarily by lipid kinase Vps34. Here we show that phosphatidylinositol-3-phosphate is selectively deficient in brain tissue from humans with Alzheimer's disease and Alzheimer's disease mouse models. Silencing Vps34 causes an enlargement of neuronal endosomes, enhances the amyloidogenic processing of amyloid precursor protein in these organelles and reduces amyloid precursor protein sorting to intraluminal vesicles. This trafficking phenotype is recapitulated by silencing components of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway, including the phosphatidylinositol-3-phosphate effector Hrs and Tsg101. Amyloid precursor protein is ubiquitinated, and interfering with this process by targeted mutagenesis alters sorting of amyloid precursor protein to the intraluminal vesicles of endosomes and enhances amyloid-beta peptide generation. In addition to establishing phosphatidylinositol-3-phosphate deficiency as a contributing factor in Alzheimer's disease, these results clarify the mechanisms of amyloid precursor protein trafficking through the endosomal system in normal and pathological states.

Compartmentalizing the neuronal plasma membrane from axon initial segments to synapses.

Many membrane proteins localize to restricted domains in neurons, such as axons, dendrites, synapses, or axon initial segments. The exquisite subcellular compartmentalization of adhesion molecules, growth factor receptors, signaling receptors, voltage-gated and ligand-gated channels, and others underlies the complex functioning of neurons and ultimately vectorial propagation of signaling in neuronal circuits. This chapter discusses the cellular mechanisms for compartmentalizing the neuronal plasma membrane. Among the mechanisms contributing to protein segregation in the membrane are sorting and targeting in the Golgi/TGN, endocytosis, recycling, and degradation, and control of membrane protein diffusion. The molecular underpinnings of these cellular mechanisms are reviewed in the first part. The second part discusses the contribution of each cellular mechanism to targeting proteins to axons and dendrites, to synapses, to axon initial segments, and to Nodes of Ranvier. For most, if not all proteins and locations, all four mechanisms are in effect and additively contribute to the precise localization of membrane proteins in neurons. Since disruption of proper protein distribution results in defects in neuronal function and can lead to neurodegenerative diseases, a full understanding of the cellular mechanisms of plasma membrane compartmentalization is an important goal for the future.

The somatodendritic endosomal regulator NEEP21 facilitates axonal targeting of L1/NgCAM.

Correct targeting of proteins to axons and dendrites is crucial for neuronal function. We showed previously that axonal accumulation of the cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) depends on endocytosis (Wisco, D., E.D. Anderson, M.C. Chang, C. Norden, T. Boiko, H. Folsch, and B. Winckler. 2003. J. Cell Biol. 162:1317-1328). Two endocytosis-dependent pathways to the axon have been proposed: transcytosis and selective retrieval/retention. We show here that axonal accumulation of L1/NgCAM occurs via nondegradative somatodendritic endosomes and subsequent anterograde axonal transport, which is consistent with transcytosis. Additionally, we identify the neuronal-specific endosomal protein NEEP21 (neuron-enriched endosomal protein of 21 kD) as a regulator of L1/NgCAM sorting in somatodendritic endosomes. Down-regulation of NEEP21 leads to missorting of L1/NgCAM to the somatodendritic surface as well as to lysosomes. Importantly, the axonal accumulation of endogenous L1 in young neurons is also sensitive to NEEP21 depletion. We propose that small endosomal carriers derived from somatodendritic recycling endosomes can serve to redistribute a distinct set of membrane proteins from dendrites to axons.