Distinct Neuronal Compartments Research Articles

The fates of internalized NaV1.7 channels in sensory neurons: Retrograde cotransport with other ion channels, axon-specific recycling, and degradation

Neuronal function relies on the maintenance of appropriate levels of various ion channels at the cell membrane, which is accomplished by balancing secretory, degradative, and recycling pathways. Neuronal function further depends on membrane specialization through polarized distribution of specific proteins to distinct neuronal compartments such as axons. Voltage-gated sodium channel NaV1.7, a threshold channel for firing action potentials in nociceptors, plays a major role in human pain, and its abundance in the plasma membrane is tightly regulated. We have recently characterized the anterograde axonal trafficking of NaV1.7 channels in Rab6A-positive vesicles, but the fate of internalized channels is not known. Membrane proteins that have undergone endocytosis can be directed into multiple pathways including those for degradation, recycling to the membrane, and transcytosis. Here, we demonstrate NaV1.7 endocytosis and dynein-dependent retrograde trafficking in Rab7-containing late endosomes together with other axonal membrane proteins using real-time imaging of live neurons. We show that some internalized NaV1.7 channels are delivered to lysosomes within the cell body, and that there is no evidence for NaV1.7 transcytosis. In addition, we show that NaV1.7 is recycled specifically to the axonal membrane as opposed to the soma membrane, suggesting a novel mechanism for the development of neuronal polarity. Together, these results shed light on the mechanisms by which neurons maintain excitable membranes and may inform efforts to target ion channel trafficking for the treatment of disorders of excitability.

Early Mitochondrial Fragmentation and Dysfunction in a Drosophila Model for Alzheimer's Disease.

Many different cellular systems and molecular processes become compromised in Alzheimer's disease (AD) including proteostasis, autophagy, inflammatory responses, synapse and neuronal circuitry, and mitochondrial function. We focused in this study on mitochondrial dysfunction owing to the toxic neuronal environment produced by expression of Aβ42, and its relationship to other pathologies found in AD including increased neuronal apoptosis, plaque deposition, and memory impairment. Using super-resolution microscopy, we have assayed mitochondrial status in the three distinct neuronal compartments (somatic, dendritic, axonal) of mushroom body neurons of Drosophila expressing Aβ42. The mushroom body neurons comprise a major center for olfactory memory formation in insects. We employed calcium imaging to measure mitochondrial function, immunohistochemical and staining techniques to measure apoptosis and plaque formation, and olfactory classical conditioning to measure learning. We found that mitochondria become fragmented at a very early age along with decreased function measured by mitochondrial calcium entry. Increased apoptosis and plaque deposition also occur early, yet interestingly, a learning impairment was found only after a much longer period of time-10 days, which is a large fraction of the fly's lifespan. This is similar to the pronounced delay between cellular pathologies and the emergence of a memory dysfunction in humans. Our studies are consistent with the model that mitochondrial dysfunction and/or other cellular pathologies emerge at an early age and lead to much later learning impairments. The results obtained further develop this Drosophila model as a useful in vivo system for probing the mechanisms by which Aβ42 produces mitochondrial and other cellular toxicities that produce memory dysfunction.

Roles for the Endoplasmic Reticulum in Regulation of Neuronal Calcium Homeostasis.

By influencing Ca2+ homeostasis in spatially and architecturally distinct neuronal compartments, the endoplasmic reticulum (ER) illustrates the notion that form and function are intimately related. The contribution of ER to neuronal Ca2+ homeostasis is attributed to the organelle being the largest reservoir of intracellular Ca2+ and having a high density of Ca2+ channels and transporters. As such, ER Ca2+ has incontrovertible roles in the regulation of axodendritic growth and morphology, synaptic vesicle release, and neural activity dependent gene expression, synaptic plasticity, and mitochondrial bioenergetics. Not surprisingly, many neurological diseases arise from ER Ca2+ dyshomeostasis, either directly due to alterations in ER resident proteins, or indirectly via processes that are coupled to the regulators of ER Ca2+ dynamics. In this review, we describe the mechanisms involved in the establishment of ER Ca2+ homeostasis in neurons. We elaborate upon how changes in the spatiotemporal dynamics of Ca2+ exchange between the ER and other organelles sculpt neuronal function and provide examples that demonstrate the involvement of ER Ca2+ dyshomeostasis in a range of neurological and neurodegenerative diseases.

Calcium-Handling Defects and Neurodegenerative Disease.

Calcium signaling is critical to neuronal function and regulates highly diverse processes such as gene transcription, energy production, protein handling, and synaptic structure and function. Because there are many common underlying calcium-mediated pathological features observed across several neurological conditions, it has been proposed that neurodegenerative diseases have an upstream underlying calcium basis in their pathogenesis. With certain diseases such as Alzheimer's, Parkinson's, and Huntington's, specific sources of calcium dysregulation originating from distinct neuronal compartments or channels have been shown to have defined roles in initiating or sustaining disease mechanisms. Herein, we will review the major hallmarks of these diseases, and how they relate to calcium dysregulation. We will then discuss neuronal calcium handling throughout the neuron, with special emphasis on channels involved in neurodegeneration.

Autophagy in the presynaptic compartment in health and disease.

Synapses are functionally distinct neuronal compartments that are critical for brain function, with synaptic dysfunction being an early pathological feature in aging and disease. Given the large number of proteins needed for synaptic function, the proliferation of defective proteins and the subsequent loss of protein homeostasis may be a leading cause of synaptic dysfunction. Autophagic mechanisms are cellular digestion processes that recycle cellular components and contribute to protein homeostasis. Autophagy is important within the nervous system, but its function in specific compartments such as the synapse has been unclear. Evidence from research on both autophagy and synaptic function suggests that there are links between the two and that synaptic homeostasis during aging requires autophagy to regulate protein homeostasis. Exciting new work on autophagy-modulating proteins that are enriched at the synapse has begun to link autophagy to synapses and synaptic dysfunction in disease. A better understanding of these links will help us harness the potential therapeutic benefits of autophagy in combating age-related disorders of the nervous system.

Compartment-Specific Regulation of Autophagy in Primary Neurons

Autophagy is an essential degradative pathway that maintains neuronal homeostasis and prevents axon degeneration. Initial observations suggest that autophagy is spatially regulated in neurons, but how autophagy is regulated in distinct neuronal compartments is unclear. Using live-cell imaging in mouse hippocampal neurons, we establish the compartment-specific mechanisms of constitutive autophagy under basal conditions, as well as in response to stress induced by nutrient deprivation. We find that at steady state, the cell soma contains populations of autophagosomes derived from distinct neuronal compartments and defined by differences in maturation state and dynamics. Axonal autophagosomes enter the soma and remain confined within the somatodendritic domain. This compartmentalization likely facilitates cargo degradation by enabling fusion with proteolytically active lysosomes enriched in the soma. In contrast, autophagosomes generated within the soma are less mobile and tend to cluster. Surprisingly, starvation did not induce autophagy in either the axonal or somatodendritic compartment. While starvation robustly decreased mTORC1 signaling in neurons, this decrease was not sufficient to activate autophagy. Furthermore, pharmacological inhibition of mammalian target of rapamycin with Torin1 also was not sufficient to markedly upregulate neuronal autophagy. These observations suggest that the primary physiological function of autophagy in neurons may not be to mobilize amino acids and other biosynthetic building blocks in response to starvation, in contrast to findings in other cell types. Rather, constitutive autophagy in neurons may function to maintain cellular homeostasis by balancing synthesis and degradation, especially within distal axonal processes far removed from the soma. Autophagy is an essential homeostatic process in neurons, but neuron-specific mechanisms are poorly understood. Here, we compare autophagosome dynamics within neuronal compartments. Axonal autophagy is a vectorial process that delivers cargo from the distal axon to the soma. The soma, however, contains autophagosomes at different maturation states, including input received from the axon combined with locally generated autophagosomes. Once in the soma, autophagosomes are confined to the somatodendritic domain, facilitating cargo degradation and recycling of biosynthetic building blocks to primary sites of protein synthesis. Neuronal autophagy is not robustly upregulated in response to starvation or mammalian target of rapamycin inhibition, suggesting that constitutive autophagy in neurons maintains homeostasis by playing an integral role in regulating the quality of the neuronal proteome.

Sculpting neuronal connectivity

It emerges that a transcription program differentially regulates inhibitory inputs in distinct neuronal compartments — an unexpected coordinated switch for achieving experience-dependent 'plasticity' in neural circuits. See Letter p.121 Individual neurons distinguish synaptic inputs received at their soma and dendrites, but how behaviour may affect their balance has been unclear. Now Michael Greenberg and colleagues show that mouse hippocampus neurons respond to sensory enrichment with increased levels of the transcription factor NPAS4 and its target-gene product, brain derived neurotrophic factor (BDNF), which then promotes inhibitory synapses on the cell body while destabilizing those on dendrites. Thus individual neurons respond to sensory stimulation by redrawing the map of their inhibitory inputs, restricting their somatic output while promoting plasticity at their dendrites.

Ultrastructural relationship between the AMPA-GluR2 receptor subunit and the mu-opioid receptor in the mouse central nucleus of the amygdala

Activation of GluR2-expressing non-calcium-permeable AMPA-type glutamate receptors in the central nucleus of the amygdala (CeA) may play an important role in integrating emotion and memory with goal-directed behaviors involved in opioid addiction. The location of non-calcium-permeable AMPA receptors within distinct neuronal compartments (i.e., soma, dendrite, or axon) is an important functional feature of these proteins; however, their ultrastructural location and subcellular relationship with mu-opioid receptors (μOR) in the CeA are unknown. Immunocytochemical electron microscopy was used to characterize the ultrastructural distribution of GluR2 and its association with μOR in the mouse CeA. A single-labeling analysis of GluR2 distribution employing immunoperoxidase or immunogold markers revealed that this protein was frequently affiliated with intracellular vesicular organelles, as well as the plasma membrane of CeA neuronal profiles. Among all GluR2-labeled neuronal structures, over 85% were dendrites or somata. Unlabeled axon terminals frequently formed asymmetric excitatory-type synaptic junctions with GluR2-labeled dendritic profiles. Dual-labeling immunocytochemical analysis showed that GluR2 and μOR were co-localized in neuronal compartments. Among all dual-labeled structures, approximately 80% were dendritic. Synaptic inputs to these dual-labeled dendrites were frequently from unlabeled axon terminals forming asymmetric excitatory-type synapses. The presence of GluR2 in dendritic profiles receiving asymmetric synapses suggests that activation of the non-calcium-permeable AMPA receptor plays a role in the postsynaptic modulation of excitatory signaling involving CeA neuronal circuits that coordinate sensory, affective, and behavioral processes involved in drug addiction. Given the critical role of non-calcium-permeable AMPA receptor function in neural and behavioral adaptability, their dendritic association with μOR in CeA dendrites provides a neuronal substrate for opioid-mediated plasticity.

Role of the synprint site in presynaptic targeting of the calcium channel CaV2.2 in hippocampal neurons

Sequences in the cytoplasmic II-III loop of CaV2 voltage-gated calcium channels, termed the synaptic protein interaction (synprint) site, are considered important for the functional incorporation of presynaptic calcium channels into the synaptic vesicle fusion apparatus. Two novel CaV2.2 splice variants lack large parts of the cytoplasmic II-III loop (Delta1 R756-L1139, Delta2 K737-A1001) including the synprint protein-protein interaction domain. Here we expressed green fluorescent protein (GFP)-alpha1B subunit fusion constructs of CaV2.2 splice variants in mouse hippocampal neurons to study their distribution in distinct neuronal compartments and to address the question of whether and how the synprint site functions in the presynaptic targeting of N-type calcium channels. Similar to full-length GFP-alpha1B but divergent from the somatodendritic alpha1C-HA (CaV1.2) channel type, the splice variants GFP-alpha1B-Delta1 and GFP-alpha1B-Delta2 were targeted into the axons. Nevertheless, their ability to form bona fide presynaptic clusters was almost abolished for GFP-alpha1B-Delta1 and significantly reduced for GFP-alpha1B-Delta2. Thus, the synprint site is important for normal synaptic targeting of CaV2.2 but not essential. Conversely, insertion of the synprint site into the II-III loop of alpha1C-HA did not restore axonal targeting or synaptic clustering. Together these results indicate that protein-protein interactions with the synprint site must cooperate with other targeting mechanisms in the incorporation of CaV2.2 into presynaptic specializations of hippocampal neurons but are neither necessary nor sufficient for axonal targeting. The unique targeting properties of the splice variants lacking the synprint site are suggestive of specific functions of these calcium channels apart from activating fast synaptic transmission.

Anterograde Axonal Transport, Transcytosis, and Recycling of Neurotrophic Factors

Traditional views of neurotrophic factor biology held that trophic factors are released from target cells, retrogradely transported along their axons, and rapidly degraded upon arrival in cell bodies. Increasing evidence indicates that several trophic factors such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF-2), glial cell-line derived neurotrophic factor (GDNF), insulin-like growth factor (IGF-I), and neurotrophin-3 (NT-3), can move anterogradely along axons. They can escape the degradative pathway upon internalization and are recycled for future uses. Internalized ligands can move through intermediary cells by transcytosis, presumably by endocytosis via endosomes to the Golgi system, by trafficking of the factor to dendrites or by sorting into anterograde axonal transport with subsequent release from axon terminals and uptake by second- or third-order target neurons. Such data suggest the existence of multiple "trophic currencies," which may be used over several steps in neural networks to enable nurturing relationships between connected neurons or glial cells, not unlike currency exchanges between trading partners in the world economy. Functions of multistep transfer of trophic material through neural networks may include regulation of neuronal survival, differentiation of phenotypes and dendritic morphology, synapse plasticity, as well as excitatory neurotransmission. The molecular mechanisms of sorting, trafficking, and release of trophic factors from distinct neuronal compartments are important for an understanding of neurotrophism, but they present challenging tasks owing to the low levels of the endogenous factors.

Region-specific mRNA expression of phosphatidylinositol 3-kinase regulatory isoforms in the central nervous system of C57BL/6J mice

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) by receptor tyrosine kinases for growth factors is crucial for neuronal cell survival and proliferation. This class of kinases is comprised of heterodimers, each consisting of one regulatory and one catalytic subunit. Multiple isoforms of regulatory subunits exist, including p85alpha and its alternative splice products p50alpha and AS53/p55alpha, and p85beta and p55(PIK), which are derived from different genes. The regional distribution of these PI 3-kinase regulatory isoforms was mapped in the adult murine brain by in situ hybridization histochemistry. All isoforms were demonstrated in abundance in choroid plexus and anterior pituitary. In neuronal compartments, however, PI 3-kinase isoforms were distributed in a regionally specific manner. In general, the mRNAs for p85alpha, p50alpha, AS53, and p85beta were widespread, with the highest level in the olfactory system, in neuronal groups of the forebrain and hypothalamus, in the hippocampus, cortex, inferior and superior colliculus, pituitary, and cerebellum. However, each isoform had specific variations. Lower expression levels of these isoforms were found in the thalamus, diencephalon, mesencephalon, and brainstem. In contrast, abundant mRNA expression of p55(PIK) was limited to cerebellum and anterior pituitary, with moderate levels of p55(PIK) in the olfactory bulb and hippocampus and low levels elsewhere. The distribution pattern of PI 3-kinase isoforms in the brain indicates pluripotent signaling properties for PI 3-kinase isoforms p85alpha, p50alpha, AS53/p55alpha, and p85beta for a variety of receptor tyrosine kinases, whereas the restricted expression of p55(PIK) implies a regionally specific role for this isoform in neuronal signaling. The unique integrated expression profiles of PI 3-kinase isoforms in distinct neuronal compartments denote complex intracellular signaling pathways for each neuronal region to ensure specificity of receptor tyrosine kinase signal transduction.

Region‐specific mRNA expression of phosphatidylinositol 3‐kinase regulatory isoforms in the central nervous system of C57BL/6J mice

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) by receptor tyrosine kinases for growth factors is crucial for neuronal cell survival and proliferation. This class of kinases is comprised of heterodimers, each consisting of one regulatory and one catalytic subunit. Multiple isoforms of regulatory subunits exist, including p85α and its alternative splice products p50α and AS53/p55α, and p85β and p55PIK, which are derived from different genes. The regional distribution of these PI 3-kinase regulatory isoforms was mapped in the adult murine brain by in situ hybridization histochemistry. All isoforms were demonstrated in abundance in choroid plexus and anterior pituitary. In neuronal compartments, however, PI 3-kinase isoforms were distributed in a regionally specific manner. In general, the mRNAs for p85α, p50α, AS53, and p85β were widespread, with the highest level in the olfactory system, in neuronal groups of the forebrain and hypothalamus, in the hippocampus, cortex, inferior and superior colliculus, pituitary, and cerebellum. However, each isoform had specific variations. Lower expression levels of these isoforms were found in the thalamus, diencephalon, mesencephalon, and brainstem. In contrast, abundant mRNA expression of p55PIK was limited to cerebellum and anterior pituitary, with moderate levels of p55PIK in the olfactory bulb and hippocampus and low levels elsewhere. The distribution pattern of PI 3-kinase isoforms in the brain indicates pluripotent signaling properties for PI 3-kinase isoforms p85α, p50α, AS53/p55α, and p85β for a variety of receptor tyrosine kinases, whereas the restricted expression of p55PIK implies a regionally specific role for this isoform in neuronal signaling. The unique integrated expression profiles of PI 3-kinase isoforms in distinct neuronal compartments denote complex intracellular signaling pathways for each neuronal region to ensure specificity of receptor tyrosine kinase signal transduction. J. Comp. Neurol. 415:105–120, 1999. © 1999 Wiley-Liss, Inc.