When activity levels are altered over days, a network of cells is capable of recognizing this perturbation and triggering several distinct compensatory changes that should help to recover and maintain the original activity levels homeostatically. One feature commonly observed after activity blockade has been a compensatory increase in excitatory quantal amplitude. The sensing machinery that detects altered activity levels is a central focus of the field currently, but thus far it has been elusive. The vast majority of studies that reduce network activity also reduce neurotransmission. We address the possibility that reduced neurotransmission can trigger increases in quantal amplitude. In this work, we blocked glutamatergic or GABAA transmission in ovo for 2 days while maintaining relatively normal network activity. We found that reducing GABAA transmission triggered compensatory increases in both GABA and AMPA quantal amplitude in embryonic spinal motoneurons. Glutamatergic blockade had no effect on quantal amplitude. Therefore, GABA binding to the GABAA receptor appears to be a critical step in the sensing machinery for homeostatic synaptic plasticity. The findings suggest that homeostatic increases in quantal amplitude may normally be triggered by reduced levels of activity, which are sensed in the developing spinal cord by GABA, via the GABAA receptor. Therefore, GABA appears to be serving as a proxy for activity levels.
In this study, we examined the tissue-specific expression of two electroneutral Na/HCO3 cotransporter (NBCn1) variants that differ from each other by the presence of the N-terminal 123 amino acids (cassette II). A rat Northern blot with the probe to nucleotides encoding cassette II detected a 9 kb NBCn1 mRNA strongly in the heart and weakly in skeletal muscles, but absent from most of the tissues including kidney, brain, and pancreas. In the rat heart, PCR with primers flanking cassette II preferentially amplified a DNA fragment that lacked cassette II. However, in the human heart, PCR preferentially amplified a fragment that contained cassette II. This larger PCR product was found virtually in all regions of the human cardiovascular system with strong amplification in the apex, atrium, and atrioventricular nodes. These findings indicate that the variant containing cassette II is almost absent in tissues including brain, kidney, and pancreas, where NBCn1 has been extensively examined.
A role for clathrin in AP-3–dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3–dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain–FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and colocalizes with endogenous and recombinant FKBP chimeric clathrin polypeptides in PC12-cell endosomes. AP-3 displays, however, a divergent behavior from AP-1, AP-2, and clathrin chains. AP-3 cofractionates with clathrin-coated vesicle fractions isolated from PC12 cells even after clathrin function is acutely inhibited by AP20187. We predicted that AP20187 would inhibit AP-3 vesicle formation from endosomes after a brefeldin A block. AP-3 vesicle formation continued, however, after brefeldin A wash-out despite impairment of clathrin function by AP20187. These findings indicate that AP-3–clathrin association is dispensable for endosomal AP-3 vesicle budding and suggest that endosomal AP-3–clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis.
Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize 'individual' poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (<10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and α-actinin, and the elongation factor, EF-1α (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a 'microdomain' where mRNA is attached and functionally expressed.
The trunk plays a pivotal role in limbed locomotion. Yet, little is known about how the brain stem controls trunk activity during walking. In this study, we assessed the spatiotemporal activity patterns of axial and hindlimb motoneurons (MNs) during drug-induced fictive locomotor-like activity (LLA) in an isolated brain stem-spinal cord preparation of the neonatal mouse. We also evaluated the extent to which these activity patterns are affected by removal of brain stem. Recordings were made in the segments T7, L2, and L5 using calcium imaging from individual axial MNs in the medial motor column (MMC) and hindlimb MNs in lateral motor column (LMC). The MN activities were analyzed during both the rhythmic and the tonic components of LLA, the tonic component being used as a readout of generalized increase in excitability in spinal locomotor networks. The most salient effect of brain stem removal was an increase in locomotor rhythm frequency and a concomitant reduction in burst durations in both MMC and LMC MNs. The lack of effect on the tonic component of LLA indicated specificity of action during the rhythmic component. Cooling-induced silencing of the brain stem reproduced the increase in rhythm frequency and accompanying decrease in burst durations in L2 MMC and LMC, suggesting a dependency on brain stem neuron activity. The work supports the idea that the brain stem locomotor circuits are operational already at birth and further suggests an important role in modulating trunk activity. The brain stem may influence the axial and hindlimb spinal locomotor rhythm generating circuits by extending their range of operation. This may represent a critical step of locomotor development when learning how to walk in different conditions and environments is a major endeavor.
NBCn1 (SLC4A7) plays a role in transepithelial HCO3− movement and intracellular pH maintenance in many tissues. In this study, we searched PDZ proteins capable of binding to NBCn1. We screened a protein array membrane, on which 96 different class I PDZ protein peptides were blotted, with the C‐terminal domain of NBCn1 fused to GST. Thirteen proteins were identified in these screens: MAGI‐3, NHERF‐1, NHERF‐2, PSD‐95, chapsyn‐110, ERBIN, MALS‐1, densin‐180, syntrophins α1, β2, γ2, MUPP1, and PDZK1. After determining these binding partners, we analyzed the database of known and predicted protein interactions to obtain an NBCn1 interaction network. The network shows NBCn1 being physically and functionally associated with a variety of membrane and cytosolic proteins via the binding partners. We then focused on syntrophin γ2 to examine the molecular and functional interaction between NBCn1 and one of the identified binding partners in the Xenopus oocyte expression system. GST/NBCn1 pulled down syntrophin γ2 and conversely GST/syntrophin γ2 pulled down NBCn1. Moreover, syntrophin γ2 increased intracellular pH recovery, from acidification, mediated by NBCn1's Na/HCO3 cotransport. Syntrophin γ2 also increased an ionic conductance produced by NBCn1 channel‐like activity. Thus, syntrophin γ2 regulates NBCn1 activity. In conclusion, this study demonstrates that NBCn1 binds to many PDZ proteins, which in turn may allow the transporter to associate with other physiologically important proteins.
Recent studies suggest that the epithelial sodium channel (ENaC) is expressed in the endothelial cells. To test whether high salt affects the NO production via regulation of endothelial ENaC, human umbilical vein endothelial cells (HUVECs) were incubated in solutions containing either normal or high sodium (additional 20 mM NaCl). Our data showed that high sodium treatment significantly increased α-, β-, and γ-ENaC expression levels in HUVECs. Using the cell-attached patch-clamp technique, we demonstrated that high sodium treatment significantly increased ENaC open probability (PO). Moreover, nitric oxide synthase (eNOS) phosphorylation (Ser 1177) levels and NO production were significantly decreased by high sodium in HUVECs; the effects of high sodium on eNOS phosphorylation and NO production were inhibited by a specific ENaC blocker, amiloride. Our results showed that high sodium decreased AMP-activated kinase (AMPK) phosphorylation in endothelial cells. On the other hand, metformin, an AMPK activator, prevented high sodium-induced upregulation of ENaC expression and PO. Moreover, metformin prevented high salt-induced decrease in NO production and eNOS phosphorylation. These results suggest that high sodium stimulates ENaC activation by negatively modulating AMPK activity, thereby leading to reduction in eNOS activity and NO production in endothelial cells.
Background
Cultured spinal motor neurons are a valuable tool to study basic mechanisms of development, axon growth and pathfinding, and, importantly, to analyze the pathomechanisms underlying motor neuron diseases. However, the application of this cell culture model is limited by the lack of efficient gene transfer techniques which are available for other neurons. To address this problem, we have established magnetofection as a novel method for the simple and efficient transfection of mouse embryonic motor neurons. This technique allows for the study of the effects of gene expression and silencing on the development and survival of motor neurons.
Results
We found that magnetofection, a novel transfection technology based on the delivery of DNA-coated magnetic nanobeads, can be used to transfect primary motor neurons. Therefore, in order to use this method as a new tool for studying the localization and transport of axonal proteins, we optimized conditions and determined parameters for efficient transfection rates of >45% while minimizing toxic effects on survival and morphology. To demonstrate the potential of this method, we have used transfection with plasmids encoding fluorescent fusion-proteins to show for the first time that the spinal muscular atrophy-disease protein Smn is actively transported along axons of live primary motor neurons, supporting an axon-specific role for Smn that is different from its canonical function in mRNA splicing. We were also able to show the suitability of magnetofection for gene knockdown with shRNA-based constructs by significantly reducing Smn levels in both cell bodies and axons, opening new opportunities for the study of the function of axonal proteins in motor neurons.
Conclusions
In this study we have established an optimized magnetofection protocol as a novel transfection method for primary motor neurons that is simple, efficient and non-toxic. We anticipate that this novel approach will have a broad applicability in the study of motor neuron development, axonal trafficking, and molecular mechanisms of motor neuron diseases.
We have previously discovered the naturally occurring antitussive alkaloid noscapine as a tubulin-binding agent that attenuates microtubule dynamics and arrests mammalian cells at mitosis via activation of the c-Jun NH2-terminal kinase pathway. It is well established that the p53 protein plays a crucial role in the control of tumor cell response to chemotherapeutic agents and DNA-damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. In this study, we compared chemosensitivity, cell cycle distribution, and apoptosis on noscapine treatment in four cell lines derived from the colorectal carcinoma HCT116 cells: p53+/+ (p53-wt), p53−/− (p53-null), p21−/− (p21-null), and BAX−/− (BAX-null). Using these isogenic variants, we investigated the roles of p53, BAX, and p21 in the cellular response to treatment with noscapine. Our results show that noscapine treatment increases the expression of p53 over time in cells with wild-type p53 status. This increase in p53 is associated with an increased apoptotic BAX/Bcl-2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. Conversely, loss of p53 and p21 alleles had a counter effect on both BAX and Bcl-2 expression and the p53-null and p21-null cells were significantly resistant to the antiproliferative and apoptotic effects of noscapine. All but the p53-null cells displayed p53 protein accumulation in a time-dependent manner on noscapine treatment. Interestingly, despite increased levels of p53, p21-null cells were resistant to apoptosis, suggesting a proapoptotic role of p21 and implying that p53 is a necessary but not sufficient condition for noscapine-mediated apoptosis.
The localization of β-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3′ untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate premRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of β-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of β-actin mRNA.