Biomedical subjects

Guoping Feng

Publications and source records attributed to Guoping Feng.

At least 19 recordsLinked to original sources

PubMed2021

AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset.

Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood-brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.

Administration, Intravenous↗

PubMed2006

Next-generation optical technologies for illuminating genetically targeted brain circuits.

Emerging technologies from optics, genetics, and bioengineering are being combined for studies of intact neural circuits. The rapid progression of such interdisciplinary "optogenetic" approaches has expanded capabilities for optical imaging and genetic targeting of specific cell types. Here we explore key recent advances that unite optical and genetic approaches, focusing on promising techniques that either allow novel studies of neural dynamics and behavior or provide fresh perspectives on classic model systems.

Animals↗

PubMed2006

Calcium channel alpha2delta1 subunit mediates spinal hyperexcitability in pain modulation.

Mechanisms of chronic pain, including neuropathic pain, are poorly understood. Upregulation of voltage-gated calcium channel (VGCC) alpha2delta1 subunit (Ca(v)alpha2delta1) in sensory neurons and dorsal spinal cord by peripheral nerve injury has been suggested to contribute to neuropathic pain. To investigate the mechanisms without the influence of other injury factors, we have created transgenic mice that constitutively overexpress Ca(v)alpha2delta1 in neuronal tissues. Ca(v)alpha2delta1 overexpression resulted in enhanced currents, altered kinetics and voltage-dependence of VGCC activation in sensory neurons; exaggerated and prolonged dorsal horn neuronal responses to mechanical and thermal stimulations at the periphery; and pain behaviors. However, the transgenic mice showed normal dorsal horn neuronal responses to windup stimulation, and behavioral responses to tissue-injury/inflammatory stimuli. The pain behaviors in the transgenic mice had a pharmacological profile suggesting a selective contribution of elevated Ca(v)alpha2delta1 to the abnormal sensations, at least at the spinal cord level. In addition, gabapentin blocked VGCC currents concentration-dependently in transgenic, but not wild-type, sensory neurons. Thus, elevated neuronal Ca(v)alpha2delta1 contributes to specific pain states through a mechanism mediated at least partially by enhanced VGCC activity in sensory neurons and hyperexcitability in dorsal horn neurons in response to peripheral stimulation. Modulation of enhanced VGCC activity by gabapentin may underlie at least partially its antihyperalgesic actions.

Animals↗

PubMed2006

The chloride transporter Na(+)-K(+)-Cl- cotransporter isoform-1 contributes to intracellular chloride increases after in vitro ischemia.

Ischemic episodes in the CNS cause significant disturbances in neuronal ionic homeostasis. To directly measure changes in intracellular Cl- concentration ([Cl-]i) during and after ischemia, we used Clomeleon, a novel ratiometric optical indicator for Cl-. Hippocampal slices from adult transgenic mice expressing Clomeleon in hippocampal neurons were subjected to 8 min of oxygen-glucose deprivation (OGD) (an in vitro model for ischemia) and reoxygenated in the presence of glucose. This produced mild neuronal damage 3 h later that was prevented when the extracellular [Cl-] was maintained at 10 mm during reoxygenation. OGD induced a transient decrease in fluorescence resonance energy transfer within Clomeleon, indicating an increase in [Cl-]i. During reoxygenation, there was a partial recovery in [Cl-]i, but [Cl-]i rose again 45 min later. To investigate sources of Cl- accumulation, we examined the effects of Cl- transport inhibitors on the rises in [Cl-]i during and after OGD. Bumetanide and furosemide, which inhibit Cl- influx through the Na(+)-K(+)-Cl- cotransporter isoform-1 (NKCC-1) and efflux through the K(+)-Cl- cotransporter isoform-2, were unable to inhibit the first rise in [Cl-]i, yet entirely prevented the secondary rise in [Cl-]i during reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl- channel, did not inhibit the secondary rise in [Cl-]i after OGD. [Cl-]i increases during reoxygenation were accompanied by an increase in phosphorylation of NKCC-1, an indication of increased NKCC-1 activity after OGD. We conclude that NKCC-1 plays an important role in OGD-induced Cl- accumulation and subsequent neuronal damage.

Animals↗

PubMed2006

In vivo imaging of juxtaglomerular neuron turnover in the mouse olfactory bulb.

As a consequence of adult neurogenesis, the olfactory bulb (OB) receives a continuous influx of newborn neurons well into adulthood. However, their rates of generation and turnover, the factors controlling their survival, and how newborn neurons intercalate into adult circuits are largely unknown. To visualize the dynamics of adult neurogenesis, we produced a line of transgenic mice expressing GFP in approximately 70% of juxtaglomerular neurons (JGNs), a population that undergoes adult neurogenesis. Using in vivo two-photon microscopy, time-lapse analysis of identified JGN cell bodies revealed a neuronal turnover rate of approximately 3% of this population per month. Although new neurons appeared and older ones disappeared, the overall number of JGNs remained constant. This approach provides a dynamic view of the actual appearance and disappearance of newborn neurons in the vertebrate central nervous system, and provides an experimental substrate for functional analysis of adult neurogenesis.

Animals↗

PubMed2006

Two-photon imaging reveals somatodendritic chloride gradient in retinal ON-type bipolar cells expressing the biosensor Clomeleon.

A somatodendritic gradient of Cl(-) concentration ([Cl(-)](i)) has been postulated to generate GABA-evoked responses of different polarity in retinal bipolar cells, hyperpolarizing in OFF cells with low dendritic [Cl(-)](i), and depolarizing in ON cells with high dendritic [Cl(-)](i). As glutamate released by the photoreceptors depolarizes OFF cells and hyperpolarizes ON cells, the bipolars' antagonistic receptive field (RF) could be computed by simply integrating glutamatergic inputs from the RF center and GABAergic inputs from horizontal cells in the RF surround. Using ratiometric two-photon imaging of Clomeleon, a Cl(-) indicator transgenically expressed in ON bipolar cells, we found that dendritic [Cl(-)](i) exceeds somatic [Cl(-)](i) by up to 20 mM and that GABA application can lead to Cl(-) efflux (depolarization) in these dendrites. Blockers of Cl(-) transporters reduced the somatodendritic [Cl(-)](i) gradient. Hence, our results support the idea that ON bipolar cells employ a somatodendritic [Cl(-)](i) gradient to invert GABAergic horizontal cell input.

Animals↗

PubMed2005

Dynamic remodeling of dendritic arbors in GABAergic interneurons of adult visual cortex.

Despite decades of evidence for functional plasticity in the adult brain, the role of structural plasticity in its manifestation remains unclear. To examine the extent of neuronal remodeling that occurs in the brain on a day-to-day basis, we used a multiphoton-based microscopy system for chronic in vivo imaging and reconstruction of entire neurons in the superficial layers of the rodent cerebral cortex. Here we show the first unambiguous evidence (to our knowledge) of dendrite growth and remodeling in adult neurons. Over a period of months, neurons could be seen extending and retracting existing branches, and in rare cases adding new branch tips. Neurons exhibiting dynamic arbor rearrangements were GABA-positive non-pyramidal interneurons, while pyramidal cells remained stable. These results are consistent with the idea that dendritic structural remodeling is a substrate for adult plasticity and they suggest that circuit rearrangement in the adult cortex is restricted by cell type-specific rules.

Aging↗

PubMed2005

Ubiquilin-1 regulates nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors.

Chronic exposure to nicotine, as in tobacco smoking, up-regulates nicotinic acetylcholine receptor surface expression in neurons. This up-regulation has been proposed to play a role in nicotine addiction and withdrawal. The regulatory mechanisms behind nicotine-induced up-regulation of surface nicotinic acetylcholine receptors remain to be determined. It has recently been suggested that nicotine stimulation acts through increased assembly and maturation of receptor subunits into functional pentameric receptors. Studies of muscle nicotinic acetylcholine receptors suggest that the availability of unassembled subunits in the endoplasmic reticulum can be regulated by the ubiquitin-proteosome pathway, resulting in altered surface expression. Here, we describe a role for ubiquilin-1, a ubiquitin-like protein with the capacity to interact with both the proteosome and ubiquitin ligases, in regulating nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors. Ubiquilin-1 interacts with unassembled alpha3 and alpha4 subunits when coexpressed in heterologous cells and interacts with endogenous nicotinic acetylcholine receptors in neurons. Coexpression of ubiquilin-1 and neuronal nicotinic acetylcholine receptors in heterologous cells dramatically reduces the expression of the receptors on the cell surface. In cultured superior cervical ganglion neurons, expression of ubiquilin-1 abolishes nicotine-induced up-regulation of nicotinic acetylcholine receptors but has no effect on the basal level of surface receptors. Coimmunostaining shows that the interaction of ubiquilin-1 with the alpha3 subunit draws the receptor subunit and proteosome into a complex. These data suggest that ubiquilin-1 limits the availability of unassembled nicotinic acetylcholine receptor subunits in neurons by drawing them to the proteosome, thus regulating nicotine-induced up-regulation.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Lamina-specific distribution of Synaptopodin, an actin-associated molecule essential for the spine apparatus, in identified principal cell dendrites of the mouse hippocampus.

Synaptopodin is an actin-associated molecule found in a subset of telencephalic spines. It is an essential component of the spine apparatus, a Ca(2+)-storing organelle and has been implicated in synaptic plasticity (Deller et al. [2003] Proc Natl Acad Sci U S A 100:10494-10499). In the rodent hippocampus, Synaptopodin is distributed in a characteristic region- and lamina-specific manner. To learn more about the cellular basis underlying this distribution, the regional, laminar, and cellular localization of Synaptopodin and its mRNA were analyzed in mouse hippocampus. First, Synaptopodin puncta densities were quantified after immunofluorescent labeling using confocal microscopy. Second, the dendritic distribution of Synaptopodin-positive puncta was studied using three-dimensional confocal reconstructions of Synaptopodin-immunostained and enhanced green fluorescence protein (EGFP)-labeled principal neurons. Synaptopodin puncta located within dendrites of principal neurons were primarily found in spines (>95%). Analysis of dendritic segments located in different layers revealed lamina-specific differences in the percentage of Synaptopodin-positive spines. Densities ranged between 37% (outer molecular layer) and 14% (stratum oriens; CA1). Finally, synaptopodin mRNA expression was studied using in situ hybridization, laser microdissection, and quantitative reverse transcriptase-polymerase chain reaction. Expression levels were comparable between all regions. These data demonstrate a lamina-specific distribution of Synaptopodin within dendritic segments of identified neurons. Within dendrites, the majority of Synaptopodin-positive puncta were located in spines where they represent spine apparatuses. We conclude, that this organelle is distributed in a region- and layer-specific manner in the mouse hippocampus and suggest that differences in the activity of afferent fiber systems could determine its distribution.

Animals↗

PubMed2005

The primordial, blue-cone color system of the mouse retina.

Humans and old world primates have trichromatic color vision based on three spectral types of cone [long-wavelength (L-), middle-wavelength (M-), and short-wavelength (S-) cones]. All other placental mammals are dichromats, and their color vision depends on the comparison of L- and S-cone signals; however, their cone-selective retinal circuitry is still unknown. Here, we identified the S-cone-selective (blue cone) bipolar cells of the mouse retina. They were labeled in a transgenic mouse expressing Clomeleon, a chloride-sensitive fluorescent protein, under the control of the thy1 promoter. Blue-cone bipolar cells comprise only 1-2% of the bipolar cell population, and their dendrites selectively contact S-opsin-expressing cones. In the dorsal half of the mouse retina, only 3-5% of the cones express S-opsin, and they are all contacted by blue-cone bipolar cells, whereas all L-opsin-expressing cones (approximately 95%) are avoided. In the ventral mouse retina, the great majority of cones express both S- and L-opsin. They are not contacted by blue-cone bipolar cells. A minority of ventral cones express S-opsin only, and they are selectively contacted by blue-cone bipolar cells. We suggest that these are genuine S-cones. In contrast to the other cones, their pedicles contain only low amounts of cone arrestin. The blue-cone bipolar cells of the mouse retina and their cone selectivity are closely similar to primate blue-cone bipolars, and we suggest that they both represent the phylogenetically ancient color system of the mammalian retina.

Animals↗

PubMed2005

LNX1 is a perisynaptic Schwann cell specific E3 ubiquitin ligase that interacts with ErbB2.

Non-myelinating perisynaptic Schwann cells wrap motor axon terminals and are required for both functional and structural integrity of the neuromuscular junction. Several lines of evidence indicate that fine-tuning of neuregulin-1/ErbB signaling is critical for maintaining perisynaptic Schwann cells at synapses and that this control may be achieved by the developmental downregulation of the ErbB2 receptor. Here, we identify a direct interaction between ErbB2 and LNX1, an E3 ubiquitin ligase that can target interacting proteins for degradation through ubiquitination. Immunostaining shows that LNX1 is specifically localized in perisynaptic Schwann cells but not in Schwann cells along the motor axon. Developmentally, levels of LNX1 protein are inversely correlated with the responsiveness of perisynaptic Schwann cells to neuregulin-1. Furthermore, the LNX1 staining disappears upon denervation, whereas ErbB2 reappears in Schwann cells after denervation. Taken together, these data suggest that LNX1 may play a role in regulating neuregulin-1/ErbB signaling in perisynaptic Schwann cells.

Animals↗

PubMed2004

Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex.

We show that the neural cell recognition molecule Close Homolog of L1 (CHL1) is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior region of the developing mouse neocortex. CHL1 was expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex. Deep layer pyramidal neurons of CHL1-minus mice were shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented, often inverted apical dendrites. Impaired migration of CHL1-minus cortical neurons was suggested by strikingly slower rates of radial migration in cortical slices, failure to potentiate integrin-dependent haptotactic cell migration in vitro, and accumulation of migratory cells in the intermediate and ventricular/subventricular zones in vivo. The restriction of CHL1 expression and effects of its deletion in posterior neocortical areas suggests that CHL1 may regulate area-specific neuronal connectivity and, by extension, function in the visual and somatosensory cortex.

Animals↗

PubMed2004

Differential mRNA expression and protein localization of the SAP90/PSD-95-associated proteins (SAPAPs) in the nervous system of the mouse.

The supramolecular anchoring/signaling complex at the postsynaptic density of glutamatergic synapses has been proposed to play a key role in regulating synaptic function and plasticity. One class of proteins present in the complex is the SAP90/PSD-95-associated protein family (SAPAPs). The SAPAPs, identified by their direct interaction with PSD-95 family proteins, were initially proposed to function in the anchoring/signaling complex as linker proteins between glutamate receptor binding proteins and the cytoskeleton. However, recent studies have indicated that the SAPAPs also bind to signaling molecules and may thus have multiple roles at synapses. Four homologous genes encoding SAPAP proteins have been previously identified. As a first step toward understanding the physiological function of the SAPAPs, we have investigated in detail, at both the mRNA and protein levels, the localization of the individual SAPAP genes in the adult murine nervous system. We find that the SAPAP mRNAs are highly, yet differentially, expressed in many regions of the brain, including the hippocampus and cerebellum. Furthermore, SAPAP3 mRNA is targeted to dendrites, whereas SAPAP1, -2, and -4 mRNAs are detected mainly in cell bodies. The SAPAP proteins are localized at synapses in a manner consistent with mRNA expression. Surprisingly, in addition to glutamatergic synapse localization, antibody staining also reveals that the SAPAP proteins are localized at cholinergic synapses, including neuronal cholinergic synapses and the neuromuscular junction. Together, these results indicate that the SAPAPs are general components of excitatory synapses and that each of these proteins may perform a distinct function.

Animals↗

PubMed2004

PSD93 regulates synaptic stability at neuronal cholinergic synapses.

Neuronal cholinergic synapses play important roles in both the PNS and CNS. However, the mechanisms that regulate the formation, maturation, and stability of neuronal cholinergic synapses are poorly understood. In this study, we use the readily accessible mouse superior cervical ganglion (SCG) and submandibular ganglion (SMG) to examine the assembly of the postsynaptic complex of neuronal cholinergic synapses. We find that novel splicing forms of PSD93 (postsynaptic density 93) are expressed in SCG. By immunostaining, we show that PSD93 proteins precisely colocalize with neuronal nicotinic acetylcholine receptors (nAChRs) at synapses of the SCG and SMG. Subcellular fractionation demonstrates that PSD93 is enriched in the PSD fraction of SCG, and coimmunoprecipitation shows that PSD93 and neuronal nAChRs form a complex in vivo. Furthermore, two additional components of the well characterized glutamatergic postsynaptic complex, GKAP/SAPAP (guanylate kinase domain-associated protein/synapse-associated protein-associated protein) and Shank/ProSAP family proteins, are also present at neuronal cholinergic synapses. To assess the function of this protein complex at neuronal cholinergic synapses in vivo, we examined ganglia in mice that lack PSD93. We find that neuronal cholinergic synapses form properly in PSD93 null mice. After denervation, however, synaptic clusters of nAChRs disassemble much faster in mice lacking PSD93 than those in wild-type mice. These results demonstrate that PSD93 is a key component of the postsynaptic scaffold at neuronal cholinergic synapses and plays an important role in synaptic stability. In addition, these results suggest that the mechanism of postsynaptic scaffolding is conserved between neuronal cholinergic and glutamatergic synapses.

Alternative Splicing↗

PubMed2004

Labeling neurons in vivo for morphological and functional studies.

Increasingly sophisticated strategies for labeling cells in vivo are providing unprecedented opportunities to study neurons in living animals. Transgenic expression of genetically encoded reporters enables us to monitor changes in neuronal activity in response to sensory stimuli, and the labeling of single neurons with fluorescent proteins allows the dynamics of neuronal connectivity to be observed in transgenic animals over periods ranging from minutes to months. Advances in transient labeling techniques such as viral infection and electroporation provide a rapid means by which to analyze neuronal gene function in vivo. These new approaches to labeling, manipulating and imaging neurons in intact organisms are transforming the way in which the nervous system is studied.

Animals↗

PubMed2004

A rat pain model of vincristine-induced neuropathy.

Vincristine belongs to the family of vinca alkaloids used for treatment of malignant tumors. Clinical application of these agents is often associated with dose-dependent painful neuropathy due to damages to the peripheral axons. A rat model of vincristine-induced hyperalgesia was developed through intravenous injection of vincristine by Aley et al. (1996) and was later modified by Nozaki-Taguchi et al. (2001) using continuous intravenous infusion of vincristine. This model provides consistent and long-lasting neuropathic pain states mimicking vincristine-induced pain conditions in human patients. Therefore, this model is a valuable means of studying the mechanisms and pharmacology of vincristine-induced neuropathic pain. In this chapter we describe in detail steps the generation of vincristine-induced neuropathy in rats through continuous intravenous infusion of vincristine.

Animals↗

PubMed2003

Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition.

Synaptic activity drives synaptic rearrangement in the vertebrate nervous system; indeed, this appears to be a main way in which experience shapes neural connectivity. One rearrangement that occurs in many parts of the nervous system during early postnatal life is a competitive process called 'synapse elimination'. At the neuromuscular junction, where synapse elimination has been analysed in detail, muscle fibres are initially innervated by multiple axons, then all but one are withdrawn and the 'winner' enlarges. In support of the idea that synapse elimination is activity dependent, it is slowed or speeded when total neuromuscular activity is decreased or increased, respectively. However, most hypotheses about synaptic rearrangement postulate that change depends less on total activity than on the relative activity of the competitors. Intuitively, it seems that the input best able to excite its postsynaptic target would be most likely to win the competition, but some theories and results make other predictions. Here we use a genetic method to selectively inhibit neurotransmission from one of two inputs to a single target cell. We show that more powerful inputs are strongly favoured competitors during synapse elimination.

Animals↗

PubMed2003

Postsynaptic requirement for Abl kinases in assembly of the neuromuscular junction.

Agrin signals through the muscle-specific receptor tyrosine kinase (MuSK) to cluster acetylcholine receptors (AChRs) on the postsynaptic membrane of the neuromuscular junction (NMJ). This stands as the prevailing model of synapse induction by a presynaptic factor, yet the agrin-dependent MuSK signaling cascade is largely undefined. Abl1 (previously known as Abl) and the Abl1-related gene product Abl2 (previously known as Arg) define a family of tyrosine kinases that regulate actin structure and presynaptic axon guidance. Here we show that the Abl kinases are critical mediators of postsynaptic assembly downstream of agrin and MuSK. In mouse muscle, Abl kinases were localized to the postsynaptic membrane of the developing NMJ. In cultured myotubes, Abl kinase activity was required for agrin-induced AChR clustering and enhancement of MuSK tyrosine phosphorylation. Moreover, MuSK and Abl kinases effected reciprocal tyrosine phosphorylation and formed a complex after agrin engagement. Our findings suggest that Abl kinases provide the developing synapse with the kinase activity required for signal amplification and the intrinsic cytoskeletal regulatory capacity required for assembly and remodeling.

Adaptor Proteins, Signal Transducing↗