Zuyuan Qian

Regional and cellular distribution of protein kinase C in rat cerebellar purkinje cells

Protein kinase C (PCK) is a family of isoforms that are implicated in subcellular signal transduction. The authors investigated the distribution of several PKC isoforms (PKC‐α, PKC‐β, PKC‐γ, PKC‐δ, and PKC‐ϵ) within major cerebellar cell types as well as cerebellar projection target neurons, including Purkinje neurons, cerebellar nuclear neurons, and secondary vestibular neurons. PKC‐α, PKC‐β, PKC‐γ, PKC‐δ, and PKC‐ϵ are found within the cerebellum. Of these isoforms, PKC‐γ and PKC‐δ are highly expressed in Purkinje cells. PKC‐γ is expressed in all Purkinje cells, whereas the expression of PKC‐δ is restricted to sagittal bands of Purkinje cells in the posterior cerebellar cortex. In the lower folia of the uvula and nodulus, Purkinje cell expression of PKC‐δ is uniformly high, and the sagittal banding for PKC‐δ expression is absent. Within the cerebellar nuclei, PKC‐δ‐immunolabeled axons terminate within the medial aspect of the caudal half of the ipsilateral interpositus nucleus. PKC δ‐immunolabeled axons also terminated within the caudal medial and descending vestibular nuclei (MVN and DVN, respectively), the parasolitary nucleus (Psol), and the nucleus prepositus hypoglossi (NPH). PKC‐γ‐immunolabeled axons terminated in all of the cerebellar nuclei as well as in the lateral and superior vestibular nuclei and the MVN, DVN, Psol, and NPH. The projection patterns of PKC‐immunolabeled Purkinje cells were confirmed by lesion‐depletion studies in which unilateral uvula‐nodular lesions caused depletion of PKC‐immunolabeled terminals ipsilateral to the lesion in the vestibular complex. These data identify circuitry that is unique to cerebellar‐vestibular interactions. J. Comp. Neurol. 427:235–254, 2000.

Acyl coenzyme A-binding protein (ACBP) is phosphorylated and secreted by retinal Müller astrocytes following protein kinase C activation.

Horizontal optokinetic stimulation of rabbit retina in vivo evokes increased expression of acyl coenzyme A‐binding protein (ACBP), also known as ‘diazepam binding inhibitor,’ from retinal Müller cells. If the expressed ACBP were also secreted by Müller cells, then stimulus‐evoked secretion of ACBP could influence the activity of GABAA receptor‐expressing retinal neurons. In this study, we examine in vitro whether ACBP is secreted by Müller glial cells and Müller‐like QNR/K2 cells following stimulation with elevated levels of KCl and phorbol myristic acetate (PMA). KCl and PMA stimulation evoked secretion of threonine‐phosphorylated ACBP. A sequence analysis of ACBP shows that it has five potential phosphorylation sites: Two threonine sites fit a protein kinase C phosphorylation pattern. Two threonine sites fit a casein kinase II (CK2) pattern. One serine site fits a CK2 pattern. As CK2 is not expressed in QNR/K2 cells, it is probable that protein kinase C accounts for the phosphorylation of ACBP in these cells and for the PMA‐evoked secretion of ACBP. Serine phosphorylation was constitutive. Horizontal optokinetic stimulation increased threonine‐phosphorylated ACBP in rabbit retina. Phosphorylation of ACBP may influence its target affinity. We used a proteolytic fragment of ACBP, octadecaneuropeptide (ODN), to investigate how threonine phosphorylation influences its affinity for GABAA receptors. Threonine‐phosphorylated ODN had a stronger affinity for GABAA receptors than did unphosphorylated ODN or unphosphorylated ACBP. We conclude that stimulus‐induced Müller cell secretion of phosphorylated ACBP could influence the GABAergic transmission in neighboring retinal neurons.

Climbing fibers induce microRNA transcription in cerebellar Purkinje cells.

The coordinated expression of as many as 100 proteins may be required to sustain simple changes in synaptic transmission. While each protein may be regulated separately, the translation of multiple proteins could be regulated by microRNAs. MicroRNAs are short non-coding RNAs that translationally repress cognate sequences in targeted mRNAs. If these targeted sequences are shared across several genes, then a single microRNA could, effectively regulate the activity of several genes in parallel. Here we investigate whether microRNA transcription is influenced by naturally evoked synaptic activity at the climbing fiber-Purkinje cell synapse in the mouse cerebellar flocculus. Mice received 24 h of binocular horizontal optokinetic stimulation (HOKS) evoking sustained increases in climbing fiber activity to Purkinje cells in one flocculus and decreases to Purkinje cells in the other. Increased climbing fiber activity increased transcription of 12 microRNAs in the flocculus. The transcription of one of these microRNAs, miR335, was proportional to duration of stimulation, increasing 18-fold after 24 h of HOKS. We localized miR335 transcripts to Purkinje cells using hybridization histochemistry. Transcripts of miR335 decayed to baseline within 3 h after HOKS was stopped. We identified mRNA targets for miR335 using multiple screens: sequence analysis, microinjection of miR335 inhibitors and identification of mRNAs whose transcription decreased during HOKS. Two genes, calbindin and 14-3-3-θ passed these screens. Our data suggest that microRNA transcription could provide an important synaptic or homeostatic mechanism for the regulation of proteins that contribute to Purkinje cell plasticity.

Activity-Dependent Expression of Acyl-Coenzyme A-Binding Protein in Retinal Muller Glial Cells Evoked by Optokinetic Stimulation

Long-term horizontal optokinetic stimulation (HOKS) decreases the gain of the horizontal optokinetic reflex and evokes the second phase of optokinetic afternystagmus (OKAN-II). We investigated the possible molecular constituents of this adaptation. We used a differential display reverse transcriptase-PCR screen for mRNAs isolated from retinas of rabbits that received HOKS. In each rabbit, we compared mRNAs from the retina stimulated in the posterior→anterior (preferred) direction with mRNAs from the retina stimulated in the anterior→posterior (null) direction. Acyl-CoA-binding protein (ACBP) mRNA was one of four mRNAs selected by this screen, the proteins of which interact with GABA receptors. HOKS in the preferred direction increased ACBP mRNA transcription and ACBP protein expression. ACBP was localized to Muller glial cells by hybridization histochemistry and by immunohistochemistry. ACBP interacts with the α1-subunit of the GABAA receptor, as determined by a yeast two-hybrid technique. This interaction was confirmed by coimmunoprecipitation of ACBP and the α1-subunit of the GABAA receptor using an antibody to GABAAα1. The interaction was also confirmed by a “pull-down” assay in which histidine-tagged ACBP was used to pull down the GABAAα1. ACBP does not cross the blood–brain barrier. However, smaller truncated proteolytic fragments of ACBP do, increasing the excitability of central cortical neurons. Muller cells may secrete ACBP in the inner plexiform layer, thereby decreasing the sensitivity of GABAA receptors expressed on the surface of ganglion cell dendrites. Because retinal directional sensitivity is linked to GABAergic transmission, HOKS-induced expression of ACBP could provide a molecular basis for adaptation to HOKS and for the genesis of OKAN-II.

Activity-dependent expression of calbindin in rabbit floccular Purkinje cells modulated by optokinetic stimulation

Optokinetic stimulation activates visual climbing fiber pathways that synapse upon contralateral floccular Purkinje cells. Long-term horizontal optokinetic stimulation causes a progressive decrease in gain of the optokinetic reflex and leads to the subsequent genesis of a prolonged negative optokinetic afternystagmus. Since the flocculus is involved in adaptation to optokinetic stimulation, we used the technique of differential display reverse transcription-polymerase chain reaction to explore transcriptional changes in the flocculus evoked by long-term optokinetically evoked climbing fiber discharge. Several differentially transcribed gene products were isolated and sequenced. One of these, calbindin mRNA, was expressed in relatively decreased abundance in the flocculus that received increased climbing fiber input. Decreased transcription of calbindin mRNA was confirmed by northern blots. Hybridization histochemistry was used to localize calbindin mRNA to Purkinje cells and confirmed decreased transcription of calbindin mRNA in Purkinje cells located in folium 1 of the flocculus. Western blots and immunohistochemistry localized the climbing fiber-evoked decreased expression of calbindin to Purkinje cells in folia 1 of the flocculus. The expression of four other calcium-binding proteins in the flocculus was not influenced by optokinetic stimulation. Changes in expression of calbindin could be evoked by decreases in intracellular calcium associated with climbing fiber-evoked decreases in Purkinje cell simple spike activity.The application of differential display reverse transcription-polymerase chain reaction has provided a positive screen for several molecules in addition to calbindin whose expression is affected by naturally evoked activity in a major synaptic pathway to the cerebellum. Further experiments will be required to specify the functional role of each of these molecules.

Neuronally modulated transcription of a glycine transporter in rat dorsal cochlear nucleus and nucleus of the medial trapezoid body.

Neurotransmitter transporters limit transmitter concentration at the postsynaptic membrane by removing neurotransmitters from the synaptic cleft. Not only do neurotransmitter transporters contribute to the regulation of synaptic transmission, but they themselves might be dynamically regulated by neuronal activity of the neurons in which they are expressed. In this experiment, we investigated the question of whether the transcription of two different glycine transporters, Glyt1 and Glyt2, is influenced by neuronal activity. These transporters are found in the dorsal cochlear nucleus (DCN) and medial nucleus of the trapezoid body. Glyt1 and Glyt2 mRNA were measured by using hybridization histochemistry and a semiquantitative reverse transcription polymerase chain reaction. Decreases in auditory primary afferent activity, caused by either unilateral labyrinthectomy or disruption of the middle ear ossicles, caused a reduction in Glyt2, but not Glyt1 mRNA in the ipsilateral DCN and in the contralateral medial nucleus of the trapezoid body. Acoustic stimulation at either 10 kHz or 40 kHz was used to provide controlled increases in primary afferent activity, evoking localized increases in Glyt2 mRNA in clusters of neurons in the DCN. The location of these clusters corresponded to the regions of the auditory tonotopic map devoted to these frequencies. The duration of changes in Glyt2 mRNA evoked by unilateral labyrinthectomy, measured with the reverse transcription polymerase chain reaction, was 5–10 days. These data provide the first example of in vivo regulation of transporter transcription by neuronal activity. J. Comp. Neurol. 415:175–188, 1999.

Climbing fiber activity reduces 14-3-3-θ regulated GABAA receptor phosphorylation in cerebellar Purkinje cells

Cerebellar adaptive plasticity regulates posture and movement in response to changing conditions of sensory stimulation. Study of adaptive plasticity of cerebellar circuitry in vitro confines experimental interest to mechanisms with a time scale of minutes. However, cerebellar plasticity, measured behaviorally or electrophysiologically in vivo, occurs over a time scale of tens of minutes and hours. Here we investigate how optokinetically-evoked increases in climbing fiber activity influence expression of key subcellular signaling proteins that regulate the accumulation of GABA(A) receptors (GABA(A)Rs) in the cytoplasm of Purkinje cells and their insertion into the plasma membrane. We used long-term horizontal optokinetic stimulation (HOKS) to activate climbing fibers that project to the flocculus of mice. Although long-term increases in climbing fiber activity in vivo do not alter the expression of any of the subunits of GABA(A)Rs expressed by Purkinje cells, they do influence other subcellular events such as transcription and interaction of signaling proteins. Specifically, increased climbing fiber activity evoked decreased expression of 14-3-3-θ, reduced serine phosphorylation of GABA(A)g(2), and reduced the interaction of 14-3-3-θ with protein kinase C-γ (PKC-γ). Knockdown of 14-3-3-θ in vivo reduced the serine phosphorylation of GABA(A)γ(2). Conversely, treatment of cerebellar lysates with phorbol 12-myristate-13-acetate (PMA), a PKC activator, increased serine phosphorylation of GABA(A)γ(2). Knockdown of 14-3-3-θ or PKC-γ in N2a cells in vitro reduced serine phosphorylation of GABA(A)γ(2) and reduced its cell-surface expression. We interpret these data to mean that a prolonged increase in climbing fiber activity decreases the cell-surface expression of GABA(A)Rs in Purkinje cells and thereby reduces their sensitivity to GABAergic inhibition. This provides a homeostatic mechanism by which Purkinje cells become less sensitive to stellate cell inhibition also evoked by climbing fiber activity.

Long-term climbing fibre activity induces transcription of microRNAs in cerebellar Purkinje cells

Synaptic activation of central neurons is often evoked by electrical stimulation leading to post-tetanic potentiation, long-term potentiation or long-term depression. Even a brief electrical tetanus can induce changes in as many as 100 proteins. Since climbing fibre activity is often associated with cerebellar behavioural plasticity, we used horizontal optokinetic stimulation (HOKS) to naturally increase synaptic input to floccular Purkinje cells in mice for hours, not minutes, and investigated how this activity influenced the transcription of microRNAs, small non-coding nucleotides that reduce transcripts of multiple, complementary mRNAs. A single microRNA can reduce the translation of as many as 30 proteins. HOKS evoked increases in 12 microRNA transcripts in floccular Purkinje cells. One of these microRNAs, miR335, increased 18-fold after 24 h of HOKS. After HOKS stopped, miR335 transcripts decayed with a time constant of approximately 2.5 h. HOKS evoked a 28-fold increase in pri-miR335 transcripts compared with an 18-fold increase in mature miR335 transcripts, confirming that climbing fibre-evoked increases in miR335 could be attributed to increases in transcription. We used three screens to identify potential mRNA targets for miR335 transcripts: (i) nucleotide complementarity, (ii) detection of increased mRNAs following microinjection of miR335 inhibitors into the cerebellum, and (iii) detection of decreased mRNAs following HOKS. Two genes, calbindin and 14-3-3-θ, passed these screens. Transfection of N2a cells with miR335 inhibitors or precursors inversely regulated 14-3-3-θ transcripts. Immunoprecipitation of 14-3-3-θ co-immunoprecipitated PKC-γ and GABAAγ2. Knockdown of either 14-3-3-θ or PKC-γ decreased the serine phosphorylation of GABAAγ2, suggesting that 14-3-3-θ and PKC-γ under the control of miR335 homeostatically regulate the phosphorylation and insertion of GABAAγ2 into the Purkinje cell post-synaptic membrane.

Climbing fiber‐evoked Purkinje cell discharge reduces expression of GABAa receptor‐associated protein and decreases its interaction with GABAa receptors

J. Neurochem. (2011) 117, 197–208.