Wangcai Gao

Bidirectional expression of CUG and CAG expansion transcripts and intranuclear polyglutamine inclusions in spinocerebellar ataxia type 8

We previously reported that a (CTG)n expansion causes spinocerebellar ataxia type 8 (SCA8), a slowly progressive ataxia with reduced penetrance. We now report a transgenic mouse model in which the full-length human SCA8 mutation is transcribed using its endogenous promoter. (CTG)116 expansion, but not (CTG)11 control lines, develop a progressive neurological phenotype with in vivo imaging showing reduced cerebellar-cortical inhibition. 1C2-positive intranuclear inclusions in cerebellar Purkinje and brainstem neurons in SCA8 expansion mice and human SCA8 autopsy tissue result from translation of a polyglutamine protein, encoded on a previously unidentified antiparallel transcript (ataxin 8, ATXN8 ) spanning the repeat in the CAG direction. The neurological phenotype in SCA8 BAC expansion but not BAC control lines demonstrates the pathogenicity of the (CTG-CAG)n expansion. Moreover, the expression of noncoding (CUG)n expansion transcripts (ataxin 8 opposite strand, ATXN8OS ) and the discovery of intranuclear polyglutamine inclusions suggests SCA8 pathogenesis involves toxic gain-of-function mechanisms at both the protein and RNA levels.

RNA Gain-of-Function in Spinocerebellar Ataxia Type 8

Microsatellite expansions cause a number of dominantly-inherited neurological diseases. Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins. Bi-directional expression of the spinocerebellar ataxia type 8 (SCA8) CTG CAG expansion produces CUG expansion RNAs (CUGexp) from the ATXN8OS gene and a nearly pure polyglutamine expansion protein encoded by ATXN8 CAGexp transcripts expressed in the opposite direction. Here, we present three lines of evidence that RNA gain-of-function plays a significant role in SCA8: 1) CUGexp transcripts accumulate as ribonuclear inclusions that co-localize with MBNL1 in selected neurons in the brain; 2) loss of Mbnl1 enhances motor deficits in SCA8 mice; 3) SCA8 CUGexp transcripts trigger splicing changes and increased expression of the CUGBP1-MBNL1 regulated CNS target, GABA-A transporter 4 (GAT4/Gabt4). In vivo optical imaging studies in SCA8 mice confirm that Gabt4 upregulation is associated with the predicted loss of GABAergic inhibition within the granular cell layer. These data demonstrate that CUGexp transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUGexp disorders. Moreover, our demonstration that relatively short CUGexp transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.

Cerebellar Cortical Molecular Layer Inhibition Is Organized in Parasagittal Zones

Molecular layer inhibitory interneurons generate on-beam and off-beam inhibition in the cerebellar cortex that is hypothesized to control the timing and/or spatial patterning of Purkinje cell discharge. On- and off-beam inhibition has been assumed to be spatially uniform and continuous within a folium. Using flavoprotein autofluorescence optical imaging in the mouse cerebellar cortex in vivo, this study demonstrates that the inhibition evoked by parallel fiber and peripheral stimulation results in parasagittal bands of decreases in fluorescence that correspond to zebrin II-positive bands. The parasagittal bands of decreased fluorescence are abolished by GABAA antagonists and reflect the activity of molecular layer interneurons on their targets. The same banding pattern was observed using Ca2+ imaging. The bands produce spatially specific decreases in the responses to peripheral input. Therefore, molecular layer inhibition is compartmentalized into zebrin II parasagittal domains that differentially modulate the spatial pattern of cerebellar cortical activity.

Flavoprotein autofluorescence imaging in the cerebellar cortex in vivo

Autofluorescence optical imaging is rapidly becoming a widely used tool for mapping activity in the central nervous system function in vivo and investigating the coupling among neurons, glia, and metabolism. This paper provides a brief review of autofluorescence and of our recent work using flavoprotein imaging in the cerebellar cortex. Stimulation of the parallel fibers evokes an intrinsic fluorescence signal that is tightly coupled to neuronal activation and primarily generated postsynaptically. The signal originates from mitochondrial flavoproteins. The signal is biphasic, with the initial increase in fluorescence (light phase) resulting from the oxidation of flavoproteins and the subsequent decrease (dark phase) from the reduction of flavoproteins. The light phase is primarily neuronal, and the dark phase is primarily glial. Exploiting the spatial properties of molecular layer inhibition in the cerebellar cortex, we show that flavoprotein autofluorescence can monitor both excitatory and inhibitory activity in the cerebellar cortex. Furthermore, flavoprotein autofluorescence has revealed that molecular layer inhibition is organized into parasagittal domains that differentially modulate the spatial pattern of cerebellar cortical activity. The reduction in flavoprotein autofluorescence occurring in the inhibitory bands most likely reflects a decrease in intracellular Ca2+ in the neurons inhibited by the molecular layer interneurons. Therefore, flavoprotein autofluorescence imaging is providing new insights into cerebellar cortical function and neurometabolic coupling.

Abnormalities in the Climbing Fiber-Purkinje Cell Circuitry Contribute to Neuronal Dysfunction in ATXN1[82Q] Mice

One fundamental unanswered question in the field of polyglutamine diseases concerns the pathophysiology of neuronal dysfunction. Is there dysfunction in a specific neuronal population or circuit initially that contributes the onset of behavioral abnormalities? This study used a systems-level approach to investigate the functional integrity of the excitatory cerebellar cortical circuitry in vivo from several transgenic ATXN1 mouse lines. We tested the hypotheses that there are functional climbing fiber (CF)–Purkinje cell (PC) and parallel fiber (PF)–PC circuit abnormalities using flavoprotein autofluorescence optical imaging and extracellular field potential recordings. In early-symptomatic and symptomatic animals expressing ATXN1[82Q], there is a marked reduction in PC responsiveness to CF activation. Immunostaining of vesicular glutamate transporter type 2 demonstrated a decrement in CF extension on PC dendrites in symptomatic ATXN1[82Q] mice. In contrast, responses to PF stimulation were relatively normal. Importantly, the deficits in CF–PC synaptic transmission required expression of pathogenic ataxin-1 (ATXN1[82Q]) and for its entrance into the nucleus of PCs. Loss of endogenous mouse Atxn1 had no discernible effects. Furthermore, the abnormalities in CF–PC synaptic transmission were ameliorated when mutant transgene expression was prevented during postnatal cerebellar development. The results demonstrate the preferential susceptibility of the CF–PC circuit to the effects of ATXN1[82Q]. Further, this deficit likely contributes to the abnormal motor phenotype of ATXN1[82Q] mice. For polyglutamine diseases generally, the findings support a model whereby specific neuronal circuits suffer insults that alter function before cell death.

Low-Frequency Oscillations in the Cerebellar Cortex of the Tottering Mouse

The tottering mouse is an autosomal recessive disorder involving a missense mutation in the gene encoding P/Q-type voltage-gated Ca2+ channels. The tottering mouse has a characteristic phenotype consisting of transient attacks of dystonia triggered by stress, caffeine, or ethanol. The neural events underlying these episodes of dystonia are unknown. Flavoprotein autofluorescence optical imaging revealed transient, low-frequency oscillations in the cerebellar cortex of anesthetized and awake tottering mice but not in wild-type mice. Analysis of the frequencies, spatial extent, and power were used to characterize the oscillations. In anesthetized mice, the dominant frequencies of the oscillations are between 0.039 and 0.078 Hz. The spontaneous oscillations in the tottering mouse organize into high power domains that propagate to neighboring cerebellar cortical regions. In the tottering mouse, the spontaneous firing of 83% (73/88) of cerebellar cortical neurons exhibit oscillations at the same low frequencies. The oscillations are reduced by removing extracellular Ca2+ and blocking L-type Ca2+ channels. The oscillations are likely generated intrinsically in the cerebellar cortex because they are not affected by blocking AMPA receptors or by electrical stimulation of the parallel fiber-Purkinje cell circuit. Furthermore, local application of an L-type Ca2+ agonist in the tottering mouse generates oscillations with similar properties. The beam-like response evoked by parallel fiber stimulation is reduced in the tottering mouse. In the awake tottering mouse, transcranial flavoprotein imaging revealed low-frequency oscillations that are accentuated during caffeine-induced attacks of dystonia. During dystonia, oscillations are also present in the face and hindlimb electromyographic (EMG) activity that become significantly coherent with the oscillations in the cerebellar cortex. These low-frequency oscillations and associated cerebellar cortical dysfunction demonstrate a novel abnormality in the tottering mouse. These oscillations are hypothesized to be involved in the episodic movement disorder in this mouse model of episodic ataxia type 2.

Parasagittally aligned, mGluR1-dependent patches are evoked at long latencies by parallel fiber stimulation in the mouse cerebellar cortex in vivo.

The parallel fibers (PFs) in the cerebellar cortex extend several millimeters along a folium in the mediolateral direction. The PFs are orthogonal to and cross several parasagittal zones defined by the olivocerebellar and corticonuclear pathways and the expression of molecular markers on Purkinje cells (PCs). The functions of these two organizations remain unclear, including whether the bands respond similarly or differentially to PF input. By using flavoprotein imaging in the anesthetized mouse in vivo, this study demonstrates that high-frequency PF stimulation, which activates a beamlike response at short latency, also evokes patches of activation at long latencies. These patches consist of increased fluorescence along the beam at latencies of 20-25 s with peak activation at 35 s. The long-latency patches are completely blocked by the type 1 metabotropic glutamate receptor (mGluR(1)) antagonist LY367385. Conversely, the AMPA and NMDA glutamate receptor antagonists DNQX and APV have little effect. Organized in parasagittal bands, the long-latency patches align with zebrin II-positive PC stripes. Additional Ca(2+) imaging demonstrates that the patches reflect increases in intracellular Ca(2+). Both the PLCβ inhibitor U73122 and the ryanodine receptor inhibitor ryanodine completely block the long-latency patches, indicating that the patches are due to Ca(2+) release from intracellular stores. Robust, mGluR(1)-dependent long-term potentiation (LTP) of the patches is induced using a high-frequency PF stimulation conditioning paradigm that generates LTP of PF-PC synapses. Therefore, the parasagittal bands, as defined by the molecular compartmentalization of PCs, respond differentially to PF inputs via mGluR(1)-mediated release of internal Ca(2+).

Long-Term Potentiation of the Responses to Parallel Fiber Stimulation in Mouse Cerebellar Cortex in Vivo

Long-term potentiation (LTP) of parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum has been suggested to underlie aspects of motor learning. Previous in vitro studies have primarily used low frequency PF stimulation conditioning paradigms to generate either presynaptic PF-PC LTP (4-8 Hz) or postsynaptic PF-PC LTP (1 Hz). Little is known about the conditions that evoke PF-PC LTP in vivo. High frequency stimulation in vivo increases PC responsiveness to peripheral stimuli; however, neither the site of action nor the signaling pathways involved have been examined. Using flavoprotein autofluorescence optical imaging in the FVB mouse in vivo, this report describes that a conditioning stimulation consisting of a high frequency burst of PF stimulation (100 Hz, 15 pulse trains every 3 s for 5 min) evokes a long-term increase in the response to PF stimulation. Following the conditioning stimulation, the response to PF stimulation increases over 20 min to approximately 130% above baseline and this potentiation persists for at least 2 h. Field potential recordings of the responses to PF stimulation show that the postsynaptic component is potentiated but the presynaptic, parallel fiber volley is not. Paired-pulse facilitation does not change after the conditioning stimulation, suggesting the potentiation occurs postsynaptically. Blocking non-NMDA (N-methyl-d-aspartic acid) ionotropic glutamate receptors with DNQX (6,7-dinitroquinoxaline-2,3-dione disodium salt, 50 muM, bath application) during the conditioning stimulation has no effect on the long-term increase in fluorescence. However, blocking subtype I metabotropic glutamate receptors (mGLuR(1)) with LY367385 (200 muM) during the conditioning stimulation abolishes the long-term increase in fluorescence. Blocking GABAergic neurotransmission is not required to evoke this long-term potentiation. Blocking GABA(A) receptors reduces but does not eliminate the long-term potentiation. Therefore, this study demonstrates that high frequency PF stimulation generates long-term potentiation of PF-PC synapses in vivo. This novel form of LTP is generated primarily postsynaptically and is mediated by mGluR(1) receptors.

Parasagittal Zones in the Cerebellar Cortex Differ in Excitability, Information Processing, and Synaptic Plasticity

At the molecular and circuitry levels, the cerebellum exhibits a striking parasagittal zonation as exemplified by the spatial distribution of molecules expressed on Purkinje cells and the topography of the afferent and efferent projections. The physiology and function of the zonation is less clear. Activity-dependent optical imaging has proven a useful tool to examine the physiological properties of the parasagittal zonation in the intact animal. Recent findings show that zebrin II-positive and zebrin II-negative zones differ markedly in their responses to parallel fiber inputs. These findings suggest that cerebellar cortical excitability, information processing, and synaptic plasticity depend on the intrinsic properties of different parasagittal zones.

Cellular and Metabolic Origins of Flavoprotein Autofluorescence in the Cerebellar Cortex in vivo

Flavoprotein autofluorescence imaging, an intrinsic mitochondrial signal, has proven useful for monitoring neuronal activity. In the cerebellar cortex, parallel fiber stimulation evokes a beam-like response consisting of an initial, short-duration increase in fluorescence (on-beam light phase) followed by a longer duration decrease (on-beam dark phase). Also evoked are parasagittal bands of decreased fluorescence due to molecular layer inhibition. Previous work suggests that the on-beam light phase is due to oxidative metabolism in neurons. The present study further investigated the metabolic and cellular origins of the flavoprotein signal in vivo, testing the hypotheses that the dark phase is mediated by glia activation and the inhibitory bands reflect decreased flavoprotein oxidation and increased glycolysis in neurons. Blocking postsynaptic ionotropic and metabotropic glutamate receptors abolished the on-beam light phase and the parasagittal bands without altering the on-beam dark phase. Adding glutamate transporter blockers reduced the dark phase. Replacing glucose with lactate (or pyruvate) or adding lactate to the bathing media abolished the on-beam dark phase and reduced the inhibitory bands without affecting the light phase. Blocking monocarboxylate transporters eliminated the on-beam dark phase and increased the light phase. These results confirm that the on-beam light phase is due primarily to increased oxidative metabolism in neurons. They also show that the on-beam dark phase involves activation of glycolysis in glia resulting in the generation of lactate that is transferred to neurons. Oxidative savings in neurons contributes to the decrease in fluorescence characterizing the inhibitory bands. These findings provide strong in vivo support for the astrocyte–neuron lactate shuttle hypothesis.