Generation of Astrocyte-Specific MAOB Conditional Knockout Mouse with Minimal Tonic GABA Inhibition

Monoamine oxidase B (MAOB) is a key enzyme for GABA production in astrocytes in several brain regions. To date, the role of astrocytic MAOB has been studied in MAOB null knockout (KO) mice, although MAOB is expressed throughout the body. Therefore, there has been a need for genetically engineered mice in which only astrocytic MAOB is targeted. Here, we generated an astrocyte-specific MAOB conditional KO (cKO) mouse line and characterized it in the cerebellar and striatal regions of the brain. Using the CRISPR-Cas9 gene-editing technique, we generated Maob floxed mice (B6-Maob em1Cjl /Ibs) which have floxed exons 2 and 3 of Maob with two loxP sites. By crossing these mice with hGFAP-CreER T2 , we obtained Maob floxed::hGFAP-CreER T2 mice which have a property of tamoxifen-inducible ablation of Maob under the human GFAP (hGFAP) promoter. When we treated Maob floxed::hGFAP-CreER T2 mice with tamoxifen for 5 consecutive days, MAOB and GABA immunoreactivity were significantly reduced in striatal astrocytes as well as in Bergmann glia and lamellar astrocytes in the cerebellum, compared to sunflower oil-injected control mice. Moreover, astrocyte-specific MAOB cKO led to a 74.6% reduction in tonic GABA currents from granule cells and a 76.8% reduction from medium spiny neurons. Our results validate that astrocytic MAOB is a critical enzyme for the synthesis of GABA in astrocytes. We propose that this new mouse line could be widely used in studies of various brain diseases to elucidate the pathological role of astrocytic MAOB in the future.

Differential Proximity of Perisynaptic Astrocytic Best1at the Excitatory and Inhibitory Tripartite Synapses in APP/PS1 and MAOB-KO Mice Revealed by Lattice Structured Illumination Microscopy

Bestrophin-1 (Best1) is a GABA- and glutamate-permeable, Ca 2+ -activated Cl - channel, which is mainly expressed in astrocytes and localized at the microdomain or perisynaptic junction of the tripartite synapse. Distribution of Best1 is dramatically changed in pathological conditions such as Alzheimer’s disease. However, it is still unknown whether Best1 is located at the glutamatergic or GABAergic tripartite synapses. Here, we utilized the Lattice structured illumination microscopy (Lattice SIM) to visualize Best1 expression at the perisynaptic junctions of the tripartite synapses in CA1 of mouse hippocampus. We performed co-labeling with antibodies against 1) Best1 and vesicular glutamate transporter-2 (vGLUT2) or 2) Best1 and vesicular GABA transporter (vGAT) to measure the proximity of Best1-containing perisynapse to glutamatergic or GABAergic presynapse, respectively. In addition, we examined two transgenic mouse lines of 1) APP/PS1 mouse showing high astrocytic MAOB activity and cytosolic GABA and 2) MAOB-KO mouse showing low astrocytic GABA. Lattice SIM images were further processed by Imaris, which allowed 3Drendering and spot identification. We found that astrocytic Best1 was distributed closer to the glutamatergic synapses than GABAergic synapses in the wild-type mice. In APP/PS1 mice, Best1 distribution was significantly changed by moving away from the glutamatergic synapses while moving closer to the GABAergic synapses. On the contrary, in MAOB-KO mice, the Best1 distribution was dramatically changed by moving closer to the glutamatergic synapses and moving far away from the GABAergic synapses. Our findings propose that the proximity of Best1-containing perisynapses to presynapses dynamically changes according to the level of astrocytic cytosolic GABA.

Adenovirus-induced Reactive Astrogliosis Exacerbates the Pathology of Parkinson’s Disease

Parkinson’s disease (PD) is the most prevalent neurodegenerative motor disorder. While PD has been attributed to dopaminergic neuronal death in substantia nigra pars compacta (SNpc), accumulating lines of evidence have suggested that reactive astrogliosis is critically involved in PD pathology. These pathological changes are associated with α-synuclein aggregation, which is more prone to be induced by an A53T mutation. Therefore, the overexpression of A53T-mutated α-synuclein (A53T-α-syn) has been utilized as a popular animal model of PD. However, this animal model only shows marginal-to-moderate extents of reactive astrogliosis and astrocytic α-synuclein accumulation, while these phenomena are prominent in human PD brains. Here we show that Adeno-GFAP-GFP virus injection into SNpc causes severe reactive astrogliosis and exacerbates the A53Tα-syn-mediated PD pathology. In particular, we demonstrate that AAV-CMV-A53T-α-syn injection, when combined with Adeno-GFAP-GFP, causes more significant loss of dopaminergic neuronal tyrosine hydroxylase level and gain of astrocytic GFAP and GABA levels. Moreover, the combination of AAV-CMV-A53T-α-syn and Adeno-GFAP-GFP causes an extensive astrocytic α-syn expression, just as in human PD brains. These results are in marked contrast to previous reports that AAV-CMV-A53T-α-syn alone causes α-syn expression mostly in neurons but rarely in astrocytes. Furthermore, the combination causes a severe PD-like motor dysfunction as assessed by rotarod and cylinder tests within three weeks from the virus injection, whereas Adeno-GFAP-GFP alone or AAV-CMV-A53T-α-syn alone does not. Our findings implicate that inducing reactive astrogliosis exacerbates PD-like pathologies and propose the virus combination as an advanced strategy for developing a new animal model of PD.

The Pathological Role of Astrocytic MAOB in Parkinsonism Revealed by Genetic Ablation and Over-expression of MAOB

The cause of Parkinson’s disease has been traditionally believed to be the dopaminergic neuronal death in the substantia nigra pars compacta (SNpc).This traditional view has been recently challenged by the proposal that reactive astrocytes serve as key players in the pathology of Parkinson’s disease through excessive GABA release. This aberrant astrocytic GABA is synthesized by the enzymatic action of monoamine oxidase B (MAOB), whose pharmacological inhibition and gene-silencing are reported to significantly alleviate parkinsonian motor symptoms in animal models of Parkinson’s disease. However, whether genetic ablation and over-expression of MAOB can bidirectionally regulate parkinsonian motor symptoms has not been tested. Here we demonstrate that genetic ablation of MAOB blocks the MPTP-induced augmentation of astrocytic GABA-mediated tonic inhibition of neighboring dopaminergic neurons as well as parkinsonian motor symptoms, indicating the necessity of MAOB for parkinsonian motor symptoms. Furthermore, we demonstrate that GFAP-MAOB transgenic mice, in which MAOB is over-expressed under the GFAP promoter for astrocyte-specific over-expression, display exacerbated MPTP-induced tonic inhibition and parkinsonian motor symptoms compared to wild-type mice, indicating the importance of astrocytic MAOB for parkinsonian motor symptoms. Our study provides genetic pieces of evidence for the causal link between the pathological role of astrocytic MAOB-dependent tonic GABA synthesis and parkinsonian motor symptoms.

Differential Proximity of Perisynaptic Astrocytic Best1at the Excitatory and Inhibitory Tripartite Synapses in APP/PS1 and MAOB-KO Mice Revealed by Lattice Structured Illumination Microscopy

Bestrophin-1 (Best1) is a GABA- and glutamate-permeable, Ca 2+ -activated Cl - channel, which is mainly expressed in astrocytes and localized at the microdomain or perisynaptic junction of the tripartite synapse. Distribution of Best1 is dramatically changed in pathological conditions such as Alzheimer’s disease. However, it is still unknown whether Best1 is located at the glutamatergic or GABAergic tripartite synapses. Here, we utilized the Lattice structured illumination microscopy (Lattice SIM) to visualize Best1 expression at the perisynaptic junctions of the tripartite synapses in CA1 of mouse hippocampus. We performed co-labeling with antibodies against 1) Best1 and vesicular glutamate transporter-2 (vGLUT2) or 2) Best1 and vesicular GABA transporter (vGAT) to measure the proximity of Best1-containing perisynapse to glutamatergic or GABAergic presynapse, respectively. In addition, we examined two transgenic mouse lines of 1) APP/PS1 mouse showing high astrocytic MAOB activity and cytosolic GABA and 2) MAOB-KO mouse showing low astrocytic GABA. Lattice SIM images were further processed by Imaris, which allowed 3Drendering and spot identification. We found that astrocytic Best1 was distributed closer to the glutamatergic synapses than GABAergic synapses in the wild-type mice. In APP/PS1 mice, Best1 distribution was significantly changed by moving away from the glutamatergic synapses while moving closer to the GABAergic synapses. On the contrary, in MAOB-KO mice, the Best1 distribution was dramatically changed by moving closer to the glutamatergic synapses and moving far away from the GABAergic synapses. Our findings propose that the proximity of Best1-containing perisynapses to presynapses dynamically changes according to the level of astrocytic cytosolic GABA.

Adenovirus-induced Reactive Astrogliosis Exacerbates the Pathology of Parkinson’s Disease

Parkinson’s disease (PD) is the most prevalent neurodegenerative motor disorder. While PD has been attributed to dopaminergic neuronal death in substantia nigra pars compacta (SNpc), accumulating lines of evidence have suggested that reactive astrogliosis is critically involved in PD pathology. These pathological changes are associated with α-synuclein aggregation, which is more prone to be induced by an A53T mutation. Therefore, the overexpression of A53T-mutated α-synuclein (A53T-α-syn) has been utilized as a popular animal model of PD. However, this animal model only shows marginal-to-moderate extents of reactive astrogliosis and astrocytic α-synuclein accumulation, while these phenomena are prominent in human PD brains. Here we show that Adeno-GFAP-GFP virus injection into SNpc causes severe reactive astrogliosis and exacerbates the A53Tα-syn-mediated PD pathology. In particular, we demonstrate that AAV-CMV-A53T-α-syn injection, when combined with Adeno-GFAP-GFP, causes more significant loss of dopaminergic neuronal tyrosine hydroxylase level and gain of astrocytic GFAP and GABA levels. Moreover, the combination of AAV-CMV-A53T-α-syn and Adeno-GFAP-GFP causes an extensive astrocytic α-syn expression, just as in human PD brains. These results are in marked contrast to previous reports that AAV-CMV-A53T-α-syn alone causes α-syn expression mostly in neurons but rarely in astrocytes. Furthermore, the combination causes a severe PD-like motor dysfunction as assessed by rotarod and cylinder tests within three weeks from the virus injection, whereas Adeno-GFAP-GFP alone or AAV-CMV-A53T-α-syn alone does not. Our findings implicate that inducing reactive astrogliosis exacerbates PD-like pathologies and propose the virus combination as an advanced strategy for developing a new animal model of PD.

The Pathological Role of Astrocytic MAOB in Parkinsonism Revealed by Genetic Ablation and Over-expression of MAOB

The cause of Parkinson’s disease has been traditionally believed to be the dopaminergic neuronal death in the substantia nigra pars compacta (SNpc).This traditional view has been recently challenged by the proposal that reactive astrocytes serve as key players in the pathology of Parkinson’s disease through excessive GABA release. This aberrant astrocytic GABA is synthesized by the enzymatic action of monoamine oxidase B (MAOB), whose pharmacological inhibition and gene-silencing are reported to significantly alleviate parkinsonian motor symptoms in animal models of Parkinson’s disease. However, whether genetic ablation and over-expression of MAOB can bidirectionally regulate parkinsonian motor symptoms has not been tested. Here we demonstrate that genetic ablation of MAOB blocks the MPTP-induced augmentation of astrocytic GABA-mediated tonic inhibition of neighboring dopaminergic neurons as well as parkinsonian motor symptoms, indicating the necessity of MAOB for parkinsonian motor symptoms. Furthermore, we demonstrate that GFAP-MAOB transgenic mice, in which MAOB is over-expressed under the GFAP promoter for astrocyte-specific over-expression, display exacerbated MPTP-induced tonic inhibition and parkinsonian motor symptoms compared to wild-type mice, indicating the importance of astrocytic MAOB for parkinsonian motor symptoms. Our study provides genetic pieces of evidence for the causal link between the pathological role of astrocytic MAOB-dependent tonic GABA synthesis and parkinsonian motor symptoms.

Tweety-homolog (Ttyh) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRAC(swell)) in the Brain

In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl⁻- and osmolytes-efflux via volume-regulated anion channel (VRAC). Although LRRC8 family has been recently proposed as the pore-forming VRAC which is activated by low cytoplasmic ionic strength but not by swelling, the molecular identity of the pore-forming swelling-dependent VRAC (VRAC(swell)) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRAC(swell) in astrocytes. Gene-silencing of all Ttyh1/2/3 eliminated hypo-osmotic-solution-induced Cl⁻ conductance (I(Cl,swell)) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant I(Cl,swell) with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as I(Cl,swell) observed in native astrocytes. Mutagenesis-based structure-activity analysis revealed that positively charged arginine residue at 165 in TTYH1 and 164 in TTYH2 is critical for the formation of the channel-pore. Our results demonstrate that TTYH family confers the bona fide VRAC(swell) in the brain.

Pharmacological Dissection of Intrinsic Optical Signal Reveals a Functional Coupling between Synaptic Activity and Astrocytic Volume Transient

The neuronal activity-dependent change in the manner in which light is absorbed or scattered in brain tissue is called the intrinsic optical signal (IOS), and provides label-free, minimally invasive, and high spatial (~100 µm) resolution imaging for visualizing neuronal activity patterns. IOS imaging in isolated brain slices measured at an infrared wavelength (>700 nm) has recently been attributed to the changes in light scattering and transmittance due to aquaporin-4 (AQP4)-dependent astrocytic swelling. The complexity of functional interactions between neurons and astrocytes, however, has prevented the elucidation of the series of molecular mechanisms leading to the generation of IOS. Here, we pharmacologically dissected the IOS in the acutely prepared brain slices of the stratum radiatum of the hippocampus, induced by 1 s/20 Hz electrical stimulation of Schaffer-collateral pathway with simultaneous measurement of the activity of the neuronal population by field potential recordings. We found that 55% of IOSs peak upon stimulation and originate from postsynaptic AMPA and NMDA receptors. The remaining originated from presynaptic action potentials and vesicle fusion. Mechanistically, the elevated extracellular glutamate and K⁺ during synaptic transmission were taken up by astrocytes via a glutamate transporter and quinine-sensitive K2P channel, followed by an influx of water via AQP-4. We also found that the decay of IOS is mediated by the DCPIB- and NPPB-sensitive anion channels in astrocytes. Altogether, our results demonstrate that the functional coupling between synaptic activity and astrocytic transient volume change during excitatory synaptic transmission is the major source of IOS.

Astrocytic proBDNF and Tonic GABA Distinguish Active versus Reactive Astrocytes in Hippocampus

Astrocytes are the most abundant cell type in the brain and they make close contacts with neurons and blood vessels. They respond dynamically to various environmental stimuli and change their morphological and functional properties. Both physiological and pathological stimuli can induce versatile changes in astrocytes, as this phenomenon is referred to as ‘astrocytic plasticity’. However, the molecular and cellular mechanisms of astrocytic plasticity in response to various stimuli remain elusive, except for the presence of hypertrophy, a conspicuous structural change which is frequently observed in activated or reactive astrocytes. Here, we investigated differential characteristics of astrocytic plasticity in a stimulus-dependent manner. Strikingly, a stab wound brain injury lead to hypertrophy of astrocytes accompanied by increased GABA expression and tonic GABA release in mouse CA1 hippocampus. In contrast, the mice experiencing enriched environment exhibited astrocytic hypertrophy with enhanced proBDNF immunoreactivity but without GABA signal. Based on the results, we define proBDNF-positive/GABA-negative hypertrophic astrocytes as ‘active’ astrocytes and GABA-positive hypertrophic astrocytes as ‘reactive’ astrocytes, respectively. We propose for the first time that astrocytic proBDNF can be a bona fide molecular marker of the active astrocytes, which are distinct from the reactive astrocytes which show hypertrophy but with aberrant GABA.

Expression of µ-Opioid Receptor in CA1 Hippocampal Astrocytes

µ-opioid receptor (MOR) is a class of opioid receptors with a high affinity for enkephalins and beta-endorphin. In hippocampus, activation of MOR is known to enhance the neuronal excitability of pyramidal neurons, which has been mainly attributed to a disinhibition of pyramidal neurons via activating Gαi subunit to suppress the presynaptic release of GABA in hippocampal interneurons. In contrast, the potential role of MOR in hippocampal astrocytes, the most abundant cell type in the brain, has remained unexplored. Here, we determine the cellular and subcellular distribution of MOR in different cell types of the hippocampus by utilizing MOR-mCherry mice and two different antibodies against MOR. Consistent with previous findings, we demonstrate that MOR expression in the CA1 pyramidal layer is co-localized with axon terminals from GABAergic inhibitory neurons but not with soma of pyramidal neurons. More importantly, we demonstrate that MOR is highly expressed in CA1 hippocampal astrocytes. The ultrastructural analysis further demonstrates that the astrocytic MOR is localized in soma and processes, but not in microdomains near synapses. Lastly, we demonstrate that astrocytes in ventral tegmental area and nucleus accumbens also express MOR. Our results provide the unprecedented evidence for the presence of MOR in astrocytes, implicating potential roles of astrocytic MOR in addictive behaviors.

Orai1 and Orai3 in Combination with Stim1 Mediate the Majority of Store-operated Calcium Entry in Astrocytes

Astrocytes are non-excitable cells in the brain and their activity largely depends on the intracellular calcium (Ca²⁺) level. Therefore, maintaining the intracellular Ca²⁺ homeostasis is critical for proper functioning of astrocytes. One of the key regulatory mechanisms of Ca²⁺ homeostasis in astrocytes is the store-operated Ca²⁺ entry (SOCE). This process is mediated by a combination of the Ca²⁺-store-depletion-sensor, Stim, and the store-operated Ca²⁺-channels, Orai and TrpC families. Despite the existence of all those families in astrocytes, previous studies have provided conflicting results on the molecular identification of astrocytic SOCE. Here, using the shRNA-based gene-silencing approach and Ca²⁺-imaging from cultured mouse astrocytes, we report that Stim1 in combination with Orai1 and Orai3 contribute to the major portion of astrocytic SOCE. Gene-silencing of Stim1 showed a 79.2% reduction of SOCE, indicating that Stim1 is the major Ca²⁺-store-depletion-sensor. Further gene-silencing showed that Orai1, Orai2, Orai3, and TrpC1 contribute to SOCE by 35.7%, 20.3%, 26.8% and 12.2%, respectively. Simultaneous gene-silencing of all three Orai subtypes exhibited a 67.6% reduction of SOCE. Based on the detailed population analysis, we predict that Orai1 and Orai3 are expressed in astrocytes with a large SOCE, whereas TrpC1 is exclusively expressed in astrocytes with a small SOCE. This analytical approach allows us to identify the store operated channel (SOC) subtype in each cell by the degree of SOCE. Our results propose that Stim1 in combination with Orai1 and Orai3 are the major molecular components of astrocytic SOCE under various physiological and pathological conditions.

Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey brain. One month after injection, monkeys were sacrificed, and then the presence of viral infection by expression of reporter fluorescence proteins was examined. Tissues were sectioned and stained with NeuN and GFAP antibodies for identifying neuronal cells or astrocytes, respectively, and viral reporter GFP-expressing cells were counted. We found that while lentivirus infected mostly astrocytes, AAV infected neurons at a higher rate than astrocytes. Moreover, astrocytes showed reactiveness when cells were infected by virus, likely due to virus-mediated neuroinflammation. The Sholl analysis was done to compare the hypertrophy of infected and uninfected astrocytes by virus. The lentivirus infected astrocytes showed negligible hypertrophy whereas AAV infected astrocytes showed significant changes in morphology, compared to uninfected astrocytes. In the brain of cynomolgus monkey, lentivirus shows tropism for astrocytes over neurons without much reactivity in astrocytes, whereas AAV shows tropism for neurons over glial cells with a significant reactivity in astrocytes. We conclude that AAV is best-suited for gene delivery to neurons, whereas lentivirus is the best choice for gene delivery to astrocytes in the brain of cynomolgus monkeys.