Exp Neurobiol 2017; 26(6): 350-361
Published online December 31, 2017
© The Korean Society for Brain and Neural Sciences
Wuhyun Koh1,2, Yongmin Mason Park1,2, Seung Eun Lee3* and C. Justin Lee1,2,4*
1Division of Bio-Medical Science &Technology, Department of Neuroscience, KIST School, Korea University of Science and Technology, Seoul 02792, 2Center for Neuroscience and Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul 02792, 3Virus Facility, Research Animal Resource Center, Korea Institute of Science and Technology (KIST), Seoul 02792, 4Center for Glia-Neuron Interaction, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea
Correspondence to: *To whom correspondence should be addressed.
C. Justin Lee, TEL: 82-2-958-6940, FAX: 82-2-958-6937
Seung Eun Lee, TEL: 82-2-958-6959, FAX: 82-2-958-6937
Adeno-associated virus (AAV)-mediated gene delivery has been proposed to be an essential tool of gene therapy for various brain diseases. Among several cell types in the brain, astrocyte has become a promising therapeutic target for brain diseases, as more and more contribution of astrocytes in pathophysiology has been revealed. Until now, genetically targeting astrocytes has been possible by utilizing the
In the central nervous system, the most abundant cell type is astrocyte, which has been revealed to have a number of functions, including modulation of synaptic transmission [1,2], relay for nutrients to adjacent neuron , clearance of waste molecules . To study functions of astrocyte in many brain regions, researchers have been relying heavily on the conventional astrocyte-specific marker, glial fibrillary acidic protein (GFAP) and its promoter after the discovery of its specificity for astrocytes . However, it also has been reported that GFAP expression is low in several brain areas, including thalamus . GFAP also has been known to be expressed transiently in neural progenitor cells, which could lead to neuronal expression near neurogenic areas . Therefore, utilizing GFAP may not be optimal in those brain areas. Moreover, there is a pressing need for an alternative astrocyte-specific marker to study astrocytic functions in those brain areas.
There are two superb examples of utilizing astrocyte-specific promoter to study and target astrocytes in the mouse brain. First one is the generation of transgenic mouse lines such as GFAP-EGFP mouse  and GFAP-Cre/ERT2 mouse . Although utilizing transgenic mouse is one of the best choices to target astrocytes, it could be very resource- and time-consuming when they need to be transferred or maintained. It is also difficult to extend the study to other species beyond mouse. Another example is to apply astrocyte specific promoter in virus applications such as in GFAP-GFP virus . Virus containing astrocyte-specific promoter allows researchers to examine the effects of a gene of interest in local astrocytes in a specific brain area where the virus is injected. Moreover, virus is easy to apply to different species, such as mouse, marmoset, monkey or even human. As more and more contribution of astrocytes in pathophysiology of brain diseases has been revealed, astrocyte-specific targeting with virus would be critically essential as a potential therapeutic tool . Among viruses, adeno-associated virus (AAV) has become very popular with many prominent advantages: AAV is able to infect both dividing and non-dividing cell types with less immunogenic effect . Ultimately, AAV promises to be the best available option for gene therapy for brain diseases [13,14].
As an alternative to GFAP promoter, aldehyde dehydrogenase family 1, member L1 (ALDH1L1) was recently identified as a new astrocyte-specific marker in the brain . The promoter of ALDH1L1 should be useful in brain regions where GFAP expression is low. There has been attempts to characterize the
In this study, we constructed and characterized human
The viral vectors were pseudotyped, where the transgene of interest was flanked by inverted terminal repeats of the AAV2 packaged in an AAV-DJ capsid. AAV-DJ was engineered via DNA family shuffling technology which created a hybrid capsid from 8 AAV serotype. AAV-hALDH1L1-Cre vector was thereafter purified by iodixanol gradients by the KIST Virus Facility (http://virus.kist.re.kr). The production titer was 1.5×1013 genome copies/ml (GC/ml).
Adult (aged 8~10 weeks) male and female Ai14 mice (RCL-tdTomato; Rosa-CAG-LSL-tdTomato-WPRE::deltaNeo) in C57BL/6J strain were used as transgenic reporter line. Mice had free access to food and water and were kept on a 12 hours light-dark cycle. All experimental procedures described below were performed in accordance with the Institutional guidelines for experimental animal care and use of the Korea Institute of Science and Technology (KIST; Seoul, Korea).
Mice were anesthetized with 2% avertin (20 µl/g, i.p.) and placed on stereotaxic apparatus (Kopf instrument, USA). AAV-hALDH1L1-Cre virus was injected through 33 gauge blunt NanoFil needle (World Precision Instruments, USA), connected with 10 µl Hamilton micro syringe (Hamilton, Switzerland) filled with distilled water. Syringe pump (KD Scientific, USA) was used to inject the virus with the rate of 0.2 µl per minute. Needle was gently placed into target regions (thalamic VB: -1.8 mm AP, ±1.6 mm ML, 3.75 mm (1 µl) and 3.25 mm (1 µl) from brain surface; hippocampal CA1: -1.8 mm AP, ±1.6 mm ML, 1.6 mm (2 µl) from brain surface; BLA: -1.8 mm AP, ±3.4 mm ML, 4.55 mm (2 µl) from brain surface; mPFC: 1.8 mm AP, ±0.3 mm ML, 1.7 mm (2 µl) from brain surface), bilaterally. Mice were sacrificed after 10 days recovery.
Mice were anesthetized with 2% avertin (20 µl/g, i.p.) and perfused with 0.1 M phosphate buffered saline (PBS) at room temperature followed by ice-cold 4% paraformaldehyde. Extracted brains were post-fixed in 4% paraformaldehyde at 4℃ overnight. Post-fixed brains were incubated in 30% sucrose at 4℃ for more than 24 hours. Coronal brain sections were prepared with 30 µm thickness in cryostat microtome (Thermo Scientific). Brain sections were rinsed three times with PBS, and blocking step was performed with a blocking solution (0.3% Triton X-100 (Sigma), 2% donkey serum (Millipore) and 2% goat serum (Abcam) in 0.1 M PBS) for 1.5 hours. Blocking step was followed by application of a mixture of primary antibodies as follows, rabbit anti-Aldh1L1 antibody (Novus), mouse anti-NeuN antibody (Millipore). Sections were incubated overnight at 4℃ with shaking for primary antibodies. After overnight incubation, the sections were washed three times in PBS and then incubated in corresponding fluorescent secondary antibodies for 1.5 hours. In three times wash process of secondary antibodies, they also were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, Pierce) before they were mounted onto slide glass (Thermo Scientific). A series of fluorescence images were obtained with Nikon A1 confocal microscope, and 30 µm Z stack images in 3-µm steps were processed for further analysis using NIS-Elements (Nikon) software and ImageJ (NIH) program. Reference atlases of mouse brain in the figure were obtained from Allen Brain Atlas (Reference Atlas, Version 2 (2011)) .
Primary cortical astrocytes were cultured from P0 to P3 C57BL/6N mouse pups. The cerebral cortex was dissected free meninges and softly triturating to single cell unit. Culture media was prepared as follow. Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 25 mM glucose, 2 mM glutamine, 10% heat-inactivated horse serum, 10% heat-inactivated fetal bovine serum and 1,000 units/ml penicillin-streptomycin. On the third day after culture, cells were rinsed with repeated gentle pipetting and the media was replaced to get rid of debris. The day before treatment of viruses, cells were transferred to coverslip (1×104 per coverslip) coated with 0.1 mg/ml Poly D-Lysine (PDL, Sigma). Cultured cells were maintained at 37℃ in a humidified 5% CO2 incubator.
Three days after virus transduction, cultured astrocytes on coverslip were rinsed with PBS and fixed with 4% paraformaldehyde for 20 minutes. After three times wash with PBS, immunocytochemistry was performed as similar manner in IHC as describe above. Briefly, blocking step was performed with a blocking solution (same with IHC, but no goat serum) for 1 hour. Blocking step was followed by application of primary antibody, rabbit anti-Aldh1L1 antibody for 2 hours, RT. After that, the cells were washed in PBS and then incubated in corresponding fluorescent secondary antibodies for 1 hour. In three times wash process of secondary antibodies, they also were counterstained with DAPI before coverslips were mounted onto slide glass.
Aldh1L1 has been shown to be a highly specific marker for astrocytes with a substantially broader pattern of astrocytic expression than the conventional astrocyte marker, GFAP . We sought to develop h
To test cell-type specific expression of Cre recombinase by h
Next, we tested astrocyte-specific Cre expression in amygdala and hippocampus. AAV-hALDH1L1-Cre virus was delivered to BLA and hippocampal CA1, respectively (Fig. 3A and 4A). In BLA, both cell types, astrocytes and neurons, showed tdTomato fluorescence (Fig. 3B). Average percentage of Aldh1L1+/tdTomato+ cells in each image was lower than that of NeuN+/tdTomato+ cells (Aldh1L1+: 26.5±7.5% versus NeuN+: 69.5±8.7%) (Fig. 3C). Among the analyzed total Aldh1L1+ cells, tdTomato+ cells were 28.35%, and NeuN+ cells were 68.13% (Fig. 3D and E). These results indicate that AAV-hALDH1L1-Cre virus expresses Cre recombinase in both astrocytes and neurons in BLA.
Similar to BLA, in hippocampal CA1, AAV-hALDH1L1-Cre virus induced tdTomato fluorescence from astrocytes and neurons in Ai14 mouse (Fig. 4B). Average percentage of Aldh1L1+/tdTomato+ cells in each image was 20.4±8.8%, which is lower than that of NeuN+/tdTomato+ cells (75.3±8.6%) (Fig. 4C). In total of analyzed tdTomato+ cells in hippocampal CA1 area, a population of Aldh1L1+ cells was 18.00%, and a population of NeuN+ cells was 76.25% (Fig. 4D and E). These results indicate that AAV-hALDH1L1-Cre virus expresses Cre recombinase in both astrocytes and neurons in hippocampal CA1.
We further investigated activity pattern of h
Taken together, expression pattern of AAV-hALDH1L1-Cre virus-mediated tdTomato fluorescence was different in different brain areas (Table 1). Additionally, we further analyzed a proportion of astrocytes that express tdTomato in these brain regions. To measure the proportion of astrocytes expressing tdTomato fluorescence, we counted all Aldh1L1+ cells near the infected areas from each brain region, and the number of tdTomato+ cells with Aldh1L1+ was divided by the total number of Aldh1L1+ cells. Calculated proportions of astrocytes expressing tdTomato were as following, VB: 81.91%; BLA: 51.03%; CA1: 25.55%; mPFC: 13.26%. These results indicate that astrocytes in VB, BLA, CA1 and mPFC are heterogeneous in virus transduction efficacy or h
As a summary, AAV-hALDH1L1-Cre virus showed highly specific expression in astrocyte of thalamic area. The virus-mediated tdTomato fluorescence could be observed in both astrocyte and neuron in amygdala and hippocampus. Finally, AAV-hALDH1L1-Cre virus in mPFC showed highly specific expression in neuron.
We further investigated the efficiency of AAV-hALDH1L1-Cre virus in cultured system. To examine the Cre recombinase expression under h
Our study is the first development and characterization of h
One possible reason is that serotype of AAV virus could affect cell-type specificity of virus infection, as many studies previously reported [22,23,24]. Natural AAV viruses have a specific pattern of infection that reflects the interaction and recognition between the viral capsid, envelope and receptors expressed in susceptible cells. In particular, although AAV serotype 4 and AAV rh43 are known to target astrocytes [7,25], the receptor of this serotype has not yet been identified. Moreover, pseudotype-dependent lentivirus has been utilized to target for astrocyte. In the current study, we utilized AAV-DJ serotype to target astrocytes with h
Another possible reason could be that h
Lastly, the neurons that expressed tdTomato fluorescence by h
In conclusion, AAV-mediated gene expression under h
|Cell types||Thalamic ventrobasal nucleus (VB)||Basolateral amygdala (BLA)||Hippocampal CA1||Medial prefrontal cortex (mPFC)|
|Astrocytic transduction efficiency (%)||81.91||51.03||25.55||13.26|