ABSTRACT
Viral tracers that enable efficient retrograde labeling of projection neurons are powerful vehicles for structural and functional dissections of the neural circuit and for the treatment of brain diseases. Currently, some recombinant adeno-associated viruses (rAAVs) based on capsid engineering are widely used for retrograde tracing, but display undesirable brain area selectivity due to inefficient retrograde transduction in certain neural connections. Here we developed an easily editable toolkit to produce high titer AAV11 and demonstrated that it exhibits potent and stringent retrograde labeling of projection neurons in adult male wild-type or Cre transgenic mice. AAV11 can function as a powerful retrograde viral tracer complementary to AAV2-retro in multiple neural connections. In combination with fiber photometry, AAV11 can be used to monitor neuronal activities in the functional network by retrograde delivering calcium-sensitive indicator under the control of a neuron-specific promoter or the Cre-lox system. Furthermore, we showed that GfaABC1D promoter embedding AAV11 is superior to AAV8 and AAV5 in astrocytic tropism in vivo, combined with bidirectional multi-vector axoastrocytic labeling, AAV11 can be used to study neuron-astrocyte connection. Finally, we showed that AAV11 allows for analyzing circuit connectivity difference in the brains of the Alzheimer's disease and control mice. These properties make AAV11 a promising tool for mapping and manipulating neural circuits and for gene therapy of some neurological and neurodegenerative disorders.
Subject(s)
Astrocytes , Neurons , Mice , Male , Animals , Mice, Transgenic , Interneurons , Brain , Dependovirus/genetics , Genetic Vectors/geneticsABSTRACT
As powerful tools for local gene delivery, adeno-associated viruses (AAVs) are widely used for neural circuit studies and therapeutical purposes. However, most of them have the characteristics of large diffusion range and retrograde labeling, which may result in off-target transduction during in vivo application. Here, in order to achieve precise gene delivery, we screened AAV serotypes that have not been commonly used as gene vectors and found that AAV13 can precisely transduce local neurons in the brain, with a smaller diffusion range than AAV2 and rigorous anterograde labeling. Then, AAV13-based single-viral and dual-viral strategies for sparse labeling of local neurons in the brains of C57BL/6 or Cre transgenic mice were developed. Additionally, through the neurobehavioral test in the ventral tegmental area, we demonstrated that AAV13 was validated for functional monitoring by means of carrying Cre recombinase to drive the expression of Cre-dependent calcium-sensitive indicator. In summary, our study provides AAV13-based toolkits for precise local gene delivery, which can be used for in situ small nuclei targeting, sparse labeling and functional monitoring.
Subject(s)
Dependovirus , Genetic Vectors , Animals , Mice , Mice, Inbred C57BL , Dependovirus/metabolism , Genetic Vectors/genetics , Gene Transfer Techniques , Mice, Transgenic , Transduction, GeneticABSTRACT
Cumulative evidence suggests that dysfunction of ubiquitinating enzymes is responsible for multiple types of diseases including cancer. However, what role the ubiquitinating enzyme plays in gastric cancer remains unknown. In this study, using bioinformatics analysis and a series of experimental analyses, we found that an E3 ubiquitin-protein, MKRN2 was down-regulated in gastric cancer tissues. Kaplan-Meier survival analysis showed the low MKRN2 expression significantly indicated poor prognosis. Overexpression of MKRN2 notably inhibited cell proliferation in vitro and in vivo. Conversely, knockdown of MKRN2 had the opposite effects in vitro. Additionally, the mechanical analysis indicated that MKRN2 promoted ubiquitination-mediated degradation of PKM2 and attenuated its effect on ERK. Overall, the present study suggests that MKRN2 may be a potential therapeutic target for gastric cancer.
Subject(s)
Stomach Neoplasms , Cell Line, Tumor , Cell Proliferation/physiology , Gene Expression Regulation, Neoplastic , Humans , Ribonucleoproteins/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Ubiquitin/metabolism , UbiquitinationABSTRACT
Retrograde tracers based on viral vectors are powerful tools for the imaging and manipulation of upstream neural networks projecting to a specific brain region, and they play important roles in structural and functional studies of neural circuits. However, currently reported retrograde viral tracers have many limitations, such as brain area selectivity or the inability to retrograde label genetically defined brain-wide projection neurons. To overcome these limitations, a new retrograde tracing method, AAV-PHP.eB assisted retrograde tracing systems (PARTS) based on rabies virus, was established through brain-wide TVA-dependent targeting using an AAV-PHP.eB that efficiently crosses the blood-brain barrier in C57BL/6 J mice, and complementation of EnvA-pseudotyped defective rabies virus that specifically recognizes the TVA receptor. Furthermore, combined with Cre transgenic mice, cell-type-specific PARTS (cPARTS) was developed, which can retrograde label genetically defined brain-wide projection neurons. Our research provides new tools and technical support for the analysis of neural circuits.
