Insulin, insulin-like growth factor and target of rapamycin (TOR) signaling pathways are critical for proper regulation of systemic organismal growth under variable nutritional and environmental conditions (1). In mammals, insulin receptor (INSR) and insulin-like growth factor receptors (IGF-1R and IGF-2R) are present ubiquitously pre- and postnatally. The
Insulin/Insulin-like growth factor signaling pathways have primarily been investigated in liver, fat and skeletal muscles as they were reported to be direct target tissues of the signal. Recently the role of insulin signaling in central nervous system (CNS) to integrate nutrients, metabolites and hormonal signal, and to maintain glucose and lipid homeostasis and neurogenesis is emerging (4-7). Nervous system development requires proper organization, specification and interaction between neural and non-neural cells. The blood-brain barrier (BBB) arbitrates communication between the brain and the body. The Drosophila and vertebrate species BBBs are similar in many structural and functional aspects (8, 9). CNS physiology is sensitive to energy balance and it is reported that IGF-1 plays a neuroprotective role (10, 11). Despite its significance, little is known about the mechanism that regulates traffic of growth factors across the BBB. In this paper, we show that
We made polyclonal antibody against SDR protein. Because
Because SDR is expressed in the glial cells in the CNS and the eyes of adult flies, we used
To investigate the permeability and the integrity of the BBB in
To determine whether apoptosis is in progress in
The human genome encodes 58 RTKs belonging to 20 RTK protein families. The
Recently, numerous effects of insulin signaling in brain are emerging. They include regulation of appetite, body temperature, reproductive function, systemic glucose metabolism (15, 16) and learning and memory (17, 18). Decreased expression of
In fact, we isolated
Brain activity depends on access to circulating oxygen and nutrients. Endothelial transport of oxygen and glucose and brain activity is facilitated by a neurovascular mechanism (26). Insulin signaling in the adult brain is required as insulin inhibits neuronal apoptosis and tau phosphorylation (19). SDR may continuously bind to and dissociate from ILPs (
Flies were collected as newborn adults over a 12-hour time window. Ten newborn flies were placed in a fresh vial and stored for 1 day at 25°C before being used in climbing and survival tests. During the experiment, flies were maintained at 29°C. After negative geotactic pressure was applied, the number of flies that climbed to the top of the vial within 20 seconds was counted and recorded.
To construct an SDR overexpression plasmid, a 730 nucleotide DNA fragment was amplified by PCR using LD44769 (the EST clone of the SDR 5’ region) as template DNA and Pfu polymerase (Agilent/Strategene) and the following two primers: 5’ CGGGATCCGTGGCAGATGTATC3’ and 5’GGAATTCTGCGA TAGTCGTGATTG3’.
GE22018 flies were purchased from Genexel (Taejun, Korea). In the genome of GE22018 flies, a P-element is inserted in the 1st exon of SDR at location 14918660 (RefSeq NT_033777). To mobilize the P-element in the GE22018 genome, GE22018 flies were crossed to flies harboring P[Δ2-3] transposase, and progeny of this cross were monitored for loss of eye pigmentation as a marker of P-element excision. To determine whether the P-element was excised precisely or imprecisely, we bred homozygous flies with putative P-element excisions, isolated their genomic DNA, and PCR amplified and analyzed the remaining
The heads of adult flies were fixed in 4% paraformaldehyde for 10 min, transferred to embedding media, and then cryosectioned at a thickness of 15 μm. Sections were placed on lysine-coated slides (Superior Marienfield, Lauda-Konigshfen, Germany) and immunostained as described in Kim
A solution containing tetramethylrhodamine-labeled dextran conjugates (10,000 MW, Molecular Probes) was loaded into a glass capillary prepared with a needle puller (PN-30, Narishige Scientific Instrument Lab, Tokyo), and injected into the abdomen of adult flies (150 nl/fly). After 2 h, injected flies were dissected, and the brains of 2 flies were placed into a single well containing 50 μl 0.1% SDS. The fluorescence from each well was measured using a SpectraMax Gemini EM microplate spectrofluorometer (Excitation = 555 nm, Emission = 580 nm). Each experiment was repeated at least 8 times. The amount of fluorescent dye in eyes of injected flies was estimated by examining eyes under a Zeiss Microscope (Axioplan2, Carl-Zeiss, Oberkochen). Experiments were repeated at least 10 times for each experimental group.
This work was supported by grant NRF-2017R1D1A1B06029547 funded by the Ministry of Education, Science, and Technology of Korea to J. Kim-Ha.
The authors have no conflicting interests.