Supplementary Figures

Supplementary Figure 1 Diagrams of the mouse Bdnf gene and the Bdnfklox allele. (a) The mouse Bdnf gene contains 9 exons and two alternative polyadenylation (pA) sites. Curved lines linking boxes (exons) indicate alternative splicing from the first eight exons to exon 9. Filled box in exon 9 represents the BDNF coding sequence. All transcripts in theory could be polyadenylated at either pA site to give rise to two Bdnf mRNA species: one with a short 3ʹ′UTR and the other with a long 3ʹ′UTR. (b) The Bdnfklox allele has two insertions: one lox P site inserted into the 5ʹ′UTR within exon 9 and a sequence containing three tandem SV40 polyadenylation signals, a loxP site, and a LacZ gene that is inserted at a site 3ʹ′ to the first Bdnf polyadenylation site.

Nature Medicine doi:10.1038/nm.2687

Supplementary Figure 2 Obesity phenotypes of Bdnfklox/klox mice. (a) Female +/k mice (n=8) had significantly higher body weights than female +/+ mice (n=6) at 6 months of age. (b) Levels of serum leptin. Sera were collected from fed female mice at 16 weeks of age (n=4 mice for each genotype). (c) Weights of fat pads from three pairs of male +/+ and k/k and three pairs of female +/+ and k/k mice at 5 months of age. (d) Blood glucose levels were measured in 16-week-old mice fasted overnight (n=11, 10, 6, and 7 mice for male +/+, male k/k, female +/+, and female k/k, respectively). (e) Glucose tolerance tests were performed in female mice at 16 weeks of age (n=6 +/+ mice and 7 k/k mice). There was a significant effect of genotype (F(1,66)=56.89, P < 0.001). (f) Glucose tolerance tests were performed in male mice at 16 weeks of age (n=5 +/+ mice and 3 k/k mice). There was a significant effect of genotype (F(1,36)=12.13, P < 0.01). Error bars in all panels represent the standard error of the mean. Student’s t test: * P < 0.05, ** P < 0.01, and *** P < 0.001.

Nature Medicine doi:10.1038/nm.2687

Supplementary Figure 3 Insulin stimulates local translation of mRNA containing the long Bdnf 3ʹ′UTR in dendrites of hypothalamic neurons. (a) Insulin stimulated local protein synthesis via the long Bdnf 3'UTR in distal dendrites of hypothalamic neurons. GFP fluorescence intensity on distal dendrites (100-150 µm and 150-200 µm away from the soma) was measured and normalized to control levels (myr-d1GFP-nls-A: n=17 neurons for vehicle and 19 neurons for insulin; myr-d1GFP-nls-A*B: n=15 neurons for vehicle and 22 neurons for insulin). (b) Insulin treatment did not alter somatic levels of GFP in neurons transfected by the myr-d1GFP-nls-A*B construct. Error bars in all panels represent the standard error of the mean. Student’s t test: ** P < 0.01.

Nature Medicine doi:10.1038/nm.2687

Supplementary Figure 4 Activation and localization of STAT3. (a-f) Localization of TrkB and pSTAT3 in the DMH and VMH of leptin-injected TrkBLacZ/+ mice. The mice were euthanized 45 min after leptin injection (5 µg/g of body weight). Coronal brain sections were stained with antibodies to β-galactosidase and pSTAT3. The β-galactosidase and pSTAT3 immunoreactivity serves as the indicator for the expression of TrkB and LepRb, respectively. White arrows denote neurons expressing both TrkB and LepRb, white arrowheads neurons expressing TrkB but not LepRb, yellow arrows TrkB-expressing glia, and yellow arrowheads neurons expressing LepRb but not TrkB. Note that there are many TrkB-expressing neurons in the DMH but few in the VMH. Scale bar, 50 µm. (g-j) Leptin-induced activation of STAT3 in the VMH of WT and Bdnfklox/klox mice. Leptin injection significantly increased pSTAT3 immunoreactivity in the VMH of both WT and Bdnfklox/klox mice at 6 weeks of age. Scale bar, 100 µm.

Nature Medicine doi:10.1038/nm.2687

Supplementary Figure 5 Expression of SOCS3, POMC, NPY, and AgRP in the arcuate nucleus of WT and Bdnfklox/klox mice at 5 weeks of age. (a) Representative images of radioactive in situ hybridization show SOCS3 expression in the brain. Mice were injected intraperitoneally with leptin (5 mg/kg of body weight) or vehicle and euthanized 45 min post injection. The sense probe did not reveal any signals. (b) Quantification of radioactive in situ hybridization signals in the arcuate nucleus of +/+ and k/k mice. Error bars indicate standard errors. Student’s t test: *** P < 0.0001 (n=4 mice for each group). There is no significant difference in levels of ARC SOCS3 mRNA between +/+ and k/k treated with either vehicle (P=0.457) or leptin (P=0.515). (c) Representative images of radioactive in situ hybridization show the expression of Pomc, Npy, and Agrp in the brain. The sense probe did not reveal any signals. Arrows denote the arcuate nucleus. (d) Quantification of radioactive in situ hybridization signals in the arcuate nucleus of +/+ and k/k mice. Error bars indicate standard errors. Student’s t test: P=0.656 for Pomc mRNA, P=0.395 for Npy mRNA, and P=0.906 for Agrp mRNA (n=4 mice for each genotype).

Nature Medicine doi:10.1038/nm.2687

Supplementary Figure 6 Induction of c-Fos in the VMH. Leptin injection significantly increased c-Fos immunoreactivity in the VMH of both WT and Bdnfklox/klox mice. The degree of c-Fos induction was reduced in Bdnfklox/klox mutant. Scale bar, 100 µm.

Supplementary Figure 7 Projections of POMC and AgRP neurons to the DMH. Brain sections from +/+ and k/k mice at 5 weeks of age were stained with antibodies to alpha melanocyte-stimulating hormone (αMSH) or AgRP. Alpha MSH is a peptide derived from its precursor POMC. A z stack of images of immunoreactivity was captured at 4-µm intervals to cover a depth of 30 µm using a scanning confocal microscope. (a, b) Projected images of αMSH immunoreactivity in the DMH of +/+ and k/k mice. (c, d) Projected images of AgRP immunoreactivity in the DMH of +/+ and k/k mice. Scale bar, 50 µm. (e) Quantification of the area covered by αMSH- or AgRP-immunoreactive fibers in projected images. The size of each image was 1.06 x 105 µm2. Student’s t test: * P < 0.005 (n=8 sections and 4 mice for each genotype).

Nature Medicine doi:10.1038/nm.2687

Supplementary Figure 8 Schematic model of food intake regulation by dendritic BDNF synthesis. Neuron A expresses the LepRb and innervates neuron B. Neuron B expresses BDNF and its receptor TrkB. Both neurons B and C do not express the LepRb, and their activation inhibits food intake. The three subsets of neurons likely reside in more than one hypothalamic nuclei. When leptin binds to the LepRb in neuron A, it activates signaling cascades downstream the LepRb, including the pSTAT3 cascade. Leptin signaling in neuron A induces action potential and c-Fos expression in neuron B. Activation of neuron B sometimes also induces c-Fos expression in neuron C through synaptic connection. Dendritic BDNF synthesis in neuron B is required for the formation, maintenance, and/or function of synapses between neuron A and neuron B. When dendritic BDNF synthesis is disrupted, the synapses are defective and thus leptin fails to inhibit food intake through neurons B and C.

Nature Medicine doi:10.1038/nm.2687

Supplementary Methods In situ hybridization probes. To generate riboprobes, mouse cDNA sequences for Pomc (GenBank accession number NM_008895, nucleotides 150-857), Npy (GenBank accession number NM_023456, nucleotides 74-367), Agrp (GenBank accession number NM_007427, nucleotides 208-603), and SOCS3 (GenBank accession number U88328, nucleotides 241-687) were amplified by PCR and cloned into the pBluscript II KS (-) plasmid (Stratagene, Cedar Creek, TX, USA). Bdnf probes were from the mouse Bdnf coding region. Analysis of serum leptin and blood glucose. Serum leptin levels of fed animals at 5 or 16 weeks of age were measured using Mouse Leptin ELISA kit according to the manufacturer’s instruction (BioVendor, Modrice, Czech Republic). Fasting blood glucose levels were measured from tail vein of 16-week-old animals using OneTouch Ultra2 glucose meter and test strip (LifeScan, Inc., Milpitas, CA). For glucose tolerance tests, animals at 16 weeks of age were fasted overnight and received a single glucose injection (1.5 g/kg body weight) intraperitoneally in the next morning. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min post injection. Hypothalamic neuronal culture. Hypothalami were dissected from E18 rat embryos, followed by digestion with 0.25 mg/ml trypsin and 0.28 mg/ml DNase I in 1X Hanks’ balanced salt solution at 37 °C for 15 minutes. Dissociated cells were plated onto glass coverslips coated with poly-D-lysine at 37.5 µg/ml at a density of 5x105 cells per coverslip in Neurobasal medium containing 2% B27 supplement, 2 mM glutamine, 0.25% glucose, 3.3 mg/ml insulin, 100 µM ascorbic acid, 1% vitamin solutions, 100 U/ml penicillin, and 100 µg/ml streptomycin. All culture reagents were purchased from either Invitrogen Corporation (Carlsbad, CA) or Sigma-Aldrich (St. Louis, MO). AAV production and injection. HEK-293 cells cultured in DMEM growth medium (containing 4.5 g/L glucose, 110 mg/L sodium pyruvate, and 4 mM L-glutamine; Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum were used for viral packaging. Plasmid pAAV-GFP, pAAV-BDNF-A, or pAAV-BDNF-A*B was co-transfected with helper plasmid pDC2 into HEK-293 cells at >90% confluence. Media were refreshed 24 h post transfection. Cells and media were harvested 48 h post transfection. Viral particles were purified using the ViraKit (Virapur, LLC, San Diego, CA) according to the manufacture’s protocol and concentrated through VIVASPIN 6 (Sartrius Stedim Biotech, Goettingen, Germany). Virus titers were determined by quantitative PCR, and 1µl of viral preparation (~1 × 108 viral particles) was stereotaxically injected to each VMH of animals at P14 using the following coordinates: anteroposterior, −1.2 mm; mediolateral, ±0.5 mm; dorsoventral, −5.9 mm. Virus was administered bilaterally using a 10 µl Hamilton syringe with a 33-gauge needle attached to a stereotaxic apparatus (Stoelting Co., Wood Dale, Illinois) and an infusion microsyringe pump (kd Scientific, Holliston, MA) at a rate of 6 µl/h. Injected pups were returned to their original cages for continuous parental care and weaned at P21. Body weight was measured weekly starting at 4 weeks of age. Mice were singly-caged at 12 weeks of age for food intake measurement. Body length and fasting blood glucose level were measured at 14 weeks of age. Animals were then perfused and their brain sections were stained with antibodies against GFP or

Nature Medicine doi:10.1038/nm.2687

Myc to examine the injection sites and to verify AAV-mediated GFP or BDNF expression, respectively. Projections of α-MSH or AgRP fibers from ARC to DMH. Four pairs of Bdnfklox/klox and their WT littermates were perfused at 5-6 weeks of age and their brains were sectioned at 30 µm using a sliding microtome (Leica Microsystems Inc. Buffalo Grove, IL). Every fourth brain sections were stained using antibodies against α-MSH (1:4,000 dilution, Millipore, Billerica, MA) or AgRP (1:2,000 dilution, Phoenix Pharmaceuticals, Inc. Burlingame, CA), followed by incubation with DyLight 488 or 594 conjugated secondary antibody (1:1,000 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA). Series of confocal images were acquired and projected for quantification of fiber projections from POMC or AgRP neurons to the DMH. The area covered by immunoreactive fibers in the DMH was quantified using ImageJ software.

Nature Medicine doi:10.1038/nm.2687