Developmental NMDA receptor dysregulation in the infantile neuronal ceroid lipofuscinosis mouse model
- University of Illinois at Chicago, United States
Figures
Figure 1
ALs deposit immediately following eye opening in visual cortex of Ppt1-/- mice.
(A) Representative composite confocal images through area V1 of visual cortex in WT (top) and Ppt1-/- mice (bottom) during development and into adulthood. DAPI nuclear stain (blue, 405 nm …
Figure 2 with 1 supplement
NMDAR subunit composition is biased toward immaturity in Ppt1-/- visual cortex.
(A) Representative immunoblots from synaptosomes of GluN2 subunits, GluN2A and GluN2B across age (P11–P60) and genotype as indicated (top) and quantification of band density (bottom) normalized to …
Figure 2—figure supplement 1
NMDAR subunit composition is immature in whole lysates from Ppt1-/- visual cortex.
(A) Representative immunoblots of the GluN2A in whole lysates across age and genotype as indicated (top) and quantification of band density (bottom) normalized to β-actin loading control within …
Figure 3
NMDAR-mediated EPSCs are altered in Ppt1-/- visual cortex.
(A) Representative traces of amplitude-scaled NMDAR-EPSCs recorded from pyramidal neurons in layer II/III of the visual cortex (V1) of WT and Ppt1-/- mice. Black and red arrows above traces indicate …
Figure 4
Dendritic spine morphology is immature in Ppt1-/- layer II/III visual cortical neurons.
(A) Schematic of in utero electroporation procedure and timeline (bottom) (B) coronal diagram from Paxinos’ mouse brain atlas demonstrating areas of visual cortex (left) and representative …
Figure 5
GluN2B to GluN2A NMDAR switch and Ppt1-/--induced synaptic deficits are recapitulated in primary cortical neurons.
(A) Representative immunoblot (top) and quantification of GluN2B levels in WT and Ppt1-/- neurons at DIV7, 10, and 18. (B) Representative immunoblot (top) and quantification of GluN2A levels …
Figure 6
Dendritic spine morphology is immature in Ppt1-/- neurons in vitro.
(A) Representative composite confocal images of live DIV15 (left) and DIV20 (right) GFP-transfected, cultured WT and Ppt1-/- neurons. Insets represent dendrite segments within dotted line. Scale bar …
Figure 7
Calcium imaging reveals extrasynaptic calcium dynamics in Ppt1-/- neurons.
DIV16-18, WT and Ppt1-/- cortical neurons transfected with GCaMP3 and imaged in the absence of Mg2+ for 5 min. (A) Single frames from Videos 1 and 2 of WT (left, note that cell is rotated 90° from Vi…
Figure 8
Ppt1-/- cultured neurons show enhanced vulnerability to NMDA-mediated excitotoxicity.
(A) Schematic of cellular toxicity experimental design. Briefly, neurons were grown to DIV11, treated with vehicle of palmitoylation inhibitors for 7 days (every 48 hr) and neuronal viability was …
Figure 9 with 1 supplement
Palmitoylation inhibitors rescue enhanced vulnerability to NMDA-mediated excitotoxicity in Ppt1-/- cultured neurons.
(A) 3D reconstructions of a WT and Ppt1-/- neuron at DIV20. Arrows point to AL deposits. Scale bar = 5 μm (B) Representative collapsed z-stacks of WT and Ppt1-/- DIV20 neurons, demonstrating …
Figure 9—figure supplement 1
ALs accumulate in enlarged lysosomes of Ppt1-/- neurons.
(A) Single-channel greyscale images of the WT, vehicle-treated representative composite image from Figure 9B displaying LAMP2, 561 nm (AL), MAP2, and DAPI signals individually. Red lines in images …
Figure 10
Palmitoylation inhibitor treatment partially reverses pathological calcium dynamics in Ppt1-/- neurons.
A subset of Ppt1-/- cells treated with the palmitoylation inhibitors, 2 BP (1 μM) or cerulenin (1 μM) from DIV12-18 were transfected with GCaMP3 and imaged in the absence of Mg2+ for 5 min. Sites of …
Figure 11
Hyperpalmitoylation of Fyn kinase and GluN2B is reversed in Ppt1-/- primary cortical neurons by palmitoylation inhibitor treatment.
(A) Representative post-APEGS immunoblot of PSD-95 with β-actin loading control. (B) Quantification of total PSD-95 levels following chronic (7d) treatment with vehicle or the palmitoylation …
Videos
Video 1
Spontaneous calcium activity in DIV16-18 WT neuron.
Representative video of spontaneous neuronal calcium activity in a WT cultured neuron at DIV16-18.
Video 2
Spontaneous calcium activity in DIV16-18 Ppt1-/- neuron.
Representative video of spontaneous neuronal calcium activity in a Ppt1-/- cultured neuron at DIV16-18.
Video 3
Spontaneous calcium activity in DIV16-18 WT neuron before and after treatment with Ro 25–6981.
Representative video of spontaneous neuronal calcium activity in a WT cultured neuron at DIV16-18 prior to, and following, bath application of Ro 25–6981 (1 μM, after 30 s).
Video 4
Spontaneous calcium activity in DIV16-18 Ppt1-/- neuron before and after treatment with Ro 25–6981.
Representative video of spontaneous neuronal calcium activity in a Ppt1-/- cultured neuron at DIV16-18 prior to, and following, bath application of Ro 25–6981 (1 μM, after 31 s).
Video 5
Spontaneous calcium activity in DIV18 Ppt1-/- neuron treated with 2 BP (1 μM, from DIV12-18).
Representative video of spontaneous neuronal calcium activity in a Ppt1-/- cultured neuron that was treated from DIV12-18 with 2 BP, at a dose of 1 μM. The last treatment was 4–6 hr before the …
Video 6
Spontaneous calcium activity in DIV18 Ppt1-/- neuron treated with cerulenin (1 μM, from DIV12-18).
Representative video of spontaneous neuronal calcium activity in a Ppt1-/- cultured neuron that was treated from DIV12-18 with cerulenin, at a dose of 1 μM. The last treatment was 4–6 hr before the …
Video 7
Spontaneous calcium activity in DIV18 Ppt1-/- neuron treated with vehicle (DMSO, from DIV12-18).
Representative video of spontaneous neuronal calcium activity in a Ppt1-/- cultured neuron that was treated from DIV12-18 with DMSO as vehicle. The last treatment was 4–6 hr before the imaging …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | B6;129-Ppt1tm1Hof/J | Jax stock #: 004313 | Gupta et al., 2001; RRID:MGI:004313 | |
| Antibody | Rabbit polyclonal anti-GluN2A | Novus Biologicals | Cat: NB300-105; RRID:AB_10001400 | (1:1000) |
| Antibody | Mouse monoclonal anti-GluN2B | UC Davis/NIH NeuroMab Facility | Cat: 75/097; RRID:AB_10673405 | (1:1000) |
| Antibody | Mouse monoclonal anti-GluN1 | UC Davis/NIH NeuroMab Facility | Cat: 75/272; RRID:AB_11000180 | (1:1000) |
| Antibody | Mouse monoclonal anti-PSD-95 | UC Davis/NIH NeuroMab Facility | Cat: K28/74; RRID:AB_2315909 | (1:2000) |
| Antibody | Mouse monoclonal anti-SAP102 | UC Davis/NIH NeuroMab Facility | Cat: N19/2; RRID:AB_2261666 | (1:2000) |
| Antibody | Rabbit polyclonal anti-Fyn | Cell Signaling | Cat: 4032 | (1:1000) |
| Antibody | Rabbit polyclonal anti-PPT1 | Gift from Sandra Hofmann | (1:500); Dr. Hofmann | |
| Antibody | Mouse monoclonal anti-β-actin-HRP | ThermoFisher Scientific | Cat: MA5-15739-HRP; RRID:AB_2537667 | (1:2000) |
| Antibody | Rabbit polyclonal anti-MAP2 | Millipore Sigma | Cat: AB5622; RRID:AB_91939 | (1:400) |
| Antibody | Rat monoclonal anti-LAMP2 | abcam | Cat: ab13524; RRID:AB_2134736 | (1:400) |
| Recombinant DNA reagent | pEF-GFP | Addgene | Plasmid: 11154 | Drs. Matsuda and Cepko |
| Recombinant DNA reagent | G-CaMP3 | Addgene | Plasmid: 22692 | Dr. Looger |
| Commercial assay or kit | PrestoBlue cell viability assay | ThermoFisher Scientific | Cat: A13261 | |
| Chemical compound, drug | 2-bromopalmitate | Sigma | Cat: 238422 | Treatment: 1 μM |
| Chemical compound, drug | cerulenin | Cayman Chemicals | Cat: 10005647 | Treatment: 1 μM |
| Software, algorithm | Fiji |
Additional files
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Supplementary file 1
Mean values, s.e.m., and n for the bar chart in Figure 1C.
Values are represented for each layer, at each age in WT and Ppt1-/- mice.
- https://doi.org/10.7554/eLife.40316.022
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Transparent reporting form
- https://doi.org/10.7554/eLife.40316.023
