All experiments involving mice were conducted in accordance with policies and procedures described in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Animal Care and Use Committees at the Rowan University School of Osteopathic Medicine. The results and experiments of this study that involves animals are also reported in accordance with ARRIVE guidelines. The C57BL/6J-Npc1nmf164/J mouse strain (Jax stock number 004817) was provided by Dr. Robert Burgess at The Jackson Laboratory. Npc1nmf164 heterozygous mice were bred and housed in a 12/12-hour light/dark cycle to generate both WT and Npc1nmf164 homozygous mutant mice. To produce NPC1-deficient mice with PCs expressing GFP (Npc1nmf164-Pcp2EGF), Npc1nmf164 heterozygous mice were intercrossed with the B6;FVB-Tg(Pcp2-EGFP)2Yuza/J (Jax stock number 004690). To study changes in mouse PCs dendrites during postnatal development caused by haploinsufficiency of Pten, the Ptenflox mouse strain (B6.129S4-Ptentm1Hwu/J, Jackson stock number 006440) was crossed to the Pcp2-cre strain (B6.129-Tg(Pcp2-cre)2Mpin/J, Jackson stock number 004146) to generate F1 heterozygous mice for both Ptenflox/-/Pcp2-Cre+/- (here will be referred as Pten-cHet). Both males and females were used in this study, at a ratio of 2:2 when 4 mice were used. The tissue and results from WT mice used for the NPC1 deficiency studies were also used as a control for the experiments with Pten-cHet mice.
Golgi-Cox staining technique
The Golgi-Cox staining technique was performed using and following the instructions of a commercially available kit (FD Rapid GolgiStain™ Kit, FD NeuroTechnologies Inc.) Briefly, after mice were euthanized with CO2, brains were dissected, immersed, and incubated in the impregnating solution (mixed solution A and B) for two weeks. After the two weeks of incubation, the impregnated solution was replaced by the 30% sucrose solution (solution C) and incubated for 72hrs. Then, brains were removed from the solution, quick-froze, and stored at -80° C. For tissue sectioning, the brain was immersed and frozen in optimal compound temperature (OCT) media. Cryostat sections of 160 µm were collected in solution C, then rinsed in distilled water prior to being immersed in the developing solution (solutions D and E). After rinsing the tissue slices in distilled water, they were mounted onto slides, dried, and dehydrated using 75%, 85%, and 100% ethanol prior to being immersed in Histo-Clear II (National Diagnostics). Slides were mounted using Permount and allowed to dry overnight prior to analysis.
Mouse Perfusion and Tissue Preparation
After mice were euthanized with CO2, transcardial perfusion with 1X PBS followed by 4% paraformaldehyde was performed. Brains were carefully dissected and fixed by immersion in 4% paraformaldehyde overnight. After fixation, brains were rinsed in 1X PBS, immersed in 30% sucrose/PBS solution overnight at 4°C, frozen in OCT, and cryosectioned as 40μm and 50μm floating sections.
For immunostaining experiments, 40-50μm floating sections were collected in 1X PBS, then rinsed once in 1X PBT (PBS + 1% Triton 100X), and incubated overnight at 4°C in a cocktail of primary antibodies that were diluted in 1X PBT + 20% normal donkey serum. After the overnight incubation, sections were rinsed three times with 1X PBT for 10 min and incubated for 1.5 hours in the corresponding secondary antibodies (1:500, Jackson-ImmunoResearch or Invitrogen). Cerebellar sections were then washed three times with 1X PBT for 10–15 min, incubated with DAPI, and mounted in Poly-aquamount (Polysciences). The following primary antibodies were used: rabbit anti-IBA1 (1:200, Wako, # 019-19741), mouse anti-CALB (calbindin,1:200, Sigma-Aldrich, #C9848), rat anti-CD68 (1:200, Bio-Rad, #MCA1957), Lycopersicon esculentum(Tomato)-Lectin (1:200, Sigma-Aldrich, #L0401), guinea-pig anti-VGLUT1 (1:800, Synaptic Systems, #135404), rabbit anti-phosphorylated S6R (1:200, Cell Signaling, #2211), rabbit anti-phosphorylated AKT (1:200, Cell Signaling, #8112), rabbit anti-TFEB (1:200, Bethyl Laboratories, A303-673A), rabbit anti-FASN (1:200, Cell Signaling, #3180), rabbit anti-pyruvate dehydrogenase E 1 alpha (1:200, GeneTex, # GTX104015 ), rat anti-CD107a (LAMP-1) (1:200, BioLegend, # 121602).
Microscopy image analysis
For all the image analyses described below, investigators were blind to the genotype of the mice. PCs stained by the Golgi-Cox technique were imaged with the Keyence BZ-X800 imaging system using the 40X objective and the Quick Full Focus tool. To measure the total length and perform the Sholl-Analysis of these Golgi-Cox-stained PCs, the Simple Neurite Tracer plugin from the ImageJ software was used.
For the quantification of GFP+ spines that were colocalizing or not with VGLUT1+ presynaptic inputs from parallel fibers, a Nikon A1R Confocal System equipped with Live Cell 6 Laser Line and Resonant Dual Scanner was used to take 0.8mm images with a 63X objective. To increase the visual magnification of these structures and facilitate the manual quantification of them, high magnified snapshots of the confocal images were taken using the Bitplane ImarisÔ software (Oxford Instruments). Then, these snapshots with scale bars (see images in Figure 1e) were used to measure the length of dendritic processes and manually count (Cell counter plugin) the number of spines per mm using the ImageJ software. Two to three images were taken per mouse (n=4 mice).
For 3D image reconstructions and analyses, three sagittal 50μm cerebellar sections were immunostained by free-floating immunohistochemistry. All the images analyzed by the Bitplane ImarisÔ software were acquired using the Nikon A1R Confocal System. Confocal image stacks were acquired using a 40X objective lens with a 1μm interval through a 40μm z-depth of the tissue. Two to three confocal images per mouse were taken in the cerebellar cortex from the first 4 lobes (anterior region of the cerebellum). To quantify the total length and terminal points of cerebellar capillaries in the cerebellar cortex of postnatally developing mice, the Filament Tracer plugin from the ImarisÔ software was used. Two to three images per mouse (n=4 mice) were used for the quantifications. Quantitative analysis of 3D images to determine the volume of GFP or VGLUT1 inside microglia was performed using the ImarisÔ Surface rendering tool. First, IBA1+ microglia were segregated using the Surface rendering tool. Then to quantify GFP from PC dendrites or VGLUT1 synaptic terminals contacted or engulfed by microglia, the “Mask all” tool was used to create a new channel of the GFP+ or VGLUT1+ areas inside of the created surface (in this case IBA1 surface) by clearing all the fluorescence that was not found overlapping/contacting the IBA1 rendering surface. The sum of the GFP or VGLUT1 volume contacted or inside the IBA1 surface was calculated and provided by the software then used for the data analysis presented here.
Quantifications of the PDE and LAMP1 total volume inside CALB+ dendrites were performed by cropping a region in the ML (300μm height X 400μm wide) in 40X confocal images and creating a 3D surface rendering for CALB that was used to obtain the sum of the volume of the CALB+ dendrites. Then, the “Mask all” tool was used to create a new channel for the PDE+ or LAMP1+ areas inside of the created surface (in this case CALB surface) by clearing all the fluorescence that is not found overlapping/contacting the rendering surface. 3D surface renderings were created for the newly created PDE and LAMP1 channels in order to determine the volume of this staining that was inside the CALB+ dendrites. The ImarisÔ software calculated and provided the measurements of the respective volumes for PDE and LAMP1, and the percentage of these markers in PC dendrites were calculated by dividing them by the CALB volume of the dendrites.
The ImarisÔ surface rendering tool was also used to measure the levels of pS6R and pAKT volume in PC dendrites and soma respectively. FASN fluorescence raw intensity per PC soma was measured using ImageJ, where the soma of the cells was manually traced to select the region of interest for the measurement. The intensity value was then divided by the area of the traced cell. PCs with nuclear labeling of TFEB were manually counted after selecting a region of interest using a box of 50 X 40 pixels and divided by the total number of PCs inside the box to calculate the percentage of PCs with TFEB nuclear labeling. NeuroTrace was used to label the PC soma when FASN and TFEB antibodies were used.
For electron microscopy, WT and Npc1nmf164 mice (12 weeks of age) were perfused with 4% PFA, then brains were dissected and hemisected in the midsagittal plane. One of the hemisections was fixed overnight (2% paraformaldehyde + 2% glutaraldehyde diluted in 0.1 M cacodylate buffer with 0.05% CaCl2) for electron microscopy. The process of resin embedding was performed as previously described7,56. Briefly, small pieces of the processed cerebella were infiltrated with 50/50 Epon-Araldite resin and propylene oxide for 1 h, then in 100% Epon-Araldite and left in the desiccator overnight. The next day the cerebellum samples were placed in cubic molds and embedded in 100% resin. The resin block was trimmed and, using an ultramicrotome (Sorvall MT-2), longitudinal sections were cut; semi-thin sections (1μm thick) for light microscopy, and ultrathin (90 nm) for electron microscopy. For light microscopy, semi-thin sections were stained using methylene blue-azure II and basic fuchsin. Thin sections were examined with a JEOL JEM-1011 electron microscope equipped with a Gatan digital camera (Model-832) to describe the ultrastructural features of the cerebellar ML.
Data were analyzed using GraphPad Prism software. Significance was calculated using unpaired t-tests for comparisons between two groups. p-values are provided as stated by GraphPad Prism software and significance was determined with p-values less than 0.05.