Fig 2: Synapse loss in ALS frontal cortex. Representative electron micrographs showing two excitatory synapses in the frontal cortex of a healthy control (a) and an ALS case (b). Edges of the post-synaptic density (PSD) are marked by red arrowheads and pre-synaptic terminals by black asterisks. The white asterisk in b highlights an electron dense, degenerating presynaptic terminal. Scale bars 100 nm. Decrease in synaptic density in the frontal cortex [(c) 2-tailed unpaired t test; p = 0.016], and increased numbers of degenerating synapses [(d) 2-tailed Mann–Whitney test; p = 0.014] in ALS patients (n = 20) compared to controls (n = 5), measured using TEM. Each data point represents the mean total synapse (c) or degenerating synapse (d) count per 100 µm2 for each individual. Three-dimensional reconstructions of fifteen 70 nm array tomography sections from a control frontal cortex (e) and an ALS frontal cortex (f), stained for synaptophysin (green) and DAPI (white). Scale bar is 10 µm. g Decrease in synaptic puncta (2-tailed unpaired t test; p = 0.017) in ALS patients compared to controls, measured using array tomography. Each data point represents the mean synapse count per mm3 of frontal cortex, for each individual (control n = 14; ALS n = 29). Blue icons = C9orf72 expansions, Red icons = SOD1 variant and Green icons = NEK1 variant. h Cortical thickness comparisons between control (n = 7) and ALS (n = 34) revealed no difference (2-tailed unpaired t test; p = 0.76). Each data point represents the mean of ten measurements per cortical block. All histogram bars plot the mean ± SD. i 3D reconstruction of 25 array tomography sections (each 70 nm thick), stained for synaptophysin (red), PSD95 (blue), pTDP-43 (green) and DAPI (white). A large perinuclear pTDP-43 aggregate (white arrow), numerous pTDP-43 + ve neuritic threads (white arrowheads) and small pTDP-43 puncta (open arrows) are clearly visible. Scale bar 10 µm. By cropping the image in i, compressing 3 consecutive sections and then splitting the channels, it is evident in panel (j) that pTDP-43 aggregates are found both in some pre (synaptophysin, red) and post (PSD95, blue) synaptic compartments. Scale bar 1 µm

Fig 3: Quantitative STED analysis reveals highly variable Munc13-1 signal in excitatory synapses on FSIN dendrites.(A) Confocal maximum intensity projection image of a biocytin-filled FSIN (Cell1, soma, and basal dendrites in the str. oriens are shown). The dendritic segments that were re-sectioned and analyzed are highlighted in yellow. (B) Reconstruction of the re-sectioned dendritic segments (from twenty 200 nm thick sections) shown in (A). Colored circles indicate en face excitatory synapses (n = 33) identified by Munc13-1 and PSD-95 double immunolabeling. (C) STED analysis of Munc13-1 and PSD-95 immunofluorescent signals on a single 200 nm thick section (shown in the boxed area in (B)). The biocytin-filled dendrite shown in a confocal image (top left) is outlined by a white line. Colored regions of interests (ROIs) represent en face synapses based on the Munc13-1 and PSD-95 immunosignals. (D) Relative Munc13-1 intensity as a function of relative PSD-95 signal in individual synapses. Symbols represent mean normalized integrated fluorescent intensities in individual synapses (Cell1, n = 33; Cell2, n = 26). Magenta and green filled symbols indicate the corresponding color-coded synapses shown in (C). Note that the two synapses have very similar Munc13-1 content although their PSD-95 reactivity is ~2.5-fold different. (E) Munc13-1 to PSD-95 ratio as a function of relative PSD-95 intensity in individual synapses. Magenta and green filled symbols indicate the corresponding color-coded synapses shown in (C). rs, Spearman’s rank correlation coefficient.Figure 6—source data 1.Quantitative STED analysis reveals highly variable Munc13-1 signal in excitatory synapses on FSIN dendrites.

Fig 4: Ab-dependent synaptic loss and neuritic dystrophies in adult APP/PSEN1 and APP/PSEN1/APOEnull mice.(A) Representative images of array tomography ribbons after immunostaining for Ab (1C22), presynaptic terminals (antisynapsin I, red), postsynaptic densities (anti-PS95, green), and nuclei (DAPI, blue). It is possible to appreciate how diffuse is the amyloid in the brain of APP/APOEnull mouse as compared with the compact staining observed in the APP mouse. Scale bar = 10 μm. (B) Scatter dot plots summarizing the density of PSD95 (left panel) and synapsin (right panel)-positive puncta in the cortex of wild-type, APOEnull, APP/PSEN1, and APP/PSEN1/APOEnull mice. A significant decrease in synaptic density was only observed in the close vicinity of amyloid plaques in AD transgenic mice. n = 6 mice/group (10–12 mo “adult” cohort); Two-way ANOVA and Tukey’s post hoc test. *P < 0.05 and ***P < 0.0001. (C) Representative images of neuritic dystrophies (arrows) around amyloid plaques observed in APP/PSEN1 and APP/PSEN1/APOEnull mice, showing that most sprouting axons are found in mice expressing endogenous murine apoE. Scale bar = 50 μm. (D) Scatter dot plot summarizing the stereological quantification of the number of dystrophies observed per area of amyloid (evaluated by immunohistological staining). n = 6 mice per group (10–12 mo “adult” cohort); Mann–Whitney test. *P < 0.005.

Fig 5: Mapping excitatory synapses in the spinal cord revealed early symptomatic stage synaptic changes in SOD1G93a mice. a High-resolution mapping of PSD95-eGFP was performed to analyse synaptic changes in SOD1G93a mice at different time points of the disease. b Example images of postsynaptic puncta in control and SOD1G93a mice at different time points of disease. c Heat maps of PSD density in male and female SOD1G93a mice compared to their respective controls at different ages. d Heat maps of PSD size in male and female SOD1G93a mice compared to their respective controls at different ages. e Chart plotting PSD density in all 8 laminae of 16-week-old controls, WTSOD1 controls and SOD1G93a males. Sample sizes denoted as n = number of WTSOD1’s/number of controls/number of SOD1G93a’s. f Chart plotting PSD size in all 8 laminae of 16-week-old controls, WTSOD1 controls and SOD1G93a males. g Chart plotting PSD density in all 8 laminae of 16-week-old controls, WTSOD1 controls and SOD1G93a females. h Chart plotting PSD size in all 8 laminae of 16-week-old controls, WTSOD1 controls and SOD1G93a females