PWS T1 deletion is associated with impaired cellular integrity in the hypothalamus

To comprehensively profile both gene and protein expression using FFPE sections of postmortem brain tissues collected since 1983, we divided the sections into three distinct sets. The first set underwent Nissl staining to define anatomical orientation, and immunohistochemistry or immunofluorescence techniques were applied to visualize the expression of specific target proteins (Fig. 1a). A second set of sections was dedicated to determining PWS genetic subtypes through the MLPA assay, effectively identifying deletions in genes located on chromosome 15q11-q13 (Fig. 1b). For the third set of FFPE sections, RNA extraction was performed to facilitate subsequent bulk RNA-sequencing analysis (Fig. 1c).

Fig. 1

Schematic flow of the experimental setup and genetic profiling. a Postmortem hypothalamic tissues were formalin-fixed paraffin-embedded (FFPE) sections, and the consecutive FFPE sections were used for morphological profiling by immunohistochemistry or immunofluorescence. DNA and RNA isolated from these sections were used for genotyping and next-generation RNA sequencing. b Multiplex Ligation-dependent Probe Amplification (MLPA)-assisted genotyping of PWS with copy number. No deletion was observed in control subjects; PWS type 1 (PWS T1) subjects showed a 50% gene dose from BP1 to BP3 (red circles, starting from NIPA1 and TUBGCP5 between BP1 and BP2 (NIPA2 and CYFIP1 between BP1 and BP2 were not included in the MLPA analysis)), PWS type 2 (PWS T2) subjects showed a 50% gene dose from BP2 to BP3 (red circles, starting from MKRN3 and MAGEL2). c The significant differentially expressed genes (DEGs) are depicted as within-gene Z-scores in the heatmap, representing all the genes that are significantly up- or down-regulated when comparing PWS T1 and T2. d The majority of the biological processes down-regulated in PWS T1 compared to PWS T2. e Brain non-neuronal cell-type-specific genes among the DEGs within-gene Z-score. Genes in red color are down-regulated in PWS T1 comparing to PWS T2, genes in black color are down-regulated in PWS T1 comparing to controls

The genotypic assessment allowed the categorization of subjects into three groups: Controls, PWS T1, and PWS T2. In all PWS subjects, copy number reduction was observed for genes situated on the long arm of chromosome 15q11-q13 between break point 2 (BP2) and BP3, encompassing NECDIN, MRKN3, and MAGEL2. Notably, the distinction between the two genotypes relied on the profiling of NIPA1 and TUBGCP5 genes positioned between BP1 and BP2.

These genes exhibited normal levels in PWS T2 but displayed reduced-gene copies in PWS T1 (Fig. 1b). In contrast, control samples exhibited no alterations in copy number. Altogether, three PWS T1 and seven PWS T2 subjects were successfully identified. These PWS subjects were subsequently compared to 32 control subjects, that were matched in terms of age, postmortem delay time, and tissue fixation time (Table 1). Within the subset of three PWS T1 subjects, the body mass index (BMI) data were available for subjects aged 6 months and 4 years. Given that hyperphagia and obesity typically onset after 6 months of age, it is reasonable that the average BMI of the PWS T1 group did not display statistically significant differences when compared to the control subjects. However, as anticipated, subjects in the PWS T2 category exhibited a notably higher average BMI compared to the control group, aligning with the expected phenotype.

Delving into the transcriptomic analysis, we identified the most prominently DEGs within the context of PWS T1 deletion (Fig. 1c, Supplementary Fig. 3). Specifically, upon comparing T1 and T2, we observed a substantial downregulation of genes related to cellular integrity, including those associated with cytoskeletal structure, morphology, adhesion, migration, protein transport, and vesicle trafficking. Furthermore, we noted a decrease in the expression of genes linked to phagocytosis, lysosomal activity, as well as neuronal development and communication in PWS T1 subjects (Fig. 1d). To investigate whether these transcriptional changes were associated with specific cell types, we used publicly available processed scRNA seq data from the mammary, tuberal, and supraoptic regions of the hypothalamus from Human Brain Cell Atlas [43]. Using FindAllMarkers from the Seurat package [19], we identified cell-type-specific genes and intersected these with our DEGs [43]. This analysis revealed a strong oligodendrocyte and, to a lesser extent, microglia-specific gene profile among the DEGs that are down-regulated in PWS T1 compared to PWS T2 or controls (Fig. 1e). In addition, some of the down-regulated genes are also specifically expressed in astrocytes, vascular, or ependymal cells (Fig. 1e). Furthermore, we also discovered more than 200 neurons-enriched genes that are down-regulated in PWS T1 compared to PWS T2 or controls (Supplementary Fig. 4). Collectively, these transcriptomic signatures unveiled robust gene expression differences between PWS T1, T2 subtypes and the controls. Conjectural interpretation of these data hinted for glial and neuronal dysfunction as a pathophysiological mechanism that underlies the worsened physiological and behavioral traits in patients with a PWS T1 deletion.

PWS T1 subjects present dysmorphic microglia

Among the four haploinsufficient genes unique to PWS T1, CYFIP1 is highly expressed in microglial cells in the brain [18, 42] and is involved in actin cytoskeleton remodelling [14]. Our transcriptomic analysis revealed pathway changes in the cytoskeleton, combined with enrichment of downregulation of DEGs in PWS T1 in comparison to PWS T2 or controls that are microglia-specific. Consequently, we first evaluated microglial morphology using Iba1-immunoreactivity (Iba1-ir). Strikingly, Iba1-ir cells in PWS T1 subjects exhibited aberrant morphology characterized by cytoplasmic deterioration and fragmentation (16.45 ± 2.8 fragments/cell), a process known as cytorhexis [48] (Fig. 2a - c). This PWS T1-associated microglial dysmorphism was not found in controls and PWS T2 subjects, in which the Iba1-ir microglia throughout the hypothalamus were morphologically intact, with few visible primary processes (Control: 1.48 ± 0.31/cell, PWS T2: 1.70 ± 0.26/cell, p = 0.62). Moreover, we observed an increased number of Iba1-ir cells and a larger relative area of coverage in the mediobasal hypothalamus of PWS T2 subjects compared to controls, indicating heightened immune activity in microglia among subjects with PWS T2 deletion (Fig. 2d, e).

Fig. 2

PWS T1 deletion is associated with dysmorphic microglia that are partially driven by Cyfip1 haploinsufficiency. a-c Representative images of Iba1-ir cells in the mediobasal hypothalamus of the control (n = 32), PWS T1 (n = 3), and PWS T2 (n = 7) subjects. Dark arrow-pointed microglia in the upper panel of a are shown at a higher magnification in the lower panel. m, months; y, years. d, e Comparison of the hypothalamic Iba1 soma number and relative area of coverage. f Immunohistochemistry for Iba1-ir microglia in the mediobasal hypothalamus of wild-type (n = 6) and Cyfip1 haploinsufficient (n = 8) male rats. Dark arrow-pointed microglia in the left panel of each genotype in f are shown with higher magnification in the two right panels. g-i Iba1-ir cell number and soma size and primary processes in Cyfip1+/− male rats. j Iba1-ir microglia in the mediobasal hypothalamus of control mice (Cx3cr1Cre−ERT+/−Cyfip1fl−/−) (n = 8) or Cx3cr1Cre−ERT+/−Cyfip1fl+/− mice (n = 7) at the age of 32 weeks. Dark arrow-pointed microglia in the left panel of each genotype in j are shown at higher magnification in the right panels. Scale bar: 20 µm in a-c upper panel, 100 µm in f, 50 µm in j. Data are represented as mean ± SEM. Significance in d and e was calculated using the Kruskal–Wallis test, significance in i and m was calculated using the Student’s t-test. * p < 0.05, ** p < 0.01

Next, we investigated whether disruptive microglial morphology could be observed using other microglial functional markers. TMEM 119 and P2Y12R were used as microglial homeostatic identifiers [45]. Our findings showed that the disruption in microglial morphology observed in the Iba1-ir subset was also present in TMEM 119-ir and P2Y12R-ir microglia (Supplementary Fig. 5). However, unlike the Iba1-ir results, we did not observe any differences in the cell number or relative area of coverage between the controls and PWS T2 with TMEM 119-ir and P2Y12R-ir (Supplementary Fig. 5). Furthermore, we found that Iba1-ir microglia in the hippocampal CA1 area of PWS T1 individuals exhibited dysmorphic features similar to those observed in the hypothalamus of these subjects, with significantly lower total area of coverage (Supplementary Fig. 6).

CYFIP1 is involved in actin cytoskeleton remodelling [14]. To determine whether the absence of CYFIP1 can be a causal factor in microglial dysmorphism, we evaluated Iba1-ir microglial cells in the mediobasal hypothalamus of wild-type (+/+) rats and global Cyfip1 haploinsufficient (Cyfip1+/−) male rats (Fig. 2f–i). We found a reduced number of primary processes due to fragmentation in the microglia of Cyfip1+/− rats compared to their littermates (Fig. 2i), but no significant alterations in Iba1-ir cell count or relative covered area (Fig. 2g, h). Thus, the microglial morphological disruption in global Cyfip1 +/− rats partially recapitulates our findings in PWS T1. Of importance, microglial morphological alterations were also detected in female Cyfip1 haploinsufficient (Cyfip1+/−) rats (Supplementary Fig. 7a—d). Given the robust expression of Cyfip1 within microglial cells and the early emergence of microglial dysmorphism by the age of 6 months in PWS subjects, we generated a specialized Cx3cr1Cre−ERT+/−Cyfip1fl+/−mouse model targeting myeloid cells, including microglia in the brain. We evaluated microglial morphology in adult (32 weeks of age) male mice. Although the number of Iba1-ir microglia and soma size in Cx3cr1Cre−ERT+/−Cyfip1fl+/− mice were comparable to the controls (Fig. 2k, l), the number of primary branches on each cell were profoundly reduced in the Cx3cr1Cre−ERT+/−Cyfip1fl+/− mice (Fig. 2m). Thus, Cyfip1 haploinsufficiency has a detrimental impact on microglial morphology in different rodent models.

However, it is important to emphasize that in the Cx3cr1Cre−ERT+/−Cyfip1fl+/− mice, we did not observe cytoplasmic fragmentation in microglial cells in the brain, as was found in the brains of PWS T1 subjects. This suggests that Cyfip1 haploinsufficiency may not be the sole driver behind the microglial dysmorphisms observed in human brains afflicted with PWS T1 deletion. This leads us to consider the possibility that other genes within the PWS T1 deletion region, such as TUBGCP5, which is known to be involved in microtubule dynamics [24], or common neuronal dysfunctions shared by both PWS T1 and T2 deletions, involving genes in the core PWS region, may be acting synergistically to contribute to the microglial dysmorphic changes witnessed in PWS T1 deletion brains.

PWS Type 1 is associated with defective microglial phagolysosomal activity

The microglial immune surveillance and scavenging functions heavily depend on phagocytosis and lysosome activity [10]. Given the mechanical changes that microglial cells undergo to engulf and digest particles, we hypothesized that dysmorphic microglia in PWS T1 might exhibit significant phagocytosis defects. Therefore, we evaluated their phagocytic capacity by co-immunostaining for Iba1 and CD68, a phagosome surface marker (Fig. 3a–c). The ratio of phagosome volume to soma volume was used to assess microglial phagocytic capacity. Interestingly, we found a significantly higher CD68-ir/Iba1-ir volume ratio in PWS T1 microglia compared to controls, primarily due to an enlargement of CD68-ir phagosome particles in these cells (Fig. 3d). In contrast, PWS T2 microglia exhibited a CD68-ir/Iba1-ir volume ratio comparable to controls (Fig. 3d). However, we observed in PWS T2 a higher proportion of CD68-ir microglial cells among the total Iba1-ir microglia compared to controls (Fig. 3e), consistent with the elevated number of microglia in PWS T2 (as shown in Fig. 2d, e).

Fig. 3

Dysmorphic microglia with PWS T1 deletion are defective in phagolysosome activity. a-c Representative images of CD68 expression in Iba1-ir microglia in the mediobasal hypothalamus of control (n = 32), PWS T1 (n = 3), and PWS T2 (n = 7) subjects. Yellow arrow-pointed microglia in the upper panel are shown at higher magnification in the lower panel of a-c. d-e Quantitative analysis of CD68-ir positive microglia among total Iba1-ir cells and CD68-ir volume relative to the Iba1-ir volume. f–h Representative images of CTSS expression in Iba1-ir microglia. i Quantitative analysis of CTSS-ir volume relative to Iba1-ir volume. j-l Representative images of LAMP1 expression in Iba1-ir microglia. m Quantitative analysis of LAMP1-ir volume relative to Iba1-ir volume. m, months; y, years. Scale bar: 30 µm in the upper panel of a-c, 10 µm in the lower panel of a-c, f–h and j-l. Data are presented as mean ± SEM. Significance was calculated using the Kruskal–Wallis test for all comparisons. * p < 0.05

Next, we assessed whether the abnormal phagosome volume in PWS T1 was coupled with a higher capacity for debris degradation within phagolysosomes. To examine this, we investigated two lysosomal markers: CTSS, a protease belonging to the cathepsin family, and LAMP1, a cardinal lysosomal indicator marker [23, 53]. We found that, in contrast to CD68, PWS T1 microglia exhibited a significant reduction in CTSS-ir (Fig. 3f–i) and LAMP1-ir (Fig. 3j-m), indicating a decreased debris degradation capacity within lysosomes. Microglia in PWS T2 brains had CTSS-ir and LAMP1-ir levels comparable to those in the control group (Fig. 3i, m).

As CYFIP1-involved actin cytoskeleton remodelling plays an important role in the fusion process during the phagolysosome maturation [29, 31], we assessed the microglial phagocytic capacity in Cx3cr1Cre−ERT+/−Cyfip1fl+/− mice. We found a significantly higher CD68-ir/Iba1-ir volume ratio in the microglial cells of the Cx3cr1Cre−ERT+/−Cyfip1fl+/− mice compared to control mice (Supplementary Fig. 7e, f). These data suggest that CYFIP1 deficiency may directly impact the phagocytic function of microglia in PWS.

PWS T1 is associated with increased glymphatic system aquaporin 4 in the hypothalamus

Given the compromised phagolysosome activity of microglia in PWS T1 brains, we hypothesized that the brain microenvironment might accumulate more cell debris and waste compared to PWS T2 brains, necessitating enhanced cleaning mechanisms. Consequently, we investigated AQP4, a critical component of the brain’s glymphatic drainage system [22, 33]. In the human brain, AQP4-ir is exclusively found in astrocytic cells, with punctate staining primarily attributed to astroglial end-feet processes, spanning the parenchymal area (Fig. 4a, b). These end-feet processes create a perivascular space, known as the glymphatic system. We detected a significantly larger AQP4-ir covered area in the hypothalamus of PWS T1 subjects compared to controls, while no significant difference was observed between PWS T2 and controls (Fig. 4c–f).

Fig. 4

Enhanced glymphatic component aquaporin 4 expression in PWS T1 deletion. a, b Illustration of AQP4-ir astrocytes surrounding alpha-SMA-ir vessels that form the perivascular glymphatic system. The white dashed line-framed area in a is shown with higher magnification in b, and white arrows indicate the space between the AQP4-ir astrocytes and the alpha-SMA-ir vessel. c-e Representative images of AQP4-expressing astrocytes in control, PWS T1, and PWS T2 subjects. f Quantitative analysis of AQP4-ir covered area in the hypothalamus. g-i Representative images of alpha-SMA-ir vessels in control, PWS T1, and PWS T2 subjects. j Quantitative analysis of the number of alpha-SMA-ir vessels in the hypothalamus. k-m Representative images of the AQP4-ir astrocytes surrounding the alpha-SMA-ir vessels in control, PWS T1, and PWS T2 subjects. n Quantitative analysis of the area of AQP4-ir surrounding the alpha-SMA-ir vessels. m, months; y, years. Scale bar: 30 µm in a, 5 µm in b, 50 µm in c-e, 150 µm in g-i, 10 µm in k-m. Data are presented as mean ± SEM. Significance was calculated using the Kruskal–Wallis test for all comparisons. * p < 0.05

Next, we assessed hypothalamic vasculature through alpha-SMA-ir, an endothelial marker for arteries and arterioles. We found an increased number of alpha-SMA-ir vessels in PWS, irrespective of the subgenotype (Fig. 4g–i), indicating hypothalamic angiogenesis in this pathology. We further investigated the topographic association between AQP4-ir astroglia and alpha-SMA-ir vessels by evaluating the AQP4-ir surrounding the alpha-SMA vessels within a 20 µm radius from each individual vessel. We found an increased presence of AQP4-expressing astroglia in the perivascular space in PWS T1 compared to controls and PWS T2 subjects (Fig. 4k–n). These findings point towards heightened-glymphatic system activity in PWS T1 individuals requiring closer interaction with vasculature and suggesting an increased demand for the removal of harmful molecules from their brain. This response likely arises due to the defective immune scavenging and cleaning function of microglia within the brain microenvironment.

Different PWS sub-genotype displays distinct white matter patterns

Given the downregulation of genes associated with microglia, oligodendrocytes and myelination capacity in our transcriptomic analysis, along with previous findings in Cyfip1+/− rats showing abnormal white matter structure—characterized by a reduced number of oligodendrocytes and decreased myelin thickness in the corpus callosum [44]—we conducted an assessment of white matter integrity in PWS brains, with a primary focus on the fornix, a major white matter tract originating from the hippocampus, passing through the hypothalamus, and ending in the hypothalamus and mammillary body [51] (Fig. 5a–c). In control and PWS T2 subjects, using PLP-ir, we observed a uniform distribution myelin ring across the fornix, suggesting normal structural and homeostatic myelination capacity. However, in PWS T1, the myelin rings were sporadic, with a significantly lower total number of myelin rings compared to controls and PWS T2 (Fig. 5d), potentially indicating the presence of underdeveloped myelin. No difference was found in the intensity of PLP-ir observed in both the fornix and the gray matter adjacent to the fornix among all the groups (Fig. 5e, f). To ascertain whether white matter deterioration in PWS T1 was exclusive to the hypothalamus, we also evaluated myelin microstructure in the anterior commissure and hippocampus. However, we did not observe structural abnormalities in any of the evaluated white matter landmarks, regardless of the PWS subgenotype (Supplementary Fig. 8 and Supplementary Fig. 9).

Fig. 5

Abnormal white matter microstructure in the fornix of PWS T1 subjects. a-c Representative images of PLP-ir at the level of the fornix in the hypothalamus of controls (n = 32), PWS T1 (n = 3), and PWS T2 (n = 7) individuals. Framed areas in upper and middle panels are displayed in details in their lower panels respectively. Individuals with PWS T1 deletion have aberrant white matter structures, as shown by a drastic reduction in PLP-ir nodes (myelin rings, indicated by white arrowheads) throughout the fornix. d-f Comparison of PLP-ir myelin rings and optical density in the fornix or gray matter outside the fornix. g Fornix outlined by PLP-ir in the hypothalamus of wild-type (n = 4) and Cyfip1 haploinsufficient (n = 4) rats. h Comparison of the PLP-ir optical density in the fornix. Fx, fornix; O.D., optical density; m, months; y, years. Scale bar in a-c, 100 µm in upper panel, 20 µm in middle panel, and 5 µm in lower panel; 100 µm in g. Data are represented as the mean ± SEM. Significance was calculated using the Kruskal–Wallis test for d, e, and f. * p < 0.05

Microglia are closely associated with myelin sheath organization and have intimate interactions with oligodendrocytes [41]. Therefore, we examined Iba1-ir microglia within the fornix (Supplementary Fig. 10). Compared to controls (Supplementary Fig. 10a, e), microglial fragmentation was observed in the fornix of PWS T1 (Supplementary Fig. 10b, f), while an increased Iba1-ir covered area was noted in the fornix of PWS T2 (Supplementary Fig. 10c, d). Since myelin-laden microglia are a common feature of demyelinating pathologies, we investigated whether there was differential myelin phagocytosis in the PWS subtypes. However, Iba1-ir cells co-localized with PLP-ir particles were only occasionally found in microglia cell in general (Supplementary Fig. 10e–g), making it challenging to conduct a comprehensive quantitative analysis.

During myelin sheath development, the turnover of actin filaments in oligodendrocytes plays a critical role in regulating repetitive cycles of leading-edge protrusion and spreading [36]. In alignment with a previous study on Cyfip1+/− rats that demonstrated aberrant white matter structure [44], we found a reduction in PLP-ir in the fornix of Cyfip1+/− rats (Fig. 5g, h). Our data suggest a possibility that the loss of myelin rings in the fornix in the PWS T1 hypothalamus might be due to CYFIP1 haploinsufficiency-induced actin disarrangement within oligodendrocytes.

Both PWS T1 and T2 show hypothalamic neuropeptidergic imbalance

Functional microglia play a crucial role in supporting neural development and maintaining health. Therefore, we meticulously profiled neurons in the hypothalamus, with a focus on those producing neuropeptides that regulate energy homeostasis. Regrettably, one PWS T1 subject lacked sections for neuropeptide analysis in the infundibular nucleus and paraventricular nucleus (PVN) in the hypothalamus. We initiated our analysis at the infundibular nucleus level, particularly examining anorexigenic POMC and orexigenic NPY neurons. We noted a decrease in the POMC-ir covered area and cell count in the hypothalami of PWS T2 subjects compared to controls (Supplementary Fig. 11a–e). Interestingly, the PWS T1 infant (6 months old) exhibited increased POMC-ir parameters, while the adult did not differ from PWS T2 subjects. Due to the reduction in anorexigenic POMC-ir neurons, we also evaluated orexigenic NPY-ir neurons in the same area, finding an overall diminished number of NPY-ir cells in both PWS T1 and T2 (Supplementary Fig. 11f–j). Moving on, we examined neuronal populations within the PVN, focusing on OXT and AVP producing neurons. We found no alterations in AVP-ir neurons in the PVN of patients with PWS (Supplementary Fig. 12a–e). However, OXT-ir mirrored the pattern found in POMC-ir expressing cells, displaying a clear reduction in PWS T2 (Supplementary Fig. 12f–j). In sum, our findings confirm that dysfunction in hypothalamic neuropeptidergic machinery is a hallmark of PWS, likely driven by the PWS T2 deletion.

PWS T1 presents worsened neural communication

One of the major outcomes of our transcriptome analysis is the downregulation of pathways involved in synaptic function and neuronal communication. Given the critical role of microglial scavenging and cleaning function in synaptic pruning, coupled with Cyfip1’s implication in synaptic homeostasis [13] and its enrichment in cortical inhibitory synaptic sites [44], we posited that PWS T1 might suffer from impaired synaptic stabilization and function. We, therefore, assessed the expression of the synaptic integrity marker synaptophysin. In controls, synaptophysin-ir exhibited a punctual pattern, evenly distributed throughout the tissue (Fig. 6a). Brains of PWS T2 did not show alterations in synaptophysin expression compared to controls (Fig. 6a, c, d). In contrast, PWS T1 brains displayed a marked reduction in synaptophysin-ir (Fig. 6b, d). Furthermore, we observed no significant changes in synaptophysin expression in the hippocampal CA1 region (Supplementary Fig. 13). Our data suggest that deleted genes in PWS T1 may contribute to the disrupted synaptic integrity in the hypothalamus.

Fig. 6

Reduced hypothalamic synaptophysin expression in PWS T1 subjects. a-c Representative images of synaptophysin immunoreactivity in the hypothalamus of control (n = 32), PWS T1 (n = 3), and PWS T2 individuals (n = 7). PWS T1 hypothalami showed a reduction in synaptophysin-ir compared to controls indicating defective neuron-neuron communication. Dotted lines frame the fornix. d Quantitative analysis of the hypothalamic synaptophysin, as demonstrated by the relative area of coverage. Fx, fornix; m, months; y, years. Scale bar: 100 µm. Data are represented as mean ± SEM. Significance was calculated using the Kruskal–Wallis test in d. * p < 0.05

Confounder analysis

Considering the substantial heterogeneity in postmortem human brains, we performed a confounder analysis, considering age, tissue fixation time, postmortem delay, and BMI. The limited number of PWS T1 specimens precluded sex analysis in this group, and no sex differences were observed in the controls or PWS T2. All potential confounders were matched between the control and PWS groups, except those with data not available (see in Supplementary Table 1). Linear regression analysis revealed incidental significance for several parameters, including Iba1-ir soma number/mm2 vs. BMI in controls, Iba-ir relative area of coverage vs. age in PWS T1 (Supplementary Fig. 14d, e); TMEM119-ir soma number/mm2 and relative area of coverage vs. age in controls (Supplementary Fig. 15a, e); LAMP1-ir/Iba1-ir volume (%) vs. fixation time (Supplementary Fig. 19b); number of alpha-SMA-ir vessels vs. BMI in PWS T2 (Supplementary Fig. 21d); % of AQP4-ir surrounding the alpha-SMA vessels vs. BMI in PWS T2 (Supplementary Fig. 21e); AVP-ir soma number/mm2 and percentage of coverage vs. age in PWS T2 (Supplementary Fig. 27a, e); synaptophysin-ir relative area of coverage vs. fixation time in controls and PWS T2 (Supplementary Fig. 29b). However, these findings did not alter the overall implications of our study (Supplementary Figs. 14—29).