Elevated ganglioside GM2 activator (GM2A) in human brain tissue reduces neurite integrity and spontaneous neuronal activity

Overview of the study

Multiple studies have explored the proteomic landscape of the human brain, with the purpose of identifying proteins and pathways differentially altered in AD [16, 29,30,31]. While highly valuable, the impact of those studies has been limited by the availability of assays for measuring the functional consequences of differentially expressed proteins in the human brain. Therefore, a challenge in the field has been to disentangle protein changes that are driving synaptic loss and neurodegeneration from those that are indirect consequences of neuronal death. We aimed to identify the proteins present in the aged brain that may have neurotoxic or neuroprotective activities, using unbiased proteomics coupled to NI and MEA assays of structural and functional integrity of neuron.

Brain tissue was acquired postmortem from 43 individuals, 14 with low AD neuropathology who were not cognitively impaired (LP-NCI), 14 with high AD neuropathology who were not cognitively impaired (HP-NCI), and 15 with clinical and pathological diagnoses of AD. Table 1 outlines distributions of sex, age at death, and postmortem interval (PMI) across brain samples. The mean age at death was 81 and was not significantly different across diagnostic categories. Tris buffered saline (TBS)-soluble extracts were prepared from the prefrontal cortex and analyzed by unbiased proteomics. The same TBS-soluble brain extracts also were used in either 1 or 2 assays of neuronal structure and function: an imaging-based morphological NI assay and/or an MEA-based spontaneous firing assay (limiting amounts of available brain tissue precluded the use of all extracts in both assays). Proteins identified were those associated with neurotoxicity or neuroprotection in each assay. An overview of the experimental design of the study is outlined in Fig. 1.

Fig. 1figure 1

Overview of study. Brain tissue from the prefrontal cortex was acquired for 43 individuals, and TBS-soluble extracts prepared. Tissue was extensively dialyzed in BrainPhys media to remove small molecules, such as glutamate, present in the extracts. Extracts were analyzed by proteomic profiling, and 1841 proteins quantified. These extracts were used either in a neurite integrity assay and/or in an MEA-based spontaneous activity assay. Correlations were then calculated between proteins quantified via proteomics and activity in each assay

Table 1. Brain samples used in this studyDifferentially expressed proteins in the TBS-soluble AD brain proteome

The proteome in each of the 43 brain extracts was examined by unbiased proteomic analysis using liquid chromatography/tandem mass-spectroscopy (LC/MS-MS), followed by label-free quantification (LFQ) to detect 1841 proteins across the brain samples [16, 18]. Principal components analysis (PCA) demonstrates that AD samples tend to cluster together and away from LP-NCI samples, while HP-NCI samples intermix with each. This may be because the category of HP-NCI is likely to include both those individuals who have sustained, strong cognitive resilience even in the presence of high amyloid pathology and individuals who may have developed AD had they lived longer. While there is some separation between these categories in PCA space (Fig. 2A), there is clearly a lot of overlap across categories in the proteome-wide profile.

Fig. 2figure 2

Differentially expressed proteins in the TBS-soluble AD brain proteome. A PCA plot of brain tissue samples using proteomic data. AD TBS-soluble proteomes are heterogeneous within categories, with overlap across categories. B, C Differential expression of individual proteins was analyzed between AD vs. LP-NCI (B) and AD vs. HP-NCI (C). Many more proteins were differentially expressed in the LP-NCI vs. AD comparison than in the HP-NCI vs. AD comparison. Unpaired t-test with Welch correction, two-stage step-up, dotted line at q = 0.05. D Scatter plots of top DEPs across categories. Units are normalized abundance ratios. One-way ANOVA with Dunn’s multiple comparisons test, p-values as listed. E Top hits in the REACTOME database for GSEA of analyses in B&C. Size = number of components in that gene set, ES = enrichment score, NES = normalized enrichment score

An analysis of differentially expressed proteins (DEPs) by category revealed 601 DEPs between AD vs. LPNCI and 80 DEPs between AD and HP-NCI (FDR < 0.05; Fig. 2B, C). Importantly, these proteome profiles may show substantial differences compared to profiles previously reported from similarly aged individuals [16, 30, 31], because here we are only measuring the TBS-soluble proteome, while most other studies reported the urea-soluble proteome. The top DEP between AD and NCI was VGF, which was lower in the AD brain relative to both LP-NCI and HP-NCI brain (Fig. 2B-E). VGF levels in the brain have previously been reported to significantly associate with AD [32]. In addition to VGF, some DEPs identified here validate findings in prior studies, such as MSN and GFAP [33, 34], while other DEPs have not previously been linked to AD, such as PGAM2 and RENBP (Fig. 2D). Gene set enrichment analysis (GSEA) of DEPs using the REACTOME database showed significant associations (False Discovery Rate [FDR] < 0.05) with specific terms including extracellular matrix organization, transmission across chemical synapses, Rho GTPases activate WASPs and WAVEs, and diseases of metabolism (Fig. 2E).

Assessing effects of human brain extracts on spontaneous neuronal firing using multi-electrode arrays

The generation of action potentials in neuronal cultures can be assessed using a multi-electrode array (MEA) based spontaneous firing assay. We used MEA recordings to assess the effect of TBS-soluble brain extracts on spontaneous firing of primary rat neurons in dense, mixed-cortical cultures (treatments on Day 21 of culture).

TBS-soluble brain extracts were dialyzed 1:10,000 and buffer exchanged into BrainPhys media after centrifugation over a 3-kDa cutoff column (StemCell Technologies). These brain extracts were normalized by protein concentration across all samples, such that extracts contained 1 mg/mL of brain derived total protein. First, to characterize the experimental system and optimize conditions, extracts were prepared from tissue samples of an unaffected, non-cognitively impaired control individual (MGH1887) and an AD diagnosed individual (ALB01). These extracts were diluted 1:1 in BrainPhys media, and then used to treat Day 21 primary rat cortical cultures grown on MEAs. While similar AD brain extracts have been shown to inhibit LTP within rodent brain organotypic slice cultures within minutes to hours [11, 35, 36], the maximal effect on spontaneous firing induced from the AD brain extract emerged over 4 days (Fig. 3A). After 4 days of treatment with AD brain extract, we observed significantly decreased spontaneous firing by approximately an order of magnitude compared to those treated with control brain extract (− 0.993 ∆log10Hz, p = 2.07 × 10− 5; Fig. 3A, B).

Fig. 3figure 3

Effect of human brain extract on spontaneous firing of cultured cortical neurons. A Time course of firing activity within rat cortical cultures prior to, during, and following treatment with human brain extract from 2 individuals. Each dot represents a single multi-electrode array (8 electrodes per array) during each recording. Solid line tracks the mean firing frequency per condition at each recording, and dashed lines indicate mean  ± SEM. B Comparison of firing frequencies across treatment conditions prior to, 4-days post-treatment, and 2-days following washout. Error bars represent mean  ± SEM. Significant differences between conditions indicated by brackets, *, p < 0.05, ***, p < 0.001. C Effects on spontaneous firing elicited across a panel of human brain extracts. An estimated effect on spontaneous firing of individual rat neurons at baseline (Day 0), 1-day, and 4-days following treatment with human brain extracts. Error bars represent mean  ± SEM as predicted by linear-mixed effect model based on treatment data from 19 individuals across 1 to 9 replicate experiments per individual. Numbers below indicate number of experiments (“N”) and number of independent arrays (“n”) tested for each brain sample

After the removal and washout of the brain extract treatment, there was a partial recovery in spontaneous firing within the AD brain extract treated cultures, peaking 2-days following the exchange of extract-containing media with fresh, untreated culture media (Fig. 3B). While the firing activity was still significantly diminished in the AD brain extract-treated culture 2-days following the washout of the treatment, compared to the activity in the cultures previously treated with control brain extract (0.646 ∆log10Hz, p = 0.0158), the extent of recovery within the AD brain extract treated cultures was itself significant (0.648 ∆log10Hz, p = 0.0152). This recovery in spontaneous firing activity is consistent with previous reports that synaptic structures within cultured neurons are able to regenerate following exposure to recombinant Aβ species [37]. Further, examination of the cultures after 4-days of treatment by immunocytochemistry showed that the integrity of cells within the AD brain extract treatment condition was largely intact (Supplemental Fig. 1A).

Subsequent experiments showed that the potency of the effects on spontaneous firing induced by the AD brain extract extended over a narrow range (Supplemental Fig. 1B). This was determined by treating cultures with brain extract that had been further diluted in culture media using a half-log (1:3) dilution series. Within a 10-fold dilution of the AD brain extract, the difference in firing between the AD brain extract treated cultures, and those treated control brain extract was completely attenuated.

While the brain extracts were dialyzed, it is possible that residual pharmacological agents, metabolites, or neurotransmitters could be responsible for the inhibition of firing. In order to determine whether the effect was likely due to proteinaceous species within the extracts, cultures were treated with brain extracts from two AD individuals (ALB01 & MGH1892) in either their native form or following denaturation by boiling at 100 °C for five minutes. After a 4-day treatment, activity remained significantly higher in the cultures treated with boiled extract compared to those treated with native extract (ALB01: ∆log10Hz = 1.04, p = 1.67 × 10− 6; MGH1892: ∆log10Hz = 0.804, p = 0.0364; Supplemental Fig. 1C).

To understand whether the effects on spontaneous activity were representative of AD and unaffected patient populations as a whole, these experiments were extended to test a panel of human brain extracts from 19 individuals (Fig. 3C, see also Table 1). The individuals within this panel were designated to 1 of 3 classes defined by neuropathology and clinical diagnosis (n = 7 LP-NCI, n = 5 HP-NCI, and n = 7 AD). The primary samples within this panel were acquired periodically over a period of 2 years, and the amount of tissue obtained from each individual ranged from a few grams to an entire hemisphere of the brain. The consequence of this is that it was impossible to test all subjects in all experiments in a head-to-head fashion or to test all individuals the same number of times given the disparities in the material available. In total, the brain extracts from the 19 individuals have been tested across 22 experiments.

To make inferences about the effect of each brain extract on spontaneous firing, we used a linear mixed effect model [23] comprised of data from all experiments to estimate the firing frequencies observed in the treated cultures as function of individual extract and time. Interestingly, despite the brain extracts representing three distinct patient populations, the degree of reduction of firing is continuous across the panel of individuals. AD and HP-NCI individuals tended to induce a greater decrease in firing activity than those of extracts of LP-NCI individuals, however, the differences do not reach the significance threshold of p < 0.05 (LP-NCI vs. AD p = 0.185; LP-NCI vs. HP-NCI p = 0.138).

Both HP-NCI and AD brain extracts induce degeneration of neurites compared to LP-NCI brain extracts

A recent study established and characterized a live-cell, morphological neurite integrity (NI) assay to monitor changes to neuronal processes in a dynamic fashion in response to exposure to aqueous-soluble brain extracts [6]. Using the same experimental procedure as reported by Jin and colleagues [6], neurite integrity in cultured human iPSC-derived neurons (iNs) was monitored at 2-hour intervals for 78 hours following treatment with TBS-soluble human brain extracts from 27 individuals (Fig. 4). At the end of the treatment time, neurites treated with some brain extracts remained intact (Fig. 4A, C) while cultures treated with other brain extracts became fragmented (Fig. 4B, C).

Fig. 4figure 4

Influence of human brain extracts on neurite integrity in vitro. A, B Representative images of human iPSC-derived neurons (iNs) treated with an LP-NCI brain extract (A) and neurons treated with AD brain extract (B) at 72 hr. post-treatment. Scale bars = 200 μm. C Quantification of data for each individual brain extract over 78 hours. Data show mean  ± SEM; n = 6 for each brain extract. D Estimated neurite integrity following 72-hr treatment by diagnosis class, combining all data from (C). N = 36 for vehicle-treated wells, and n = 36 for no treatment condition. Neurite integrity in human iNs normalized to baseline (time 0), examined at 2-hr intervals over 78-hr treatment with human brain extracts. One-way ANOVA with Tukey’s multiple comparisons test, comparing LP-NCI, HP-NCI, and AD-extract treated conditions. Significance is shown relative to the LP-NCI extract-treated condition. *, adjusted p < 0.005; **** p < 0.0001

Human iNs showed a continuous response across the brain extract treatments, with some extracts causing negligible effect and others causing a near complete atrophy of neurites at the end of the recording (Fig. 4C). Examining the effects on neurites in the context of diagnosis shows that extracts from AD and HP-NCI individuals induced significantly stronger effects than LP-NCI brain extracts (Fig. 4D) (78-hr-timepoint, ANOVA with Dunnett’s multiple comparisons test: HP-NCI p < 0.0001; AD p < 0.0001). However, there was no significant difference between populations of HP-NCI and AD brain extracts.

Levels of specific Aβ and tau peptides in brain extracts correlate with effect size in the MEA and NI assays

Having tested brain extracts from 43 individuals in two assays, we next aimed to identify candidate factors that may be contributing to the effects observed in each assay. PMI and age at death were not significantly associated with effect size in either assay (Fig. 5A, B, D, E). Sex of the brain tissue donor was associated with effect-size in the NI assay (t-test, p = 0.04), but not in the MEA assay (t-test, p = 0.64). While intriguing, the distribution across categories was not the same between males and females, with fewer males in the LP-NCI category (Fig. 5C, F). Thus, any interpretations of this potential effect of sex must be made with caution.

Fig. 5figure 5

Associations of Aβ and tau peptides with MEA and neurite integrity. A-F Effect size on neurite integrity and spontaneous activity was examined as a function of age at death (A&D), postmortem interval (B&E), and sex (C&F). Data points in C&F are colored by category: LP-NCI (blue), HP-NCI (green), and AD (red). P-values were calculated by Spearman correlations (A-B&D-E) or Mann-Whitney test (C&F). G-J Aβ levels were measured by ELISA in TBS-soluble brain extracts and compared across categories. Aβ42 (G) and Aβ42:40 (H) were measured by 6E10 MSD ELISA, and Aβ oligomers were measured using two different ELISAs: both used 3D6 for detection and for capture antibody one used 71A1 and the other used 1C22. ANOVA with Dunnett T3 multiple comparisons test, p-values as listed. K-O Tau peptides were measured using mass spectrometry and ELISA and compared across categories. Scatter plots show the relative abundance of all tau peptides (K), tau peptides of the N1 and N2 domains “N1,N2” (L), tau peptides of the second MTBR, “MTBR2” (M), and tau phosphorylated at T181 (pT181) and T217 (pT217) (N-O). One-way ANOVA with Dunnett T3 multiple comparisons test, p-values as listed. P Heatmap of Spearman correlation coefficients calculated between the measures in (G-M) and effect size in the MEA and neurite integrity assays. *, p < 0.05. Q Graph of the strongest association between tau peptides with the N-terminal inserts and neurite integrity. p = 0.0004

The AD brain accumulates Aβ and tau in plaques and tangles, respectively. Previous studies have suggested that certain forms of soluble Aβ and tau are neurotoxic, and some AD brains induce neurite degeneration in vitro and inhibit long-term potentiation (LTP) in an Aβ-dependent manner [11, 35]. Therefore, we first measured Aβ and tau levels in this collection of brain extracts. Aβ was measured via ELISA before and after treating the extracts with the denaturing agent guanidine hydrochloride (GuHCl), which allows for epitope exposure within Aβ. As shown in Supplemental Table 1, the concentration of aqueous soluble Aβ42 is in the range of hundreds of pg/mL within the TBS-soluble extracts prepared from these individuals. These values are similar to the concentrations of Aβ42 in CSF observed within a similarly aged population [38]. Additionally, the aqueous insoluble Aβ42 concentration, especially amongst the HP-NCI and AD individuals, falls within the low ng/mL range. This mirrors the concentration of Aβ42 found within the tissue of symptomatic AD individuals processed using a denaturing extraction protocol [39]. It has been previously suggested that it is the oligomeric form of Aβ rather than the monomeric form that is the neurotoxic species present in the brain [11, 36, 40]. Therefore, we measured Aβ oligomer levels using two oligomer-specific ELISA platforms [12]. Both Aβ42 levels and oligomeric Aβ levels were significantly higher in AD and HP-NCI TBS-soluble brain extracts compared to LP-NCI TBS-soluble brain extracts, and not significantly different from one another (Fig. 5G-I). The ratio of Aβ42:Aβ40 is consistently elevated with hundreds of familial AD mutations. Here, Aβ42:Aβ40 levels were significantly higher in HP-NCI extracts compared to both LP-NCI and AD extracts in this brain tissues that were not from FAD carriers (Fig. 5J). This was a surprising observation, as our previous analyses of another cohort of TBS-soluble brain tissue showed significant elevation of Aβ42:Aβ40 in both HP-NCI and AD compared to LP-NCI [14]. Here, in addition to being a separate cohort of brains, there also were differences in the preparation of the brain samples including the addition of the 3 kDa molecular weight cutoff filter and the dialysis step. In addition to Aβ, tau was quantified via mass spectrometry. Overall tau levels were unchanged across categories (Fig. 5K). Tau is alternatively spliced in the brain to produce 6 isoforms which contain either 3 or 4 microtubule-binding repeats (“MTBRs”) and 0, 1, or 2 N-terminal inserts (“N”). Here, we identified and quantified peptides encoding the N inserts (1 N, 2 N) and peptides encoding the alternatively spliced MTBR (the second MTBR, MTBR2). Overall tau and MTBR2-containing tau levels were not significantly different across categories, while levels of peptides encoding the N inserts were significantly higher in the AD brain extracts compared to LP-NCI (Fig. 5K-M).

We next examined whether these Aβ and/or tau measures in the TBS-soluble extracts are associated with effect size in either the NI assay or the MEA assay by calculating Spearman correlation coefficients. Higher levels of each of these measures of Aβ and tau trended toward stronger effect size in each assay (Fig. 5P). The ratio of long to short Aβ (42:40) was associated with effect size in the MEA assay (Spearman r = − 0.58, p = 0.017). In the NI assay, both measures of oligomeric Aβ were significantly associated with effect size (71A1: Spearman r = − 0.46, p = 0.016; 1C22: Spearman r = − 0.42, p = 0.03). Two phosphorylated tau isoforms, pT181 and pT217 used as biomarkers for Alzheimer’s disease in CSF [41, 42], also were significantly associated with effect size (pT181: Spearman r = 0.418, p = 0.03; pT217: Spearman r = − 0.389, p = 0.045) (Fig. 5N-P). Intriguingly, as pT217 went up, neurite integrity went down, and pT181 showed the opposite association, and the meaning underlying this difference will be a subject of future studies. Interestingly, tau peptides with N-terminal inserts showed the strongest association with effect size in the NI assay (Spearman r = − 0.63; p = 0.0004; Fig. 5Q).

Identification of proteins in the human brain associated with neurite degeneration and loss of spontaneous activity

While associations of Aβ and tau were observed with effect size in the MEA and NI assays, neither could fully account for the magnitude of effects across brain samples in either assay. This is in accord with previous findings which showed that while Aβ and tau levels in human brain were elevated in those with AD, neither could fully account for the extent or rate of cognitive decline [43, 44]. Therefore, it is likely that additional factors in the human brain differentially impinge upon the toxicity of Aβ and tau either to accelerate degeneration or to protect against their effects. Therefore, we next used the same unbiased proteomic profiling data of these TBS-soluble brain extracts to identify proteins associated with neurotoxicity and neuroprotection in the MEA and NI assays. To assess the relationship between each of the 1841 proteins detected in this analysis and the in vitro phenotypes, Spearman’s correlation coefficients were calculated between the normalized LFQ value for each protein within each extract and the effect size of each extract in both the neurite integrity and spontaneous firing assays. Top correlations in each assay are shown in the waterfall plots (Fig. 6A, B). A positive correlation represents a protein that exhibits higher expression in extracts with less effect on neurite integrity or spontaneous firing, while a negative correlation represents a protein that exhibits higher expression in extracts with greater effect on these phenotypes. Shared effects were observed across both assays for 18 proteins (Fig. 6C).

Fig. 6figure 6

Associations between individual proteins across brain samples and estimated effect size in assays of neuronal structure and function. A, B Waterfall plots showing top associated proteins in the neurite integrity (A) and spontaneous activity (B) assays, calculated with the Spearman correlation coefficient. C Table of proteins meeting a p-value of < 0.05 across both assays. Top associations in both assays included ABI1, which showed a protective association in the assay, and GM2A which showed a neurotoxic association. D GSEA analyses using the REACTOME database for associations with the MEA and NI assays identify Diseases of metabolism and Effectors of Rho GTPase signaling/WASP and WAVE complexes with effect size in both assays. E Schematic of WAVE regulatory complex (WRC) and ARP complexes, gene symbols in red were identified as leading-edge genes in the GSEA analysis in (D). F Scatter plot of ABI1/ABI2 across TBS-brain extracts by category. Error bars represent mean. Statistical analyses used one-way-ANOVA with Dunn’s multiple comparisons test. *, p < 0.05. G Schematic of GM2A removing GM2 from the membrane for hydrolysis mediated by HEXA/HEXB. All 3 proteins were in the leading edge of the Diseases of metabolism term. H Scatter plot of GM2A across TBS-brain extracts by category reveal higher levels in AD. Error bars represent mean  ± SEM. Statistical analyses used one-way-ANOVA with Dunn’s multiple comparisons test. *, p < 0.05. I GM2A levels are elevated in CSF of AD compared to controls (data from [39]). Statistical analyses used unpaired t-test with Welch correction. *, p < 0.05

Gene set enrichment analysis (GSEA) using the REACTOME database revealed that proteins associated with protection from toxicity shared terms that contained Rho GTPase effectors/activation of WASPs and WAVEs (Fig. 6D). Proteins driving protective associations include components of the WAVE regulatory complex (WRC). The WRC is a heteropentameric complex that interacts with the ARP2/3 complex to regulate actin polymerization. Interactions between the WRC and ARP2/3 complex mediate actin polymerization and branching, impacting neurite morphogenesis, plasticity, endocytosis, and trafficking. Leading edge proteins driving the GSEA enrichment include multiple proteins that are core components of both complexes (Fig. 6E, leading edge gene highlighted in red), and 4 of the top 18 shared associations between the NI and MEA assays were included under this category (ABI1, ABI2, BRK1, and ARPC1A). Intriguingly, TBS-soluble protein levels of both ABI1 and ABI2 were reduced in AD brain (Fig. 6F). ABI1 and ABI2 are not predicted to act extracellularly under physiological circumstances, but rather to be functioning inside cells. Thus, it is unclear here if the extracellular additions of these specific proteins present in brain extracts are driving a protective influence on neurons, or if the observed associations are simply biomarkers of the presence of other factors present in the brain that are mediating the effects. Future studies will further probe the meaning of these interesting associations.

Proteins associated with neurotoxic effects in both assays shared the enrichment term “Diseases of metabolism” (Fig. 6D). Intriguingly, many of the leading edge proteins driving this association have previously been implicated in neurodegenerative lysosomal glycosphingolipid storage disorders including GM2A, HEXA, HEXB, ASAH1, and GAA. Loss-of-function mutations in GM2A, HEXA and HEXB cause GM2 gangliosidosis, such as Tay-Sachs disease, B1 variant, Sandhoff disease, and GM2A deficiency (AB variant) (Fig. 6G, reviewed in [8, 45,46,47]. Of these proteins, the strongest association shared across the MEA and NI assays was with GM2A (Fig. 6C). GM2A can be found both in the cytoplasm and in the extracellular space, and its levels are elevated in TBS-soluble AD brain extracts (Fig. 6H) and in AD CSF (Fig. 6I, original CSF data from [48]), suggesting that extracellular GM2A may be physiologically relevant [8, 45,46,47].

GM2A is sufficient to reduce mean firing rate and neurite integrity of human neurons

GM2A arose as a top candidate factor for exerting a causative influence on effect size in the MEA and NI assays, due to its association with neurotoxicity in both assays, its previously described role in neurodegenerative diseases, and its presence in the extracellular space in vivo. In order to test whether high levels of extracellular GM2A impart neurotoxic effects, we first treated human iNs with recombinant GM2A in the NI assay. Based on western blot quantification of GM2A in the human brain, we estimate that GM2A levels range from 0.5–1 ng/uL in this brain extract collection (Supplemental Fig. 2A). However, treatment with 2 or 5 ng/uL of recombinant GM2A had no effect in the NI assay (Supplemental Fig. 2B). GM2A has been shown to be post-translationally modified in multiple ways in mammalian cells [49], so we next tested human cell-derived GM2A in this ass

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