Neuronal endolysosomal alterations induced by Apolipoprotein E4 emerge over time in primary neurons[image]

Emma Nyberg; Sabine C Konings; Nils Lindblom; Bodil Israelsson; Oxana Klementieva; Isak Martinsson; Gunnar K Gouras

doi:10.1016/j.jbc.2025.110479

. 2025 Jul 15;301(8):110479. doi: 10.1016/j.jbc.2025.110479

Neuronal endolysosomal alterations induced by Apolipoprotein E4 emerge over time in primary neurons

Emma Nyberg ^1,^∗, Sabine C Konings ^2,³, Nils Lindblom ¹, Bodil Israelsson ¹, Oxana Klementieva ², Isak Martinsson ^1,⁴, Gunnar K Gouras ^1,^∗

PMCID: PMC12357307 PMID: 40675218

Abstract

Apolipoprotein E4 (ApoE4), the major genetic risk factor for Alzheimer’s disease (AD), is vital for understanding cellular processes involved in AD pathogenesis. Evidence implicates endosomes as a central player in AD, where endosomal enlargement in neurons is among the earliest changes in AD. This enlargement was reported to be enhanced in APOE4 carriers. Cells internalize ApoE into endosomes for lipid delivery, and previous studies indicate that ApoE4 influences endosomes. However, the effect of ApoE4 on endosome function seems different depending on cell type, and our understanding of how ApoE4 influences endosomes in mature neurons, the cell type degenerating in AD, remains limited. We aimed to increase understanding of the impact ApoE4 has on endosomal dynamics in primary neurons and whether external triggers, such as time-in-culture/aging, synaptic activity, and cholesterol, influence these endosomal changes. We show that without external triggers, mature primary neurons from ApoE knockout (KO), ApoE3, and ApoE4 mice show no major differences in endosomal appearance and function and adapt similarly to increased synaptic activity. However, with prolonged time in culture, neurons with ApoE4 show reduced degradative ability, along with a decreased number of active lysosomal compartments. Moreover, when supplying aged cultures with cholesterol, ApoE4 neurons have a predisposition to accumulate cholesterol in the endolysosomal system. Taken together, we show that ApoE4 impacts endolysosome function in primary neurons, but that changes emerge only after prolonged time in culture. A better understanding of how ApoE4 impacts neurons could provide important insights into ApoE4-directed therapy for AD.

Keywords: Apolipoprotein E (ApoE), neuron, endosome, lysosome, cholesterol

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease, with the number of affected individuals escalating in our aging society. Scientific advances in biomarkers have revealed that the disease process of AD initiates decades before the onset of clinical symptoms (1, 2). Nevertheless, our comprehension of the mechanisms during the early cellular phase of AD remains limited.

As ApoE4 is the predominant genetic risk factor for AD, a better understanding of its fundamental cellular pathways implicated in the development of AD is important to understand how AD develops and progresses. In humans, ApoE exists in three primary isoforms: ApoE2, ApoE3, and ApoE4. While ApoE3, the most common variant, is considered neutral in AD, ApoE4 is highly prevalent among the population with AD (40–60%), although prevalence rates vary among different ethnic groups (3, 4). Conversely, the rarer ApoE2 isoform offers protection against AD (3, 5). ApoE functions as the major lipid carrier in the brain, supplying phospholipids and cholesterol to various cells in the brain, including neurons. While astrocytes are the predominant producer of ApoE, other cell types, such as microglia and neurons, can express ApoE under certain stress-induced circumstances (6, 7). Previous research has underscored ApoE’s significance in diverse cellular pathways, including metabolism, endocytosis, synaptic activity, and inflammation (8). While harboring ApoE4 has been shown to alter several of these cellular processes (8), our understanding remains incomplete, particularly regarding how the cellular pathways affected by ApoE4 might be connected to each other, how ApoE4 affects different cell types of the brain, and more importantly, how ApoE4 raises the risk for AD. Multiple lines of evidence have shown that ApoE4 detrimentally affects amyloid-β (Aβ) metabolism and plaque formation (9), important drivers in AD pathogenesis. Cumulative research suggests that ApoE4 influences AD pathology at very early stages before plaques appear (10, 11). Thus, turning off ApoE4 expression in AD transgenic mice prior to plaque formation prevents amyloid deposition in their brains (10, 11).

Endosomal enlargement (12) as well as synaptic impairments (13) represent two of the earliest detected changes in AD. The endosomal system is a fine-tuned machinery that regulates a multitude of cellular pathways, including synaptic activity. Furthermore, endosomal compartments serve as sites where amyloid precursor protein (APP) is processed to Aβ, with Aβ beginning to accumulate prominently in a subtype of endosomes called multivesicular bodies (MVBs) during early stages of AD, preceding plaque formation (14). Notably, endosomes are cellular hubs, where many important players in both AD and normal neuronal function intersect, including ApoE and Aβ (15) and their interaction can influence the intracellular accumulation of Aβ (16). Before plaque formation enlarged endosomes were detected in both cases with sporadic AD and Trisomy-21 (Down syndrome; DS), who harbor three copies of the APP gene and thus have higher risk of developing AD (12). Moreover, APOE4 carriers exhibited an enhanced and accelerated endosomal phenotype (12). Another early feature of AD and DS is intraneuronal accumulation of Aβ₄₂ in AD vulnerable neurons (17, 18, 19, 20), which preferentially occurs in endosomes-lysosomes (14, 21). It is therefore probable that AD pathogenesis initiates with endosomal alterations, making them a potential therapeutic target. Nevertheless, the knowledge of the initial triggers for Aβ accumulation and potentially aggregation in endosomes remains limited.

The question of whether the enhanced endosomal enlargement observed with ApoE4 is driven by Aβ and/or if ApoE4 itself initially alters endosomal function and size to then influence Aβ remains unclear. Previous studies provide robust evidence that Aβ induces endosomal impairments in various cell types. Brains with AD pathology exhibit dystrophic neurites filled with endosomal compartments, including autophagosomes, lysosomes, and MVBs (22). Additionally, primary neurons from AD transgenic mice and neurons treated with Aβ develop enlarged MVBs and impaired endolysosomal function (23, 24). Moreover, mouse AD transgenic neurons display Aβ-dependent deficiencies in functional MVB sorting (25). Astrocytes also struggle to handle Aβ; when exposed to Aβ protofibrils, astrocytes readily take up Aβ, but degrade it ineffectively, resulting in enlarged endosomes (26).

ApoE loaded with lipids uses the endosomal route to deliver various lipid species to cells, including neurons. The endosome system is responsible for dissociating and distributing lipids within the receiving cell (27). Failure in lipid trafficking, for example, in Niemann Pick disease, causes endolysosomal impairments together with AD-like pathological phenotypes (28). It is therefore a possibility that ApoE acts through a similar mechanism in AD, causing endosomal deficits that later induce AD with age.

Targeted-replacement mice, in which the mouse APOE coding region has been replaced by the human APOE gene-without the presence of aggregation-prone human Aβ-suggest that ApoE4 influences the endosomal system independently of human Aβ pathology. These studies have reported that when ApoE4 mice reach 12 months, they show reduced levels of exosomes in the brain (29), an alternative endocytic pathway for clearing intracellular cargo (30). When ApoE4 mice reached 14 to 15 months, they showed elevated expression of numerous genes involved in endosomal function in an AD-vulnerable brain region (entorhinal cortex), with no changes in an AD-resistant region (primary visual cortex) (31). Changes in early endosome and cathepsin D morphology in AD vulnerable regions have been shown to appear in neurons in 18-month-old ApoE4 mice (31). These studies suggest that ApoE, independent of elevated Aβ and/or Aβ pathology, can influence the endosomal system but requires aging and extended time to manifest morphologically.

Both the molecular effect of ApoE4 and ApoE intracellular trafficking have been reported to differ between different cell types (15, 32). Cellular models have been used to study the endosomal effects of ApoE4 and differences between cell types. ApoE4 expressing astrocytes have been reported to have reduced levels of endosomes, specifically early endosomes, together with reduced acute uptake of extracellular cargo (33). It has further been shown that ApoE4 expressing astrocytes show cargo specific uptake/trafficking alterations (34) as well as reduced autophagy induction (35). Both alkalinization (36) and acidification (37) of lysosomes have been reported in ApoE4 compared to ApoE3 astrocytes as well as altered pH depending on subtype of endosomes/lysosome (34). However, opposite to the reduced number of early endosomes in astrocytes (33), ApoE4 expressing yeast show an elevated number of early endosomes (33). It has been reported that ApoE4 expressing microglia show an elevated number of early and late endosomes as well as an increased number of lysosomes (38) which has also been reported in HEK293 cells (39). An increase in early endosome area has further been reported in ApoE4 human induced pluripotent stem cell (IPSC)-derived neurons, alongside an elevated number of synapses, increased synaptic activity, and elevated Aβ₄₂ secretion (32). Additionally, in primary neuron cultures several research groups have shown that ApoE4 appears to exert an effect on neuronal endosome recycling and affect numerous receptors and cellular functions including long-term potentiation (LTP) and lipid metabolism (40, 41, 42). In addition to the reported ApoE4 effects on early endosomes and recycling, studies have additionally reported increased calcium levels in neuronal lysosomes with ApoE4 (43).

The above findings underscore the disruptive influence ApoE4 can have on endosomal machinery but also highlight the complexity of the effects of ApoE4 on endosomal function in different cell types. The endosomal system is highly dynamic, and our understanding of how ApoE4 precisely alters this intricate system remains incomplete, especially in mature and functional neurons. Targeting endosomes therapeutically has the potential to modify AD progression but requires an in-depth understanding of these ApoE4 changes and what drives them in different experimental cellular models. In our study, our objective was to delve into how the ApoE4 isoform impacts the endolysosomal system in primary mouse neurons harboring different human ApoE variants (ApoE3, ApoE4, ApoE KO), allowing us to study the effect of ApoE isoforms without the influence of human Aβ, which can impact endosome function (23). Our investigations sought to provide deeper insights into temporal and contextual factors, including neuronal activity and elevated cholesterol, by which ApoE4 can induce endosomal alterations.

Results

Mature ApoE4 primary neurons exhibit only minor alterations in late endolysosome appearance in dendrites

Previous studies have demonstrated that ApoE4 appears to influence the endosomal system, leading to an increased number and enlargement of early endosomes in the cell soma of neurons in 18-month-old mice (31). Notably, Nuriel and colleagues identified changes in mRNA levels of endosomal markers in brains already in 14 to 15-month-old ApoE4 targeted-replacement mice (31), and Peng et al. reported changes in exosome release in 12-month-old ApoE4 mice (29). We hypothesized that there might be early changes in the endosomal machinery in ApoE4 primary neurons. To study neuronal mechanisms induced by ApoE4, we utilized rodent primary neurons, a model that is widely used by numerous research groups in diverse areas of neuroscience to study underlying cellular mechanisms and intrinsic neuronal function in health and disease (44, 45, 46).

During time in culture, primary neurons start forming processes and create synaptic connections with each other. Initially, we studied potential ApoE-dependent cellular changes in the endolysosomal machinery in what are typically regarded as mature neurons. Therefore, primary hippocampal/cortical mixed brain cultures were isolated from human ApoE4 and ApoE3 targeted-replacement mouse embryos as well as ApoE KO mouse embryos and maintained in vitro for 18 days (18 DIV) (Fig. 1A). At 18 DIV, the neurons have extended their processes, established extensive neuronal networks, and are considered mature (44, 45). Immunostaining with various cellular markers (MAP2 = neurons, s100β = astrocytes, CD140a = oligodendrocyte progenitor cells) shows that our ApoE primary cultures are comprised not only of neurons but also contain astrocytes and oligodendrocyte progenitor cells (Fig. S1A), as previously described (15). Hence, our cultured human ApoE knock-in neurons are exposed to both secreted astrocytic and neuronal human ApoE, given that primary neurons are under more stress (45). At 18 DIV, there was no difference in cellular stress between the different ApoE groups, as assessed by the lactate dehydrogenase (LDH) assay (Fig. S1C). Moreover, levels of ApoE in cell lysates were similar between ApoE3 and ApoE4 cultures (Fig. S1D). We further evaluated mitochondria between the ApoE neurons and could not detect major differences in levels of either Tomm40 or Cytochrome C in cell bodies or dendrites (Fig. S1, C–L). When evaluating the levels of mitochondria complexes in the lysates, there were no significant changes in the levels of mitochondria Complex V, III, or II, but ApoE4 showed significantly reduced levels of Complex I compared to ApoE KO cultures (Fig. S1, J–N). A previous study from our group had shown that at this maturation stage (19 DIV), ApoE KO, ApoE3, and ApoE4 primary neurons have similar dendritic arborization (47). The same study showed that ApoE4 primary neurons had reduced dendritic density of vGlut1 and vGAT as well as altered neuronal activity (47). In the present study, we further showed that ApoE4 primary cultures have significantly higher levels of the presynaptic marker synaptophysin compared to ApoE3 (Fig. S1, O and P), and a trend toward higher levels of synapsin I in ApoE4 cultures (Fig. S1, O and Q). Together with the prior study (47), this highlights synaptic differences between the ApoE cultures at 18 DIV.

**Mature ApoE4 primary neurons exhibit only minor alterations in late endolysosome appearance in dendrites.**A, schematic overview of study design, where brain cells from cortex and hippocampus are isolated from mouse embryos (ApoE KO, ApoE3, and ApoE4) and cultured in-vitro for 18 days (18 DIV). B, representative epifluorescence images of early endosomes (EEA1 antibody) together with MAP2 at neuronal cell bodies (scale bar: 10 μm) and dendrites (scale bar: 20 μm). *C-D*, Quantification of number of early endosomes (EEA1) in neuronal cell bodies (C) and along 100 μm of dendrites (D). 6 to 10 fields of view per N, N (embryo) = 3, one-way ANOVA (C): p = 0.572, one-way ANOVA (D): p = 0.862. Bar graph presented as mean ± SD, C: KO = 123 ± 10, E3 = 120 ± 28, E4 = 106 ± 18, D: KO = 162 ± 20, E3 = 148 ± 43, E4 = 153 ± 28. *E-F*, quantification of average size of early endosomes (EEA1) in neuronal cell bodies (E) and along 100 μm dendrite dendrites (F). 6 to 10 fields of view per N, N (embryo) = 3, one-way ANOVA (E): p = 0.836, Kruskal-Wallis (F): p = 0.289. Bar graph presented as mean ± SD, E: KO = 0.187 ± 0.028, E3 = 0.182 ± 0.018, E4 = 0.192 ± 0.012, F: KO = 0.103 ± 0.010, E3 = 0.097 ± 0.002, E4 = 0.107 ± 0.013. G, representative epifluorescence images showing LAMP1 together with MAP2 in the cell body and dendrites of 18 DIV ApoE KO, ApoE3, and ApoE4 neurons. Scale bar: 20 μm. *H-I,* quantification of number of late endosomes/lysosomes (LAMP1+ vesicles) in neuronal cell bodies (H) and along 100 μm of dendrites (I). 6 to 10 fields of view per N, N (embryo) = 3, one-way ANOVA (H): p = 0.98, one-way ANOVA (I): p = 0.044, *post hoc* Tukey’s E3 *versus* E4 p = 0.039 (15.1% mean difference). Bar graph presented as mean ± SD, H: KO = 40 ± 12, E3 = 40 ± 9, E4 = 41 ± 5, I: KO = 153 ± 5.0, E3 = 138 ± 2, E4 161 ± 14. *J-K*, quantification of average size of LAMP1+ vesicles in neuronal cell bodies (J) and along 100 μm of dendrites (K). 6 to 10 fields of view per N, N (embryo) = 3, one-way ANOVA (J): p = 0.115, one-way ANOVA (K): p = 0.823. Bar graph presented as mean ± SD, J: KO = 0.705 ± 0.088, E3 = 0.832 ± 0.046, E4 = 0.791 ± 0.043, K: KO = 0.246 ± 0.004, E3 = 0.258 ± 0.027, E4 = 0.257 ± 0.036.

To explore endolysosomal changes, we examined the appearance of early endosomes and late endosomes/lysosomes by immunolabeling the primary cultures with antibodies against early endosomal antigen 1 (EEA1) and lysosome associated membrane protein 1 (LAMP1, which detects late endosomes/lysosomes) together with microtubule-associated protein 2 (MAP2) to mark dendrites. Neurons are large and polar cells, and their dendritic system consists of several regions. Endosomes mature along the dendrite as they traffic retrogradely, resulting in mature lysosomes in the cell soma (48). Due to this gradient, we analyzed endosomes in the cell bodies and primary dendrites (Fig. S1B). We did not detect any differences in either the size or number of early endosomes between the ApoE groups (EKO, E3, and E4) (Fig. 1, B–F). However, we did detect a significantly higher number of LAMP1+ vesicles in ApoE4 compared to ApoE3 neuronal dendrites, although there were no detectable differences in LAMP1 average vesicle size nor differences in number of LAMP1+ vesicles in the neuronal cell bodies between the ApoE groups (Fig. 1, G–K).

We next wondered whether there might be disparities between the ApoE isoforms in protein levels of these endosomal markers: EEA1 (early endosomes), LAMP1 (late endosomes/lysosomes), Rab7 (late endosomes), and LC3β (autophagosomes) (Fig. S1, R–V). However, we could not detect any significant differences in protein levels of endosomal markers between the different ApoE neuron cultures.

Mature neurons traffic and degrade endocytosed cargo equally between ApoE isoforms and adapt similar to increased neuronal activity

While endosomal morphology can offer insights into the state of the endosomal system, it falls short in assessing functionality. Despite no major differences in the appearance of endosomes between the ApoE groups, we wondered whether there could be potential differences in endosomal cargo trafficking. For this purpose, we evaluated how the neurons trafficked EGF-rhodamine. EGF-rhodamine follows a well-defined traffic route (Fig. 2A), being internalized upon binding to its receptor and transported to early endosomes before eventual degradation in lysosomes (25, 49). The primary cultures were incubated with 0.5 μg/ml EGF-rhodamine for 4 h at 18 DIV. The different ApoE neurons (EKO, E3, and E4) showed no variations in how much EGF localized to the cell body after 4 h (Fig. 2, B and C), suggesting similar trafficking of EGF to the cell body at 18 DIV.

**Mature neurons traffic and degrade endocytosed cargo equally between ApoE isoforms.**A, Illustration showing the trafficking pathway of EGF and EGFR. EGF binds to the receptor and is internalized into endosomes and subsequently is trafficked through early endosomes to late endosomes with final destination in lysosomes. B. Representative epifluorescence images of EGF-rhodamine (4 h, 0.5 μM) in 18 DIV old neuronal cell bodies. Scale bar: 10 μm. C. Quantification of EGF-rhodamine area in neuronal cell bodies at 18 DIV. The values were normalized to the total sum of all three groups for each independent experiment. 6 to 10 fields of view per N, N (embryo) = 4, Kruskal-Wallis: p = 0.235. Bar graph presented as mean ± SD, KO = 0.308 ± 0.036, E3 = 0.351 ± 0.028, E4 = 0.341 ± 0.025. D, illustration showing the property of DQ BSA, which is non-fluorescent until proteolytic cleavage. DQ BSA is internalized by the cells, and when reaching the lysosomes and become degraded starts to emit fluorescent light. Thus, increased fluorescent signals are equivalent to increased degradation. E, representative epifluorescence image showing DQ BSA in LAMP1+ compartments in a neuronal cell body. Scale bar: 10 μm. F, representative epifluorescence images of 18 DIV neurons treated 4 h (*left panel*) or 8 h (*right panel*) with DQ BSA. Scale bar: 20 μm. *G-H*, quantification of DQ BSA (8 h) integrated density in the neuronal cell bodies (G) and dendrites (H) of 18 DIV old neurons, normalized to the background. The values were normalized to the total sum of all three groups for each independent experiment. 6 to 10 fields of view per N, N (embryo) = 4, one-way ANOVA (G): p = 0.582, Kruskal-Wallis (H) p = 0.219. Bar graph presented as mean ± SD, G: KO = 0.316 ± 0.033, E3 = 0.333 ± 0.057, E4 = 0.351 ± 0.048, H: KO = 0.292 ± 0.064, E3 = 0.326 ± 0.109, E4 = 0.382 ± 0.059.

We next wanted to assess whether the different ApoE neurons display differences in degradative capacity. Therefore, we used DQ BSA, which remains quenched until proteolytic cleavage, yielding a fluorescent signal upon degradation by lysosomal proteases, thereby serving as a tool to evaluate degradative capacity (Fig. 2D) (50, 51). Using neuroblastoma (N2A) cells, we first established that pre-treatment with 5 nM bafilomycin A1 overnight, thus blocking lysosomal function, prevented fluorescent signal from DQ BSA (Fig. S1W). When the primary cultures were treated with 25 μg/ml DQ BSA, we could detect a fluorescent signal in neuronal cell bodies, but also to a lower extent in dendrites. After 4 h of treatment, some DQ BSA had already reached the cell body and started to be degraded. However, after 8 h, the fluorescent signal from DQ BSA in the cell body was visually much higher (Fig. 2F), and we therefore continued evaluating the degradation of DQ BSA after 8 h. As expected, DQ BSA was present within LAMP1+ compartments (Fig. 2E), showing that DQ BSA trafficked to lysosomes and started to be degraded. When we compared our ApoE groups at 18 DIV there were no differences in the total intensities of DQ BSA in dendrites or cell bodies between ApoE KO, ApoE3, and ApoE4 neurons after 8 h (Fig. 2, G and H). Together, our findings suggest that at a mature age (18 DIV), ApoE4 does not seem to have a profound effect on the endolysosomal system of neurons, only showing a modest difference in the number of LAMP1 compartments in dendrites as noted above.

Compared to neurons in a dish, neurons within the brain encounter a multitude of stimuli and external factors, forming an environment that is constantly changing. We hypothesized that certain external factors could, in synergy with ApoE4, trigger effects on the endosomal machinery. Regions vulnerable to AD pathology have high synaptic activity and high metabolic demand (52, 53). In addition, endosomes and neuronal activity are tightly linked, and altering synaptic activity in primary neuronal cultures modulates protein turnover and endosome trafficking (51, 54, 55, 56). Therefore, we asked if a prolonged increase in neuronal excitability could impact the endolysosomal system differently between the ApoE groups (ApoE KO, E3, E4). At 18 DIV, we treated cultures for 48 h with 20 μM bicuculline, which blocks GABAergic input and thereby increases neuronal excitability (Fig. S2A). To assess whether the cultures indeed increased their excitability and to determine whether the different ApoE groups had a different activity response, we measured calcium fluctuations using Fluo-4 after both acute (0.5–2 h) and 48 h of bicuculline. We note that relative to baseline controls for neurons from each embryo, both the frequency (Fig. S2B) and the number of active neurons (Fig. S2C) increased across all ApoE groups following acute and 48 h of bicuculline treatment, with no significant difference observed among the ApoE groups. Consistent with chronic elevation in activity, cell lysate analysis 48 h after bicuculline treatment revealed markedly elevated levels of Fos-B, a marker for chronic neuronal activity, in the treated groups compared to their untreated controls (Fig. S2D). Furthermore, we studied whether the endosomal system changed from its non-stimulated controls after the prolonged synaptic activity, and whether it changed differently depending on ApoE genotype. However, we did not detect an ApoE-dependent alteration in either endosomal morphology (Fig. S2, E–I), EGF trafficking (Fig. S2J), or DQ-BSA degradation (Fig. S2, K and L) after prolonged neuronal activity. This suggests that using neuronal activity as an external trigger did not drive ApoE-dependent changes in the endosomal machinery.

Early endosomal enlargement is not evident in ApoE4 neurons with a more prolonged time in culture

As reported in vivo, ApoE4-dependent early endosome morphological changes developed with aging (18 months), while before that, the endosomal appearance did not differ between ApoE3 and ApoE4 mice (31). This led us to consider that time-in-culture might be a crucial factor for developing endosomal problems with ApoE4. It is well-established that primary neurons undergo an accelerated maturation process, manifesting age-like stress over time in culture (45, 46, 50). Moreover, recent work utilizing primary brain cultures has unveiled deficits in the endolysosomal pathway in aging wild-type mouse neurons (45, 46, 50, 57). We hypothesized that these time-dependent changes in the cultures might be required to trigger an endosomal change with ApoE4. Therefore, we allowed the cultures to age until 25 to 26 DIV (Fig. 3A). The LDH assay revealed significantly heightened stress levels in ApoE KO cultures compared to ApoE3 (Fig. S3A). There was no difference in ApoE levels in cell lysates between ApoE3 and ApoE4 neurons (Fig. S3B). However, ApoE KO neurons showed trends for lower levels of Tomm40 and cytochrome C compared to ApoE3 (Fig. S3, C–G), together with lower levels of mitochondrial complexes with significantly lower levels of Complex II in ApoE KO compared to ApoE3 lysates (Fig. S3, H–L). At 25 to 26 DIV, ApoE4 cultures continued to show a notable trend toward reduced levels of Complex I, but at this time point, so did ApoE KO cultures, and ApoE3 cultures showed the highest levels of Complex I, although these did not reach significance. Together, this suggests potential differences in mitochondrial function between the ApoE cultures. However, unlike 18 DIV, no differences in the levels of synaptophysin and synapsin I could be detected between the cultures at 25 DIV (Fig. S3, M–O).

**Early endosomal enlargement is not evident in ApoE4 neurons with more prolonged time in culture.**A, schematic overview of experimental design. Primary brain cultures were incubated in-vitro for 25 to 26 days (25–26 DIV). *B-C*, representative epifluorescence images demonstrating early endosomes (EEA1) with MAP2 in cell body (B, scale bar: 10 μm) and dendrites (C, scale bar: 20 μm). *D-F*, quantification of number of early endosomes (EEA1) in neuronal cell bodies (D) and along 100 μm of dendrites (F). 6 to 10 fields of view per N, N (embryo) = 4 Kruskal-Wallis (D): p = 0.065, one-way ANOVA (F): p = 0.503. Bar presented as mean ± SD, D: KO = 113 ± 10, E3 = 92 ± 11, E4 = 92 ± 10, F: KO = 169 ± 38, E3 = 141 ± 18, E4 = 164 ± 43. *E-G*, quantification of average size of early endosomes (EEA1) in neuronal cell bodies (E) and along 100 μm of dendrites (G). 6 to 10 fields of view per N, N (embryo) = 4, one-way ANOVA (E): p = 0.472, Kruskal-Wallis (G): p = 0.289. Bar graph presented as mean ± SD, E: KO = 0.205 ± 0.015, E3 = 0.184 ± 0.03, E4 = 0.191 ± 0.023, G: KO = 0.119 ± 0.010, E3 = 0.122 ± 0.014, E4 = 0.120 ± 0.011.

Examining EEA1+ vesicles, we still did not detect major differences in the appearance of early endosomes between the ApoE groups, in either cell bodies or dendrites (Fig. 3, B–G), suggesting changes in the early endosomal morphology that has been observed in vivo does not develop in our primary culture model even by 25 to 26 DIV. We next explored whether protein expression of endosomal markers (EEA1, Rab7, LAMP1, and LC3 β) had changed between the ApoE groups at 25 DIV. However, Western blot analysis revealed no significant differences in levels of these proteins among the ApoE groups (Fig. S3, P–T). However, we noted that in aged 25 DIV cultures, the ratio of LC3β I to II was lower than at 18 DIV (Fig. S3T), suggesting elevation and/or accumulation of LC3β II with time in culture. In fact, accumulation of LC3β II has been previously shown to increase in aged primary neurons (46).

With time in culture, ApoE4 neurons develop reduced degradative capacity in association with a lower number of active degradative compartments

Although we did not detect significant differences in LAMP1+ vesicle number and size (Fig. 4, A–E) or trafficking of EGF to the cell body (Fig. S3W) between the ApoE isoforms at 25 to 26 DIV, remarkably the degradation of DQ BSA after 8 h was reduced with ApoE4, consistent with a significant reduction in DQ BSA integrated intensity in the cell bodies compared to ApoE3 (Fig. 4, F and G, 21.9%, p = 0.0495). There was also a trend toward reduced DQ BSA integrated intensity in ApoE KO neurons compared to ApoE3 (Fig. 4, F and G). We further noticed that the number of vesicles containing DQ BSA in the cell bodies was significantly lower both in ApoE4 (20%, p = 0.0195) and ApoE KO (17.8%, p = 0.0048) compared to ApoE3 neurons (Fig. 4H). There was, however, no difference in mean puncta DQ BSA intensity in the neuronal cell bodies (Fig. S3U) nor a difference in DQ BSA intensity in dendrites (Fig. S3V) between the ApoE groups. Despite no apparent difference in the number and size of LAMP1+ late endosomes/lysosomes, our findings are consistent with the scenario that aged ApoE4 neurons (25–26 DIV) show lower degradative capacity that could reflect a lower number of active degradative compartments.

To further evaluate lysosomal function, we next used the pH-sensitive dye LysoTracker to evaluate the degradative compartments with low pH. As LysoTracker is most optimal to evaluate using live cells, we seeded primary cultures on 96-well plates with an optical surface. At 15 DIV, we transfected the cultures with lenti-viral vectors containing a plasmid with the synapsin I-promoter and GFP expression to visualize the neurons. At 25 to 26 DIV, the cultures were treated with LysoTracker and Hoechst dye for 10 to 15 min before live-cell image acquisition using a High Content Analysis System (Operetta CLS). Remarkedly, similar to the DQ BSA results, we detected a 19.8% lower number of LysoTracker positive vesicles (p = 0.011) and a 35.6% lower total LysoTracker intensity per neuronal cell body (p = 0.0059) in ApoE4 compared to ApoE3 neurons (Fig. 4, I–K), supporting the scenario that ApoE4 neurons, after longer time in culture, have lower number of active degradative compartments.

After detecting these changes in degradative capacity in ApoE4 neurons, we wondered whether there were any differences in appearance of late endosomes and their protease levels. We immunolabeled 25 to 26 DIV old cultures for CD63, a known late endosomal marker that resides in intraluminal vesicles and an antibody to Cathepsin D, which detects both the inactivated and activated protease. Although non-significant, we could detect a trend toward higher levels of CD63 (Figs. 4L, N and S3X) and Cathepsin D (Figs. 4M, O and S3Y) in the cell body and dendrites of ApoE4 and ApoE KO neurons compared to ApoE3, which might reflect reduced degradation in ApoE4 neurons.

Alterations in astrocytes, ApoE levels, and lipofuscin between 18 and 25 DIV primary cultures

The maturation of primary neuronal cultures has been extensively studied by various groups, and with prolonged culturing in vitro the cellular stress is known to increase. These stress phenotypes with time in culture include lipofuscin accumulation, DNA damage, increased stress signalling, endosome impairment, and increased levels of reactive oxygen species (44, 46, 50, 58). Because we detected ApoE-dependent endosome alterations after a longer time in culture, we wanted to further explore the potential changes that occur in our primary cultures between 18 and 25 DIV. As astrocytes are the main producers of ApoE and provide support to neurons, we wondered whether the astrocyte population changed with time in culture. At 25 DIV, we could detect higher protein levels of GFAP in cell lysates compared to their 18 DIV matched controls, which was significant for ApoE3 and ApoE4 (Fig. 5, A and B). We wondered whether this was caused by astrocytic proliferation and thus a higher number of astrocytes. However, immunofluorescence against astrocyte marker GFAP together with neuronal marker MAP2 showed that the proportion of neurons and astrocytes did not change significantly between 18 and 25 DIV (Fig. 5, C and D). Thus, a change in astrocyte number would not explain the increased GFAP levels. These data are consistent with a scenario where the astrocytes mature and/or become more reactive with longer time in culture.

Due to the increase in GFAP levels with time in culture, we next wondered if this change in astrocyte phenotype might alter the levels of ApoE and secretion. Therefore, we collected conditioned media from the primary cultures at 18 and 25 DIV and evaluated whether ApoE levels had changed with time in culture. There was, however, not a significant change in ApoE levels in the media between 18 and 25 DIV, although there was a trend toward higher ApoE levels at 25 DIV, especially for ApoE4 cultures (Fig. 5, E and F). Notably, we did detect a lower ApoE molecular band, slightly above 28 kDa, that was more pronounced, although not significantly, at 25 DIV compared to 18 DIV for both ApoE3 and ApoE4 cultures (Fig. 5, E and G). A similar-sized fragment of ApoE has previously been reported (59).

Lipofuscin accumulation is a well-established fate of postmitotic cells as they age and is composed of various compounds, including in particular oxidized lipids and proteins that cannot be degraded and thus aggregate in lysosomes (60, 61). Previous research has shown increased lipofuscin accumulation in primary neurons as they age (44, 46, 58), and furthermore, lipofuscin formation was recently reported to be ApoE-dependent (62). Therefore, we next sought to study whether lipofuscin accumulation changed in our cultures between 18 and 25 DIV. We utilized the green filter channel to detect autofluorescent granules as a means to assess lipofuscin accumulation. Using immunolabeling for MAP2 and GFAP to distinguish between neurons and astrocytes, respectively, we could detect autofluorescent granules in both cell types, the latter probably caused by limited astrocyte division in our medium optimized for neurons (Fig. 5, H and I). Although there were considerable variations between independent experiments, and therefore these results were nonsignificant, we observed that the area of autofluorescent granules increased at 25 DIV compared to 18 DIV in ApoE3 and ApoE4 neurons (Fig. 5J) and astrocytes (Fig. 5K). Interestingly, the same trend was not observed for the ApoE KO neurons or astrocytes. These results led us to consider whether there was a detectable difference in lipofuscin accumulation between the ApoE groups at 25 DIV. When comparing the ApoE groups at 25 DIV, we could detect a trend toward higher levels of lipofuscin in ApoE4 compared to ApoE KO and ApoE3 neurons (Fig. 5L) and significantly higher levels of lipofuscin accumulation in ApoE4 compared to ApoE KO and ApoE3 astrocytes (Fig. 5M).

ApoE isoform-dependent cholesterol alterations in neurons

Extensive research has shown that alterations in lipids, such as in the pediatric neurodegenerative Niemann-Pick disease, can lead to enlarged endosomes and neurodegeneration (28, 63). This highlights the intricate interplay between lipid metabolism and endosome morphology and function. In addition, cholesterol accumulation has been reported in both astrocytes and oligodendrocytes with ApoE4 (37, 64, 65). Therefore, after detecting that with time in culture, ApoE4 neurons develop lysosomal alterations, we were curious if this might alter cholesterol trafficking and hypothesized that certain lipids, such as cholesterol, may influence endosomal dynamics in an ApoE isoform-dependent manner. We therefore at 22 to 23 DIV treated our ApoE primary cultures with cholesterol (12.5 μg/ml), slightly higher levels than found in CSF (66), for 3 days (Fig. 6D). Using Filipin to detect cholesterol, we revealed that without cholesterol supplementation, there was significantly lower Filipin intensity in dendrites of ApoE3 compared to ApoE KO neurons and a trend for lower levels compared to ApoE4. Furthermore, a similar distinct trend of lower membrane Filipin intensity in ApoE3 was detected in neuronal cell bodies (Fig. 6, A–C).

Without cholesterol supplementation, Filipin dye is only seen in cell membranes. However, when supplying the cultures with cholesterol for 3 days, we could detect droplets of accumulated cholesterol that, to a large extent, overlapped with LAMP1 labeling (Figs. 6, E, F and S4B). When evaluating how the cholesterol levels, measured by Filipin intensity, changed after cholesterol supplementation, we could detect a nonsignificant trend for ApoE3 neurons showing an increase in their membrane cholesterol levels, with the most in neuronal cell bodies, while there was no appreciable change in dendrites (Fig. 6, G and H). Interestingly, we detected two different outcomes of cholesterol accumulation in endolysosomes in ApoE4 neurons. Half of the time, similar to ApoE3 neurons, ApoE4 neurons accumulated very low amounts of cholesterol in endolysosomes (LAMP1+). Remarkably, however, when cholesterol did accumulate in ApoE4 neurons, it reached a considerably higher level than either ApoE KO or ApoE3 (Figs. 6, F, I, J and S4A). Thus, when only comparing cultures with detected cholesterol accumulation (cultures with an average of ≥4 Filipin accumulated droplets per neuronal cell body), there was a significant difference between ApoE4 compared to ApoE KO and ApoE3 (Fig. 6J).

We were further curious about whether the endosome system was affected by cholesterol supplementation (Figs. 6, K–M and S4, A–E). Even though cholesterol seemed to have an impact on endosomes, including a lower number of early endosomes (Fig. 6K), larger LAMP1+ vesicles (Fig. 6L) without altering the number (Fig. S4E), and reduced degradation of DQ-BSA after 8 h (Fig. 6M), we detected similar changes between ApoE KO, ApoE3, and ApoE4. Due to the quite striking effect in DQ-BSA degradation after cholesterol supplementation (Fig. 6M), we wondered how the degradation was affected by cholesterol at 18 DIV. Therefore, at 15 DIV, we treated primary cultures with 12.5 μg/ml cholesterol for 3 days. Unlike 25 DIV, the number of EEA1+ vesicles tended to increase with cholesterol treatment compared to untreated E3 controls, but there were no significant differences between the ApoE groups (Fig. S4F). Similar to at 25 DIV, cholesterol supplementation also occasionally reduced the degradation of DQ-BSA, most often in ApoE4 neurons, although this was a nonsignificant difference (Fig. S4G).

Discussion

Understanding the fundamental cellular mechanisms in neurons altered by ApoE4 could be crucial for comprehending why endosome enlargement occurs early in AD and why Aβ initially accumulates within endosomes of neurons and later transitions into plaques in AD. Given that endosome alterations represent one of the earliest phenotypes in AD (12) and that Aβ42 initially accumulates in endosomes prior to amyloid plaques (14), we view AD pathobiology as initiating within endosomes. Given the highly dynamic nature of the endosomal machinery, particularly in neurons, we sought to elucidate whether ApoE isoforms influence endolysosomal dynamics in primary mouse neurons, also in the setting of relevant external triggers related to AD, such as time-in-culture/aging, synaptic activity, and cholesterol. We show that under basal conditions, ApoE3, ApoE4, and ApoE KO primary neurons exhibit comparable endosomal morphologies and functions at 18 DIV, which are considered to represent mature neurons. However, we then demonstrate that with further time in culture, ApoE4 neurons reduced their degradative capacity and lowered their number of active degradative compartments compared to ApoE3. We further show that at this later time point, ApoE4 neurons, but not ApoE3 or ApoE KO neurons, can accumulate large amounts of administered cholesterol in degradative compartments.

The reported impact of ApoE4 on endosome function in the literature ranges from changes to early endosomes, endocytic recycling, exosomes, and lysosomes detected in vivo and in different in vitro cell models (29, 31, 32, 40, 41, 42, 43). Two fundamental questions remain. Firstly, is there a particular order of endosomal compartments that ApoE4 affects, and if so, which is the one that is initially impaired? The second question is what precisely is it that leads ApoE4 to impact the endosomal machinery? We hypothesize that ApoE4 impacts lipids and/or other proteins to disturb endosomes.

Although ApoE4 is established as the major genetic risk factor for AD, its disease penetrance is not complete as is the case for familial AD mutations. Moreover, there are no obvious detrimental phenotypes seen in children and young adult ApoE4 individuals. It is thus reasonable that under normal physiological conditions, ApoE4 neurons may not manifest clearly distinct phenotypes. Analogous to the findings by Nuriel et al. (2017) showing that endosome alterations occurred only in aged mice (31), we observed that mature 18 DIV ApoE3, ApoE4, and ApoE KO neurons show no obvious differences in endosome morphology. Together, this suggests that ApoE might not have a large impact on the endosome system without an additional trigger. Of note, Chen and colleagues reported that ApoE4 has an immediate biological impact on endocytic recycling. After internalization, ApoE4 together with its receptor were not immediately recycled back but experienced a delay and thereby impacted LTP (40). Such a recycling delay induced by ApoE4 might however not be detected as changes in endosome appearance or trafficking of cargo through the endolysosomal system. However, it is possible that such initial problems in recycling might eventually develop into further endosomal dysfunctions. It is important to keep in mind that the different parts of the endosomal system are highly connected to each other and for instance, problems in retromer function, that prevent lysosomal cargo degradation, cause endolysosomal deficits (67).

Curiously, it seems that ApoE4 influences endosomes uniquely depending on cell type. In IPSC-derived astrocytes, expression of ApoE4 resulted in a reduced number of early endosomes, whereas ApoE4-expressing yeast showed an elevated number of early endosomes compared to ApoE3 (33). These findings highlight the complexity of ApoE4 and its effect on the endosomal system, and that ApoE4 might only together with other factors, influence endosomes and thereby causing different effects depending on cellular context and cell types. Nuriel et al. (2017) showed endosomal changes in two AD vulnerable regions (entorhinal cortex and cingulate cortex) that are part of the default network, while there were no changes in more resistant regions (primary visual cortex) (31). The differences in activity and energy demand might be important external influences for age-related endosomal changes with ApoE4. Similarly, Lin et al. (2018) showed higher synaptic activity in ApoE4 neurons alongside early endosomal enlargement (32). However, here we detected that increasing excitability in our culture for 48 h did not result in ApoE-dependent endosomal changes. It is possible that these highly complex differences in activity and energy demand cannot effectively be modeled in culture. Another aspect is, of course, time, and our 48 h of increased neuronal activity might be insufficient to trigger ApoE4-dependent early endosomal alterations.

Importantly, we demonstrate in this study that with time-in-culture/aging, ApoE4 neurons have lower degradative capacity, evaluated by DQ BSA and lower acidification of their degradative compartments as shown with LysoTracker, without altering the number of LAMP1 positive vesicles. As fewer numbers of both DQ BSA and LysoTracker positive compartments were detected in ApoE4 neurons, we speculate that ApoE4 neurons have a lower number of lysosomes/autolysosomes that are degradation active. We detected minor changes in late endosomes/lysosomes with ApoE4 already at 18 DIV, so it is possible that there are subtle initial problems early on that later manifest in a more profound way and then affect the functionality of the endolysosome system. Many changes occur in the aged brain, from changes in metabolism, oxidative stress to degradation capacity (61), and some of these effects have also been described in aging neurons in culture (45, 46, 50, 57, 68). Both Burrinha et al. and Guix et al. recently showed that the degradative system becomes dysfunctional in neurons as they age (45, 50), and our results suggest that ApoE4 might enhance such effects. In addition, a recent study showed that IPSC-derived ApoE4 neurons present reduced calcium efflux from their lysosomes (43), supporting our data that ApoE4 disrupts lysosomal function in neurons. Lipofuscin has been shown to accumulate in neurons with time in culture (44, 46, 58), and lipofuscin accumulation is ApoE-dependent (62). Here we have shown that not only neurons in our culture accumulate lipofuscin, but astrocytes as well. Lipofuscin is a sign of senescence (69, 70), and as there is limited cell division of astrocytes in our brain cultures due to the neuronal-growth-promoting media, our model might recapitulate a senescence state. We show that ApoE4 astrocytes accumulate more lipofuscin with time in culture compared to ApoE KO and ApoE3 astrocytes, supporting a previous study (62) suggesting that accumulation of lipofuscin is ApoE4 dependent in different cell types.

Niemann Pick disease is a lysosomal storage disorder that is highly relevant for AD research. Through genetic mutations, Niemann Pick disease develops during childhood and results in failure to transport cholesterol out of endosomes, causing cholesterol accumulation in late endosomes/lysosomes. Intriguingly, certain disease phenotypes in Niemann Pick are comparable to cellular alterations in AD, including Aβ accumulation, neurofibrillary tangles, endosome dysfunction, and neurodegeneration (28, 71). In addition, cholesterol metabolism is known to be important in APP processing and Aβ generation (72, 73, 74). In this study, we show that older ApoE3 neurons present lower levels of membrane cholesterol, which is of interest given that previous research has reported that lowering cholesterol levels with aging is important for neuronal health and survival (58). We further show that ApoE4 primary neurons have the predisposition to accumulate large amounts of supplemented cholesterol in endosomal compartments. As ApoE3 neurons increased their membrane cholesterol the most after cholesterol supplementation, we speculate that ApoE3 neurons incorporate the overflow of cholesterol into the membrane. Cholesterol supplementation can reduce the degradation capacity in all three ApoE groups, both in aged neurons and younger neurons, which shows that cholesterol can affect the endolysosome system, but cannot explain the ApoE4-dependent predisposition to accumulate cholesterol. Interestingly, Guix et al., next to showing that as primary rat neurons age, they develop lysosome deficits, reported that aged neurons also accumulate supplemented cholesterol in their endosome system (45), thus highlighting that cholesterol sequestering can be age-dependent in neurons. Importantly, various studies have reported disrupted cholesterol metabolism with ApoE4 in different cell types and experimental models (64, 65). Our research on neurons, together with previous research, highlights that ApoE4’s impact on cholesterol trafficking and metabolism might be a universal problem for several cell types within the brain.

To put our results in a broader context it is important to acknowledge that rather than existing in isolation, alterations in endosomal pathways in neurons may both influence and be influenced by other cellular functions and mechanisms, including amongst other, mitochondrial function, metabolic state, ER dynamics, and synaptic integrity. Both in-vivo and in-vitro studies have reported that ApoE4-dependent alterations in neurons seem to occur at different stages, where, for example, synaptic differences can be observed already at an early stage (47, 75) and endosomal alterations emerge later (29, 31). This temporal pattern highlights the need to understand not only individual molecular disruptions, but also how they interact and evolve over time.

In conclusion, we show that primary ApoE4 neurons develop time-dependent alterations in their degradative compartments/lysosomes and have a predisposition to accumulate cholesterol and lipofuscin. This work supports the scenario of endolysosomal impairment driven by ApoE4 occurring in the early cellular phase of AD pathogenesis. To target these ApoE4-induced differences for therapy, a better understanding of the precise molecular mechanism(s) altered in endolysosomes in AD and how this might be therapeutically modulated would be important.

Experimental procedures

Animals and ethical considerations

Human ApoE targeted-replacement mice used: ApoE KO (B6.129P2-Apoe<tm1Unc>/J, the Jackson Laboratory) (76), ApoE3 (B6.Cg-Apoeem2(APOE∗)Adiuj/J, the Jackson Laboratory) (77) and ApoE4 (B6(SJL)-Apoetm1.1(APOE∗4)Adiuj/J, the Jackson Laboratory) (77). All animal experiments were approved by the Malmö/Lund Ethical Committee for Animal Research (permit number: 5.8.18-05983/2019 and 5.8.18-13038/2024)

Primary mouse brain cultures and induction of activity

Primary hippocampal/cortical mouse brain cultures were harvested from ApoE3-KI, ApoE4-KI, and ApoE-KO mice as previously described (23). In brief, pregnant mice were sacrificed at embryonic day 15 to 17, and hippocampus and cortex regions were dissected from the embryonic brain and placed in cooled (∼4 °C) Hank’s balanced salt solution (Thermo Fisher Scientific, #SH30588.1). If necessary, a biopsy was taken from the embryos for genotyping. The tissue was dissociated using 0.25% trypsin (Thermo Fisher Scientific, #15090046), washed with Hank’s balanced salt solution, and manually triturated using glass Pasteur pipettes in Dulbecco’s modified Eagle medium (DMEM: Thermo Fisher Scientific, #SH30243.01) supplemented with 10% fetal bovine serum (FBS; Gibco, #10082147) and 1% penicillin/streptomycin. The dissociated brain cells were seeded on poly-D-lysine (Sigma-Aldrich, #P7405) coated coverslips, 6-well plates, 96-lumox multiwell (Sarstedt, 94.6120.096), or PhenoPlate 96-well plates (PerkinElmer, #6055302). One embryo represents one set of cultures. After 4 to 7 h, the media were changed to Neurobasal medium (Gibco, #21103049) supplemented with B27 (Gibco, #17504044), 1% penicillin-streptomycin, and 1.4 mM L-glutamine (Gibco, #25030081). The brain cultures were kept in vitro for 18 or 25 days (18 DIV or 25 DIV). For studies analyzing changes following prolonged neuronal activity, 20 μM bicuculline (Sigma-Aldrich, #14340) was added for 48 h at 18 DIV or 2 h at 20 DIV, and the control cultures were handled in the same way as the bicuculline-treated ones. At the described time points, the cultures were observed with widefield microscopy, and dead/unhealthy cultures were excluded.

Genotyping

For ApoE3-KI mice, which do not always breed homozygously, genotyping was performed on biopsies. For this, the PCRbio Rapid Extract PCR kit (PCRbiosystem, #PB10.24) was used. In brief, DNA was extracted by incubating biopsies with 70 μl distilled H₂O, 20 μl 5× PCRbio buffer A, and 10 μl 10× PCRbio buffer B first 5 min at 75 °C and then 10 min at 95 °C. The samples were cooled, vortexed, and centrifuged (10,000g, 1 min). The DNA-containing supernatant was transferred to new vials and either stored at - 20 °C or used directly. For the PCR, 1 μl DNA supernatant was mixed with 9.5 μl distilled H₂O, 12.5 μl 2× PCRbio rapid PCR mix, 1 μl primer-set (1. AAT TTT TCC CTC CGC AGA CT, 2. ACA GCT GCT CAG GGC TAT TG, 3. AGG AGG TTG AGG TGA GGA TG). The DNA was amplified by temperature cycles (30 s 95 °C, 30 s 60 °C, and 30 s 72 °C). The DNA was visualized using agarose gel electrophoresis and gel red (Biotium, #41003).

Cholesterol treatment

Highly purified cholesterol (EMD Millipore, #228111) was diluted in DMSO (10 mg/ml) and heated to 70 °C with shaking until the cholesterol was dissolved (DMSO was used instead of ethanol, as even low concentration of ethanol killed the primary neurons). Subsequently, a small volume (10 μl) of the cholesterol-DMSO solution was added to fatty acid free BSA solution (∼0.3 g/ml: Thermo Fisher Scientific, #BP9704–100) every 10 min at 37 °C followed by vortexing and repeating until a final concentration of 1 mg/ml was reached, thereby conjugating the cholesterol to the BSA. The stock solution was stored in aliquots (in glass vials) at −20 °C. On the day of treatment cholesterol solution or vehicle control (BSA-DMSO) was heated to 37 °C, vortexed and added to the cultures to a final concentration of 12.5 μg/ml and incubated for 3 days. Thereafter, the cultures were washed with PBS and fixed for 15 min at room temperature (RT) using PBS supplemented with 4% PFA and 4% sucrose, followed by storage at 4 °C until further use. For cholesterol detection, Filipin complex (Merck, #SAE008) was used. As Filipin is sensitive to light and oxygen, the stock solution was aliquoted into small 200 μl tubes (∼100 μl per tube) to prevent a larger volume to be in contact with the air. After aliquoting as well as after each use, the tubes were flushed with nitrogen gas, closed, wrapped in aluminum foil and stored at - 20 °C. For the staining Filipin (1:500) was added in the blocking step (2 h incubation at RT in the dark) of the immunolabelling protocol (see below). To prevent lipid washing away, saponin was only used in the blocking step and at minimal concentrations (0.05%). As Filipin is highly sensitive to light, during image acquisition the coverslips that were not imaged were covered in aluminum foil.

EGF-tetra rhodamine and DQ-BSA treatments

After set time points as described above, EGF-tetra rhodamine (500 nM: Thermo Fisher Scientific, #E3481) was added to the cultures and incubated for 4 h. For studying degradation, DQ BSA (25 μg/ml: Thermo Fisher Scientific, #D12051) was added to the cultures and incubated for 4 or 8 h. Subsequently, the cultures were washed with PBS and fixed for 15 min at RT using PBS supplemented with 4% PFA and 4% sucrose. For testing DQ BSA, neuroblastoma cells (N2a, ATCC CCL-131) were cultured and seeded on glass coverslips, described previously (15). In this round of culture, mycoplasma was not tested in the culture. The next day they were treated overnight with 5 nM Bafilomycin A1 (Sigma Aldrich, #B1793) and subsequently incubated with DQ BSA (25 μg/ml) for 8 h followed by fixation. The plates were protected from light and stored at 4 °C until further immunofluorescence and image acquisition.

LDH assay

To assess the cellular stress in the primary brain cultures a CyQUANT LDH cytotoxicity Assay kit (Invitrogen, C20300 & C20301) was used in accordance with the manufacturer’s protocol. Briefly, at the described time points media from the primary brain cultures were collected, and centrifuged for 10 min at 10,000g (4 °C); supernatants were snap frozen in dry ice and stored at −80 °C until further use. The substrate mix was diluted in 11.4 ml distilled H₂O and combined with 600 μl Assay Buffer Stock Solution to prepare the Reaction Mixture (protected from light). A positive control was prepared by diluting 1.5 μl LDH Positive Control solution with 1 ml of 1% BSA in PBS. 50 μl samples (duplicates); negative neurobasal control (triplicates) and positive control (triplicates) were loaded into a 96-well plate, followed by 50 μl Reaction Mixture. After incubating the plate at RT for 30 min 50 μl Stop Solution was added and the absorbance was measured at 490 nm (true signal) and 680 nm (background).

Western blot and SDS-PAGE

At the described time point, cells cultured on 6-well plates were placed on ice. Media was collected, centrifuged at 10,000g for 10 min and supernatant was collected, frozen in dry ice and stored at −20 °C until use. The plates containing the cells were washed with ice-cold PBS and lysed in 300 μl NP40 buffer containing 20 mM Tris, 150 mM KCl, 5 mM MgCl₂, 1% NP40 supplemented with halt-protease inhibitors (Thermo Fisher Scientific, #78430) and halt-phosphatase inhibitors (Thermo Fisher Scientific, #78428) for 10 min at 4 °C. The lysate was collected, and centrifuged at 20,000g for 20 min and the supernatant was stored at −20 °C. The protein concentrations in the lysates were determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, #23227) in accordance with the manufacturer's protocol and used for loading the same amount of protein for lysates. For media and evaluation of ApoE concentrations, the same media volume was loaded for each sample. Samples (media and lysate) were prepared by mixing the sample, corrected for protein concentration, with NuPAGE reducing agent (Invitrogen, NP0004), NuPAGE LDS sample buffer (NP0007), and distilled H₂O. The samples were heated at 70 °C for 10 min except for detection of mitochondria complexes which was heated at 37 °C for 15 min, centrifuged, and loaded on NuPAGE 4 to 12% Bis-Tris gels (Invitrogen, NP0323BOX & NP0321BOX), and SeeBlue Plus2 (Invitrogen, LC5925) was used as ladder. The proteins were separated in NuPAGE MES SDS running buffer at 150V, and 100 V for mitochondria complexes. The proteins were transferred to an iBLOT2 PVDF Mini Stack membrane (Invitrogen, IB24002) using iBlot2 program P0 (1 min 20 V, 4 min 23 V, 2 min 25 V). Afterwards, the membranes were cut and blocked in PBS 0.05% Tween supplemented with 5% milk or bovine serum albumin. Subsequently, the membranes were incubated in primary antibodies (see Table 1) diluted in PBS-Tween containing 5% milk or bovine serum albumin for 15 to 40 h at 4 °C. After primary antibodies, the membranes were washed with PBS-Tween, incubated with HRP conjugated secondary antibodies (1:2000) and diluted in PBS-Tween supplemented with 5% milk or bovine serum albumin for 1 to 2 h at RT. Thereafter, the membranes were washed, and bands visualized using Clarity Western ECL Substrate (BIORAD, 170-5061) or SuperSignal West Pico PLUS (Thermo Fischer Scientific, #1863097 and #1863096) and imaged using ChemiDoc XRS+ with ImageLab software and band area under the curve was measure using ImageLab software.

Table 1.

List of antibodies and dyes (all company verified)

Antibody	Company	Identifier	Dilution
Rabbit polyclonal anti-early endosomal antigen 1 (EEA1)	Sigma-Aldrich	Cat#E4156	WB 1:1000 IF 1:500
Rat monoclonal anti-Lysosome associated protein 1 (LAMP1)	Abcam	Cat#ab25245	WB 1:1000 IF 1:1000
Chicken polyclonal anti-MAP2	Abcam	Cat#ab92434	IF 1:800
Rabbit polyclonal anti-LC3β	Cell Signaling	Cat#2775	WB 1:1000
Mouse monoclonal anti-Rab7	Abcam	Cat#ab50533	WB 1: 1000
Rabbit monoclonal FosB (5G4)	Cell Signaling	Cat#2251	WB 1:1000
Rat monoclonal anti-CD140a	BD Bioscience	Cat#558774	IF 1:500
Rabbit monoclonal anti-S100β	Abcam	Cat#ab52642	IF 1:500
Goat polyclonal anti-ApoE	Millipore	Cat#AB947	WB 1:500
Mouse monoclonal anti-GFAP	Sigma-Aldrich	Cat#G3893	IF 1:2000-3000
Polyclonal anti-Tomm40	Proteintech	Cat# 18409-1-AP	IF 1:200
Purified mouse anti-Cytochrome C	BD Bioscience	Cat# 556432	IF 1:200
OxPhos Rodent WB Antibody Cocktail	Thermo Fisher Scientific	Cat#45–8099	WB 1:1500
Mouse Monoclonal anti-NDUFB8 antibody (20E9DH10C12)	Abcam	Cat# AB110242	WB 1:1000
Polyclonal Dylight 405-AffiniPure Goat Anti-Chicken IgY (IgG)	Jackson ImmunoResearch	Cat#103-475-155	IF 1:500-1000
Polyclonal Alexa Fluor 488-AffiniPure Goat Anti-Rabbit IgG	Jackson ImmunoResearch	Cat#111-545-144	IF 1:500
Polyclonal Alexa Fluor 568-Cross-Adsorbed Goat Anti-Rat IgG	Life technologies	Cat#A-11077	IF 1:500
Polyclonal Alexa Fluor 647-AffiniPure Goat Anti-Rat 647 IgG	Jackson ImmunoResearch	Cat#112-605-003	IF: 1:500
Polyclonal Alexa Fluor 568-Cross-Adsorbed Goat Anti-Rabbit IgG	Life technologies	Cat#A11011	IF 1:500
Polyclonal HRP-conjugated Goat Anti-Rabbit IgG	R&D systems	Cat#HAF008	WB 1:2000
Polyclonal HRP-conjugated Goat Anti-Mouse IgG	R&D systems	Cat#HAF007	WB 1:2000
Filipin Complex	Sigma-Aldrich	Cat# SAE0088	IF 1:500

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Live-cell calcium imaging and analysis

For live-cell calcium imaging, cells were grown on lumox 96 multiwells. At 18 DIV 20 μM bicuculline was added for 48 h as previously described. For acute changes in neuronal activity, at 20 DIV the cultures were treated with 20 μM bicuculline for 0.5-2 h before image acquisition. Around 0.5 to 1 h before image acquisition, the cultures were incubated at 37 °C with 3 μM of Fluo-4 AM (Thermo Fisher Scientific, #F14201), a calcium dye in the green spectrum. The cells were imaged using a Nikon Eclipse Ti microscope at 10× with 1.4 NA, equipped with an iXon Ultra CCD camera. During imaging the cells were kept in a chamber at 5% CO₂ and 37 °C. Time stacks of 2 min per well were taken, where an image was taken every 100 ms. Regions of interest of neuronal cell bodies were created using StarDist in Fiji Image J. The fluctuations in fluorescent intensities over time were processed and normalized using MatLab PeakCaller (78). The spike detection threshold was set to 10% above baseline. Active cells were determined as neurons with more than one peak per 2 min and frequency was calculated by taking the mean spike frequency of active cells.

Lysotracker and live cell imaging

Primary cultures (∼500 cells) were plated on PhenoPlate 96 well plates and cultured in Neurobasal medium without phenol red (Gibco, #12348017). At 15 DIV, the primary cultures were transfected with lentiviral vectors containing lentiviral compatible plasmid with synapsin I-promoter and GFP expression. At 25 to 26 DIV LysoTracker Red DND-99 (Invitrogen, #L7528) (1:10,000) and Hoechst 33342 (Merck, #B2261) were added for 10 to 15 min, followed live-cell image acquisition (31 frames per well and two wells per N) and image analysis using Operetta Harmony (Perkin Elmer). Cell bodies were detected using Hoechst signal and neurons were selected by neuron-specific expression of GFP. Spot detector (category B) was applied to identify and analyze LysoTracker-positive vesicles.

Immunofluorescent labeling

At set time points the cultures were fixed for 15 min in PBS containing 4% PFA and 4% sucrose and stored at 4 °C until further staining. On the day of staining the cultures were washed with PBS and subsequently blocked and permeabilized in PBS supplemented with 1% BSA, 0.1% Saponin (Sigma-Aldrich, 84510), and 2% normal goat serum (Jackson ImmunoResearch, 005-000-121) for 1 to 2 h at RT. The coverslips containing the cells were incubated overnight at 4 °C on droplets with primary antibodies (see Table 1) diluted in PBS supplemented with 2% normal goat serum and 0.1% saponin. The following day, the coverslips were washed with PBS and incubated with secondary fluorescently tagged antibodies (see Table 1) diluted in PBS containing 2% normal goat serum and 0.1% saponin for 1 to 2 h at RT protected from light. After washing with PBS, the coverslips were mounted on glass slides with ProLong Diamond Antifade Mountant (Invitrogen, P36961).

Epifluorescence microscopy

Microscopy on primary neurons on coverslips was performed using an epifluorescence Olympus IX70 microscope equipped with 405, 488, 568, and 647 nm filters/channels, X-Cite 120Q excitation light source (Excelitas Technologies), a C11440 ORCA-Flash4-oIT digital camera, and a 40X NA 1.3 oil immersion objective or 60X NA 1.4 oil immersion objective, all performed at RT using Type F Immersion liquid (Leica microsystems, #11 513 859). All image acquisition on primary neurons were performed blinded. For imaging primary neurons, MAP2 was used to locate neurons, and the neurons were selected based on morphology (larger cell body and extensive dendritic branching). Of note, MAP2 intensity varies between different neurons, especially in the 405 channel and varies within one culture as well. Therefore, because we only use MAP2 to mark the morphology of the neuron, the representative images do not always have the same image intensity settings.

Image analysis

Image analysis was performed using Fiji image J. The regions of interest; of cell body and primary dendrites were selected using the polygon selection tool in the MAP2 channel. For primary dendrite quantification, one 100-μm-long region of interest originating from the cell body was selected per neuron. For each independent N, 6 to 10 different fields of view were quantified, resulting in the quantification of 6 to 14 different neurons per independent culture. All quantifications were performed using automated macros. Endosomal appearance quantification: for quantification of endosomal appearance the images were pre-processed using background subtraction and thresholding. Image J plugin Analyze particles was used to analyze # and size of particles. Mitochondria Tomm40 and Cytochrome C: cell body and dendrites were selected and images thresholded the integrated density was calculated by combining the selected region of interest with thresholded selection and further normalized to background. For cytochrome C in dendrites, the images were pre-processed using subtract background and thresholding and further analyzed using Image J plugin Analyse particles. EGF tetra-rhodamine uptake quantification: for quantification of uptake and trafficking of fluorescently tagged EGF, the images were pre-processed using background subtraction, smoothing, and thresholding to obtain binary images. Image J plugin Analyze particles was used to quantify the total area of EGF at the cell body. Degradation of DQ-BSA_RED was analyzed by thresholding DQ-BSA signal and subsequently using Analyze particles to quantify number of DQ-BSA + vesicles. A selection of the binary DQ-BSA image was used to analyze signal intensity in the cell body and primary dendrite of the raw image. The mean intensity was measured for the background and integrated density- (mean intensity background ∗ area) was calculated. The cell composition was measured using cell counter. Auto-fluorescent granule accumulation: images from the unlabeled green filter channel were processed through background subtraction, following thresholding. The total area of autofluorescent puncta was then measured using Analyze particle. Filipin intensity quantification: regions of interest for cell body and dendrite were selected and through default thresholding and combining with the selected regions, the mean intensity in the raw image was measured and corrected to background mean intensity. Filipin accumulation quantification: the image was duplicated and filtered by Gaussian blur. Image subtraction was applied using Image calculator, resulting in removal of membrane cholesterol signal and defining the accumulated cholesterol droplets. The image was further thresholded, a LAMP1 mask was created and using Analyze particle the number of accumulated particles within the LAMP1 mask was measured.

Statistical analysis

Graph pad prism 9.5.1 was used for statistical analysis. Statistical analysis was performed on independent experiments (N), where 1 N corresponds to one embryo. Other research groups sometimes use the number of cells as independent experiments to calculate p-value. However, we are more conservative in our study and use one embryo as one independent N. In our image analysis, we take 6 to 8 different fields of view per independent N, and thus an approximate number of 10 neurons per independent N. For each analysis, normality using Shapiro–Wilks test was evaluated (best suited for lower N). If the data passed the normality test, One-way Anova was performed, and if the data did not follow normal distribution, the data were analyzed using Kruskal–Wallis test. In the outcome of significance, a post hoc test with multiple testing correction was performed (Tukey’s multiple comparison test for one-way Anova and Dunn’s test for Kruskal-Wallis). For statistical analysis to analyze the changes between 18 DIV and 25 DIV, paired t test was used to analyze the change in cell composition, while one-sample t test or one-sample Wilcoxon were used when the data was already normalized to the 18 DIV control. Mean difference = difference/average. All data in the graphs are shown as mean ± SD, where each data point reflects one independent N. ∗ p-value < 0.05, ∗∗ p-value < 0.01, ∗∗∗ p-value < 0.001, ∗∗∗∗ p-value < 0.0001.

Limitations of the study

This study uses mature and aged primary cultures to study the effects of ApoE4 on endolysosomal function. One of the advantages of this model is that we are able to study the effects of ApoE4 in mature and functional neurons. The biological nature of neurons is very different from other cell types and might elicit a different response to ApoE4. Furthermore, our model contains both astrocytes and neurons, creating a more in-vivo like scenario in our culture. This is especially important when studying ApoE, as the main producers are astrocytes. Moreover, our model does not contain human Aβ, which could be seen as both a limitation and advantage. It is known that ApoE4 impacts Aβ (9) and we further know that Aβ impacts endosome function (23). Therefore, to elucidate if ApoE4 impacts neuronal endolysosome function on its own and not through its impact on human Aβ metabolism, which is aggregation prone compared to mouse Aβ, our model is a relevant choice. The accelerated maturation causes the cultures to age and develop intracellular age-like stress (45, 46, 50); however, a limitation is that the model does not recapitulate many aspects of an in vivo environment including three-dimensional interaction, anatomical organization and contribution of multiple organs. Another limitation is that the targeted-replacement model creates a hybrid scenario, where a human protein acts in a mouse environment. Through evolution proteins in different species are adapted to each other. When surrounded by related proteins that ApoE recognizes but are slightly different from the proteins in humans, it is most probable that it does not act and behave exactly the same.

Data availability

All data presented are included within the article. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Supporting information

This article contains supportive information.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have the appearance to infuence the work reported in this paper.

Acknowledgments

We acknowledge MultiPark and Anna Hammarberg for the assisted use of the Operetta system, and thank Cecilia Lundberg and her team for producing and providing lentiviral vectors and plasmids. We further express our gratitude to our former students Catalina Merdaci and Sofia Stenström for their work on the project.

Author contributions

N. L., S. C. K., B. I., I. M., O. K., and G. G. writing–review & editing; N. L., S. C. K., B. I., I. M., and E. N. investigation; N. L. and E. N. formal analysis; S. C. K., I. M., and E. N. methodology; S. C. K., I. M., G. G., and E. N. conceptualization; G. G. supervision; G. G. resources; G. G. and E. N. project administration; G. G. funding acquisition; E. N. writing–original draft; E. N. visualization.

Funding and additional information

Swedish Research Council (Grant #2019-01125 and #2023-02630), Hjärnfonden (Grant # FO2023-0259, and # FO2024-0406), Alzheimerfonden (Grant # AF-980901) and Albert Påhlssons stiftelse för forskning och välgörenhet (Grant # F 2023/55).

Biography

graphic file with name fx1.jpg

Emma Nyberg is a PhD candidate in experimental medical science at Lund University. Her research in the Gouras lab focuses on how ApoE4, the major genetic risk factor for Alzheimer’s disease, affects neuronal function, particularly at synapses and endosomes. This study highlighted to her the complex nature of ApoE4 and the importance of uncovering its molecular mechanisms to guide the development of next-generation Alzheimer’s treatments. LinkedIn profile: https://www.linkedin.com/in/emma-nyberg-9830aba4?utm_source=share&utm_campaign=share_via&utm_content=profile&utm_medium=android_app.

Reviewed by members of the JBC Editorial Board. Edited by Elizabeth J. Coulson

Contributor Information

Emma Nyberg, Email: emma.nyberg@med.lu.se.

Gunnar K. Gouras, Email: gunnar.gouras@med.lu.se.

Supporting information

Supporting Figures

mmc1.pdf^{(2.4MB, pdf)}

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Data Availability Statement

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PERMALINK

Neuronal endolysosomal alterations induced by Apolipoprotein E4 emerge over time in primary neurons

Emma Nyberg

Sabine C Konings

Nils Lindblom

Bodil Israelsson

Oxana Klementieva

Isak Martinsson

Gunnar K Gouras

Abstract

Results

Mature ApoE4 primary neurons exhibit only minor alterations in late endolysosome appearance in dendrites

Figure 1.

Mature neurons traffic and degrade endocytosed cargo equally between ApoE isoforms and adapt similar to increased neuronal activity

Figure 2.

Early endosomal enlargement is not evident in ApoE4 neurons with a more prolonged time in culture

Figure 3.

With time in culture, ApoE4 neurons develop reduced degradative capacity in association with a lower number of active degradative compartments

Figure 4.

Alterations in astrocytes, ApoE levels, and lipofuscin between 18 and 25 DIV primary cultures

Figure 5.

ApoE isoform-dependent cholesterol alterations in neurons

Figure 6.

Discussion

Experimental procedures

Animals and ethical considerations

Primary mouse brain cultures and induction of activity

Genotyping

Cholesterol treatment

EGF-tetra rhodamine and DQ-BSA treatments

LDH assay

Western blot and SDS-PAGE

Table 1.

Live-cell calcium imaging and analysis

Lysotracker and live cell imaging

Immunofluorescent labeling

Epifluorescence microscopy

Image analysis

Statistical analysis

Limitations of the study

Data availability

Supporting information

Conflict of interest

Acknowledgments

Author contributions

Funding and additional information

Biography

Contributor Information

Supporting information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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