An increase of lysosomes through EGF-triggered endocytosis attenuated zinc-mediated lysosomal membrane permeabilization and neuronal cell death

doi:10.21203/rs.3.rs-3789670/v1

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An increase of lysosomes through EGF-triggered endocytosis attenuated zinc-mediated lysosomal membrane permeabilization and neuronal cell death

https://doi.org/10.21203/rs.3.rs-3789670/v1

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published 13 Nov, 2024

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In the context of acute brain injuries, where zinc neurotoxicity and oxidative stress are acknowledged contributors to neuronal damage, we investigated the pivotal role of lysosomes as a potential protective mechanism. Our research commenced with an exploration of epidermal growth factor (EGF) and its impact on lysosomal dynamics, particularly its neuroprotective potential against zinc-induced cytotoxicity. Using primary mouse cerebrocortical cultures, we observed the rapid induction of EGFR endocytosis triggered by EGF, resulting in a transient increase in lysosomal vesicles. Furthermore, EGF stimulated lysosomal biogenesis, evident through elevated expression of lysosomal-associated membrane protein 1 (LAMP-1) and the induction and activation of prominent lysosomal proteases, particularly cathepsin B (CTSB). This process of EGFR endocytosis was found to promote lysosomal augmentation, thus conferring protection against zinc-induced lysosomal membrane permeabilization (LMP) and subsequent neuronal death. Notably, the neuroprotective effects and lysosomal enhancement induced by EGF were almost completely reversed by the inhibition of clathrin-mediated and caveolin-mediated endocytosis pathways, along with the disruption of retrograde trafficking. Furthermore, tyrosine kinase inhibition of EGFR nullified EGFR endocytosis, resulting in the abrogation of EGF-induced lysosomal upregulation and neuroprotection. An intriguing aspect of our study is the successful replication of EGF’s neuroprotective effects through the overexpression of LAMP-1, which significantly reduced zinc-induced LMP and cell death, demonstrated in human embryonic kidney (HEK) cells. Our research extended beyond zinc-induced neurotoxicity, as we observed EGF’s protective effects against other oxidative stressors linked to intracellular zinc release, including hydrogen peroxide (H₂O₂) and 1-methyl-4-phenylpyridinium ion (MPP⁺). Collectively, our findings unveil the intricate interplay between EGF-triggered EGFR endocytosis, lysosomal upregulation, an increase in the regulatory capacity for zinc homeostasis, and the subsequent alleviation of zinc-induced neurotoxicity. These results present promising avenues for therapeutic interventions to enhance neuroprotection by targeting lysosomal augmentation.

Biological sciences/Neuroscience/Cell death in the nervous system

Biological sciences/Neuroscience/Diseases of the nervous system/Stroke

Zinc, an essential metal ion, exerts a crucial regulatory role over numerous proteins within cells and organisms. Proteomic analysis reveals that around 10% of proteins possess zinc-binding motifs, and their functions are under the influence of zinc [1, 2]. In the central nervous system (CNS), zinc is highly concentrated within the synaptic vesicles of glutamatergic neurons, primarily in its free ion form, unbound to proteins [3, 4, 5]. During synaptic activity, zinc is co-released with glutamate into the synaptic cleft, potentially influencing the activity of post-synaptic neurons [6, 7].

In cases of acute brain injuries, such as stroke, traumatic brain injury, and epilepsy, synaptic zinc is not promptly reuptake into cells, resulting in its excessive entry into post-synaptic neurons [3, 8–10]. This excessive zinc influx triggers the over-activation of kinases like Protein Kinase C (PKC), Src, and Extracellular signal-regulated kinase (ERK), as well as NADPH oxidase and poly(ADP-ribose) polymerase, ultimately leading to neuronal death [11–14]. Zinc-induced neurotoxicity, combined with calcium-mediated excitotoxicity, oxidative stress, and apoptosis, constitutes a central mechanism in acute brain injuries [8, 13, 15–17].

Lysosomes are cellular organelles responsible for intracellular digestion and degradation. Their function can deteriorate due to factors like aging or various genetic causes [18–20]. When lysosomal function declines, specific proteins, including tau, α-synuclein, and transactive response DNA binding protein of 43 (TDP-43), can accumulate within nerve cells, contributing to the onset of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS). Maintaining an acidic pH within lysosomes is crucial, and this process relies on the action of the zinc transporters [21, 22]. Zinc also plays a role in rapidly facilitating the assembly of v-ATPase on lysosomes, promoting acidification. Additionally, zinc induces the activation of the transcription factor EB (TF-EB), which, in turn, leads to lysosomal biosynthesis [23]. Similar to mitochondria, lysosomes play a crucial role in maintaining zinc homeostasis by sequestering excess zinc when cytosolic zinc concentrations rise. However, in pathological conditions, a sudden and significant increase in intracellular zinc can accelerate its movement into lysosomes, overwhelming and damaging them, ultimately leading to lysosomal membrane permeabilization (LMP) [24]. When LMP occurs, lysosomal degradative enzymes like cathepsin B (CTSB) are released into the cytoplasm, triggering additional mechanisms of neuronal damage, including the activation of caspase-3 to execute apoptosis and the formation of inflammasomes to induce neuroinflammation [25–30].

Elevated intracellular zinc levels can originate from external sources or extracellular space, and they can also be a consequence of increased oxidative stress, which releases zinc from zinc-binding proteins. This process results in an increased concentration of labile zinc in the cytoplasm [31]. Although Metallothionein (MT) proteins are typically responsible for binding zinc and maintaining zinc homeostasis in the cytoplasm, heightened oxidative stress, such as elevated hydrogen peroxide (H₂O₂) levels, can lead to the release of zinc from MT proteins [32–35]. Consequently, this release triggers LMP and subsequent neuronal cell death [36]. In the context of PD, mitochondrial toxins like 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone are implicated in causing damage to dopaminergic neurons, potentially contributing to the disease’s progression [37–41]. Studies have indicated that the administration of MPP⁺ in in vitro neuronal cell models or MPTP in in vivo mouse animal models results in increased oxidative stress and the induction of LMP [42, 43]. This, in turn, can lead to lysosomal deficiency and expedite the accumulation of α-synuclein proteins. In essence, lysosomes are involved in the mechanisms of neuronal cell death, both in cases of acute brain injury mediated by LMP and in instances of lysosomal dysfunction and the accumulation of protein aggregates in neurodegenerative diseases.

In our study, we aimed to investigate whether the quantitative regulation of lysosomes could effectively control zinc-induced neurotoxicity. Epidermal Growth Factor (EGF) is a well-known growth factor that stimulates cell proliferation. It is recognized for its ability to bind to its receptor and, upon initiating signal transduction, undergoes rapid endocytosis along with its receptor EGFR, ultimately being transported to lysosomes for degradation [44, 45]. This process involves an increase in vesicles such as endosomes, resulting in a temporary rise in the number of lysosomes. Furthermore, studies have demonstrated that promoting endocytosis can activate the transcriptional activity of TF-EB, a transcription factor responsible for lysosomal biogenesis [46]. Hence, we chose EGF as a stimulant for increasing lysosomal quantity, given its potential to contribute to the upregulation of lysosomes via endocytosis and TF-EB-mediated lysosomal biogenesis.

In the initial phase of our research, we conducted experiments to determine whether the application of EGF to primary mouse cerebrocortical neuronal cultures could indeed lead to an increase in lysosomes. Once we confirmed this increase, our investigation delved further into whether EGF could mitigate zinc toxicity and whether this protective effect was linked to improved regulation of zinc homeostasis achieved through a quantitative augmentation of lysosomes, resulting in a reduction in LMP. Additionally, we explored whether the EGF-induced increase in lysosomes could also contribute to the suppression of oxidative damage, which is responsible for the release of cytosolic zinc. These findings shed light on the critical role of lysosomes in neuronal death, not only in the context of neurodegenerative diseases but also in acute brain injury. Ultimately, these insights may pave the way for the development of therapeutic strategies targeting lysosomal activation for acute brain diseases.

EGF treatment enhances lysosomal dynamics in mouse cerebrocortical cultures.

Our investigation began with an exploration of the impact of EGF treatment on lysosomal dynamics in mouse cerebrocortical cultures. EGF is widely recognized for its ability to induce EGFR endocytosis and the subsequent increase in endocytic vesicles [44]. We aimed to establish whether this led to a surge in lysosomal vesicle abundance. Following EGF treatment, a significant increase in the number of acidic vesicles, as indicated by LysoTracker Red (LTR) fluorescence, was observed as early as 15 minutes post-treatment (Fig. 1A). However, starting from 2 hours after EGF exposure, the count of acidic lysosomes returned to basal levels, suggesting that EGF-induced lysosomal upregulation is a transient phenomenon (Fig. 1A).

We then explored the possibility of EGF triggering lysosomal biogenesis. At 15 minutes post-treatment, we detected an induction of lysosomal-associated membrane protein 1 (LAMP-1), with more pronounced changes becoming evident at the 30-minute mark (Fig. 1B). This effect persisted for up to 4 hours after EGF exposure (Fig. 1B). Additionally, the levels of cathepsin B (CTSB), a representative lysosomal protease, exhibited rapid increments in response to EGF treatment, along with their swift activation into their mature forms (Fig. 1C). We also confirmed heightened CTSB activity (Fig. 1D). In summary, our findings suggest that the rapid endocytosis of EGFR triggered by EGF results in a prompt increase in the population of acidic lysosomal vesicles. Moreover, this process induces the synthesis of proteins necessary for lysosomal function, leading to a significant enhancement in both the quantity and functionality of lysosomes.

EGF treatment attenuates zinc-induced neuronal death in mouse cerebrocortical cultures.

After establishing the role of EGF in enhancing functional lysosomes, we aimed to determine whether EGF could mitigate zinc-induced neuronal death in mouse cerebrocortical cultures. Exposure to zinc resulted in an increase in Propidium iodide (PI)-stained damaged cells and LDH release into media, both of which were alleviated by EGF treatment (Fig. 2A & B).

Exposure to lethal dose of zinc within the culture media resulted in an elevation of cytosolic labile zinc levels, with subsequent infiltration into intracellular organelles, notably lysosomes [24]. The rapid and excessive increase in free zinc concentration within lysosomes led to the rupture of the lysosomal membrane, a phenomenon known as lysosomal membrane permeabilization (LMP). This process, in turn, resulted in the release of lysosomal proteases, including CTSB, triggering further detrimental effects such as caspase-3 activation and inflammasome formation [47, 48]. To confirm the involvement of LMP in zinc-induced neuronal death, we employed pretreatment with leupeptin (a cysteine protease inhibitor) or CA-074 methyl ester (CA074, a cathepsin B inhibitor). Remarkably, leupeptin, CA074 as well as tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a zinc chelator, substantially alleviated zinc-induced neuronal death (Fig. 2C & D). Furthermore, when EGF was co-administered with a CTSB inhibitor, it failed to provide additional neuroprotective effects, indicating a common protective mechanism mediated by EGF and lysosomal proteases (Fig. 2E). Thus, EGF significantly reduced zinc-induced neuronal death by inhibiting LMP and subsequent protease-mediated actions within the cytosol.

EGF treatment prevents zinc-induced lysosomal membrane permeabilization (LMP) in mouse cerebrocortical cultures.

To observe LMP occurrence after zinc exposure and determine whether EGF could counteract it, we performed double staining with Fluozin-3 and LysoTracker Red (LTR). Our investigation revealed dynamic changes in cellular zinc distribution and lysosomal integrity. Initially, there was an increase in zinc-containing vesicles within the first 2 hours, followed by a subsequent decline, coinciding with an increase in cytoplasmic zinc concentration (Fig. 3A, Fluozin-3 green color). Similarly, LTR-stained lysosomal vesicles showed a peak increase at 1–2 hours post zinc exposure, gradually decreasing below control levels from 4 hours onwards (Fig. 3A, LTR red color). Merged yellow puncta, representing zinc-containing lysosomes, emphasized this pattern. Notably, the increased presence of zinc-containing lysosomes at 1 or 2 hours after zinc exposure was followed by their diminishment at 4 or 8 hours, signifying the onset of LMP (Fig. 3A, merged yellow color).

The confirmation of zinc-induced LMP was achieved through western blot analysis of cathepsin B (CTSB) release into the cytosol. A significant increase in released CTSB levels was observed from 6 hours after zinc treatment (Fig. 3B).

To evaluate the potential of EGF in mitigating zinc-induced LMP, we examined the effect of EGF on zinc-treated cultures. At 4 hours after zinc treatment, there was only an increase in diffused cytosolic zinc, and intact lysosomes were notably diminished (Fig. 3C), consistent with Fig. 1A. However, EGF administration preserved intact lysosomes (indicated by red arrowheads) and zinc-containing lysosomes (depicted by yellow arrowheads) compared to cultures treated solely with zinc (Fig. 3C). This protective effect was accompanied by a reduction in the levels of CTSB release, as shown through western blot analysis (Fig. 3D left), emphasizing the mitigatory impact of EGF on zinc-induced LMP. Furthermore, we confirmed that LMP and CTSB release are induced by increased intracellular zinc, as TPEN, an intracellular zinc chelator, did not induce LMP or CTSB release (Fig. 3C & D). Considering the neuroprotective effects of leupeptin and CA074 against zinc-induced cytotoxicity (Fig. 2C-E), we examined their influence on zinc-induced LMP in mouse cerebrocortical cultures. However, the presence of lysosomal protease inhibitors, leupeptin or CA074, did not maintain the integrity of LTR-positive dots, resembling the conditions seen in cultures exposed solely to zinc (Fig. 3C). Additionally, leupeptin or CA074 was ineffective in reducing zinc-induced CTSB release into the cytosol (Fig. 3D). On the contrary, CA-074 seemed to augment zinc-induced CTSB release into cytosol, a result of heightened stability by inhibiting CTSB activity. These results suggest that while CTSB inhibitors may not directly impede LMP during zinc neurotoxicity, they may exert their protective effect against zinc-induced neurotoxicity by suppressing CTSB activity released from lysosomes to the cytosol following LMP.

The protective effects of EGF against zinc-induced LMP and neuronal cell death are mediated by endocytosis and retrograde trafficking processes.

Having established EGF’s capacity to enhance functional lysosomes and reduce LMP-associated neuronal death, we aimed to elucidate the significance of endocytic processes in facilitating EGF’s protective effects. The internalization of EGFR involves both clathrin-mediated endocytosis (CME) and non-clathrin endocytosis (NCE, specifically caveolin-mediated endocytosis), along with subsequent degradation processes [46, 49]. Our initial focus was on evaluating the potential of inhibiting CME and NCE pathways as strategies to counteract EGF’s protective influence on zinc-induced LMP and neuronal death.

Pretreatment with methyl-β-cyclodextrin (MβCD), a caveolin-dependent endocytosis inhibitor, and chlorpromazine (CP), a clathrin-dependent endocytosis blocker, before concurrent zinc and EGF treatment, effectively reversed the neuroprotective effect conferred by EGF against zinc-induced neurotoxicity (Fig. 4A left). We subsequently confirmed that the reversal effect of MβCD and CP was a result of inhibiting EGFR endocytosis. Notably, these agents, MβCD and CP, significantly preserved EGFR levels even after EGF treatment, and EGF-induced LAMP-1 expression returned to its control baseline (Fig. 4B left). MβCD and CP also led to a reduction in the number of acidified lysosomal vesicles (Fig. 4C). Furthermore, the improvement of CTSB release by EGF was significantly reversed by MβCD and CP (Fig. 4D). All these results underscore the contribution of CME and NCE to EGF-induced lysosomal enhancement and subsequent protection against zinc-induced LMP and neuronal death.

Subsequently, we investigated the impact of Compound 56 (Cpd56), a tyrosine kinase inhibitor targeting EGFR, on EGF-induced lysosomal upregulation and neuroprotection. Inhibiting EGFR’s tyrosine kinase activity with Cpd56 led to a reversal of EGF-mediated neuroprotection (Fig. 4A middle). We confirmed that Cpd56’s effect was mediated by the blockade of EGFR endocytosis, as demonstrated by the maintenance of EGFR (Fig. 4B middle). EGF-triggered LAMP-1 induction and the elevation in lysosomal vesicles were also reversed by Cpd56 (Fig. 4B & C). Finally, we observed that the blocking of zinc-induced CTSB release by EGF was effectively counteracted by the EGFR tyrosine kinase inhibitor (Fig. 4D).

Next, we assessed the role of retrograde trafficking in EGF-mediated neuroprotection by using ciliobrevin A (ciliob), a dynein-inhibiting chemical agent. Disrupting endosome maturation via retrograde transport inhibition with ciliobrevin A significantly reversed EGF-triggered neuroprotection against zinc toxicity (Fig. 4A right). Notably, ciliobrevin A also reversed EGF-triggered EGFR degradation and subsequent lysosomal formation (Fig. 4B & C). Ciliobrevin A also reversed the EGF-induced attenuation of CTSB release (Fig. 4D). Taken together, our findings underscore the pivotal role of EGF-triggered EGFR endocytosis in promoting lysosomal upregulation and providing neuroprotection against zinc-induced neurotoxicity.

LAMP-1 overexpression also attenuates zinc-induced LMP and cell death in HEK cells.

Having previously observed that EGF-triggered lysosomal upregulation effectively attenuates zinc-induced LMP and cell death, we tested whether genetic overexpression of LAMP-1 could replicate these protective effects. To achieve this, we transiently transfected the RFP-LAMP-1 plasmid into human embryonic kidney (HEK) cells. Due to the limited transfection efficiency observed in primary mouse cerebrocortical neurons, we opted for HEK cells. The overexpression of LAMP-1 notably augmented the population of RFP-positive organelles, corresponding to lysosomes. Interestingly, we confirmed that transfection of the LAMP-1 plasmid not only elevated the number of lysosomes but also enhanced lysosomal acidification, as indicated by lysosensor green (LSG) staining (Fig. 5A).

Considering EGF’s ability to mitigate zinc neurotoxicity (Fig. 2) and zinc-induced LMP in mouse cerebrocortical neurons (Fig. 3C & D), we aimed to determine if LAMP-1 overexpression would similarly confer protective effects against zinc toxicity in HEK cells. Remarkably, elevated LAMP-1 expression resulted in a substantial reduction in zinc-induced toxicity (Fig. 5B) and the prevention of zinc-induced LMP and CTSB release into the cytosol (Fig. 5C) within HEK cells. These findings collectively indicate that LAMP-1 overexpression enhances both the quantity and activity of lysosomes, thereby contributing to the regulation of intracellular zinc homeostasis.

EGF does not protect against glutamate-induced excitotoxicity and STSP-induced apoptosis.

Zinc-induced neuronal death plays a crucial role in neuronal loss in the cortex and hippocampus following acute brain injuries such as stroke, trauma, and epilepsy [4, 10–12, 50]. Since excess calcium influx and excitotoxicity are also significant contributors to neuronal death in these conditions [51], we investigated whether EGF-induced lysosome upregulation could reduce calcium-mediated toxicity. Initially, we observed that neuronal death induced by glutamate was not inhibited by the zinc chelator TPEN, but significantly decreased by the calcium chelator EDTA (Fig. 6A & B). In addition, EGF did not exhibit a reduction in calcium-mediated excitotoxicity (Fig. 6A & B).

CTSB released into the cytoplasm after LMP can activate caspase-3 and contribute to cell death in the form of apoptosis [48, 52–54]. Therefore, we further investigated whether lysosomal upregulation induced by EGF treatment could reduce staurosporine (STSP)-induced apoptosis. STSP-induced neuronal death was effectively inhibited by the pan-caspase inhibitor zVAD but remained unaffected by the zinc chelator TPEN or EGF (Fig. 6C & D). In summary, EGF-mediated lysosomal upregulation appears to specifically target zinc-related neuronal cell death mechanisms.

Oxidative damage mediated by zinc, following treatment with H ₂ O ₂ or MPP⁺, is also reduced under conditions of lysosome upregulation.

Numerous studies have emphasized the accumulation of zinc within lysosomes, leading to LMP upon exposure to hydrogen peroxide (H₂O₂) in cerebrocortical and hippocampal neuronal cultures [24]. Additionally, the complex Ⅰ inhibitor, 1-methyl-4-phenylpyridinium ion (MPP⁺), has been shown to induce intracellular zinc accumulation both in vitro and in vivo. Furthermore, lysosomal breakdown has been observed in dopaminergic neuronal cells exposed to MPP⁺ [43]. Given these findings, we investigated whether EGF could confer protective effects against other types of oxidative stress, such as H₂O₂ or MPP⁺. Before assessing the impact of EGF, we confirmed that cell death induced by H₂O₂ and MPP⁺ could be prevented by the zinc chelator TPEN in mouse cerebrocortical cultures (Fig. 6A & B), thus confirming the role of zinc in mediating neuronal death. Subsequently, we observed that EGF effectively prevented H₂O₂ and MPP⁺-induced neuronal death (Fig. 6C & D). Notably, the overexpression of LAMP-1 in HEK cells significantly suppressed H₂O₂ or MPP⁺-induced cytotoxicity (Fig. 6E & F), reinforcing the applicability of our findings to diverse forms of neurotoxicity arising from intracellular zinc elevation and subsequent LMP.

In our study, we delved into the crucial role of lysosomes in guarding against zinc-mediated neurotoxicity, a significant contributor to acute brain injuries. Firstly, we observed a rapid increase in the number of lysosomal vesicles and the promotion of lysosomal biogenesis triggered by EGF. This augmentation, induced by EGF, effectively shielded against zinc-induced LMP and subsequent neuronal death. We confirmed that EGF’s protective effects were mediated through clathrin- and caveolin-mediated endocytosis pathways, in conjunction with retrograde trafficking. Remarkably, the overexpression of LAMP-1 replicated EGF’s protective effects in HEK cells. Also noted was EGF-induced lysosomal enhancement extending protection to other oxidative stress associated with intracellular zinc release. These findings highlight the intricate interplay between EGF-triggered EGFR endocytosis, lysosomal upregulation, enhanced regulation of zinc homeostasis, and the alleviation of zinc-induced neurotoxicity.

Our study demonstrated that EGFR endocytosis triggered by EGF leads to a transient escalation in lysosomal vesicles characterized by a lower pH. This surge is accompanied with increased expressions of both LAMP-1, a representative lysosomal marker protein, and CTSB, a prototypical lysosomal protease. Notably, while sustained elevation of LAMP-1 and inactive CTSB proform persist for up to 4 hours post-EGF treatment, the count of acidic vesicles returns to baseline after 2 hours (Fig. 1A-C). Lysosomal biogenesis continues for up to 4 hours, but a significant increase in acidic lysosomal vesicles begins to decrease after 2 hours. Corresponding to this lysosomal dynamics, CTSB activity peaks at 1 hour post-EGF treatment, diminishes, and peak again at 4 hours (Fig. 1D). In essence, these CTSB activity fluctuations post-EGF treatment represent a combined outcome from the initial surge in low pH vesicles and subsequent delayed, sustained rises in CTSB expression.

Additionally, we confirmed that EGF-induced lysosomal upregulation is not dependent on the activation of intracellular signaling cascade, but rather mediated by EGFR's endocytosis process (Fig. 4). Blocking endocytosis and retrograde trafficking preserved intact EGFR without degradation, halting lysosomal upregulation. Consequently, inhibiting the endocytosis erased the neuroprotective effect against zinc-induced LMP and neuronal death. Furthermore, compound 56, an EGFR tyrosine kinase inhibitor, when co-treated with EGF, impeded EGFR endocytosis, thus nullifying lysosomal upregulation and the protective effects against zinc neurotoxicity. These results suggest that EGF’s neuroprotective effect against zinc neurotoxicity is due to the facilitation of endocytosis rather than activation of signaling cascade. Validating that endocytosis promotion effectively regulates cytosolic free zinc levels via lysosomal upregulation, inhibiting zinc-mediated neuronal cell death, could establish it as an acute brain injury therapeutic strategy.

An important finding in EGF-induced lysosomal upregulation is the rapid increase in LAMP-1 expression within 15 minutes after EGF treatment, along with CTSB. Surprisingly, LAMP-1 overexpression alone augmented the count of low-pH lysosomes and reduced zinc-induced neurotoxicity. However, the mechanism driving the swift induction of LAMP-1 expression via endocytosis remains unclear. Transcription factor TF-EB is widely recognized for orchestrating lysosomal biogenesis, involving the activation of various lysosomal proteins like CTSB, cathepsin D, and v-ATPase subunits through dephosphorylation and nuclear translocation [23]. Despite the rapid increase in LAMP-1 and CTSB expression, we observed no significant alterations in TF-EB dephosphorylation or nuclear translocation (data not shown). Further investigations are needed to elucidate how endocytosis promotion precisely triggers lysosomal biogenesis, particularly the mechanisms driving increased LAMP-1 and CTSB expressions.

Recent research has shed light on the impact of LAMP-1 overexpression on lysosomal function. To maintain a low pH inside vesicles, the action of v-ATPase is essential [55]. Jiang et al. demonstrated that when LAMP-1 or LAMP-2 binds to the TMEM175 channel in lysosomes, it forms a complex inhibiting TMEM175 function, thereby contributing to lysosomal acidification (Mol Cell, 2023) [56]. TMEM175 acts as a proton leak channel in acidic environments, releasing protons out of lysosomes, alongside v-ATPase, maintaining lysosomal pH balance. Increased LAMP-1 expression inhibits TMEM175 function, resulting in v-ATPase predominance, acidifying lysosomes, and creating an environment where lysosomal hydrolases can be actively functional. To comprehensively understand the role of LAMP-1, further research investigating how LAMP-1 promotes lysosomal functions is warranted.

Our observations highlight that increased intracellular free zinc, triggered by oxidative damage or excessive zinc intake, accelerates zinc entry into lysosomes, initiating sudden LMP. Following LMP, released lysosomal enzymes potentially induce cellular damage by degrading cytoplasmic proteins or cytoskeleton matrix [57–59]. Additionally, cytosolic release of CTSB may contribute to initiating an inflammatory response by promoting the assembly of the inflammasome—a complex comprising NLRP3, ACS, and pro-caspase-1 [60–62]. This provokes caspase-1 activation, leading to the cleavage of pro IL-1β and pro IL-18, subsequently releasing IL-1β and IL-18 into the extracellular space [63]. Furthermore, cytosolic release of CTSB might directly induce apoptotic cell death by cleaving caspase-3 [64]. Zinc-mediated neuronal cell death exhibits feature of both necrosis and apoptosis [8, 65], characterized by increased oxidative stress via nNOS, NADPH oxidase, etc., heightened caspase-3 activity facilitated by p75NTR and Egr-1 activation, and typical apoptotic traits including DNA fragmentation [11, 14, 15, 66–68]. Consequently, the release of CTSB following LMP in zinc-mediated neuronal death is hypothesized to expedite the apoptotic process through caspase-3 activity. Given the unknown precise mechanism of CTSB in zinc-mediated neuronal death, further research is needed to determine if the neuroprotective mechanism of CTSB inhibition involves reducing cytosolic proteins or cytoskeletal degradation, suppressing inflammasome formation, or inhibiting caspase-3 activation.

Intracellular calcium elevation acts as a secondary messenger, typically maintained at low level of around ~ 100 nM [69]. During synaptic activity or cellular signaling, elevated calcium ions should be expelled from cytoplasm via energy-dependent transporter action [70]. Proteins like calbindin also contribute to intracellular calcium homeostasis [71]. In the case of zinc ions, typically at around 100 pM in the cytoplasm—much lower than cytoplasmic calcium concentration—can transiently surge to micromolar levels during brain ischemic damage [72, 73]. Ten known zinc transporters, ZnT1 to ZnT10, are present in organelles like lysosomes, ER, and synaptic vesicles, allowing zinc influx using proton gradients [3]. Studies indicated that as intracellular zinc levels rise, lysosomes uptake zinc ions, potentially causing LMP and subsequent cell death if excessive [36, 74]. Our study validated lysosomes as critical intracellular organelles regulating zinc levels. Augmenting lysosomes via facilitated endocytosis enhances zinc homeostasis, reducing zinc-mediated neuronal cell death. Besides from calcium, ER and mitochondria also uptake zinc ions to regulate cytoplasmic zinc levels [75, 76]. However, given the significant difference in concentrations—cytoplasmic calcium being approximately 10³ times higher than zinc—segregation of representative intracellular organelles regulating calcium and zinc seems advantageous. Lysosomes likely act as representative intracellular organelles in zinc homeostasis regulation. Furthermore, since an increase in reactive oxygen species (ROS) triggers zinc release from zinc-bound proteins, an elevation in zinc could be a cause in ROS-induced neuronal death. Hence, understanding lysosomal quantitative control mechanisms might alleviate neuronal cell death induced by ROS as well as intracellular zinc increase. Research on lysosomal upregulation mechanisms could be crucial in preventing and treating neurological disorders such as stroke, epilepsy, traumatic brain injury—conditions involving increased ROS and zinc levels—underscoring the importance of comprehending lysosomal regulation.

Our findings indicate that EGF treatment attenuated neuronal death caused by the mitochondrial toxicant MPP⁺. Mitochondrial impairment stands as a significant factor in PD [77]. Although our study observed LMP induction by MPP⁺ toxicity solely in a cellular model, in the progression of PD, recurrent LMP gradually leads to lysosome deficiency. Therefore, exploring strategies to inhibit LMP could not only attenuate acute neuronal death but also tackle neurodegenerative diseases such as PD. Recurrent LMP gradually leads to lysosome deficiency, impeding the clearance of α-synuclein aggregates, resulting in damage to dopaminergic neurons via the formation of Lewy bodies—a potential contributor to PD development.

In conclusion, EGF-triggered endocytosis enhances lysosomes, providing protection against zinc-induced neurotoxicity by regulating zinc homeostasis. This intricate interplay among endocytosis activation, lysosomal upregulation, and zinc homeostasis suggests potential strategies for mitigating acute brain injuries by targeting endocytosis pathways and suppressing LMP. Furthermore, these findings emphasize the significance of lysosomal regulation, not only in oxidative stress or zinc-mediated neurotoxicity but also as potential therapeutic targets for managing neurodegenerative diseases like PD, addressing lysosome-related issues in the context of mitochondrial impairment and α-synuclein aggregation.

Cultures of primary mouse cerebrocortical neurons and HEK cells

Primary cerebrocortical neuronal cells were cultured from ICR mouse embryos collected at embryonic day 13–14 (ORIENT BIO, Gyeonggi, South Korea). In brief, dissociated cortical cells were seeded onto poly-D-lysine (Sigma, St. Louis, MO, USA)-coated plates (SPL Life Sciences, Gyeonggi-do, South Korea), with 4 ~ 5 embryos per plate. These cultures were maintained in a growth medium, consisting of glutamine-free Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA), supplemented with 2 mM glutamine, 5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), and 5% horse serum (HS; Invitrogen). The cultures were incubated at 37°C in a humidified 5% CO₂ atmosphere. Experiments, except for confocal microscopy, were conducted at 10–12 days in vitro (DIV). For confocal imaging, primary cerebrocortical cultures were treated with 10 µM cytosine arabinoside (Sigma-Aldrich) at DIV 3 to prepare near-pure neuronal cultures, which were used at DIV 7. All animal experimental procedures were carried out under the guidelines for Care and Use of Laboratory Animals and were reviewed and approved by the Animal Care and Use Committee of Sejong University.

Human embryonic kidney (HEK) cells were maintained in high glucose DMEM (Welgene, Gyeongsan, South Korea) supplemented with 10% FBS (Invitrogen) and incubated at 37°C in a humidified 5% CO₂ atmosphere.

Chemical treatments

The growth medium, containing 5% FBS and 5% HS, was replaced with a minimal essential medium (MEM; Invitrogen) before chemical exposure. Various compounds, including 100 ng/ml recombinant mouse epithelial growth factor (EGF; R&D, Minneapolis, MN, USA), 1 µM N,N,N’,N’-tetrakis (2-pyridylmethyl)-ethylenediamine (TPEN; Merck, Burlington, MA, USA), 20 µM CA074 methyl ester (CA074, Merck), or 100 µM leupeptin (Thermo Fisher, Waltham, MA, USA), were administered as a 30-minute pre-treatment before exposure to 40 µM zinc (Sigma-Aldrich), 140 µM hydrogen peroxide (H₂O₂; Thermo Fisher), or 300 µM 1-methyl-4-phenylpyridinium ion (MPP⁺; Abcam, Cambridge, UK). HEK cells were subjected to 80 µM zinc, 280 µM H₂O₂, or 3 mM MPP⁺ to induce cytotoxicity.

To inhibit the endocytosis of EGF receptor (EGFR), 2 mM methyl-beta-cyclodextrin (MβCD; Sigma-Aldrich), 2 µM chlorpromazine (CP; Sigma-Aldrich), 1 µM compound 56 (Cpd56; Merck), or 500 nM ciliobrevin A (Ciliob; GlpBio, Montclair, CA, USA) were administered as a 30-minute pre-treatment before EGF treatment.

LDH release assay

To assess cell death, we measured lactate dehydrogenase (LDH) activity in the culture medium as an indicator of cellular damage, following a previously published protocol [78]. In brief, at the desired time points after zinc exposure, 50 µl of culture medium containing extracellular LDH was mixed with 25 µl of 23 mM pyruvate and 125 µl of 0.03% NADH. The reduction of NADH was kinetically measured for 5 minutes at 340 nm using a VersaMax absorbance microplate reader (Molecular Devices, San Jose, CA, USA). The LDH value was normalized, with sham-wash set to 0%, and treatment with 100 µM N-methyl-D-aspartic acid (NMDA; Abcam) for cortical neuronal cultures or 500 µM zinc for HEK cells set to 100% in sister cultures.

Propidium iodide (PI) staining

We also employed propidium iodide (PI) staining to quantify dead cells. After a designated time period following zinc exposure, cells were treated with 2.5 µg/ml PI (Sigma-Aldrich) for 10 minutes at 37℃, followed by washing with MEM. Stained dead cells were observed using a fluorescence microscope (EVOS Cell Imaging System, Thermo Fisher). Images were randomly selected, and the fluorescence intensity was quantified using the Image J program.

Cell Counting Kit-8 (CCK-8) assay

To assess the survival rate of HEK cells, we added CCK-8 solution (Abbkine, Wuhan, China) to 1/20 of the medium’s volume at the designated time point after MPP⁺ exposure. The mixture was then incubated at 37℃ for 3–4 hours. The change in color was measured at 450 nm using a VersaMax absorbance microplate reader (Molecular Devices). Cell viability was calculated as 100% for untreated conditions (sham-wash) and 0% using 500 µM zinc as the condition representing complete cell death.

Microscopic detection of lysosomal activity

To monitor lysosomal activity, neuronal cultures were exposed to 75 nM LysoTracker Red DND-99 (LTR; Invitrogen) or 1 µM Lysosensor Green DND-189 (LSG; Invitrogen) for 30 minutes at 37℃. After a wash with MEM, the treated cells were exposed to the respective chemicals for the indicated duration. The intensity of fluorescence directly correlated with the lysosome’s acidity. Fluorescent signals were observed in live cells under a fluorescence microscope, and their intensity was quantified using the Image J program. Fluorescent images were selected randomly, and phase-contrast images from the same area were also provided.

In situ microscopic detection of cathepsin B activity

To analyze in situ cathepsin B activity, neuronal cultures were exposed to the 1X Magic Red Cathepsin B Detection Kit (Immunochemistry, Minneapolis, MN, USA) following the manufacturer’s protocol. After washing with MEM, the cells were incubated with EGF (100 ng/ml) for the indicated times. Red fluorescence was detected in live cells under a fluorescence microscope, and the fluorescence intensity was measured using the Image J software. The fluorescent images were chosen randomly.

Confocal imaging for lysosomal zinc

To visualize lysosomal zinc, primary near-pure neuronal cultures plated on poly-D-lysine-coated cover glasses were subjected to double staining with 5 µM Fluozin-3 (Invitrogen) and 75 nM Lysotracker Red for 30 minutes at 37℃ before drug treatment. After removing excess dyes with MEM, cells were exposed to the specified chemical treatments. Subsequently, the cells were fixed in 4% paraformaldehyde for 15 minutes, followed by twice washing with cold PBS. Post-washing, the cells were immediately mounted and examined using the Leica TCS SP5 confocal laser scanning microscope (Wetzlar, Germany). The fluorescent cells were selected randomly, with a minimum of three fields analyzed for each condition.

Western blots

Total protein extracts were prepared with RIPA lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], and 5 mM ethylene-diamine-tetraacetic acid [EDTA] with freshly added 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM phenylmethanesulfonyl fluoride [PMSF], 1 mM sodium orthovanadate [Na₃VO₄], 5 mM sodium fluoride, and 10 mM sodium pyrophosphate [Na₄P₂O₇]).

The cytosol fraction was obtained following a previously described method¹. In brief, after removing the culture medium, cytosol extraction buffer (250 mM glucose, 20 mM hydroxyethyl piperazine ethanesulfonic acid (HEPES), 10 mM KCl, 1.5 mM MgCl₂, 2 mM EDTA, and 25 µg/ml digitonin) was added to just cover the cell surface. The plate was gently shaken on ice at 100 rpm for 15 minutes to allow cytosolic components to flow out into the extraction buffer. Acetone was added in a volume four times that of the buffer, and the cytosolic proteins were precipitated overnight at -20℃. The precipitated protein was collected by centrifugation at 3,000 rpm for 20 minutes at 4℃, and the pellets were resuspended in cytosol lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 2.5 mM Na₄P₂O₇, 1 µM Na₃VO₄, 1 µg/ml leupeptin, 1 mM PMSF). Cytosolic proteins were used without quantification.

For electrophoresis, the protein samples were boiled at 95℃ for 5 minutes, loaded onto SDS-polyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membranes. Antibodies against EGFR (Santa Cruz Biotechnologies, Dallas, TX, USA), cathepsin B (Cell signaling Technology, Danvers, MA, USA), and LAMP-1 (Merck) were used. An anti-actin antibody (Abbkine) served as the loading control. Western blot bands were visualized through a chemical reaction involving horseradish peroxidase (HRP) and an enhanced chemiluminescence (ECL; Abbkine) solution, and a gel documentation system (BIS 303 PC, DNF Bio-Imaging Systems, Israel) was employed for documentation. Band densities were analyzed using the Image J program.

Lysosomal associated membrane protein-1 (LAMP-1) overexpression

HEK cells were transfected with 250 ng/ml pCMV3-untagged negative control vector (Sino Biological, Beijing, China) or Lamp1-RFP plasmid for 4 hours using Lipofectamine 2000 (Invitrogen). The bathing medium was then replaced with DMEM containing 10% FBS and 100 µg/ml gentamicin. The cells were allowed to stabilize for an additional day before the experiment. We gratefully acknowledge Dr. Jung-Jin Hwang (University of Ulsan College of Medicine, Seoul, South Korea) for providing the RFP-LAMP-1 plasmid.

Statistical analysis

All quantitative data were presented as mean ± standard error of the mean (SEM). Two-group comparisons were performed using a two-tailed Student’s t-test. For comparisons involving multiple groups, the entire quantitative dataset was analyzed using an ANOVA test with Dunnett’s multiple comparison post-hoc analysis. The figure legend provides the p-value for statistical significance. All data were analyzed using the GraphPad Prism program.

ACKNOWLEDGEMENTS

This study was supported by the National Research Foundation of Korea (NRF) grants NRF-2021R1A2C2008234 and RS-2023-00242206.

AUTHOR CONTRIBUTIONS

Y-HK and J-WE designed the experiments. J-WE and J-YL performed the experiments. Y-HK, J-WE, and J-YL analyzed the data and wrote the manuscript. All authors reviewed and approved the manuscript.

CONFLICT OF INTERESTS

The authors declare no conflict of interests.

ETHICS APPROVAL

This study was approved by the Animal Care and Use Committee of Sejong University.

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An increase of lysosomes through EGF-triggered endocytosis attenuated zinc-mediated lysosomal membrane permeabilization and neuronal cell death

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Abstract

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INTRODUCTION

RESULTS

DISCUSSION

MATERIALS AND METHODS

Cultures of primary mouse cerebrocortical neurons and HEK cells

Chemical treatments

LDH release assay

Propidium iodide (PI) staining

Cell Counting Kit-8 (CCK-8) assay

Microscopic detection of lysosomal activity

In situ microscopic detection of cathepsin B activity

Confocal imaging for lysosomal zinc

Western blots

Lysosomal associated membrane protein-1 (LAMP-1) overexpression

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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