Carlos Velez-Pardo

Familial Alzheimer’s disease (FAD) is an accelerated form of dementia affecting cholinergic neurons. Despite several efforts, no single drug or treatment has demonstrated complete efficacy. Therefore, finding effective therapeutic agents is imperative. Previous studies have shown that the PSEN1 I416T variant induces FAD-like neuropathology in cholinergic-like neurons (ChLNs), characterized by the intracellular accumulation of the Aβ (iAβ) peptide, the oxidation of the stress sensor protein DJ-1, the abnormal phosphorylation of the tau protein at serine 202/threonine 205 (pS202/T205), the loss of mitochondrial membrane potential (ΔΨ_m), and activation of the pro-apoptotic proteins tumor protein 53 (TP53), Jun proto-oncogene, AP-1 transcription factor subunit (c-JUN), p53 upregulated modulator of apoptosis (PUMA), and cleaved caspase-3 (CC3). We report for the first time that PSEN1 I416T induces abnormal phosphorylation of Leucine-rich repeat kinase 2 (LRRK2) kinase at residue serine 935 (S935), concomitant with phosphorylated alpha-synuclein (αSYN) at residue serine 129 (S129) and abnormal accumulation of autophagosomes and atypical increase in vesicular lysosomal pH, thereby provoking a profound alteration in autophagy in ChLNs. Here, we also demonstrate for the first time that the potent LRRK2 inhibitor PF-06447475 (hereafter referred to as PF475) almost completely attenuated PSEN1 I416T-induced proteinopathy, oxidative stress (OS), and apoptosis and improve autophagy to an activity comparable to untreated wild-type (WT) ChLNs. Overall, PF475 restored the survival of mutant ChLNs. Taken together, these findings suggest that PF475 is an excellent pharmacological tool with which to investigate the regulation of LRRK2-associated pathological signaling in the PSEN1 I416T familial Alzheimer’s disease (FAD) model.

Acute lymphoblastic (ALL) and chronic myeloid (CML) leukemias are blood cancers that often resist traditional chemotherapy and other treatments. This is likely due to their ability to evade apoptosis. Therefore, inducing apoptosis in leukemia cells using innovative drug combinations may be the most effective therapeutic approach. Methods for multidrug combinations involving three or more drugs are scarce and much more complex to analyze. To address this issue, we propose an effective concentration 50 (EC ₅₀ )-based, three-step method. The first step determines the lowest EC ₅₀ for each drug (e.g., artemisinin, chloroquine, primaquine, mefloquine, ivermectin, moxidectin, doxorubicin, and minocycline) by analyzing four cell endpoints (e.g., cell cycle, sub-G1, mitochondrial membrane potential (ΔΨ _m ), autophagy (lysosomes), and cleaved caspase 3 (CC3)) on K562 cells. Step two involves establishing the deleterious effect of the EC ₅₀ -based drug combination at concentrations of single drugs at 1-, ½-, and ¼-EC ₅₀ , respectively, on K562 leukemia cells. Step three involves using the optimal combined drugs to evaluate the same cellular endpoints in other non-leukemic and leukemic cells. We found that the combination of AM (1 µM), MD (10 µM), and DR (1.5 µM), i.e., at ½ EC ₅₀ , induced cell cycle arrest in the S (25% ± 13, N = 4) and G2/M (55% ± 18, N = 4) phases, a drastic loss of ΔΨ _m (81% ± 6, N = 4), high lysosome accumulation (82% ± 10, N = 4), and CC3 (83% ± 13, N = 4), as evidence of apoptosis in K562 and Jurkat cells and ex vivo ALL and CML cells. The combined drugs were innocuous to peripheral blood lymphocytes (PBLs) (S phase = 40%; G2/M = 26%; ΔΨ _m = 4%; lysosomes = 3%; CC3 = 4%; n = 3). Our approach to combining drugs has the potential to provide a new pharmacological treatment for leukemias.

The intracellular accumulation of amyloid beta 42 (iAβ42) has been proposed as an early pathological indicator of familial Alzheimer’s disease (FAD). DJ-1 is a multifunctional protein sensitive to oxidative stress (OS) that has been associated with neurodegeneration; however, its role in iAβ42 pathology is unclear. In this study, we examined whether oxidized (sulfonic) DJ-1 (Cys¹⁰⁶-SO₃H) drives iAβ42 accumulation using postmortem brain samples and in vitro 3D iPSC-derived cerebral organoids (COs) or 2D induced pluripotent stem cells (iPSC)-derived ChLNs (cholinergic-like neurons) models from a PSEN1 E280A patient and a healthy volunteer (as a control sample). Post-mortem analyses of the temporal and frontal cortices and hippocampus from FAD PSEN1 E280A patients revealed strong intracellular co-localization of sulfonic DJ-1 and iAβ42, which was absent in control samples. To validate these findings, we generated COs from an iPSC PSEN1 E280A FAD patient and a healthy donor. In these organoids, we observed the co-localization of oxidized DJ-1 and Aβ42 in the absence of extracellular fibrils or plaques, as confirmed by BTA-1 staining. To further support these observations, 2D iPSC PSEN1 E280A-derived ChLNs cultures showed that intracellular Aβ42 accumulates progressively in direct correlation with increasing DJ-1 oxidation, as demonstrated by immunofluorescence microscopy and Western blotting analysis. These results indicate that DJ-1 oxidation accompanies the earliest intracellular stages of Aβ42 pathology. Furthermore, complementary in silico molecular docking analysis revealed a higher affinity between Aβ42 and oxidized sulfonic DJ-1 (DJ-1 Cys¹⁰⁶-SO₃H) compared to sulfenic (DJ-1 Cys¹⁰⁶-SOH) or sulfinic acid (DJ-1 Cys¹⁰⁶-SO₂H) forms. Likewise, ELISA tests and seeding assays confirmed that oxidized DJ-1 binds to and decelerates Aβ42 aggregation kinetics. Together, our results identify DJ-1 oxidation as a critical molecular event in the accumulation of iAβ42 in FAD. These findings suggest that oxidized DJ-1 represents not only a potential early biomarker of intracellular pathology but also a pharmacological target. Preventing the oxidation of DJ-1 or its pathological aggregation could provide new biomarkers and therapeutic strategies for reducing the intracellular accumulation of Aβ42 and neurodegeneration in FAD.

The intracellular accumulation of amyloid beta 42 (iAβ42) has been proposed as an early pathological indicator of familial Alzheimer’s disease (FAD). DJ-1 is a multifunctional protein sensitive to oxidative stress (OS) that has been associated with neurodegeneration; however, its role in iAβ42 pathology is unclear. In this study, we examined whether oxidized (sulfonic) DJ-1 (Cys¹⁰⁶-SO₃) drives iAβ42 accumulation using postmortem brain samples and in vitro 3D (iPSC-derived cerebral organoids, COs) or 2D induced pluripotent stem cells (iPSC)-derived ChLNs (cholinergic-like neurons) models from a PSEN1 E280A patient and a healthy volunteer (as a control sample). Post-mortem analyses of the temporal and frontal cortices and hippocampus from FAD PSEN1 E280A patients revealed strong intracellular co-localization of sulfonic DJ-1 and iAβ42, which was absent in control samples. To validate these findings, we generated cerebral organoids (COs) from an iPSCs PSEN1 E280A FAD patient and a healthy donor. In these organoids, we observed the co-localization of oxidized DJ-1 and Aβ42 in the absence of extracellular fibrils or plaques, as confirmed by BTA-1 staining. To further support these observations, 2D iPSC PSEN1 E280A-derived ChLNs cultures showed that intracellular Aβ42 accumulates progressively in direct correlation with increasing DJ-1 oxidation, as demonstrated by immunofluorescence microscopy and Western blotting analysis. These results indicate that DJ-1 oxidation accompanies the earliest intracellular stages of Aβ42 pathology. Furthermore, complementary in silico molecular docking analysis revealed a higher affinity between Aβ42 and oxidized sulfonic DJ-1 (DJ-1 1Cys¹⁰⁶-SO₃) compared to sulfenic (DJ-1 Cys¹⁰⁶-SOH) or sulfinic acid (DJ-1 Cys¹⁰⁶-SO₂H) forms. Likewise, ELISA tests and seeding assays confirmed that oxidized DJ-1 binds to and decelerates Aβ42 aggregation kinetics. Together, our results identify DJ-1 oxidation as a critical molecular event in the accumulation of iAβ42 in FAD. These findings suggest that oxidized DJ-1 represents not only a potential early biomarker of intracellular pathology but also a pharmacological target. Preventing the oxidation of DJ-1 or its pathological aggregation could provide new biomarkers and therapeutic strategies for reducing the intracellular accumulation of Aβ42 and neurodegeneration in FAD.

Familial Alzheimer’s disease (FAD) is a progressive neurodegenerative disorder associated with loss of cholinergic neurons, intra- and extracellular accumulation of amyloid beta (iAβ, eAβ), hyperphosphorylated tau, and neuroinflammation i.e., reactive glia. The precise contribution of astrocytes to neurodegeneration in FAD is still incompletely understood. Therefore, there is a need for a reliable and simple in vitro cell culture model of neuroinflammation. The aim of the present investigation was to determine the pathophysiological behavior of astrocyte-like cells (ALCs) derived from wild-type (WT) and PSEN1 E280A menstrual stromal cells (MenSCs). We found that WT and PSEN1 E280A MenSCs displayed similar cellular mesenchymal lineage markers and similar capacity to differentiate into mesenchymal lineage osteocytes, adipocytes, and chondrocytes. In addition, WT and mutant ALCs exhibited similar percentages of astrocyte lineage markers such as GFAP+ (~ 90%) and S100β+ (70%). However, compared to WT ALCs, PSEN1 E280A ALCs (i) showed a complete lack of response to Glu-induced Ca ²⁺ influx; (ii) presented a high percentage of iAβ-positive cells (+ 3900% increase); (iii) exhibited a markedly dysfunctional phagocytosis activity (-70% reduction); (iv) revealed a dysregulation of apoptotic body clearance (-64% Lysotracker+/PI + cells); (v) showed abnormal accumulation of autophagosomes (+ 543% LC3-II + cells); (vi) revealed an increased TNF-α-induced activation of NF-κB (+ 37% MFI increase); (vii) an increased secretion of IL-6 (+ 160%), and (viii) secreted high amounts of Aβ42 (extracellular Aβ42, ~ 57 pg/mL). Notably, there was no change in TREM2 expression, mitochondrial membrane potential (ΔΨm, 100%), or induced cleaved caspase 3 (CC3, 0%) in PSEN1 E280A and WT ALCs. These findings highlight the importance of MenSCs-derived ALCs as a promising model to study the pathophysiological basis of FAD. Graphical abstract

Familial Alzheimer’s disease (FAD) is an accelerated form of dementia that affects cholinergic neurons. Unfortunately, despite several efforts, no single drug or treatment has shown complete efficacy. Therefore, it is imperative to find effective therapeutic agents. Previously, it has been shown that the PSEN1 I416T variant induces FAD-like neuropathology in cholinergic-like neurons (ChLNs), which is characterized by intracellular accumulation of the A(beta) peptide, oxidation of the stress sensor protein DJ-1, abnormal phosphorylation of the TAU protein at residue S202/T205, loss of mitochondrial membrane potential (deltapsi(m)), and activation of the pro-apoptotic proteins TP53, c-JUN, PUMA, and cleaved caspase 3 (CC3). We report for the first time that PSEN1 I416T induces (i) abnormal phosphorylation of LRRK2 kinase at residue S935, concomitant with (ii) phosphorylated alpha-synuclein (aSyn) at residue S129 and abnormal accumulation of autophagosomes and atypical increase (alkaline) in vesicular lysosomal pH, thereby provoking a profound alteration in autophagy in ChLNs. Here, we also demonstrate for the first time that the potent LRRK2 inhibitor PF-06447475 (hereafter referred to as PF475) almost completely attenuated PSEN1 I416T-induced proteinopathy, oxidative stress (OS), and apoptosis and restored autophagy to an activity comparable to untreated wild-type (WT) ChLNs. Overall, PF475 restored the survival of mutant ChLNs. Since LRRK2 kinase inhibitors are promising therapeutic drugs for the treatment of Parkinson’s disease (PD), our findings suggest that LRRK2 may also be a potential target for therapeutic intervention in FAD.

In familial Alzheimer’s disease (FAD), presenilin 1 (PSEN1) E280A cholinergic-like neurons (ChLNs) induce aberrant secretion of extracellular amyloid beta (eAβ). How PSEN1 E280A ChLNs-eAβ affects microglial activity is still unknown. We obtained induced microglia-like cells (iMG) from human peripheral blood cells (hPBCs) in a 15-day differentiation process to investigate the effect of bolus addition of Aβ42, PSEN1 E280A cholinergic-like neuron (ChLN)-derived culture supernatants, and PSEN1 E280A ChLNs on wild type (WT) iMG, PSEN1 E280A iMG, and sporadic Alzheimer’s disease (SAD) iMG. We found that WT iMG cells, when challenged with non-cellular (e.g., lipopolysaccharide, LPS) or cellular (e.g., Aβ42, PSEN1 E280A ChLN-derived culture supernatants) microenvironments, closely resemble primary human microglia in terms of morphology (resembling an “amoeboid-like phenotype”), expression of surface markers (Ionized calcium-binding adapter molecule 1, IBA-1; transmembrane protein 119, TMEM119), phagocytic ability (high pHrodo™ Red E. coli BioParticles™ phagocytic activity), immune metabolism (i.e., high generation of reactive oxygen species, ROS), increase in mitochondrial membrane potential (ΔΨm), response to ATP-induced transient intracellular Ca²⁺ influx, cell polarization (cluster of differentiation 68 (CD68)/CD206 ratio: M1 phenotype), cell migration activity according to the scratch wound assay, and especially in their inflammatory response (secretion of cytokine interleukin-6, IL-6; Tumor necrosis factor alpha, TNF-α). We also found that PSEN1 E280A and SAD iMG are physiologically unresponsive to ATP-induced Ca²⁺ influx, have reduced phagocytic activity, and diminished expression of Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) protein, but when co-cultured with PSEN1 E280A ChLNs, iMG shows an increase in pro-inflammatory phenotype (M1) and secretes high levels of cytokines IL-6 and TNF-α. As a result, PSEN1 E280A and SAD iMG induce apoptosis in PSEN1 E280A ChLNs as evidenced by abnormal phosphorylation of protein TAU at residue T205 and cleaved caspase 3 (CC3). Taken together, these results suggest that PSEN1 E280A ChLNs initiate a vicious cycle between damaged neurons and M1 phenotype microglia, resulting in excessive ChLN death. Our findings provide a suitable platform for the exploration of novel therapeutic approaches for the fight against FAD.

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder for which there are currently no curative therapies. Therefore, the need for innovative treatments for this illness is critical. The glucosylceramidase beta 1 (GBA1) and leucine-rich repeated kinase 2 (LRRK2) genes have been postulated as potential genetically defined drug targets. We report for the first time that the LRRK2 inhibitor PF-06447475 (PF-475) not only restores GCase enzyme activity, but also increases mitochondrial membrane potential, significantly decreases DJ-1 Cys106-SO ₃ , reduces lysosome accumulation, and diminishes cleaved caspase-3 (CC3) in GBA1 K198E fibroblasts. Furthermore, in addition to a significant reduction in p-Ser935 LRRK2 kinase, we found that PF-475 reduced p-Thr73 RAB 10 and p-Ser129 α-Syn in mutant skin fibroblasts. In addition, we found that the GCase activator GCA (NCGC00188758) increased GCase activity and decreased lysosomal accumulation, but did not affect p-Ser935 LRRK2, ∆Ψm, p-Ser129 α-Syn, DJ-1 Cys106-SO ₃ , or CC3 in K198E GBA1 fibroblasts. The GCase inhibitor conduritol-β-epoxide (CBE), used as an internal control, significantly reduced GCase and left the other pathological markers largely unaltered in GBA1 K198E, but reduced GCase and increased the accumulation of lysosomes only in WT GBA1 fibroblasts. Taken together, these results suggest that LRRK2 is a critical signaling kinase in the pathogenic mechanism associated with the lysosomal GBA1/GCase K198E variant. Our findings suggest that the use of LRRK2 inhibitors in PD patients with GBA1 mutations, such as K198E, may be effective in reversing GBA1/GCase deficiency, autophagy impairment, oxidative stress, and neuronal death. Graphical Abstract

Familial Alzheimer’s disease (FAD) is a complex multifactorial disorder clinically characterized by cognitive impairment and memory loss. Pathologically, FAD is characterized by intracellular accumulation of the protein fragment Aβ42 (iAβ), hyperphosphorylated microtubule-associated protein TAU (p-TAU), and extensive degeneration of basal forebrain cholinergic neurons of the nucleus basalis of Meynert (NbM) and the medial septal nucleus (MSN), mainly caused by mutations in the amyloid precursor protein (APP), presenilin 1 (PSEN1), and PSEN2 gene. Since the dopaminergic system may contribute to FAD symptoms, alterations in the nigro-hippocampal pathway may be associated with cognitive impairment in FAD. Interestingly, p-α-synuclein (p-α-Syn), Aβ, and p-TAU have been found to coexist in vulnerable regions of postmortem AD brains. However, the mechanism by which Aβ, p-TAU, and α-Syn coexist in DAergic neurons in AD brains has not been determined. We generated PSEN1 I416T dopaminergic-like neurons (DALNs) from I416T menstrual stromal cells (MenSCs) in NeuroForsk 2.0 medium for 7 days and then cultured them in minimal culture medium (MCm) for another 4 days. On day 11, DALNs were analyzed for molecular and pathological markers by flow cytometry and fluorescence microscopy. We found that mutant DALNs showed increased accumulation of iAβ as well as increased phosphorylation of TAU at S202/T205 compared to WT DALNs. Thus, mutant DALNs exhibited typical pathological hallmarks of Alzheimer’s disease. Furthermore, PSEN1 I416T DALNs showed concomitant signs of OS as evidenced by the appearance of oxidized sensor protein DJ-1 (i.e., DJ-1C106-SO₃) and apoptotic markers TP53, pS63-c-JUN, PUMA, and cleavage caspase 3 (CC3). Notably, these DALNs exhibited PD-associated proteins such as intracellular accumulation of α-Syn (detected as aggregates of pS129-α-Syn) and phosphorylation of LRRK2 kinase at residue S935. In addition, mutant DALNs showed a 17.16- and 6.17-fold decrease in DA-induced Ca²⁺ flux, compared to WT DALNs. These observations suggest that iAβ and p-TAU, together with p-α-Syn, and p-LRRK2 kinase, may damage DAergic neurons and thereby contribute to the exacerbation of neuropathologic processes in FAD. Remarkably, the LRRK2 inhibitor PF-06447475 (PF-475) significantly reversed PSEN1 I416T-induced neuropathological markers in DAergic neurons. PF-465 inhibitor reduced iAβ, oxDJ-1C106-SO₃, and p-TAU. In addition, this inhibitor reduced pS935-LRRK2, pS129-αSYN, pS63-c-JUN, and CC3. We conclude that the observed neuroprotective effects of PF-475 are due to direct inhibition of LRRK2 activity and that the LRRK2 protein is upstream of the molecular cascade of apoptosis and proteinopathy. Our results suggest that PF-475 is an effective neuroprotective agent against endogenous PSEN1 I416T-induced neurotoxicity in DALNs coexisting with Parkinson’s disease markers. Therefore, PF-475 may be of great therapeutic value in FAD.