Parkinson’s disease (PD) is characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta and by pathological accumulation of misfolded alpha-synuclein (α-syn) and other aggregation-prone proteins (Lewy bodies). Multiple, intersecting mechanisms contribute to neurodegeneration in PD, including proteasomal dysfunction, mitochondrial stress, impaired dopamine release, and oxidative damage.
Recent mechanistic and genetic studies have highlighted lysosomal dysfunction as a central contributor to PD pathogenesis. Lysosomes maintain cellular proteostasis by degrading macromolecules via acidic hydrolases (eg, cathepsins) and participate in autophagy, endocytosis, and phagocytosis. Because large protein aggregates such as α-syn are primarily processed by the lysosomal-autophagy system, disruptions in lysosomal homeostasis can impair clearance of aggregation-prone proteins and promote neuronal injury.
Progranulin (PGRN) is a widely expressed growth factor that supports lysosomal function by enhancing lysosomal enzyme activity, including cathepsin D. PGRN is secreted by microglia in the central nervous system and is internalized and trafficked to lysosomes in neurons and glia. PGRN deficiency impairs microglial function and promotes neurodegeneration, whereas increasing PGRN levels in animal models enhances neuronal survival and reduces oxidative stress. The lysosomal delivery of PGRN depends on interactions with sorting receptors such as sortilin and with Prosaposin (PSAP). PSAP itself is a lysosomal regulatory protein and precursor of saposins; loss of PSAP diminishes neuronal PGRN and exacerbates lysosomal dysfunction. Collectively, the PGRN/PSAP complex is mechanistically implicated in lysosomal maintenance and clearance of proteins like α-syn, making it an appealing therapeutic target in PD.
AZP2006 (Ezeprogind) is a small, lysosomotropic molecule originally identified for its capacity to reduce amyloidogenic processing and protect neurons in other neurodegenerative disease models. Prior work demonstrated that AZP2006 binds the PGRN/PSAP complex and that its neuroprotective effects are lost without PGRN or PSAP, suggesting the compound restores lysosomal function through this interaction. AZP2006 is in clinical development for tauopathies, including Progressive Supranuclear Palsy, and was evaluated here for potential benefit in PD-relevant models.
The study evaluated AZP2006 in multiple PD-like systems that reproduce key pathological features: mitochondrial injury induced by toxins (6-OHDA, MPP+, rotenone), toxicity and spreading of α-syn preformed fibrils (PFF), and α-syn accumulation in neurons. Both in vitro (primary mesencephalic cultures with or without microglia; WT and PGRN-KO SH-SY5Y human neuroblastoma cells) and in vivo (aged mice injured with α-syn PFF) approaches were used to test AZP2006’s neuroprotective activity and its mechanistic link to lysosomal homeostasis and the PGRN/PSAP complex.
Primary mesencephalic neurons were prepared from embryonic rat or mouse midbrains and cultured with defined media; some protocols included microglial cells to assess microglia-dependent effects. Neuronal cultures were injured on day 6 with mitochondrial toxins (20 µM 6-OHDA, 4 µM MPP+, or 10 nM rotenone) in the presence of AZP2006 for defined intervals. For α-syn PFF experiments, cultures were exposed to a quantified α-syn preparation (containing monomers, oligomers, PFF and fibrils) at 250 nM for 48–96 h depending on microglial presence.
A PGRN (GRN gene) knockout (KO) SH-SY5Y cell line was generated using CRISPR-Cas9 and selected clones were banked. WT and PGRN-KO SH-SY5Y cells were differentiated with retinoic acid before intoxication. MPP+ concentrations were adjusted between WT (1 mM) and KO (500 µM) to produce comparable survival after intoxication. AZP2006 was administered as a co-treatment to probe PGRN-dependent effects.
Human α-syn peptide was aggregated into a characterized preparation containing monomeric forms, oligomers, PFFs and fibrils by agitation at 37 °C for 3 days. Cultures were exposed to this α-syn mix to model chronic α-syn–driven toxicity and spreading, with AZP2006 present during exposure periods.
Functional lysosomes were assessed in live SH-SY5Y cells using a fluorescent acidic-vesicle dye (PhenoVue 577), where increased fluorescence indicated enhanced lysosomal function. Fixed-cell immunostaining targeted markers including tyrosine hydroxylase (TH) for dopaminergic neurons, α-syn, LAMP2 for lysosomes, LC3b for autophagy, PSD95 for synapses, and OX-41 for microglial activation. High-content automated microscopy acquired multiple fields per well and quantitative image analysis measured TH(+) neuron counts, neurite length, areas of cytoplasmic α-syn, LAMP2(+) vesicles, overlap between α-syn and LAMP2, LC3b vesicles, PhenoVue(+) area, and microglial activation area.
AZP2006 reduced neuronal damage induced by acute mitochondrial toxins (6-OHDA, MPP+, rotenone) and attenuated chronic toxicity from α-syn PFF exposure. Treatment effects included protection of TH(+) neurons and neurite networks, reduction of α-syn accumulation in neuronal cytoplasm, and decreases in microglial activation markers when cultures included microglia. Live-cell lysosomal dye intensity and LAMP2-associated readouts supported restoration of lysosomal function with AZP2006 treatment in these models.
The protective in vitro observations translated in vivo: AZP2006 treatment protected dopaminergic neurons and reduced α-syn accumulation in aged mice injured with α-syn PFF. The study reports that AZP2006’s effects were associated with markers consistent with improved lysosomal homeostasis in these animals.
Using lysosomal markers, PGRN-KO cell lines, and prior evidence that AZP2006 requires PGRN/PSAP for effect, the study provides mechanistic support that AZP2006 mediates neuroprotection by restoring lysosomal homeostasis through interaction with the PGRN/PSAP complex. Where PGRN was absent or neutralized, AZP2006’s neuroprotective actions were attenuated, indicating dependence on PGRN-mediated pathways.
All experiments followed relevant animal care guidelines. Funding was provided by Alzprotect and Neuro-Sys; Alzprotect employed two coauthors and contracted Neuro-Sys to perform experiments. Philippe Verwaerde, CEO of Alzprotect, received a related grant and is a coauthor; the funding and company involvement are declared as competing interests. The article states that all relevant data are within the manuscript and supporting information. Specific quantitative results, statistical details, and full datasets are reported in the original manuscript and supporting files.
The data support a strategy of enhancing PGRN and PSAP levels or activity to restore lysosomal homeostasis and mitigate α-syn–related pathology. AZP2006, by targeting the PGRN/PSAP complex, represents a candidate approach for neuroprotection in PD and related proteinopathies. Further investigation, including detailed mechanistic work and clinical evaluation, is warranted to define therapeutic potential and safety in PD populations.