Dysregulation of autophagic pathways leads to accumulation of abnormal proteins and damaged organelles in many neurodegenerative disorders, including Parkinson's disease (PD) and Lewy body dementia (LBD). Autophagy-related dysfunction may also trigger secretion and spread of misfolded proteins, such as α-synuclein (α-syn), the major misfolded protein found in PD/LBD. However, the mechanism underlying these phenomena remains largely unknown. Here, we used cell-based models, including human induced pluripotent stem cell-derived neurons, CRISPR/Cas9 technology, and male transgenic PD/LBD mice, plus vetting in human postmortem brains (both male and female). We provide mechanistic insight into this pathologic pathway. We find that aberrant S-nitrosylation of the autophagic adaptor protein p62 causes inhibition of autophagic flux and intracellular buildup of misfolded proteins, with consequent secretion resulting in cell-to-cell spread. Thus, our data show that pathologic protein S-nitrosylation of p62 represents a critical factor not only for autophagic inhibition and demise of individual neurons, but also for α-syn release and spread of disease throughout the nervous system.SIGNIFICANCE STATEMENT In Parkinson's disease and Lewy body dementia, dysfunctional autophagy contributes to accumulation and spread of aggregated α-synuclein. Here, we provide evidence that protein S-nitrosylation of p62 inhibits autophagic flux, contributing to α-synuclein aggregation and spread.

Human Alzheimer's disease (AD) brains and transgenic AD mouse models manifest hyperexcitability. This aberrant electrical activity is caused by synaptic dysfunction that represents the major pathophysiological correlate of cognitive decline. However, the underlying mechanism for this excessive excitability remains incompletely understood. To investigate the basis for the hyperactivity, we performed electrophysiological and immunofluorescence studies on hiPSC-derived cerebrocortical neuronal cultures and cerebral organoids bearing AD-related mutations in presenilin-1 or amyloid precursor protein vs. isogenic gene corrected controls. In the AD hiPSC-derived neurons/organoids, we found increased excitatory bursting activity, which could be explained in part by a decrease in neurite length. AD hiPSC-derived neurons also displayed increased sodium current density and increased excitatory and decreased inhibitory synaptic activity. Our findings establish hiPSC-derived AD neuronal cultures and organoids as a relevant model of early AD pathophysiology and provide mechanistic insight into the observed hyperexcitability.

Beginning at early stages, human Alzheimer's disease (AD) brains manifest hyperexcitability, contributing to subsequent extensive synapse loss, which has been linked to cognitive dysfunction. No current therapy for AD is disease-modifying. Part of the problem with AD drug discovery is that transgenic mouse models have been poor predictors of potential human treatment. While it is undoubtedly important to test drugs in these animal models, additional evidence for drug efficacy in a human context might improve our chances of success. Accordingly, in order to test drugs in a human context, we have developed a platform of physiological assays using patch-clamp electrophysiology, calcium imaging, and multielectrode array (MEA) experiments on human (h)iPSC-derived 2D cortical neuronal cultures and 3D cerebral organoids. We compare hiPSCs bearing familial AD mutations vs. their wild-type (WT) isogenic controls in order to characterize the aberrant electrical activity in such a human context. Here, we show that these AD neuronal cultures and organoids manifest increased spontaneous action potentials, slow oscillatory events (~1 Hz), and hypersynchronous network activity. Importantly, the dual-allosteric NMDAR antagonist NitroSynapsin, but not the FDA-approved drug memantine, abrogated this hyperactivity. We propose a novel model of synaptic plasticity in which aberrant neural networks are rebalanced by NitroSynapsin. We propose that hiPSC models may be useful for screening drugs to treat hyperexcitability and related synaptic damage in AD.

Metabolic syndrome (MetS) and Type 2 diabetes mellitus (T2DM) increase risk for Alzheimer's disease (AD). The molecular mechanism for this association remains poorly defined. Here we report in human and rodent tissues that elevated glucose, as found in MetS/T2DM, and oligomeric β-amyloid (Aβ) peptide, thought to be a key mediator of AD, coordinately increase neuronal Ca(2+) and nitric oxide (NO) in an NMDA receptor-dependent manner. The increase in NO results in S-nitrosylation of insulin-degrading enzyme (IDE) and dynamin-related protein 1 (Drp1), thus inhibiting insulin and Aβ catabolism as well as hyperactivating mitochondrial fission machinery. Consequent elevation in Aβ levels and compromise in mitochondrial bioenergetics result in dysfunctional synaptic plasticity and synapse loss in cortical and hippocampal neurons. The NMDA receptor antagonist memantine attenuates these effects. Our studies show that redox-mediated posttranslational modification of brain proteins link Aβ and hyperglycaemia to cognitive dysfunction in MetS/T2DM and AD.

Recent studies have pointed to protein S-nitrosylation as a critical regulator of cellular redox homeostasis. For example, S-nitrosylation of peroxiredoxin-2 (Prx2), a peroxidase widely expressed in mammalian neurons, inhibits both enzymatic activity and protective function against oxidative stress. Here, using in vitro and in vivo approaches, we identify a role and reaction mechanism of the reductase sulfiredoxin (Srxn1) as an enzyme that denitrosylates (thus removing -SNO) from Prx2 in an ATP-dependent manner. Accordingly, by decreasing S-nitrosylated Prx2 (SNO-Prx2), overexpression of Srxn1 protects dopaminergic neural cells and human-induced pluripotent stem cell (hiPSC)-derived neurons from NO-induced hypersensitivity to oxidative stress. The pathophysiological relevance of this observation is suggested by our finding that SNO-Prx2 is dramatically increased in murine and human Parkinson's disease (PD) brains. Our findings therefore suggest that Srxn1 may represent a therapeutic target for neurodegenerative disorders such as PD that involve nitrosative/oxidative stress.

A causal relationship between mitochondrial metabolic dysfunction and neurodegeneration has been implicated in synucleinopathies, including Parkinson disease (PD) and Lewy body dementia (LBD), but underlying mechanisms are not fully understood. Here, using human induced pluripotent stem cell (hiPSC)-derived neurons with mutation in the gene encoding α-synuclein (αSyn), we report the presence of aberrantly S-nitrosylated proteins, including tricarboxylic acid (TCA) cycle enzymes, resulting in activity inhibition assessed by carbon-labeled metabolic flux experiments. This inhibition principally affects α-ketoglutarate dehydrogenase/succinyl coenzyme-A synthetase, metabolizing α-ketoglutarate to succinate. Notably, human LBD brain manifests a similar pattern of aberrantly S-nitrosylated TCA enzymes, indicating the pathophysiological relevance of these results. Inhibition of mitochondrial energy metabolism in neurons is known to compromise dendritic length and synaptic integrity, eventually leading to neuronal cell death. Our evidence indicates that aberrant S-nitrosylation of TCA cycle enzymes contributes to this bioenergetic failure.

Mutations in PARK6 (PINK1) and PARK2 (Parkin) are linked to rare familial cases of Parkinson's disease (PD). Mutations in these genes result in pathological dysregulation of mitophagy, contributing to neurodegeneration. Here, we report that environmental factors causing a specific posttranslational modification on PINK1 can mimic these genetic mutations. We describe a molecular mechanism for impairment of mitophagy via formation of S-nitrosylated PINK1 (SNO-PINK1). Mitochondrial insults simulating age- or environmental-related stress lead to increased SNO-PINK1, inhibiting its kinase activity. SNO-PINK1 decreases Parkin translocation to mitochondrial membranes, disrupting mitophagy in cell lines and human-iPSC-derived neurons. We find levels of SNO-PINK1 in brains of α-synuclein transgenic PD mice similar to those in cell-based models, indicating the pathophysiological relevance of our findings. Importantly, SNO-PINK1-mediated deficits in mitophagy contribute to neuronal cell death. These results reveal a direct molecular link between nitrosative stress, SNO-PINK1 formation, and mitophagic dysfunction that contributes to the pathogenesis of PD.

In Alzheimer's disease (AD), dysfunctional mitochondrial metabolism is associated with synaptic loss, the major pathological correlate of cognitive decline. Mechanistic insight for this relationship, however, is still lacking. Here, comparing isogenic wild-type and AD mutant human induced pluripotent stem cell (hiPSC)-derived cerebrocortical neurons (hiN), evidence is found for compromised mitochondrial energy in AD using the Seahorse platform to analyze glycolysis and oxidative phosphorylation (OXPHOS). Isotope-labeled metabolic flux experiments revealed a major block in activity in the tricarboxylic acid (TCA) cycle at the α-ketoglutarate dehydrogenase (αKGDH)/succinyl coenzyme-A synthetase step, metabolizing α-ketoglutarate to succinate. Associated with this block, aberrant protein S-nitrosylation of αKGDH subunits inhibited their enzyme function. This aberrant S-nitrosylation is documented not only in AD-hiN but also in postmortem human AD brains versus controls, as assessed by two separate unbiased mass spectrometry platforms using both SNOTRAP identification of S-nitrosothiols and chemoselective-enrichment of S-nitrosoproteins. Treatment with dimethyl succinate, a cell-permeable derivative of a TCA substrate downstream to the block, resulted in partial rescue of mitochondrial bioenergetic function as well as reversal of synapse loss in AD-hiN. These findings have therapeutic implications that rescue of mitochondrial energy metabolism can ameliorate synaptic loss in hiPSC-based models of AD.

Parkinson's disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn-antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.