Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production.

Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS-based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III, and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits-including NDUFA8, NDUFB4, and NDUFS8-decreased complex I activity, mitochondria-derived ATP, and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

Quantitative Proteomic Analysis Reveals apoE4-Dependent Phosphorylation of the Actin-Regulating Protein VASP.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease. While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here, we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in vasodilator-stimulated phosphoprotein (VASP) S235 phosphorylation in a protein kinase A (PKA)-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.

Suppressors of superoxide production from mitochondrial complex III.

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

Astrocytes in selective vulnerability to neurodegenerative disease.

Selective vulnerability of specific brain regions and cell populations is a hallmark of neurodegenerative disorders. Mechanisms of selective vulnerability involve neuronal heterogeneity, functional specializations, and differential sensitivities to stressors and pathogenic factors. In this review we discuss the growing body of literature suggesting that, like neurons, astrocytes are heterogeneous and specialized, respond to and integrate diverse inputs, and induce selective effects on brain function. In disease, astrocytes undergo specific, context-dependent changes that promote different pathogenic trajectories and functional outcomes. We propose that astrocytes contribute to selective vulnerability through maladaptive transitions to context-divergent phenotypes that impair specific brain regions and functions. Further studies on the multifaceted roles of astrocytes in disease may provide new therapeutic approaches to enhance resilience against neurodegenerative disorders.

Hippocampal astrocytes induce sex-dimorphic effects on memory.

Astrocytic receptors influence cognitive function and can promote behavioral deficits in disease. These effects may vary based on variables such as biological sex, but it is not known if the effects of astrocytic receptors are dependent on sex. We leveraged in vivo gene editing and chemogenetics to examine the roles of astrocytic receptors in spatial memory and other processes. We show that reductions in metabotropic glutamate receptor 3 (mGluR3), the main astrocytic glutamate receptor in adults, impair memory in females but enhance memory in males. Similarly, increases in astrocytic mGluR3 levels have sex-dependent effects and enhance memory in females. mGluR3 manipulations also alter spatial search strategies during recall in a sex-specific manner. In addition, acute chemogenetic stimulation of Gi/o-coupled or Gs-coupled receptors in hippocampal astrocytes induces bidirectional and sex-dimorphic effects on memory. Thus, astrocytes are sex-dependent modulators of cognitive function and may promote sex differences in aging and disease.

A refined analysis of superoxide production by mitochondrial sn-glycerol 3-phosphate dehydrogenase.

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H(2)O(2) at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H(2)O(2) production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H(2)O(2). Importantly, we demonstrate that a substantial portion of H(2)O(2) production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.

Mitochondrial complex II can generate reactive oxygen species at high rates in both the forward and reverse reactions.

Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.

A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells.

Here, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESC-derived neural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATP supply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77% of their ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both in absolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs is consistent with the expression levels of the mitochondrial gene regulators peroxisome-proliferator-activated receptor γ coactivator (PGC)-1α, PGC-1ß and receptor-interacting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types. Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energy-demanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higher rate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media. Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somatic cell types, and supports important roles for mitochondria and secretion in hESC biology.

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production.

Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS- based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits-including NDUFA8, NDUFB4, and NDUFS8-decreased complex I activity, mitochondria-derived ATP and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

Astrocytic TDP-43 dysregulation impairs memory by modulating antiviral pathways and interferon-inducible chemokines.

Transactivating response region DNA binding protein 43 (TDP-43) pathology is prevalent in dementia, but the cell type-specific effects of TDP-43 pathology are not clear, and therapeutic strategies to alleviate TDP-43-linked cognitive decline are lacking. We found that patients with Alzheimer's disease or frontotemporal dementia have aberrant TDP-43 accumulation in hippocampal astrocytes. In mouse models, induction of widespread or hippocampus-targeted accumulation in astrocytic TDP-43 caused progressive memory loss and localized changes in antiviral gene expression. These changes were cell-autonomous and correlated with impaired astrocytic defense against infectious viruses. Among the changes, astrocytes had elevated levels of interferon-inducible chemokines, and neurons had elevated levels of the corresponding chemokine receptor CXCR3 in presynaptic terminals. CXCR3 stimulation altered presynaptic function and promoted neuronal hyperexcitability, akin to the effects of astrocytic TDP-43 dysregulation, and blockade of CXCR3 reduced this activity. Ablation of CXCR3 also prevented TDP-43-linked memory loss. Thus, astrocytic TDP-43 dysfunction contributes to cognitive impairment through aberrant chemokine-mediated astrocytic-neuronal interactions.

Neuronal RING finger protein 11 (RNF11) regulates canonical NF-κB signaling.

BACKGROUND: The RING domain-containing protein RING finger protein 11 (RNF11) is a member of the A20 ubiquitin-editing protein complex and modulates peripheral NF-κB signaling. RNF11 is robustly expressed in neurons and colocalizes with a population of α-synuclein-positive Lewy bodies and neurites in Parkinson disease patients. The NF-κB pathway has an important role in the vertebrate nervous system, where the absence of NF-κB activity during development can result in learning and memory deficits, whereas chronic NF-κB activation is associated with persistent neuroinflammation. We examined the functional role of RNF11 with respect to canonical NF-κB signaling in neurons to gain understanding of the tight association of inflammatory pathways, including NF-κB, with the pathogenesis of neurodegenerative diseases. METHODS AND RESULTS: Luciferase assays were employed to assess NF-κB activity under targeted short hairpin RNA (shRNA) knockdown of RNF11 in human neuroblastoma cells and murine primary neurons, which suggested that RNF11 acts as a negative regulator of canonical neuronal NF-κB signaling. These results were further supported by analyses of p65 translocation to the nucleus following depletion of RNF11. Coimmunoprecipitation experiments indicated that RNF11 associates with members of the A20 ubiquitin-editing protein complex in neurons. Site-directed mutagenesis of the myristoylation domain, which is necessary for endosomal targeting of RNF11, altered the impact of RNF11 on NF-κB signaling and abrogated RNF11's association with the A20 ubiquitin-editing protein complex. A partial effect on canonical NF-κB signaling and an association with the A20 ubiquitin-editing protein complex was observed with mutagenesis of the PPxY motif, a proline-rich region involved in Nedd4-like protein interactions. Last, shRNA-mediated reduction of RNF11 in neurons and neuronal cell lines elevated levels of monocyte chemoattractant protein 1 and TNF-α mRNA and proteins, suggesting that NF-κB signaling and associated inflammatory responses are aberrantly regulated in the absence of RNF11. CONCLUSIONS: Our findings support the hypothesis that, in the nervous system, RNF11 negatively regulates canonical NF-κB signaling. Reduced or functionally compromised RNF11 could influence NF-κB-associated neuronal functions, including exaggerated inflammatory responses that may have implications for neurodegenerative disease pathogenesis and progression.

Impaired mitochondrial trafficking in Huntington's disease.

Impaired mitochondrial function has been well documented in Huntington's disease. Mutant huntingtin is found to affect mitochondria via various mechanisms including the dysregulation of gene transcription and impairment of mitochondrial function or trafficking. The lengthy and highly branched neuronal processes constitute complex neural networks in which there is a large demand for mitochondria-generated energy. Thus, the impaired mitochondrial trafficking in neuronal cells may play an important role in the selective neuropathology of Huntington's disease. Here we discuss the evidence for the effect of the Huntington's disease protein huntingtin on the intracellular trafficking of mitochondria and the involvement of this defective trafficking in the pathogenesis of Huntington's disease.

Accumulation of N-terminal mutant huntingtin in mouse and monkey models implicated as a pathogenic mechanism in Huntington's disease.

A number of mouse models expressing mutant huntingtin (htt) with an expanded polyglutamine (polyQ) domain are useful for studying the pathogenesis of Huntington's disease (HD) and identifying appropriate therapies. However, these models exhibit neurological phenotypes that differ in their severity and nature. Understanding how transgenic htt leads to variable neuropathology in animal models would shed light on the pathogenesis of HD and help us to choose HD models for investigation. By comparing the expression of mutant htt at the transcriptional and protein levels in transgenic mice expressing N-terminal or full-length mutant htt, we found that the accumulation and aggregation of mutant htt in the brain is determined by htt context. HD mouse models demonstrating more severe phenotypes show earlier accumulation of N-terminal mutant htt fragments, which leads to the formation of htt aggregates that are primarily present in neuronal nuclei and processes, as well as glial cells. Similarly, transgenic monkeys expressing exon-1 htt with a 147-glutamine repeat (147Q) died early and showed abundant neuropil aggregates in swelling neuronal processes. Fractionation of HD150Q knock-in mice brains revealed an age-dependent accumulation of N-terminal mutant htt fragments in the nucleus and synaptosomes, and this accumulation was most pronounced in the striatum due to decreased proteasomal activity. Our findings suggest that the neuropathological phenotypes of HD stem largely from the accumulation of N-terminal mutant htt fragments and that this accumulation is determined by htt context and cell-type-dependent clearance of mutant htt.

The use of site-specific suppressors to measure the relative contributions of different mitochondrial sites to skeletal muscle superoxide and hydrogen peroxide production.

Reactive oxygen species are important signaling molecules crucial for muscle differentiation and adaptation to exercise. However, their uncontrolled generation is associated with an array of pathological conditions. To identify and quantify the sources of superoxide and hydrogen peroxide in skeletal muscle we used site-specific suppressors (S1QELs, S3QELs and NADPH oxidase inhibitors). We measured the rates of hydrogen peroxide release from isolated rat muscle mitochondria incubated in media mimicking the cytosol of intact muscle. By measuring the extent of inhibition caused by the addition of different site-specific suppressors of mitochondrial superoxide/hydrogen peroxide production (S1QELs for site IQ and S3QELs for site IIIQo), we determined the contributions of these sites to the total signal. In media mimicking resting muscle, their contributions were each 12-18%, consistent with a previous method. In C2C12 myoblasts, site IQ contributed 12% of cellular hydrogen peroxide production and site IIIQo contributed about 30%. When C2C12 myoblasts were differentiated to myotubes, hydrogen peroxide release increased five-fold, and the proportional contribution of site IQ doubled. The use of S1QELs and S3QELs is a powerful new way to measure the relative contributions of different mitochondrial sites to muscle hydrogen peroxide production under different conditions. Our results show that mitochondrial sites IQ and IIIQo make a substantial contribution to superoxide/hydrogen peroxide production in muscle mitochondria and C2C12 myoblasts. The total hydrogen peroxide release rate and the relative contribution of site IQ both increase substantially upon differentiation to myotubes.

N-terminal mutant huntingtin associates with mitochondria and impairs mitochondrial trafficking.

Huntington's disease (HD) is caused by polyglutamine (polyQ) expansion in huntingtin (htt), a large (350 kDa) protein that localizes predominantly to the cytoplasm. Proteolytic cleavage of mutant htt yields polyQ-containing N-terminal fragments that are prone to misfolding and aggregation. Disease progression in HD transgenic models correlates with age-related accumulation of soluble and aggregated forms of N-terminal mutant htt fragments, suggesting that multiple forms of mutant htt are involved in the selective neurodegeneration in HD. Although mitochondrial dysfunction is implicated in the pathogenesis of HD, it remains unclear which forms of cytoplasmic mutant htt associate with mitochondria to affect their function. Here we demonstrate that specific N-terminal mutant htt fragments associate with mitochondria in Hdh(CAG)150 knock-in mouse brain and that this association increases with age. The interaction between soluble N-terminal mutant htt and mitochondria interferes with the in vitro association of microtubule-based transport proteins with mitochondria. Mutant htt reduces the distribution and transport rate of mitochondria in the processes of cultured neuronal cells. Reduced ATP level was also found in the synaptosomal fraction isolated from Hdh(CAG)150 knock-in mouse brain. These findings suggest that specific N-terminal mutant htt fragments, before the formation of aggregates, can impair mitochondrial function directly and that this interaction may be a novel target for therapeutic strategies in HD.

Neuronal Apolipoprotein E4 Expression Results in Proteome-Wide Alterations and Compromises Bioenergetic Capacity by Disrupting Mitochondrial Function.

Apolipoprotein (apo) E4, the major genetic risk factor for Alzheimer's disease (AD), alters mitochondrial function and metabolism early in AD pathogenesis. When injured or stressed, neurons increase apoE synthesis. Because of its structural difference from apoE3, apoE4 undergoes neuron-specific proteolysis, generating fragments that enter the cytosol, interact with mitochondria, and cause neurotoxicity. However, apoE4's effect on mitochondrial respiration and metabolism is not understood in detail. Here we used biochemical assays and proteomic profiling to more completely characterize the effects of apoE4 on mitochondrial function and cellular metabolism in Neuro-2a neuronal cells stably expressing apoE4 or apoE3. Under basal conditions, apoE4 impaired respiration and increased glycolysis, but when challenged or stressed, apoE4-expressing neurons had 50% less reserve capacity to generate ATP to meet energy requirements than apoE3-expressing neurons. ApoE4 expression also decreased the NAD+/NADH ratio and increased the levels of reactive oxygen species and mitochondrial calcium. Global proteomic profiling revealed widespread changes in mitochondrial processes in apoE4 cells, including reduced levels of numerous respiratory complex subunits and major disruptions to all detected subunits in complex V (ATP synthase). Also altered in apoE4 cells were levels of proteins related to mitochondrial endoplasmic reticulum-associated membranes, mitochondrial fusion/fission, mitochondrial protein translocation, proteases, and mitochondrial ribosomal proteins. ApoE4-induced bioenergetic deficits led to extensive metabolic rewiring, but despite numerous cellular adaptations, apoE4-expressing neurons remained vulnerable to metabolic stress. Our results provide insights into potential molecular targets of therapies to correct apoE4-associated mitochondrial dysfunction and altered cellular metabolism.

To be or not to be pink(1): contradictory findings in an animal model for Parkinson's disease.

The PTEN-induced putative kinase 1 knockout rat (Pink1-/-) is marketed as an established model for Parkinson's disease, characterized by development of motor deficits and progressive degeneration of half the dopaminergic neurons in the substantia nigra pars compacta by 8 months of age. In this study, we address our concerns about the reproducibility of the Pink1-/- rat model. We evaluated behavioural function, number of substantia nigra dopaminergic neurons and extracellular striatal dopamine concentrations by in vivo microdialysis. Strikingly, we and others failed to observe any loss of dopaminergic neurons in 8-month-old male Pink1-/- rats. To understand this variability, we compared key experimental parameters from the different studies and provide explanations for contradictory findings. Although Pink1-/- rats developed behavioural deficits, these could not be attributed to nigrostriatal degeneration as there was no loss of dopaminergic neurons in the substantia nigra and no changes in neurotransmitter levels in the striatum. To maximize the benefit of Parkinson's disease research and limit the unnecessary use of laboratory animals, it is essential that the research community is aware of the limits of this animal model. Additional research is needed to identify reasons for inconsistency between Pink1-/- rat colonies and why degeneration in the substantia nigra is not consistent.

A FRET-based method to study protein thiol oxidation in histological preparations.

Cysteine residues in proteins have important biological roles. For example, disulfide bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage. Because organs are heterogeneous structures composed of diverse cell types, there is a compelling need for a histological approach to investigate thiol oxidation in situ in order to address the role of specific cell types in oxidative imbalance. Here we describe a fluorescence technique-which can be used in association with standard immunological staining procedures-to detect variations in disulfides in histological preparations. Moreover, by monitoring the fluorescence resonance energy transfer (FRET) between a labeled specific primary antibody and the thiol probe described here, this method can detect thiol oxidation in candidate proteins of interest. When applied to an animal model of Parkinson's disease, our technique demonstrated that thiol oxidation occurs selectively in the dopaminergic neurons of the substantia nigra, the same neurons that are lost selectively in the disease. In summary, this technique provides a new, powerful tool for providing further understanding of oxidative imbalance, a phenomenon common to many diseases.

Plate-Based Measurement of Superoxide and Hydrogen Peroxide Production by Isolated Mitochondria.

Superoxide and hydrogen peroxide produced by mitochondria play important roles in various physiological and pathological processes. This chapter describes a plate-based method to measure rates of superoxide and/or hydrogen peroxide production at specific sites in isolated mitochondria.

Long-term oral kinetin does not protect against α-synuclein-induced neurodegeneration in rodent models of Parkinson's disease.

Mutations in the mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) cause Parkinson's disease (PD), likely by disrupting PINK1's kinase activity. Although the mechanism(s) underlying how this loss of activity causes degeneration remains unclear, increasing PINK1 activity may therapeutically benefit some forms of PD. However, we must first learn whether restoring PINK1 function prevents degeneration in patients harboring PINK1 mutations, or whether boosting PINK1 function can offer protection in more common causes of PD. To test these hypotheses in preclinical rodent models of PD, we used kinetin triphosphate, a small-molecule that activates both wild-type and mutant forms of PINK1, which affects mitochondrial function and protects neural cells in culture. We chronically fed kinetin, the precursor of kinetin triphosphate, to PINK1-null rats in which PINK1 was reintroduced into their midbrain, and also to rodent models overexpressing α-synuclein. The highest tolerated dose of oral kinetin increased brain levels of kinetin for up to 6 months, without adversely affecting the survival of nigrostriatal dopamine neurons. However, there was no degeneration of midbrain dopamine neurons lacking PINK1, which precluded an assessment of neuroprotection and raised questions about the robustness of the PINK1 KO rat model of PD. In two rodent models of α-synuclein-induced toxicity, boosting PINK1 activity with oral kinetin provided no protective effects. Our results suggest that oral kinetin is unlikely to protect against α-synuclein toxicity, and thus fail to provide evidence that kinetin will protect in sporadic models of PD. Kinetin may protect in cases of PINK1 deficiency, but this possibility requires a more robust PINK1 KO model that can be validated by proof-of-principle genetic correction in adult animals.