High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

Manganese and Vanadium Co-Exposure Induces Severe Neurotoxicity in the Olfactory System: Relevance to Metal-Induced Parkinsonism.

Chronic environmental exposure to toxic heavy metals, which often occurs as a mixture through occupational and industrial sources, has been implicated in various neurological disorders, including Parkinsonism. Vanadium pentoxide (V2O5) typically presents along with manganese (Mn), especially in welding rods and high-capacity batteries, including electric vehicle batteries; however, the neurotoxic effects of vanadium (V) and Mn co-exposure are largely unknown. In this study, we investigated the neurotoxic impact of MnCl2, V2O5, and MnCl2-V2O5 co-exposure in an animal model. C57BL/6 mice were intranasally administered either de-ionized water (vehicle), MnCl2 (252 µg) alone, V2O5 (182 µg) alone, or a mixture of MnCl2 (252 µg) and V2O5 (182 µg) three times a week for up to one month. Following exposure, we performed behavioral, neurochemical, and histological studies. Our results revealed dramatic decreases in olfactory bulb (OB) weight and levels of tyrosine hydroxylase, dopamine, and 3,4-dihydroxyphenylacetic acid in the treatment groups compared to the control group, with the Mn/V co-treatment group producing the most significant changes. Interestingly, increased levels of α-synuclein expression were observed in the substantia nigra (SN) of treated animals. Additionally, treatment groups exhibited locomotor deficits and olfactory dysfunction, with the co-treatment group producing the most severe deficits. The treatment groups exhibited increased levels of the oxidative stress marker 4-hydroxynonenal in the striatum and SN, as well as the upregulation of the pro-apoptotic protein PKCδ and accumulation of glomerular astroglia in the OB. The co-exposure of animals to Mn/V resulted in higher levels of these metals compared to other treatment groups. Taken together, our results suggest that co-exposure to Mn/V can adversely affect the olfactory and nigral systems. These results highlight the possible role of environmental metal mixtures in the etiology of Parkinsonism.

Mito-metformin protects against mitochondrial dysfunction and dopaminergic neuronal degeneration by activating upstream PKD1 signaling in cell culture and MitoPark animal models of Parkinson's disease.

Impaired mitochondrial function and biogenesis have strongly been implicated in the pathogenesis of Parkinson's disease (PD). Thus, identifying the key signaling mechanisms regulating mitochondrial biogenesis is crucial to developing new treatment strategies for PD. We previously reported that protein kinase D1 (PKD1) activation protects against neuronal cell death in PD models by regulating mitochondrial biogenesis. To further harness the translational drug discovery potential of targeting PKD1-mediated neuroprotective signaling, we synthesized mito-metformin (Mito-Met), a mitochondria-targeted analog derived from conjugating the anti-diabetic drug metformin with a triphenylphosphonium functional group, and then evaluated the preclinical efficacy of Mito-Met in cell culture and MitoPark animal models of PD. Mito-Met (100-300 nM) significantly activated PKD1 phosphorylation, as well as downstream Akt and AMPKα phosphorylation, more potently than metformin, in N27 dopaminergic neuronal cells. Furthermore, treatment with Mito-Met upregulated the mRNA and protein expression of mitochondrial transcription factor A (TFAM) implying that Mito-Met can promote mitochondrial biogenesis. Interestingly, Mito-Met significantly increased mitochondrial bioenergetics capacity in N27 dopaminergic cells. Mito-Met also reduced mitochondrial fragmentation induced by the Parkinsonian neurotoxicant MPP+ in N27 cells and protected against MPP+-induced TH-positive neurite loss in primary neurons. More importantly, Mito-Met treatment (10 mg/kg, oral gavage for 8 week) significantly improved motor deficits and reduced striatal dopamine depletion in MitoPark mice. Taken together, our results demonstrate that Mito-Met possesses profound neuroprotective effects in both in vitro and in vivo models of PD, suggesting that pharmacological activation of PKD1 signaling could be a novel neuroprotective translational strategy in PD and other related neurocognitive diseases.

Epitranscriptomic Reader YTHDF2 Regulates SEK1( MAP2K4 )-JNK-cJUN Inflammatory Signaling in Astrocytes during Neurotoxic Stress.

As the most abundant glial cells in the CNS, astrocytes dynamically respond to neurotoxic stress, however, the key molecular regulators controlling the inflammatory status of these sentinels during neurotoxic stress have remained elusive. Herein, we demonstrate that the m6A epitranscriptomic mRNA modification tightly regulates the pro-inflammatory functions of astrocytes. Specifically, the astrocytic neurotoxic stresser, manganese (Mn), downregulated the m6A reader YTHDF2 in human and mouse astrocyte cultures and in the mouse brain. Functionally, YTHDF2 knockdown augmented, while its overexpression dampened, neurotoxic stress induced proinflammatory response, suggesting YTHDF2 serves as a key upstream regulator of inflammatory responses in astrocytes. Mechnistically, YTHDF2 RIP-sequencing identified MAP2K4 ( MKK4; SEK1) mRNA as a YTHDF2 target influencing inflammatory signaling. Our target validation revealed Mn-exposed astrocytes mediates proinflammatory response by activating the phosphorylation of SEK1, JNK, and cJUN signaling. Collectively, YTHDF2 serves a key upstream 'molecular switch' controlling SEK1( MAP2K4 )-JNK-cJUN proinflammatory signaling in astrocytes.

Therapeutic Potential of Targeting Prokineticin Receptors in Diseases.

The prokineticins (PKs) were discovered approximately 20 years ago as small peptides inducing gut contractility. Today, they are established as angiogenic, anorectic, and proinflammatory cytokines, chemokines, hormones, and neuropeptides involved in variety of physiologic and pathophysiological pathways. Their altered expression or mutations implicated in several diseases make them a potential biomarker. Their G-protein coupled receptors, PKR1 and PKR2, have divergent roles that can be therapeutic target for treatment of cardiovascular, metabolic, and neural diseases as well as pain and cancer. This article reviews and summarizes our current knowledge of PK family functions from development of heart and brain to regulation of homeostasis in health and diseases. Finally, the review summarizes the established roles of the endogenous peptides, synthetic peptides and the selective ligands of PKR1 and PKR2, and nonpeptide orthostatic and allosteric modulator of the receptors in preclinical disease models. The present review emphasizes the ambiguous aspects and gaps in our knowledge of functions of PKR ligands and elucidates future perspectives for PK research. SIGNIFICANCE STATEMENT: This review provides an in-depth view of the prokineticin family and PK receptors that can be active without their endogenous ligand and exhibits "constitutive" activity in diseases. Their non- peptide ligands display promising effects in several preclinical disease models. PKs can be the diagnostic biomarker of several diseases. A thorough understanding of the role of prokineticin family and their receptor types in health and diseases is critical to develop novel therapeutic strategies with safety concerns.

Aggregation-Inhibiting scFv-Based Therapies Protect Mice against AAV1/2-Induced A53T-α-Synuclein Overexpression.

To date, there is no cure for Parkinson's disease (PD). There is a pressing need for anti-neurodegenerative therapeutics that can slow or halt PD progression by targeting underlying disease mechanisms. Specifically, preventing the build-up of alpha-synuclein (αSyn) and its aggregated and mutated forms is a key therapeutic target. In this study, an adeno-associated viral vector loaded with the A53T gene mutation was used to induce rapid αSyn-associated PD pathogenesis in C57BL/6 mice. We tested the ability of a novel therapeutic, a single chain fragment variable (scFv) antibody with specificity only for pathologic forms of αSyn, to protect against αSyn-induced neurodegeneration, after unilateral viral vector injection in the substantia nigra. Additionally, polyanhydride nanoparticles, which provide sustained release of therapeutics with dose-sparing properties, were used as a delivery platform for the scFv. Through bi-weekly behavioral assessments and across multiple post-mortem immunochemical analyses, we found that the scFv-based therapies allowed the mice to recover motor activity and reduce overall αSyn expression in the substantia nigra. In summary, these novel scFv-based therapies, which are specific exclusively for pathological aggregates of αSyn, show early promise in blocking PD progression in a surrogate mouse PD model.

Microglial immune regulation by epigenetic reprogramming through histone H3K27 acetylation in neuroinflammation.

Epigenetic reprogramming is the ability of innate immune cells to form memories of environmental stimuli (priming), allowing for heightened responses to secondary stressors. Herein, we explored microglial epigenetic marks using the known inflammagen LPS as a memory priming trigger and Parkinsonian-linked environmental neurotoxic stressor manganese (Mn) as the secondary environmental trigger. To mimic physiological responses, the memory priming trigger LPS treatment was removed by triple-washing to allow the cells' acute inflammatory response to reset back before applying the secondary insult. Our results show that after the secondary Mn insult, levels of key proinflammatory markers, including nitrite release, iNOS mRNA and protein expression, Il-6, Il-α and cytokines were exaggerated in LPS-primed microglia. Our paradigm implies primed microglia retain immune memory that can be reprogrammed to augment inflammatory response by secondary environmental stress. To ascertain the molecular underpinning of this neuroimmune memory, we further hypothesize that epigenetic reprogramming contributes to the retention of a heightened immune response. Interestingly, Mn-exposed, LPS-primed microglia showed enhanced deposition of H3K27ac and H3K4me3 along with H3K4me1. We further confirmed the results using a PD mouse model (MitoPark) and postmortem human PD brains, thereby adding clinical relevance to our findings. Co-treatment with the p300/H3K27ac inhibitor GNE-049 reduced p300 expression and H3K27ac deposition, decreased iNOS, and increased ARG1 and IRF4 levels. Lastly, since mitochondrial stress is a driver of environmentally linked Parkinson's disease (PD) progression, we examined the effects of GNE-049 on primary trigger-induced mitochondrial stress. GNE-049 reduced mitochondrial superoxide, mitochondrial circularity and stress, and mitochondrial membrane depolarization, suggesting beneficial consequences of GNE-049 on mitochondrial function. Collectively, our findings demonstrate that proinflammatory primary triggers can shape microglial memory via the epigenetic mark H3K27ac and that inhibiting H3K27ac deposition can prevent primary trigger immune memory formation and attenuate subsequent secondary inflammatory responses.

Central and peripheral α-synuclein in Parkinson disease detected by seed amplification assay.

OBJECTIVES: Detection of α-synuclein aggregates by seed amplification is a promising Parkinson disease biomarker assay. Understanding intraindividual relationships of α-synuclein measures could inform optimal biomarker development. The objectives were to test accuracy of α-synuclein seed amplification assay in central (cerebrospinal fluid) and peripheral (submandibular gland) sources, compare to total α-synuclein measures, and investigate within-subject relationships. METHODS: The Systemic Synuclein Sampling Study aimed to characterize α-synuclein in multiple tissues and biofluids within Parkinson disease subjects (n = 59) and compared to healthy controls (n = 21). Motor and non-motor measures and dopamine transporter scans were obtained. Four measures of α-synuclein were compared: seed amplification assay in cerebrospinal fluid and formalin-fixed paraffin-embedded submandibular gland, total α-synuclein quantified in biofluids using enzyme-linked immunoassay, and aggregated α-synuclein in submandibular gland detected by immunohistochemistry. Accuracy of seed amplification assay for Parkinson disease diagnosis was examined and within-subject α-synuclein measures were compared. RESULTS: Sensitivity and specificity of α-synuclein seed amplification assay for Parkinson disease diagnosis was 92.6% and 90.5% in cerebrospinal fluid, and 73.2% and 78.6% in submandibular gland, respectively. 25/38 (65.8%) Parkinson disease participants were positive for both cerebrospinal fluid and submandibular gland seed amplification assay. Comparing accuracy for Parkinson disease diagnosis of different α-synuclein measures, cerebrospinal fluid seed amplification assay was the highest (Youden Index = 83.1%). 98.3% of all Parkinson disease cases had ≥1 measure of α-synuclein positive. INTERPRETATION: α-synuclein seed amplification assay (cerebrospinal fluid>submandibular gland) had higher sensitivity and specificity compared to total α-synuclein measures, and within-subject relationships of central and peripheral α-synuclein measures emerged.

New protocol for kinetic assay seeding ability recovery "KASAR" from formalin-fixed paraffin-embedded tissues.

The real-time quaking-induced conversion (RT-QuIC) alpha-synuclein (aSyn) protein kinetic seeding assay has been very useful for detecting pathological aggregates in various synucleinopathies including Parkinson's disease (PD). This biomarker assay relies on fresh frozen tissue to effectively seed and amplify aSyn aggregating protein. With vast repositories of formalin-fixed paraffin-embedded (FFPE) tissues, it is paramount to harness the power of kinetic assays to unlock the diagnostic potential of archived FFPE biospecimens. However, the major challenge posed by significantly reduced amplification of formalin-fixed tissues in the assay suggests that formalin fixation deterred monomer interaction with the sample seed and depressed subsequent protein aggregation. To overcome this challenge, we developed a kinetic assay seeding ability recovery (KASAR) protocol to maintain the integrity of the tissue and seeding protein. For this, we implemented a series of heating steps with the brain tissue suspended in a buffer composed of 500 mM tris-HCl (pH 7.5) and 0.02% SDS after the standard deparaffinization of the tissue sections. Initially, samples from seven human brain samples, including four samples from patients diagnosed with dementia with Lewy bodies (DLB) and three samples from healthy controls without DLB, were compared to fresh frozen samples under three different, but clinically common sample storage conditions: formalin-fixed, FFPE, and FFPE slices cut 5 µm thick. The KASAR protocol was able to recover seeding activity for all positive samples in all storage conditions. Next, 28 FFPE samples from the submandibular gland (SMG) of patients diagnosed with PD, incidental Lewy body disease (ILBD), or healthy controls were tested with 93% of results replicating when blinded. With samples of only a few milligrams, this protocol recovered the same quality of seeding in formalin-fixed tissue as fresh frozen tissue. Moving forward, protein aggregate kinetic assays, in conjunction with the KASAR protocol, can be used to understand and diagnose neurodegenerative diseases more comprehensively. Overall, our KASAR protocol unlocks and restores the seeding ability of formalin-fixed paraffin-embedded tissues for the amplification of biomarker protein aggregates in kinetic assays.

Impact of Environmental Risk Factors on Mitochondrial Dysfunction, Neuroinflammation, Protein Misfolding, and Oxidative Stress in the Etiopathogenesis of Parkinson's Disease.

As a prevalent progressive neurodegenerative disorder, Parkinson's disease (PD) is characterized by the neuropathological hallmark of the loss of nigrostriatal dopaminergic (DAergic) innervation and the appearance of Lewy bodies with aggregated α-synuclein. Although several familial forms of PD have been reported to be associated with several gene variants, most cases in nature are sporadic, triggered by a complex interplay of genetic and environmental risk factors. Numerous epidemiological studies during the past two decades have shown positive associations between PD and several environmental factors, including exposure to neurotoxic pesticides/herbicides and heavy metals as well as traumatic brain injury. Other environmental factors that have been implicated as potential risk factors for PD include industrial chemicals, wood pulp mills, farming, well-water consumption, and rural residence. In this review, we summarize the environmental toxicology of PD with the focus on the elaboration of chemical toxicity and the underlying pathogenic mechanisms associated with exposure to several neurotoxic chemicals, specifically 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat (PQ), dichloro-diphenyl-trichloroethane (DDT), dieldrin, manganese (Mn), and vanadium (V). Our overview of the current findings from cellular, animal, and human studies of PD provides information for possible intervention strategies aimed at halting the initiation and exacerbation of environmentally linked PD.

Environmental neurotoxic pesticide exposure induces gut inflammation and enteric neuronal degeneration by impairing enteric glial mitochondrial function in pesticide models of Parkinson's disease: Potential relevance to gut-brain axis inflammation in Parkinson's disease pathogenesis.

Despite the growing recognition that gastrointestinal (GI) dysfunction is prevalent in Parkinson's disease (PD) and occurs as a major prodromal symptom of PD, its cellular and molecular mechanisms remain largely unknown. Among the various types of GI cells, enteric glial cells (EGCs), which resemble astrocytes in structure and function, play a critical role in the pathophysiology of many GI diseases including PD. Thus, we investigated how EGCs respond to the environmental pesticides rotenone (Rot) and tebufenpyrad (Tebu) in cell and animal models to better understand the mechanism underlying GI abnormalities. Both Rot and Tebu induce dopaminergic neuronal cell death through complex 1 inhibition of the mitochondrial respiratory chain. We report that exposing a rat enteric glial cell model (CRL-2690 cells) to these pesticides increased mitochondrial fission and reduced mitochondrial fusion by impairing MFN2 function. Furthermore, they also increased mitochondrial superoxide generation and impaired mitochondrial ATP levels and basal respiratory rate. Measurement of LC3, p62 and lysosomal assays revealed impaired autolysosomal function in ECGs during mitochondrial stress. Consistent with our recent findings that mitochondrial dysfunction augments inflammation in astrocytes and microglia, we found that neurotoxic pesticide exposure also enhanced the production of pro-inflammatory factors in EGCs in direct correlation with the loss in mitochondrial mass. Finally, we show that pesticide-induced mitochondrial defects functionally impaired smooth muscle velocity, acceleration, and total kinetic energy in a mixed primary culture of the enteric nervous system (ENS). Collectively, our studies demonstrate for the first time that exposure to environmental neurotoxic pesticides impairs mitochondrial bioenergetics and activates inflammatory pathways in EGCs, further augmenting mitochondrial dysfunction and pro-inflammatory events to induce gut dysfunction. Our findings have major implications in understanding the GI-related pathogenesis and progression of environmentally linked PD.

Mitoapocynin Attenuates Organic Dust Exposure-Induced Neuroinflammation and Sensory-Motor Deficits in a Mouse Model.

Increased incidences of neuro-inflammatory diseases in the mid-western United States of America (USA) have been linked to exposure to agriculture contaminants. Organic dust (OD) is a major contaminant in the animal production industry and is central to the respiratory symptoms in the exposed individuals. However, the exposure effects on the brain remain largely unknown. OD exposure is known to induce a pro-inflammatory phenotype in microglial cells. Further, blocking cytoplasmic NOX-2 using mitoapocynin (MA) partially curtail the OD exposure effects. Therefore, using a mouse model, we tested a hypothesis that inhaled OD induces neuroinflammation and sensory-motor deficits. Mice were administered with either saline, fluorescent lipopolysaccharides (LPSs), or OD extract intranasally daily for 5 days a week for 5 weeks. The saline or OD extract-exposed mice received either a vehicle or MA (3 mg/kg) orally for 3 days/week for 5 weeks. We quantified inflammatory changes in the upper respiratory tract and brain, assessed sensory-motor changes using rotarod, open-field, and olfactory test, and quantified neurochemicals in the brain. Inhaled fluorescent LPS (FL-LPS) was detected in the nasal turbinates and olfactory bulbs. OD extract exposure induced atrophy of the olfactory epithelium with reduction in the number of nerve bundles in the nasopharyngeal meatus, loss of cilia in the upper respiratory epithelium with an increase in the number of goblet cells, and increase in the thickness of the nasal epithelium. Interestingly, OD exposure increased the expression of HMGB1, 3- nitrotyrosine (NT), IBA1, glial fibrillary acidic protein (GFAP), hyperphosphorylated Tau (p-Tau), and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-positive cells in the brain. Further, OD exposure decreased time to fall (rotarod), total distance traveled (open-field test), and olfactory ability (novel scent test). Oral MA partially rescued olfactory epithelial changes and gross congestion of the brain tissue. MA treatment also decreased the expression of HMGB1, 3-NT, IBA1, GFAP, and p-Tau, and significantly reversed exposure induced sensory-motor deficits. Neurochemical analysis provided an early indication of depressive behavior. Collectively, our results demonstrate that inhalation exposure to OD can cause sustained neuroinflammation and behavior deficits through lung-brain axis and that MA treatment can dampen the OD-induced inflammatory response at the level of lung and brain.

Clostridioides difficile Infection Dysregulates Brain Dopamine Metabolism.

Gastrointestinal illnesses and dysbiosis are among the most common comorbidities reported in patients with neurodevelopmental disorders. The manuscript reports that C. difficile infection (CDI), predisposed by antibiotic-induced gut dysbiosis, causes significant alterations in dopamine metabolism in major dopaminergic brain regions in mice (P < 0.05). In addition, C. difficile infected mice exhibited significantly reduced dopamine beta-hydroxylase (DBH) activity compared to controls (P < 0.01). Moreover, a significantly increased serum concentration of p-cresol, a DBH inhibiting gut metabolite produced by C. difficile, was also observed in C. difficile infected mice (P < 0.05). Therefore, this study suggests a potential mechanistic link between CDI and alterations in the brain dopaminergic axis. Such alterations may plausibly influence the precipitation and aggravation of dopamine dysmetabolism-associated neurologic diseases in infected patients. IMPORTANCE The gut-brain axis is thought to play a significant role in the development and manifestation of neurologic diseases. This study reports significant alterations in the brain dopamine metabolism in mice infected with C. difficile, an important pathogen that overgrows in the gut after prolonged antibiotic therapy. Such alterations in specific brain regions may have an effect on the precipitation or manifestation of neurodevelopmental disorders in humans.

Mechanistic Insights Into Gut Microbiome Dysbiosis-Mediated Neuroimmune Dysregulation and Protein Misfolding and Clearance in the Pathogenesis of Chronic Neurodegenerative Disorders.

The human gut microbiota is a complex, dynamic, and highly diverse community of microorganisms. Beginning as early as in utero fetal development and continuing through birth to late-stage adulthood, the crosstalk between the gut microbiome and brain is essential for modulating various metabolic, neurodevelopmental, and immune-related pathways. Conversely, microbial dysbiosis - defined as alterations in richness and relative abundances - of the gut is implicated in the pathogenesis of several chronic neurological and neurodegenerative disorders. Evidence from large-population cohort studies suggests that individuals with neurodegenerative conditions have an altered gut microbial composition as well as microbial and serum metabolomic profiles distinct from those in the healthy population. Dysbiosis is also linked to psychiatric and gastrointestinal complications - comorbidities often associated with the prodromal phase of Parkinson's disease (PD) and Alzheimer's disease (AD). Studies have identified potential mediators that link gut dysbiosis and neurological disorders. Recent findings have also elucidated the potential mechanisms of disease pathology in the enteric nervous system prior to the onset of neurodegeneration. This review highlights the functional pathways and mechanisms, particularly gut microbe-induced chronic inflammation, protein misfolding, propagation of disease-specific pathology, defective protein clearance, and autoimmune dysregulation, linking gut microbial dysbiosis and neurodegeneration. In addition, we also discuss how pathogenic transformation of microbial composition leads to increased endotoxin production and fewer beneficial metabolites, both of which could trigger immune cell activation and enteric neuronal dysfunction. These can further disrupt intestinal barrier permeability, aggravate the systemic pro-inflammatory state, impair blood-brain barrier permeability and recruit immune mediators leading to neuroinflammation and neurodegeneration. Continued biomedical advances in understanding the microbiota-gut-brain axis will extend the frontier of neurodegenerative disorders and enable the utilization of novel diagnostic and therapeutic strategies to mitigate the pathological burden of these diseases.

Functionalized polyanhydride nanoparticles for improved treatment of mitochondrial dysfunction.

Parkinson's disease (PD) is a devastating neurodegenerative disease affecting a large proportion of older adults. Exposure to pesticides like rotenone is a leading cause for PD. To reduce disease progression and prolong life expectancy, it is important to target disease mechanisms that contribute to dopaminergic neuronal atrophy, including mitochondrial dysfunction. Achieving targeted mitochondrial delivery is difficult for many therapeutics by themselves, necessitating higher therapeutic doses that could lead to toxicity. To minimize this adverse effect, targeted nano-carriers such as polyanhydride nanoparticles (NPs) can protect therapeutics from degradation and provide sustained release, enabling fewer administrations and lower therapeutic dose. This work expands upon the use of the polyanhydride NP platform for targeted drug delivery by functionalizing the polymer with a derivative of triphenylphosphonium called (3-carboxypropyl) triphenylphosphonium (CPTP) using a novel method that enables longer CPTP persistence on the NPs. The extent to which neurons internalized both nonfunctionalized and functionalized NPs was tested. Next, the efficacy of these nanoformulations in treating rotenone-induced mitochondrial dysfunction in the same cell line was evaluated using a novel neuroprotective drug, mito-metformin. CPTP functionalization significantly improved NP internalization by neuronal cells. This was correlated with significant protection by CPTP-functionalized, mito-metformin encapsulated NPs against rotenone-induced mitochondrial dysfunction. However, nonfunctionalized, mito-metformin encapsulated NPs and soluble mito-metformin administered at the same dose did not significantly protect cells from rotenone-induced toxicity. These results indicate that the targeted NP platform can provide enhanced dose-sparing and potentially reduce the occurrence of systemic side-effects for PD therapeutics.

Mitochondrial dysfunction-induced H3K27 hyperacetylation perturbs enhancers in Parkinson's disease.

Mitochondrial dysfunction is a major pathophysiological contributor to the progression of Parkinson's disease (PD); however, whether it contributes to epigenetic dysregulation remains unknown. Here, we show that both chemically and genetically driven mitochondrial dysfunctions share a common mechanism of epigenetic dysregulation. Under both scenarios, lysine 27 acetylation of likely variant H3.3 (H3.3K27ac) increased in dopaminergic neuronal models of PD, thereby opening that region to active enhancer activity via H3K27ac. These vulnerable epigenomic loci represent potential transcription factor motifs for PD pathogenesis. We further confirmed that mitochondrial dysfunction induces H3K27ac in ex vivo and in vivo (MitoPark) neurodegenerative models of PD. Notably, the significantly increased H3K27ac in postmortem PD brains highlights the clinical relevance to the human PD population. Our results reveal an exciting mitochondrial dysfunction-metabolism-H3K27ac-transcriptome axis for PD pathogenesis. Collectively, the mechanistic insights link mitochondrial dysfunction to epigenetic dysregulation in dopaminergic degeneration and offer potential new epigenetic intervention strategies for PD.

Interleukin-6 and lactate dehydrogenase expression in a novel ex vivo rocking model of equine corneal epithelial wound healing.

PURPOSE: To establish a physiologically relevant ex vivo model of equine corneal epithelial wound healing. METHODS: Fourteen equine corneas were randomly assigned to one of two groups: wounded (n = 8) or unwounded (n = 6) controls. In the wounded group, the axial corneal epithelium was removed by applying a 6 mm filter paper disk soaked in 1N-NaOH for 60 s. Corneas were subsequently cultured using an air-liquid interface model. Evaluation of corneal healing was performed daily, and culture medium was collected. Corneas were randomly assigned to undergo processing via histopathology and RNAscope in situ hybridization for interleukin-6 (IL-6) and alpha-smooth muscle actin (αSMA) expression at T24, T48, and T72 h after wounding. Media of the cultured corneas were evaluated for the presence of lactate dehydrogenase (LDH) by a colorimetric assay. RESULTS: The ulcerated area of the wounded corneas decreased over time and all corneas healed within 72 h. Histologically, normal corneal architecture was observed including healthy epithelium (in areas other than the ulcerated ones), minimal stromal edema, intact endothelium, and Descemet's membrane. IL-6 expression was increased in wounded corneas compared with unwounded controls. LDH expression was elevated for both wounded and unwounded corneas at T24 but decreased substantially and was not detected at T48 in media from wounded and unwounded corneas, respectively. No αSMA expression was detected from either wounded or unwounded corneas. CONCLUSIONS: The equine air-liquid interface, ex vivo, corneal epithelial wound healing model is effective and physiologically relevant. This model can be used in future studies evaluating various corneal therapies.

Chronic Manganese Exposure and the Enteric Nervous System: An in Vitro and Mouse in Vivo Study.

BACKGROUND: Chronic environmental exposure to manganese (Mn) can cause debilitating damage to the central nervous system. However, its potential toxic effects on the enteric nervous system (ENS) have yet to be assessed. OBJECTIVE: We examined the effect of Mn on the ENS using both cell and animal models. METHOD: Rat enteric glial cells (EGCs) and mouse primary enteric cultures were exposed to increasing concentrations of Mn and cell viability and mitochondrial health were assessed using various morphological and functional assays. C57BL/6 mice were exposed daily to a sublethal dose of Mn (15mg/kg/d) for 30 d. Gut peristalsis, enteric inflammation, gut microbiome profile, and fecal metabolite composition were assessed at the end of exposure. RESULTS: EGC mitochondria were highly susceptible to Mn neurotoxicity, as evidenced by lower mitochondrial mass, adenosine triphosphate-linked respiration, and aconitase activity as well as higher mitochondrial superoxide, upon Mn exposure. Minor differences were seen in the mouse model: specifically, longer intestinal transit times and higher levels of colonic inflammation. CONCLUSION: Based on our findings from this study, Mn preferentially induced mitochondrial dysfunction in a rat EGC line and in vivo resulted in inflammation in the ENS. https://doi.org/10.1289/EHP7877.