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.

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.

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.

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.

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.

Transcriptomic profile analysis of brain inferior colliculus following acute hydrogen sulfide exposure.

Hydrogen sulfide (H2S) is a gaseous molecule found naturally in the environment, and as an industrial byproduct, and is known to cause acute death and induces long-term neurological disorders following acute high dose exposures. Currently, there is no drug approved for treatment of acute H2S-induced neurotoxicity and/or neurological sequelae. Lack of a deep understanding of pathogenesis of H2S-induced neurotoxicity has delayed the development of appropriate therapeutic drugs that target H2S-induced neuropathology. RNA sequencing analysis was performed to elucidate the cellular and molecular mechanisms of H2S-induced neurodegeneration, and to identify key molecular elements and pathways that contribute to H2S-induced neurotoxicity. C57BL/6J mice were exposed by whole body inhalation to 700 ppm of H2S for either one day, two consecutive days or 4 consecutive days. Magnetic resonance imaging (MRI) scan analyses showed H2S exposure induced lesions in the inferior colliculus (IC) and thalamus (TH). This mechanistic study focused on the IC. RNA Sequencing analysis revealed that mice exposed once, twice, or 4 times had 283, 193 and 296 differentially expressed genes (DEG), respectively (q-value < 0.05, fold-change> 1.5). Hydrogen sulfide exposure modulated multiple biological pathways including unfolded protein response, neurotransmitters, oxidative stress, hypoxia, calcium signaling, and inflammatory response in the IC. Hydrogen sulfide exposure activated PI3K/Akt and MAPK signaling pathways. Pro-inflammatory cytokines were shown to be potential initiators of the modulated signaling pathways following H2S exposure. Furthermore, microglia were shown to release IL-18 and astrocytes released both IL-1ß and IL-18 in response to H2S. This transcriptomic analysis data revealed complex signaling pathways involved in H2S-induced neurotoxicity and may provide important associated mechanistic insights.

Utilization of the CRISPR-Cas9 Gene Editing System to Dissect Neuroinflammatory and Neuropharmacological Mechanisms in Parkinson's Disease.

Chronic and debilitating neurodegenerative diseases, such as Parkinson's disease (PD), impose an immense medical, emotional, and economic burden on patients and society. Due to a complex interaction between genetic and environmental risk factors, the etiology of PD remains elusive. However, the cumulative evidence emerging from clinical and experimental research over the last several decades has identified mitochondrial dysfunction, oxidative stress, neuroinflammation, and dysregulated protein degradation as the main drivers of PD neurodegeneration. The genome-editing system CRISPR (clustered regularly interspaced short palindromic repeats) has recently transformed the field of biotechnology and biomedical discovery and is poised to accelerate neurodegenerative disease research. It has been leveraged to generate PD animal models, such as Parkin, DJ-1, and PINK1 triple knockout miniature pigs. CRISPR has also allowed the deeper understanding of various PD gene interactions, as well as the identification of novel apoptotic pathways associated with neurodegenerative processes in PD. Furthermore, its application has been used to dissect neuroinflammatory pathways involved in PD pathogenesis, such as the PKCδ signaling pathway, as well as the roles of novel compensatory or protective pathways, such as Prokineticin-2 signaling. This review aims to highlight the historical milestones in the evolution of this technology and attempts to illustrate its transformative potential in unraveling disease mechanisms as well as in the development of innovative treatment strategies for PD. Graphical Abstract.

Midazolam Efficacy Against Acute Hydrogen Sulfide-Induced Mortality and Neurotoxicity.

Hydrogen sulfide (H2S) is a colorless, highly neurotoxic gas. It is not only an occupational and environmental hazard but also of concern to the Department of Homeland Security for potential nefarious use. Acute high-dose H2S exposure causes death, while survivors may develop neurological sequelae. Currently, there is no suitable antidote for treatment of acute H2S-induced neurotoxicity. Midazolam (MDZ), an anti-convulsant drug recommended for treatment of nerve agent intoxications, could also be of value in treating acute H2S intoxication. In this study, we tested the hypothesis that MDZ is effective in preventing/treating acute H2S-induced neurotoxicity. This proof-of-concept study had two objectives: to determine whether MDZ prevents/reduces H2S-induced mortality and to test whether MDZ prevents H2S-induced neurological sequelae. MDZ (4 mg/kg) was administered IM in mice, 5 min pre-exposure to a high concentration of H2S at 1000 ppm or 12 min post-exposure to 1000 ppm H2S followed by 30 min of continuous exposure. A separate experiment tested whether MDZ pre-treatment prevented neurological sequelae. Endpoints monitored included assessment of clinical signs, mortality, behavioral changes, and brain histopathological changes. MDZ significantly reduced H2S-induced lethality, seizures, knockdown, and behavioral deficits (p < 0.01). MDZ also significantly prevented H2S-induced neurological sequelae, including weight loss, behavior deficits, neuroinflammation, and histopathologic lesions (p < 0.01). Overall, our findings show that MDZ is a promising drug for reducing H2S-induced acute mortality, neurotoxicity, and neurological sequelae.

Expression pattern of an evolutionarily conserved splice variant in the rat Tacc2 gene.

The transforming acidic coiled-coil containing protein 2 (Tacc2) gene and its paralogs, Tacc1 and Tacc3 encode proteins that are associated with the centrosome and involved in microtubule assembly during the cell cycle. Tacc2 produces several splice variants, which are poorly characterized, especially in the rat. Characterization of the temporal/spatial expression patterns of these isoforms would be useful in understanding their distinct and overlapping functions. By comparative sequence analyses of Tacc2 in multiple species, we identified a third splice variant in rat, which is much shorter in size (1,021 aa) than the longest isoform (2,834 aa). This newly identified Tacc2 splice variant (isoform 3) uses a distinct first exon and generates a different open reading frame. Although Isoform 3 is expressed predominantly during developmental stages, the long Tacc2 isoform (isoform 1) is distributed mainly in adult tissues. Multiple protein sequence analyses revealed that Tacc2 Isoform 3 could be the ancient form, as it is conserved in mammals, birds, and amphibians; whereas the long Tacc2 isoforms may have evolved in the mammalian lineage by adding exons toward the 5' region of the ancient isoform.