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Excessive exposure to manganese (Mn) increases the risk of chronic neurological diseases, including Parkinson's disease (PD) and other related Parkinsonisms. Aggregated α-synuclein (αSyn), a hallmark of PD, can spread to neighboring cells by exosomal release from neurons. We previously discovered that Mn enhances its spread, triggering neuroinflammatory and neurodegenerative processes. To better understand the Mn-induced release of exosomal αSyn, we examined the effect of Mn on endosomal trafficking and misfolded protein degradation. Exposing MN9D dopaminergic neuronal cells stably expressing human wild-type (WT) αSyn to 300 μM Mn for 24 h significantly suppressed protein and mRNA expression of Rab11a, thereby downregulating endosomal recycling, forcing late endosomes to mature into multivesicular bodies (MVBs). Ectopic expression of WT Rab11a significantly mitigated exosome release, whereas ectopic mutant Rab11a (S25N) increased it. Our in vitro and in vivo studies reveal that Mn exposure upregulated (1) mRNA and protein levels of endosomal Rab27a, which mediates the fusion of MVBs with the plasma membrane; and (2) expression of the autophagosomal markers Beclin-1 and p62, but downregulated the lysosomal marker LAMP2, thereby impairing autophagolysosome formation as confirmed by LysoTracker, cathepsin, and acridine orange assays. Our novel findings demonstrate that Mn promotes the exosomal release of misfolded αSyn by impairing endosomal trafficking and protein degradation.

When exposed to X-rays, scintillators emit visible luminescence. X-ray-mediated optogenetics employs scintillators for remotely activating light-sensitive proteins in biological tissue through X-ray irradiation. This approach offers advantages over traditional optogenetics, allowing for deeper tissue penetration and wireless control. Here, we assessed the short-term safety and efficacy of candidate scintillator materials for neuronal control. Our analyses revealed that lead-free halide scintillators, such as Cs 3 Cu 2 I 5 , exhibited significant cytotoxicity within 24 h and induced neuroinflammatory effects when injected into the mouse brain. In contrast, cerium-doped gadolinium aluminum gallium garnet (Ce:GAGG) nanoparticles showed no detectable cytotoxicity within the same period, and injection into the mouse brain did not lead to observable neuroinflammation over four weeks. Electrophysiological recordings in the cerebral cortex of awake mice showed that X-ray-induced radioluminescence from Ce:GAGG nanoparticles reliably activated 45% of the neuronal population surrounding the implanted particles, a significantly higher activation rate than europium-doped GAGG (Eu:GAGG) microparticles, which activated only 10% of neurons. Furthermore, we established the cell-type specificity of this technique by using Ce:GAGG nanoparticles to selectively stimulate midbrain dopamine neurons. This technique was applied to freely behaving mice, allowing for wireless modulation of place preference behavior mediated by midbrain dopamine neurons. These findings highlight the unique suitability of Ce:GAGG nanoparticles for X-ray-mediated optogenetics. The deep tissue penetration, short-term safety, wireless neuronal control, and cell-type specificity of this system offer exciting possibilities for diverse neuroscience applications and therapeutic interventions.

Rotenone, as a common pesticide and insecticide frequently found in environmental samples, may be present in aquatic habitats worldwide. Exposure to low concentrations of this compound may cause alterations in the nervous system, thus contributing to Parkinsonian motor symptoms in both vertebrates and invertebrates. However, the effects of chronic exposure to low doses of rotenone on the activity of neurotransmitters that govern motor functions and on the specific molecular mechanisms leading to movement morbidity remain largely unknown for many aquatic invertebrates. In this study, we analyzed the effects that rotenone poisoning exerts on the activity of dopamine (DA) and acetylcholine (ACh) synthesis enzymes in the central nervous system (CNS) of Asian shore crab, Hemigrapsus sanguineus (de Haan, 1835), and elucidated the association of its locomotor behavior with Parkinson's-like symptoms. An immunocytochemistry analysis showed a reduction in tyrosine hydroxylase (TH) in the median brain and the ventral nerve cord (VNC), which correlated with the subsequent decrease in the locomotor activity of shore crabs. We also observed a variation in cholinergic neurons' activity, mostly in the ventral regions of the VNC. Moreover, the rotenone-treated crabs showed signs of damage to ChAT-lir neurons in the VNC. These data suggest that chronic treatment with low doses of rotenone decreases the DA level in the VNC and the ACh level in the brain and leads to progressive and irreversible reductions in the crab's locomotor activity, life span, and changes in behavior.

Adenosine A 2A receptor (A 2A R) antagonists are the leading nondopaminergic therapy to manage Parkinson's disease (PD) since they afford both motor benefits and neuroprotection. PD begins with a synaptic dysfunction and damage in the striatum evolving to an overt neuronal damage of dopaminergic neurons in the substantia nigra. We tested if A 2A R antagonists are equally effective in controlling these two degenerative processes. We used a slow intracerebroventricular infusion of the toxin MPP + in male rats for 15 days, which caused an initial loss of synaptic markers in the striatum within 10 days, followed by a neuronal loss in the substantia nigra within 30 days. Interestingly, the initial loss of striatal nerve terminals involved a loss of both dopaminergic and glutamatergic synaptic markers, while GABAergic markers were preserved. The daily administration of the A 2A R antagonist SCH58261 (0.1 mg/kg, i.p.) in the first 10 days after MPP + infusion markedly attenuated both the initial loss of striatal synaptic markers and the subsequent loss of nigra dopaminergic neurons. Strikingly, the administration of SCH58261 (0.1 mg/kg, i.p. for 10 days) starting 20 days after MPP + infusion was less efficacious to attenuate the loss of nigra dopaminergic neurons. This prominent A 2A R-mediated control of synaptotoxicity was directly confirmed by showing that the MPTP-induced dysfunction (MTT assay) and damage (lactate dehydrogenase release assay) of striatal synaptosomes were prevented by 50 nM SCH58261. This suggests that A 2A R antagonists may be more effective to counteract the onset rather than the evolution of PD pathology.

There are currently no disease-modifying therapies for Parkinson's disease (PD), a progressive neurodegenerative disorder associated with dopaminergic neuronal loss. There is increasing evidence that endogenous dopamine (DA) can be a pathological factor in neurodegeneration in PD. Tyrosine hydroxylase (TH) is the key rate-limiting enzyme for DA generation. Drugs that inhibit TH, such as alpha-methyltyrosine (α-MT), have recently been shown to protect against neurodegeneration in various PD models. DA receptor agonists can activate post-synaptic DA receptors to alleviate DA-deficiency-induced PD symptoms. However, DA receptor agonists have no therapeutic effects against neurodegeneration. Thus, a combination therapy with DA receptor agonists plus TH inhibitors may be an attractive therapeutic approach. TH inhibitors can protect and promote the survival of remaining dopaminergic neurons in PD patients' brains, whereas DA receptor agonists activate post-synaptic DA receptors to alleviate PD symptoms. Additionally, other PD drugs, such as N-acetylcysteine (NAC) and anticholinergic drugs, may be used as adjunctive medications to improve therapeutic effects. This multi-drug cocktail may represent a novel strategy to protect against progressive dopaminergic neurodegeneration and alleviate PD disease progression., Competing Interests: The authors declare no conflicts of interest.

The etiology underlying most sporadic Parkinson's' disease (PD) cases is unknown. Environmental exposures have been suggested as putative causes of the disease. In cell models and in animal studies, certain chemicals can destroy dopaminergic neurons. However, the mechanisms of how these chemicals cause the death of neurons is not understood. Several of these agents are mitochondrial toxins that inhibit the mitochondrial complex I of the electron transport chain. Familial PD genes also encode proteins with important functions in mitochondria. Mitochondrial dysfunction of the respiratory chain, in combination with the presence of redox active dopamine molecules in these cells, will lead to the accumulation of reactive oxygen species (ROS) in dopaminergic neurons. Here, I propose a mechanism regarding how ROS may lead to cell killing with a specificity for neurons. One rarely considered hypothesis is that ROS produced by defective mitochondria will lead to the formation of oxidative DNA damage in nuclear DNA. Many genes that encode proteins with neuron-specific functions are extraordinary long, ranging in size from several hundred kilobases to well over a megabase. It is predictable that such long genes will contain large numbers of damaged DNA bases, for example in the form of 8-oxoguanine (8-oxoG), which is a major DNA damage type produced by ROS. These DNA lesions will slow down or stall the progression of RNA polymerase II, which is a term referred to as transcription stress. Furthermore, ROS-induced DNA damage may cause mutations, even in postmitotic cells such as neurons. I propose that the impaired transcription and mutagenesis of long, neuron-specific genes will lead to a loss of neuronal integrity, eventually leading to the death of these cells during a human lifetime.

Several theories have been proposed to explain the mechanisms of substance use in schizophrenia. Brain neurons pose a potential to provide novel insights into the association between opioid addiction, withdrawal, and schizophrenia. Thus, we exposed zebrafish larvae at 2 days post-fertilization (dpf) to domperidone (DPM) and morphine, followed by morphine withdrawal. Drug-induced locomotion and social preference were assessed, while the level of dopamine and the number of dopaminergic neurons were quantified. In the brain tissue, the expression levels of genes associated with schizophrenia were measured. The effects of DMP and morphine were compared to vehicle control and MK-801, a positive control to mimic schizophrenia. Gene expression analysis revealed that α1C , α1Sa , α1Aa , drd2a , and th1 were up-regulated after 10 days of exposure to DMP and morphine, while th2 was down-regulated. These two drugs also increased the number of positive dopaminergic neurons and the total dopamine level but reduced the locomotion and social preference. The termination of morphine exposure led to the up-regulation of th2 , drd2a , and c-fos during the withdrawal phase. Our integrated data implicate that the dopamine system plays a key role in the deficits in social behavior and locomotion that are common in the schizophrenia-like symptoms and opioid dependence.

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain. Restoration of nigrostriatal dopamine neurons has been proposed as a potential therapeutic strategy for PD. Because currently used PD therapeutics only help relieve motor symptoms and do not treat the cause of the disease, highly effective drugs are needed. Vildagliptin, a dipeptidyl peptidase 4 (DPP4) inhibitor, is an anti-diabetic drug with various pharmacological properties including neuroprotective effects. However, the detailed effects of vildagliptin against PD are not fully understood. We investigated the effects of vildagliptin on PD and its underlying molecular mechanisms using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model and a 1-methyl-4-phenylpyridium (MPP + )-induced cytotoxicity model. Vildagliptin (50 mg/kg) administration significantly attenuated MPTP-induced motor deficits as evidenced by rotarod, pole, and nest building tests. Immunohistochemistry and Western blot analysis revealed that vildagliptin increased tyrosine hydroxylase-positive cells in the SNpc and striatum, which was reduced by MPTP treatment. Furthermore, vildagliptin activated MPTP-decreased PI3k/Akt and mitigated MPTP-increased ERK and JNK signaling pathways in the striatum. Consistent with signaling transduction in the mouse striatum, vildagliptin reversed MPP + -induced dephosphorylation of PI3K/Akt and phosphorylation of ERK and JNK in SH-SY5Y cells. Moreover, vildagliptin attenuated MPP + -induced conversion of LC3B-II in SH-SY5Y cells, suggesting its role in autophagy inhibition. Taken together, these findings indicate that vildagliptin has protective effects against MPTP-induced motor dysfunction by inhibiting dopaminergic neuronal apoptosis, which is associated with regulation of PI3k/Akt, ERK, and JNK signaling transduction. Our findings suggest vildagliptin as a promising repurposing drug to treat PD.

Hericium erinaceus (HE) is a common edible mushroom consumed in several Asian countries and considered to be a medicinal mushroom with neuroprotective effects. Erinacine A (EA) is a bioactive compound in Hericium erinaceus mycelium (HEM) that has been shown to have a neuroprotective effect against neurodegenerative diseases, e.g., Parkinson's disease (PD). Although the etiology of PD is still unclear, neuroinflammation may play an important role in causing dopaminergic neuron loss, which is a pathological hallmark of PD. However, glial cell activation has a close relationship with neuroinflammation. Thus, this study aimed to investigate the anti-neuroinflammatory and neuroprotective effects of EA on lipopolysaccharide (LPS)-induced glial cell activation and neural damage in vitro and in vivo. For the in vitro experiments, glial cells, BV-2 microglial cells and CTX TNA2 astrocytes were pretreated with EA and then stimulated with LPS and/or IFN-γ. The expression of proinflammatory factors in the cells and culture medium was analyzed. In addition, differentiated neuro-2a (N2a) cells were pretreated with EA or HEM and then stimulated with LPS-treated BV-2 conditioned medium (CM). The cell viability and the amount of tyrosine hydroxylase (TH) and mitogen-activated protein kinases (MAPKs) were analyzed. In vivo, rats were given EA or HEM by oral gavage prior to injection of LPS into the substantia nigra (SN). Motor coordination of the rats and the expression of proinflammatory mediators in the midbrain were analyzed. EA pretreatment prevented LPS-induced iNOS expression and NO production in BV-2 cells and TNF-α expression in CTX TNA2 cells. In addition, both EA and HEM pretreatment significantly increased cell viability and TH expression and suppressed the phosphorylation of JNK and NF- κB in differentiated N2a cells treated with CM. In vivo, both EA and HEM significantly improved motor dysfunction in the rotarod test and the amphetamine-induced rotation test and reduced the expression of TNF-α, IL-1β and iNOS in the midbrain of rats intranigrally injected with LPS. The results demonstrate that EA ameliorates LPS-induced neuroinflammation and has neuroprotective properties.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia, responsible for the onset of several long-term complications. Recent evidence suggests that cognitive dysfunction represents an emerging complication of DM, but the underlying molecular mechanisms are still obscure. Dopamine (DA), a neurotransmitter essentially known for its relevance in the regulation of behavior and movement, modulates cognitive function, too. Interestingly, alterations of the dopaminergic system have been observed in DM. This review aims to offer a comprehensive overview of the most relevant experimental results assessing DA's role in cognitive function, highlighting the presence of dopaminergic dysfunction in DM and supporting a role for glucotoxicity in DM-associated dopaminergic dysfunction and cognitive impairment. Several studies confirm a role for DA in cognition both in animal models and in humans. Similarly, significant alterations of the dopaminergic system have been observed in animal models of experimental diabetes and in diabetic patients, too. Evidence is accumulating that advanced glycation end products (AGEs) and their precursor methylglyoxal (MGO) are associated with cognitive impairment and alterations of the dopaminergic system. Further research is needed to clarify the molecular mechanisms linking DM-associated dopaminergic dysfunction and cognitive impairment and to assess the deleterious impact of glucotoxicity.

Parkinson's disease (PD) is characterized by the progressive accumulation of neuronal intracellular aggregates largely composed of alpha-Synuclein (αSyn) protein. The process of αSyn aggregation is induced during aging and enhanced by environmental stresses, such as the exposure to pesticides. Paraquat (PQ) is an herbicide which has been widely used in agriculture and associated with PD. PQ is known to cause an increased oxidative stress in exposed individuals but the consequences of such stress on αSyn conformation remains poorly understood. To study αSyn pathogenic modifications in response to PQ, we exposed Drosophila expressing human αSyn to a chronic PQ protocol. We first showed that PQ exposure and αSyn expression synergistically induced fly mortality. The exposure to PQ was also associated with increased levels of total and phosphorylated forms of αSyn in the Drosophila brain. Interestingly, PQ increased the detection of soluble αSyn in highly denaturating buffer but did not increase αSyn resistance to proteinase K digestion. These results suggest that PQ induces the accumulation of toxic soluble and misfolded forms of αSyn but that these toxic forms do not form fibrils or aggregates that are detected by the proteinase K assay. Collectively, our results demonstrate that Drosophila can be used to study the effect of PQ or other environmental neurotoxins on αSyn driven pathology.

The exact etiology of Parkinson's disease (PD) remains largely unknown, but more and more research suggests the involvement of the gut microbiota. Interestingly, idiopathic PD patients were shown to have at least a 10 times higher prevalence of Helicobacter suis ( H. suis ) DNA in gastric biopsies compared to control patients. H. suis is a zoonotic Helicobacter species that naturally colonizes the stomach of pigs and non-human primates but can be transmitted to humans. Here, we investigated the influence of a gastric H. suis infection on PD disease progression through a 6-hydroxydopamine (6-OHDA) mouse model. Therefore, mice with either a short- or long-term H. suis infection were stereotactically injected with 6-OHDA in the left striatum and sampled one week later. Remarkably, a reduced loss of dopaminergic neurons was seen in the H. suis/ 6-OHDA groups compared to the control/6-OHDA groups. Correspondingly, motor function of the H. suis -infected 6-OHDA mice was superior to that in the non-infected 6-OHDA mice. Interestingly, we also observed higher expression levels of antioxidant genes in brain tissue from H. suis -infected 6-OHDA mice, as a potential explanation for the reduced 6-OHDA-induced cell loss. Our data support an unexpected neuroprotective effect of gastric H. suis on PD pathology, mediated through changes in oxidative stress.

Dopamine is likely the most studied modulatory neurotransmitter, in great part due to characteristic motor deficits in Parkinson's disease that arise after the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc). The SNc, together with the ventral tegmental area (VTA), play a key role modulating motor responses through the basal ganglia. In contrast to the large amount of existing literature addressing the mammalian dopaminergic system, comparatively little is known in other vertebrate groups. However, in the last several years, numerous studies have been carried out in basal vertebrates, allowing a better understanding of the evolution of the dopaminergic system, especially the SNc/VTA. We provide an overview of existing research in basal vertebrates, mainly focusing on lampreys, belonging to the oldest group of extant vertebrates. The lamprey dopaminergic system and its role in modulating motor responses have been characterized in significant detail, both anatomically and functionally, providing the basis for understanding the evolution of the SNc/VTA in vertebrates. When considered alongside results from other early vertebrates, data in lampreys show that the key role of the SNc/VTA dopaminergic neurons modulating motor responses through the basal ganglia was already well developed early in vertebrate evolution.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta, leading to classical PD motor symptoms. Current therapies are purely symptomatic and do not modify disease progression. Cannabidiol (CBD), one of the main phytocannabinoids identified in Cannabis Sativa, which exhibits a large spectrum of therapeutic properties, including anti-inflammatory and antioxidant effects, suggesting its potential as disease-modifying agent for PD. The aim of this study was to evaluate the effects of chronic treatment with CBD (10 mg/kg, i.p.) on PD-associated neurodegenerative and neuroinflammatory processes, and motor deficits in the 6-hydroxydopamine model. Moreover, we investigated the potential mechanisms by which CBD exerted its effects in this model. CBD-treated animals showed a reduction of nigrostriatal degeneration accompanied by a damping of the neuroinflammatory response and an improvement of motor performance. In particular, CBD exhibits a preferential action on astrocytes and activates the astrocytic transient receptor potential vanilloid 1 (TRPV1), thus, enhancing the endogenous neuroprotective response of ciliary neurotrophic factor (CNTF). These results overall support the potential therapeutic utility of CBD in PD, as both neuroprotective and symptomatic agent.

A real-life environment during pregnancy involves multiple and simultaneous exposures to toxic chemicals. Perinatal exposures to toxic chemicals have been reported to exert an inhibitory effect on mouse neural development and behaviors. However, the effect of combined exposures of organophosphate and nicotine has not been previously reported. In this study, we investigated whether a combined exposure of diazinon and nicotine can have a synergistic effect. The effects of the combined chemical exposure on cell viability and neuronal differentiation were examined using mouse Sox1-GFP cells. Additionally, mice were maternally administered 0.18 mg/kg diazinon, a no adverse effect level (NOAEL) dose, combined with 0.4, 1, and 2 mg/kg nicotine. Mice offspring underwent behavior tests to assess locomotor, depressive, cognitive, and social behaviors. Morphological change in the brain was investigated with immunolocalization. We revealed that the combined exposure to diazinon and nicotine can have a synergistic adverse effect in vitro. In addition, the chemical-treated mouse offspring showed abnormalities in motor learning, compulsive-like behaviors, spatial learning, and social interaction patterns. Moreover, 0.18 mg/kg diazinon and 2 mg/kg nicotine co-exposure resulted in an increase in tyrosine hydroxylase (TH)-positive dopaminergic neurons. Thus, the findings suggest that perinatal co-exposure to nicotine and diazinon can result in abnormal neurodevelopment and behavior, even at low-level administration.

The benefits of a ketogenic diet in childhood epilepsy steered up hope for neuroprotective effects of hyperketonemia in Parkinson's disease (PD). There are multiple theoretical reasons but very little actual experimental proof or clinical trials. We examined the long-term effects of the ketogenic diet in an animal model of early PD. A progressive, selective dopaminergic medium size lesion was induced by 6-OHDA injection into the medial forebrain bundle. Animals were kept on the stringent ketogenic diet (1% carbohydrates, 8% protein, 70% fat) for 3 weeks prior and 4 weeks after the brain operation. Locomotor activity, neuron count, dopaminergic terminal density, dopamine level, and turnover were analyzed at three time-points post-lesion, up to 4 weeks after the operation. Energy metabolism parameters (glycogen, mitochondrial complex I and IV, lactate, beta-hydroxybutyrate, glucose) were analyzed in the brain and liver or plasma. Protein expression of enzymes essential for gluconeogenesis (PEPCK, G6PC) and glucose utilization (GCK) was analyzed in the liver. Despite long-term hyperketonemia pre- and post-lesion, the ketogenic diet did not protect against 6-OHDA-induced dopaminergic neuron lesions. The ketogenic diet only tended to improve locomotor activity and normalize DA turnover in the striatum. Rats fed 7 weeks in total with a restrictive ketogenic diet maintained normoglycemia, and neither gluconeogenesis nor glycogenolysis in the liver was responsible for this effect. Therefore, potentially, the ketogenic diet could be therapeutically helpful to support the late compensatory mechanisms active via glial cells but does not necessarily act against the oxidative stress-induced parkinsonian neurodegeneration itself. A word of caution is required as the stringent ketogenic diet itself also carries the risk of unwanted side effects, so it is important to study the long-term effects of such treatments. More detailed metabolic long-term studies using unified diet parameters are required, and human vs. animal differences should be taken under consideration.

Parkinson's disease (PD) is the most common movement disorder, characterized by progressive degeneration of the nigrostriatal pathway, which consists of dopaminergic cell bodies in substantia nigra and their neuronal projections to the striatum. Moreover, PD is associated with an array of non-motor symptoms such as olfactory dysfunction, gastrointestinal dysfunction, impaired regulation of the sleep-wake cycle, anxiety, depression, and cognitive impairment. Inflammation and concomitant oxidative stress are crucial in the pathogenesis of PD. Thus, this study aimed to model PD via intrastriatal injection of the inflammagen lipopolysaccharide (LPS)to investigate if the lesion causes olfactory and motor impairments, inflammation, oxidative stress, and alteration in synaptic proteins in the olfactory bulb, striatum, and colon. Ten µg of LPS was injected unilaterally into the striatum of 27 male C57BL/6 mice, and behavioural assessment was conducted at 4 and 8 weeks post-treatment, followed by tissue collection. Intrastriatal LPS induced motor impairment in C57BL/6 mice at 8 weeks post-treatment evidenced by reduced latency time in the rotarod test. LPS also induced inflammation in the striatum characterized by increased expression of microglial marker Iba-1 and astrocytic marker GFAP, with degeneration of dopaminergic neuronal fibres (reduced tyrosine hydroxylase immunoreactivity), and reduction of synaptic proteins and DJ-1 protein. Additionally, intrastriatal LPS induced inflammation, oxidative stress and alterations in synaptic proteins within the olfactory bulb, although this did not induce a significant impairment in olfactory function. Intrastriatal LPS induced mild inflammatory changes in the distal colon, accompanied by increased protein expression of 3-nitrotyrosine-modified proteins. This model recapitulated the major features of PD such as motor impairment and degeneration of dopaminergic neuronal fibres in the striatum, as well as some pathological changes in the olfactory bulb and colon; thus, this model could be suitable for understanding clinical PD and testing neuroprotective strategies.

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson's disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, mainly characterized by motor deficits correlated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SN). Necroptosis is a caspase-independent form of regulated cell death mediated by the concerted action of receptor-interacting protein 3 (RIP3) and the pseudokinase mixed lineage domain-like protein (MLKL). It is also usually dependent on RIP1 kinase activity, influenced by further cellular clues. Importantly, necroptosis appears to be strongly linked to several neurodegenerative diseases, including PD. Here, we aimed at identifying novel chemical inhibitors of necroptosis in a PD-mimicking model, by conducting a two-step screening. Firstly, we phenotypically screened a library of 31 small molecules using a cellular model of necroptosis and, thereafter, the hit compound effect was validated in vivo in a sub-acute 1-methyl-1-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) PD-related mouse model. From the initial compounds, we identified one hit-Oxa12-that strongly inhibited necroptosis induced by the pan-caspase inhibitor zVAD-fmk in the BV2 murine microglia cell line. More importantly, mice exposed to MPTP and further treated with Oxa12 showed protection against MPTP-induced dopaminergic neuronal loss in the SN and striatum. In conclusion, we identified Oxa12 as a hit compound that represents a new chemotype to tackle necroptosis. Oxa12 displays in vivo effects, making this compound a drug candidate for further optimization to attenuate PD pathogenesis.

The diurnal rodent Octodon degus ( O. degus ) is considered an attractive natural model for Alzheimer's disease and other human age-related features. However, it has not been explored so far if the O. degus could be used as a model to study Parkinson's disease. To test this idea, 10 adult male O. degus were divided into control group and MPTP-intoxicated animals. Motor condition and cognition were examined. Dopaminergic degeneration was studied in the ventral mesencephalon and in the striatum. Neuroinflammation was also evaluated in the ventral mesencephalon, in the striatum and in the dorsal hippocampus. MPTP animals showed significant alterations in motor activity and in visuospatial memory. Postmortem analysis revealed a significant decrease in the number of dopaminergic neurons in the ventral mesencephalon of MPTP animals, although no differences were found in their striatal terminals. We observed a significant increase in neuroinflammatory responses in the mesencephalon, in the striatum and in the hippocampus of MPTP-intoxicated animals. Additionally, changes in the subcellular expression of the calcium-binding protein S100β were found in the astrocytes in the nigrostriatal pathway. These findings prove for the first time that O. degus are sensitive to MPTP intoxication and, therefore, is a suitable model for experimental Parkinsonism in the context of aging.

Human induced pluripotent stem (iPS) cells have the potential to give rise to a new era in Parkinson's disease (PD) research. As a unique source of midbrain dopaminergic (DA) neurons, iPS cells provide unparalleled capabilities for investigating the pathogenesis of PD, the development of novel anti-parkinsonian drugs, and personalized therapy design. Significant progress in developmental biology of midbrain DA neurons laid the foundation for their efficient derivation from iPS cells. The introduction of 3D culture methods to mimic the brain microenvironment further expanded the vast opportunities of iPS cell-based research of the neurodegenerative diseases. However, while the benefits for basic and applied studies provided by iPS cells receive widespread coverage in the current literature, the drawbacks of this model in its current state, and in particular, the aspects of differentiation protocols requiring further refinement are commonly overlooked. This review summarizes the recent data on general and subtype-specific features of midbrain DA neurons and their development. Here, we review the current protocols for derivation of DA neurons from human iPS cells and outline their general weak spots. The associated gaps in the contemporary knowledge are considered and the possible directions for future research that may assist in improving the differentiation conditions and increase the efficiency of using iPS cell-derived neurons for PD drug development are discussed.

Oxidative stress, mitochondrial dysfunction, and neuroinflammation are strongly associated with the pathogenesis of Parkinson's disease (PD), which suggests that anti-oxidative and anti-inflammatory compounds might provide an alternative treatment for PD. Here, we evaluated the neuroprotective effects of evernic aid (EA), which was screened from a lichen library provided by the Korean Lichen Research Institute at Sunchon National University. EA is a secondary metabolite generated by lichens, including Ramalina, Evernia, and Hypogymnia , and several studies have described its anticancer, antifungal, and antimicrobial effects. However, the neuroprotective effects of EA have not been studied. We found that EA protected primary cultured neurons against 1-methyl-4-phenylpyridium (MPP + )-induced cell death, mitochondrial dysfunction, and oxidative stress, and effectively reduced MPP + -induced astroglial activation by inhibiting the NF-κB pathway. In vivo, EA ameliorated MPTP-induced motor dysfunction, dopaminergic neuronal loss, and neuroinflammation in the nigrostriatal pathway in C57BL/6 mice. Taken together, our findings demonstrate that EA has neuroprotective and anti-inflammatory effects in PD models and suggest that EA is a potential therapeutic candidate for PD.

Retinal aging is the result of accumulating molecular and cellular damage with a manifest decline in visual functions. Somatostatin (SST) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been implicated in neuroprotection through regulating disparate aspects of neuronal activity (survival, proliferation and renewal). The aim of the present study was to validate a transgenic model for SST-expressing amacrine cells and to investigate the chronic effect of PACAP on the aging of SSTergic and dopaminergic cells of the retina. SST-tdTomato transgenic mice that were 6, 12 and 18 months old were treated intravitreally with 100 pmol of PACAP every 3 months. The density of SST and dopaminergic amacrine cells was assessed in whole-mounted retinas. Cells displaying the transgenic red fluorescence were identified as SST-immunopositive amacrine cells. By comparing the three age groups. PACAP treatment was shown to induce a moderate elevation of cell densities in both the SST and dopaminergic cell populations in the 12- and 18-month-old animals. By contrast, the control untreated and saline-treated retinas showed a minor cell loss. In conclusion, we report a reliable transgenic model for examining SSTergic amacrine cells. The fundamental novelty of this study is that PACAP could increase the cell density in matured retinal tissue, anticipating new therapeutic potential in age-related pathological processes.

There are two well-characterized cannabinoid receptors (CB1R and CB2R and other candidates): the central nervous system (CNS) enriched CB1R and peripheral tissue enriched CB2R with a wide dynamic range of expression levels in different cell types of human tissues. Hepatocytes and neurons express low baseline CB1R and CB2R, respectively, and their cell-type-specific functions are not well defined. Here we report inducible expression of CB1R in the liver by high-fat and high sugar diet and CB2R in cortical neurons by methamphetamine. While there is less controversy about hepatocyte CB1R, the presence of functional neuronal CB2R is still debated to date. We found that neuron CB2R basal expression was higher than that of hepatocyte CB1R by measuring mRNA levels of specific isoform CB2A in neurons isolated by fluorescence-activated cell sorting (FACS) and CB1A in hepatocytes isolated by collagenase perfusion of liver. For in vivo studies, we generated hepatocyte, dopaminergic neuron, and microglia-specific conditional knockout mice (Abl-Cnr1 Δ , Dat-Cnr2 Δ , and Cx3cr1-Cnr2 Δ ) of CB1R and CB2R by crossing Cnr1 f/f and Cnr2 f/f strains to Abl-Cre, Dat-Cre, and Cx3cr1-Cre deleter mouse strains, respectively. Our data reveals that neuron and microglia CB2Rs are involved in the "tetrad" effects of the mixed agonist WIN 55212-2, CB1R selective agonist arachidonyl-2'-chloroethylamide (ACEA), and CB2R selective agonist JWH133. Dat-Cnr2 Δ and Cx3cr1-Cnr2 Δ mice showed genotypic differences in hypomobility, hypothermia, analgesia, and catalepsy induced by the synthetic cannabinoids. Alcohol conditioned place preference was abolished in DAT-Cnr2 Δ mice and remained intact in Cx3cr1-Cnr2 Δ mice in comparison to WT mice. These Cre-loxP recombinant mouse lines provide unique approaches in cannabinoid research for dissecting the complex endocannabinoid system that is implicated in many chronic disorders.

Atypical antipsychotic drugs were introduced in the early 1990s. Unlike typical antipsychotics, which are effective only against positive symptoms of schizophrenia, atypical antipsychotics are effective against negative and cognitive symptoms as well. Furthermore, they are effective not only in psychotic but also in affective disorders, on their own or as adjuncts to antidepressant drugs. This review presents the neural mechanisms of currently existing atypical antipsychotics and putative antipsychotics currently being investigated in preclinical and clinical studies and how these relate to their effectiveness in mood disorders such as depression, anxiety, and post-traumatic stress disorder (PTSD). Typical antipsychotics act almost exclusively on the dopamine system. Atypical drugs, however, modulate serotonin (5-HT), norepinephrine, and/or histamine neurotransmission as well. This multimodal mechanism of action putatively underlies the beneficial effect of atypical antipsychotics in mood and anxiety disorders. Interestingly, novel experimental drugs having dual antipsychotic and antidepressant therapeutic potential, such as histamine, adenosine, and trace amine-associated receptors (TAAR) ligand, are also characterized by a multimodal stimulatory effect on central 5-HT, norepinephrine, and/or histamine transmission. The multimodal stimulatory effect on central monoamine neurotransmission may be thus primarily responsible for the combined antidepressant and antipsychotic therapeutic potential of certain central nervous system (CNS) drugs.

Schizophrenia is a multifactorial developmental neuropsychiatric disorder. This study examined the interplay of maternal infection and postweaning social isolation, which are prenatal and postnatal risk factors, respectively. Pregnant mice received poly I:C or saline injection on gestation day 9 and the pups were weaned at postnatal day 28. After weaning, male offspring were randomly assigned into group-rearing and isolation-rearing groups. In their adulthood, we performed behavioral tests and characterized the histochemical features of their mesocorticolimbic structures. The sociability and anxiety levels were not affected by either manipulation, but synergistic effects of the two hits on stress-coping behavior was observed. Either of the single manipulations caused defects in sensorimotor gating, novel object recognition and spatial memory tests, but the combination of the two hits did not further exacerbate the disabilities. Prenatal infection increased the number of dopaminergic neurons in midbrain, whereas postweaning isolation decreased the GABAergic neurons in cortex. Single manipulation reduced the dendritic complexity and spine densities of neurons in the medial prefrontal cortex (mPFC) and dentate gyrus. Our results support the current perspective that disturbances in brain development during the prenatal or postnatal period influence the structure and function of the brain and together augment the susceptibility to mental disorders, such as schizophrenia.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Several brain-gut peptides are able to exert neuroprotective effects on the nigrostriatal dopaminergic system. Apelin-13 is a neuropeptide, conveying potential neuroprotective activities. However, whether, and how, apelin-13 could antagonize rotenone-induced neurotoxicity has not yet been elucidated. In the present study, rotenone-treated SH-SY5Y cells and rats were used to clarify whether apelin-13 has protective effects on dopaminergic neurons, both in vivo and in vitro. The results showed that apelin-13 could protect SH-SY5Y cells from rotenone-induced injury and apoptosis. Apelin-13 was able to activate autophagy, and restore rotenone induced autophagy impairment in SH-SY5Y cells, which could be blocked by the autophagy inhibitor 3-Methyladenine. Apelin-13 activated AMPK/mTOR/ULK-1 signaling, AMPKα inhibitor compound C, as well as apelin receptor blockage via siRNA, which could block apelin-13-induced signaling activation, autophagy activation, and protective effects, in rotenone-treated SH-SY5Y cells. These results indicated that apelin-13 exerted neuroprotective properties against rotenone by stimulating AMPK/mTOR/ULK-1 signaling-mediated autophagy via the apelin receptor. We also observed that intracerebroventricular injection of apelin-13 could alleviate nigrostriatal dopaminergic neuron degeneration in rotenone-treated rats. Our findings provide new insights into the mechanism by which apelin-13 might attenuate neurotoxicity in PD.

Parkinson's disease, the second common neurodegenerative disease is clinically characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) with upregulation of neuroinflammatory markers and oxidative stress. Autophagy lysosome pathway (ALP) plays a major role in degradation of damaged organelles and proteins for energy balance and intracellular homeostasis. However, dysfunction of ALP results in impairment of α-synuclein clearance which hastens dopaminergic neurons loss. In this study, we wanted to understand the neuroprotective efficacy of Val in rotenone induced PD rat model. Animals received intraperitoneal injections (2.5 mg/kg) of rotenone daily followed by Val (40 mg/kg, i.p) for four weeks. Valeric acid, a straight chain alkyl carboxylic acid found naturally in Valeriana officianilis have been used in the treatment of neurological disorders. However, their neuroprotective efficacy has not yet been studied. In our study, we found that Val prevented rotenone induced upregulation of pro-inflammatory cytokine oxidative stress, and α-synuclein expression with subsequent increase in vital antioxidant enzymes. Moreover, Val mitigated rotenone induced hyperactivation of microglia and astrocytes. These protective mechanisms prevented rotenone induced dopaminergic neuron loss in SNpc and neuronal fibers in the striatum. Additionally, Val treatment prevented rotenone blocked mTOR-mediated p70S6K pathway as well as apoptosis. Moreover, Val prevented rotenone mediated autophagic vacuole accumulation and increased lysosomal degradation. Hence, Val could be further developed as a potential therapeutic candidate for treatment of PD.

In Parkinson's disease, mitochondrial oxidative stress-mediated apoptosis is a major cause of dopaminergic neuronal loss in the substantia nigra (SN). G protein-coupled receptor 4 (GPR4), previously recognised as an orphan G protein coupled-receptor (GPCR), has recently been claimed as a member of the group of proton-activated GPCRs. Its activity in neuronal apoptosis, however, remains undefined. In this study, we investigated the role of GPR4 in the 1-methyl-4-phenylpyridinium ion (MPP + ) and hydrogen peroxide (H 2 O 2 )-treated apoptotic cell death of stably GPR4-overexpressing and stably GPR4-knockout human neuroblastoma SH-SY5Y cells. In GPR4-OE cells, MPP + and H 2 O 2 were found to significantly increase the expression levels of both mRNA and proteins of the pro-apoptotic Bcl-2-associated X protein (Bax) genes, while they decreased the anti-apoptotic B-cell lymphoma 2 (Bcl-2) genes. In addition, MPP + treatment activated Caspase-3, leading to the cleavage of poly (ADP-ribose) polymerase (PARP) and decreasing the mitochondrial membrane potential (ΔΨm) in GPR4-OE cells. In contrast, H 2 O 2 treatment significantly increased the intracellular calcium ions (Ca 2+ ) and reactive oxygen species (ROS) in GPR4-OE cells. Further, chemical inhibition by NE52-QQ57, a selective antagonist of GPR4, and knockout of GPR4 by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 decreased the Bax/Bcl-2 ratio and ROS generation, and stabilised the ΔΨm, thus protecting the SH-SY5Y cells from MPP + - or H 2 O 2 -induced apoptotic cell death. Moreover, the knockout of GPR4 decreased the proteolytic degradation of phosphatidylinositol biphosphate (PIP 2 ) and subsequent release of the endoplasmic reticulum (ER)-stored Ca 2+ in the cytosol. Our results suggest that the pharmacological inhibition or genetic deletion of GPR4 improves the neurotoxin-induced caspase-dependent mitochondrial apoptotic pathway, possibly through the modulation of PIP 2 degradation-mediated calcium signalling. Therefore, GPR4 presents a potential therapeutic target for neurodegenerative disorders such as Parkinson's disease.

Fabry disease, an X-linked recessive lysosomal disease, results from mutations in the GLA gene encoding lysosomal α-galactosidase A (α-Gal A). Due to these mutations, there is accumulation of globotriaosylceramide (GL-3) in plasma and in a wide range of cells throughout the body. Like other lysosomal enzymes, α-Gal A is synthesized on endoplasmic reticulum (ER) bound polyribosomes, and upon entry into the ER it undergoes glycosylation and folding. It was previously suggested that α-Gal A variants are recognized as misfolded in the ER and undergo ER-associated degradation (ERAD). In the present study, we used Drosophila melanogaster to model misfolding of α-Gal A mutants. We did so by creating transgenic flies expressing mutant α-Gal A variants and assessing development of ER stress, activation of the ER stress response and their relief with a known α-Gal A chaperone, migalastat. Our results showed that the A156V and the A285D α-Gal A mutants underwent ER retention, which led to activation of unfolded protein response (UPR) and ERAD. UPR could be alleviated by migalastat. When expressed in the fly's dopaminergic cells, misfolding of α-Gal A and UPR activation led to death of these cells and to a shorter life span, which could be improved, in a mutation-dependent manner, by migalastat.

Parkinson's disease (PD) is a common neurodegenerative disorder, characterized by selective degeneration of dopaminergic nigrostriatal neurons. Most of the existing pharmacological approaches in PD consider replenishing striatal dopamine. It has been reported that activation of the cholinergic system has neuroprotective effects on dopaminergic neurons, and human α7-nicotinic acetylcholine receptor (α7-nAChR) stimulation may offer a potential therapeutic approach in PD. Our recent in-vitro studies demonstrated that curcumin causes significant potentiation of the function of α7-nAChRs expressed in Xenopus oocytes. In this study, we conducted in vivo experiments to assess the role of the α7-nAChR on the protective effects of curcumin in an animal model of PD. Intra-striatal injection of 6-hydroxydopmine (6-OHDA) was used to induce Parkinsonism in rats. Our results demonstrated that intragastric curcumin treatment (200 mg/kg) significantly improved the abnormal motor behavior and offered neuroprotection against the reduction of dopaminergic neurons, as determined by tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra and caudoputamen. The intraperitoneal administration of the α7-nAChR-selective antagonist methyllycaconitine (1 µg/kg) reversed the neuroprotective effects of curcumin in terms of both animal behavior and TH immunoreactivity. In conclusion, this study demonstrates that curcumin has a neuroprotective effect in a 6-hydroxydopmine (6-OHDA) rat model of PD via an α7-nAChR-mediated mechanism.

The zebrafish ( Danio rerio ) is routinely used in biological studies as a vertebrate model system that provides unique strengths allowing applications in studies of neurodevelopmental and neurodegenerative diseases. One specific advantage is that the neurotransmitter systems are highly conserved throughout vertebrate evolution, including between zebrafish and humans. Disruption of the dopaminergic signaling pathway is linked to multiple neurological disorders. One of the most common is Parkinson's disease, a neurodegenerative disease associated with the loss of dopaminergic neurons, among other neuropathological characteristics. In this review, the development of the zebrafish's dopaminergic system, focusing on genetic control of the dopaminergic system, is detailed. Second, neurotoxicant models used to study dopaminergic neuronal loss, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the pesticides paraquat and rotenone, and 6-hydroxydopamine (6-OHDA), are described. Next, zebrafish genetic knockdown models of dj1 , pink1 , and prkn established for investigating mechanisms of Parkinson's disease are discussed. Chemical modulators of the dopaminergic system are also highlighted to showcase the applicability of the zebrafish to identify mechanisms and treatments for neurodegenerative diseases such as Parkinson's disease associated with the dopaminergic system.

Alzheimer's disease (AD) is a progressive neurodegenerative disease. In this study, to investigate the effect of microglial elimination on AD progression, we administered PLX3397, a selective colony-stimulating factor 1 receptor inhibitor, to the mouse model of AD (5xFAD mice). Amyloid-beta (Aβ) deposition and amyloid precursor protein (APP), carboxyl-terminal fragment β, ionized calcium-binding adaptor molecule 1, synaptophysin, and postsynaptic density (PSD)-95 levels were evaluated in the cortex and hippocampus. In addition, the receptor density changes in dopamine D2 receptor (D2R) and metabotropic glutamate receptor 5 were evaluated using positron emission tomography (PET). D2R, tyrosine hydroxylase (TH), and dopamine transporter (DAT) levels were analyzed in the brains of Tg (5xFAD) mice using immunohistochemistry. PLX3397 administration significantly decreased Aβ deposition following microglial depletion in the cortex and hippocampus of Tg mice. In the neuro-PET studies, the binding values for D2R in the Tg mice were lower than those in the wild type mice; however, after PLX3397 treatment, the binding dramatically increased. PLX3397 administration also reversed the changes in synaptophysin and PSD-95 expression in the brain. Furthermore, the D2R and TH expression in the brains of Tg mice was significantly lower than that in the wild type; however, after PLX3397 administration, the D2R and TH levels were significantly higher than those in untreated Tg mice. Thus, our findings show that administering PLX3397 to aged 5xFAD mice could prevent amyloid pathology, concomitant with the rescue of dopaminergic signaling, suggesting that targeting microglia may serve as a useful therapeutic option for neurodegenerative diseases, including AD.

Parkinson's Disease (PD) is a neurological disease characterized by the progressive degeneration of the nigrostriatal dopaminergic pathway with consequent loss of neurons in the substantia nigra pars compacta and dopamine depletion. The cytoplasmic inclusions of α-synuclein (α-Syn), known as Lewy bodies, are the cytologic hallmark of PD. The presence of α-Syn aggregates causes mitochondrial degeneration, responsible for the increase in oxidative stress and consequent neurodegeneration. PD is a progressive disease that shows a complicated pathogenesis. The current therapies are used to alleviate the symptoms of the disease without changing its clinical course. Recently, phytocompounds with neuroprotective effects and antioxidant properties such as caffeine have aroused the interest of researchers. The purpose of this review is to summarize the preclinical studies present in the literature and clinical trials recorded in ClinicalTrial.gov, aimed at illustrating the effects of caffeine used as a nutraceutical compound combined with the current PD therapies. Therefore, the preventive effects of caffeine in the neurodegeneration of dopaminergic neurons encourage the use of this alkaloid as a supplement to reduce the progress of the PD., Competing Interests: The authors declare there no conflict of interest.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. The neurodegeneration leading to incapacitating motor abnormalities mainly occurs in the nigrostriatal pathway due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Several animal models have been developed not only to better understand the mechanisms underlying neurodegeneration but also to test the potential of emerging disease-modifying therapies. However, despite aging being the main risk factor for developing idiopathic PD, most of the studies do not use aged animals. Therefore, this study aimed at assessing the effect of aging in the unilateral 6-hydroxydopamine (6-OHDA)-induced animal model of PD. For this, female young adult and aged rats received a unilateral injection of 6-OHDA into the medial forebrain bundle. Subsequently, the impact of aging on 6-OHDA-induced effects on animal welfare, motor performance, and nigrostriatal integrity were assessed. The results showed that aging had a negative impact on animal welfare after surgery. Furthermore, 6-OHDA-induced impairments on skilled motor function were significantly higher in aged rats when compared with their younger counterparts. Nigrostriatal histological analysis further revealed an increased 6-OHDA-induced dopaminergic cell loss in the SNpc of aged animals when compared to young animals. Overall, our results demonstrate a higher susceptibility of aged animals to 6-OHDA toxic insult.

Calcium homeostasis is a cellular process required for proper cell function and survival, maintained by the coordinated action of several transporters, among them members of the Na + /Ca 2+ -exchanger family, such as SLC8A3. Transforming growth factor beta (TGF-β) signaling defines neuronal development and survival and may regulate the expression of channels and transporters. We investigated the regulation of SLC8A3 by TGF-β in a conditional knockout mouse with deletion of TGF-β signaling from Engrailed 1-expressing cells, i.e., in cells from the midbrain and rhombomere 1, and elucidated the underlying molecular mechanisms. The results show that SLC8A3 is significantly downregulated in developing dopaminergic and dorsal raphe serotonergic neurons in mutants and that low SLC8A3 abundance prevents the expression of the anti-apoptotic protein Bcl-xL. TGF-β signaling affects SLC8A3 via the canonical and p38 signaling pathway and may increase the binding of Smad4 to the Slc8a3 promoter. Expression of the lipid peroxidation marker malondialdehyde (MDA) was increased following knockdown of Slc8a3 expression in vitro. In neurons lacking TGF-β signaling, the number of MDA- and 4-hydroxynonenal (4-HNE)-positive cells was significantly increased, accompanied with increased cellular 4-HNE abundance. These results suggest that TGF-β contributes to the regulation of SLC8A3 expression in developing dopaminergic and dorsal raphe serotonergic neurons, thereby preventing oxidative stress.

Increasing reports of neurological and psychiatric complications due to psychostimulant synthetic cathinones (SCs) have recently raised public concern. However, the precise mechanism of SC toxicity is unclear. This paucity of understanding highlights the need to investigate the in-vitro toxicity and mechanistic pathways of three SCs: butylone, pentylone, and 3,4-Methylenedioxypyrovalerone (MDPV). Human neuronal cells of SH-SY5Y were cultured in supplemented DMEM/F12 media and differentiated to a neuronal phenotype using retinoic acid (10 μM) and 12- O -tetradecanoylphorbol-13-acetate (81 nM). Trypan blue and lactate dehydrogenase assays were utilized to assess the neurotoxicity potential and potency of these three SCs. To investigate the underlying neurotoxicity mechanisms, measurements included markers of oxidative stress, mitochondrial bioenergetics, and intracellular calcium (Ca 2+ ), and cell death pathways were evaluated at two doses (EC 15 and EC 40 ), for each drug tested. Following 24 h of treatment, all three SCs exhibited a dose-dependent neurotoxicity, characterized by a significant ( p < 0.0001 vs. control) production of reactive oxygen species, decreased mitochondrial bioenergetics, and increased intracellular Ca 2+ concentrations. The activation of caspases 3 and 7 implicated the orchestration of mitochondrial-mediated neurotoxicity mechanisms for these SCs. Identifying novel therapeutic agents to enhance an altered mitochondrial function may help in the treatment of acute-neurological complications arising from the illicit use of these SCs., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

The ventral tegmental area (VTA) plays an important role in the reward and motivational processes that facilitate the development of drug addiction. Presynaptic α1-AR activation modulates glutamate and Gamma-aminobutyric acid (GABA) release. This work elucidates the role of VTA presynaptic α1-ARs and their modulation on glutamatergic and GABAergic neurotransmission during cocaine sensitization. Excitatory and inhibitory currents (EPSCs and IPSCs) measured by a whole cell voltage clamp show that α1-ARs activation increases EPSCs amplitude after 1 day of cocaine treatment but not after 5 days of cocaine injections. The absence of a pharmacological response to an α1-ARs agonist highlights the desensitization of the receptor after repeated cocaine administration. The desensitization of α1-ARs persists after a 7-day withdrawal period. In contrast, the modulation of α1-ARs on GABA neurotransmission, shown by decreases in IPSCs' amplitude, is not affected by acute or chronic cocaine injections. Taken together, these data suggest that α1-ARs may enhance DA neuronal excitability after repeated cocaine administration through the reduction of GABA inhibition onto VTA dopamine (DA) neurons even in the absence of α1-ARs' function on glutamate release and protein kinase C (PKC) activation. α1-AR modulatory changes in cocaine sensitization increase our knowledge of the role of the noradrenergic system in cocaine addiction and may provide possible avenues for therapeutics.

Chronic neuroinflammation contributes to the pathogenesis of Parkinson's disease (PD). However, cellular and molecular mechanisms by which chronic neuroinflammation is formed and maintained remain elusive. This study aimed to explore detailed mechanisms by which anti-inflammatory cytokine interleukin-10 (IL-10) prevented chronic neuroinflammation and neurodegeneration. At 24 h after an intranigral injection of lipopolysaccharide (LPS), levels of NLRP3, pro-caspase-1, pro-IL-1β, active caspase-1, and mature IL-1β in the midbrain were much higher in IL-10 -/- mice than wildtype mice. Mechanistically, IL-10 -/- microglia produced more intracellular reactive oxygen species (iROS) and showed more profound activation of NADPH oxidase (NOX2) than wildtype microglia. Meanwhile, suppression of NOX2-derived iROS production blocked LPS-elicited caspase-1 activation and IL-1β maturation in IL-10 -/- microglia in vitro and in vivo. One month after intranigral LPS injection, IL-10 -/- mice revealed more profound microglial activation and dopaminergic neurodegeneration in the substantia nigra than wildtype mice. Importantly, such PD-like pathological changes were prevented by IL-1β neutralization. Collectively, IL-10 inhibited LPS-elicited production of NOX2-derived iROS thereby suppressing synthesis of NLRP3, pro-caspase-1 and pro-IL-1β and their activation and cleavage. By this mechanism, IL-10 prevented chronic neuroinflammation and neurodegeneration. This study suggested boosting anti-inflammatory effects of IL-10 and suppressing NLRP3 inflammasome activation could be beneficial for PD treatment.

In our ongoing research to discover natural products with neuroprotective effects, hyperoside (quercetin 3-O-galactoside) was isolated from Acer tegmentosum, which has been used in Korean traditional medicine to treat liver-related disorders. Here, we demonstrated that hyperoside protects cultured dopaminergic neurons from death via reactive oxygen species (ROS)-dependent mechanisms, although other relevant mechanisms of hyperoside activity remain largely uncharacterized. For the first time, we investigated the neuroprotective effects of hyperoside on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in neurons, and the possible underlying mechanisms. Hyperoside significantly ameliorated the loss of neuronal cell viability, lactate dehydrogenase release, excessive ROS accumulation and mitochondrial membrane potential dysfunction associated with 6-OHDA-induced neurotoxicity. Furthermore, hyperoside treatment activated the nuclear erythroid 2-related factor 2 (Nrf2), an upstream molecule of heme oxygenase-1 (HO-1). Hyperoside also induced the expression of HO-1, an antioxidant response gene. Remarkably, we found that the neuroprotective effects of hyperoside were weakened by an Nrf2 small interfering RNA, which blocked the ability of hyperoside to inhibit neuronal death, indicating the vital role of HO-1. Overall, we show that hyperoside, via the induction of Nrf2-dependent HO-1 activation, suppresses neuronal death caused by 6-OHDA-induced oxidative stress. Moreover, Nrf2-dependent HO-1 signaling activation represents a potential preventive and therapeutic target in Parkinson's disease management., Competing Interests: The authors declare no conflict of interest.

α-Synuclein is an abundant neuronal protein that accumulates in insoluble inclusions in Parkinson's disease and other synucleinopathies. Fatty acids partially regulate α-Synuclein accumulation, and mesencephalic dopaminergic neurons highly express fatty acid-binding protein 3 (FABP3). We previously demonstrated that FABP3 knockout mice show decreased α-Synuclein oligomerization and neuronal degeneration of tyrosine hydroxylase (TH)-positive neurons in vivo . In this study, we newly investigated the importance of FABP3 in α-Synuclein uptake, 1-methyl-4-phenylpyridinium (MPP + )-induced axodendritic retraction, and mitochondrial dysfunction. To disclose the issues, we employed cultured mesencephalic neurons derived from wild type or FABP3 -/- C57BL6 mice and performed immunocytochemical analysis. We demonstrated that TH + neurons from FABP3 +/+ mice take up α-Synuclein monomers while FABP3 -/- TH + neurons do not. The formation of filamentous α-Synuclein inclusions following treatment with MPP + was observed only in FABP3 +/+ , and not in FABP3 -/- neurons. Notably, detailed morphological analysis revealed that FABP -/- neurons did not exhibit MPP + -induced axodendritic retraction. Moreover, FABP3 was also critical for MPP + -induced reduction of mitochondrial activity and the production of reactive oxygen species. These data indicate that FABP3 is critical for α-Synuclein uptake in dopaminergic neurons, thereby preventing synucleinopathies, including Parkinson's disease.

Caspases are a family of conserved cysteine proteases that play key roles in multiple cellular processes, including programmed cell death and inflammation. Recent evidence shows that caspases are also involved in crucial non-apoptotic functions, such as dendrite development, axon pruning, and synaptic plasticity mechanisms underlying learning and memory processes. The activated form of caspase-3, which is known to trigger widespread damage and degeneration, can also modulate synaptic function in the adult brain. Thus, in the present study, we tested the hypothesis that caspase-3 modulates synaptic plasticity at corticostriatal synapses in the phosphatase and tensin homolog (PTEN) induced kinase 1 (PINK1) mouse model of Parkinson's disease (PD). Loss of PINK1 has been previously associated with an impairment of corticostriatal long-term depression (LTD), rescued by amphetamine-induced dopamine release. Here, we show that caspase-3 activity, measured after LTD induction, is significantly decreased in the PINK1 knockout model compared with wild-type mice. Accordingly, pretreatment of striatal slices with the caspase-3 activator α-(Trichloromethyl)-4-pyridineethanol (PETCM) rescues a physiological LTD in PINK1 knockout mice. Furthermore, the inhibition of caspase-3 prevents the amphetamine-induced rescue of LTD in the same model. Our data support a hormesis-based double role of caspase-3; when massively activated, it induces apoptosis, while at lower level of activation, it modulates physiological phenomena, like the expression of corticostriatal LTD. Exploring the non-apoptotic activation of caspase-3 may contribute to clarify the mechanisms involved in synaptic failure in PD, as well as in view of new potential pharmacological targets.

Current therapeutics for Parkinson's disease (PD) are only effective in providing relief of symptoms such as rigidity, tremors and bradykinesia, and do not exert disease-modifying effects by directly modulating mitochondrial function. Here, we investigated auraptene (AUR) as a potent therapeutic reagent that specifically protects neurotoxin-induced reduction of mitochondrial respiration and inhibits reactive oxygen species (ROS) generation. Further, we explored the mechanism and potency of AUR in protecting dopaminergic neurons. Treatment with AUR significantly increased the viability of substantia nigra (SN)-derived SN4741 embryonic dopaminergic neuronal cells and reduced rotenone-induced mitochondrial ROS production. By inducing antioxidant enzymes AUR treatment also increased oxygen consumption rate. These results indicate that AUR exerts a protective effect against rotenone-induced mitochondrial oxidative damage. We further assessed AUR effects in vivo, investigating tyrosine hydroxylase (TH) expression in the striatum and substantia nigra of MPTP-induced PD model mice and behavioral changes after injection of AUR. AUR treatment improved movement, consistent with the observed increase in the number of dopaminergic neurons in the substantia nigra. These results demonstrate that AUR targets dual pathogenic mechanisms, enhancing mitochondrial respiration and attenuating ROS production, suggesting that the preventative potential of this natural compound could lead to improvement in PD-related neurobiological changes.

Background: Parkinson's disease (PD) is one of the most common neurodegenerative disorders involving devastating loss of dopaminergic neurons in the substantia nigra. Early steps in PD pathogenesis include mitochondrial dysfunction, and mutations in mitochondrial genes have been linked to familial forms of the disease. However, low penetrance of mutations indicates a likely important role for environmental factors in PD risk through gene by environment interactions. Herein, we study how genetic deficiencies in mitochondrial dynamics processes including fission, fusion, and mitophagy interact with environmental exposures to impact neurodegeneration., Methods: We utilized the powerful model organism Caenorhabditis elegans to study ultraviolet C radiation (UVC)- and 6-hydroxydopamine-induced degeneration of fluorescently-tagged dopaminergic neurons in the background of fusion deficiency (MFN1/2 homolog, fzo-1 ), fission deficiency (DMN1L homolog, drp-1 ), and mitochondria-specific autophagy (mitophagy) deficiency (PINK1 and PRKN homologs, pink-1 and pdr-1 )., Results: Overall, we found that deficiency in either mitochondrial fusion or fission sensitizes nematodes to UVC exposure (used to model common environmental pollutants) but protects from 6-hydroxydopamine-induced neurodegeneration. By contrast, mitophagy deficiency makes animals more sensitive to these stressors with an interesting exception- pink-1 deficiency conferred remarkable protection from 6-hydroxydopamine. We found that this protection could not be explained by compensatory antioxidant gene expression in pink-1 mutants or by differences in mitochondrial morphology., Conclusions: Together, our results support a strong role for gene by environment interactions in driving dopaminergic neurodegeneration and suggest that genetic deficiency in mitochondrial processes can have complex effects on neurodegeneration.

Background: The neurological effects of short-term dioxin exposure during the fetal period is an important health risk in humans. Here, we investigated the effects of dioxin on neural differentiation using human embryonic stem cells (hESCs) to evaluate human susceptibility to dioxin., Methods: Using an enzymatic bulk passage, neural differentiation from human ESCs was carried out. 2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) was added to various stages of culture. The expression levels of the neuronal markers microtubule-associated protein 2 (MAP2) and thyroxine hydroxylase (TH) were measured by RT-qPCR and image analysis of immunostaining., Results: Although early-stage neuronal cells are quite resistant to TCDD, the numbers of neural rosettes and increases in mRNA expression levels and the number of cells positive for MAP2 and TH were significant by temporal exposure at embryoid body stage (Day9-exposure group). In contrast, the TCDD exposures against ESCs (Day0-exposure group) and differentiated neural cells (Day35-exposure group) were not affected at all. The increment was similarly observed by continuous exposure of TCDD from Day9 through Day60., Conclusions: These results indicated that dioxin exposure during the early stage of differentiation from hESCs increases the contents of neuronal cells, especially TH-positive neuronal cells. Regulations of aryl hydrocarbon receptor (AHR) signaling in an early stage of embryogenesis should be investigated extensively to understand the mechanism underlying the increase in neuronal cell populations and to apply the knowledge to regenerative medicine.

Ras homolog enriched in striatum (Rhes) is a protein that exerts important physiological functions and modulates psychostimulant drug effects. On this basis, the object of this study was to assess 3,4-methylenedioxymethamphetamine (MDMA) effects on microglial (CD11b) and astroglial (GFAP) activation and on dopamine neuron degeneration (TH) in wild-type (WT) and Rhes knockout (KO) male and female mice of different ages. Motor activity was also evaluated. Adult (3 months) MDMA-treated mice displayed an increase in GFAP-positive cells in striatum (STR), whereas the substantia nigra pars compacta (SNc) was affected only in male mice. In these mice, the increase of CD11b was more extensive including STR, SNc, motor cortex (CTX), ventral tegmental area (VTA), and nucleus accumbens (NAc). MDMA administration also affected TH immunoreactivity in both STR and SNc of male but not female WT and Rhes KO mice. In middle-aged mice (12 months), MDMA administration further increased GFAP and CD11b and decreased TH immunoreactivity in STR and SNc of all mice. Finally, MDMA induced a higher increase of motor activity in adult Rhes KO male, but not female mice. The results show that Rhes protein plays an important role on MDMA-mediated neuroinflammation and neurodegeneration dependent on gender and age, and confirm the important role of Rhes protein in neuroinflammatory and neurodegenerative processes.

Parkinson's disease (PD), a multifactorial movement disorder that involves progressive degeneration of the nigrostriatal system affecting the movement ability of the patient. Oxidative stress and neuroinflammation both are shown to be involved in the etiopathogenesis of PD. The aim of this study was to evaluate the therapeutic potential of thymol, a dietary monoterpene phenol in rotenone (ROT)-induced neurodegeneration in rats that precisely mimics PD in humans. Male Wistar rats were injected ROT at a dose of 2.5 mg/kg body weight for 4 weeks, to induce PD. Thymol was co-administered for 4 weeks at a dose of 50 mg/kg body weight, 30 min prior to ROT injection. The markers of dopaminergic neurodegeneration, oxidative stress and inflammation were estimated using biochemical assays, enzyme-linked immunosorbent assay, western blotting and immunocytochemistry. ROT challenge increased the oxidative stress markers, inflammatory enzymes and cytokines as well as caused significant damage to nigrostriatal dopaminergic system of the brain. Thymol treatment in ROT challenged rats appears to significantly attenuate dopaminergic neuronal loss, oxidative stress and inflammation. The present study showed protective effects of thymol in ROT-induced neurotoxicity and neurodegeneration mediated by preservation of endogenous antioxidant defense networks and attenuation of inflammatory mediators including cytokines and enzymes.

Several mechanisms underlying 3,4-Methylenedioxy- N -methylamphetamine (MDMA) neurotoxicity have been proposed, including neurochemical alterations and excitotoxicity mediated by reactive oxygen species (ROS), nitric oxide (NO), and reactive nitrogen species (RNS). However, ROS, NO, and RNS sources in the brain are not fully known. We aimed to investigate possible alterations in the expression of the ROS producer NOX enzymes (NOX2, NOX1, and NOX4), NO generators (iNOS, eNOS, and nNOS), markers of oxidative (8-hydroxy-2'-deoxyguanosine, 8OHdG), and nitrosative (3-nitrotyrosine, NT) stress, as well as the colocalization between cells positive for the dopamine transporter (DT1) and cells expressing the neuronal nuclei (NeuN) marker, in the frontal cortex of rats receiving saline or MDMA, sacrificed 6 h, 16 h, or 24 h after its administration. MDMA did not affect NOX2, NOX1, and NOX4 immunoreactivity, whereas iNOS expression was enhanced. The number of NT-positive cells was increased in MDMA-exposed animals, whereas no differences were detected in 8OHdG expression among experimental groups. MDMA and NT markers colocalized with DT1 positive cells. DT1 immunostaining was found in NeuN-positive stained cells. Virtually no colocalization was observed with microglia and astrocytes. Moreover, MDMA immunostaining was not found in NOX2-positive cells. Our results suggest that iNOS-derived nitrosative stress, but not NOX enzymes, may have a crucial role in the pathogenesis of MDMA-induced neurotoxicity, highlighting the specificity of different enzymatic systems in the development of neuropathological alterations induced by the abuse of this psychoactive compound.

In previous studies, we found regional differences in the induction of antioxidative molecules in astrocytes against oxidative stress, postulating that region-specific features of astrocytes lead region-specific vulnerability of neurons. We examined region-specific astrocytic features against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) as an oxidative stress using co-culture of mesencephalic neurons and mesencephalic or striatal astrocytes in the present study. The 6-OHDA-induced reduction of mesencephalic dopamine neurons was inhibited by co-culturing with astrocytes. The co-culture of midbrain neurons with striatal astrocytes was more resistant to 6-OHDA than that with mesencephalic astrocytes. Furthermore, glia conditioned medium from 6-OHDA-treated striatal astrocytes showed a greater protective effect on the 6-OHDA-induced neurotoxicity and oxidative stress than that from mesencephalic astrocytes. The cDNA microarray analysis showed that the number of altered genes in both mesencephalic and striatal astrocytes was fewer than that changed in either astrocyte. The 6-OHDA treatment, apparently up-regulated expressions of Nrf2 and some anti-oxidative or Nrf2-regulating phase II, III detoxifying molecules related to glutathione synthesis and export in the striatal astrocytes but not mesencephalic astrocytes. There is a profound regional difference of gene expression in astrocytes induced by 6-OHDA. These results suggest that protective features of astrocytes against oxidative stress are more prominent in striatal astrocytes, possibly by secreting humoral factors in striatal astrocytes.

Neuroinflammation has been demonstrated to be linked with Parkinson's disease (PD), Alzheimer's disease, and cerebral ischemia. Our previous investigation had identified that kaempferol (KAE) exerted protective effects on cortex neuron injured by LPS. In this study, the effects and possible mechanism of KAE on striatal dopaminergic neurons induced by LPS in mice were further investigated. The results showed that KAE improved striatal neuron injury, and increased the levels of tyrosine hydroxylase (TH) and postsynaptic density protein 95 (PSD95) in the striatum of mice. In addition, KAE inhibited the production of pro-inflammatory cytokines, including interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), reduced the level of monocyte chemotactic protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1), and cyclooxygenase-2 (COX-2) in the striatum tissues. Furthermore, KAE protected blood-brain barrier (BBB) integrity and suppressed the activation of the HMGB1/TLR4 inflammatory pathway induced by LPS in striatum tissues of mice. In conclusion, these results suggest that KAE may have neuroprotective effects against striatum injury that is induced by LPS and the possible mechanisms are involved in anti-neuroinflammation, maintaining BBB integrity, and down-regulating the HMGB1/TLR4 pathway.