Tyrosine Hydroxylase Protein Research Articles

Alterations in Hypothalamic Mechanisms Associated with Reduced Suckling-Induced Prolactin Secretion in the Lactation-Deficient OFA hr/hr Rat

Introduction: OFA hr/hr rats have deficient lactation with impaired suckling-induced PRL release. Unlike their background strain, Sprague-Dawley (SD) rats, OFA rats display abnormal mediobasal hypothalamus (MBH) dopaminergic tone during late pregnancy and lactation. We explored if the expression of MBH components, including various receptors (R) and proteins that regulate the dopaminergic system, is altered in mid-lactating OFA compared to SD rats, which may be associated with the abnormality. Methods: Four groups of mid-lactating rats were used: continuous lactation; pups separated overnight; 30-min suckling (S); and 2 h or 4 h S after separation. Mothers were sacrificed to obtain serum for PRL RIA and MBHs to determine tyrosine hydroxylase (TH), PRL-R, PRL signaling molecules (activator: STAT5b; inhibitors: SOCS1, SOCS3, CIS), opioids (PENK, PDYN), and µ- and κ-opioid R (MOR, KOR) mRNA expression by qPCR and phospho-TH (p-TH) and TH proteins by Western blot. Results: Suckling-induced PRL was lower in OFA and p-TH expression diminished in both strains. Separation increased TH mRNA and protein in SD, which decreased after 4 h S, but OFA protein levels remained unchanged. Separation of pups also resulted in decreased PRL-R and CIS expression in SD but increased PRL-R and SOCS3 in OFA. Despite the lower PRL-R, STAT5b, SOCS1, and SOCS3 levels in OFA compared to SD, suckling diminished them further. We observed subtle changes in SD opioids and their R, but in OFA, suckling decreased PENK, KOR, and MOR. Conclusion: The different patterns of TH, opioids, their R, and PRL signaling inhibitor expression with conserved TH activation by suckling may disturb the balance between stimulation and inhibition of PRL release resulting in impaired suckling-induced PRL secretion in OFA rats.

The prominin-like Gene Expressed in a Subset of Dopaminergic Neurons Regulates Locomotion in Drosophila.

CD133, also known as prominin-1, was first identified as a biomarker of mammalian cancer and neural stem cells. Previous studies have shown that the prominin-like (promL) gene, an orthologue of mammalian CD133 in Drosophila, plays a role in glucose and lipid metabolism, body growth, and longevity. Because locomotion is required for food sourcing and ultimately the regulation of metabolism, we examined the function of promL in Drosophila locomotion. Both promL mutants and pan-neuronal promL inhibition flies displayed reduced spontaneous locomotor activity. As dopamine is known to modulate locomotion, we also examined the effects of promL inhibition on the dopamine concentration and mRNA expression levels of tyrosine hydroxylase (TH) and DOPA decarboxylase (Ddc), the enzymes responsible for dopamine biosynthesis, in the heads of flies. Compared with those in control flies, the levels of dopamine and the mRNAs encoding TH and Ddc were lower in promL mutant and pan-neuronal promL inhibition flies. In addition, an immunostaining analysis revealed that, compared with control flies, promL mutant and pan-neuronal promL inhibition flies had lower levels of the TH protein in protocerebral anterior medial (PAM) neurons, a subset of dopaminergic neurons. Inhibition of promL in these PAM neurons reduced the locomotor activity of the flies. Overall, these findings indicate that promL expressed in PAM dopaminergic neurons regulates locomotion by controlling dopamine synthesis in Drosophila.

Obesity in male volcano mice Neotomodon alstoni affects the daily rhythm of metabolism and thermoregulation.

The mouse N. alstoni spontaneously develops the condition of obesity in captivity when fed regular chow. We aim to study the differences in metabolic performance and thermoregulation between adult lean and obese male mice. The experimental approach included indirect calorimetry using metabolic cages for VO2 intake and VCO2 production. In contrast, the body temperature was measured and analyzed using intraperitoneal data loggers. It was correlated with the relative presence of UCP1 protein and its gene expression from interscapular adipose tissue (iBAT). We also explored in this tissue the relative presence of Tyrosine Hydroxylase (TH) protein, the rate-limiting enzyme for catecholamine biosynthesis present in iBAT. Results indicate that obese mice show a daily rhythm persists in estimated parameters but with differences in amplitude and profile. Obese mice presented lower body temperature, and a low caloric expenditure, together with lower VO2 intake and VCO2 than lean mice. Also, obese mice present a reduced thermoregulatory response after a cold pulse. Results are correlated with a low relative presence of TH and UCP1 protein. However, qPCR analysis of Ucp1 presents an increase in gene expression in iBAT. Histology showed a reduced amount of brown adipocytes in BAT. The aforementioned indicates that the daily rhythm in aerobic metabolism, thermoregulation, and body temperature control have reduced amplitude in obese mice Neotomodon alstoni.

The proSAAS Chaperone Provides Neuroprotection and Attenuates Transsynaptic α-Synuclein Spread in Rodent Models of Parkinson's Disease.

Parkinson's disease involves aberrant aggregation of the synaptic protein alpha-synuclein (aSyn) in the nigrostriatal tract. We have previously shown that proSAAS, a small neuronal chaperone, blocks aSyn-induced dopaminergic cytotoxicity in primary nigral cultures. To determine if proSAAS overexpression is neuroprotective in animal models of Parkinson's disease. proSAAS- or GFP-encoding lentivirus was injected together with human aSyn-expressing AAV unilaterally into the substantia nigra of rats and motor asymmetry assessed using a battery of motor performance tests. Dopamine neuron survival was assessed by nigral stereology and striatal tyrosine hydroxylase (TH) densitometry. To examine transsynaptic spread of aSyn, aSyn AAV was injected into the vagus of mice in the presence of AAVs encoding either GFP or proSAAS; the spread of aSyn-positive neurites into rostral nuclei was quantified following immunohistochemistry. Coinjection of proSAAS-encoding lentivirus profoundly reduced the motor asymmetry caused by unilateral nigral AAV-mediated human aSyn overexpression. This was accompanied by significant amelioration of the human aSyn-induced loss of both nigral TH-positive cells and striatal TH-positive terminals, demonstrating clear proSAAS-mediated protection of the nigrostriatal tract. ProSAAS overexpression reduced human aSyn protein levels in nigra and striatum and reduced the loss of TH protein in both regions. Following vagal administration of human aSyn-encoding AAV, the number of human aSyn-positive neurites in the pons and caudal midbrain was considerably reduced in mice coinjected with proSAAS-, but not GFP-encoding AAV, supporting proSAAS-mediated blockade of transsynaptic aSyn transmission. The proSAAS chaperone may represent a promising target for therapeutic development in Parkinson's disease.

Embelin and levodopa combination therapy for improved Parkinson's disease treatment.

Parkinson’s disease (PD), a progressive neurodegenerative disorder, affects dopaminergic neurons. Oxidative stress and gut damage play critical roles in PD pathogenesis. Inhibition of oxidative stress and gut damage can prevent neuronal death and delay PD progression. The objective of this study was to evaluate the therapeutic effect of embelin or the combination with levodopa (LD) in a rotenone-induced PD mouse model. At the end of experimentation, the mice were sacrificed and the midbrain was used to evaluate various biochemical parameters, such as nitric oxide, peroxynitrite, urea, and lipid peroxidation. In the substantia nigra (midbrain), tyrosine hydroxylase (TH) expression was examined by immunohistochemistry, and Nurr1 expression was evaluated by western blotting. Gut histopathology was evaluated on tissue sections stained with hematoxylin and eosin. In silico molecular docking studies of embelin and α-synuclein (α-syn) fibrils were also performed. Embelin alone or in combination with LD ameliorated oxidative stress and gut damage. TH and Nurr1 protein levels were also significantly restored. Docking studies confirmed the affinity of embelin toward α-syn. Taken together, embelin could be a promising drug for the treatment of PD, especially when combined with LD.

M6A methyltransferase METTL3 participated in sympathetic neural remodeling post-MI via the TRAF6/NF-κB pathway and ROS production

ObjectiveSudden cardiac death caused by ventricular arrhythmias (VAs) is the main cause of high mortality in patients with myocardial infarction (MI). Sympathetic neural remodeling caused by inflammation after MI is closely associated with the occurrence of VAs. METTL3, the earliest identified m6A methyltransferase, is critical in mediating inflammatory responses. Our aim was to investigate whether the m6A methyltransferase METTL3 was involved in sympathetic remodeling post-MI and its specific mechanism. Methods and resultsA rat MI model was established via left coronary artery ligation. The expression of METTL3, TRAF6, NOX2, and NF-κB increased at 3 days and remained elevated at 7 days after MI, as determined via Western blotting. METTL3 was primarily present in macrophages, as determined via immunofluorescence. Intramyocardial injection of lentivirus carrying METTL3-shRNA inhibited METTL3 expression in vivo. Methylated immunoprecipitation-qPCR determined the METTL3 knockdown inhibited the m6A level of TRAF6 mRNA 3′-UTR. The co-immunoprecipitation experiment proved that METTL3 combines with TRAF6. Western blotting showed that silencing METTL3 inhibited TRAF6 level, NF-κB activation, and ROS production; decreased cytokine release (TNF-α and IL-1β); and downregulated nerve growth factor expression. Finally, METTL3 knockdown reduced sympathetic remodeling after MI, as determined via immunofluorescence assays of tyrosine hydroxylase and growth-associated protein 43. Programmed electrical stimulation, renal sympathetic nerve activity recording, and haemodynamic measurements showed that METTL3 inhibition decreased sympathetic activity and improved cardiac function. ConclusionsDownregulation of METTL3 expression attenuated the excessive sympathetic neural remodeling induced by MI, further reducing the incidence of VAs and improving cardiac function. This was partly associated with the inhibition of the TRAF6/NF-κB pathway and ROS production.

The Effects of Novel Formulations of Edaravone and Curcumin in the Mouse Intrastriatal Lipopolysaccharide Model of Parkinson's Disease.

The major hallmark of Parkinson's disease (PD) is the degeneration of dopaminergic neurons in the substantia nigra (SN), which is responsible for the core motor symptoms of PD. Currently, there is no cure for PD, and its prevalence is increasing, prompting the search for novel neuroprotective treatments. Neuroinflammation is a core pathological process in PD, evident by increased inflammatory biomarkers in the SN and cerebrospinal fluid. Interestingly, epidemiological studies have reported a reduced risk of PD in users of non-steroidal anti-inflammatory drugs compared to non-users, suggesting the neuroprotective potential of anti-inflammatory drugs. Therefore, this study aimed to: (1) test the efficacy of novel oral formulations of edaravone (EDR) and curcumin (CUR) (which possess anti-inflammatory and anti-oxidative properties) to alleviate motor and non-motor symptoms, and associated pathology in the intrastriatal lipopolysaccharide (LPS) model of PD; (2) investigate the expression of proteins linked to familial PD and markers of autophagy in the intrastriatal LPS model treated with EDR and CUR. Fifty-two C57BL/6 mice were divided into 4 groups, namely; (1) control + vehicle; (2) LPS + vehicle; (3) LPS + EDR (made in vehicle) and (4) LPS + CUR (made in vehicle). 10 μg of LPS was administered stereotaxically into the right striatum, and EDR and CUR treatments were initiated 2-weeks after the LPS injections. Behavioural tests were carried out at 4- and 8-weeks after LPS injection followed by tissue collection at 8-weeks. Intrastriatal administration of LPS induced motor deficits and anxiety-like behaviours at 4- and 8-weeks, which were accompanied by astroglial activation, increased protein expression of α-synuclein, heat shock cognate protein of 70 kDa (HSC-70) and Rab-10, and reduced levels of tyrosine hydroxylase (TH) protein in the striatum. Additionally, LPS induced astroglial activation in the olfactory bulb, along with changes in the protein expression of HSC-70. The changes associated with EDR and CUR in the striatum and olfactory bulb were not statistically significant compared to the LPS group. Intrastriatal administration of LPS induced pathological changes of PD such as motor deficits, reduced expression of TH protein and increased α-synuclein protein, as well as some alterations in proteins linked to familial PD and autophagy in the olfactory bulb and striatum, without pronounced therapeutic effects of EDR and CUR. Our results may suggest that EDR and CUR lack therapeutic effects when administered after the disease process was already initiated. Thus, our treatment regimen or the physicochemical properties of EDR and CUR could be further refined to elevate the therapeutic effects of these formulations.

Reactive medullary astrocytes are associated with RVLM neuroinflammation in heart failure rats

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome. Brain inflammation has been suggested to play a key role in HFpEF progression by modulating the activity of several brainstem circuits encompassing cardiovascular and respiratory regulation. Indeed, we recently reported RVLM‐C1 catecholaminergic neurons hyperactivity in rats with HFpEF. However, the precise mechanism underpinning changes in C1 activity in HFpEF remains to be determined. Interestingly, glial cell activation within the RVLM (particularly astrocytes) has been shown to modulate C1 neuron activity. Despite this evidence whether astrocytes may become activated in HFPEF has not been previously studied. Accordingly, we aimed to determine changes in astrocyte activity within the RVLM of HFpEF rats and its relation with RVLM neuroinflammatory status. Adult male Sprague‐Dawley rats (250 g) underwent volume overload to induce HFpEF. Eight weeks after surgical induction of HFpEF, rats underwent respiratory function recordings in freely moving conditions using whole‐body plethysmography. At the end of the physiological experiments, brains were harvested, fixed in 4% PFA and processed for immunohistochemical analysis. Immunofluorescence for tyrosine hydroxylase and glial fibrillary acidic protein (GFAP) were performed in 25 μm free‐floating brainstem sections containing the RVLM. Sholl analysis was performed to evaluate astrocytic morphological differences between HFpEF and Sham rats (total length of processes, # of processes, maximum intersection radius, # of intersections). RVLM neuroinflammatory status was measured in micropunches by immunoblot (GFAP) and qPCR (TNF‐α, IL‐1β). Compared to Sham rats, HFpEF animals showed clear signs of astrocyte reactivity evidenced by increases in the number of astrocyte processes (11.27 ± 1.096 vs. 18.64 ± 1.333; HFpEF vs. Sham, respectively), length of astrocyte processes (267.5 ± 17.92μm vs. 392.8 ± 22.53μm; HFpEF vs. Sham, respectively) and number of intersections (3.410 ± 0,25 vs. 4.538 ± 0.317; HFpEF vs. Sham, respectively). Consistently, we found an increased GFAP expression within the RVLM of HFpEF rats compared with Sham rats (99.9 ± 5.9 vs. 135.3 ± 15.8; HFpEF vs. Sham, respectively. In addition, we found that TNF‐α and IL‐1β were both augmented in HFpEF (~7 and ~3 fold, respectively) compared to Sham. Notably, respiratory dysfunction observed in HFpEF were significantly and positively correlated with the degree of activation of RVLM astrocytes. Indeed, respiratory irregularity score (R2 = 0.98; p<0.05), short‐term variability of the breath‐to‐breath interval (R2 = 0.98; p<0.05), and apneas/hypopneas index (R2 = 0.94; p<0.05)) were all related to GFAP expression in HFpEF rats. Our findings showed the presence of reactive astrocytes within the RVLM of HFpEF animals and that the degree of astrocyte activity was associated with RVLM neuroinflammation. Together our results suggest that increases in astrocyte reactivity in HFpEF may help to induce/maintain a pro‐iflammatory niche within the RVLM that ultimately lead to C1 neuron activity alterations.

Mitochondrial Dysfunction Contributes To Zinc-induced Neurodegeneration: a Link with NADPH Oxidase.

Mitochondrial dysfunction and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) are the major sources of augmentation in free radical generation leading to neurodegeneration. Although NADPH oxidase involvement is reported in zinc (Zn)-induced neurodegeneration, contribution of the mitochondrial dysfunction and its association with NADPH oxidase are not known. Therefore, the study was aimed to decipher the role of mitochondrial dysfunction and its link with NADPH oxidase in Zn-induced Parkinsonism. Zn reduced the motor activities, the number of tyrosine hydroxylase (TH)-positive neurons, and level of TH protein. Conversely, Zn increased the mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LPO), and superoxide dismutase (SOD) activity and reduced the mitochondrial membrane potential and catalytic activities of complex I and III. Zn also attenuated B-cell lymphoma-2 (Bcl-2) and pro-caspase 9/3 levels and augmented the translocation of cytosolic Bcl-2 associated X (Bax) protein to the mitochondria and cytochrome c release into cytosol from the mitochondria. Cyclosporine A, a mitochondrial outer membrane transition pore inhibitor and apocynin, a NADPH oxidase inhibitor, independently, ameliorated the Zn-induced changes. Similarly, Zn reduced cell viability through mitochondrial dysfunction and apoptosis in human neuroblastomaSH-SY5Y cells, which were notably normalized in the presence of cyclosporine or apocynin. The results demonstrate that mitochondrial dysfunction contributes to Zn-induced neurodegeneration, which could be partially aided by the NADPH oxidase.

Impact of Yiqi Huoxue Decoction on the Relationship between Remodeling of Cardiac Nerves and Macrophages after Myocardial Infarction in Rats.

Sympathetic nerve remodeling after myocardial infarction (MI) has an indispensable role in cardiac remodeling. Numerous works have shown that sympathetic nerve remodeling can be delayed by inhibition of inflammatory response. Earlier studies have shown improvement in ventricular remodeling and inhibited chronic stage neural remodeling by Yiqi Huoxue decoction (YQHX). Therefore, the current study looked at the inhibitory effect of YQHX prescription on proinflammatory mediators and macrophages and the effect on neural remodeling at 3 and 7 days after MI. YQHX inhibited the expression of Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κB) proteins and macrophage infiltration within 7 days after myocardial infarction. YQHX could decrease Th-positive nerve fiber density in the area around infarction and reduce the expression of growth-associated protein 43 (GAP43), nerve growth factor (NGF), and tyrosine hydroxylase (TH) proteins, which was associated with the remodeling of sympathetic nerves. Thus, the nerve remodeling inhibition after MI due to YQHX may be through its anti-inflammatory action. These data provide direct evidence for the potential application of traditional Chinese medicine (TCM) in the remodeling of sympathetic nerves after MI.

Tyrosine hydroxylase messenger RNA corroborates protein localization in the nervous system of the pond snail, Lymnaea stagnalis

AbstractCatecholaminergic neurons are abundant in molluscs and are involved in a variety of behaviors such as feeding, respiration, learning, and locomotion. However, previous identification of these neurons has relied almost exclusively on immunohistochemistry using antibodies, which have not been fully validated for use in molluscs. We employed tissue‐specific quantitative PCR in adults of Lymnaea stagnalis (a pulmonate gastropod) and whole‐mount in situ hybridization in larvae to both quantify and visualize messenger RNA of the catecholamine synthesis enzyme, tyrosine hydroxylase (TH). TH messenger RNA was found to localize primarily in the foot and the central nervous system, with smaller quantities present in the cephalic sensory organs. Additionally, we performed western blots that validated a popular antibody used as a marker for catecholaminergic neurons in molluscs. Taken together, these data indicate that TH messenger RNA is present in the central and peripheral nervous system of L. stagnalis and support the specificity of past immunohistochemical labeling of the TH protein. These findings have potentially broad implications, given the wide range of biological processes that have been studied in L. stagnalis.

Attenuation of the Counter-Regulatory Glucose Response in CVLM C1 Neurons: A Possible Explanation for Anorexia of Aging

This study aimed to determine the effect of age on CVLM C1 neuron glucoregulatory proteins in the feeding pathway. Male Sprague Dawley rats aged 3 months and 24 months old were divided into two subgroups: the treatment group with 2-deoxy-d-glucose (2DG) and the control group. Rat brains were dissected to obtain the CVLM region of the brainstem. Western blot was used to determine protein expression of tyrosine hydroxylase (TH), phosphorylated TH at Serine40 (pSer40TH), AMP-activated protein kinase (AMPK), phosphorylated AMPK (phospho AMPK), and neuropeptide Y Y5 receptors (NPY5R) in CVLM samples. Immunofluorescence was used to determine TH-, AMPK-, and NPY5R-like immunoreactivities among other brain coronal sections. Results obtained denote a decrease in basal TH phosphorylation levels and AMPK proteins and an increase in TH proteins among aged CVLM neurons. Increases in the basal immunoreactivity of TH+, AMPK+, NPY5R+, TH+/AMPK+, and TH+/NPY5R+ were also observed among old rats. Young treatment-group rats saw a decrease in TH phosphorylation and AMPK proteins following 2DG administration, while an increase in AMPK phosphorylation and a decrease in TH proteins were found among the old-treatment-group rats. These findings suggest the participation of CVLM C1 neurons in counter-regulatory responses among young and old rats. Altering protein changes in aged CVLM C1 neurons may attenuate responses to glucoprivation, thus explaining the decline in food intake among the elderly.

Neuropathological investigation of patients with prolonged anorexia nervosa.

ObjectivesRecent neuroimaging studies have indicated that the mesolimbic pathway, known to work as reward neuronal circuitry, regulates cognitive–behavioral flexibility in prolonged anorexia nervosa (AN). Although AN is associated with the highest mortality rate among psychiatric disorders, there have been few neuropathological studies on this topic. This study aims to identify alterations of the reward circuitry regions, especially in the nucleus accumbens (NAcc), using AN brain tissues.MethodsThe neuronal networks in AN cases and controls were examined by immunohistochemistry directed at tyrosine hydroxylase (TH; dopaminergic neuron marker) and glial fibrillary acidic protein (GFAP; astrocyte marker). We also immunochemically analyzed frozen samples presenting astrogliosis, especially in the NAcc and striatum.ResultsHistologically, neuronal deformation with cytoplasmic shrinkage was seen in reward‐related brain regions, such as the orbitofrontal cortex/anterior cingulate cortex. The NAcc showed massive GFAP‐positive astrocytes and dot‐like protrusions of astrocytes in the shell compartment. In the shell, TH and GFAP immunoreactivities revealed prominent astrogliosis within striosomes, which receive projection from the ventral tegmental area (VTA). The numbers of GFAP‐positive astrocytes in the NAcc (P = 0.0079) and VTA (P = 0.0025) of AN cases were significantly higher than those of controls. Strongly immunoreactive 18 to 25 kDa bands, which might represent degradation products, were detected only in the NAcc of AN cases. Clinically, all cases presented cognitive rigidity, which might reflect a deficit of the reward pathway.ConclusionOur findings suggest impaired dopaminergic innervation between the NAcc and VTA in AN. Functional dysconnectivity in the reward‐related network might induce neuropsychiatric symptoms associated with AN.

Priapism caused by partial deficiency of tetrahydrobiopterin through hypofunction of the sympathetic neurons in sepiapterin reductase gene‐disrupted mice

6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for aromatic L-amino acid hydroxylases, including tyrosine hydroxylase (TH), alkylglycerol monooxygenase, and three types of nitric oxide (NO) synthases (NOS). Sepiapterin reductase (SPR) catalyzes the third step of BH4 biosynthesis. SPR gene-disrupted (Spr-/- ) mice exhibit a dystonic posture, low body weight, hyperphenylalaninemia, and unstable hypertension with endothelial dysfunction. In this study, we found that Spr-/- mice suffered from a high incidence of severe priapism. Their erections persisted for months. The biopterin, BH4, and norepinephrine contents, and TH protein levels in the penile tissue of Spr-/- mice without and with priapism were significantly reduced compared to those of Spr+/+ mice. In contrast, their neural NOS (nNOS) protein levels were increased, and the cyclic guanosine monophosphate (cGMP) levels were remarkably elevated in the penises of Spr-/- mice with priapism. The symptoms were relieved by repeated administration of BH4. The biopterin, BH4, and norepinephrine contents were increased in penile homogenates from BH4-supplemented Spr-/- mice, and the TH protein levels tended to increase, and their nitrite plus nitrate levels were significantly lower than those of vehicle-treated Spr-/- mice and were approximately the same as vehicle- and BH4-supplemented Spr+/+ mice. Thus, we deduced that the priapism of Spr-/- mice is primarily caused by hypofunction of the sympathetic neurons due to cofactor depletion and the loss of TH protein and, further, dysregulation of the NO/cGMP signaling pathway, which would be caused by disinhibition of nNOS-containing neurons and/or abnormal catabolism of cyclic nucleotides is suggested.

Polymannuronic acid prebiotic plus Lacticaseibacillus rhamnosus GG probiotic as a novel synbiotic promoted their separate neuroprotection against Parkinson’s disease

Gut microbiota (GM) dysbiosis plays key roles in aggravating Parkinson’s disease (PD) and discovery of agents targeting GM may open new avenues for PD therapy. This study aims to investigate the potentially neuroprotective effects and underlying mechanisms of polymannuronic acid (PM) or Lacticaseibacillus rhamnosus GG (LGG), or their combination in a chronic PD mice model. Our results found oral administration of prebiotic PM or LGG separately or in combination for 5 weeks could prevent dopaminergic neuronal loss via improving reduced walking distance and activity or weakened muscle strength in behavior tests by enhancing tyrosine hydroxylase (TH) gene and/or protein expressions in the midbrain and striatum of PD mice. Strikingly, PM and LGG in combination had a much better neuroprotective effects than separate PM or LGG. PM provided neuroprotection via a short chain fatty acids (SCFAs)-mediated anti-inflammation and anti-apoptosis mechanism. The neuroprotective effects of LGG might be associated with its ability to improve the expression of striatal glial cell-derived neurotrophic factor (GDNF) and to increase bacteria abundance of Clostridiales. When PD mice were administered with PM + LGG, PM as prebiotic favored bacterial growth (from Bacilli class to Lactobacillus genus) in the colon, which helped to improve blood brain barrier (BBB) integrity and increase brain-derived neurotrophic factor (BDNF) and GDNF expressions, thereby inhibiting apoptosis in the striatum. In conclusion, PM and LGG in combination promoted their separate neuroprotection against PD. Our study discovered and testified a novel synbiotic that might be one of the ideal oral agents for PD therapy.

Erinacine A Prevents Lipopolysaccharide-Mediated Glial Cell Activation to Protect Dopaminergic Neurons against Inflammatory Factor-Induced Cell Death In Vitro and In Vivo.

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.

GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis.

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant (gch1-/-), using CRISPR/Cas technology. gch1-/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1-/- larvae improved survival without ameliorating the motor phenotype. RNAseq of gch1-/- larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1-/- The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.

Time dependent alteration of locomotor behavior in rat with acute liver failure induced cerebellar neuroinflammation and neuro-astroglial damage

Hepatic encephalopathy (HE) is a neurophysiological syndrome secondary to acute or chronic liver failure. Studies showed that HE patients exhibit a deficit in motor coordination, which may result from cerebellar functional impairment. The aim of this study is to assess the time-dependent alteration of locomotor behavior and the glial and neuronal alteration in rat with acute HE induced chemically. The study was carried out in male Sprague-Dawley rats with thioacetamide (TAA) induced acute liver failure at different stages 12 h, 24 h and 36 h. Hepatic and renal functions were assessed via various biochemical and histopathological examinations, while the cerebellum and the midbrain were examined using histology and immunohistochemistry for tyrosine hydroxylase (TH), cyclooxygenase-2 (COX-2) and glial fibrillary acidic protein (GFAP). We used as well, the open field test and the Rotarod test for assessing the locomotor activity and coordination. Our data showed a progressive loss of liver function and a progressive alteration in locomotor behavior and motor coordination in acute HE rats. In the cerebellum, we noted an increase in the degeneration of cerebellar Purkinje neurons parallel to increased COX-2 immunoreactivity together with astrocytic morphology and density changes. Likewise, in substantia nigra pars compacta, TH levels were reduced. We showed through the current study, a progressive deterioration in locomotor behavior in acute HE rats, as a result of Purkinje neurons death and a deficient dopaminergic neurotransmission, together with the morpho-functional astroglial modifications involving the oxidative stress and neuroinflammation.

Transcriptional responses of skeletal stem/progenitor cells to hindlimb unloading and recovery correlate with localized but not systemic multi-systems impacts

Disuse osteoporosis (DO) results from mechanical unloading of weight-bearing bones and causes structural changes that compromise skeletal integrity, leading to increased fracture risk. Although bone loss in DO results from imbalances in osteoblast vs. osteoclast activity, its effects on skeletal stem/progenitor cells (SSCs) is indeterminate. We modeled DO in mice by 8 and 14 weeks of hindlimb unloading (HU) or 8 weeks of unloading followed by 8 weeks of recovery (HUR) and monitored impacts on animal physiology and behavior, metabolism, marrow adipose tissue (MAT) volume, bone density and micro-architecture, and bone marrow (BM) leptin and tyrosine hydroxylase (TH) protein expression, and correlated multi-systems impacts of HU and HUR with the transcript profiles of Lin−LEPR+ SSCs and mesenchymal stem cells (MSCs) purified from BM. Using this integrative approach, we demonstrate that prolonged HU induces muscle atrophy, progressive bone loss, and MAT accumulation that paralleled increases in BM but not systemic leptin levels, which remained low in lipodystrophic HU mice. HU also induced SSC quiescence and downregulated bone anabolic and neurogenic pathways, which paralleled increases in BM TH expression, but had minimal impacts on MSCs, indicating a lack of HU memory in culture-expanded populations. Although most impacts of HU were reversed by HUR, trabecular micro-architecture remained compromised and time-resolved changes in the SSC transcriptome identified various signaling pathways implicated in bone formation that were unresponsive to HUR. These findings indicate that HU-induced alterations to the SSC transcriptome that persist after reloading may contribute to poor bone recovery.

Flavin-containing monooxygenase 1 deficiency promotes neuroinflammation in dopaminergic neurons in mice

A growing body of evidence indicates an association between flavin-containing monooxygenase (FMO) and neurodegeneration, including Parkinson’s disease (PD); however, the details of this association are unclear. We previously showed that the level of Fmo1 mRNA is decreased in an in vitro rotenone model of parkinsonism. To further explore the potential involvement of FMO1 deficiency in parkinsonism, we generated Fmo1 knockout (KO) mice and examined the survival of dopaminergic neurons and relative changes. Fmo1 KO mice exhibited loss of tyrosine hydroxylase-positive neurons, decreased levels of tyrosine hydroxylase and Parkin proteins, and increased levels of pro-inflammatory cytokines (IL1β and IL6) in the nigrostriatal region. Moreover, the protein levels of PTEN induced kinase 1 (PINK1) and p62, and the Microtubule associated protein 1 light chain 3 (LC3)-II/I ratio were not significantly altered in Fmo1 KO mice (P > 0.05). FMO1 deficiency promotes neuroinflammation in dopaminergic neurons in mice, thus may plays a potential pathological role in dopaminergic neuronal loss. These findings may provide new insight into the pathogenesis of PD.