Calcium-dependent activator protein for secretion 2 is involved in dopamine release in mouse midbrain neurons.

The Ca2+-dependent activator protein for secretion (CAPS/CADPS) family protein facilitates catecholamine release through the dense-core vesicle exocytosis in model neuroendocrine cell lines. However, it remains unclear if it induces dopamine release in the central neurons. This study aimed to examine the expression and function of CADPS2, one of the two CADPS paralogs, in dopamine neurons of the mouse midbrain. This study shows that CADPS2 was expressed in tyrosine hydroxylase and the vesicular monoamine transporter 2 (VMAT2)-positive dopaminergic neurons of the midbrain samples and primary mesencephalic cell cultures. Subcellular fractions rich in dopamine were collected using immunoaffinity for CADPS2 from midbrain protein extracts. Cell imaging using fluorescent false neurotransmitter FFN511 as a substrate for VMAT2 showed decreased activity-dependent dopamine release in Cadps2-deficient cultures, compared to that in wild-type cultures. These results suggest that CADPS2 is involved in dopamine release from the central neurons, indicating its involvement in the central dopamine pathway.

Hypothalamic neurons fully or partially expressing the dopaminergic phenotype: development, distribution, functioning and functional significance. A review.

The hypothalamus is a key link in neuroendocrine regulations, which are provided by neuropeptides and dopamine. Until the late 1980 s, it was believed that, along with peptidergic neurons, hypothalamus contained dopaminergic neurons. Over time, it has been shown that besides dopaminergic neurons expressing the dopamine transporter and dopamine-synthesizing enzymes - tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) - the hypothalamus contains neurons expressing only TH, only AADC, both enzymes or only dopamine transporter. The end secretory product of TH neurons is L-3,4-dihydroxyphenylalanine, while that of AADC neurons and bienzymatic neurons lacking the dopamine transporter is dopamine. During ontogenesis, especially in the perinatal period, monoenzymatic neurons predominate in the hypothalamic neuroendocrine centers. It is assumed that L-3,4-dihydroxyphenylalanine and dopamine are released into the neuropil, cerebral ventricles, and blood vessels, participating in the regulation of target cell differentiation in the perinatal period and the functioning of target cells in adulthood.

l-theanine, the unique constituent of tea, improves neuronal survivability by curtailing inflammatory responses in MPTP model of Parkinson's disease.

Discrete components of tea possess multitude of health advantages. Escalating evidence advocate a consequential association between habitual tea consumption and a subsided risk of Parkinson's disease (PD). l-theanine is a non-protein amino acid inherent in tea plants, which exhibits structural resemblance with glutamate, the copious excitatory neurotransmitter in brain. Neuromodulatory effects of l-theanine are evident from its competency in traversing the blood brain barrier, promoting a sense of calmness beyond enervation, and enhancing cognition and attention. Despite the multifarious reports on antioxidant properties of l-theanine and its potential to regulate brain neurotransmitter levels, it is obligatory to understand its exact contribution in ameliorating the pathophysiology of PD. In this study, MPTP-induced mouse model was established and PD-like symptoms were developed in test animals where an increasing dosage of l-theanine (5, 25, 50, 100 and 250 mg/kg) was intraperitoneally administered for 23 days. 50 and 100 mg/kg dosage of l-theanine alleviated motor impairment and specific non-motor symptoms in Parkinsonian mice. The dosage of 100 mg/kg of l-theanine also improved striatal dopamine and serotonin level and tyrosine-hydroxylase positive cell count in the substantia nigra. Most crucial finding of the study is the proficiency of l-theanine to diminish astroglial injury as well as nitric oxide synthesis, which suggests its possible credential to prevent neurodegeneration by virtue of its anti-inflammatory attribute.

A combined MRI, histological and immunohistochemical rendering of the rhesus macaque locus coeruleus (LC) enables the differentiation of three distinct LC subcompartments.

Locus coeruleus (LC) neurons send their noradrenergic axons across multiple brain regions, including neocortex, subcortical regions, and spinal cord. Many aspects of cognition are known to be dependent on the noradrenergic system, and it has been suggested that dysfunction in this system may play central roles in cognitive decline associated with both normative aging and neurodegenerative disease. While basic anatomical and biochemical features of the LC have been examined in many species, detailed characterizations of the structure and function of the LC across the lifespan are not currently available. This includes the rhesus macaque, which is an important model of human brain function because of their striking similarities in brain architecture and behavioral capacities. In the present study, we describe a method to combine structural MRI, Nissl, and immunofluorescent histology from individual monkeys to reconstruct, in 3 dimensions, the entire macaque LC nucleus. Using these combined methods, a standardized volume of the LC was determined, and high-resolution confocal images of tyrosine hydroxylase-positive neurons were mapped into this volume. This detailed representation of the LC allows definitions to be proposed for three distinct subnuclei, including a medial region and a lateral region (based on location with respect to the central gray, inside or outside, respectively), and a compact region (defined by densely packed neurons within the medial compartment). This enabled the volume to be estimated and cell density to be calculated independently in each LC subnucleus for the first time. This combination of methods should allow precise characterization of the LC and has the potential to do the same for other nuclei with distinct molecular features.

Viscerosensory signalling to the nucleus accumbens via the solitary tract nucleus.

The nucleus of the solitary tract (NTS) receives direct viscerosensory vagal afferent input that drives autonomic reflexes, neuroendocrine function and modulates behaviour. A subpopulation of NTS neurons project to the nucleus accumbens (NAc); however, the function of this NTS-NAc pathway remains unknown. A combination of neuroanatomical tracing, slice electrophysiology and fibre photometry was used in mice and/or rats to determine how NTS-NAc neurons fit within the viscerosensory network. NTS-NAc projection neurons are predominantly located in the medial and caudal portions of the NTS with 54 ± 7% (mice) and 65 ± 3% (rat) being TH-positive, representing the A2 NTS cell group. In horizontal brainstem slices, solitary tract (ST) stimulation evoked excitatory post-synaptic currents (EPSCs) in NTS-NAc projection neurons. The majority (75%) received low-jitter, zero-failure EPSCs characteristic of monosynaptic ST afferent input that identifies them as second order to primary sensory neurons. We then examined whether NTS-NAc neurons respond to cholecystokinin (CCK, 20 µg/kg ip) in vivo in both mice and rats. Surprisingly, there was no difference in the number of activated NTS-NAc cells between CCK and saline-treated mice. In rats, just 6% of NTS-NAc cells were recruited by CCK. As NTS TH neurons are the primary source for NAc noradrenaline, we measured noradrenaline release in the NAc and showed that NAc noradrenaline levels declined in response to cue-induced reward retrieval but not foot shock. Combined, these findings suggest that high-fidelity afferent information from viscerosensory afferents reaches the NAc. These signals are likely unrelated to CCK-sensitive vagal afferents but could interact with other sensory and higher order inputs to modulate learned appetitive behaviours.

Cysteamine HCl Administration Impedes Motor and Olfactory Functions, Accompanied by a Reduced Number of Dopaminergic Neurons, in Experimental Mice: A Preclinical Mimetic Relevant to Parkinson's Disease.

Cysteamine hydrochloride (Cys-HCl) has been established as a potent ulcerogenic agent of the gastrointestinal (GI) system. GI dysfunction and olfactory deficits are the most common clinical symptoms of many movement disorders, including Parkinson's disease (PD). Cys-HCl has been shown to interfere with dopamine, a neurotransmitter crucial for motor, olfactory, and cognitive functions. However, the reports on the effect of Cys-HCl treatment on the behavioral aspects and functions of the dopamine system appear to be inconsistent. Therefore, we revisited the impact of Cys-HCl on the motor function in experimental mice using a battery of behavioral tests, such as the pole test (PT), beam-walking test (BWT), and rotarod test (RDT), while the olfactory ability and cognitive functions were examined through the buried-food test (BFT) and Y-maze test. Furthermore, we investigated the effect of Cys-HCl on the number of dopaminergic tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and olfactory bulb (OB) of the experimental mice using immunohistochemistry. The results revealed that Cys-HCl administration in the mice induced significant impairments in their motor balance and coordination, as their movement-related performances were markedly reduced in terms of the behavioral tasks. Mice exposed to Cys-HCl showed pronounced reductions in their odor discrimination abilities as well as cognitive impairments. Strikingly, the number of TH-positive neurons was found to be reduced in the SN and OB of the Cys-HCl-treated group, which is a bonafide neuropathogenic hallmark of PD. This study highlights the potential neurotoxic effects of Cys-HCl in experimental brains and suggests further investigation into its role in the pathogenesis of Parkinsonism.

The C886T Mutation in the Th Gene Reduces the Activity of Tyrosine Hydroxylase in the Mouse Brain.

Tyrosine hydroxylase (TH) catalyzes hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine, the initial and rate-limiting step in the synthesis of dopamine, noradrenaline, and adrenaline. Mutations in the human TH gene are associated with hereditary motor disorders. The common C886T mutation identified in the mouse Th gene results in the R278H substitution in the enzyme molecule. We investigated the impact of this mutation on the TH activity in the mouse midbrain. The TH activity in the midbrain of Mus musculus castaneus (CAST) mice homozygous for the 886C allele was higher compared to C57BL/6 and DBA/2 mice homozygous for the 886T allele. Notably, this difference in the enzyme activity was not associated with changes in the Th gene mRNA levels and TH protein content. Analysis of the TH activity in the midbrain in mice from the F2 population obtained by crossbreeding of C57BL/6 and CAST mice revealed that the 886C allele is associated with a high TH activity. Moreover, this allele showed complete dominance over the 886T allele. However, the C886T mutation did not affect the levels of TH protein in the midbrain. These findings demonstrate that the C886T mutation is a major genetic factor determining the activity of TH in the midbrain of common laboratory mouse strains. Moreover, it represents the first common spontaneous mutation in the mouse Th gene whose influence on the enzyme activity has been demonstrated. These results will help to understand the role of TH in the development of adaptive and pathological behavior, elucidate molecular mechanisms regulating the activity of TH, and explore pharmacological agents for modulating its function.

Time association study on a sub-acute mouse model of Parkinson's disease.

Parkinson's disease (PD) is a severe neurodegenerative disease that disturbs human health. In the laboratory researches about PD, the mice model induced by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was widely used. However, there has been controversy about the model effectiveness to simulate PD symptoms and pathology, and the time-varying development of behavioral and pathological characteristic after MPTP treatment remains unclear. In order to solve these problems, we designed a series of experiments to evaluate this PD model at different time points. We constructed the subacute PD mouse model by intraperitoneal injection of MPTP for 5 consecutive days. The rotarod test, open field test and the immunohistochemical staining of tyrosine hydroxylase were conducted at -5, 1, 5, 7, 14, 21 and 28 days after the last injection of MPTP. The results showed that 5 days after the last MPTP administration, typical motor disorders with significant balance function damage in rotarod test began to appear and remained stable throughout the entire experiment. Simultaneously, we also observed the loss of tyrosine hydroxylase (TH) positive cells in the substantia nigra compacta and reduction of TH content in the striatum but this pathological change in the substantia nigra compacta reversed 21 days after injection. Besides, the spontaneous movement of mice in open field test remained unchanged by MPTP. This research indicated the time-dependence of MPTP neurotoxicity that impair the motor function and histological features and confirmed the symptom occurrence time after MPTP injection, which provides a reference for the future research about MPTP-induced PD.

Prolactin mitigates chronic stress-induced maladaptive behaviors and physiology in ovariectomized female rats.

Stress is a major risk factor for several neuropsychiatric disorders in women, including postpartum depression. During the postpartum period, diminished ovarian hormone secretion increases susceptibility to developing depressive symptoms. Pleiotropic peptide hormones, like prolactin, are markedly released during lactation and suppress hypothalamic-pituitary-adrenal axis responses in women and acute stress-induced behavioral responses in female rodents. However, the effects of prolactin on chronic stress-induced maladaptive behaviors remain unclear. Here, we used chronic variable stress to induce maladaptive physiology in ovariectomized female rats and concurrently administered prolactin to assess its effects on several depression-relevant behavioral, endocrine, and neural characteristics. We found that chronic stress increased sucrose anhedonia and passive coping in saline-treated, but not prolactin-treated rats. Prolactin treatment did not alter stress-induced thigmotaxis, corticosterone (CORT) concentrations, hippocampal cell activation or survival. However, prolactin treatment reduced basal CORT concentrations and increased dopaminergic cells in the ventral tegmental area. Further, prolactin-treated rats had reduced microglial activation in the ventral hippocampus following chronic stress exposure. Together, these data suggest prolactin mitigates chronic stress-induced maladaptive behaviors and physiology in hypogonadal females. Moreover, these findings imply neuroendocrine-immune mechanisms by which peptide hormones confer stress resilience during periods of low ovarian hormone secretion.

Impact of Serotonin Deficiency on Circadian Dopaminergic Rhythms.

Physiology and behavior are structured temporally to anticipate daily cycles of light and dark, ensuring fitness and survival. Neuromodulatory systems in the brain-including those involving serotonin and dopamine-exhibit daily oscillations in neural activity and help shape circadian rhythms. Disrupted neuromodulation can cause circadian abnormalities that are thought to underlie several neuropsychiatric disorders, including bipolar mania and schizophrenia, for which a mechanistic understanding is still lacking. Here, we show that genetically depleting serotonin in Tph2 knockout mice promotes manic-like behaviors and disrupts daily oscillations of the dopamine biosynthetic enzyme tyrosine hydroxylase (TH) in midbrain dopaminergic nuclei. Specifically, while TH mRNA and protein levels in the Substantia Nigra (SN) and Ventral Tegmental Area (VTA) of wild-type mice doubled between the light and dark phase, TH levels were high throughout the day in Tph2 knockout mice, suggesting a hyperdopaminergic state. Analysis of TH expression in striatal terminal fields also showed blunted rhythms. Additionally, we found low abundance and blunted rhythmicity of the neuropeptide cholecystokinin (Cck) in the VTA of knockout mice, a neuropeptide whose downregulation has been implicated in manic-like states in both rodents and humans. Altogether, our results point to a previously unappreciated serotonergic control of circadian dopamine signaling and propose serotonergic dysfunction as an upstream mechanism underlying dopaminergic deregulation and ultimately maladaptive behaviors.

Boosting endogenous dopamine production: a novel therapeutic approach for Parkinson's disease.

Innovative therapeutic strategies are urgently needed for Parkinson's disease due to limited efficacy of current treatments and a weak therapeutic pipeline. In this forum article, we propose targeting tyrosine hydroxylase phosphorylation as a novel mechanism of action to address this critical need.

Unveiling the Journey from the Gut to the Brain: Decoding Neurodegeneration-Gut Connection in Parkinson's Disease.

Parkinson's disease, a classical motor disorder affecting the dopaminergic system of the brain, has been as a disease of the brain, but this classical notion has now been viewed differently as the pathology begins in the gut and then gradually moves up to the brain regions. The microorganisms in the gut play a critical role in maintaining the physiology of the gut from maintaining barrier integrity to secretion of microbial products that maintain a healthy gut state. The pathology subsequently alters the normal composition of gut microbes and causes deleterious effects that ultimately trigger strong neuroinflammation and nonmotor symptoms along with characteristic synucleopathy, a pathological hallmark of the disease. Understanding the complex pathomechanisms in distinct and established preclinical models is the primary goal of researchers to decipher how exactly gut pathology has a central effect; the quest has led to many answered and some open-ended questions for researchers. We summarize the popular opinions and some contrasting views, concise footsteps in the treatment strategies targeting the gastrointestinal system.

Aging disrupts the coordination between mRNA and protein expression in mouse and human midbrain.

Age-related dopamine (DA) neuron loss is a primary feature of Parkinson's disease. However, it remains unclear whether similar biological processes occur during healthy aging, albeit to a lesser degree. We therefore determined whether midbrain DA neurons degenerate during aging in mice and humans. In mice, we identified no changes in midbrain neuron numbers throughout aging. Despite this, we found age-related decreases in midbrain mRNA expression of tyrosine hydroxylase (Th), the rate limiting enzyme of DA synthesis. Among midbrain glutamatergic cells, we similarly identified age-related declines in vesicular glutamate transporter 2 (Vglut2) mRNA expression. In co-transmitting Th +/Vglut2 + neurons, Th and Vglut2 transcripts decreased with aging. Importantly, striatal Th and Vglut2 protein expression remained unchanged. In translating our findings to humans, we found no midbrain neurodegeneration during aging and identified age-related decreases in TH and VGLUT2 mRNA expression similar to mouse. Unlike mice, we discovered diminished density of striatal TH+ dopaminergic terminals in aged human subjects. However, TH and VGLUT2 protein expression were unchanged in the remaining striatal boutons. Finally, in contrast to Th and Vglut2 mRNA, expression of most ribosomal genes in Th + neurons was either maintained or even upregulated during aging. This suggests a homeostatic mechanism where age-related declines in transcriptional efficiency are overcome by ongoing ribosomal translation. Overall, we demonstrate species-conserved transcriptional effects of aging in midbrain dopaminergic and glutamatergic neurons that are not accompanied by marked cell death or lower striatal protein expression. This opens the door to novel therapeutic approaches to maintain neurotransmission and bolster neuronal resilience.

Persistent Neurological Deficits in Mouse PASC Reveal Antiviral Drug Limitations.

Post-Acute Sequelae of COVID-19 (PASC) encompasses persistent neurological symptoms, including olfactory and autonomic dysfunction. Here, we report chronic neurological dysfunction in mice infected with a virulent mouse-adapted SARS-CoV-2 that does not infect the brain. Long after recovery from nasal infection, we observed loss of tyrosine hydroxylase (TH) expression in olfactory bulb glomeruli and neurotransmitter levels in the substantia nigra (SN) persisted. Vulnerability of dopaminergic neurons in these brain areas was accompanied by increased levels of proinflammatory cytokines and neurobehavioral changes. RNAseq analysis unveiled persistent microglia activation, as found in human neurodegenerative diseases. Early treatment with antivirals (nirmatrelvir and molnupiravir) reduced virus titers and lung inflammation but failed to prevent neurological abnormalities, as observed in patients. Together these results show that chronic deficiencies in neuronal function in SARS-CoV-2-infected mice are not directly linked to ongoing olfactory epithelium dysfunction. Rather, they bear similarity with neurodegenerative disease, the vulnerability of which is exacerbated by chronic inflammation.

Rethinking Parkinson's disease: could dopamine reduction therapy have clinical utility?

Following reports of low striatal dopamine content in Parkinson's disease, levodopa was shown to rapidly reverse hypokinesis, establishing the model of disease as one of dopamine deficiency. Dopaminergic therapy became standard of care, yet it failed to reverse the disease, suggesting the understanding of disease was incomplete. The literature suggests the potential for toxicity of dopamine and its metabolites, perhaps more relevant given the recent evidence for elevated cytosolic dopamine levels in the dopaminergic neurons of people with Parkinson's. To understand the relevance of these data, multiple investigations are reviewed that tested dopamine reduction therapy as an alternative to dopaminergic agents. The data from use of an inhibitor of dopamine synthesis in experimental models suggest that such an approach could reverse disease pathology, which suggests that cytosolic dopamine excess is a primary driver of disease. These data support clinical investigation of dopamine reduction therapy for Parkinson's disease. Doing so will determine whether these experimental models are predictive and this treatment strategy is worth pursuing further. If clinical data are positive, it could warrant reconsideration of our disease model and treatment strategies, including a shift from dopaminergic to dopamine reduction treatment of the disease.

Lead specifically declines tyrosine hydroxylase activity to induce the onset of Parkinson's disease through disrupting dopamine biosynthesis in fly models.

Parkinson's disease (PD) is one of the fastest-growing neurodegenerative diseases and has been linked to the exposure to numerous environmental neurotoxins. Although lead (Pb) exposure has been related to the development of PD, the molecular target of Pb to cause the onset of PD is insufficiently investigated. Herein, we explored the effects of Pb exposure on behavior, pathophysiology, and gene expression of wild-type (WT) fly (Drosophila melanogaster) by comparison with its PD model. After exposure to Pb, the WT flies showed PD-like locomotor impairments and selective loss of dopaminergic (DAergic) neurons, displaying similar phenotypes to fly PD model (PINK1). Transcriptomic analysis showed the similarity in gene expression profiles between Pb treatment WT flies and PINK1 mutant flies. Moreover, Pb exposure resulted in endogenous dopamine deficits in WT flies. Analyses of gene expression and enzyme activity confirmed that Pb exposure reduced tyrosine hydroxylase (TH) activity and led to failure of dopamine synthesis. Furthermore, molecular dynamics simulation confirmed that Pb was adsorbed by TH and subsequently inhibited the enzymatic activity. Exogenous injection of L-dopa and melatonin could partially rescue the pathological phenotypes of Pb-exposed flies and PD fly model. Antagonist injection of microRNA-133, which negatively regulated the expression of TH gene, ultimately rescued in the manifestation of PD phenotypes in flies. Involvement of TH overexpression mutants of fly strongly promoted the resistance to Pb exposure and rescued both behavior and the number of DAergic neurons. Therefore, our study elucidates the Pb molecular target in dopamine pathway and mechanism underlying the risks of Pb exposure on the occurrence of PD at environmentally-relevant concentrations.

Immunophenotyping Tracks Motor Progression in Parkinson's Disease Associated with a TH Mutation.

Background: Parkinson's disease (PD) is the second most common neurodegenerative disorder, with genetic factors accounting for about 15% of cases. There is a significant challenge in tracking disease progression and treatment response, crucial for developing new therapies. Traditional methods like imaging, clinical monitoring, and biomarker analysis have not conclusively tracked disease progression or treatment response in PD. Our previous research indicated that PD patients with increased dopamine transporter (DAT) and tyrosine hydroxylase (TH) in peripheral blood mononuclear cells (PBMCs) might show disease progression and respond to levodopa treatment. Objective: This study evaluates whether DAT- and TH-expressing PBMCs can monitor motor progression in a PD patient with a heterozygous TH mutation. Methods: We conducted a longitudinal follow-up of a 46-year-old female PD patient with a TH mutation, assessing her clinical features over 18 months through DaT scans and PBMC immunophenotyping. This was compared with idiopathic PD patients (130 subjects) and healthy controls (80 age/sex-matched individuals). Results: We found an increase in DAT+ immune cells concurrent with worsening motor scores (UPDRS-III). Following levodopa therapy, unlike idiopathic PD patients, TH+ immune cell levels in this patient remained high even as her motor scores improved. Conclusions: Longitudinal immunophenotyping in this PD patient suggests DAT+ and TH+ PBMCs as potential biomarkers for tracking PD progression and treatment efficacy, supporting further exploration of this approach in PD research.

Tyrosine Hydroxylase Inhibitors and Dopamine Receptor Agonists Combination Therapy for Parkinson's Disease.

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.

Immobilization of human tyrosine hydroxylase onto magnetic nanoparticles - A novel formulation of a therapeutic enzyme.

Human tyrosine hydroxylase (hTH) has key role in the production of catecholamine neurotransmitters. The structure, function and regulation of hTH has been extensively researched area and the possibility of enzyme replacement therapy (ERT) involving hTH through nanocarriers has been raised as well. However, our understanding on how hTH may interact with nanocarriers is still lacking. In this work, we attempted to investigate the immobilization of hTH on magnetic nanoparticles (MNPs) with various surface linkers in quantitative and mechanistic detail. Our results showed that the activity of hTH was retained after immobilization via secondary and covalent interactions as well. The colloidal stability of hTH could be also enhanced proved by Dynamic light scattering and Zeta potential analysis and a homogenous enzyme layer could be achieved, which was investigated by Raman mapping. The covalent attachment of hTH on MNPs via aldehyde or epoxy linkers provide irreversible immobilization and 38.1 % and 16.5 % recovery (ER). The hTH-MNPs catalyst had 25 % ER in average in simulated nasal electrolyte solution (SNES). This outcome highlights the relevance of immobilization applying MNPs as a potential formulation tool of sensitive therapeutic enzymes offering new opportunities for ERT related to neurodegenerative disorders.

IGFBP5 Promotes Neuronal Apoptosis in a 6-OHDA-Toxicant Model of Parkinson's Disease by Inhibiting the Sonic Hedgehog Signaling Pathway.

INTRODUCTION: Parkinson's disease (PD) is the most common neurodegenerative disease worldwide. Studies have shown that insulin-like growth factor-binding protein 5 (IGFBP5) may contribute to methamphetamine-induced neurotoxicity and neuronal apoptosis in PC-12 cells and rat striatum. Here, we studied the expression and role of IGFBP5 in the 6-OHDA-toxicant model of PD. METHODS: PC-12 and SH-SY5Y cells were exposed to 50 µm 6-OHDA for 24 h. qRT-PCR, western blotting, CCK-8 assay, EdU staining, annexin V staining, and immunofluorescence were performed to study the effects of IGFBP5-specific siRNAs. The effects of IGFBP5 on a rat 6-OHDA model of PD were confirmed by performing behavioral tests, tyrosine hydroxylase (TH) immunofluorescence staining, and western blotting. RESULTS: In the GSE7621 dataset, IGFBP5 was highly expressed in the substantia nigra tissues of PD patients compared to healthy controls. In PC-12 and SH-SY5Y cells, IGFBP5 was upregulated following 6-OHDA exposure in a dose-dependent manner. Silencing of IGFBP5 promoted PC-12 and SH-SY5Y proliferation and inhibited apoptosis under 6-OHDA stimulation. Silencing of IGFBP5 relieved 6-OHDA-induced TH-positive neuron loss. Hedgehog signaling pathway was predicted as a downstream signaling pathway of IGFBP5. Negative regulation between IGFBP5 and sonic hedgehog (SHH) signaling pathway was confirmed in vitro. The effects of IGFBP5 silencing on SH-SY5Y cells were partially reversed using cyclopamine, a direct inhibitor of the SHH signaling pathway. In addition, silencing of IGFBP5 attenuated motor deficits and neuronal damage in 6-OHDA-induced PD rats. CONCLUSION: Elevated IGFBP5 expression may be involved in 6-OHDA-induced neurotoxicity through regulation of the SHH signaling pathway.