sFlt-1 Impairs Neurite Growth and Neuronal Differentiation in SH-SY5Y Cells and Human Neurons.

Pre-eclampsia (PE) is a hypertensive disorder of pregnancy which is associated with increased risk of neurodevelopmental disorders in exposed offspring. The pathophysiological mechanisms mediating this relationship are currently unknown, and one potential candidate is the anti-angiogenic factor soluble Fms-like tyrosine kinase 1 (sFlt-1), which is highly elevated in PE. While sFlt-1 can impair angiogenesis via inhibition of VEGFA signalling, it is unclear whether it can directly affect neuronal development independently of its effects on the vasculature. To test this hypothesis, the current study differentiated the human neural progenitor cell (NPC) line ReNcell® VM into a mixed culture of mature neurons and glia, and exposed them to sFlt-1 during development. Outcomes measured were neurite growth, cytotoxicity, mRNA expression of nestin, MBP, GFAP, and ßIII-tubulin, and neurosphere differentiation. sFlt-1 induced a significant reduction in neurite growth and this effect was timing- and dose-dependent up to 100 ng/mL, with no effect on cytotoxicity. sFlt-1 (100 ng/mL) also reduced ßIII-tubulin mRNA and neuronal differentiation of neurospheres. Undifferentiated NPCs and mature neurons/glia expressed VEGFA and VEGFR-2, required for endogenous autocrine and paracrine VEGFA signalling, while sFlt-1 treatment prevented the neurogenic effects of exogenous VEGFA. Overall, these data provide the first experimental evidence for a direct effect of sFlt-1 on neurite growth and neuronal differentiation in human neurons through inhibition of VEGFA signalling, clarifying our understanding of the potential role of sFlt-1 as a mechanism by which PE can affect neuronal development.

Human α-synuclein overexpression upregulates SKOR1 in a rat model of simulated nigrostriatal ageing.

Parkinson's disease (PD) is characterised by progressive loss of dopaminergic (DA) neurons from the substantia nigra (SN) and α-synuclein (αSyn) accumulation. Age is the biggest risk factor for PD and may create a vulnerable pre-parkinsonian state, but the drivers of this association are unclear. It is known that ageing increases αSyn expression in DA neurons and that this may alter molecular processes that are central to maintaining nigrostriatal integrity. To model this, adult female Sprague-Dawley rats received a unilateral intranigral injection of adeno-associated viral (AAV) vector carrying wild-type human αSyn (AAV-αSyn) or control vector (AAV-Null). AAV-αSyn induced no detrimental effects on motor behaviour, but there was expression of human wild-type αSyn throughout the midbrain and ipsilateral striatum at 20 weeks post-surgery. Microarray analysis revealed that the gene most-upregulated in the ipsilateral SN of the AAV-αSyn group was the SKI Family Transcriptional Corepressor 1 (SKOR1). Bioenergetic state analysis of mitochondrial function found that SKOR1 overexpression reduced the maximum rate of cellular respiration in SH-SY5Y cells. Furthermore, experiments in SH-SY5Y cells revealed that SKOR1 overexpression impaired neurite growth to the same extent as αSyn, and inhibited BMP-SMAD-dependent transcription, a pathway that promotes DA neuronal survival and growth. Given the normal influence of ageing on DA neuron loss in human SN, the extent of αSyn-induced SKOR1 expression may influence whether an individual undergoes normal nigrostriatal ageing or reaches a threshold for prodromal PD. This provides new insight into mechanisms through which ageing-related increases in αSyn may influence molecular mechanisms important for the maintenance of neuronal integrity.

Maternal hypertensive disorders of pregnancy and depression or anxiety in adolescence: Findings from the Millennium Cohort Study.

BACKGROUND: The short-term effects of hypertensive disorders of pregnancy (HDP) on the health of the fetus are well known; however, their impacts on the risk of mental health in the exposed offspring are not fully understood. Our aim was to examine the association between HDP and depression/anxiety at age 17 years. METHODS: We used data from The Millennium Cohort Study, a nationally representative longitudinal study of children born in the United Kingdom. Data on HDP and potential confounders were collected when children were 9-months. Data on depression and anxiety were collected as one variable when children were aged 17 years using self-reported doctor diagnosis, and reclassified as depression/anxiety (overall), depression/anxiety with treatment, and depression/anxiety without treatment. Crude and adjusted logistic regression models were performed to examine the association between HDP and depression/anxiety, adjusting for several maternal and socio-economic factors. RESULTS: There were 9517 singleton mother-child pairs included in the analyses. Adjusted logistic regression suggested an association between HDP and depression/anxiety (adjusted odds ratio, (aOR):1.30 [95 % CI, 1.02-1.66]) at age 17 years. A similar association was observed for HDP and depression/anxiety with treatment (aOR:1.33 [95 % CI, 1.01-1.73]) and HDP and depression/anxiety without treatment (aOR: 1.30 [95 % CI, 0.80-2.12]), although the latter did not reach statistical significance. LIMITATIONS: Data on severity and classifications of HDP were not available. CONCLUSION: Exposure to HDP may be associated with an increased likelihood of depression or anxiety at age 17 years. Future research should consider severity and different classifications of HDP.

A combined proteomics and bioinformatics analysis of ZNHIT1-interacting proteins reveals a significant enrichment in proteins associated with mitochondrial function.

Adenosine 5'-triphosphate (ATP) is the principal source of cellular energy, which is essential for neuronal health and maintenance. Parkinson's disease (PD) and other neurodegenerative disorders are characterised by impairments in mitochondrial function and reductions in cellular ATP levels. Thus there is a need to better understand the biology of intracellular regulators of ATP production, in order to inform the development of new neuroprotective therapies for diseases such as PD. One such regulator is Zinc finger HIT-domain containing protein 1 (ZNHIT1). ZNHIT1 is an evolutionarily-conserved component of a chromatin-remodelling complex, which has been recently shown to increase cellular ATP production in SH-SY5Y cells and to protect against impairments in mitochondrial function caused by alpha-synuclein, a protein which is integral to PD pathophysiology. This effect of ZNHIT1 on cellular ATP production is thought to be due to increased expression of genes associated with mitochondrial function, but it is also possible that ZNHIT1 regulates mitochondrial function by binding to mitochondrial proteins. To examine this question, we performed a combined proteomics and bioinformatics analysis to identify ZNHIT1-interacting proteins in SH-SY5Y cells. We report that ZNHIT1-interacting proteins are significantly enriched in multiple functional categories, including mitochondrial transport, ATP synthesis and ATP-dependent activity. Furthermore we also report that the correlation between ZNHIT1 and dopaminergic markers is reduced in the PD brain. These data suggest that the reported beneficial effects of ZNHIT1 on ATP production may be mediated, at least in part, by its direct interaction with mitochondrial proteins and suggest that potential alterations in ZNHIT1 in PD may contribute to the known impairments in ATP generation in midbrain dopaminergic neurons in PD.

Regulator of G-Protein Signalling 4 (RGS4) negatively modulates nociceptin/orphanin FQ opioid receptor signalling: Implication for l-Dopa-induced dyskinesia.

BACKGROUND AND PURPOSE: Regulator of G-protein signalling 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gαi/o and Gαq subunits, suppressing GPCR signalling. Here, we investigate whether RGS4 modulates nociceptin/orphanin FQ (N/OFQ) opioid (NOP) receptor signalling and if this modulation has relevance for l-Dopa-induced dyskinesia. EXPERIMENTAL APPROACH: HEK293T cells transfected with NOP, NOP/RGS4 or NOP/RGS19 were challenged with N/OFQ and the small-molecule NOP agonist AT-403, using D1-stimulated cAMP levels as a readout. Primary rat striatal neurons and adult mouse striatal slices were challenged with either N/OFQ or AT-403 in the presence of the experimental RGS4 chemical probe, CCG-203920, and D1-stimulated cAMP or phosphorylated extracellular signal regulated kinase 1/2 (pERK) responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and l-Dopa to 6-hydroxydopamine hemilesioned rats, and dyskinetic movements, striatal biochemical correlates of dyskinesia (pERK and pGluR1 levels) and striatal RGS4 levels were measured. KEY RESULTS: RGS4 expression reduced NOFQ and AT-403 potency and efficacy in HEK293T cells. CCG-203920 increased N/OFQ potency in primary rat striatal neurons and potentiated AT-403 response in mouse striatal slices. CCG-203920 enhanced AT-403-mediated inhibition of dyskinesia and its biochemical correlates, without compromising its motor-improving effects. Unilateral dopamine depletion caused bilateral reduction of RGS4 levels, which was reversed by l-Dopa. l-Dopa acutely up-regulated RGS4 in the lesioned striatum. CONCLUSIONS AND IMPLICATIONS: RGS4 physiologically inhibits NOP receptor signalling. CCG-203920 enhanced NOP responses and improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of striatal RGS4 up-regulation occurring during dyskinesia expression. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Diet quality, sleep and quality of life in Parkinson's disease: a cross-sectional study.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder characterised by motor and non-motor symptoms that impact quality of daily life, including diet and sleep. However, relatively little is known about dietary intake and quality in people with PD (PwP). Lifestyle factors, and how they relate to diet, are also insufficiently understood. The aims of this study were to investigate dietary intake and quality, sleep and quality of life in PwP, and to explore the relationships between these factors. METHODS: Forty-five community-dwelling participants with PD (n = 45) were recruited to this cross-sectional study through the Cork Parkinson's Association, Ireland. Dietary intake was assessed using the EPIC food frequency questionnaire, and diet quality was assessed using the Healthy Diet Indicator. Dietary intakes were compared to Irish RDAs for adults > 65 years. Sleep duration and quality were subjectively measured using the PD Sleep Scale and Pittsburgh sleep quality index and objectively measured by actigraphy in a subset of participants (n = 27). QOL was measured using the validated PDQ-39 questionnaire. RESULTS: Energy intake in PwP was significantly higher than that of the general population (2013 vs 1755 kcal/d, p = 0.01), despite their lower mean BMI (25.9 vs 27.7 kg/m2, p = 0.02). Intakes of carbohydrate, protein and fruits and vegetables were significantly higher in PwP compared to recommended and population intakes (all p < 0.01), but fibre intake was significantly lower than recommended (17.3 vs 25 g/d, p [Formula: see text] 0.05). Seventy-eight percent of participants had poor dietary quality, and poor sleep quality was associated with poor QOL. CONCLUSIONS: Carbohydrates, protein, fruit and vegetable intakes were greater in PwP than population norms, but overall diet quality was low. Interventions to improve dietary and lifestyle factors may improve health and QOL in PwP.

Characterisation of the consequences of maternal immune activation on distinct cell populations in the developing rat spinal cord.

Maternal immune activation (MIA) during gestation has been implicated in the development of neurological disorders such as schizophrenia and autism. Epidemiological studies have suggested that the effect of MIA may depend on the gestational timing of the immune challenge and the region of the central nervous system (CNS) in question. This study investigated the effects of MIA with 100 µg/kg lipopolysaccharide at either Embryonic days (E)12 or E16 on the oligodendrocytes, microglia and astrocytes of the offspring spinal cord. At E16, MIA decreased the number of olig2+ and Iba-1+ cells in multiple grey and white matter regions of the developing spinal cord 5 h after injection. These decreases were not observed at postnatal day 14. In contrast, MIA at E12 did not alter Olig2+ or Iba-1+ cell number in the developing spinal cord 5 h after injection, however, Olig2+ cell number was decreased in the ventral grey matter of the P14 spinal cord. No changes were observed in glial fibrillary acidic protein (GFAP) expression at P14 following MIA at either E12 or E16. These data suggest that E16 may be a window of immediate vulnerability to MIA during spinal cord development, however, the findings also suggest that the developmental process may be capable of compensation over time. Potential changes in P14 animals following the challenge at E12 are indicative of the complexity of the effects of MIA during the developmental process.

Maternal Immune Activation and Interleukin 17A in the Pathogenesis of Autistic Spectrum Disorder and Why It Matters in the COVID-19 Era.

Autism spectrum disorder (ASD) is the commonest neurodevelopmental disability. It is a highly complex disorder with an increasing prevalence and an unclear etiology. Consensus indicates that ASD arises as a genetically modulated, and environmentally influenced condition. Although pathogenic rare genetic variants are detected in around 20% of cases of ASD, no single factor is responsible for the vast majority of ASD cases or that explains their characteristic clinical heterogeneity. However, a growing body of evidence suggests that ASD susceptibility involves an interplay between genetic factors and environmental exposures. One such environmental exposure which has received significant attention in this regard is maternal immune activation (MIA) resulting from bacterial or viral infection during pregnancy. Reproducible rodent models of ASD are well-established whereby induction of MIA in pregnant dams, leads to offspring displaying neuroanatomical, functional, and behavioral changes analogous to those seen in ASD. Blockade of specific inflammatory cytokines such as interleukin-17A during gestation remediates many of these observed behavioral effects, suggesting a causative or contributory role. Here, we review the growing body of animal and human-based evidence indicating that interleukin-17A may mediate the observed effects of MIA on neurodevelopmental outcomes in the offspring. This is particularly important given the current corona virus disease-2019 (COVID-19) pandemic as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during pregnancy is a potent stimulator of the maternal immune response, however the long-term effects of maternal SARS-CoV-2 infection on neurodevelopmental outcomes is unclear. This underscores the importance of monitoring neurodevelopmental outcomes in children exposed to SARS-CoV-2-induced MIA during gestation.

Gene Co-expression Analysis of the Human Substantia Nigra Identifies ZNHIT1 as an SNCA Co-expressed Gene that Protects Against α-Synuclein-Induced Impairments in Neurite Growth and Mitochondrial Dysfunction in SH-SY5Y Cells.

Parkinson's disease (PD) is neurodegenerative disorder with the pathological hallmarks of progressive degeneration of midbrain dopaminergic neurons from the substantia nigra (SN), and accumulation and spread of inclusions of aggregated α-synuclein (α-Syn). Since current PD therapies do not prevent neurodegeneration, there is a need to identify therapeutic targets that can prevent α-Syn-induced reductions in neuronal survival and neurite growth. We hypothesised that genes that are normally co-expressed with the α-Syn gene (SNCA), and whose co-expression pattern is lost in PD, may be important for protecting against α-Syn-induced dopaminergic degeneration, since broken correlations can be used as an index of functional misregulation. Gene co-expression analysis of the human SN showed that nuclear zinc finger HIT-type containing 1 (ZNHIT1) is co-expressed with SNCA and that this co-expression pattern is lost in PD. Overexpression of ZNHIT1 was found to increase deposition of the H2A.Z histone variant in SH-SY5Y cells, to promote neurite growth and to prevent α-Syn-induced reductions in neurite growth and cell viability. Analysis of ZNHIT1 co-expressed genes showed significant enrichment in genes associated with mitochondrial function. In agreement, bioenergetic state analysis of mitochondrial function revealed that ZNHIT1 increased cellular ATP synthesis. Furthermore, α-Syn-induced impairments in basal respiration, maximal respiration and spare respiratory capacity were not seen in ZNHIT1-overexpressing cells. These data show that ZNHIT1 can protect against α-Syn-induced degeneration and mitochondrial dysfunction, which rationalises further investigation of ZNHIT1 as a therapeutic target for PD.

Peripheral administration of the Class-IIa HDAC inhibitor MC1568 partially protects against nigrostriatal neurodegeneration in the striatal 6-OHDA rat model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterised by nigrostriatal dopaminergic (DA) neurodegeneration. There is a critical need for neuroprotective therapies, particularly those that do not require direct intracranial administration. Small molecule inhibitors of histone deacetylases (HDIs) are neuroprotective in in vitro and in vivo models of PD, however it is unknown whether Class IIa-specific HDIs are neuroprotective when administered peripherally. Here we show that 6-hydroxydopamine (6-OHDA) treatment induces protein kinase C (PKC)-dependent nuclear accumulation of the Class IIa histone deacetylase (HDAC)5 in SH-SY5Y cells and cultured DA neurons in vitro. Treatment of these cultures with the Class IIa-specific HDI, MC1568, partially protected against 6-OHDA-induced cell death. In the intrastriatal 6-OHDA lesion in vivo rat model of PD, MC1568 treatment (0.5 mg/kg i.p.) for 7 days reduced forelimb akinesia and partially protected DA neurons in the substantia nigra and their striatal terminals from 6-OHDA-induced neurodegeneration. MC1568 treatment prevented 6-OHDA-induced increases in microglial activation in the striatum and substantia nigra. Furthermore, MC1568 treatment decreased 6-OHDA-induced increases in nuclear HDAC5 in nigral DA neurons. These data suggest that peripheral administration of Class IIa-specific HDIs may be a potential therapy for neuroprotective in PD.

The association between hypertensive disorders of pregnancy and childhood asthma.

BACKGROUND: Asthma is a chronic long-term inflammatory disease of the airways and is a major global health challenge. The primary aim of this study was to investigate the association between hypertensive disorders of pregnancy (HDP) and the risk of asthma at or before the age of seven years using the UK Millennium Cohort Study (MCS). METHODS: Overall, 18,552 families were recruited for wave one of the MCS when the child was 9 months old, and subsequently participated in waves two, three and four when they were three, five and seven years respectively. HDP were self-reported by mothers in wave one. The primary outcome was a parent-reported diagnosis of "ever asthma" at seven years, based on responses to a validated questionnaire. RESULTS: Following adjustment for a range of potential confounding factors, HDP was found to be associated with asthma in the offspring (adjusted odds ratio (AOR 1.35; 95% CI: 1.15-1.59)). A larger effect estimate was observed amongst children exposed to HDP and born preterm (AOR 1.81; 95% CI: 1.25-2.61) or small for gestational age (SGA) (AOR = 1.58; (95% CI: 1.15-2.18)). CONCLUSIONS: In conclusion, children exposed to HDP may be at increased odds of asthma diagnosis by age seven, particularly if they were born preterm or SGA. IMPACT: There is a paucity of data investigating the relationship between hypertensive disorders of pregnancy and childhood asthma, with recent studies showing conflicting results. Our study investigated this relationship using a large cohort with ample information on a wide range of confounding factors. Our study showed that individuals exposed to HDP may be at increased odds of asthma diagnosis by age seven years, particularly if they were also born SGA or preterm.

NME1 Protects Against Neurotoxin-, α-Synuclein- and LRRK2-Induced Neurite Degeneration in Cell Models of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disease characterised by the progressive degeneration of midbrain dopaminergic neurons, coupled with the intracellular accumulation of α-synuclein. Axonal degeneration is a central part of the pathology of PD. While the majority of PD cases are sporadic, some are genetic; the G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is the most common genetic form. The application of neurotrophic factors to protect dopaminergic neurons is a proposed experimental therapy. One such neurotrophic factor is growth differentiation factor (GDF)5. GDF5 is a dopaminergic neurotrophic factor that has been shown to upregulate the expression of a protein called nucleoside diphosphate kinase A (NME1). However, whether NME1 is neuroprotective in cell models of axonal degeneration of relevance to PD is unknown. Here we show that treatment with NME1 can promote neurite growth in SH-SY5Y cells, and in cultured dopaminergic neurons treated with the neurotoxin 6-hydroxydopamine (6-OHDA). Similar effects of NME1 were found in SH-SY5Y cells and dopaminergic neurons overexpressing human wild-type α-synuclein, and in stable SH-SY5Y cell lines carrying the G2019S LRRK2 mutation. We found that the effects of NME1 require the RORα/ROR2 receptors. Furthermore, increased NF-κB-dependent transcription was partially required for the neurite growth-promoting effects of NME1. Finally, a combined bioinformatics and biochemical analysis of the mitochondrial oxygen consumption rate revealed that NME1 enhanced mitochondrial function, which is known to be impaired in PD. These data show that recombinant NME1 is worthy of further study as a potential therapeutic agent for axonal protection in PD.

Growth differentiation factor 5: a neurotrophic factor with neuroprotective potential in Parkinson's disease.

Parkinson's disease is the most common movement disorder worldwide, affecting over 6 million people. It is an age-related disease, occurring in 1% of people over the age of 60, and 3% of the population over 80 years. The disease is characterized by the progressive loss of midbrain dopaminergic neurons from the substantia nigra, and their axons, which innervate the striatum, resulting in the characteristic motor and non-motor symptoms of Parkinson's disease. This is paralleled by the intracellular accumulation of α-synuclein in several regions of the nervous system. Current therapies are solely symptomatic and do not stop or slow disease progression. One promising disease-modifying strategy to arrest the loss of dopaminergic neurons is the targeted delivery of neurotrophic factors to the substantia nigra or striatum, to protect the remaining dopaminergic neurons of the nigrostriatal pathway. However, clinical trials of two well-established neurotrophic factors, glial cell line-derived neurotrophic factor and neurturin, have failed to meet their primary end-points. This failure is thought to be at least partly due to the downregulation by α-synuclein of Ret, the common co-receptor of glial cell line-derived neurorophic factor and neurturin. Growth/differentiation factor 5 is a member of the bone morphogenetic protein family of neurotrophic factors, that signals through the Ret-independent canonical Smad signaling pathway. Here, we review the evidence for the neurotrophic potential of growth/differentiation factor 5 in in vitro and in vivo models of Parkinson's disease. We discuss new work on growth/differentiation factor 5's mechanisms of action, as well as data showing that viral delivery of growth/differentiation factor 5 to the substantia nigra is neuroprotective in the α-synuclein rat model of Parkinson's disease. These data highlight the potential for growth/differentiation factor 5 as a disease-modifying therapy for Parkinson's disease.

Maternal pre-eclampsia serum increases neurite growth and mitochondrial function through a potential IL-6-dependent mechanism in differentiated SH-SY5Y cells.

Introduction: Pre-eclampsia (PE) is a common and serious hypertensive disorder of pregnancy, which affects 3%-5% of first-time pregnancies and is a leading cause of maternal and neonatal morbidity and mortality. Prenatal exposure to PE is associated with an increased risk of neurodevelopmental disorders in affected offspring, although the cellular and molecular basis of this increased risk is largely unknown. Methods: Here, we examined the effects of exposure to maternal serum from women with PE or a healthy uncomplicated pregnancy on the survival, neurite growth and mitochondrial function of neuronally differentiated human SH-SY5Y neuroblastoma cells, which are commonly used to study neurite growth. Neurite growth and mitochondrial function are two strongly linked neurodevelopmental parameters in which alterations have been implicated in neurodevelopmental disorders. Following this, we investigated the pleiotropic cytokine interleukin-6 (IL-6) levels as a potential mechanism. Results: Cells exposed to 3% (v/v) PE serum for 72 h exhibited increased neurite growth (p < 0.05), which was validated in the human neural progenitor cell line, ReNcell® VM (p < 0.01), and mitochondrial respiration (elevated oxygen consumption rate (p < 0.05), basal mitochondrial respiration, proton leak, ATP synthesis, and non-mitochondrial respiration) compared to control serum-treated cells. ELISA analysis showed elevations in maternal IL-6 in PE sera (p < 0.05) and placental explants (p < 0.05). In support of this, SH-SY5Y cells exposed to 3% (v/v) PE serum for 24 h had increased phospho-STAT3 levels, which is a key intracellular mediator of IL-6 signalling (p < 0.05). Furthermore, treatment with anti-IL-6 neutralizing antibody blocked the effects of PE serum on neurite growth (p < 0.05), and exposure to IL-6 promoted neurite growth in SH-SY5Y cells (p < 0.01). Discussion: Collectively these data show elevated serum levels of maternal IL-6 in PE, which increases neurite growth and mitochondrial function in SH-SY5Y cells. This rationalizes the further study of IL-6 as a potential mediator between PE exposure and neurodevelopmental outcome in the offspring.

Mid-gestation cytokine profiles in mothers of children affected by autism spectrum disorder: a case-control study.

Autism Spectrum disorder is one of the commonest and most important neurodevelopmental conditions affecting children today. With an increasing prevalence and an unclear aetiology, it is imperative we find early markers of autism, which may facilitate early identification and intervention. Alterations of gestational cytokine profiles have been reported in mothers of autistic children. Increasing evidence suggests that the intrauterine environment is an important determinant of autism risk. This study aims to examine the mid-gestational serum cytokine profiles of the mothers of autistic children from a well-characterised birth cohort. A nested sub-cohort within a large mother-child birth cohort were identified based on a confirmed multi-disciplinary diagnosis of autism before the age 10 years and neuro-typical matched controls in a 2:1 ratio. IFN-γ, IL-1ß, IL-4, IL-6, IL-8, IL-17A, GMCSF and TNFα were measured in archived maternal 20-week serum using MesoScale Diagnostics multiplex technology and validation of our IL-17A measurements was performed using an ultrasensitive assay. From a cohort of 2137 children, 25 had confirmed autism before 10 years and stored maternal serum from mid-gestation. We examined the sera of these 25 cases and 50 matched controls. The sex ratio was 4:1 males to females in each group, and the mean age at diagnosis was 5.09 years (SD 2.13). We found that concentrations of IL-4 were significantly altered between groups. The other analytes did not differ significantly using either multiplex or ultra-sensitive assays. In our well-characterised prospective cohort of autistic children, we confirmed mid-gestational alterations in maternal IL-4 concentrations in autism affected pregnancies versus matched controls. These findings add to promising evidence from animal models and retrospective screening programmes and adds to the knowledge in this field.

LMK235, a small molecule inhibitor of HDAC4/5, protects dopaminergic neurons against neurotoxin- and α-synuclein-induced degeneration in cellular models of Parkinson's disease.

Epigenetic modifications in neurodegenerative disease are under investigation for their roles in disease progression. Alterations in acetylation rates of certain Parkinson's disease (PD)-linked genes have been associated with the pathological progression of this disorder. In light of this, and given the lack of disease-modifying therapies for PD, HDAC inhibitors (HDIs) are under consideration as potential pharmacological agents. The neuroprotective effects of pan-HDACs and some class-specific inhibitors have been tested in in vivo and in vitro models of PD, with varying outcomes. Here we used gene co-expression analysis to identify HDACs that are associated with human dopaminergic (DA) neuron development. We identified HDAC3, HDAC5, HDAC6 and HDAC9 as being highly correlated with the DA markers, SLC6A3 and NR4A2. RT-qPCR revealed that mRNA expression of these HDACs exhibited similar temporal profiles during embryonic mouse midbrain DA (mDA) neuron development. We tested the neuroprotective potential of a number of class-specific small molecule HDIs on human SH-SY5Y cells, using neurite growth as a phenotypic readout of neurotrophic action. Neither the class I-specific HDIs, RGFP109 and RGFP966, nor the HDAC6 inhibitor ACY1215, had significant effects on neurite outgrowth. However, the class IIa HDI, LMK235 (a HDAC4/5 inhibitor), significantly increased histone acetylation and neurite outgrowth. We found that LMK235 increased BMP-Smad-dependent transcription in SH-SY5Y cells and that this was required for its neurite growth-promoting effects on SH-SY5Y cells and on DA neurons in primary cultures of embryonic day (E) 14 rat ventral mesencephalon (VM). These effects were also seen in SH-SY5Y cells transfected with HDAC5 siRNA. Furthermore, LMK235 treatment exerted neuroprotective effects against degeneration induced by the DA neurotoxin 1-methyl-4-phenylpyridinium (MPP+), in both SH-SY5Y cells and cultured DA neurons. Treatment with LMK235 was also neuroprotective against axonal degeneration induced by overexpression of wild-type (WT) or A53T mutant α-synuclein in both SH-SY5Y cells and primary cultures of DA neurons. In summary, these data show the neuroprotective potential of the class IIa HDI, LMK235, in cell models of relevance to PD.

Quinacrine and Niclosamide Promote Neurite Growth in Midbrain Dopaminergic Neurons Through the Canonical BMP-Smad Pathway and Protect Against Neurotoxin and α-Synuclein-Induced Neurodegeneration.

Parkinson's disease is a neurodegenerative disorder characterised by nigrostriatal dopaminergic degeneration, and intracellular α-synuclein aggregation. Current pharmacological treatments are solely symptomatic so there is a need to identify agents that can slow or stop dopaminergic degeneration. One proposed class of therapeutics are neurotrophic factors which promote the survival of nigrostriatal dopaminergic neurons. However, neurotrophic factors need to be delivered directly to the brain. An alternative approach may be to identify pharmacological agents which can reach the brain to stimulate neurotrophic factor expression and/or their signalling pathways in dopaminergic neurons. BMP2 is a neurotrophic factor that is expressed in the human substantia nigra; exogenous BMP2 administration protects against dopaminergic degeneration in in vitro models of PD. In this study, we investigated the neurotrophic potential of two FDA-approved drugs, quinacrine and niclosamide, that are modulators of BMP2 signalling. We report that quinacrine and niclosamide, like BMP2, significantly increased neurite length, as a readout of neurotrophic action, in SH-SY5Y cells and dopaminergic neurons in primary cultures of rat ventral mesencephalon. We also show that these effects of quinacrine and niclosamide require the activation of BMP-Smad signalling. Finally, we demonstrate that quinacrine and niclosamide are neuroprotective against degeneration induced by the neurotoxins, MPP+ and 6-OHDA, and by viral-mediated overexpression of α-synuclein in vitro. Collectively, this study identifies two drugs, that are safe for use in patients' to 'are approved for human use, that exert neurotrophic effects on dopaminergic neurons through modulation of BMP-Smad signalling. This rationalises the further study of drugs that target the BMP-Smad pathway as potential neuroprotective pharmacotherapy for Parkinson's disease.