Selective regulation of nerve growth factor expression in developing cutaneous tissue by early sensory innervation

Background: In the developing vertebrate peripheral nervous system, the survival of sympathetic neurons and the majority of sensory neurons depends on a supply of nerve growth factor (NGF) from tissues they innervate. Although neurotrophic theory presupposes, and the available evidence suggests, that the level of NGF expression is completely independent of innervation, the possibility that innervation may regulate the timing or level of NGF expression has not been rigorously investigated in a sufficiently well-characterized developing system. Results: To address this important question, we studied the influence of innervation on the regulation of NGF mRNA expression in the embryonic mouse maxillary process in vitro and in vivo. The maxillary process receives its innervation from predominantly NGF-dependent sensory neurons of the trigeminal ganglion and is the most densely innervated cutaneous territory with the highest levels of NGF in the embryo. When early, uninnervated maxillary processes were cultured alone, the level of NGF mRNA rose more slowly than in maxillary processes cultured with attached trigeminal ganglia. In contrast to the positive influence of early innervation on NGF mRNA expression, the levels of brain-derived neurotrophic factor (BDNF) mRNA and neurotrophin-3 (NT3) mRNA rose to the same extent in early maxillary processes grown with and without trigeminal ganglia. The level of NGF mRNA, but not BDNF mRNA or NT3 mRNA, was also significantly lower in the maxillary processes of erbB3-/- mice, which have substantially fewer trigeminal neurons than wild-type mice. Conclusions: This selective effect of initial innervation on target field NGF mRNA expression provokes a re-evaluation of a key assertion of neurotrophic theory that the level of NGF expression is independent of innervation

Region-specific role of growth differentiation factor-5 in the establishment of sympathetic innervation

Background Nerve growth factor (NGF) is the prototypical target-derived neurotrophic factor required for sympathetic neuron survival and for the growth and ramification of sympathetic axons within most but not all sympathetic targets. This implies the operation of additional target-derived factors for regulating terminal sympathetic axon growth and branching. Results Here report that growth differentiation factor 5 (GDF5), a widely expressed member of the transforming growth factor beta (TGFβ) superfamily required for limb development, promoted axon growth from mouse superior cervical ganglion (SCG) neurons independently of NGF and enhanced axon growth in combination with NGF. GDF5 had no effect on neuronal survival and influenced axon growth during a narrow window of postnatal development when sympathetic axons are ramifying extensively in their targets in vivo. SCG neurons expressed all receptors capable of participating in GDF5 signaling at this stage of development. Using compartment cultures, we demonstrated that GDF5 exerted its growth promoting effect by acting directly on axons and by initiating retrograde canonical Smad signalling to the nucleus. GDF5 is synthesized in sympathetic targets, and examination of several anatomically circumscribed tissues in Gdf5 null mice revealed regional deficits in sympathetic innervation. There was a marked, highly significant reduction in the sympathetic innervation density of the iris, a smaller though significant reduction in the trachea, but no reduction in the submandibular salivary gland. There was no reduction in the number of neurons in the SCG. Conclusions These findings show that GDF5 is a novel target-derived factor that promotes sympathetic axon growth and branching and makes a distinctive regional contribution to the establishment of sympathetic innervation, but unlike NGF, plays no role in regulating sympathetic neuron survival

Zeb2 is a negative regulator of midbrain dopaminergic axon growth and target innervation

Neural connectivity requires neuronal differentiation, axon growth, and precise target innervation. Midbrain dopaminergic neurons project via the nigrostriatal pathway to the striatum to regulate voluntary movement. While the specification and differentiation of these neurons have been extensively studied, the molecular mechanisms that regulate midbrain dopaminergic axon growth and target innervation are less clear. Here we show that the transcription factor Zeb2 cell-autonomously represses Smad signalling to limit midbrain dopaminergic axon growth and target innervation. Zeb2 levels are downregulated in the embryonic rodent midbrain during the period of dopaminergic axon growth, when BMP pathway components are upregulated. Experimental knockdown of Zeb2 leads to an increase in BMP-Smad-dependent axon growth. Consequently there is dopaminergic hyperinnervation of the striatum, without an increase in the numbers of midbrain dopaminergic neurons, in conditional Zeb2 (Nestin-Cre based) knockout mice. Therefore, these findings reveal a new mechanism for the regulation of midbrain dopaminergic axon growth during central nervous system development

Expression of endogenous Mkp1 in 6-OHDA rat models of Parkinson's disease.

We have previously demonstrated that mitogen-activated protein kinase phosphatase 1, Mkp1, is expressed in the developing and rat adult substantia nigra and striatum, where it promotes the growth of nigral dopaminergic neurons. Mkp1 may therefore have therapeutic potential for Parkinson's disease. In the present study, we have assessed the expression of Mkp1 and TH in the substantia nigra and striatum of parkinsonian rat models. Expression was measured at 4 and 10 days post-lesion in the 6-hydroxydopamine (6-OHDA) medial forebrain bundle lesion model and after 4, 10 and 28 days in the 6-OHDA striatal lesion model. Our results show that Mkp1 expression was transiently up-regulated in the substantia nigra at 4 days post-6-OHDA administration in the two models while TH expression was decreased at the later time-points examined. These data suggest that Mkp1 may play a role in counteracting the neurotoxic effects of 6-OHDA in nigral dopaminergic neurons

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

Drug discovery for male subfertility using high-throughput screening:a new approach to an unsolved problem

STUDY QUESTIONCan pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility?SUMMARY ANSWERHigh-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility.WHAT IS KNOWN ALREADYThere is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform.STUDY DESIGN, SIZE, DURATIONSpermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016.PARTICIPANTS/MATERIALS, SETTING, METHODSA HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 μM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping.MAIN RESULTS AND THE ROLE OF CHANCEOf the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase in fluorescence in the primary screen were rescreened and evaluated further, resulting in 48 hit compounds that produced a concentration-dependent increase in [Ca2+]i. Sperm penetration studies confirmed in vitro exposure to two hit compounds (A and B) resulted in significant improvement in functional motility in spermatozoa from healthy volunteer donors (A: 1 cm penetration index 2.54, 2 cm penetration index 2.49; P &lt; 0.005 and B: 1 cm penetration index 2.1, 2 cm penetration index 2.6; P &lt; 0.005), but crucially, also in patient samples from those undergoing fertility treatment (A: 1 cm penetration index 2.4; P = 0.009, 2 cm penetration index 3.6; P = 0.02 and B: 1 cm penetration index 2.2; P = 0.0004, 2 cm penetration index 3.6; P = 0.002). This was primarily as a result of direct or indirect CatSper channel action, supported by evidence from electrophysiology studies of individual sperm.LIMITATIONS, REASONS FOR CAUTIONIncrease and fluxes in [Ca2+]i are fundamental to the regulation of sperm motility and function, including acrosome reaction. The use of calcium signalling as a surrogate for sperm motility is acknowledged as a potential limitation in this study.WIDER IMPLICATIONS OF THE FINDINGSWe conclude that HTS can robustly, efficiently, identify novel compounds that increase [Ca2+]i in human spermatozoa and functionally modify motility, and propose its use as a cornerstone to build and transform much-needed drug discovery for male infertility.</p

STRAP and NME1 mediate the neurite growth-promoting effects of the neurotrophic factor GDF5

Loss of midbrain dopaminergic (mDA) neurons and their axons is central to Parkinson's disease (PD). Growth differentiation factor (GDF)5 is a potential neurotrophic factor for PD therapy. However, the molecular mediators of its neurotrophic action are unknown. Our proteomics analysis shows that GDF5 increases the expression of serine threonine receptor-associated protein kinase (STRAP) and nucleoside diphosphate kinase (NME)1 in the SH-SY5Y neuronal cell line. GDF5 overexpression increased NME1 expression in adult rat brain in vivo. NME and STRAP mRNAs are expressed in developing and adult rodent midbrain. Expression of both STRAP and NME1 is necessary and sufficient for the promotion of neurite growth in SH-SY5Y cells by GDF5. NME1 treatment increased neurite growth in both SH-SY5Y cells and cultured mDA neurons. Expression patterns of NME and STRAP are altered in PD midbrain. NME1 and STRAP are thus key mediators of GDF5's neurotrophic effects, rationalizing their future study as therapeutic targets for PD

Association of distinct type 1 bone morphogenetic protein receptors with different molecular pathways and survival outcomes in neuroblastoma

Neuroblastoma (NB) is a paediatric cancer that arises in the sympathetic nervous system. Patients with stage 4 tumours have poor outcomes and 20% of high-risk cases have MYCN amplification. The bone morphogenetic proteins (BMPs) play roles in sympathetic neuritogenesis, by signalling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB; it is unknown if the expression of BMPR1A or BMPR1B is altered. We report lower BMPR2 and BMPR1B, and higher BMPR1A, expression in stage 4 and in MYCN-amplified NB. Kaplan–Meier plots showed that high BMPR2 or BMPR1B expression was linked to better survival, while high BMPR1A was linked to worse survival. Gene ontology enrichment and pathway analyses revealed that BMPR2 and BMPR1B co-expressed genes were enriched in those associated with NB differentiation. BMPR1A co-expressed genes were enriched in those associated with cell proliferation. Moreover, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This suggested that differentiation should decrease BMPR1A and increaseBMPR1Bexpression.Inagreement,nervegrowthfactortreatmentofculturedsympatheticneuronsdecreasedBmpr1aexpressionandincreasedBmpr1bexpression.Overexpression of dominant negative BMPR1B, treatment with a BMPR1B inhibitor and treatment with GDF5, which signals via BMPR1B, showed that BMPR1B signalling is required for optimal neuritogenesis in NB cells, suggesting that loss of BMPR1B may alter neuritogenesis. The present study shows that expression of distinct BMPRs is associated with different survival outcomes in NB

The origin and evolution of the normal Type Ia SN 2018aoz with infant-phase reddening and excess emission

SN~2018aoz is a Type Ia SN with a $B$-band plateau and excess emission in the infant-phase light curves $\lesssim$ 1 day after first light, evidencing an over-density of surface iron-peak elements as shown in our previous study. Here, we advance the constraints on the nature and origin of SN~2018aoz based on its evolution until the nebular phase. Near-peak spectroscopic features show the SN is intermediate between two subtypes of normal Type Ia: Core-Normal and Broad-Line. The excess emission could have contributions from the radioactive decay of surface iron-peak elements as well as ejecta interaction with either the binary companion or a small torus of circumstellar material. Nebular-phase limits on H$\alpha$ and He~I favour a white dwarf companion, consistent with the small companion size constrained by the low early SN luminosity, while the absence of [O~I] and He~I disfavours a violent merger of the progenitor. Of the two main explosion mechanisms proposed to explain the distribution of surface iron-peak elements in SN~2018aoz, the asymmetric Chandrasekhar-mass explosion is less consistent with the progenitor constraints and the observed blueshifts of nebular-phase [Fe~II] and [Ni~II]. The helium-shell double-detonation explosion is compatible with the observed lack of C spectral features, but current 1-D models are incompatible with the infant-phase excess emission, $B_{\rm max}-V_{\rm max}$ color, and absence of nebular-phase [Ca~II]. Although the explosion processes of SN~2018aoz still need to be more precisely understood, the same processes could produce a significant fraction of Type Ia SNe that appear normal after $\sim$ 1 day.Comment: Submitted for publication in ApJ. 35 pages, 16 figures, 7 table

Rapid Evolution of Sex Pheromone-Producing Enzyme Expression in Drosophila

Rapid evolution of gene expression patterns responsible for pheromone production in 24 species of Drosophila was mapped to simple mutations within the regulatory domain of the desatF gene