HoxB8 in noradrenergic specification and differentiation of the autonomic nervous system.

Different prespecification of mesencephalic and trunk neural crest cells determines their response to environmental differentiation signals and contributes to the generation of different autonomic neuron subtypes, parasympathetic ciliary neurons in the head and trunk noradrenergic sympathetic neurons. The differentiation of ciliary and sympathetic neurons shares many features, including the initial BMP-induced expression of noradrenergic characteristics that is, however, subsequently lost in ciliary but maintained in sympathetic neurons. The molecular basis of specific prespecification and differentiation patterns has remained unclear. We show here that HoxB gene expression in trunk neural crest is maintained in sympathetic neurons. Ectopic expression of a single HoxB gene, HoxB8, in mesencephalic neural crest results in a strongly increased expression of sympathetic neuron characteristics like the transcription factor Hand2, tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) in ciliary neurons. Other subtype-specific properties like RGS4 and RCad are not induced. HoxB8 has only minor effects in postmitotic ciliary neurons and is unable to induce TH and DBH in the enteric nervous system. Thus, we conclude that HoxB8 acts by maintaining noradrenergic properties transiently expressed in ciliary neuron progenitors during normal development. HoxC8, HoxB9, HoxB1 and HoxD10 elicit either small and transient or no effects on noradrenergic differentiation, suggesting a selective effect of HoxB8. These results implicate that Hox genes contribute to the differential development of autonomic neuron precursors by maintaining noradrenergic properties in the trunk sympathetic neuron lineage.

Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. Here, we focus on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivo inhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression.

The transcription factors AP-2ß and AP-2α are required for survival of sympathetic progenitors and differentiated sympathetic neurons.

Differentiation of sympathetic neurons is controlled by a group of transcription factors, including Phox2b, Ascl1, Hand2 and Gata3, induced by bone morphogenetic proteins (BMPs) in progenitors located in ganglion primordia at the dorsal aorta. Here, we address the function of the transcription factors AP-2ß and AP-2α, expressed in migrating neural crest cells (NCC) and maintained in sympathetic progenitors and differentiated neurons. The elimination of both AP-2α and AP-2ß results in the virtually complete absence of sympathetic and sensory ganglia due to apoptotic cell death of migrating NCC. In the AP-2ß knockout only sympathetic ganglia (SG) are targeted, leading to a reduction in ganglion size by about 40%, which is also caused by apoptotic death of neural crest progenitors. The conditional double knockout of AP-2α and AP-2ß in sympathetic progenitors and differentiated noradrenergic neurons results in a further decrease in neuron number, leading eventually to small sympathetic ganglion rudiments postnatally. The elimination of AP-2ß also leads to the complete absence of noradrenergic neurons of the Locus coeruleus (LC). Whereas AP-2α/ß transcription factors are in vivo not required for the onset or maintenance of noradrenergic differentiation, their essential survival functions are demonstrated for sympathetic progenitors and noradrenergic neurons.

Experimental induction of recurrent airway obstruction with inhaled fungal spores, lipopolysaccharide, and silica microspheres in horses.

OBJECTIVE: To evaluate experimental induction of recurrent airway obstruction (RAO) with inhaled fungal spores, lipopolysaccharide, and silica microspheres in horses. ANIMALS: 7 horses with and 3 horses without a history of RAO. PROCEDURES: RAO-susceptible horses ranged in age from 17 to approximately 30 years, and control horses ranged in age from 7 to approximately 15 years. Pure mold cultures were derived from repeated culture of hay and identified via gene amplification and sequencing. Pulmonary function testing and bronchoalveolar lavage were performed before and after nebulization with a suspension of spores derived from 3 fungi, lipopolysaccharide, and 1-microm silica microspheres in all horses. This was followed by a 4-month washout period and a further pulmonary function test followed by saline (0.9% NaCl) solution challenge and bronchoalveolar lavage. RESULTS: Lichtheimia corymbifera, Aspergillus fumigatus, and Eurotium amstelodami were consistently identified in cultures of moldy hay. Nebulization with fungal spores, lipopolysaccharide, and microspheres induced significant increases in pleural pressure in RAO-susceptible but not control horses. Airway neutrophilia developed in both groups of horses with exposure to challenge material but more severely in RAO-susceptible horses. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that inhalation of fungal spores in combination with lipopolysaccharide and silica microspheres can induce disease exacerbation in susceptible horses and may thus be a useful model for future standardized studies of RAO in horses.

The cortistatin gene PSS2 rather than the somatostatin gene PSS1 is strongly expressed in developing avian autonomic neurons.

Somatostatin and cortistatin are neuromodulators with divergent expression patterns and biological roles. Whereas expression and function of genes encoding somatostatin (PSS1) and the related peptide cortistatin (PSS2) have been studied in detail for the central nervous system (CNS) and immune system, relatively little is known about their expression patterns in the peripheral nervous system (PNS). We compare the expression patterns of PSS1 and PSS2 in chicken embryos. At E14, PSS1 is higher in the CNS versus PNS, whereas PSS2 is higher in the PNS. During early development, PSS1 is transiently expressed in lumbar sympathetic ganglia and is detectable at low levels throughout the development of dorsal root and ciliary ganglia. In contrast, PSS2 expression increases as development progresses in sympathetic and dorsal root ganglia, whereas levels in ciliary ganglia by E8 are more than 100-fold higher than in sympathetic ganglia. Activin, which induces somatostatin-like immunoreactivity in ciliary ganglion neurons in vivo and in vitro, controls PSS2 expression by stabilizing PSS2 but not PSS1 mRNA. We conclude that much of the somatostatin-like immunoreactivity in the developing avian peripheral nervous system is actually cortistatin, the PSS2 product, as opposed to true somatostatin, which is the PSS1 product. The identification of PSS2 as the predominantly expressed somatostatin gene family member in avian autonomic neurons provides a molecular basis for further functional and pharmacological studies.

Parkinson phenotype in aged PINK1-deficient mice is accompanied by progressive mitochondrial dysfunction in absence of neurodegeneration.

BACKGROUND: Parkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. METHODOLOGY/PRINCIPAL FINDINGS: Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of alpha-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. CONCLUSION: Thus, aging Pink1(-/-) mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.