BackgroundParkinson'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 FindingsNow 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 α-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.ConclusionThus, aging Pink1−/− mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.

SUMMARYNeuroblastoma (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. Activation of Alk signaling, in particular overexpression of ALK wt and NB ALK mutants resulted in the upregulation of NMyc and trkB, which may contribute to continued proliferation and NB predisposition. MATERIALS AND METHODS Expression plasmidsPcDNA3.1 expression plasmids for human ALK wt , ALK F1174L and ALK R1275Q, and pCAGGS expression plasmids for Hand2, Phox2b wt and Phox2b K155X have been described previously (Janoueix-Lerosey et al., 2008;Reiff et al., 2010). shRNA constructsThe shRNA against Gallus gallus Alk and Mk were designed using BLOCK-iT RNAi Designer (Invitrogen, Karlsruhe, Germany) and target the following sequences: shAlk, 5Ј-AAU GGU UUC UCU CUA UGU CCA ACU C-3Ј; shMk, 5Ј-GAG CUG ACU GCA AGU ACA AGU UUG A-3Ј. Scrambled shRNAs (sc-shRNA, Invitrogen, Karlsruhe, Germany) served as controls. In situ hybridizationNon-radioactive in situ hybridization on cryosections and preparation of digoxigenin labeled probes for chick Phox2b was carried out as described previously (Ernsberger et al., 1997;Stanke et al., 1999). qPCR analysisEqual amounts of RNA were used to synthesize cDNA with Oligo(dT)-primers and Superscript-III-reverse-transcriptase according to manufacturer's instructions (Invitrogen, Karlsruhe, Germany). The PCR was carried out using Abgene's Absolute Blue SYBR-Green qPCR Mix (Abgene, Epsom, UK) in a Stratagene Mx3000p Light Cycler (Stratagene, Waldbronn, Germany). All primers (MWG Biotech AG, Ebersberg, Germany) were designed to anneal optimally at 58°C with PerlPrimer (Marshall, 2004) (95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, repeated for 50 cycles and a subsequent dissociation curve). The primer pairs (see Table S1 in the supplementary material) were ...

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-2β 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-2β results in the virtually complete absence of sympathetic and sensory ganglia due to apoptotic cell death of migrating NCC. In the AP-2β 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-2β 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-2β also leads to the complete absence of noradrenergic neurons of the Locus coeruleus (LC). Whereas AP-2α/β 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.

Abstract.A study of acute respiratory disease in horses in Ontario was undertaken to determine the identity of current causative infectious agents. A nasopharyngeal swab was designed and utilized to maximize isolation of viruses, mycoplasma, and pathogenic bacteria. Serum samples were collected for parallel determination of antibody titers to equine influenza virus type A subtype 1 (H7N7) and subtype 2 (H3N8), equine rhinovirus types 1 and 2, equine herpesvirus type 1, Mycoplasma equirhinius, and Mycoplasma felis. Equine rhinovirus type 2 was recovered from 28/92 horses tested, and equine influenza virus type A, subtype 2, was recovered from 5. The mycoplasma and bacteria isolated were consistent with those commonly associated with nonspecific respiratory diseases in horses, except that Streptococcus pneumoniae capsular type 3 was isolated from 10 horses.Contagious respiratory infections in horses are major causes of both acute and chronic respiratory diseases resulting in impaired pulmonary function and reduced performance. 12,13,39 The chronic sequelae are important to the horse industry and include bronchopneumonia, chronic obstructive pulmonary disease, and exerciseinduced hemorrhage. 26,39 Earlier studies conducted in Ontario on thoroughbred 8 and standardbred horses used isolation and serodiagnosis to demonstrate that equine influenza virus type A subtypes 1 (AE1; H7N7) and 2 (AE2; H3N8), equine rhinovirus types 1 (ERhV-1) and 2 (ERhV-2), and equine rhinopneumonitis virus, now termed equine herpesvirus type 4 (EHV-4), were the major causes of respiratory disease. Other infectious agents that can induce respiratory disease in the horse are equine abortion virus 1 (distinguished as equine herpesvirus type 1 [EHV-1] 33 ), equine cytomegalovirus (equine herpesvirus type 2 10 ), equine arteritis virus (EAV), 34 equine adenovirus, 14,26 equine rhinovirus-3, 26 mycoplasmas, 16,40 and bacteria. 18,22 Vaccines are currently available in Ontario only for AE1, AE2, EHV-1, EHV-4, and EAV. These viral agents are believed to be the most common causes of clinical respiratory disease in North America. 26 Received for publication March 18, 1996. cious vaccination for AE1, 39 AE2, 39 EHV-1 , 24 and EHV-4 24 using modern vaccines. This continuing disease problem may be due to partial vaccine-induced immunity, genetic diversity of 26 or the antigenic drift of influenza viruses. 13,26,39 This study was conducted to identify the infectious agents more recently implicated in respiratory disease in horses in Ontario . 37 We used patient data, clinical examination, isolation, and paired serology for specific infectious agents. Nasal swabs from 92 horses with acute clinical respiratory disease from 29 different premises were cultured for viruses, mycoplasma, and pathogenic bacteria. Paired sera were collected and tested for antibodies to AE1, AE2, ERhV-1, ERhV-2, and EHV-1. For an additional 89 horses from 13 stables where respiratory disease occurred annually, these same serologic tests and antibody tests for EAV were perf...

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.