Construction and validation of a Parkinson's disease mutation genotyping array for the Parkin gene.

Parkin mutations account for the majority of familial and sporadic early onset Parkinson's disease (EOPD) cases with a known genetic association. More than 100 mutations have been described in the Parkin gene that includes homozygous, compound heterozygous, and single heterozygous mutations. We have designed a Parkin mutation genotyping array (gene chip) that includes published Parkin sequence variants and allows their simultaneous detection. The chip was validated by screening 85 PD cases and 47 controls previously tested for Parkin mutations. Similar genotyping microarrays have been developed for other genetically heterogeneous diseases including age-related macular degeneration. Here, we show the utility of a genotyping array for Parkinson's disease by analysis of 60 subjects from the Genetic Epidemiology of Parkinson Disease (GEPD) study that includes 15 early-onset PD case probands and 45 relatives.     

18F]-Dopa positron emission tomography imaging in early-stage, non-parkin juvenile parkinsonism.

There are few reports of positron emission tomography (PET) in juvenile parkinsonism (JP). We report on the results of (18)F-6-fluoro-L-dopa (FD) PET in a 14-year-old patient with JP of 5 years duration associated with atypical features. This is the youngest subject to be investigated to date. There was a severe asymmetric reduction in striatal FD uptake, with a rostrocaudal gradient in the putamen similar to that seen in adult-onset idiopathic parkinsonism. Extensive DNA analysis in this patient did not show mutations in the parkin gene.     

Lumbar plexus block in children: a comparison of two procedures in 50 patients

Two techniques for blocking the lumbar plexus were prospectively evaluated in 50 children undergoing surgery in the hip region and randomly allocated to one of two equal groups. A variant of the "psoas compartment block" and the classic technique were used in groups 1 (n = 25) and 2 (n = 25), respectively. All procedures were carried out under light general anesthesia with the patients in the lateral position using insulated needles and electrical stimulation. Both procedures were effective, allowing completion of surgery without additional treatment in almost all patients. However, the distribution of analgesia differed: 23 (ipsilateral) lumbar and sacral plexus blocks and 2 (ipsilateral) lumbar blocks alone were produced in group 2, compared to 22 areas of anesthesia comparable to those that might be associated with a lumbar epidural block and two ipsilateral lumbar plexus blocks in group 1. The two techniques are not, therefore, mere variants of the same basic approach to the lumbar plexus. The procedure described by Winnie et al. (Anesthesiol Rev 1974;1:11-6) was more suitable for providing unilateral blockade than the "psoas compartment block."     

Detection of Parkin (PARK2) and DJ1 (PARK7) mutations in early-onset Parkinson disease: Parkin mutation frequency depends on ethnic origin of patients

Mutations in the Parkin (PARK2) and the DJ1 (PARK7) gene cause early-onset Parkinson disease (EOPD). We tested 75 Serbian EOPD patients for mutations in both genes by conventional mutational screening (SSCP/dHPLC/sequencing) to detect small sequence alterations and by gene dosage studies (quantitative PCR) to reveal deletions or multiplications of one or more exons. A compound heterozygous Parkin mutation (exon deletion and point mutation; [c.836_972del]+[c.1411C>T]; +1 is first nucleotide of GenBank AB009973.1) was identified in a patient who showed a relatively benign course after a disease onset at 41 years. Another case had a heterozygous exon deletion in DJ1 ([c.253_322del]+[?]) and presented with an age at onset of 45 years and a rapid disease course. In conclusion, Parkin mutations are surprisingly rare in our Serbian EOPD sample, suggesting that the mutation rate depends on the ethnic origin of the patients. Although DJ1 mutations appear to be rare, we confirm their role in EOPD and demonstrate the importance of gene dosage studies.     

Matched adaptations of electrophysiological, physiological, and histological properties of skeletal muscles in response to chronic hypoxia

This study tried to differentiate the consequences of chronic hypoxia on the electrophysiological and physiological properties and the histological characteristics of slow and fast muscles in rats. Animals inhaled a 10% O₂ concentration for a 1-month period. Then, slow [soleus (SOL)] and fast [extensor digitorum longus (EDL)] muscles were analyzed in vitro by physiological and electrophysiological measurements and histological analyses. The results were compared to those obtained in corresponding muscles of an age-matched normoxic group. After exposure to hypoxia: (1) in SOL, there was a tendency to elevated F_max, a significant increase in twitch force and tetanic frequency and a shortening of M-wave duration, and a reduced percentage of type I fibres, whereas the proportion of type IIa fibres doubled; (2) in EDL, F_max and tetanic frequency were lowered, the muscle became less resistant to fatigue, and the proportion of type IId/x fibres was halved. Then, after 1 month of hypoxia, in the SOL muscle, both the contractile and histological properties resemble those of a fast muscle. By contrast, the EDL became slower, despite its histology was modestly affected. Reduced muscle use in hypoxia could explain the tendency for deteriorating adaptations in EDL, and the faster properties of SOL could result from hypoxia-induced inhibition of the growth-related fast-to-slow shift in muscle fibre types.     

Influence of epidural anesthesia on postoperative nitrogen sparing in major digestive surgery

In order to determine the role of epidural anaesthesia on protein sparing, eighteen patients undergoing major visceral surgery were randomly divided into three different groups according to the mode of anaesthesia used (general anaesthesia, epidural anaesthesia, continuous epidural anaesthesia for 24 h). These patients were intravenously fed for five days postoperatively; their nitrogen balance was studied. Compared with the "general anaesthesia" group, only the daily averages of nitrogen balance of the "continuous epidural" group were significantly better. IN order to correlate stress with nitrogen saving, the early evolution of several parameters (cortisol, glucose, prolactin) used as "stress markers" was studied : there was no evidence of any significant difference between the three groups.     

Integrins protect sensory neurons in models of paclitaxel-induced peripheral sensory neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect from cancer treatment with no known method for prevention or cure in clinics. CIPN often affects unmyelinated nociceptive sensory terminals. Despite the high prevalence, molecular and cellular mechanisms that lead to CIPN are still poorly understood. Here, we used a genetically tractable Drosophila model and primary sensory neurons isolated from adult mouse to examine the mechanisms underlying CIPN and identify protective pathways. We found that chronic treatment of Drosophila larvae with paclitaxel caused degeneration and altered the branching pattern of nociceptive neurons, and reduced thermal nociceptive responses. We further found that nociceptive neuron-specific overexpression of integrins, which are known to support neuronal maintenance in several systems, conferred protection from paclitaxel-induced cellular and behavioral phenotypes. Live imaging and superresolution approaches provide evidence that paclitaxel treatment causes cellular changes that are consistent with alterations in endosome-mediated trafficking of integrins. Paclitaxel-induced changes in recycling endosomes precede morphological degeneration of nociceptive neuron arbors, which could be prevented by integrin overexpression. We used primary dorsal root ganglia (DRG) neuron cultures to test conservation of integrin-mediated protection. We show that transduction of a human integrin β-subunit 1 also prevented degeneration following paclitaxel treatment. Furthermore, endogenous levels of surface integrins were decreased in paclitaxel-treated mouse DRG neurons, suggesting that paclitaxel disrupts recycling in vertebrate sensory neurons. Altogether, our study supports conserved mechanisms of paclitaxel-induced perturbation of integrin trafficking and a therapeutic potential of restoring neuronal interactions with the extracellular environment to antagonize paclitaxel-induced toxicity in sensory neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a prevalent adverse effect of treatment in cancer patients and survivors (1). CIPN significantly impacts quality of life as damage to sensory nerves may be permanent, and is often a dose-limiting factor during cancer treatment (2–4). Patients with CIPN report pain-related symptoms, including allodynia, hyper- or hypoalgesia, or pain that can be more severe than the pain associated with the original cancer (4). Despite increasing data on agents that protect sensory nerves, our limited understanding of the mechanisms of CIPN impedes effective treatment (5). Studies from model systems may be helpful in identifying molecules that protect sensory neuron morphology and function from the effects of chemotherapeutics.In the present study, we explored the mechanisms of CIPN induced by paclitaxel using two established models: Drosophila larval nociceptive neurons (6, 7) and primary dorsal root ganglia (DRG) neurons isolated from adult mouse (8). Similar to other peripheral neuropathies, CIPN models using paclitaxel, bortezomib, oxaliplatin, and vincristine report changes in unmyelinated intraepidermal nerve fibers (IENFs) that detect painful or noxious stimuli (9–14). These small fibers are embedded in the epidermis, and continuously turn over coincident with the turnover of skin (9, 15). Drosophila class IV nociceptive neurons are a favored model for genetic studies of nociceptive neuron development and signaling mechanisms (16). Prior studies showed that class IV neuron morphology is sensitive to paclitaxel and demonstrated morphological changes of nociceptive neurons at the onset and the end stage of paclitaxel-induced pathology (6, 7). Specifically, chronic treatment of high doses (30 μM) induce fragmentation and simplification of branching of sensory terminals (6). Additionally, acute treatments of moderate doses (10 to 20 μM) induced hyperbranching of sensory arbors without changing the branch patterns or degeneration (7). Nociceptive neurons in Drosophila larvae detect multiple qualities of noxious stimuli (17, 18), and project naked nerve terminals that are partially embedded in the epidermis (19, 20). Larvae have a stereotyped behavioral response toward noxious stimuli that can serve as a readout of nociceptive neuron function (17, 21). Nociceptive neurons in Drosophila larvae may therefore serve as a good in vivo model to study morphological and functional changes to sensory neurons induced by chemotherapeutics.Paclitaxel binds to tubulin and prevents microtubule disassembly. It is a commonly used chemotherapeutic drug for treatment of solid cancers, such as breast, ovarian, and lung cancers, by virtue of its ability to inhibit cell division. Paclitaxel causes chronic sensory neuropathy in patients and animal models (22–24). Several CIPN animal and in vitro models have also revealed acute effects of paclitaxel (7, 8, 24–26). While the mechanisms of acute and chronic neurodegeneration are likely to be distinct (27), how long-term treatment of paclitaxel can affect sensory neuron morphology and function, and how neuronal arbors can be protected against long-term toxicity is not understood.Several studies have shown that nociceptive sensory terminals share a close relationship with specific extracellular structures, most notably epidermal cells and the extracellular matrix (ECM). Thus, in addition to direct effects on neurons, paclitaxel could conceivably destabilize terminals by disrupting relationships with the extracellular environment. Indeed, a study in zebrafish indicates that epidermal cells are directly affected by paclitaxel and that epidermal changes precede neuronal degradation, indicating that degradation of neuronal substrates contributes to degeneration of adjacent arbors (25). For the most part, however, extracellular contributions to neuropathy induced by chemotherapeutics are still poorly characterized. It is therefore important to determine how sensory terminals are maintained in the context of a dynamic extracellular environment that itself may be sensitive to chemotherapeutics. Integrins are a key mediator of the interaction between cells and the ECM, and impact dendrite stabilization and maintenance in both vertebrate and invertebrate systems (20, 28, 29). Prior studies in other systems indicate that integrin levels at the surface are maintained by continuous recycling via tight regulation of the endosomal pathway rather than degradation and de novo synthesis (30). Decreased recycling or increased degradation could lead to depletion of the surface receptors (31, 32) responsible for arbor maintenance and, in turn, degeneration of nociceptive terminals. We therefore explored whether integrin–ECM interactions may impact sensory neuron maintenance upon paclitaxel-induced toxicity and how the endosomal–lysosomal pathway may be linked to the maintenance of sensory neurons.Here, we have used Drosophila and isolated mouse DRG neurons to investigate the pathological effect of paclitaxel in sensory neurons. Morphological changes in Drosophila neurons occurred at paclitaxel doses that also caused changes in thermal nociceptive behaviors. Cell-specific overexpression of integrins protected nociceptive neurons from morphological alterations and prevented the thermal nociceptive behavior deficits caused by paclitaxel in Drosophila. Transduction of integrins also protected adult mouse DRG sensory neurons from paclitaxel-induced toxicity in vitro, indicating that integrin-mediated protection is conserved in a vertebrate model of CIPN. We provide evidence that paclitaxel alters intracellular trafficking in both Drosophila and mouse models of CIPN. Furthermore, our biochemical analysis indicates a reduction of integrin surface availability, suggesting paclitaxel-induced recycling defects in mouse DRG neurons in vitro. Our study suggests that altered interactions between sensory neurons and their extracellular environment are an important contributor to paclitaxel-induced neuronal pathology, and that preventing these changes may offer a therapeutic approach.