Live-cell imaging evidence for the ciliary transport of rod photoreceptor opsin by heterotrimeric kinesin-2.

Primary cilia detect extracellular signals through membrane receptors and channels. The outer segment of a vertebrate photoreceptor cell represents the most elaborate of all primary cilia, containing extraordinarily large amounts of the visual receptor protein, opsin. Because of its high abundance, opsin represents a potential model system for the study of ciliary membrane receptors, including their transport. Here, we have analyzed the movement of ciliary opsin to test whether the highly conserved intraflagellar transport (IFT), as driven by heterotrimeric kinesin-2, is required. Results show that opsin can enter and move along the primary cilium of a nonphotoreceptor cell (an hTERT-RPE1 epithelial cell), suggesting that it can co-opt the basic anterograde motor system of cilia. Fluorescence recovery after photobleaching analysis of cilia of hTERT-RPE1 cells showed that the movement of ciliary opsin was comparable to that of the IFT protein, IFT88. Moreover, the movement of opsin in these cilia, as well as in cilia of mouse rod photoreceptor cells, was reduced significantly when KIF3A, the obligate motor subunit of heterotrimeric kinesin-2, was deficient. These studies therefore provide evidence from live-cell analysis that the conserved heterotrimeric kinesin-2 is required for the normal transport of opsin along the ciliary plasma membrane.

Huntingtin phosphorylation acts as a molecular switch for anterograde/retrograde transport in neurons.

The transport of vesicles in neurons is a highly regulated process, with vesicles moving either anterogradely or retrogradely depending on the nature of the molecular motors, kinesins and dynein, respectively, which propel vesicles along microtubules (MTs). However, the mechanisms that determine the directionality of transport remain unclear. Huntingtin, the protein mutated in Huntington's disease, is a positive regulatory factor for vesicular transport. Huntingtin is phosphorylated at serine 421 by the kinase Akt but the role of this modification is unknown. Here, we demonstrate that phosphorylation of wild-type huntingtin at S421 is crucial to control the direction of vesicles in neurons. When phosphorylated, huntingtin recruits kinesin-1 to the dynactin complex on vesicles and MTs. Using brain-derived neurotrophic factor as a marker of vesicular transport, we demonstrate that huntingtin phosphorylation promotes anterograde transport. Conversely, when huntingtin is not phosphorylated, kinesin-1 detaches and vesicles are more likely to undergo retrograde transport. This also applies to other vesicles suggesting an essential role for huntingtin in the control of vesicular directionality in neurons.

Phosphorylation of mutant huntingtin at S421 restores anterograde and retrograde transport in neurons.

Huntingtin (htt), the protein mutated in Huntington's disease, is a positive regulatory factor for vesicular transport whose function is lost in disease. Here, we demonstrate that phosphorylation of htt at serine 421 (S421) restores its function in axonal transport. Using a strategy involving RNA (ribonucleic acid) interference and re-expression of various constructs, we show that polyQ (polyglutamine)-htt is unable to promote transport of brain-derived neurotrophic factor (BDNF)-containing vesicles, but polyQ-htt constitutively phosphorylated at S421 is as effective as the wild-type (wt) as concerns transport of these vesicles. The S421 phosphorylated polyQ-htt displays the wt function of inducing BDNF release. Phosphorylation restores the interaction between htt and the p150(Glued) subunit of dynactin and their association with microtubules in vitro and in cells. We also show that the IGF-1 (insulin growth factor type I)/Akt pathway by promoting htt phosphorylation compensates for the transport defect. This is the first description of a mechanism that restores the htt function altered in disease.

Inhibition of calcineurin by FK506 protects against polyglutamine-huntingtin toxicity through an increase of huntingtin phosphorylation at S421.

Huntington's disease (HD) is caused by an abnormal expanded polyglutamine (polyQ) repeat in the huntingtin protein. Insulin-like growth factor-1 acting through the prosurvival kinase Akt mediates the phosphorylation of huntingtin at S421 and inhibits the toxicity of polyQ-expanded huntingtin in cell culture, suggesting that compounds enhancing phosphorylation are of therapeutic interest. However, it is not clear whether phosphorylation of S421 is crucial in vivo. Using a rat model of HD based on lentiviral-mediated expression of a polyQ-huntingtin fragment in the striatum, we demonstrate here that phosphorylation of S421 is neuroprotective in vivo. We next demonstrate that calcineurin (CaN), a calcium/calmodulin-regulated Ser/Thr protein phosphatase, dephosphorylates S421 in vitro and in cells. Inhibition of calcineurin activity, either by overexpression of the dominant-interfering form of CaN or by treatment with the specific inhibitor FK506, favors the phosphorylation of S421, restores the alteration in huntingtin S421 phosphorylation in HD neuronal cells, and prevents polyQ-mediated cell death of striatal neurons. Finally, we show that administration of FK506 to mice increases huntingtin S421 phosphorylation in brain. Collectively, these data highlight the importance of CaN in the modulation of S421 phosphorylation and suggest the potential use of CaN inhibition as a therapeutic approach to treat HD.

Atlastin1 mutations are frequent in young-onset autosomal dominant spastic paraplegia.

BACKGROUND: Hereditary spastic paraplegias are disorders that are very heterogeneous, both clinically and genetically. The atlastin1 gene has recently been implicated in SPG3A, a form of autosomal dominant pure spastic paraplegia. Atlastin1 mutations have been identified in 8 families so far. OBJECTIVES: To determine the relative frequency, phenotype, and mutation spectrum of SPG3A in patients with pure autosomal dominant spastic paraplegia and onset before age 20 years. PATIENTS AND METHODS: We sequenced the atlastin1 gene in a large series of patients (31 families) in which mutations in the spastin gene, corresponding to the frequent SPG4 locus, had previously been excluded. The phenotype was compared with 126 SPG4 patients. RESULTS: We identified 12 families (39%) including 34 patients with 9 different missense atlastin1 mutations, 7 of which are newly described. The main clinical characteristic of these SPG3A patients was pure spasticity with very young onset of symptoms (mean age, 4.6 +/- 3.9 years) and slow progression. However, additional signs such as decreased vibration sense and wasting in lower limbs, sphincter disturbances, and scoliosis were found in a minority of patients. In addition, several gene carriers were clinically affected but still asymptomatic (n = 5) or had no clinical signs (n = 2), indicating incomplete penetrance. Compared with patients from other families meeting the same diagnostic criteria (43 patients) and families with SPG4 (126 patients), the major form of autosomal dominant spastic paraplegia, SPG3A patients had earlier symptom onset, less frequently increased reflexes in the upper limbs, decreased vibration sense in the lower limbs, and fewer sphincter disturbances, but more frequently observed wasting in the lower limbs and scoliosis. These particularities, as well as frequent abnormal motor evoked potentials, could help identify patients to be screened for atlastin1 gene mutations. CONCLUSIONS: This study enables us to estimate the frequency of the SPG3A mutations in France at 39% in families with young-onset autosomal dominant spastic paraplegia after exclusion of SPG4 cases. So far, most mutations have been private, although they were all found in exons 7, 8, 12, and 13. These exons should be given priority when performing molecular diagnoses for SPG3A.

Phosphorylation of arfaptin 2 at Ser260 by Akt Inhibits PolyQ-huntingtin-induced toxicity by rescuing proteasome impairment.

Huntington disease (HD) is caused by an abnormal expanded polyglutamine repeat in the huntingtin protein. Insulin-like growth factor-1 is of particular interest in HD because it strongly inhibits polyQ-huntingtin-induced neurotoxicity. This neuroprotective effect involves the phosphorylation of huntingtin at Ser(421) by the prosurvival kinase Akt (Humbert, S., Bryson, E. A., Cordelieres, F. P., Connors, N. C., Datta, S. R., Finkbeiner, S., Greenberg, M. E., and Saudou, F. (2002) Dev. Cell 2, 831-837). Here, we report that Akt inhibits polyQ-huntingtin-induced toxicity in the absence of phosphorylation of huntingtin at Ser(421), suggesting that Akt also acts on other downstream effector(s) to prevent neuronal death in HD. We show that this survival effect involves the ADP-ribosylation factor-interacting protein arfaptin 2, the levels of which are increased in HD patients. Akt phosphorylated arfaptin 2 at Ser(260). Lack of phosphorylation of arfaptin 2 at this site substantially modified its subcellular distribution and increased neuronal death and intranuclear inclusions caused by polyQ-huntingtin. In contrast, arfaptin 2 had a neuroprotective effect on striatal neurons when phosphorylated by Akt. This effect is mediated through the proteasome, as phosphorylated arfaptin 2 inhibited the blockade of the proteasome induced by polyQ-huntingtin. This study points out a new mechanism by which Akt promotes neuroprotection in HD, emphasizing the potential therapeutic interest of this pathway in the disease.

Akt is altered in an animal model of Huntington's disease and in patients.

The insulin-like growth factor I (IGF-1)/Akt pathway plays a crucial role in Huntington's disease by phosphorylating the causative protein, polyQ-huntingtin, and abolishing its toxic properties [Humbert et al. (2002)Dev. Cell, 2, 831-837; Rangone et al. (2004)Eur. J. Neurosci., 19, 273-279]. Therefore, dysregulation of this pathway may be essential for disease progression. In the present report, we thus aimed to analyse the status of Akt in brain or in peripheral tissues in Huntington's disease. Using a genetic model of Huntington's disease in rat that reproduces neuronal dysfunction and death, we show a progressive alteration of Akt during neuronal dysfunction and prior neurodegeneration. By analysing a limited number of lymphoblasts and lymphocytes, we detected modifications of Akt in Huntington's disease patients confirming a dysregulation of Akt in the disease process. Finally, we demonstrate that during late stages of the disease, Akt is cleaved into an inactive form by caspase-3. These observations demonstrate a progressive but marked alteration of this pro-survival pathway in Huntington's disease, and further implicate it as a key transduction pathway regulating the toxicity of huntingtin.