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Inclusions containing Fused in Sarcoma (FUS) are found in familial and sporadic cases of the incurable progressive motor neuron disease amyotrophic lateral sclerosis and in a common form of dementia, frontotemporal dementia. Most disease-associated mutations are located in the C-terminal proline–tyrosine nuclear localization sequence (PY-NLS) of FUS and impair its nuclear import. It has been shown in cell culture that the nuclear import of FUS is mediated by transportin, which binds the PY-NLS and the last arginine/glycine/glycine-rich (RGG) domain of FUS. Methylation of this last RGG domain by protein arginine methyltransferases (PRMTs) weakens transportin binding and therefore impairs nuclear translocation of FUS. To investigate the requirements for the nuclear import of FUS in an in vivo model, we generated different transgenic Drosophila lines expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. To rule out effects caused by heterologous hFUS expression, we analysed the corresponding variants for the Drosophila FUS orthologue Cabeza (Caz wt, P398L, Q349X). Expression of these variants in eyes and motor neurons confirmed the PY-NLS-dependent nuclear localization of FUS/Caz and caused neurodegenerative effects. Surprisingly, FUS/Caz toxicity was correlated to the degree of its nuclear localization in this overexpression model. High levels of nuclear FUS/Caz became insoluble and reduced the endogenous Caz levels, confirming FUS autoregulation in Drosophila. RNAi-mediated knockdown of the two transportin orthologues interfered with the nuclear import of FUS/Caz and also enhanced the eye phenotype. Finally, we screened the Drosophila PRMT proteins (DART1-9) and found that knockdown of Dart1 led to a reduction in methylation of hFUS P525L and aggravated its phenotype.These findings show that the molecular mechanisms controlling the nuclear import of FUS/Caz and FUS autoregulation are conserved between humans and Drosophila. In addition to the well-known neurodegenerative effects of FUS loss-of function, our data suggest toxic potential of overexpressed FUS in the nucleus and of insoluble FUS.

Several lines of evidence suggest that accumulation of aggregated alpha-synuclein (α-synuclein) in the central nervous system (CNS) is an early pathogenic event in Parkinson's disease and other Lewy body disorders. In recent years, animal studies have indicated immunotherapy with antibodies directed against α-synuclein as a promising novel treatment strategy. Since large α-synuclein oligomers, or protofibrils, have been demonstrated to possess pronounced cytotoxic properties, such species should be particularly attractive as therapeutic targets. In support of this, (Thy-1)-h[A30P] α-synuclein transgenic mice with motor dysfunction symptoms were found to display increased levels of α-synuclein protofibrils in the CNS. An α-synuclein protofibril-selective monoclonal antibody (mAb47) was evaluated in this α-synuclein transgenic mouse model. As measured by ELISA, 14 month old mice treated for 14 weeks with weekly intraperitoneal injections of mAb47 displayed significantly lower levels of both soluble and membrane-associated protofibrils in the spinal cord. Besides the lower levels of pathogenic α-synuclein demonstrated, a reduction of motor dysfunction in transgenic mice upon peripheral administration of mAb47 was indicated. Thus, immunotherapy with antibodies targeting toxic α-synuclein species holds promise as a future disease-modifying treatment in Parkinson's disease and related disorders.

Mutations in the N-terminus of the gene encoding α-synuclein (α-syn) are linked to autosomal dominantly inherited Parkinson's disease (PD). The vast majority of PD patients develop neuropsychiatric symptoms preceding motor impairments. During this premotor stage, synucleinopathy is first detectable in the olfactory bulb (OB) and brain stem nuclei; however its impact on interconnected brain regions and related symptoms is still less far understood. Using a novel conditional transgenic mouse model, displaying region-specific expression of human mutant α-syn, we evaluated effect and reversibility of olfactory synucleinopathy. Our data showed that induction of mutant A30P α-syn expression increased transgenic deposition into somatodendritic compartment of dopaminergic neurons, without generating fibrillar inclusions. We found reversibly reduced levels of dopamine and metabolites in the OB, suggesting an impact of A30P α-syn on olfactory neurotransmitter content. We further showed that mutant A30P expression led to neurodegenerative changes on an ultrastructural level and a behaviorally hyperactive response correlated with novelty, odor processing and stress associated with an increased dopaminergic tone in midbrain regions. Our present data indicate that mutant (A30P) α-syn is directly implicated in reduction of dopamine signaling in OB interneurons, which mediates further alterations in brain regions without transgenic expression leading functionally to a hyperactive response. These modulations of neurotransmission may underlie in part some of the early neuropsychiatric symptoms in PD preceding dysfunction of the nigrostriatal dopaminergic system.

Cyclin-dependent kinases and mitogen-activated protein kinases have been implicated in the regulation of cellular survival and apoptosis. We tested the effect of two mitogen-activated/cyclin-dependent kinase inhibitors, olomoucine and butyrolactone I, on thein vitrosurvival of chick embryonic neurons. Sensory, sympathetic, and ciliary neurons, when prepared at their respective time point of programmed cell death, could be rescued from apoptosis by both inhibitors in a dose-dependent fashion. In contrast, dividing sympathetic precursors underwent apoptosis when treated with olomoucine, but not butyrolactone I, at the same range of concentration. With similar potency, olomoucine and butyrolactone I inhibited immunocomplex c-Jun kinase activity. Both substances inhibited neurite outgrowth in a dose-dependent manner; developmentally younger neurons were more sensitive to this effect than older ones. These results suggest that certain mitogen-activated/cyclin-dependent kinases associated with cell division in neuronal precursors (i) may become essential components of the apoptotic machinery by the time neurons reach their phase of naturally occurring cell death and (ii) may be necessary for neurite outgrowth during development.