α-Synuclein is the major building block of Lewy bodies in PD and glial cytoplasmic inclusions in MSA 
. Abnormal deposition of α-synuclein has been linked to the pathogenesis of neurodegenerative diseases, and missense mutations of the human gene, such as A53T, increase the probability of aggregate formation (for review see, 
. It has been hypothesized that the accumulation of prefibrillary oligomers, which provide the intermediates for fibrillary aggregates or inclusion bodies, are the toxic species and cause neurodegeneration 
. Cells are capable of clearing oligomeric α-synuclein intermediates, and lysosomal pathways have been suggested to be mostly responsible for clearance of oligomers but not for fibrillar inclusion bodies 
. Thus, stimulation of lysosomal pathways may be an effective therapeutic approach to prevent α-synuclein oligomer toxicity and accumulation.
Autophagy is a lysosomal pathway for degrading organelles and long-lived proteins. The three main types of autophagy are chaperone mediated autophagy (CMA), microautophagy and macroautophagy 
. CMA involves the translocation of cytosolic proteins with a specific pentapeptide motif across the lysosomal membrane and this process requires the action of a number of cytosolic and lysosomal chaperones. In microautophagy small cytoplasmic contents are introduced into the lysosomes in a process which has been mainly characterized in yeast. Macroautophagy, often referred to only as autophagy, is a pathway by which organelles and parts of cytoplasm containing proteins are sequestered into a vesicle, termed autophagosome. After fusion of the autophagosome with the lysosome the contents are degraded 
. An equilibrium exists between autophagosome formation and lysosomal clearance, which has been termed autophagic flux 
. Autophagy can function as a cytoprotective response and is particularly crucial in the aging brain and during neurodegeneration 
. α-Synuclein can be degraded either by the proteasome or by autophagy. Both macroautophagy and CMA have been reported to contribute to α-synuclein degradation 
, however the clearance of mutant α-synuclein by CMA seems to be impaired 
In the present cell culture system, the stable expression of α-synuclein or the A53T mutated form leads to the accumulation of small punctate aggregates throughout the cytoplasm, which are more abundant in cells expressing the A53T mutation, but do not exert cytotoxic effects per se. These aggregates do not stain with thioflavine S 
and thus represent non-fibrillar inclusions which might precede and are a requirement for the formation of fibrillary deposits, as has been described in COS-7 cells transiently transfected with α-synuclein 
. Our study demonstrates that the geldanamycin analogue 17-AAG attenuates the formation of these small aggregates and that lysosomal and not proteasomal pathways are involved. By blocking the lysosomal compartment with NH4
Cl or chloroquine, the aggregate clearing effects of 17-AAG were diminished and α-synuclein deposits were even enlarged, while on the other hand inhibition of the proteasomal activity by MG-132 did not have this effect. Analysis of LC3-II immunoreactivity, which is an indicator of autophagosome formation, further revealed that induction of macroautophagy was involved in the aggregate-clearing effects of 17-AAG. This conclusion is supported by the finding that the specific inhibitor of macroautophagy 3-MA prevented 17-AAG induced occurrence of LC3 positive puncta and removal of α-synuclein aggregates.
The capability of 17-AAG to enhance macroautophagy was further demonstrated in cultured oligodendrocytes derived from the brains of newborn rats. Under normal growth conditions and in the healthy human brain oligodendrocytes do not contain α-synuclein aggregates. However, under pathological conditions and in MSA filamentous α-synuclein inclusions are present in the oligodendroglial cytoplasm and the disease has been suggested to represent an oligodendroglia synucleinopathy 
. In this respect, the finding that 17-AAG has the capacity to induce the autophagic pathway in oligodendrocytes might be of special interest as a therapeutic intervention.
The HSP90 inhibitor geldanamycin and its derivative 17-AAG modulate HSP90 function and facilitate the degradation of HSP90 client proteins 
. Geldanamycin has been demonstrated to activate a heat shock response and to suppress huntingtin protein aggregation in a cell culture model of Huntington's disease 
. The stimulation of heat shock gene transcription was also attributed to its ability to protect the brain from focal ischemia 
, and geldanamycin was shown to restore a defective heat shock response in vivo 
. Suppression of α-synuclein aggregation and toxicity by geldanamycin was observed in human H4 neuroglioma cells 
. Furthermore, it prevented from α-synuclein toxicity in a transgenic fly model despite the continuous presence of aggregate pathology 
). These reports suggested that geldanamycin exerts its effects by upregulation of HSP70 expression. In another study, HSP70 overexpression in mice has been demonstrated to reduce α-synuclein aggregation and in vitro caused a reduction in the insolubility of α-synuclein 
. Also, HSP70 may reduce α-synuclein fibril formation by binding preferentially to prefibrillar species 
. On the other hand, HSP27 and not HSP70 exerted a potent protective effect against α-synuclein mediated cell death in mammalian neuronal cells 
Our data show that HSPs and specifically HSP70 are indeed induced by 17-AAG, but to a much lesser extent than after a heat shock or by the proteasome inhibitor MG-132, and neither rapamycin nor 3-MA modulate the heat shock response. Rapamycin did not cause the induction of HSPs, and 3-MA prevented the aggregate clearing effects of 17-AAG without interfering with HSP70 induction. This suggests that HSP70 may contribute but is not the major player in this context, and that 17-AAG-induced clearance of α-synuclein aggregates is causally related mainly to its autophagy stimulating activity. The notion that in the fly model a concentration of geldanamycin not leading to the induction of HSP70 was sufficient to protect neurons against α-synuclein toxicity 
, sustains this assumption. Hence geldanamycin and its less toxic derivatives 
may provide a means to remove the pathological oligomeric species of α-synuclein, thereby ameliorating pathogenic aggregate formation and protecting the cells during disease and aging.