Alexa-fluor-488 and 555 (Invitrogen, Life Technologies, Paisley, UK) were then added at 1:1000 for 1h at RT in the dark. level was reduced in the cingulate cortex in the DLB cohort, and correlated inversely with -synuclein and -synuclein-P129. Kallikrein-6 was also reduced in DLB in the thalamus but not in relation to -synuclein or -synuclein-P129 weight and was unaltered in the frontal and parahippocampal cortex. In SH-SY5Y cells overexpressing wild-type -synuclein there was partial Droxidopa co-localisation of kallikrein-6 and calpain-1 with -synuclein, and siRNA-mediated knock-down of kallikrein-6 and calpain-1 increased the amount of -synuclein in cell lysates. Our results indicate that reductions in kallikrein-6 and calpain-1 may contribute to the accumulation of -synuclein in DLB. == Electronic supplementary material == The online version of this article (doi:10.1186/s40478-014-0164-0) contains supplementary material, which is available to authorized users. Keywords:Dementia with lewy body, Calpain-1, Kallikrein-6, -synuclein == Introduction == Abnormal aggregation of -synuclein (-syn) within neuronal perikarya (Lewy body) and neurites (Lewy neurites) are defining neuropathological hallmarks of Parkinsons disease (PD) and dementia with Lewy body (DLB). There is accumulation of both insoluble [1] and soluble oligomeric -syn [2] in sporadic PD and DLB without an increase in -syn mRNA [3,4], suggesting a major role for impaired clearance in the pathogenesis of PD and DLB. Several genetic mutations that cause PD (e.g. inPINK1,PARK2,LRRK2) involve proteins associated with the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP), which have established functions in -syn clearance (reviewed in Mouse monoclonal to BMX [5-8]) and are dysregulated in sporadic PD and DLB. Cathepsin D (CTSD) the major lysosomal protease responsible for -syn cleavage [9-11] is usually upregulated in PD and DLB [12]. A small number of non-lysosomal proteolytic enzymes have been recognized that cleave -synin vitro, including MPP-3, 9 [13], calpain-1 (CAPN1) [14,15] and kallikrein-6 (KLK6) [16,17]. These are expressed within the cytosol, plasma membrane or are secreted extracellularly. KLK6 (also known as neurosin) is usually a serine protease predominantly expressed within the brain, especially in neurons and oligodendrocytes [18-20].In vitro, KLK6 degrades monomeric -syn within the NAC region, essential for filament assembly, and protects against -syn aggregation and neurotoxicity [16,17]. KLK6 co-localises with -syn in Lewy body in post-mortem brain tissue in PD [16,21]. Direct conversation between KLK6 and -syn in mouse brain was exhibited by co-immunoprecipitation [16]. The authors also showed that KLK6 cleaved -syn when the enzyme was released from mitochondria under stress conditions. KLK6 is also secreted in an active form extracellularly and degrades extracellular -syn [22]; this may be relevant in Droxidopa preventing the transcellular spread of -syn that is thought to be important in the propagation of disease [23,24]. Phosphorylated forms of -syn which are more abundant in Lewy body diseases, and mutant forms of -syn that accumulate in familial forms of PD, are resistant to proteolytic cleavage by KLK6 [17]. A recent study showed that elevating the level of KLK6in vivoprotected against -syn aggregation and toxicity Droxidopa in a rat model of PD [25] and that KLK6 level was significantly lower in mid-temporal cortex obtained post mortem from 8 patients with DLB than from 6 controls. Calpains are calcium-dependant cysteine proteases involved in multiple and diverse cellular processes. Calpain-1 (CAPN1, -calpain) and calpain-2 (CAPN2, m-calpain) are regulated by micromolar and millimolar levels of intracellular calcium, respectively, and unlike other members of the calpain family are highly expressed within the CNS. CAPN1 and CAPN2 are dysregulated in AD and other tauopathies [26,27]. CAPN1 cleaves -synin vitro[14,15]. It is neuronally expressed and likely to interact with -syn within the pre-synaptic terminal [28]. However, to our knowledge its activity in Lewy body.