CysC is a secreted protein, targeted extracellularly via the secretory pathway and is taken up by cells (for review see [5
]). Therefore, we have studied the in vitro
effect of exogenously applied human CysC on cells of neuronal origin under neurotoxic stimuli. Here we show that CysC protects N2a neuroblastoma cells and rat primary hippocampal neurons from the toxicity induced by either oligomeric or fibrillar Aβ.
Aβ has been implicated in the pathophysiology of AD. The histopathological hallmarks of AD include the formation of Aβ amyloid plaques and neurofibrillary tangles, and the loss of synapses. While the temporal order in which these events occur and their relationship to one another is not clear, there is evidence of a direct toxic effect of Aβ on neurons [33
]. Using different promoters a variety of transgenic models have been engineered to overexpress in the brain mutant forms of amyloid β precursor protein (AβPP). Several models reproducibly deposit Aβ and develop some of the prominent pathological and behavioral features of AD [34
]. These findings resulted in focusing research on drugs that reduce the production of Aβ or enhance its degradation, and lowering cortical Aβ levels in some AD patients may be associated with stabilization of memory and cognitive decline [41
]. There are indications that both fibrillar Aβ and soluble oligomeric Aβ are neurotoxic [43
]. However, Aβ has a widespread distribution through the brain and body and there is evidence that at physiological concentrations soluble Aβ serves a variety of physiological functions, including modulation of synaptic function, facilitation of neuronal growth and survival, protection against oxidative stress, and surveillance against neuroactive compounds, toxins and pathogens (for review see [47
]). These physiological functions should be taken into account when strategies are developed to reduce Aβ load in AD, targeting oligomeric or fibrillar forms of Aβ, leaving monomeric soluble Aβ intact. Our approach does not reduce the level of soluble Aβ, focusing on enhanced binding of soluble Aβ to its carrier CysC, preventing its aggregation and fibril formation and enhancing CysC-mediated direct protection of neuronal cells from the toxicity induced by either form of Aβ.
Caspases are cysteine-dependent aspartate-specific proteases critically involved in apoptosis (for review see [48
]). The detection of cleaved caspases and the accumulation of cleaved caspase substrates in brains of AD patients support the hypothesis that apoptosis may play a role in the subsequent neuronal loss found in AD brains [49
]. Elevated mRNA expression of several caspases was shown in the brain of AD patients compared with controls [51
]. Pyramidal neurons from vulnerable regions involved in the disease showed an increase in activated caspases-3 and -6 [52
]. Synaptosomes prepared from AD brain frontal cortices showed an enrichment in caspase-9 compared with non-demented controls [54
]. Studies have shown induction of apoptosis by Aβ in multiple neuronal cell types in culture [55
]. Aβ-induced cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Aβ-induced death [28
]. Neurons from caspase-2- or 12-knockout mice are resistant to Aβ [28
]. The results indicate that caspase-2 is necessary for Aβ-induced apoptosis. Here we demonstrate that while Aβ induces activation of caspase-2 in primary hippocampal neurons, CysC inhibits this activation. This suggests that CysC protects neuronal cells from caspase-dependent apoptotic cell death induced by Aβ.
There are several indications that CysC has a role in AD: 1) Genetic studies have linked a CST3
polymorphism with an increased risk of developing AD (for review see [5
]). The amino acid exchange from Ala to Thr at the -2 position for signal peptidase cleavage [59
], causes a less efficient cleavage of the signal peptide and thus a reduced secretion of CysC [60
]. 2) Analysis of human cerebrospinal proteins by protein-chip array technology revealed that the combination of five polypeptides, including CysC, could be used for the diagnosis of AD and perhaps the assessment of its severity and progression [63
]. 3) Recent findings that low serum CysC levels predict the development of AD in subjects free of dementia at baseline [64
], suggest that low serum CysC levels precede clinical sporadic AD. 4) Immunohistochemical studies revealed strong dual staining with antibodies to Aβ and to CysC in a subpopulation of pyramidal neurons in the prefrontal cortex and hippocampus. Co-localization of CysC with Aβ was found predominantly in amyloid-laden vascular walls, and in senile plaque cores of amyloid in the brains of AD, Down syndrome, cerebral amyloid angiopathy, and cerebral infarction patients and non-demented aged individuals (for review see [5
]). Co-localization of CysC with Aβ deposits was also found in brains of transgenic mice overexpressing human AβPP [4
]. 5) Furthermore, in vitro
studies have shown that CysC binds to Aβ and inhibits fibril formation and oligomerization of Aβ in a concentration dependent manner [9
]. Such inhibitory effect was confirmed in vivo
in Aβ-depositing transgenic AβPP mice over-expressing human CysC. A reduction in Aβ amyloid load was observed in the AβPP/CysC double transgenic mice compared to single AβPP transgenic mice [11
]. In vitro
studies have shown that CysC inhibits the formation of high molecular weight Aβ oligomeric assemblies [10
]. Moreover, the binding between Aβ and CysC in human CNS was detected in brains and in cerebrospinal of neuropathologically normal controls and in AD cases. The association of CysC with Aβ in brain from control individuals and in cerebrospinal reveals an interaction of these two polypeptides in their soluble form [66
]. In addition to these previous data, showing that CysC prevents amyloid fibril formation and oligomerization of Aβ, the demonstration of direct protection of cells from the toxic forms of Aβ highlights the important defensive roles that CysC plays in AD.
Multiple studies have shown changes in CysC serum concentrations in a variety of conditions, including aging (for review see [67
]). Enhanced CysC expression occurs in human patients with epilepsy and animal models of neurodegenerative conditions, in response to injury, including facial nerve axotomy, noxious input to the sensory spinal cord, perforant path transections, hypophysectomy, transient forebrain ischemia, and induction of epilepsy (for review see [5
]). It has been suggested that this upregulation of CysC expression in response to injury and in various diseased conditions represents an intrinsic neuroprotective mechanism that may counteract progression of the disease. A reduction in CysC secretion is caused by the CST3
polymorphism in patients with late-onset sporadic AD and by two presenilin 2 mutations (PS2 M239I and T122R) linked to familial AD [68
]. We propose that CysC is a carrier of soluble Aβ in body fluids such as cerebrospinal fluid and blood, as well as in the neuropil, where it plays an ongoing role in inhibiting the association of Aβ into insoluble plaques. Furthermore, CysC directly protects neuronal cells from Aβ-induced apoptotic cell death. The inhibition of Aβ aggregation caused by binding of CysC to Aβ and the direct protection of neuronal cells from Aβ-induced death suggest two mechanisms by which a reduced CysC brain concentration is associated with AD. These protective roles of CysC in the pathogenesis of AD suggest that a novel therapeutic approach that involves modulation of CysC levels may have important disease modifying effects.