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- Cicognola, Claudia, et al.
(author)
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Tauopathy-Associated Tau Fragment Ending at Amino Acid 224 Is Generated by Calpain-2 Cleavage.
- 2020
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In: Journal of Alzheimer's disease : JAD. - 1875-8908. ; 74:4, s. 1143-1156
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Journal article (peer-reviewed)abstract
- Tau aggregation in neurons and glial cells characterizes tauopathies as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Tau proteolysis has been proposed as a trigger for tau aggregation and tau fragments have been observed in brain and cerebrospinal fluid (CSF). Our group identified a major tau cleavage at amino acid (aa) 224 in CSF; N-terminal tau fragments ending at aa 224 (N-224) were significantly increased in AD and lacked correlation to total tau (t-tau) and phosphorylated tau (p-tau) in PSP and CBD.Previous studies have shown cleavage from calpain proteases at sites adjacent to aa 224. Our aim was to investigate if calpain-1 or -2 could be responsible for cleavage at aa 224.Proteolytic activity of calpain-1, calpain-2, and brain protein extract was assessed on a custom tau peptide (aa 220-228), engineered with fluorescence resonance energy transfer (FRET) technology. Findings were confirmed with in-gel trypsination and mass spectrometry (MS) analysis of brain-derived bands with proteolytic activity on the FRET substrate. Finally, knock-down of the calpain-2 catalytic subunit gene (CAPN2) was performed in a neuroblastoma cell line (SH-SY5Y).Calpain-2 and brain protein extract, but not calpain-1, showed proteolytic activity on the FRET substrate. MS analysis of active gel bands revealed presence of calpain-2 subunits, but not calpain-1. Calpain-2 depletion and chemical inhibition suppressed proteolysis of the FRET substrate. CAPN2 knock-down caused a 76.4% reduction of N-224 tau in the cell-conditioned media.Further investigation of the calpain-2 pathway in the pathogenesis of tauopathies is encouraged.
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| 2. |
- Satir, Tugce Munise, et al.
(author)
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Accelerated neuronal and synaptic maturation by BrainPhys medium increases Aβ secretion and alters Aβ peptide ratios from iPSC-derived cortical neurons.
- 2020
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In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Journal article (peer-reviewed)abstract
- One of the neuropathological hallmarks of Alzheimer's disease (AD) is cerebral deposition of amyloid plaques composed of amyloid β (Aβ) peptides and the cerebrospinal fluid concentrations of those peptides are used as a biomarker for AD. Mature induced pluripotent stem cell (iPSC)-derived cortical neurons secrete Aβ peptides in ratios comparable to those secreted to cerebrospinal fluid in human, however the protocol to achieve mature neurons is time consuming. In this study, we investigated if differentiation of neuroprogenitor cells (NPCs) in BrainPhys medium, previously reported to enhance synaptic function of neurons in culture, would accelerate neuronal maturation and, thus increase Aβ secretion as compared to the conventional neural maintenance medium. We found that NPCs cultured in BrainPhys displayed increased expression of markers for cortical deep-layer neurons, increased synaptic maturation and number of astroglial cells. This accelerated neuronal maturation was accompanied by increased APP processing, resulting in increased secretion of Aβ peptides and an increased Aβ38 to Aβ40 and Aβ42 ratio. However, during long-term culturing in BrainPhys, non-neuronal cells appeared and eventually took over the cultures. Taken together, BrainPhys culturing accelerated neuronal maturation and increased Aβ secretion from iPSC-derived cortical neurons, but changed the cellular composition of the cultures.
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