Accumulation of ubiquitin-positive, tau- and a-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinpositive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.

TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells / Capitini, Claudia; Conti, Simona; Perni, Michele; Guidi, Francesca; Cascella, Roberta; Poli, Angela De; Penco, Amanda; Relini, Annalisa; Cecchi, Cristina; Chiti, Fabrizio. - In: THE FEBS JOURNAL. - ISSN 1742-4658. - ELETTRONICO. - 281:(2014), pp. 642-642. (Intervento presentato al convegno FEBS EMBO 2014 Conference tenutosi a Paris nel August 30- 4 September 2014).

TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells

CAPITINI, CLAUDIA
Investigation
;
CASCELLA, ROBERTA
Investigation
;
CECCHI, CRISTINA
Supervision
;
CHITI, FABRIZIO
Supervision
2014

Abstract

Accumulation of ubiquitin-positive, tau- and a-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinpositive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.
2014
FEBS EMBO 2014 Conference, Paris, France, 30 August‐4
FEBS EMBO 2014 Conference
Paris
August 30- 4 September 2014
Capitini, Claudia; Conti, Simona; Perni, Michele; Guidi, Francesca; Cascella, Roberta; Poli, Angela De; Penco, Amanda; Relini, Annalisa; Cecchi, Cristina; Chiti, Fabrizio
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1079368
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