- Email: [email protected]
CSF FERRITIN DETERMINES THE RISK OF COGNITIVE DECLINE IN PRE-CLINICAL APOE-E4 CARRIERS
Podium Presentations: Thursday, July 28, 2016 O5-04-06
P387
SUBCELLULAR TARGETING OF ENDOGENOUS AMYLOID-B OLIGOMERS IN HUMAN PRIMARY CELLS
Giovanni Meli1, Valeria La Marca1, Annalisa Manca1, Chiara Scopa1, Federica Ruggeri1, Raffaella Scardigli1,2, Antonino Cattaneo1,3, 1European Brain Research Institute, Rome, Italy; 2CNR, Rome, Italy; 3Scuola Normale Superiore, Pisa, Italy. Contact e-mail: [email protected] Background: Amyloidb oligomers (AbOs) are crucial neurotoxic species in Alzheimer’s Disease (AD) but they are still difficult targets for therapy. We demostrated that recombinant antibody fragments can be exploited as intracellular antibodies to block or modulate the toxicity of endogenous AbOs in living cells. In this way, we established a new experimental paradigm of subcellularlocalized conformational-selective interference (CSI) (Meli et al., Nature Comm 2014). Methods: The intrabody-based CSI approach, previously established in mammalian fAD cell lines, was here developed through the use of new lentiviral systems, inducible or not, exploited in different murine and human primary cell systems. In detail, we investigated: i. primary neuronal stem cells (NSC) derived from neurogenic niches of the adult brain of AD mouse model Tg2576; ii. primary human fibroblasts from different AD patients (some of them carrying the V717I fAD mutation); iii. human iPS cells derived from primary fibroblasts. NSC were mantained in vitro as neurosphere cultures and differentiated in neurons or astrocytes. Results: We demostrated that subcellular AbOs are responsible for alterations on growth and differentiation of NSC, and for dysfunctions of mitochondria and of homeostatic mechanisms in primary cells. These alterations and dysfunctions are functionally rescued by our CSI approach, in which the anti-AbOs intrabodies were targeted to the Endoplasmic Reticulum and the early secretory pathway. Conclusions: The subcellular localization of the intrabody-based CSI for AbOs in the Endoplasmic Reticulum and the early secretory pathway, strongly suggests the role of these subcellular compartments in determining the toxic activity of endogenously produced AbOs. In mammalian fAD cell lines we found an AD-relevant functional link between the ER and mitochondria, which seems to be confirmed in human primary fibroblasts. Thus, the intrabody-based CSI for AbOs is an useful approach to study AbO actions inside living cells and it gives a new perspective for in vivo immunotherapy. Supported by Alzheimer’s Association NIRG-12-237751, Human Brain Project Neuroantibodies #604102.
THURSDAY, JULY 28, 2016 ORAL SESSIONS O5-05 BIOMARKERS: NOVEL BIOMARKERS O5-05-01
CSF FERRITIN DETERMINES THE RISK OF COGNITIVE DECLINE IN PRE-CLINICAL APOE-E4 CARRIERS
Scott Ayton1, Noel G. Faux2, Ashley I. Bush2, Alzheimer’s Disease Neuroimaging Initiative, 1University of Melbourne, Melbourne, Australia; 2 The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia. Contact e-mail: [email protected]
Background: The ε4 variant of APOE is the strongest determi-
nant of late onset AD, however the neurochemical mechanisms underlying how APOE confers risk toward AD are uncertain, and there is considerable variability in clinical trajectory between patients. We recently reported (Ayton et al, Nature Comm, 2015) that CSF ferritin, a reporter of brain iron load, predicts longitudinal outcomes of AD comparable to that of the combined performance of CSF tau and Ab. Here, we explored whether CSF ferritin levels could be use in combination with other AD risk factors such as APOE ε4 to predict early cogntive changes. Methods: Subjects classified as Cognitively Normal (CN; n¼91), Mild Cognitive Impaired (MCI; n¼144) and AD (n¼67) were recruited to the Alzheimer’s Disease Neuroimaging Initiative (ADNI) study, which collected demographic, genetic (e.g. APOE isoform), clinical (e.g. cognition) and biochemical (e.g. CSF) information at baseline, and clinical appraisal was performed annually for up to seven years. Results: Baseline CSF ferritin was associated with a marked acceleration of cognitive deterioration over 7 years in CN subjects carrying the APOE ε4 risk allele (RAVLT: P¼0.0008, r2¼0.21), but there was no association in ε4-ve subjects. In contrast, the ratio of tau and Ab levels in CSF (an established biomarker for AD) was more modestly assocaited with cogntive change (RAVLT: P¼0.039, r2¼0.005), and did not vary according to ε4 genotype. A threshold value of 6.6 ng/ml CSF ferritin predicated stable and cognitively declining ε4+ve CN subjects with an accuracy (area under receiver-operator characteristic) of 0.96. Conclusions: These data show that CN ε4+ve subjects with comparatively low ferritin (<6.6 ng/ml) will not deteriorate in the foreseeable future, which could explain why w30% of ε4+ve subjects do not develop AD. Conversely, each unit increase of ferritin above this threshold predicted more rapid deterioration in ε4+ve subjects. The effect of CSF ferritin on cognitive deterioration was remarkably different between APOE ε4 carriers and non-carriers, which provides insight into the pathomechanisms of this major risk factor. These data also support lowering brain iron levels as a therapeutic strategy to slow disease progression in individuals who are ε4+ve.
P387
SUBCELLULAR TARGETING OF ENDOGENOUS AMYLOID-B OLIGOMERS IN HUMAN PRIMARY CELLS
Giovanni Meli1, Valeria La Marca1, Annalisa Manca1, Chiara Scopa1, Federica Ruggeri1, Raffaella Scardigli1,2, Antonino Cattaneo1,3, 1European Brain Research Institute, Rome, Italy; 2CNR, Rome, Italy; 3Scuola Normale Superiore, Pisa, Italy. Contact e-mail: [email protected] Background: Amyloidb oligomers (AbOs) are crucial neurotoxic species in Alzheimer’s Disease (AD) but they are still difficult targets for therapy. We demostrated that recombinant antibody fragments can be exploited as intracellular antibodies to block or modulate the toxicity of endogenous AbOs in living cells. In this way, we established a new experimental paradigm of subcellularlocalized conformational-selective interference (CSI) (Meli et al., Nature Comm 2014). Methods: The intrabody-based CSI approach, previously established in mammalian fAD cell lines, was here developed through the use of new lentiviral systems, inducible or not, exploited in different murine and human primary cell systems. In detail, we investigated: i. primary neuronal stem cells (NSC) derived from neurogenic niches of the adult brain of AD mouse model Tg2576; ii. primary human fibroblasts from different AD patients (some of them carrying the V717I fAD mutation); iii. human iPS cells derived from primary fibroblasts. NSC were mantained in vitro as neurosphere cultures and differentiated in neurons or astrocytes. Results: We demostrated that subcellular AbOs are responsible for alterations on growth and differentiation of NSC, and for dysfunctions of mitochondria and of homeostatic mechanisms in primary cells. These alterations and dysfunctions are functionally rescued by our CSI approach, in which the anti-AbOs intrabodies were targeted to the Endoplasmic Reticulum and the early secretory pathway. Conclusions: The subcellular localization of the intrabody-based CSI for AbOs in the Endoplasmic Reticulum and the early secretory pathway, strongly suggests the role of these subcellular compartments in determining the toxic activity of endogenously produced AbOs. In mammalian fAD cell lines we found an AD-relevant functional link between the ER and mitochondria, which seems to be confirmed in human primary fibroblasts. Thus, the intrabody-based CSI for AbOs is an useful approach to study AbO actions inside living cells and it gives a new perspective for in vivo immunotherapy. Supported by Alzheimer’s Association NIRG-12-237751, Human Brain Project Neuroantibodies #604102.
THURSDAY, JULY 28, 2016 ORAL SESSIONS O5-05 BIOMARKERS: NOVEL BIOMARKERS O5-05-01
CSF FERRITIN DETERMINES THE RISK OF COGNITIVE DECLINE IN PRE-CLINICAL APOE-E4 CARRIERS
Scott Ayton1, Noel G. Faux2, Ashley I. Bush2, Alzheimer’s Disease Neuroimaging Initiative, 1University of Melbourne, Melbourne, Australia; 2 The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia. Contact e-mail: [email protected]
Background: The ε4 variant of APOE is the strongest determi-
nant of late onset AD, however the neurochemical mechanisms underlying how APOE confers risk toward AD are uncertain, and there is considerable variability in clinical trajectory between patients. We recently reported (Ayton et al, Nature Comm, 2015) that CSF ferritin, a reporter of brain iron load, predicts longitudinal outcomes of AD comparable to that of the combined performance of CSF tau and Ab. Here, we explored whether CSF ferritin levels could be use in combination with other AD risk factors such as APOE ε4 to predict early cogntive changes. Methods: Subjects classified as Cognitively Normal (CN; n¼91), Mild Cognitive Impaired (MCI; n¼144) and AD (n¼67) were recruited to the Alzheimer’s Disease Neuroimaging Initiative (ADNI) study, which collected demographic, genetic (e.g. APOE isoform), clinical (e.g. cognition) and biochemical (e.g. CSF) information at baseline, and clinical appraisal was performed annually for up to seven years. Results: Baseline CSF ferritin was associated with a marked acceleration of cognitive deterioration over 7 years in CN subjects carrying the APOE ε4 risk allele (RAVLT: P¼0.0008, r2¼0.21), but there was no association in ε4-ve subjects. In contrast, the ratio of tau and Ab levels in CSF (an established biomarker for AD) was more modestly assocaited with cogntive change (RAVLT: P¼0.039, r2¼0.005), and did not vary according to ε4 genotype. A threshold value of 6.6 ng/ml CSF ferritin predicated stable and cognitively declining ε4+ve CN subjects with an accuracy (area under receiver-operator characteristic) of 0.96. Conclusions: These data show that CN ε4+ve subjects with comparatively low ferritin (<6.6 ng/ml) will not deteriorate in the foreseeable future, which could explain why w30% of ε4+ve subjects do not develop AD. Conversely, each unit increase of ferritin above this threshold predicted more rapid deterioration in ε4+ve subjects. The effect of CSF ferritin on cognitive deterioration was remarkably different between APOE ε4 carriers and non-carriers, which provides insight into the pathomechanisms of this major risk factor. These data also support lowering brain iron levels as a therapeutic strategy to slow disease progression in individuals who are ε4+ve.