Final Report Summary - STRESSAMYLOIDCASCADE (Stress cascades and Alzheimer's disease)
Increasing life expectancy calls for the urgent development of strategies to delay, attenuate or prevent Alzheimer’s disease. Several initiatives aim to identify biomarkers (i.e. Alzheimer’s Disease Neuroimaging Initiative - ADNI) for the early detection of the disease, but also the identification of preventative interventions effective at early stages because therapeutic approaches directed only at the moderate -to-late stages have been disappointing in clinical trials so far.
Our original aim was to gain insights into the stress-activated cascades that could potentially induce APP misprocessing to amyloidogenic proteins in base of previous data published by the host lab (Catania et al., 2007). Although some of the objectives stated in our proposal were accomplished as reported previously (see mid-term report), given the unexpectedly slow progression of disease symptomatology in the mice model chosen, we developed an experimental strategy that nevertheless matched with the original goal of the project.
Previous authors reported that neurotrophins can decrease the neuropathological and cognitive consequences of stress in mice (Andero et al., 2011a; 2011b). Here, it should be noted that chronic stress is a trigger of depression-like symptomatology – Bessa et al., 2009). Our experiments involved treating (in drinking water) ArcAbeta mice (human APP overexpression with Swedish and Arctic mutations) with 7,8-dihydroxyflavone (7-DHF), a small molecule that mimics the action of a neurotrophin (brain derived growth factor, BDNF) and monitoring whether this molecule altered any of the classical pathobiochemical hallmarks of Alzheimer’s disease as well as impairments in spatial learning/memory.
Synaptic degeneration is a major pathophysiological hallmark in AD. Thus promoting synaptic transmission, synaptic plasticity and synaptic growth through BDNF-based “synaptic repair” strategies, targeting the pathophysiology rather than the pathogenesis, might provide novel therapeutic approach for AD. The neurotrophic function of BDNF is mainly mediated by TrkB receptor, widely expressed in CNS. From the several ligands targeting TrkB receptor, 7,8-Dihydroxyflavone has shown a selective activity towards TrkB receptor and high penetrability to the CNS when administered peripherially. To date, only 2 other research groups have shown promising results after administration of 7,8-DHF in a mouse model of AD and in aged rats. However, those studies treated animals for more than 4 months and, in both cases, behavioral and molecular readouts were assessed immediately after the last day of administration.
Our first goal was to define when ArcAbeta mice first show obvious signs of AD pathology, allowing us to also identify asymptomatic stages in our laboratory setting. Our data indicates that arcAbeta mice, start to show cognitive dysfunction by 5 months of age, being the transgene fully disrupting at 8 months.
The behavioral characterization of the model for spatial memory and reversal learning allowed us to determine a window for therapeutic interventions, in a model predisposed to develop AD. Thus, 2 month old animals (asymptomatic) were treated orally with 7,8-DHF in drinking water for 4 weeks, coming back to standard conditions of housing (tap water) after the treatment. Five to six months later, when animals were 8-9 months old, they were assessed in the water cross maze and brains removed to evaluate Abeta levels both in hippocampus and in cortex. Our results reflect that the sub-chronic oral treatment completely prevented the age-associated cognitive decline presented by the AD-like mousee model. The treatment also reduced the levels of Abeta-40 and -42 in hippocampus and cortex; in particular, the levels of insoluble Abeta were markedly reduced. To our knowledge, this is the first demonstration that a transient, systemic pharmacological treatment, even before first signs of AD-like behavioral impairment are apparent, can prevent their age-associated appearance. It is important to point out that the effects of the drug can be observed for at least 5 months after discontinuation of the treatment.
Interestingly, the 7,8-DHF administration at 5 months old animals not only block the classical age-associated progression of the disease but also reversed completely the cognitive decline. Together, our results show that short oral administration of 7,8-Dihydroxyflavone prevents both cognitive and molecular onset of AD in arcAbeta mice, suggesting that 7,8-DHF could be a powerful disease-modifying therapy to delay or prevent Alzheimer Disease.
Given he important role of BDNF-TrkB at the synaptic level, 8 month old arcAbeta treated with 7,8-DHF (TrkB active) when they were 2 months old were sacrificed to perform volumetric analysis of hippocampal formation and PFC followed by morphological analysis by Golgi staining. Our results shown that ArcAbeta mice show a reduction in the total volume of the hippocampus, an effect that could not be reversed by 7,8-DHF. This effect on the hippocampus was also observed in the infralimic part of the prefrontal cortex (PFC);however, the volumetric reduction was reversed after administration of 7,8-DHFfor 5-6 months before the analysis. Moreover, Golgi staining revealed that in the PFC, the treatment could restore the aberrant arborization observed in ArcAbeta mice.
At present, we can only pseculate about the mechanisms through which 7,8-DHF exerts its preventative effects against beta toxicity. Likely mechanisms include improved clearance of Abeta or reduced cleavage of APP into neurotoxic fragments such as Abeta. To analyze both Abeta production and degradation, several enzymes (beta-site APP cleaving enzyme 1 or BACE1 as a principal enzyme resposible of Abeta production; and, neprilysin and insulin-degrading enzyme that participates in Abeta degradation) were analyzed by western blot in 8 months old animal that were treated at 2 months of age only for 4 weeks. Our data indicated that 7,8-DHFtreatment downregulates BACE1 protein (key player in generating Abeta from its precursor). Those effects observed by Western Blot indicate that 7,8-DHF could be protecting towards the development of the disease at multiple levels.
Results from this work may be summarized as follows:
- 7,8-DHF not only prevents but also reverses age-dependent cognitive decline in ArcAbeta mice, a transgenic model of AD.
- 7,8-DHF reduces Abeta levels in the cortex and hippocampus. This reduction involves downregulation of BACE1 levels.
- 7,8-DHF reverses Abeta-associated volumetric reductions in the hippocampus and PFC and normalizes neuronal arborization
Overall, neurotrophic stimulation with small molecules could represent a promising pharmacological tool to tackle AD. To our knowledge, this is the first demonstration that a transient, systemic pharmacological treatment, before the onset of AD-like behavioral impairments, can prevent their age-associated exacerbation. It is important to point out that the effects of the drug were seen for at least 5 months after treatment discontinuation. Thus, the results of this study uncover new pathways for the early detection and management of AD. The knowledge generated will help to identify druggable targets for the prevention of Alzheimer’s disease.
Ref.
• Andero R, Heldt SA, Ye K, Liu X, Armario A, Ressler KJ (2011a). Effect of 7,8-dihydroxyflavone, a small-molecule TrkB agonist, on emotional learning. Am J Psychiatry. 2011 Feb;168(2):163-72
• Andero R, Daviu N, Escorihuela RM, Nadal R, Armario A (2011b). 7,8-dihydroxyflavone, a TrkB receptor agonist, blocks long-term spatial memory impairment caused by immobilization stress in rats. Hippocampus. 2012 Mar;22(3):399-408
• Bessa JM, Mesquita AR, Oliveira M, Pêgo JM, Cerqueira JJ, Palha JA, Almeida OF, Sousa N (2009). A trans-dimensional approach to the behavioral aspects of depression. Front Behav Neurosci. 2009 Jan 27;3:1.
• Catania C, Sotiropoulos I, Silva R, Onofri C, Breen KC, Sousa N, Almeida OF (2007). The amyloidogenic potential and behavioral correlates of stress. Mol Psychiatry 14, 95.
Our original aim was to gain insights into the stress-activated cascades that could potentially induce APP misprocessing to amyloidogenic proteins in base of previous data published by the host lab (Catania et al., 2007). Although some of the objectives stated in our proposal were accomplished as reported previously (see mid-term report), given the unexpectedly slow progression of disease symptomatology in the mice model chosen, we developed an experimental strategy that nevertheless matched with the original goal of the project.
Previous authors reported that neurotrophins can decrease the neuropathological and cognitive consequences of stress in mice (Andero et al., 2011a; 2011b). Here, it should be noted that chronic stress is a trigger of depression-like symptomatology – Bessa et al., 2009). Our experiments involved treating (in drinking water) ArcAbeta mice (human APP overexpression with Swedish and Arctic mutations) with 7,8-dihydroxyflavone (7-DHF), a small molecule that mimics the action of a neurotrophin (brain derived growth factor, BDNF) and monitoring whether this molecule altered any of the classical pathobiochemical hallmarks of Alzheimer’s disease as well as impairments in spatial learning/memory.
Synaptic degeneration is a major pathophysiological hallmark in AD. Thus promoting synaptic transmission, synaptic plasticity and synaptic growth through BDNF-based “synaptic repair” strategies, targeting the pathophysiology rather than the pathogenesis, might provide novel therapeutic approach for AD. The neurotrophic function of BDNF is mainly mediated by TrkB receptor, widely expressed in CNS. From the several ligands targeting TrkB receptor, 7,8-Dihydroxyflavone has shown a selective activity towards TrkB receptor and high penetrability to the CNS when administered peripherially. To date, only 2 other research groups have shown promising results after administration of 7,8-DHF in a mouse model of AD and in aged rats. However, those studies treated animals for more than 4 months and, in both cases, behavioral and molecular readouts were assessed immediately after the last day of administration.
Our first goal was to define when ArcAbeta mice first show obvious signs of AD pathology, allowing us to also identify asymptomatic stages in our laboratory setting. Our data indicates that arcAbeta mice, start to show cognitive dysfunction by 5 months of age, being the transgene fully disrupting at 8 months.
The behavioral characterization of the model for spatial memory and reversal learning allowed us to determine a window for therapeutic interventions, in a model predisposed to develop AD. Thus, 2 month old animals (asymptomatic) were treated orally with 7,8-DHF in drinking water for 4 weeks, coming back to standard conditions of housing (tap water) after the treatment. Five to six months later, when animals were 8-9 months old, they were assessed in the water cross maze and brains removed to evaluate Abeta levels both in hippocampus and in cortex. Our results reflect that the sub-chronic oral treatment completely prevented the age-associated cognitive decline presented by the AD-like mousee model. The treatment also reduced the levels of Abeta-40 and -42 in hippocampus and cortex; in particular, the levels of insoluble Abeta were markedly reduced. To our knowledge, this is the first demonstration that a transient, systemic pharmacological treatment, even before first signs of AD-like behavioral impairment are apparent, can prevent their age-associated appearance. It is important to point out that the effects of the drug can be observed for at least 5 months after discontinuation of the treatment.
Interestingly, the 7,8-DHF administration at 5 months old animals not only block the classical age-associated progression of the disease but also reversed completely the cognitive decline. Together, our results show that short oral administration of 7,8-Dihydroxyflavone prevents both cognitive and molecular onset of AD in arcAbeta mice, suggesting that 7,8-DHF could be a powerful disease-modifying therapy to delay or prevent Alzheimer Disease.
Given he important role of BDNF-TrkB at the synaptic level, 8 month old arcAbeta treated with 7,8-DHF (TrkB active) when they were 2 months old were sacrificed to perform volumetric analysis of hippocampal formation and PFC followed by morphological analysis by Golgi staining. Our results shown that ArcAbeta mice show a reduction in the total volume of the hippocampus, an effect that could not be reversed by 7,8-DHF. This effect on the hippocampus was also observed in the infralimic part of the prefrontal cortex (PFC);however, the volumetric reduction was reversed after administration of 7,8-DHFfor 5-6 months before the analysis. Moreover, Golgi staining revealed that in the PFC, the treatment could restore the aberrant arborization observed in ArcAbeta mice.
At present, we can only pseculate about the mechanisms through which 7,8-DHF exerts its preventative effects against beta toxicity. Likely mechanisms include improved clearance of Abeta or reduced cleavage of APP into neurotoxic fragments such as Abeta. To analyze both Abeta production and degradation, several enzymes (beta-site APP cleaving enzyme 1 or BACE1 as a principal enzyme resposible of Abeta production; and, neprilysin and insulin-degrading enzyme that participates in Abeta degradation) were analyzed by western blot in 8 months old animal that were treated at 2 months of age only for 4 weeks. Our data indicated that 7,8-DHFtreatment downregulates BACE1 protein (key player in generating Abeta from its precursor). Those effects observed by Western Blot indicate that 7,8-DHF could be protecting towards the development of the disease at multiple levels.
Results from this work may be summarized as follows:
- 7,8-DHF not only prevents but also reverses age-dependent cognitive decline in ArcAbeta mice, a transgenic model of AD.
- 7,8-DHF reduces Abeta levels in the cortex and hippocampus. This reduction involves downregulation of BACE1 levels.
- 7,8-DHF reverses Abeta-associated volumetric reductions in the hippocampus and PFC and normalizes neuronal arborization
Overall, neurotrophic stimulation with small molecules could represent a promising pharmacological tool to tackle AD. To our knowledge, this is the first demonstration that a transient, systemic pharmacological treatment, before the onset of AD-like behavioral impairments, can prevent their age-associated exacerbation. It is important to point out that the effects of the drug were seen for at least 5 months after treatment discontinuation. Thus, the results of this study uncover new pathways for the early detection and management of AD. The knowledge generated will help to identify druggable targets for the prevention of Alzheimer’s disease.
Ref.
• Andero R, Heldt SA, Ye K, Liu X, Armario A, Ressler KJ (2011a). Effect of 7,8-dihydroxyflavone, a small-molecule TrkB agonist, on emotional learning. Am J Psychiatry. 2011 Feb;168(2):163-72
• Andero R, Daviu N, Escorihuela RM, Nadal R, Armario A (2011b). 7,8-dihydroxyflavone, a TrkB receptor agonist, blocks long-term spatial memory impairment caused by immobilization stress in rats. Hippocampus. 2012 Mar;22(3):399-408
• Bessa JM, Mesquita AR, Oliveira M, Pêgo JM, Cerqueira JJ, Palha JA, Almeida OF, Sousa N (2009). A trans-dimensional approach to the behavioral aspects of depression. Front Behav Neurosci. 2009 Jan 27;3:1.
• Catania C, Sotiropoulos I, Silva R, Onofri C, Breen KC, Sousa N, Almeida OF (2007). The amyloidogenic potential and behavioral correlates of stress. Mol Psychiatry 14, 95.