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Cannabinoids in neuroinflammation and neurogenesis: From normal aging to Alzheimer's disease models
Featured Research Session Abstract Submission F5-04: Neuroinflammation-Related Phenomenons in Alzheimer’s Disease selected brain regions are affected with the progression of the symptoms of dementia. This information might lead to more effective therapies that can slow the progression of the degeneration. The degeneration or dysfunction of the temporal lobe and forebrain cholinergic neurons are responsible for aspects of the dementia and are likely due to the selective negative effects of chronic neuroinflammation. I will present results from a series of studies in my laboratory that provide insight into how regional and cellular vulnerability is due to an interaction of three independent factors: the duration of the neuroinflammation, age and gender. Methods: We determined the time course and regional changes associated with both normal aging or the intraventricular or intrathecal infusion of LPS using a series of markers that discriminate pro- or anti-inflammatory microglia states. The time- and age-dependent effects of LPS-induced brain inflammation and the influence of normal aging upon regional changes in microglial activation will be determined using complementary methods. Young (3 months) and aged (24 months) male F-344 rats will have aCSF or LPS chronically infused into the 4th ventricle for up to eight weeks. Behavioral testing using the Morris water pool task was performed prior to sacrifice for determination of biochemical and histological evidence of the consequences of the chronic neuroinflammation. Results: The loss of forebrain acetylcholine neurons and memory impairment are time-dependent, particularly vulnerable to the action of tumor necrosis factor-alpha and can be reversed in multiple ways. In contrast to males, female rats demonstrate a unique pattern of microglia activation and show no cognitive impairment in the presence of chronic neuroinflammation. The removal of the ovaries impairs performance in females given chronic neuroinflammation. The pattern of activated microglia evolves over a period of six weeks from being uniformly distributed during the first few days to being concentrated within temporal lobe and basal forebrain regions after four weeks of LPS infusion. The microglia of aged rats are less vulnerable to the effects of LPS and less responsive to anti-inflammatory therapy. Conclusions: Chronic neuroinflammation is responsible for the selective vulnerability of specific neural systems and brain regions associated with age-related neurodegenerative changes. F5-04-05
THE MMP/TIMP SYSTEM IN ALZHEIMER’S DISEASE
S813
(Swedish) + I716V (Florida) + V717I (London) + PS1 M146L + L286V]) under the control of the Thy1 neuronal specific promoter. Molecular and cell biology: quantitative PCR, immunocytochemistry and immunoblot were used to evaluate the regulation of different metalloproteinases, TIMPs, pro-inflammatory cytokines and also the different breakdown products of APP in mice and in HEK293 APPsw cells that overproduce Ab. The latter were transiently transfected with different MMP and TIMP constructs coupled to green fluorescent protein (GFP). Results: Using quantitative PCR, we found that the hippocampus of 5xFAD mice exhibit an early inflammatory response (2-month-old), characterised by the upregulation of IL-1b and other pro-inflammatory cytokines that parallels an incipient microglial reaction and Ab accumulation confined to the subiculum. Among all the metalloproteinases and TIMPs potentially involved in the metabolism of APP/Ab, only the expression of TIMP-1 was significantly upregulated at a time where no signs of astrocyte reactivity were observed. By 4 and 6 months, a conspicuous inflammatory process was observed at the cellular and biochemical level, and mRNA and protein levels for different MMPs were upregulated in close association with reactive astrocytes around the amyloid aggregates. At these time points, the levels of TIMP-1 mRNA were much higher than in 2-month-old mice. In an attempt to emulate the in vivo situation, we incubated the HEK293 APPsw cells with different TIMPs and found that TIMP-1 induced a decrease in extracellular Ab levels without affecting the levels of sAPP^I6. Next, we transfected the cells with different TIMP constructs, including the active forms of TIMP-1 and TIMP-2 and a mutated inactive form of TIMP-1, and found that these three constructs significantly reduced the intracellular content of APP as compared with the GFP control. Furthermore, overexpression of several MMPs induced the intracellular cleavage of APP and the generation of breakdown products reminiscent of the sAPP^I6. These findings may be a first step to understand how the MMP/TIMP system, and in particular the TIMPs, affects Ab accumulation/clearance and hence the associated inflammatory processes. Conclusions: Specific MMPs and TIMPs are expressed at different states of the disease evolution in pace with the intensity of the inflammatory response. The MMP/ TIMP system affects the metabolism of APP/Ab. The TIMPs may influence the trafficking of APP through pathways that do not require MMP inhibition. In turn, MMP activity may regulate the intracellular processing of APP.
Santiago Rivera, Universite de la Mediterranee, Marseille, France. Background: The prevailing view of Alzheimer’s disease (AD) pathogenesis posits that the accumulation of neurotoxic beta-amyloid peptide (Ab), resulting from an imbalance between its generation and clearance, is one of the central events triggering neurodegeneration. Ab is generated from the sequential cleavage of its transmembrane beta amyloid precursor protein (APP) by b- and g-secretases. Alternatively, the APP can be cleaved by ^I6-secretases to generate a non-toxic soluble fragment (sAPP^I6), precluding the production of Ab. Recent experimental evidence suggests that the adamalysins (A Disintegrin And Metalloprotease, ADAM) are the physiological ^I6-secretases and matrix metalloproteinases (MMPs) degrade the oligomeric and fibrillar forms of Ab. Both, ADAMs and MMPs are inhibited by the endogenous tissue inhibitors of metalloproteinases (TIMPs), which are upregulated in astrocytes surrounding amyloid plaques and in the cerebrospinal fluid of AD patients. We and others have previously demonstrated that the MMP/TIMP system is an important modulator of neuro-inflammation and neurodegeneration in the diseased brain, but little is known about its regulation and role in AD (Rivera et al., 2010, J. Neurosci 30: 15337). Our working hypothesis is that a dysregulation of metalloproteinase activity may influence Ab generation/clearance and consequently the inflammatory status in AD brains. In order to test this hypothesis we investigated how the metalloproteinase/TIMP system affects the sAPP^I6/Ab balance and distribution, and how in turn Ab affects the expression/activity of metalloproteinases and TIMPs. Methods: Animals: the 5xFAD strain (Oakley et al., 2006) are double transgenic mice that co-express human APP and PS1 genes with five FAD mutations ([APP K670N/M671L
F5-04-06
CANNABINOIDS IN NEUROINFLAMMATION AND NEUROGENESIS: FROM NORMAL AGING TO ALZHEIMER’S DISEASE MODELS
Yannick Marchalant, Universite de la Mediterranee, Marseille, France. Background: Our recent work clearly demonstrated the benefits from low chronic infusion of the synthetic cannabinoid WIN-55,212-2 in reducing the effect of normal ageing on memory function, inflammation and neurogenesis. As no cure for Alzheimer’s disease is yet available, our project aims to evaluate the benefits of preventive WIN-55,212-2 treatment on amyloid production on cell cultures overexpressing the beta amyloid peptide (Abeta) as well as its potential on delaying the onset of AD in the 5xFAD transgenic mice model. Methods: HEK cells expressing the APP we mutation were used to determine the influence of WIN on amyloid production in the presence of inflammotion or no inflammation. 5xFAD mice were used to determine the influence of WIN on the formation of amyloid plaques and the memory impairments associated to it. Results: WIN seems to modulate the inflammatory profile as well as influencing the balance between sAPPalpha and amyloid beta production in HEK cells overexpressing Abeta. 5xFAD mice characterization of inflammatory markers, neurogenic processes and behavioral impairments are being conducted to evaluate properly the effects of WIN on the onset of Alzheimer’s disease pathology in this animal model. Conclusions: Cannabinoids, because of their anti-inflammatory, neurogenic properties, may be good candidates for a new preventive therapeutical approach aiming at delaying the onset of AD in patients and thus potentially diminishing the appearance of new cases.
THE MMP/TIMP SYSTEM IN ALZHEIMER’S DISEASE
S813
(Swedish) + I716V (Florida) + V717I (London) + PS1 M146L + L286V]) under the control of the Thy1 neuronal specific promoter. Molecular and cell biology: quantitative PCR, immunocytochemistry and immunoblot were used to evaluate the regulation of different metalloproteinases, TIMPs, pro-inflammatory cytokines and also the different breakdown products of APP in mice and in HEK293 APPsw cells that overproduce Ab. The latter were transiently transfected with different MMP and TIMP constructs coupled to green fluorescent protein (GFP). Results: Using quantitative PCR, we found that the hippocampus of 5xFAD mice exhibit an early inflammatory response (2-month-old), characterised by the upregulation of IL-1b and other pro-inflammatory cytokines that parallels an incipient microglial reaction and Ab accumulation confined to the subiculum. Among all the metalloproteinases and TIMPs potentially involved in the metabolism of APP/Ab, only the expression of TIMP-1 was significantly upregulated at a time where no signs of astrocyte reactivity were observed. By 4 and 6 months, a conspicuous inflammatory process was observed at the cellular and biochemical level, and mRNA and protein levels for different MMPs were upregulated in close association with reactive astrocytes around the amyloid aggregates. At these time points, the levels of TIMP-1 mRNA were much higher than in 2-month-old mice. In an attempt to emulate the in vivo situation, we incubated the HEK293 APPsw cells with different TIMPs and found that TIMP-1 induced a decrease in extracellular Ab levels without affecting the levels of sAPP^I6. Next, we transfected the cells with different TIMP constructs, including the active forms of TIMP-1 and TIMP-2 and a mutated inactive form of TIMP-1, and found that these three constructs significantly reduced the intracellular content of APP as compared with the GFP control. Furthermore, overexpression of several MMPs induced the intracellular cleavage of APP and the generation of breakdown products reminiscent of the sAPP^I6. These findings may be a first step to understand how the MMP/TIMP system, and in particular the TIMPs, affects Ab accumulation/clearance and hence the associated inflammatory processes. Conclusions: Specific MMPs and TIMPs are expressed at different states of the disease evolution in pace with the intensity of the inflammatory response. The MMP/ TIMP system affects the metabolism of APP/Ab. The TIMPs may influence the trafficking of APP through pathways that do not require MMP inhibition. In turn, MMP activity may regulate the intracellular processing of APP.
Santiago Rivera, Universite de la Mediterranee, Marseille, France. Background: The prevailing view of Alzheimer’s disease (AD) pathogenesis posits that the accumulation of neurotoxic beta-amyloid peptide (Ab), resulting from an imbalance between its generation and clearance, is one of the central events triggering neurodegeneration. Ab is generated from the sequential cleavage of its transmembrane beta amyloid precursor protein (APP) by b- and g-secretases. Alternatively, the APP can be cleaved by ^I6-secretases to generate a non-toxic soluble fragment (sAPP^I6), precluding the production of Ab. Recent experimental evidence suggests that the adamalysins (A Disintegrin And Metalloprotease, ADAM) are the physiological ^I6-secretases and matrix metalloproteinases (MMPs) degrade the oligomeric and fibrillar forms of Ab. Both, ADAMs and MMPs are inhibited by the endogenous tissue inhibitors of metalloproteinases (TIMPs), which are upregulated in astrocytes surrounding amyloid plaques and in the cerebrospinal fluid of AD patients. We and others have previously demonstrated that the MMP/TIMP system is an important modulator of neuro-inflammation and neurodegeneration in the diseased brain, but little is known about its regulation and role in AD (Rivera et al., 2010, J. Neurosci 30: 15337). Our working hypothesis is that a dysregulation of metalloproteinase activity may influence Ab generation/clearance and consequently the inflammatory status in AD brains. In order to test this hypothesis we investigated how the metalloproteinase/TIMP system affects the sAPP^I6/Ab balance and distribution, and how in turn Ab affects the expression/activity of metalloproteinases and TIMPs. Methods: Animals: the 5xFAD strain (Oakley et al., 2006) are double transgenic mice that co-express human APP and PS1 genes with five FAD mutations ([APP K670N/M671L
F5-04-06
CANNABINOIDS IN NEUROINFLAMMATION AND NEUROGENESIS: FROM NORMAL AGING TO ALZHEIMER’S DISEASE MODELS
Yannick Marchalant, Universite de la Mediterranee, Marseille, France. Background: Our recent work clearly demonstrated the benefits from low chronic infusion of the synthetic cannabinoid WIN-55,212-2 in reducing the effect of normal ageing on memory function, inflammation and neurogenesis. As no cure for Alzheimer’s disease is yet available, our project aims to evaluate the benefits of preventive WIN-55,212-2 treatment on amyloid production on cell cultures overexpressing the beta amyloid peptide (Abeta) as well as its potential on delaying the onset of AD in the 5xFAD transgenic mice model. Methods: HEK cells expressing the APP we mutation were used to determine the influence of WIN on amyloid production in the presence of inflammotion or no inflammation. 5xFAD mice were used to determine the influence of WIN on the formation of amyloid plaques and the memory impairments associated to it. Results: WIN seems to modulate the inflammatory profile as well as influencing the balance between sAPPalpha and amyloid beta production in HEK cells overexpressing Abeta. 5xFAD mice characterization of inflammatory markers, neurogenic processes and behavioral impairments are being conducted to evaluate properly the effects of WIN on the onset of Alzheimer’s disease pathology in this animal model. Conclusions: Cannabinoids, because of their anti-inflammatory, neurogenic properties, may be good candidates for a new preventive therapeutical approach aiming at delaying the onset of AD in patients and thus potentially diminishing the appearance of new cases.