Publication
β-amyloid-dependent neuronal dysfunction and effect on the endocytic pathway
| Summary: | Alzheimer’s disease (AD) is a major disease of aging affecting about 10% of the elderly population, is the leading cause of dementia and a major health care concern. AD patients develop a gradual and progressive decline in cognitive and functional abilities as well as behavioral and psychiatric symptoms, eventually leading to a vegetative state and ultimately death. In 1907, Dr. Alois Alzheimer published the first report linking the clinical features of AD to plaque and tangle neuropathology. Neurofibrillary tangles in the brain consist primarily of hyperphosphorylated tau. Neuritic plaques consist primarily of aggregated amyloid β (Aβ) peptides and are surrounded by dystrophic neurites, microglia, and reactive astrocytes. Plaques are increasingly viewed as tombstones, and do not correlate well with cognitive impairment. The progressive accumulation of soluble β-amyloid (Aβ) in vulnerable brain areas with aging does parallel the progressive dementia found in AD patients. Plaques are not considered to be responsible for the initial impairments. Therefore, reduction of Aβ is currently the leading approach of experimental therapies for AD. Purification and sequencing of Aβ from AD brain led to the cloning of the amyloid precursor protein (APP) gene and triggered research on trying to understand how the age-dependent accumulation of Aβ in the brain leads to the downstream pathologies and progressive dementia. Aβ is generated by two sequential cleavages of APP by β- and γ-secretases, two major forms of Aβ are produced, a long one with 42 amino acids, Aβ42, and a shorter one with 40 amino acids, Aβ40. Aβ42 is more hydrophobic; it easily aggregates and associates to membranes, likely facilitating its retention intracellularly. Aβ40 is more soluble and is mostly secreted. In 1993, the first biochemical evidence for appreciable levels of endogenous intracellular Aβ was reported in vitro. In 2000, with the development of antibodies that differentiated Aβ42 peptide from its precursor, it was reported that Aβ42 accumulated intracellularly in neurons even before plaque formation. Since then, several studies have implicated the accumulation of Aβ within neurons as important for AD pathogenesis. Loss of synapses occurs early in AD and is considered the best pathological correlate of cognitive decline. Several lines of evidence suggest a relationship between Aβ accumulation and synaptic dysfunction. Depressed synaptic transmission, including impaired LTP (long-term potentiation), has been reported in AD mouse models of β- amyloidosis, and occurs concomitantly with the appearance of intraneuronal Aβ accumulation even prior to appearance of extracellular plaques. This observation suggests that the intracellular accumulation of Aβ42 may have an important role in AD pathogenesis, especially in the initial stages of the disease. Tg2576 mice harbor the human APP transgene with the familial AD Swedish mutation and develop AD-like cerebral amyloidosis. Aβ accumulates and oligomerizes within neurons of Tg2576 mice with aging in vivo, especially within neuronal processes and synaptic compartments. To study the cell biology of Aβ-dependent neuronal dysfunction, we turned to cultured neurons from Tg2576 mice. Tg2576 neurons were previously characterized to recapitulate the accumulation of Aβ with time in culture. We set out to better characterize the consequences of this aberrant Aβ42 accumulation on synapses. In this study, we found both structural and functional alterations in pre- and postsynaptic compartments in Tg2576 compared to wild-type neurons at 19 days in vitro (DIV). The presynaptic alterations included reductions in the number of active presynaptic compartments and in the levels of synaptophysin, a synaptic vesicle protein. The postsynaptic alterations included reductions in the levels of the glutamate receptor subunit, GluR1, and of the postsynaptic density protein, PSD-95. Since Aβ42 accumulation progresses with time in culture, at 19 DIV there is higher concentration of Aβ42 with more probability of aggregation and oligomerization than at 12 DIV. We hypothesized that the changes at 19 DIV could correspond to a more severe phenotype paralleling a later stage of disease. Therefore, we studied synapses at 12 DIV in Tg2576 compared to wild-type neurons. Presynaptically there was no difference in the number of active compartments or in the levels of synaptophysin however, postsynaptically spines were reduced. Specifically, the postsynaptic changes included reduction in the levels of PSD-95 and of glutamate receptors subunits GluR1 and NR1 at the postsynaptic density but not in total cellular lysates. These results suggest that early on the alterations at synapses are due to an impaired trafficking of glutamate receptors. The glutamate receptors are critical determinants for LTP and long-term depression (LTD), forms of synaptic plasticity thought to be involved in the establishment of new memories. Our results shed light on the consequences of Aβ acumulation on synapses, specifically on the earliest alterations that lead to synaptic dysfunction and cognitive decline in AD. However, although we found Aβ42 to be responsible for the synaptic alterations in Tg2576 neurons, the mechanism whereby Aβ42 affects synapses needs further clarification. Over the past few years, increasing evidence supports that intraneuronal Aβ accumulation is involved in early AD pathogenesis. Although the precise location(s) of Aβ production in neurons remains controversial, the subcellular site of Aβ42 accumulation in AD is better accepted. Aβ42 accumulation in the brains of Tg2576 mice and human AD occurs especially in multivesicular endosomes (MVBs). Alterations in the endocytic pathway are among the earliest pathological changes in AD. The molecular mechanisms by which abnormalities in the endocytic pathway arise and are involved in the pathogenesis of AD remain unknown. In the brain, Aβ42- accumulating MVBs often localize close to degenerating synapses. Since Aβ42 accumulation with aging occurs in the outer limiting membranes of MVBs, we hypothesized that this accumulation could alter the MVB sorting pathway in neurons. MVBs are vesicular organelles morphologically defined by a limiting membrane and characteristic inner vesicles. MVBs are considered to be late endosomes, formed by maturation of early endosomes by membrane invaginations that generate inner vesicles with an acidic pH. The inner vesicles have a different content of lipids and proteins: they are rich in cholesterol, LBPA (lysobisphosphatidic acid) and lysosomal glycoproteins. MVBs are involved in many cellular functions, including the regulated trafficking of several proteins and membrane receptors. The trafficking of the epidermal growth factor receptor (EGFR) to MVBs has been the most extensively studied. EGFR upon stimulation with EGF is internalized into early endosomes. The EGF-EGFR complex is stable at the pH of early endosomes, and therefore the activated receptor follows the MVB pathway. During endosomal maturation, inward invagination of the outer membrane leads to MVB formation and traps receptors in the MVB interior, terminates signaling and promotes their subsequent degradation in the more acidic environment of inner vesicles of MVBs and lysosomes. In neurons, the functional role of MVBs is less well understood. MVBs are thought to have an important role in retrograde transport, carrying receptors from neuronal terminals to the cell body for signaling and/or degradation in lysosomes. For example, neurotrophin receptors after ligand binding are internalized presynaptically and transported in MVBs to the cell body where signaling to the nucleus can occur, thereby preventing the loss of signaling during transport. The sorting of neurotrophin receptors to MVBs has been described for TrkB, which upon brain derived neurotrophic factor (BDNF) activation is quickly removed from the cell surface by endocytosis and delivered to the MVB sorting pathway. Since Aβ42 accumulates especially in the outer limiting membrane of MVBs with AD pathogenesis, we postulated that this could impair MVB sorting. We set out to study the MVB sorting pathway in cultured Tg2576 neurons following the trafficking of EGFR after EGF binding, which is well characterized. We demonstrated that Aβ accumulating Tg2576 neurons revealed normal internalization and recycling but altered MVB sorting of EGFR upon activation. The accumulation of EGF-EGFR in MVBs of Tg2576 compared to wild-type neurons suggested reduced degradation of EGFR. We verified altered MVB sorting and degradation of another ligand-receptor complex, BDNF-TrkB, which provided similar results. To obtain more information on the trafficking of EGFR within MVBs, we assessed the levels of phosphorylated EGFR (P-EGFR), since dephosphorylation is thought to occur at the low pH of the inner vesicles of MVBs, and found elevated P-EGFR in Tg2576 neurons. These data suggested impairment in the trafficking of EGFR within MVBs that accumulate Aβ42. Ubiquitination is required for trafficking of EGFR to MVBs, subsequent translocation within MVBs, and later degradation of EGFR via the MVB sorting pathway. It was therefore possible that reduced ubiquitination of EGFR might be blocking EGFR from translocating to inner MVB membranes. Instead, we found increased ubiquitin conjugated EGFR in Tg2576 neurons, suggesting that ubiquitination of EGFR was not impaired. Therefore, we hypothesized that deubiquitination activity might be compromised. Analysis of deubiquitinating enzyme activity did reveal reduced deubiquitination in Tg2576 neurons, although the modest change seemed insufficient for the more marked elevation in ubiquitinated EGFR. Considerable evidence over the past few years has indicated that the ubiquitinproteasome system (UPS) regulates the trafficking of EGFR through the MVB sorting pathway. Proteasome activity has been described to be necessary for deubiquitination to occur prior to EGFR translocation, also since proteasome inhibition leads to decreased EGFR degradation. Therefore, we next examined proteasome activity in Tg2576 compared to wild-type neurons, which revealed a marked reduction in proteasome activity in Tg2576 neurons. Since proteasome inhibition also inhibits deubiquitination, reduced proteasome activity therefore could be responsible for the cellular alterations that we observed in the Tg2576 neurons. We provide evidence that Aβ is responsible for the alterations in the MVB sorting pathway and the UPS in Tg2576 neurons, since the inhibition of Aβ accumulation blocked the cellular alterations in Tg2576 neurons, including impairment in proteasome activity. Moreover, in support of Aβ-induced proteasome inhibition occurring in Tg2576 neurons, we found that proteasome inhibition of wild-type neurons produced alterations in the MVB sorting pathway paralleling those seen in Tg2576 neurons. The findings that the disease-linked Aβ42 peptides localize and accumulate especially in MVBs and thereby alter the trafficking of membrane receptors by inhibiting the UPS, suggest a mechanism whereby Aβ42 accumulation contributes to AD pathogenesis. The UPS is a complex system of interconnecting biochemical pathways that are increasingly being found to have major cellular functions even beyond the degradation of proteins. The UPS is being studied in the regulated recycling and degradation of membrane receptors and has been linked to the regulation of synaptic plasticity. Indeed, we hypothesize that the inhibition of the UPS by Aβ42 impairs the endocytic trafficking of neuronal receptors, and thereby may be the cause of synaptic dysfunction in AD. Our data supports a novel mechanism whereby Aβ accumulation alters the cell biology of vulnerable neurons in AD. |
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| Main Authors: | Guimas Almeida, Claudia |
| Subject: | Amilóide Beta-amilase Doença de Alzheimer Proteínas tau Proteína Beta amilóide Neurofibrilares Teses de doutoramento - 2007 |
| Year: | 2007 |
| Country: | Portugal |
| Document type: | doctoral thesis |
| Access type: | restricted access |
| Associated institution: | Universidade de Lisboa |
| Language: | English |
| Origin: | Repositório da Universidade de Lisboa |
| Summary: | Alzheimer’s disease (AD) is a major disease of aging affecting about 10% of the elderly population, is the leading cause of dementia and a major health care concern. AD patients develop a gradual and progressive decline in cognitive and functional abilities as well as behavioral and psychiatric symptoms, eventually leading to a vegetative state and ultimately death. In 1907, Dr. Alois Alzheimer published the first report linking the clinical features of AD to plaque and tangle neuropathology. Neurofibrillary tangles in the brain consist primarily of hyperphosphorylated tau. Neuritic plaques consist primarily of aggregated amyloid β (Aβ) peptides and are surrounded by dystrophic neurites, microglia, and reactive astrocytes. Plaques are increasingly viewed as tombstones, and do not correlate well with cognitive impairment. The progressive accumulation of soluble β-amyloid (Aβ) in vulnerable brain areas with aging does parallel the progressive dementia found in AD patients. Plaques are not considered to be responsible for the initial impairments. Therefore, reduction of Aβ is currently the leading approach of experimental therapies for AD. Purification and sequencing of Aβ from AD brain led to the cloning of the amyloid precursor protein (APP) gene and triggered research on trying to understand how the age-dependent accumulation of Aβ in the brain leads to the downstream pathologies and progressive dementia. Aβ is generated by two sequential cleavages of APP by β- and γ-secretases, two major forms of Aβ are produced, a long one with 42 amino acids, Aβ42, and a shorter one with 40 amino acids, Aβ40. Aβ42 is more hydrophobic; it easily aggregates and associates to membranes, likely facilitating its retention intracellularly. Aβ40 is more soluble and is mostly secreted. In 1993, the first biochemical evidence for appreciable levels of endogenous intracellular Aβ was reported in vitro. In 2000, with the development of antibodies that differentiated Aβ42 peptide from its precursor, it was reported that Aβ42 accumulated intracellularly in neurons even before plaque formation. Since then, several studies have implicated the accumulation of Aβ within neurons as important for AD pathogenesis. Loss of synapses occurs early in AD and is considered the best pathological correlate of cognitive decline. Several lines of evidence suggest a relationship between Aβ accumulation and synaptic dysfunction. Depressed synaptic transmission, including impaired LTP (long-term potentiation), has been reported in AD mouse models of β- amyloidosis, and occurs concomitantly with the appearance of intraneuronal Aβ accumulation even prior to appearance of extracellular plaques. This observation suggests that the intracellular accumulation of Aβ42 may have an important role in AD pathogenesis, especially in the initial stages of the disease. Tg2576 mice harbor the human APP transgene with the familial AD Swedish mutation and develop AD-like cerebral amyloidosis. Aβ accumulates and oligomerizes within neurons of Tg2576 mice with aging in vivo, especially within neuronal processes and synaptic compartments. To study the cell biology of Aβ-dependent neuronal dysfunction, we turned to cultured neurons from Tg2576 mice. Tg2576 neurons were previously characterized to recapitulate the accumulation of Aβ with time in culture. We set out to better characterize the consequences of this aberrant Aβ42 accumulation on synapses. In this study, we found both structural and functional alterations in pre- and postsynaptic compartments in Tg2576 compared to wild-type neurons at 19 days in vitro (DIV). The presynaptic alterations included reductions in the number of active presynaptic compartments and in the levels of synaptophysin, a synaptic vesicle protein. The postsynaptic alterations included reductions in the levels of the glutamate receptor subunit, GluR1, and of the postsynaptic density protein, PSD-95. Since Aβ42 accumulation progresses with time in culture, at 19 DIV there is higher concentration of Aβ42 with more probability of aggregation and oligomerization than at 12 DIV. We hypothesized that the changes at 19 DIV could correspond to a more severe phenotype paralleling a later stage of disease. Therefore, we studied synapses at 12 DIV in Tg2576 compared to wild-type neurons. Presynaptically there was no difference in the number of active compartments or in the levels of synaptophysin however, postsynaptically spines were reduced. Specifically, the postsynaptic changes included reduction in the levels of PSD-95 and of glutamate receptors subunits GluR1 and NR1 at the postsynaptic density but not in total cellular lysates. These results suggest that early on the alterations at synapses are due to an impaired trafficking of glutamate receptors. The glutamate receptors are critical determinants for LTP and long-term depression (LTD), forms of synaptic plasticity thought to be involved in the establishment of new memories. Our results shed light on the consequences of Aβ acumulation on synapses, specifically on the earliest alterations that lead to synaptic dysfunction and cognitive decline in AD. However, although we found Aβ42 to be responsible for the synaptic alterations in Tg2576 neurons, the mechanism whereby Aβ42 affects synapses needs further clarification. Over the past few years, increasing evidence supports that intraneuronal Aβ accumulation is involved in early AD pathogenesis. Although the precise location(s) of Aβ production in neurons remains controversial, the subcellular site of Aβ42 accumulation in AD is better accepted. Aβ42 accumulation in the brains of Tg2576 mice and human AD occurs especially in multivesicular endosomes (MVBs). Alterations in the endocytic pathway are among the earliest pathological changes in AD. The molecular mechanisms by which abnormalities in the endocytic pathway arise and are involved in the pathogenesis of AD remain unknown. In the brain, Aβ42- accumulating MVBs often localize close to degenerating synapses. Since Aβ42 accumulation with aging occurs in the outer limiting membranes of MVBs, we hypothesized that this accumulation could alter the MVB sorting pathway in neurons. MVBs are vesicular organelles morphologically defined by a limiting membrane and characteristic inner vesicles. MVBs are considered to be late endosomes, formed by maturation of early endosomes by membrane invaginations that generate inner vesicles with an acidic pH. The inner vesicles have a different content of lipids and proteins: they are rich in cholesterol, LBPA (lysobisphosphatidic acid) and lysosomal glycoproteins. MVBs are involved in many cellular functions, including the regulated trafficking of several proteins and membrane receptors. The trafficking of the epidermal growth factor receptor (EGFR) to MVBs has been the most extensively studied. EGFR upon stimulation with EGF is internalized into early endosomes. The EGF-EGFR complex is stable at the pH of early endosomes, and therefore the activated receptor follows the MVB pathway. During endosomal maturation, inward invagination of the outer membrane leads to MVB formation and traps receptors in the MVB interior, terminates signaling and promotes their subsequent degradation in the more acidic environment of inner vesicles of MVBs and lysosomes. In neurons, the functional role of MVBs is less well understood. MVBs are thought to have an important role in retrograde transport, carrying receptors from neuronal terminals to the cell body for signaling and/or degradation in lysosomes. For example, neurotrophin receptors after ligand binding are internalized presynaptically and transported in MVBs to the cell body where signaling to the nucleus can occur, thereby preventing the loss of signaling during transport. The sorting of neurotrophin receptors to MVBs has been described for TrkB, which upon brain derived neurotrophic factor (BDNF) activation is quickly removed from the cell surface by endocytosis and delivered to the MVB sorting pathway. Since Aβ42 accumulates especially in the outer limiting membrane of MVBs with AD pathogenesis, we postulated that this could impair MVB sorting. We set out to study the MVB sorting pathway in cultured Tg2576 neurons following the trafficking of EGFR after EGF binding, which is well characterized. We demonstrated that Aβ accumulating Tg2576 neurons revealed normal internalization and recycling but altered MVB sorting of EGFR upon activation. The accumulation of EGF-EGFR in MVBs of Tg2576 compared to wild-type neurons suggested reduced degradation of EGFR. We verified altered MVB sorting and degradation of another ligand-receptor complex, BDNF-TrkB, which provided similar results. To obtain more information on the trafficking of EGFR within MVBs, we assessed the levels of phosphorylated EGFR (P-EGFR), since dephosphorylation is thought to occur at the low pH of the inner vesicles of MVBs, and found elevated P-EGFR in Tg2576 neurons. These data suggested impairment in the trafficking of EGFR within MVBs that accumulate Aβ42. Ubiquitination is required for trafficking of EGFR to MVBs, subsequent translocation within MVBs, and later degradation of EGFR via the MVB sorting pathway. It was therefore possible that reduced ubiquitination of EGFR might be blocking EGFR from translocating to inner MVB membranes. Instead, we found increased ubiquitin conjugated EGFR in Tg2576 neurons, suggesting that ubiquitination of EGFR was not impaired. Therefore, we hypothesized that deubiquitination activity might be compromised. Analysis of deubiquitinating enzyme activity did reveal reduced deubiquitination in Tg2576 neurons, although the modest change seemed insufficient for the more marked elevation in ubiquitinated EGFR. Considerable evidence over the past few years has indicated that the ubiquitinproteasome system (UPS) regulates the trafficking of EGFR through the MVB sorting pathway. Proteasome activity has been described to be necessary for deubiquitination to occur prior to EGFR translocation, also since proteasome inhibition leads to decreased EGFR degradation. Therefore, we next examined proteasome activity in Tg2576 compared to wild-type neurons, which revealed a marked reduction in proteasome activity in Tg2576 neurons. Since proteasome inhibition also inhibits deubiquitination, reduced proteasome activity therefore could be responsible for the cellular alterations that we observed in the Tg2576 neurons. We provide evidence that Aβ is responsible for the alterations in the MVB sorting pathway and the UPS in Tg2576 neurons, since the inhibition of Aβ accumulation blocked the cellular alterations in Tg2576 neurons, including impairment in proteasome activity. Moreover, in support of Aβ-induced proteasome inhibition occurring in Tg2576 neurons, we found that proteasome inhibition of wild-type neurons produced alterations in the MVB sorting pathway paralleling those seen in Tg2576 neurons. The findings that the disease-linked Aβ42 peptides localize and accumulate especially in MVBs and thereby alter the trafficking of membrane receptors by inhibiting the UPS, suggest a mechanism whereby Aβ42 accumulation contributes to AD pathogenesis. The UPS is a complex system of interconnecting biochemical pathways that are increasingly being found to have major cellular functions even beyond the degradation of proteins. The UPS is being studied in the regulated recycling and degradation of membrane receptors and has been linked to the regulation of synaptic plasticity. Indeed, we hypothesize that the inhibition of the UPS by Aβ42 impairs the endocytic trafficking of neuronal receptors, and thereby may be the cause of synaptic dysfunction in AD. Our data supports a novel mechanism whereby Aβ accumulation alters the cell biology of vulnerable neurons in AD. |
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