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Abstract Alzheimer’s disease (AD) is a major public health concern worldwide, with an increasing prevalence in the elderly population. AD is a progressive neurological disorder of multi-faceted origin, where factors such as genetic mutations, biochemical changes, along with inflammatory cascade and soluble beta amyloid (Aβ) peptide, are thought to play a pivotal role in synaptic failure and neuronal death, ultimately leading to cognitive and neuropsychiatric decline in patients suffering from the disease. At present, there is no long-term cure for the disease, although there is access to pharmacotherapy that might improve cognitive and neuropsychiatric symptoms early in the course of the disease. The current pharmacological therapy for AD only provides symptomatic relief for a very short period of time. It is therefore of utmost importance to discover other pharmacological strategies that might delay the development of AD and slow down the disease progression in terms of cognitive decline and neurodegeneration. Elucidating the pathogenic mechanisms involved in AD neuropathogenesis is a major goal to find efficacious disease-modifying treatments. What remains to be understood completely are the intracellular pathways affected by Aβ protein which may lead to neurodegeneration in AD. Since phosphorylation and dephosphorylation mechanisms are crucial in the β-amyloid precursor protein (APP) metabolism, protein kinase C has emerged as one of the key regulators of the APP metabolism. Indeed, dysregulation of the PKC pathway might play a role in the intracellular mechanisms of neurodegeneration, but their effective involvement still remains elusive. Therefore, a detailed analysis of PKC pathways in established models of AD neurodegeneration is necessary and will form part of this work. Fucoidin is a sulphated polysaccharide extracted from edible brown seaweed, which has been shown to exhibit anti-inflammatory and anti-oxidant effects as well as being a neuroprotectant in various inflammatory diseases including hypoxic ischemia, atherosclerosis and Heyman nephritis. Therefore, fucoidin may have an inhibitory effect on the inflammatory mechanisms of AD. Little is known, however, about the effect of fucoidin on AD. Animal models of AD are extremely valuable for the discovery and development of new treatments. Rodents have been one of the preferred models for pharmacological and behavioural studies in AD. In this thesis, first aim was to establish a non-transgenic Aβ-induced AD model in rats. AD was induced utilising a published protocol which involved the bilateral injection of aggregated Aβ (1-42) into the CA3 subfield of the hippocampus in rat brain. Behavioural assessment with well defined tools such as the Morris water maze and T-maze were utilised to assess the impairment in spatial working memory in rats. Behavioural impairments along with increased astrocytosis and microgliosis were observed in this particular Aβ-induced AD model. In the established disease model, fucoidin (50 mg/kg/day and 25 mg/kg/day) and ibuprofen (50 mg/kg/day) were shown to provide a partial protective effect on impairment in memory function in the MWM behavioural task in rats treated prior to disease initiation and throughout the course of the study. In addition, the histopathological and quantitative analysis of AD brain sections showed a marked reduction in reactive glial fibrillary acidic protein (GFAP) and microglia in fucoidin (low and high dose) and ibuprofen treated Aβ injected rats compared to untreated Aβ injected rats. These results indicate that fucoidin may serve as a possible effective therapeutic approach to improve AD symptoms. There is strong evidence that PKC α and ε signalling pathways regulate important molecular events in memory impairment and neurodegenerative pathophysiology in AD. A possible neuroprotective mechanism of fucoidin involving attenuation of an Aβ-induced decrease in PKC ε phosphorylation using cultured SHSY5Y neuroblastoma cells as a model system was examined. Co-administration of fucoidin (2μM and 5 μM) with Aβ (1μM) abolished the inhibitory effect of Aβ on the phosphorylation of PKCε in a concentration-dependent manner as revealed by western blot analysis. These findings suggest that a possible mechanism underpinning the neuroprotective effect of fucoidin may be through prevention of A-induced inhibition of PKC phosphorylation and may serve as a possible therapeutic approach to improve AD symptoms. As cellular events that involve PKC are affected by Aβ in in vitro systems, it was necessary to examine whether PKC activity is also modulated by the Aβ treatment in vivo in our Aβ-peptide induced AD model. Therefore, the next aim was to assess the potential for fucoidin use as an intervention therapy in an established disease stage in the Aβ-peptide induced AD model. Intervention with fucoidin (50 mg/kg/day, i.p.) in the established disease stage partially prevented Aβ (1-42) mediated damage with respect to memory impairment, neuroinflammation and PKC ε phosphorylation in the in vivo AD model consistent with the in vitro findings in SHSY5Y cells.

Abstract Alzheimer’s disease (AD) is a major public health concern worldwide, with an increasing prevalence in the elderly population. AD is a progressive neurological disorder of multi-faceted origin, where factors such as genetic mutations, biochemical changes, along with inflammatory cascade and soluble beta amyloid (Aβ) peptide, are thought to play a pivotal role in synaptic failure and neuronal death, ultimately leading to cognitive and neuropsychiatric decline in patients suffering from the disease. At present, there is no long-term cure for the disease, although there is access to pharmacotherapy that might improve cognitive and neuropsychiatric symptoms early in the course of the disease. The current pharmacological therapy for AD only provides symptomatic relief for a very short period of time. It is therefore of utmost importance to discover other pharmacological strategies that might delay the development of AD and slow down the disease progression in terms of cognitive decline and neurodegeneration. Elucidating the pathogenic mechanisms involved in AD neuropathogenesis is a major goal to find efficacious disease-modifying treatments. What remains to be understood completely are the intracellular pathways affected by Aβ protein which may lead to neurodegeneration in AD. Since phosphorylation and dephosphorylation mechanisms are crucial in the β-amyloid precursor protein (APP) metabolism, protein kinase C has emerged as one of the key regulators of the APP metabolism. Indeed, dysregulation of the PKC pathway might play a role in the intracellular mechanisms of neurodegeneration, but their effective involvement still remains elusive. Therefore, a detailed analysis of PKC pathways in established models of AD neurodegeneration is necessary and will form part of this work. Fucoidin is a sulphated polysaccharide extracted from edible brown seaweed, which has been shown to exhibit anti-inflammatory and anti-oxidant effects as well as being a neuroprotectant in various inflammatory diseases including hypoxic ischemia, atherosclerosis and Heyman nephritis. Therefore, fucoidin may have an inhibitory effect on the inflammatory mechanisms of AD. Little is known, however, about the effect of fucoidin on AD. Animal models of AD are extremely valuable for the discovery and development of new treatments. Rodents have been one of the preferred models for pharmacological and behavioural studies in AD. In this thesis, first aim was to establish a non-transgenic Aβ-induced AD model in rats. AD was induced utilising a published protocol which involved the bilateral injection of aggregated Aβ (1-42) into the CA3 subfield of the hippocampus in rat brain. Behavioural assessment with well defined tools such as the Morris water maze and T-maze were utilised to assess the impairment in spatial working memory in rats. Behavioural impairments along with increased astrocytosis and microgliosis were observed in this particular Aβ-induced AD model. In the established disease model, fucoidin (50 mg/kg/day and 25 mg/kg/day) and ibuprofen (50 mg/kg/day) were shown to provide a partial protective effect on impairment in memory function in the MWM behavioural task in rats treated prior to disease initiation and throughout the course of the study. In addition, the histopathological and quantitative analysis of AD brain sections showed a marked reduction in reactive glial fibrillary acidic protein (GFAP) and microglia in fucoidin (low and high dose) and ibuprofen treated Aβ injected rats compared to untreated Aβ injected rats. These results indicate that fucoidin may serve as a possible effective therapeutic approach to improve AD symptoms. There is strong evidence that PKC α and ε signalling pathways regulate important molecular events in memory impairment and neurodegenerative pathophysiology in AD. A possible neuroprotective mechanism of fucoidin involving attenuation of an Aβ-induced decrease in PKC ε phosphorylation using cultured SHSY5Y neuroblastoma cells as a model system was examined. Co-administration of fucoidin (2μM and 5 μM) with Aβ (1μM) abolished the inhibitory effect of Aβ on the phosphorylation of PKCε in a concentration-dependent manner as revealed by western blot analysis. These findings suggest that a possible mechanism underpinning the neuroprotective effect of fucoidin may be through prevention of A-induced inhibition of PKC phosphorylation and may serve as a possible therapeutic approach to improve AD symptoms. As cellular events that involve PKC are affected by Aβ in in vitro systems, it was necessary to examine whether PKC activity is also modulated by the Aβ treatment in vivo in our Aβ-peptide induced AD model. Therefore, the next aim was to assess the potential for fucoidin use as an intervention therapy in an established disease stage in the Aβ-peptide induced AD model. Intervention with fucoidin (50 mg/kg/day, i.p.) in the established disease stage partially prevented Aβ (1-42) mediated damage with respect to memory impairment, neuroinflammation and PKC ε phosphorylation in the in vivo AD model consistent with the in vitro findings in SHSY5Y cells.

Abstract Alzheimer’s disease (AD) is a major public health concern worldwide, with an increasing prevalence in the elderly population. AD is a progressive neurological disorder of multi-faceted origin, where factors such as genetic mutations, biochemical changes, along with inflammatory cascade and soluble beta amyloid (Aβ) peptide, are thought to play a pivotal role in synaptic failure and neuronal death, ultimately leading to cognitive and neuropsychiatric decline in patients suffering from the disease. At present, there is no long-term cure for the disease, although there is access to pharmacotherapy that might improve cognitive and neuropsychiatric symptoms early in the course of the disease. The current pharmacological therapy for AD only provides symptomatic relief for a very short period of time. It is therefore of utmost importance to discover other pharmacological strategies that might delay the development of AD and slow down the disease progression in terms of cognitive decline and neurodegeneration. Elucidating the pathogenic mechanisms involved in AD neuropathogenesis is a major goal to find efficacious disease-modifying treatments. What remains to be understood completely are the intracellular pathways affected by Aβ protein which may lead to neurodegeneration in AD. Since phosphorylation and dephosphorylation mechanisms are crucial in the β-amyloid precursor protein (APP) metabolism, protein kinase C has emerged as one of the key regulators of the APP metabolism. Indeed, dysregulation of the PKC pathway might play a role in the intracellular mechanisms of neurodegeneration, but their effective involvement still remains elusive. Therefore, a detailed analysis of PKC pathways in established models of AD neurodegeneration is necessary and will form part of this work. Fucoidin is a sulphated polysaccharide extracted from edible brown seaweed, which has been shown to exhibit anti-inflammatory and anti-oxidant effects as well as being a neuroprotectant in various inflammatory diseases including hypoxic ischemia, atherosclerosis and Heyman nephritis. Therefore, fucoidin may have an inhibitory effect on the inflammatory mechanisms of AD. Little is known, however, about the effect of fucoidin on AD. Animal models of AD are extremely valuable for the discovery and development of new treatments. Rodents have been one of the preferred models for pharmacological and behavioural studies in AD. In this thesis, first aim was to establish a non-transgenic Aβ-induced AD model in rats. AD was induced utilising a published protocol which involved the bilateral injection of aggregated Aβ (1-42) into the CA3 subfield of the hippocampus in rat brain. Behavioural assessment with well defined tools such as the Morris water maze and T-maze were utilised to assess the impairment in spatial working memory in rats. Behavioural impairments along with increased astrocytosis and microgliosis were observed in this particular Aβ-induced AD model. In the established disease model, fucoidin (50 mg/kg/day and 25 mg/kg/day) and ibuprofen (50 mg/kg/day) were shown to provide a partial protective effect on impairment in memory function in the MWM behavioural task in rats treated prior to disease initiation and throughout the course of the study. In addition, the histopathological and quantitative analysis of AD brain sections showed a marked reduction in reactive glial fibrillary acidic protein (GFAP) and microglia in fucoidin (low and high dose) and ibuprofen treated Aβ injected rats compared to untreated Aβ injected rats. These results indicate that fucoidin may serve as a possible effective therapeutic approach to improve AD symptoms. There is strong evidence that PKC α and ε signalling pathways regulate important molecular events in memory impairment and neurodegenerative pathophysiology in AD. A possible neuroprotective mechanism of fucoidin involving attenuation of an Aβ-induced decrease in PKC ε phosphorylation using cultured SHSY5Y neuroblastoma cells as a model system was examined. Co-administration of fucoidin (2μM and 5 μM) with Aβ (1μM) abolished the inhibitory effect of Aβ on the phosphorylation of PKCε in a concentration-dependent manner as revealed by western blot analysis. These findings suggest that a possible mechanism underpinning the neuroprotective effect of fucoidin may be through prevention of A-induced inhibition of PKC phosphorylation and may serve as a possible therapeutic approach to improve AD symptoms. As cellular events that involve PKC are affected by Aβ in in vitro systems, it was necessary to examine whether PKC activity is also modulated by the Aβ treatment in vivo in our Aβ-peptide induced AD model. Therefore, the next aim was to assess the potential for fucoidin use as an intervention therapy in an established disease stage in the Aβ-peptide induced AD model. Intervention with fucoidin (50 mg/kg/day, i.p.) in the established disease stage partially prevented Aβ (1-42) mediated damage with respect to memory impairment, neuroinflammation and PKC ε phosphorylation in the in vivo AD model consistent with the in vitro findings in SHSY5Y cells.

Abstract Alzheimer’s disease (AD) is a major public health concern worldwide, with an increasing prevalence in the elderly population. AD is a progressive neurological disorder of multi-faceted origin, where factors such as genetic mutations, biochemical changes, along with inflammatory cascade and soluble beta amyloid (Aβ) peptide, are thought to play a pivotal role in synaptic failure and neuronal death, ultimately leading to cognitive and neuropsychiatric decline in patients suffering from the disease. At present, there is no long-term cure for the disease, although there is access to pharmacotherapy that might improve cognitive and neuropsychiatric symptoms early in the course of the disease. The current pharmacological therapy for AD only provides symptomatic relief for a very short period of time. It is therefore of utmost importance to discover other pharmacological strategies that might delay the development of AD and slow down the disease progression in terms of cognitive decline and neurodegeneration. Elucidating the pathogenic mechanisms involved in AD neuropathogenesis is a major goal to find efficacious disease-modifying treatments. What remains to be understood completely are the intracellular pathways affected by Aβ protein which may lead to neurodegeneration in AD. Since phosphorylation and dephosphorylation mechanisms are crucial in the β-amyloid precursor protein (APP) metabolism, protein kinase C has emerged as one of the key regulators of the APP metabolism. Indeed, dysregulation of the PKC pathway might play a role in the intracellular mechanisms of neurodegeneration, but their effective involvement still remains elusive. Therefore, a detailed analysis of PKC pathways in established models of AD neurodegeneration is necessary and will form part of this work. Fucoidin is a sulphated polysaccharide extracted from edible brown seaweed, which has been shown to exhibit anti-inflammatory and anti-oxidant effects as well as being a neuroprotectant in various inflammatory diseases including hypoxic ischemia, atherosclerosis and Heyman nephritis. Therefore, fucoidin may have an inhibitory effect on the inflammatory mechanisms of AD. Little is known, however, about the effect of fucoidin on AD. Animal models of AD are extremely valuable for the discovery and development of new treatments. Rodents have been one of the preferred models for pharmacological and behavioural studies in AD. In this thesis, first aim was to establish a non-transgenic Aβ-induced AD model in rats. AD was induced utilising a published protocol which involved the bilateral injection of aggregated Aβ (1-42) into the CA3 subfield of the hippocampus in rat brain. Behavioural assessment with well defined tools such as the Morris water maze and T-maze were utilised to assess the impairment in spatial working memory in rats. Behavioural impairments along with increased astrocytosis and microgliosis were observed in this particular Aβ-induced AD model. In the established disease model, fucoidin (50 mg/kg/day and 25 mg/kg/day) and ibuprofen (50 mg/kg/day) were shown to provide a partial protective effect on impairment in memory function in the MWM behavioural task in rats treated prior to disease initiation and throughout the course of the study. In addition, the histopathological and quantitative analysis of AD brain sections showed a marked reduction in reactive glial fibrillary acidic protein (GFAP) and microglia in fucoidin (low and high dose) and ibuprofen treated Aβ injected rats compared to untreated Aβ injected rats. These results indicate that fucoidin may serve as a possible effective therapeutic approach to improve AD symptoms. There is strong evidence that PKC α and ε signalling pathways regulate important molecular events in memory impairment and neurodegenerative pathophysiology in AD. A possible neuroprotective mechanism of fucoidin involving attenuation of an Aβ-induced decrease in PKC ε phosphorylation using cultured SHSY5Y neuroblastoma cells as a model system was examined. Co-administration of fucoidin (2μM and 5 μM) with Aβ (1μM) abolished the inhibitory effect of Aβ on the phosphorylation of PKCε in a concentration-dependent manner as revealed by western blot analysis. These findings suggest that a possible mechanism underpinning the neuroprotective effect of fucoidin may be through prevention of A-induced inhibition of PKC phosphorylation and may serve as a possible therapeutic approach to improve AD symptoms. As cellular events that involve PKC are affected by Aβ in in vitro systems, it was necessary to examine whether PKC activity is also modulated by the Aβ treatment in vivo in our Aβ-peptide induced AD model. Therefore, the next aim was to assess the potential for fucoidin use as an intervention therapy in an established disease stage in the Aβ-peptide induced AD model. Intervention with fucoidin (50 mg/kg/day, i.p.) in the established disease stage partially prevented Aβ (1-42) mediated damage with respect to memory impairment, neuroinflammation and PKC ε phosphorylation in the in vivo AD model consistent with the in vitro findings in SHSY5Y cells.

<p>The Amyloid beta peptide (Ab) is related to Alzheimer’s disease and is suggested to be the molecular pathogenic species of the disease, probably through the neurotoxic effect of Ab oligomers. Here the results from biophysical studies of Ab and fragments thereof, are presented. Pulsed field gradient NMR diffusion experiments show that Ab exists mainly as an unfolded monomer. In addition, the hydrodynamic radius of Ab suggests that Ab has residual secondary structure propensities. CD experiments reveal that Ab has a high propensity to adopt a polyproline type II (PII) helix at low temperature. NMR diffusion measurements as well as the 3JHNH values show that increasing the temperature from 4 C induces a structure transition from PII propensity to a beta strand propensity around 15 C and to a random coil conformation at higher temperature. The small hydrodynamic radius at low temperature may be explained by the presence of a population of a hairpin conformation as was suggested by MD simulations. 15N relaxation and secondary chemical shifts suggest that Ab consists of 6 structural regions, two regions with high PII propensity are separated by a highly mobile region located in the N-terminal part of the peptide. In the C-terminal part two regions with a propensity to adopt b-strand are located, separated by a mobile region. The structural propensities of soluble monomeric Ab agree well with the structure of the peptide in fibril aggregates as well as in SDS micelles. Ab binds zinc specifically and with high affinity. This interaction was studied using heteronuclear correlation experiments. The metal ligands were determined to be three histidines, 6,13 and 14 and the N-terminus. The Ab peptide also binds b-cyclodextrin and the combined use of NMR diffusion experiments and induced chemical shifts show that Ab has at least two binding sites for b-cyclodextrin, and the dissociation constants of these binding sites were determined.</p>

The aim of the thesis project was to examine what form(s) of Amyloid beta (Aβ) (25-­‐35) peptide interact with phospholipids in vitro and the implications of this for the mechanism of Alzheimer’s Diseases (AD). The mechanism of AD is thought to involve protein folding and misfolding. An increasing amount of evidence has shown that protein misfolding plays an important role in the biological and pathological processes of AD. Although seen as the biomedical markers of those diseases, the roles of amyloid aggregates themselves are still not fully understood. Whether the aggregates, or the monomer, or some other intermediates of Aβ cause AD is still unknown. In order to investigate the membrane-­‐interaction of Aβ and its implications for AD, two forms of Aβ, namely levorotary and dextrorotary (L-­‐ and D-­‐) Aβ isomers were used. Evidence has shown that L-­‐ and D-­‐ peptide can each form aggregates in a humid environment. However, when mixed together, L-­‐ and D-­‐ peptides tend not to form any aggregates. Using the mixtures of L-­‐ and D-­‐ peptides at different proportions and as well as using L-­‐ and D-­‐ alone can help us to determine the toxic form of Aβ. Phospholipids have been used to mimic membrane bilayers. Biological membranes in vivo are a complicated system. They contain three types of lipids, namely phospholipids, glycolipids, and steroids. Different types of cells and different membranes have different proportions of those lipids. Studying the interaction between Aβ and membranes in vivo can be extremely difficult. Artificial membranes, which only contains one kind of lipids, on the other hand, are a useful tool for the study of molecular interactions. Phospholipids are the most abundant type of membrane lipid and thus that can be seen as representative of cell membranes. The interactions of Aβ and different kinds of phospholipids have been investigated in this project. This thesis discusses the secondary structure of Aβ in different environment, the interaction between Aβ and phospholipids at the air-­‐water surface, and the location of Aβ in membranes during the interaction. The study provides useful information of the mechanisms and the origin of AD. At the end of the thesis, a discussion chapter analyses the difficulties of studying Aβ and AD and the potentials and inadequacies of this research.

The Amyloid beta peptide (Ab) is related to Alzheimer’s disease and is suggested to be the molecular pathogenic species of the disease, probably through the neurotoxic effect of Ab oligomers. Here the results from biophysical studies of Ab and fragments thereof, are presented. Pulsed field gradient NMR diffusion experiments show that Ab exists mainly as an unfolded monomer. In addition, the hydrodynamic radius of Ab suggests that Ab has residual secondary structure propensities. CD experiments reveal that Ab has a high propensity to adopt a polyproline type II (PII) helix at low temperature. NMR diffusion measurements as well as the 3JHNH values show that increasing the temperature from 4 C induces a structure transition from PII propensity to a beta strand propensity around 15 C and to a random coil conformation at higher temperature. The small hydrodynamic radius at low temperature may be explained by the presence of a population of a hairpin conformation as was suggested by MD simulations. 15N relaxation and secondary chemical shifts suggest that Ab consists of 6 structural regions, two regions with high PII propensity are separated by a highly mobile region located in the N-terminal part of the peptide. In the C-terminal part two regions with a propensity to adopt b-strand are located, separated by a mobile region. The structural propensities of soluble monomeric Ab agree well with the structure of the peptide in fibril aggregates as well as in SDS micelles. Ab binds zinc specifically and with high affinity. This interaction was studied using heteronuclear correlation experiments. The metal ligands were determined to be three histidines, 6,13 and 14 and the N-terminus. The Ab peptide also binds b-cyclodextrin and the combined use of NMR diffusion experiments and induced chemical shifts show that Ab has at least two binding sites for b-cyclodextrin, and the dissociation constants of these binding sites were determined.

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptide (Aβ), the principal component of amyloid plaques present in Alzheimer’s disease. It consists of a core complex of presenilin (PS), nicastrin, anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2) proteins. PS harbors the catalytic aspartates required for regulated intramembrane proteolysis and the paralogs (PS1 and PS2) contribute to the assembly of distinct subpopulations of γ-secretases that may fulfill distinct roles. To characterize the molecular environments of distinct γ-secretases complexes in-depth quantitative comparisons were performed on 1) wild-type PS1 and its derivative carrying point mutations known to cause heritable early-onset AD in mice, and 2) PS1- or PS2-containing γ-secretase complexes equipped with N-terminal tandem-affinity purification (TAP) tags on PS paralogs in HEK293 cells. Isobaric labeling of co-purifying peptides for quantitative mass spectrometry revealed that γ-secretase complexes interact with other protein networks, including the cellular catenin-cadherin network, the molecular machinery that targets and fuses synaptic vesicles to cellular membranes, and the H+-transporting lysosomal ATPase macro-complex. The study revealed mature γ-secretase complexes containing PS1 or mutant PS1 to be indistinguishable in their protein composition, confirmed several previously proposed γ-secretase interactors, identified many novel interactors and uncovered a subset of proteins which can engage in robust interactions with γ-secretase complexes in individual cell types but may escape detection when whole brains are used as biological source materials. Interestingly, signal peptide peptidase (SPP), a Type II TM cleaving aspartyl protease, was pre-dominantly found to co-purify with PS2-containing γ-secretase complexes and could be shown not to influence their maturation but to affect cleavage or release of cellular Aβ. A model emerged from this work that suggests PS1 and PS2 paralogs may divide up the task of handling a broad range of membrane stubs at least in part by associating with different molecular environments.

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptide (Aβ), the principal component of amyloid plaques present in Alzheimer’s disease. It consists of a core complex of presenilin (PS), nicastrin, anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2) proteins. PS harbors the catalytic aspartates required for regulated intramembrane proteolysis and the paralogs (PS1 and PS2) contribute to the assembly of distinct subpopulations of γ-secretases that may fulfill distinct roles. To characterize the molecular environments of distinct γ-secretases complexes in-depth quantitative comparisons were performed on 1) wild-type PS1 and its derivative carrying point mutations known to cause heritable early-onset AD in mice, and 2) PS1- or PS2-containing γ-secretase complexes equipped with N-terminal tandem-affinity purification (TAP) tags on PS paralogs in HEK293 cells. Isobaric labeling of co-purifying peptides for quantitative mass spectrometry revealed that γ-secretase complexes interact with other protein networks, including the cellular catenin-cadherin network, the molecular machinery that targets and fuses synaptic vesicles to cellular membranes, and the H+-transporting lysosomal ATPase macro-complex. The study revealed mature γ-secretase complexes containing PS1 or mutant PS1 to be indistinguishable in their protein composition, confirmed several previously proposed γ-secretase interactors, identified many novel interactors and uncovered a subset of proteins which can engage in robust interactions with γ-secretase complexes in individual cell types but may escape detection when whole brains are used as biological source materials. Interestingly, signal peptide peptidase (SPP), a Type II TM cleaving aspartyl protease, was pre-dominantly found to co-purify with PS2-containing γ-secretase complexes and could be shown not to influence their maturation but to affect cleavage or release of cellular Aβ. A model emerged from this work that suggests PS1 and PS2 paralogs may divide up the task of handling a broad range of membrane stubs at least in part by associating with different molecular environments.