γ-Secretase Dependent Production of Intracellular Domains Is Reduced in Adult Compared to Embryonic Rat Brain Membranes

BACKGROUND: gamma-Secretase is an intramembrane aspartyl protease whose cleavage of the amyloid precursor protein (APP) generates the amyloid beta-peptide (Abeta) and the APP intracellular domain. Abeta is widely believed to have a causative role in Alzheimer's disease pathogenesis, and therefore modulation of gamma-secretase activity has become a therapeutic goal. Besides APP, more than 50 substrates of gamma-secretase with different cellular functions during embryogenesis as well as adulthood have been revealed. Prior to gamma-secretase cleavage, substrates are ectodomain shedded, producing membrane bound C-terminal fragments (CTFs). PRINCIPAL FINDINGS: Here, we investigated gamma-secretase cleavage of five substrates; APP, Notch1, N-cadherin, ephrinB and p75 neurotrophin receptor (p75-NTR) in membranes isolated from embryonic, young or old adult rat brain by analyzing the release of the corresponding intracellular domains (ICDs) or Abeta40 by western blot analysis and ELISA respectively. The highest levels of all ICDs and Abeta were produced by embryonic membranes. In adult rat brain only cleavage of APP and Notch1 could be detected and the Abeta40 and ICD production from these substrates was similar in young and old adult rat brain. The CTF levels of Notch1, N-cadherin, ephrinB and p75-NTR were also clearly decreased in the adult brain compared to embryonic brain, whereas the APP CTF levels were only slightly decreased. CONCLUSIONS: In summary our data suggests that gamma-secretase dependent ICD production is down-regulated in the adult brain compared to embryonic brain. In addition, the present approach may be useful for evaluating the specificity of gamma-secretase inhibitors

Differences in proliferation rate between CADASIL and control vascular smooth muscle cells are related to increased TGF beta expression

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT-PCR analysis, we observed increased Transforming growth factor-beta (TGF beta) gene expression in CADASIL VSMCs. Adding TGF beta-neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGF beta-neutralizing antibody in ECs co-cultured with VSMCs. ECs co-cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co-cultured with control VSMCs, and neutralization of TGF beta normalized the proliferation rate of ECs co-cultured with CADASIL VSMCs. We suggest that increased TGF beta expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature.Peer reviewe

Differences in proliferation rate between CADASIL and control vascular smooth muscle cells are related to increased TGFβ expression

Cerebral autosomal‐dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT‐PCR analysis, we observed increased Transforming growth factor‐β (TGFβ) gene expression in CADASIL VSMCs. Adding TGFβ‐neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGFβ‐neutralizing antibody in ECs co‐cultured with VSMCs. ECs co‐cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co‐cultured with control VSMCs, and neutralization of TGFβ normalized the proliferation rate of ECs co‐cultured with CADASIL VSMCs. We suggest that increased TGFβ expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature. </p

Molecular anatomy of adult mouse leptomeninges.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology

Notch receptor processing and CNS disease

We have studied the relationship of Notch receptor processing and two diseases of the central nervous system: Alzheimer's disease (AD) and Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). The Notch receptor signalling pathway plays a crucial role during development in many different species for directing adjacent cells to adopt different fates. During maturation and ligand activation the Notch receptor undergoes a series of complex proteolytic cleavages before the intracellular domain is released and translocated to the nucleus where it affects transcription. The first cleavage (S 1) occurs during the transport out to the plasma membrane in the trans-Golgi compartment by a furin-like convertase. This cleavage is constitutive and generates a bipartite receptor, where the two Notch fragments are held together through non- covalent forces. The second cleavage (S2) occurs near the transmembrane region at the extracellular side when Notch is ligand- activated. The S2 cleavage is immediately followed by the third cleavage (S3) at the border of the transmembrane region and the intracellular side. The metalloprotease TACE (TNFalpha converting enzyme) has been shown to be responsible for the S2 cleavage, while the S3 cleavage is dependent on presenilins (PSs). The PSs have been genetically linked to familial AD and are involved in the gamma-secretase cleavage of the betaamyloid precursor protein (APP) and the production of the neurotoxic Abeta peptide, which causes senile plaques in the brain. There are four known mammalian Notch receptors (Notch I -Notch 4) with overlapping patterns of expression, but the role of the different Notch receptors is not that well understood. Mutations in the Notch 3 gene cause the genetic disorder CADASIL. CADASIL is an inherited form of stroke and dementia and the histopathological hallmarks are degeneration of vascular smooth muscle cells and deposits of granular osmiophilic material between the vascular smooth muscle cells. The mutations in the Notch 3 gene always affect a cysteine residue in the epidermal growth factor-like- repeats in the extracellular domain, but how this leads to CADASIL is poorly understood. There is an obvious need to produce therapeutical drugs that prevent the formation of Abeta peptide, in order to reduce plaque formation in AD, and inhibitors of PSs and the gamma-secretase cleavage would be useful. The fact that not only APP, but also Notch cleavage is controlled by PSs, however complicates the issue, and it will be important to find drugs that act differentially on Notch and APP. We show in a cell-based assay that PSs are absolutely required for S3/gamma-secretase processing of Notch and APP (paper I and 11). The assay was further developed and gamma-secreatse cleavage of Notch and APP could be measured by adding different gamma-secretase inhibitors and PS active site mutants. We demonstrate that the assay is specific and sensitive and can record small differences in the gamma-secretase processing of Notch and APP (paper 11). Furthermore, in an in vitro assay, we show that PSs are not the direct executors of Notch processing, but that the enzymatic activity is absolutely dependent on PSs. This notion is based on the observation that there is a specific Notch-processing activity in PS-containing cells, which is absent in PS-deficient cells, but the activity does not comigrate with PSs in protein purification (paper 11). To learn more in detail which parts of the PSI protein are crucial and responsible for gammasecretase cleavage of Notch and APP we set out to narrow down the PSI protein by deletions and mutations (paper III). We show that a five amino acid motif in the N-terminal fragment of PS I is necessary for gamma-secretase cleavage of both Notch and APP. Moreover, a tyrosine residue in position 288 is critical and the activity is restored when substituting this amino acid with hydrophobic or bulky, but not with charged residues. The endoproteolysis of PS I is in some but not all cases affected when different residues occupy the 288 position, indicating a more direct role of the Tyrosine 288 in the gamma-secretase activity. To address the cellular and biochemical mechanisms behind CADASIL, we have used a very prevalent mutation in the Notch 3 gene (R142C), which cause CADASIL in humans (paper IV). Using coculture experiments, western blot analysis and immunocytochemistry of stable cell lines expressing the CADASIL-mutated or wild type receptor, we show that the S I cleavage is impaired and less amount of the mutated receptor is expressed at the cell surface compared to wild type receptor. Furthermore, the mutated receptor is more prone to form intracellular aggregates, which are found in close vicinity to the Golgi network, but do not share the characteristics of an aggresome. In coculture experiments together with cells expressing the ligands, Serrate and Delta, we find that the Notch 3 receptor can respond to ligand activation but that there are no differences in signalling between the mutated and wild type receptor. In conclusion, we show that PSs are absolutely required for gamma-secretase cleavage of Notch and APP but that PSs are not the direct executors for Notch cleavage. The cell-based assay allows screening and detection of compounds that differentially affect processing of Notch and APP, which is crucial in the attempt to prevent AD without affecting Notch signalling. We find a critical motif in the PS I gene, tyrosine 288, which seems to affect gamma-secretase cleavage directly for both Notch and APP. Finally, cells expressing as CADASIL-mutated Notch 3 receptor show impaired S I cleavage and decreased expression at the cell surface, more intracellular aggregates but with normal ligand-induced signalling as compared to wild type Notch 3 receptor. These differences can be one of the reasons underlying the cellular mechanisms of CADASIL

The Empowerment of the European Parliament - An Analysis of its Role in the Development of the Co-decision Procedure

Abstract In this thesis the EP’s role and influence in the development of the co-decision procedure is analysed. Over the last decades the European Parliament has increased its power in the legislative process. It has been one of the central actors in the development of the co-decision procedure, established in the Maastricht Treaty and extended in the Amsterdam Treaty. The question of how the EP has enjoyed increased influence as a consequence of the extension of the co-decision procedure is also being asked. The theoretical framework is a combination of insights from both rational choice institutionalism and historical institutionalism and it is argued that a rational choice-historical institutionalist perspective is favourable in order to find a full explanation. The analysis shows that the EP due to the fact that the member states did not have full information and was not able to predict consequences of their decision of the establishment of the co-decision procedure. Hence the EP has managed to make an interpretation of how the procedure should be implemented and had a possibility to influence the development of the procedure. Trough creation of informal rules and institutions, which the EP has later managed to institutionalize in the Amsterdam Treaty, the EP has managed to maximize its interest and enjoyed an increased power by becoming co-legislator with the Council. Due to path dependency in the negotiations of Treaties, the member states have not been able to reverse decisions already taken and has had to further extend the co-decision procedure, even though the EP had interpreted the procedure in another way than originally intended. Key words: the European Parliament, historical institutionalism, rational choice institutionalism, co-decision procedure Words: 998

Upplevelsen av att vara närstående till en person i palliativ vård : En litteraturstudie

När en person närmar sig livets slut på grund av sjukdom, skador eller ålder övergår vården till att vara palliativ. Att vara närstående till en person i palliativ vård kan innebära stora förändringar. Samtidigt som den palliativa vården har som mål att erbjuda stöd till närstående att hantera sin situation framhåller tidigare forskning att närstående kan vara i stort behov av både mer och förbättrat stöd. Syftet med studien var att beskriva upplevelsen av att vara närstående till en person i palliativ vård. Metoden var en litteraturstudie av 12 kvalitativa artiklar publicerade i CINAHL som analyserades genom en kvalitativ manifest innehållsanalys med en induktiv ansats. Resultatet består av fem kategorier, 1) Att stå på sidan av vid lidande och försämring,2) Att vilja vara närvarande och ha en vårdande roll, 3) Att livet kretsar kring den sjuke och vardagen förändras, 4) Att ha behov av stöd och samhörighet, 5) Att förlora sin närstående och sin roll som vårdare. Slutsatsen är att närstående vill vara nära och stödja den sjuke personen samtidigt som de har ett eget behov av stöd. Sjuksköterskan kan möjliggöra för den närstående att vara närvarande och hantera vården av den sjuke personen genom information och utbildning och samtidigt bevara den närståendes välbefinnande genom tillgänglighet, stöd och bekräftelse