Background Traumatic brain injury (TBI) is a complex disease with a spectrum of symptoms and disabilities. Over the past decade TBI has become the focus of research due to growing epidemiological and clinical evidences that TBI incidences are strong risk factors for Alzheimer’s disease (AD). Major pathological hallmarks of AD are massive accumulations of amyloid-β peptide (Aβ) toxic oligomers and plaques. Neuroinflammation is also considered as a common denominator in AD and aging. The epidemiological and experimental studies have supported that non-steroidal anti-inflammatory drugs markedly reduce the age-related prevalence of AD and can slow amyloid deposition by mechanisms that still remain elusive. S100A9 is a multifunctional cytokine with diverse roles in the cell signaling pathways associated with inflammation and cancers. A widespread expression of S100A9 was also reported in many other ailments involving inflammatory processes, such as AD, malaria, cerebral ischemia and TBI, implying that S100A9 may be a universal biomarker of inflammation. The distinctive feature of S100A9 compared to other pro-inflammatory cytokines is its ability to self-assemble into amyloids, which may lead to the loss of its signaling functions and acquired amyloid cytotoxicity, exceeding that of Aβ. Methods S100A9 properties was studied under various ex vivo and in vitro conditions. First, human and mouse tissues with TBI and AD were subjected to microscopic, immunohistochemical and immunofluorescent techniques. Then, aged mouse treated with native, oligomeric and fibrillary S100A9 was also studied by using behavioral and neurochemical analysis. Moreover, S100A9 was established as a biomarker of dementia progression and compared with others such as Aβ42 and tau proteins, by studying cerebrospinal fluid (CSF) samples from different stages of dementia. Finally, in vitro experiments on S100A9 amyloidogenesis, co-aggregation with Aβ40 and Aβ42, digestion and cytotoxicity were also performed by using spectroscopic, atomic force microscopy and cell biology methods. Results S100A9-driven amyloid-neuroinflammatory cascade serves as a link between TBI and AD. We have found that S100A9 contributes to the plaque formation and intraneuronal responses in AD, being a part of the amyloid-neuroinflammatory cascade. In TBI we have found that extensive S100A9 neuronal production and amyloid self-assembly is triggered immediately after injury, leading to apoptotic pathways and neuronal loss. S100A9 is an integral component of both TBI precursor-plaques, formed prior to Aβ deposition, and AD plaques, characterized by different degree of amyloid maturation, indicating that all plaques are associated with inflammation. Both intra- and extracellular amyloid-neuroinflammatory cascades are intertwined and showed similar tendencies in human and mouse tissues in TBI and AD. Ex vivo findings are further supported by in vitro experiments on S100A9 amyloidogenesis, digestion and cytotoxicity. Importantly, being highly amyloidogenic itself, S100A9 can trigger and aggravate Aβ amyloid self-assembly and significantly contribute to amyloid cytotoxicity. Moreover, the CSF dynamics of S100A9 levels matches very closely the content of Aβ42 in AD, vascular dementia and mild cognitive impairment due to AD, emphasizing the involvement of S100A9 together with Aβ in the amyloid-neuroinflammatory cascade in these ailments. Conclusions The conclusions of this thesis is that the inflammatory pathways and S100A9 specifically represent a potential target for the therapeutic interventions during various post-TBI stages and far prior AD development to halt and reverse these damaging processes. / Role of pro-inflammatory S100A9 protein in amyloid-neuroinflammatory cascade in Alzheimer’s disease and traumatic brain injury

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background: Neuroinflammation is central to the aetiology of HIV-associated neurocognitive disorders (HAND) that are prevalent in late stage AIDS. ARV treatments are rolled out relatively late in the context of neuroinflammatory changes, so that their usefulness in directly preventing HAND is probably limited. It is common practice for HIV+ individuals in developing countries to make use of traditional/complimentary medicines. One such medicine is Sutherlandia frutescens - commonly consumed as a water infusion. We have also identified a new candidate complimentary medicine for use in this context - grape seed-derived proanthocyanidolic oligomers (PCO) have significant anti-inflammatory action in the peripheral compartment in the context of e.g. skeletal muscle injury, but have not been investigated in the context of either neuroinflammation or HIV/AIDS. Here the efficacy of these two substances as an anti-inflammatory modality in this context was investigated in an in vitro co-culture model of the blood-brain barrier (BBB). Methods: Single cultures of human astrocytes, HUVECs and primary human monocytes, as well as co-cultures (BBB), were stimulated with HIV-1 subtype B & C Tat protein and/or HL2/3 cell secretory proteins after pre-treatment with S. frutescens or PCO extracts. Effects of this pre-treatment on pro-inflammatory mediator expression and monocyte migration across the BBB were assessed. Results: In accordance with others, B Tat was more pro-inflammatory than C Tat, validating our model. S. frutescens decreased IL-1β secretion significantly (P<0.0001), but exacerbated both monocyte chemoattractant protein-1 (P<0001) – a major role player in HIV-associated neuroinflammation – and CD14+ monocyte infiltration across the BBB (P<0.01). PCO pre-treatment resulted in a significantly dampened IL-1β (P<0.0001) response to stimulation with HIV-associated proteins. In contrast to S. frutescens, PCO modulated monocyte chemoattractant protein-1 (P<0001) response and decreased capacity for CD14+ monocytes to migrate across the simulated BBB (P<0.0001). Additionally, PCO pre-treatment decreased both GFAP (P<0.001) and HSP-27 (P<0.001) expression in the astrocytes of the BBB. Conclusions: Current data illustrates that the combined use of HL2/3 cells and the simulated BBB presents an accurate, disease relevant in vitro model with which to study neuroinflammation in the context of HIV/AIDS. In addition, our results caution against the use of S. frutescens as anti-inflammatory modality at any stage post-HIV infection. Novel data presented here illustrate that PCO is able to blunt the MCP-1 and IL-1β response to HIV-1 proteins in single cultures of human astrocytes and HUVECs, as well as in an in vitro simulation of the BBB. In addition, PCO was able to limit monocyte transmigration across the simulated BBB in response to HIV-1 proteins generated by HL2/3 cells. This suggests that grape seed-derived PCO could be considered as complimentary anti-neuroinflammatory drug in the context of HIV/AIDS. / AFRIKAANSE OPSOMMING: Agtergrond: Neuroinflammasie staan sentraal in die ontwikkeling van MIV-verwante toestande wat gekenmerk word deur neurokognitiewe afteruitgang, veral in die later stadia van die siekte. Aangesien anti-virale middels relatief laat toegedien word in die konteks van neuroinflammasie, is hul rol in die voorkoming van neuroinflammatoriese veranderinge heel moontlik weglaatbaar. MIV+ individue, veral in ontwikkelende lande, gebruik algemeen natuurlike medisinale preparate. Sutherlandia frutescens is een so „n middel wat as „n tee ingeneem word. Verder het ons ook „n nuwe kandidaat komplimentêre medisyne identifiseer – druiwepitekstrak wat polifenole bevat (PCO) het aansienlike anti-inflammatoriese eienskappe in die periferie, bv. in die konteks van skeletspierskade, maar die middel is nog nie voorheen in die konteks van neuroinflammasie of MIV/VIGS ondersoek nie. Hier word die anti-inflammatoriese effektiwiteit van beide middels in hierdie konteks ondersoek deur gebruik te maak van „n in vitro simulasie van die bloedbreinskans (BBS). Metodes: Kulture van menslike astrosiete, menslike naelstring endoteelselle (HUVECs) en primêre menslike monosiete, sowel as gesamentlike kulture (BBS) is met MIV-1 subtipe B en C Tat proteïen en/of HL2/3 selprodukte gestimuleer na voorafbehandeling met S. frutescens of PCO ekstrakte. Effekte op pro-inflammatoriese mediator uitdrukking sowel as monosiet migrasie oor die BBS is ondersoek. Resultate: In ooreenstemming met die literatuur was B Tat meer inflammatories as C Tat, wat die akkuraatheid en gepastheid van ons model bevestig. . S. frutescens het afskeiding van IL-1β betekenisvol verminder (P<0.0001), maar het afskeiding van beide monosiet chemoaantrekkingsproteïen-1 – „n groot rolspeler in MIV-verwante neuroinflammasie – en CD14+ monosiet migrasie oor die BBS vererger (P<0.0001 en P<0.01 onderskeidelik). PCO behandeling het „n betekenisvolle demping van die IL-1β reaksie (P<0.0001) op stimulasie met MIV-geassosieerde proteïene tot gevolg gehad. Anders as S. frutescens het PCO die MCP-1 reaksie, asook CD14+ monosiet migrasie betekenisvol inhibeer. Verder het PCO ook beide GFAP en HSP-27 uitdrukking in astrosiete van die BBS verminder (beide P<0.001). Gevolgtrekkings: Huidige data wys dat die gekombineerde gebruik van HL2/3 selle en die gesimuleerde BBS „n akkurate en fisiologies relevante in vitro model daarstel, waarmee neuroinflammasie in die konteks van MIV/VIGS bestudeer kan word. Ons resultate waarsku verder teen die gebruik van S. frutescens as anti-inflammatoriese middel in selfs die vroeë stadium na MIV infeksie. Oorspronklike data wat hier aangebied word illustreer dat PCO die pro-inflammatoriese reaksie op MIV-proteïene in kulture van astrosiete en HUVECs, asook die in vitro simulasie van die BBS, effektief demp. Verder het PCO die vermoë getoon om monosiet migrasie oor die BBS, in reaksie op MIV-1 proteïene wat hul oorsprong uit HL2/3 selle het, te beperk. Hierdie bevindings beteken dat PCO dus eerder as S. frutescens oorweeg moet word as komplimentêre anti-inflammatoriese medisyne in die konteks van MIV/VIGS.

Stimulator of interferon genes (STING) is activated after detection of cytoplasmic dsDNA by cGAS (cyclic GMP-AMP synthase) as part of the innate immunity defence against viral pathogens. STING binds TANK-binding kinase 1 (TBK1). TBK1 mutations are associated with familial amyotrophic lateral sclerosis, and the STING pathway has been implicated in the pathogenesis of further neurodegenerative diseases. To test whether STING activation is sufficient to induce neurodegeneration, we analysed a mouse model that expresses the constitutively active STING variant N153S. In this model, we focused on dopaminergic neurons, which are particularly sensitive to stress and represent a circumscribed population that can be precisely quantified. In adult mice expressing N153S STING, the number of dopaminergic neurons was smaller than in controls, as was the density of dopaminergic axon terminals and the concentration of dopamine in the striatum. We also observed alpha-synuclein pathology and a lower density of synaptic puncta. Neuroinflammation was quantified by staining astroglia and microglia, by measuring mRNAs, proteins and nuclear translocation of transcription factors. These neuroinflammatory markers were already elevated in juvenile mice although at this age the number of dopaminergic neurons was still unaffected, thus preceding the degeneration of dopaminergic neurons. More neuroinflammatory markers were blunted in mice deficient for inflammasomes than in mice deficient for signalling by type I interferons. Neurodegeneration, however, was blunted in both mice. Collectively, these findings demonstrate that chronic activation of the STING pathway is sufficient to cause degeneration of dopaminergic neurons. Targeting the STING pathway could therefore be beneficial in Parkinson’s disease and further neurodegenerative diseases.

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