Unravelling the mechanisms of neurological damage during cryptococcal infection of the brain

Lay summary

The burden of infectious diseases of the brain is incapacitating to the socio-economic development of Africa. Some of the diseases are uniquely prevalent in Africa and are usually neglected in both global research and policy creating a need for African neuroscientists to prioritise and develop local solutions for these challenges. Examples of such diseases include fungal infections of the brain such as cryptococcal meningitis which kills more than 200 000 people each year in Sub-Saharan Africa and also causes neurological dysfunction and disability in survivors, most of which are of an economically productive age. Cryptococcal meningitis (CM) is the leading cause of HIV-associated meningitis, which is characterised by debilitating, inflammatory injury of the brain and a very high chance of death, even with treatment. In Sub-Saharan Africa, the number of people who die from CM and its complications is worsened by the unavailability and inaccessibility of safe and effective drugs. Although a lot of work has been done to describe the biology of the fungal pathogen that causes CM, we know very little about how the human body responds to the fungus especially at the level of the brain, which is the most affected organ in those who succumb to the disease.

One of the challenges faced by researchers intending to study brain injury in CM is the lack of appropriate experimental models that provide a good representation of the real clinical form of the disease with good enough resolution to help delineate the complex mechanisms underlying brain damage from this fatal condition. Knowledge of these mechanisms is critical to our understanding of how the disease progresses, as well as to our efforts of developing cheaper and safer drugs that can be accessed by most African people. The first part of this study therefore focusses on introducing a new organ-specific, host-specific experimental model that could be used for studying mechanisms underlying brain injury during cryptococcal infection. The proposed model is based on using both rodent and human brain tissue slices which are carefully kept alive in culture then stimulated with Cryptococcus neoformans, the causative fungus for CM. I will investigate the activation of key inflammatory machinery in immune cells of the brain including receptors for pathogen recognition, transcription factors and other chemical communication molecules released when fighting the fungus. Using state-of-the-art scientific techniques, I will also investigate the contribution of each cell type to the brain's response to the fungus. To compliment data obtained using this novel model, the second part of my proposed research aims to use a living rodent model to recapitulate the human form of CM. Using this model we will be able to describe where in the brain the fungus goes after infection and most importantly, to establish if fungal cells indeed block the fluid clearance pathways of the brain which were discovered recently.

This research will fill the existing knowledge gap on how the brain is injured in CM. This models and methods would then serve as a platform for studying the mechanisms of other infections of the CNS that are caused by bacteria, parasites and viruses to inform the development of the much-needed new therapies.

Cryptococcal meningitis (CM) is a fatal, neglected fungal infection of the brain that mainly affects immunosuppressed individuals and is associated with high mortality rates of up 70% with treatment and 100% if untreated. Due to a high burden of HIV/AIDS, Sub-Saharan Africa accounts for 73% of all global cases, and 75% of CM-associated deaths per year. Caused by Cryptococcus neoformans, CM is is often accompanied by debilitating neurological damage but the neuroimmune mechanisms underlying this brain injury remain unknown. We hypothesise that brain injury may be the result of a dysregulated excessive or inadequate neuroimmune response to the fungus and/or from disruption of immune homeostasis within the brain. In Aim 1, we will use a novel organotypic slice culture system based on both rodent and human brain tissue to characterise cell-type specific neuroimmune responses to C. neoformans. Immunofluorescence staining will be used to identify pathogen recognition and neuroimmune signalling pathways that are activated upon recognition of the fungus. We will use single nucleus RNA sequencing to determine cell-type specific changes in gene expression, and multiplexing assays for measuring cytokine release. In Aim 2, we will use an in vivo mouse model to characterise the spatiotemporal distribution of C. neoformans and the accompanying neuroimmune events that occur when it invades the brain. We will explore the hypothesis that cryptococcal cells aggregate in perivascular spaces and block the glymphatic system responsible for fluid clearance within the brain, leading to increased intracranial pressure. Using tracers, we will measure the rate of fluid clearance within the brain. Finally, we will characterise
spatiotemporal gene expression using next-generation spatial transcriptomics sequencing. Overall, this study will use novel and cutting-edge techniques to describe how brain injury occurs in CM. This knowledge is critical to inform the development of future therapies.