Dr Bradlee Heckmann and his team at the USF Health Neuroscience Institute in the US aim to develop new therapeutic avenues for treating neuroinflammatory and neurodegenerative diseases, including Alzheimer’s.
Read their original article here: http://doi.org/10.4103/1673-5374.317958
Read more about their work in Research Features
Image Credit: nobeastsofierce/Shutterstock
Hello and welcome to Research Pod! Thank you for listening and joining us today.
In this episode, we will be looking at the research of Dr Bradlee Heckmann and his team at the USF Health Neuroscience Institute in the US. The Heckmann laboratory aims to develop new therapeutic avenues for treating neuroinflammatory and neurodegenerative diseases, including Alzheimer’s.
The key phenomena involved in the inception and progression of neurodegenerative diseases, such as Alzheimer’s disease, are neuroinflammation and neurodegeneration. Conventional pathways of autophagy involve the removal of damaged organelles, protein aggregates, and other cellular components through internalisation and endocytosis. Endocytosis is a process by which a cell engulfs a foreign protein and lyses it internally. This ensures that cellular and metabolic homeostasis is maintained. Proteins like Beclin1, ATG5, and ATG7 are instrumental in regulating such autophagic pathways. They also function in the brain’s innate immune microglial cells to induce receptor-mediated endocytosis and prevent excess build-up of ß-amyloid peptide oligomers, a classic hallmark for Alzheimer’s disease progression. Additionally, autophagy regulates various immune pathways. It controls the secretion of pro-inflammatory cytokines such as type-I interferon and interleukin-1ß by targeting the interleukin-1ß precursor and pro-interleukin-1ß for degradation.
Microglial cells, the flag-bearers of immunity in the central nervous system, undergo autophagic mechanisms with the help of toll-like receptors, or TLRs, Fc receptors, immunoglobulin-superfamily receptors, scavenger receptors, and complement receptors. The expressions of Beclin1, ATG5, and ATG7 as well as the key non-canonical autophagy protein Rubicon, reduce proportionally with age. Unsurprisingly, patients with neurodegenerative disorders have shown drastically reduced levels of these proteins.
Dr Bradlee Heckmann and his collaborators have reviewed and conducted multiple studies investigating the role of the conventional autophagic mechanism and the non-canonical uses of the autophagy machinery in distinct pathways. Through these investigations, they identified the LC3-associated endocytosis, or LANDO, pathway to be a key player in modulating neuroinflammation. It uses components of the autophagic machinery and Rubicon to target LC3 to endosomes, thereby preventing exacerbated ß-amyloid accumulation and mitigating ß-amyloid induced neuroinflammation in mouse models of Alzheimer’s disease.
LC3 is pivotal in the selection of phagocytic markers and the biogenesis of autophagosomes. Previous pre-clinical studies by Heckmann and his long-time mentor and collaborator Dr Douglas Green have shown non-canonical autophagic pathways, like LANDO, to be crucial in the conjugation of LC3 to Rab5+, clathrin+ endosomes containing ß-amyloid in murine microglia. Heckmann has already considered the importance of accumulation of ß-amyloid plaques and hyperphosphorylation of the tau protein in Alzheimer’s. He and his team of researchers have previously conducted various experiments to determine the importance of LANDO in mouse models with different combinations of conventional autophagic mechanisms.
Experiments with murine models genetically engineered to lack LANDO by removing the Rubicon, a required regulator of LANDO but not canonical autophagy in microglia, showed a significant increase in pro-inflammatory cytokine production in the hippocampus. This was associated with increased levels of neurotoxic ß-amyloid protein, increased reactive microgliosis, and tau hyperphosphorylation – all of which are crucial markers of Alzheimer’s disease pathology. Reactive microgliosis is an inflammatory reaction by microglia, the primary innate immune effector cells in the CNS. Increased tau hyperphosphorylation means a process by which the otherwise integral, neuronal tau protein present in the microtubules gets detached from the parent neurons, and forms neurofibrillary tangles, leading to neurodegenerative pathologies.
Untreated, these will eventually lead to the collapse of the microtubular architecture within the neurones and axons. There were signs of accelerated neurodegeneration, impaired neuronal signalling, and memory deficits in Alzheimer’s disease-induced mice models with LANDO deficiency.
In a different experiment, another set of LANDO-deficient 5xFAD [SS1] mice had a similar fate, with severe ß-amyloid accumulation that promoted reactive microgliosis and increased tau hyperphosphorylation.
Previous research by Heckmann’s team has also explored the possibility of deficiencies of canonical autophagic pathways by genetically engineering [SS2] mice models and removing myeloid or microglia-specific FIP200 protein, which is required for conventional autophagic activation. Their data suggest that depletion of myeloid or microglia-specific FIP200 had no impact on tau phosphorylation. They thus concluded that depleting regulators of non-canonical autophagic mechanisms, like Rubicon, promoted tau phosphorylation throughout the hippocampus and the cerebral cortex. Their data emphasises the importance of LANDO in neuroinflammation and progressive neurodegenerative disease.
Another experiment was conducted with animal models whose primary microglia lacked the WD-domain of ATG16L. This is one of the most abundant domains in eukaryotic genomes, responsible for diverse cellular processes. It’s also required for LANDO activation and an important regulator of anti-inflammatory signals. As such, these LANDO-deficient models presented with a severe impairment in the recycling of TREM2, TLR4, and CD36, with increased deposition of extracellular, neurotoxic ß-amyloid, specifically neurotoxic ß-amyloid 1–40 and 1–42, in the hippocampus and cortex within two years.
Of the different non-canonical autophagic pathways discovered thus far, LC3-associated phagocytosis, or LAP, is another crucial pathway that uses the autophagic machinery with a similar mechanism as LANDO, albeit being different cellular entities. The role of LAP in mice concerning Alzheimer’s disease risks is not clearly understood.
In mouse models with FIP200-deficient microglia, there was no change as such in the development of Alzheimer’s disease-like pathology, similar to mice that are sufficient in autophagy. Researchers thus identified that a primary contributing factor for Aß accumulation in mice with LANDO-deficient microglia was not due to a defect in degradation, as is common for LAP-deficiency. Rather, it was caused by impaired recycling of receptors that recognise ß-amyloid, including TLR4 and TREM2. This indicated a difference in the importance of LAP versus LANDO in mitigating neuroinflammation, at least in the context of Alzheimer’s disease pathology.
The WD-domain of ATG16L, similar to Rubicon, interacts directly with the cytokine receptors responsible for anti-inflammatory cytokines and those responsible for adaptive immunity, which participate in downstream signalling events leading to LC3 lipidation. These include IL-10RB for IL-22R and IL-26R, and IL-2R for IL-2. Removing the WD-domain of ATG16L, similar to the Rubicon-deficient animals, reduced anti-inflammatory signalling due to delayed endocytosis and insufficient recruitment of anti-inflammatory cytokine complexes leading to increased inflammation.
Further research to differentiate between different stimuli, ranging from amyloids and extracellular aggregates to microbial pathogens, is needed to segregate specific triggers for specific pathways such as LAP and LANDO, both globally and in the context of neuroprotection against Alzheimer’s and similar diseases. However, irrespective of these differences, it is now clear that the non-canonical autophagy machinery is crucial to suppress inflammation and any error in this machinery can lead to production of pro-inflammatory cytokines and subsequent neurodegeneration.
Moreover, neuroinflammation due to impaired autophagic pathways has also been identified as a risk factor for other CNS diseases including Huntington’s and amyotrophic lateral sclerosis.
Every research experiment elaborated so far highlights the potential of non-canonical autophagic mechanisms as a therapeutic target for treating Alzheimer’s and other CNS-based diseases. This would imply targeting inflammatory cytokine production and/or signalling for mediators including IL-1ß and the NLRP3 inflammasome, as well as TNFa and IL-6. Heckmann and his team used this idea and have inhibited NLRP3 inflammasome using the NLRP3 inhibitor MCC950, which they tested on ATG16L WD-domain deficient mice with established Alzheimer’s-like disease for eight weeks. Results showed that MCC950-treated mice had comparable levels of Aß to those observed in vehicle-treated animals and restored approximately 80–90% behaviour and memory capacity. The vehicle-treated animals continued to have a decline in memory from the onset of therapy.
Mounting evidence is suggesting the root cause of Alzheimer’s disease-associated neurodegeneration is neuroinflammation, which is being addressed in future studies as well. Such studies also shed light on the importance of targeting specific inflammatory-cytokines like IL-1ß in an established disease model.
A further step in this regard would be to ensure the translational significance of determining such therapeutic targets, by implementing such studies in the human population and ensuring minimal adverse effects.
That’s all for this episode – thanks for listening, and stay subscribed to Research Pod for more of the latest science. See you again soon.