Aβ is one of the major neurotoxic factors responsible for neurodegeneration in AD. Exposure to Aβ alone is sufficient to cause progressive neuronal death. Although Aβ is indeed neurotoxic by itself, the priming of neuronal inflammasomes, including NLRP1, by an external stimulus such as MDP significantly aggravates neuronal death as shown by the drastic decrease in neuronal viability in Aβ + MDP treatment group. Interestingly, however, MDP by itself did not cause a significant change in cell viability, suggesting that inflammasome activation alone did not result in immediate cell death. It is also unlikely that Aβ cause neuronal death via the NLRP1 inflammasome pathway as, by itself, it did not significantly elevate NLRP1 level, thus having insufficient caspase-1 activity to trigger pyroptosis (Fig. 3). However, when neurons were co-exposed with MDP, Aβ was able to significantly elevate caspase-1 activity, resulting in a drastic loss of neuronal viability. These results suggest that, while inflammasome and caspase-1 activation does not necessarily lead to neuronal death, co-stimulation with an external inflammagenic substance could lead inflammasome signalling into an overdrive sufficient to induce pyroptosis, thus significantly accelerating cell death. Therefore, the exposure of the brain to immunogenic stimuli such as pathogens, which are increasingly recognised for their roles in AD , could drastically accelerate neuronal death via inflammasome activation and pyroptosis. Indeed, it was proposed that Aβ is deposited in the brain as part of an innate immune response against pathogenic infections .
The random genomic mutagenesis generated by gene trapping is a useful tool to screen for molecular mechanisms underlying a biological process. Unlike other gene silencing tools, such as siRNA, gene traps produce a permanent heterozygous (monoallelic) knockout which is equivalent to 50% reduction in gene product expression . This is particularly advantageous as homozygous knockouts could be lethal, and human mutations are often heterozygous in nature. With FDR cut-off set to 0.05, the gene trap mutagenesis phenotypic screen discovered seven significant genes which could highlight the underlying signalling pathways regulating neuronal survival and death when subjected to Aβ and MDP-primed NLRP1 inflammasome. The most significant of these ‘hit’ genes are RHOT1 and CNP with mutant allele representation of log FC = 7.11 (‘wild-type’ susceptibility) and − 7.15 (‘wild-type’ protective) respectively in the Aβ + MDP group. Both RHOT1 and CNP has roles in mitochondrial toxicity, presumably a crucial factor in Aβ-induced NLRP1-mediated neuronal death. The RHOT1 gene encodes the Ras Homolog Family Member T1 protein, also known as Mitochondrial Rho GTPase 1 (Miro1). Miro1 is a member of the Rho family of GTPases, which in turn belongs to the Ras superfamily of small GTPases. Rho GTPases are typically small G proteins which are responsible for the regulation of many essential biological processes including cell growth, transcriptional regulation, membrane trafficking and cell motility [24, 25]. Miro1 is essential in healthy neuronal function. Germline Miro1 knockout in mouse embryo die shortly after birth, and conditional knockout of neuronal Miro1 in a mouse model led to defects in mitochondria distribution and retrograde axonal mitochondrial movement, resulting in impaired motor neuron function . However, Miro1 may also contribute to the pathogenesis of neurodegenerative diseases such as Parkinson’s disease (PD) . Miro1 is known to interact with the PD-associated proteins, parkin and PINK1, where Miro1 is phosphorylated by PINK1 and subsequently ubiquitinylated by parkin, leading to its degradation by proteasomes [28, 29]. The degradation of Miro1 causes an arrest in mitochondria trafficking which quarantines defective mitochondria and prevents them from causing further damage to neighbouring healthy mitochondria. This immobilization of mitochondrial movement also facilitates its selective degradation via phagosomes and autolysosomes, a process known as mitophagy. Impaired Miro1 degradation disrupts mitophagic clearance of defective mitochondria which may elevate the production of reactive oxygen species (ROS) leading to the activation of the NLRP3 inflammasome [30, 31]. Considering that Miro1-mediated neurotoxicity was only observed at a significant level in the Aβ + MDP group, we hypothesize that Aβ and the NLRP1 inflammasome could synergistically induce neuronal death through mitochondria dysfunction as in PD. It is possible that lack of Miro1 production in the library cells encourages mitophagic clearance of defective mitochondria and enabled cell survival.
Conversely, the CNP gene which encodes 2',3'-cyclic nucleotide 3' phosphodiesterase (CNPase) was found to be highly protective against Aβ-induced NLRP1-mediated neurotoxicity as indicated by a log FC = -7.15 for mutant CNP in the Aβ + MDP group. In the 2’,3’-cAMP-adenosine pathway, CNPase is found to convert 2’,3’-cAMP to 2’-AMP which is further metabolized into adenosine . Elevated adenosine confers neuroprotective effects against excitotoxic neuronal damage . CNPase is also known to confer neuroprotection against mitochondria-induced toxicity. Specifically, CNPase regulates the opening of mitochondrial permeability transition pore (mPTP), a Ca2+-gated channel which is formed in mitochondria in response to Ca2+ overload or oxidative stress in order to increase the permeability of mitochondrial membrane [34, 35]. Open mPTP abruptly increases the permeability of mitochondrial inner membrane (which is usually almost impermeable to solutes or ions) to molecules up to 1,500 Da, leading to excessive mitochondrial swelling and cell death by apoptosis or necrosis . It is possible that mutation of CNP gene encourages the opening of mPTP in response to Ca2+ and ROS imbalance in the cells as a result of Aβ and MDP treatment.
The mutagenesis screen also identified two genes involved in TGF-β signalling, TGFBR1 and SNW1. The TGFBR1 gene encodes the TGF-β receptor type I (TGFBR1) protein which functions as a signal transduction component in TGF-β signalling. TGFBR1 is integrated to the perpetually activated TGFBR2 as a dimer which spans the entire cell membrane. Upon binding to the extracellular ligands, principally TGF-β cytokines, TGFBR1 is phosphorylated by TGFBR2, which in turn phosphorylates the intracellular SMAD family of proteins, specifically receptor-regulated SMAD proteins (R-SMADs) which consists of SMAD2 and SMAD3 . Phosphorylated R-SMADs then accumulate into complexes with SMAD4 (a common SMAD or co-SMAD) which together act as transcription factors that signal the expression of target genes. As a negative regulatory mechanism for TGF-β signalling, the transcription factor SKI acts as a co-repressor which binds to SMAD2-4, forming a complex which negatively regulates TGF-β response . The SKI-interacting protein (SKIP), encoded by the SNW1 gene, binds to SKI and antagonises its repressor effect, thereby releasing SMADs and allowing for their phosphorylation by TGFBR1, thus augmenting TGF-β signalling . Our functional screen shows that silencing TGFBR1 enabled neuronal survival in all three treatment groups at similar log FC in allele representation (mutant gene Log FC + 3, Table 1), suggesting that TGFBR1 confers susceptibility in response to toxic environment not specific to either Aβ or MDP toxicity. SNW1, however, show an opposing effect in Aβ and MDP groups with similar log FC (mutant gene Log FC = -3, Table 1) but not in the Aβ + MDP group where there are no significant log FC compared to control, suggesting that activation of other pathways override the protective effects of SNW1. TGF-β signalling is a heterogenous signalling pathway that regulates many biological aspects such as cell proliferation and immunity . The roles of TGF-β signalling in neuroinflammation and AD have been conflicting. It has been reported to promote neuroinflammation in AD by encouraging the release of pro-inflammatory cytokines such as IL-1β and TNF-α in microvessels isolated in AD cortices  and blocking TGF-β and its downstream SMAD2/3 signalling attenuates AD pathology . Other studies indicate that TGF-β is instead protective in nature, and its deficiency promotes neurodegeneration in AD . Further studies are required to evaluate the impact of TGF-β signalling, as well as its individual components, in neuroinflammation and AD pathogenesis.
Apart from TGFBR1 and SNW1 which constitute TGF-β signalling, the functional screen identified SRGAP3 as a protective gene unique to the Aβ group. The SRGAP3 gene encodes the Slit-Robo Rho GTPase Activating Protein 3 (srGAP3), also known as WAVE-Associated Rac GTPase Activating Protein (WRP) or Mental Disorder-Associated GTPase Activating Protein (MEGAP), which belongs to the Rho GAP family of Rho GTPase negative regulators. srGAP3 is involved in a variety of processes which are crucial for neuronal morphogenesis, axon guidance and synaptic functions . srGAP3 possibly confers neuroprotection against Aβ toxicity by regulating cytoskeletal reorganisation or participating in Slit-Robo signalling, both of which are vital for neurodevelopment and healthy cognitive functions . However, this neuroprotective effect is nullified in the presence of MDP, suggesting that the activation of NLRP1 inflammasome perturbs srGAP3 function and probably the regulation of cytoskeletal dynamics, which drastically reduced cellular rigidity against toxicity. This could have contributed to the synergism between Aβ and MDP-primed NLRP1 inflammasome in inducing severe loss of neuronal viability in the Aβ + MDP group.
BEND5 and GREB1 showed significance in the MDP group only, suggesting that exposure to Aβ negated the effects exerted by these genes. As with SNW1, BEND5 is involved in DNA/RNA biding and transcription regulation. The BEND5 gene encodes the protein BEN domain-containing protein 5. The mammalian BEND5 is a protein that contains a single BEN domain and is expressed in particularly high levels in pyramidal neurons . It functions as a transcriptional repressor and is likely involved neurogenesis by regulating the Notch signalling pathway. The GREB1 gene codes for the growth-regulating oestrogen receptor binding 1 protein. GREB1 is typically expressed upon binding of oestrogen response elements on oestrogen receptor 1 (ESR1) upstream of GREB1 promoter .Treatment with MDP alone did not result in significant loss of neuronal viability. It is possible that the effects of these mutant genes under MDP treatment affect cell proliferation rates rather than inducing neurodegeneration.
While the gene trap mutagenesis screening provides a convenient and high-throughput method for preliminary elucidation of biological pathways, screen efficiency is potentially limited by several factors. Firstly, the efficiency of gene trap integration is dependent on the initial nucleofection process. Secondly, although pGTIV3 does not display a 3’ intron bias, its inherent requirement for intronic integration means that genes that lack, or contain very small, introns may escape trapping. This presents a major challenge in unbiased genome-wide mutagenesis by achieving complete genome saturation. Finally, the mutagenesis screen does not inform the order of gene activities and pathways involved - whether they occur upstream or downstream of NLRP1 inflammasome activation by Aβ.