Impact of dietary vitamin B12 on amyloid-beta-induced proteotoxicity
There is currently no disease-modifying treatment for Alzheimer’s Disease (AD), the most common cause of dementia. Some AD risk factors including genetic predisposition and aging are non-modifiable while other risk factors such as diet can be altered to slow disease onset and progression. It is challenging to determine which nutrients are neuroprotective because humans and other mammals exhibit organismal complexity, genetic diversity, and consume complex diets. Pathological features of AD include accumulation of toxic amyloid-beta (Aβ), hyperphosphorylated tau, bioenergetic defects, altered mitochondrial morphology, and oxidative stress. We are using C. elegans, which eat E. coli bacteria to identify nutrients that impact Aβ-induced proteotoxicity.
Overexpression of toxic human Aβ peptides in the C. elegans body wall muscles generates oxidative stress, defects in mitochondrial morphology, reduced ATP levels, and robust time-dependent paralysis. We discovered that dietary vitamin B12 reduces the proteotoxic effects of Aβ in C. elegans (Lam et al., 2021). Supplementation of the OP50 E. coli diet with vitamin B12 delays Aβ-induced paralysis and alleviates mitochondrial fragmentation, bioenergetic defects, and oxidative stress. The protective effect of vitamin B12 requires methionine synthase, indicating that B12 in functioning as an enzyme cofactor, affecting the methionine/SAMe cycle, rather than simply as an antioxidant. Vitamin B12 is protective even when given to B12 deficient Aβ animals in adulthood, suggesting that B12 supplementation late in life could be beneficial. Our current work is focused on further defining how dietary vitamin B12 protects against Aβ proteotoxicity and utilizing a high-throughput screening pipeline to determine how loss of C. elegans homologs of genes associated with AD affects Aβ-induced paralysis and B12 protective capacity.
Mechanisms of extracellular vesicle cargo sorting and biogenesis
Secreted extracellular vesicles (EVs), membrane-wrapped structures that contain bioactive macromolecules, play a critical role in communication between cells during physiological processes as well as the progression of pathological conditions. The primary cilium is a sensory organelle protruding from cells that serves as a platform to not only receive signals, but also transmit signals via EV shedding. In C. elegans, EVs bud from sensory neuron cilia and then are either up-taken by surrounding glia or discharged into the environment where they play a role in animal-to-animal communication. Our discovery that the ion channel CLHM-1 localizes to the cilia of EV-releasing neurons and is packaged into EVs shed from the ciliary base sparked our interest in using C. elegans to answer unresolved questions in the field of EV biology.
We identified that tdTomato-tagged CLHM-1 and GFP-tagged PKD-2, another EV cargo, colocalize in cilia, but PKD-2 alone is found in the cilium distal tip and EVs shed from this site (Clupper et al., 2022). Shedding of base-derived CLHM-1 EVs increases in response to the presence of mating partners, while release of tip-derived PKD-2 EVs decreases under these conditions, suggesting that these subpopulations are differentially released in response to physiological stimulus. The heterotrimeric and homodimeric kinesin-2 motors play distinct roles in CLHM-1 EV biogenesis, colocalization of ciliary proteins, and EV cargo enrichment. Elimination of all kinesin-2 motors decreases shedding of EVs containing PKD- 2, but does not inhibit CLHM-1 cilia entry or inclusion in EVs, indicating that intraflagellar transport differentially impacts biogenesis of discrete EV subpopulations. Our current work continues to focus on how an individual cilium generates heterogeneous EVs with different signaling potentials.
Development of new genetics methods
Use of point mutants by geneticists is essential for discovery of gene function. To accelerate genotyping, we designed and optimized “SuperSelective” (SS) primers for rapid, inexpensive, end-point PCR genotyping (Touroutine and Tanis, 2020). Each SS primer contains a 5’ anchor which anneals to the template, followed by a non-complementary bridge sequence, and short 3’ foot sequence complementary to the target allele. We defined how specificity and efficiency are achieved and developed simple rules for SS primer design. This genotyping method can be used by researchers working across a wide range of disciplines and genetic systems.
Identification of regulators of postsynaptic function
Disruption of cholinergic signaling at the neuromuscular junction (NMJ) is the underlying cause of muscle weakness observed in individuals suffering from multiple neuromuscular disorders. Altered response to levamisole, a pharmacological agonist of C. elegans levamisole-sensitive ionotropic acetylcholine receptors (L-AChRs) can be used to identify genes important for postsynaptic cholinergic signaling (Davis and Tanis, 2022). We performed a genome-wide RNAi screen for altered levamisole sensitivity and identified 135 genes, including homologs of genes mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy, for which knockdown caused altered levamisole response (Chaya et al., 2021).
We have started to characterize two different classes of genes identified in our screen. Loss of the Epsin homolog epn-1 causes levamisole hypersensitivity and impacts postsynaptic receptor abundance, disrupting the proper balance of excitatory and inhibitory signaling in the body wall muscles. Knockdown of 14 different genes predicted to play a role in ATP synthesis results in levamisole hypersensitivity, suggesting that ATP depletion accelerates levamisole-induced paralysis. Consistent with this, treatment of animals with levamisole causes a L-AChR dependent reduction in ATP levels. Currently, work on this project is being carried out by students in the BISC413 Advanced Genetics lab, a course-based undergraduate research experience (CURE) designed to increase student research participation and inquiry-based learning.
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