Biology subjects

Frost, M. P.

Publications and source records attributed to Frost, M. P..

4 recordsLinked to original sources

bioRxiv2026

Adaptation of α-synuclein fibrils following multiple system atrophy transmission to mice

Synucleinopathies are a group of neurodegenerative diseases characterized by the presence of misfolded -synuclein inclusions which cause progressive disease by spreading throughout the brain in a prion-like manner. Throughout the neurodegenerative disease field, the ability of a single protein to give rise to multiple distinct clinical disorders is explained by the strain hypothesis, or the idea that the misfolded protein conformation determines the resulting disease. This was initially shown using transmission studies in cell lines and mouse models; more recently cryo-electron microscopy (cryo-EM) validated this idea by identifying distinct -synuclein filament folds in brain tissues from patients with Parkinsons disease, multiple system atrophy (MSA), and juvenile-onset synucleinopathy. However, very little is known about the -synuclein filament structures that form in animal models of these disorders, and thus their relevance to human disease and suitability as models for therapeutic development remains a question. Here we report the first atomic resolution cryo-EM structures of -synuclein fibrils from an MSA patient sample before and after transmission to a transgenic mouse model of disease. Our findings indicate that while distinct adaptations occur during fibril replication in the mouse host, key structural facets are maintained, validating the merits of this transmission model for supporting preclinical research on MSA.

neuroscience↗

bioRxiv2025

An independently tunable dual control system for RNAi complementation in Trypanosoma brucei

Trypanosoma brucei is a tractable protist parasite for which many genetic tools have been developed to study novel biology. A striking feature of T. brucei is the catenated mitochondrial DNA network called the kinetoplast DNA (kDNA) that is essential for parasite survival and life cycle completion. Maintenance of kDNA requires three independently essential paralogs that have homology to bacterial DNA polymerase I (POLIB, POLIC and POLID). We previously demonstrated that POLIB has a divergent domain architecture that displayed enzymatic properties atypical for replicative DNA polymerases. To evaluate the functional domains required for kDNA replication in vivo, we pursued an RNAi complementation approach based on the widely used tetracycline (Tet) single inducer system. Tet induction of RNAi and complementation with wildtype POLIB (POLIBWT) resulted in a 93% knockdown of endogenous POLIB mRNA but insufficient ectopic POLIBWT expression. This incomplete rescue emphasized the need for a more versatile induction system that will allow independent, tunable, and temporal regulation of gene expression. Hence, we adapted a dual control vanillic acid (Van)-Tet system that can independently control gene expression for robust RNAi complementation. Dual induction with Van and Tet (RNAi + Overexpression) resulted in 91% endogenous POLIB knockdown accompanied by robust and sustained ectopic expression of POLIBWT, and a near complete rescue of the POLIB RNAi defects. To more precisely quantify changes in kDNA size during RNAi, we also developed a semi-automated 3D image analysis tool to measure kDNA volume. Here we provide proof of principle for a dual inducer system that allows more flexible control of gene expression to perform RNAi and overexpression independently or concurrently within a single cell line. This system overcomes limitations of the single inducer system and can be valuable for elegant mechanistic studies in the field.

molecular biology↗

bioRxiv2024

Structurally targeted mutagenesis identifies key residues supporting α-synuclein misfolding in multiple system atrophy

Multiple system atrophy (MSA) and Parkinsons disease (PD) are caused by misfolded -synuclein spreading throughout the central nervous system. While familial PD is linked to several point mutations in -synuclein, there are no known mutations associated with MSA. Our previous work investigating differences in -synuclein misfolding between the two disorders showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo, providing key evidence to support the hypothesis that -synuclein adopts unique strains in patients. Here, to further interrogate -synuclein misfolding, we engineered a panel of cell lines harboring both PD-linked and novel mutations designed to identify key residues that facilitate -synuclein misfolding in MSA. These data were paired with in silico analyses using Maestro software to predict the effect of each mutation on the ability of -synuclein to misfold into one of the reported MSA cryo-electron microscopy conformations. In many cases, our modeling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation on MSA propagation in vitro, demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support -synuclein misfolding. Overall, our studies use a structure-based approach to investigate -synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.

neuroscience↗

bioRxiv2022

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Thumb Insertion That Confers Dominant Exonuclease Activity

Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might fulfill the role of a replicative, proofreading enzyme remains enigmatic. De novo modeling of POLIB revealed a structure divergent from all other Family A polymerases in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3-5 exonucleases. Here we demonstrate recombinant POLIB 3-5 exonuclease prefers DNA vs. RNA substrates and degrades single- and double-stranded DNA in a non-processive manner. The exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB is able to extend an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborate the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The unique POLIB thumb insertion influences the balance between polymerase and exonuclease activity and provides another example of exquisite diversity among DNA polymerases for specialized function.

biochemistry↗