Biology subjects

Savage, K.

Publications and source records attributed to Savage, K..

3 recordsLinked to original sources

bioRxiv2026

Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome

Pathogenic variants in the 13 protein-coding genes of the mitochondrial genome underlie clinically and biochemically heterogeneous disorders. Most mtDNA-encoded genes lack defined loss-of-function (LOF) models in vivo. To address this gap, we have generated Z-Terminator, a systematic in vivo atlas of loss-of-function alleles covering all the mtDNA-encoded OXPHOS subunits in zebrafish (Danio rerio). We used mitochondrial TALE base editors to introduce premature termination codon (PTC) alleles via C-to-T transitions across Complexes I, III, IV, and V. Larvae harboring PTC alleles displayed bioenergetic defects and elevated lactate. While mtDNA mutations are associated with sensorineural hearing loss, the cellular basis has remained unclear. We show that engineered mtDNA LOF alleles directly impair hair cell function in proportion to heteroplasmy in a living vertebrate. We investigated the germline transmission and tissue-specific heteroplasmy of these LOF alleles and observed that a subset of variants was transmitted to the F1 generation and displayed distinct mutant loads across organs. These findings establish Z-Terminator as a vertebrate platform for interrogating the role of mtDNA protein-coding genes in cellular dysfunction and for elucidating the pathophysiology of mitochondrial disorders.

genetics↗

bioRxiv2022

Comprehensive preclinical evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9ORF72 disease

Hexanucleotide G4C2 repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intracellular poly-GA and reduced aggregate formation in a poly-GA over-expressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 months was well-tolerated and led to measurable brain penetration of antibodies. Long term treatment with anti-GA antibodies produced improvement in an open field movement test in aged C9ORF72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model. SignificanceImmunotherapy has been proposed for neurodegenerative disorders including Alzheimers or Parkinsons diseases. Recent reports using antibodies against poly-GA or active immunization suggested similar immunotherapy in ALS/FTD caused by repeat expansion in the C9ORF72 gene (1, 2). Here, we systematically characterized human antibodies against multiple DPR species and tested the biological effects of antibodies targeting poly-GA in different cellular and mouse models. Target engagement was shown in three independent cellular models. Anti-GA antibodies reduced the number of intracellular poly-GA aggregates in human T98G cells but not in cultured human neurons. Whereas chronic anti-GA treatment in BAC C9ORF72450 mice did not impact poly-GA levels and modestly improved one behavioral phenotype, poly-GA levels detected by immunoassays were increased and disease progression was unaltered in AAV-(G4C2)149 mice.

neuroscience↗

bioRxiv2019

Developmental and cell-specific expression of Cacna1d splice variants

CaV1.3 is an L-type voltage-gated calcium channel implicated in several functions including gene expression, pacemaking activity, and neurotransmitter release. The gene that encodes the CaV1-pore forming subunit of CaV1.3 (Cacna1d) is a multi-exon gene that undergoes extensive alternative splicing, which provides functional versatility to this gene across tissues and cell-types. The function and expression of several Cacna1d splice variants within the C-terminus have been previously characterized. These splice variants differ in their voltage-dependence of activation, Ca2+-dependent inactivation, and their sensitivity to dihydropyridines. However, less is known about alternatively spliced exons in Cacna1d located downstream of domain I and upstream of the C-terminus (e11, e22a/e22, e31a/e31b/e32). Here, we performed a systematic study to determine the developmental and cell-specific expression of several Cacna1d splice variants. We found that the cassette e11 is upregulated during brain development, and in adult cortical tissue is more abundant in excitatory neurons relative to inhibitory interneurons. This exon is also upregulated upon nerve growth factor (NGF) induced differentiation of pheochromocytoma cells, PC12. At the functional level, the splice variants resulting from e11 alternative splicing (+e11-Cacna1d and {Delta}e11-Cacna1d) form functional CaV1.3 channels with similar biophysical properties in expression mammalian systems. Of the pair of mutually exclusive exons, e22a and e22, the later dominates at all stages. However, we observed a slight upregulation of e22 from embryonic to adult human brain. A second pair of mutually exclusive exons, e31a and e31b, was also studied. We found that e31a increases during brain development. Finally, the cassette exon 32 is repressed in adult brain tissue.

neuroscience↗