Biology subjects

Huang, H.-W.

Publications and source records attributed to Huang, H.-W..

5 recordsLinked to original sources

bioRxiv2026

A single-domain antibody targets aggregation-prone region of α-synuclein to reduce synucleinopathy, rescue neurodegeneration and improve function

Synucleinopathies are a group of neurodegenerative disorders characterized by the accumulation of aggregated -synuclein (-syn), including Parkinsons disease, Dementia with Lewy Bodies, and Multiple System Atrophy. These diseases are marked by locomotor and non-motor impairments, as well as mitochondrial dysfunction and the loss of dopaminergic (DA) neurons. We have developed several anti--syn single-domain antibodies (sdAbs) and demonstrated the diagnostic imaging potential of two of them and the acute therapeutic benefit of one in clearing -syn in a mouse model. However, whether these sdAbs can suppress -syn-mediated neuronal loss and locomotor impairment in vivo remains unclear. We evaluated the therapeutic potential of five anti--syn sdAbs to clear pathological -syn in mouse neuronal culture and then demonstrated their in vivo efficacy in a Drosophila model of synucleinopathy. The sdAbs differed in their efficacy to lower levels of phospho-serine 129 -syn, prevent loss of DA neurons, alleviate mitochondrial dysfunction, improve motor function, and prolong survival in synucleinopathy flies. The most effective sdAb, 2H1, has not been reported before. It binds strongly to the aggregation prone region of -syn and robustly improves all these disease parameters. Additionally, that sdAb is associated with -syn in the fly neurons, as shown through proximity dependent turboID biotinylation assays. The sdAb-turboID also biotinylated -syn-associated proteins involved in synapse/vesicle trafficking pathways, pinpointing the location of their intracellular interaction. Our findings provide an insight into the therapeutic mechanism of action of these sdAbs and strongly support their clinical development.

neuroscience↗

bioRxiv2026

NECAP antagonizes light-induced Rhodopsin-1 internalization to promote photoreceptor homeostasis

AP-2 is a key mediator of clathrin-mediated endocytosis (CME) that internalizes cargo from the plasma membrane. NECAP proteins physically bind phosphorylated AP-2 during CME, but their roles during endocytosis remain unresolved, with conflicting reports about their function. Here, we report that Drosophila NECAP is dispensable for development, but antagonizes light-dependent Rhodopsin-1 (Rh1) internalization, a process that occurs through AP-2-mediated endocytosis. Specifically, loss of Drosophila NECAP causes excessive light-dependent Rh1 internalization and an age-related retinal degeneration that can be rescued by photoreceptor-specific expression of a wild-type transgenic NECAP. A Drosophila NECAP mutant transgene, equivalent to a canine NECAP1 variant associated with retinal atrophy, failed to rescue the NECAP loss-of-function phenotype in the eye. Furthermore, overexpression of wild-type NECAP suppressed massive Rh1 internalization in a distinct Drosophila model of light-dependent Rh1 endocytosis and retinal degeneration. These results establish NECAP as a negative regulator of light-dependent Rh1 internalization essential for photoreceptor survival.

cell biology↗

bioRxiv2023

Cell-based production of Fc-GlcNAc and Fc-alpha-2,6 sialyl glycan enriched antibody with improved effector functions through glycosylation pathway engineering

Glycosylation of antibody plays an important role in Fc-mediated killing of tumor cells and virus-infected cells through effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP) and vaccinal effect. Previous studies showed that therapeutical humanized antibodies with 2-6 sialyl complex type (SCT) glycan attached to Fc-Asn297 exhibited optimal binding to the Fc receptors on effector cells associated with ADCC, ADCP and vaccinal effect. However, the production of antibodies with homogeneous Fc-SCT needs multiple in vitro enzymatic and purification steps. In this study, we report two different approaches to shorten the processes to produce SCT-enriched antibodies. First, we expressed a bacterial endoglycosidase in GNT1-KO EXPI293 cells to trim all N-glycans to mono-GlcNAc glycoforms for in vitro transglycosylation to generate homogeneous SCT antibody. Second, we engineered the glycosylation pathway of HEK293 cells through knockout of the undesired glycosyltransferases and expression of the desired glycosyltransferases to produce SCT enriched antibodies with similar binding affinity to Fc receptors and ADCC activity to homogenous SCT antibody.

biochemistry↗

bioRxiv2021

Drosophila fabp is a retinoid-inducible gene required for Rhodopsin-1 homeostasis and photoreceptor survival

Retinoids act as chromophore co-factors for light-detecting rhodopsin proteins. In vertebrates, retinoids also actively regulate gene expression. Whether retinoids regulate gene expression in Drosophila for a specific biological function remains unclear. Here, we report that Drosophila fatty acid binding protein (fabp) is a retinoid-inducible gene required for Rhodopsin-1 (Rh1) protein homeostasis and photoreceptor survival. Specifically, we performed a photoreceptor-specific gene expression profiling study in flies bearing a misfolding-prone Rhodopsin-1 (Rh1) mutant, ninaEG69D, which serves as a Drosophila model for Retinitis Pigmentosa. ninaEG69D photoreceptors showed increased expression of genes that control Rh1 protein levels, along with a poorly characterized gene, fabp. We found that in vivo fabp expression was reduced when the retinoids were deprived through independent methods. Conversely, fabp mRNA was induced when we challenged cultured Drosophila cells with retinoic acid. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-activated Rh1 degradation. These observations indicate that a retinoid-inducible gene expression program regulates fabp that is required for Rh1 proteostasis and photoreceptor survival. Author SummaryRhodopsins are light-detecting proteins that use retinoids as chromophore co-factors. In vertebrates, retinoids also actively regulate gene expression. Whether retinoids regulate Rhodopsin function aside from its role as a chromophore remains unclear. Here, we report that Drosophila fatty acid binding protein (fabp) is a retinoid-inducible gene required for Rhodopsin-1 (Rh1) protein homeostasis and photoreceptor survival. Specifically, we found that fabp is among the genes induced by a misfolding-prone Rhodopsin-1 (Rh1) mutant, ninaEG69D, which serves as a Drosophila model for Retinitis Pigmentosa. We further found that fabp induction in ninaEG69D photoreceptors required retinoids. fabp was required in photoreceptors to help degrade light-activated Rh1. In the absence of fabp, Rh1 accumulated in cytoplasmic vesicles in a light-dependent manner, and exhibited light-dependent retinal degeneration. These observations indicate that a retinoid-inducible gene expression program regulates fabp that is required for Rh1 proteostasis and photoreceptor survival.

cell biology↗

bioRxiv2017

highroad is induced by retinoids and clears mutant Rhodopsin-1 in Drosophila Retinitis Pigmentosa models.

The light detecting protein, Rhodopsin, requires retinoid chromophores for their function. In vertebrates, retinoids also serve as signaling molecules, but whether these molecules similarly regulate gene expression in Drosophila remains unclear. Here, we report the identification of a retinoid-inducible gene in Drosophila, highroad, which is required for photoreceptors to clear folding-defective mutant Rhodopsin-1 proteins. Specifically, we identified highroad through an in vivo RNAi based genetic interaction screen with one such folding defective Rhodopsin-1 mutant, ninaEG69D. CRISPR-Cas9-mediated deletion of highroad results in the stabilization of folding-defective mutant Rhodopsin-1 proteins, and acceleration of the age-related retinal degeneration phenotype of ninaEG69D mutants. Elevated highroad transcript levels are detected ninaEG69D flies, and interestingly, deprivation of retinoids in the fly diet blocks this effect. Consistently, mutations in the retinoid transporter santa maria impairs the induction of highroad in ninaEG69D flies. In cultured S2 cells, highroad expression is induced by retinoic acid treatment. These results indicate that cellular quality control mechanism against misfolded Rhodopsin-1 involves regulation of gene expression by retinoids.

genetics↗