Biology subjects

Durham, H. D.

Publications and source records attributed to Durham, H. D..

4 recordsLinked to original sources

bioRxiv2024

Interactors of sacsin DNAJ domain identify function in organellar transport and membrane composition relevant to ARSACS pathogenesis

Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS) is caused by loss of function mutations in the SACS gene encoding sacsin, a 520kDa protein with multiple functional domains. The goal of this study was to identify client proteins interacting with the J domain, a cochaperone domain interacting with Hsp70 chaperones, to gain insights into sacsins function and its disruption in experimental models of ARSACS. Pull downs from mouse brain identified Rabs and Rab-associated proteins including Rab1b, ARF5 and endophilin B2, involved in organelle trafficking. In cell and mouse models of ARSACS, higher molecular weight species of Rab1b were identified on SDS-PAGE in addition to the normal 25kDa band and Rab1was retained in the soma along with membranous organelles (i.e., ER, Golgi and ATG9 autophagic vesicles). These changes were reversed by expression of the DNAJ domain or the Ubl domain of sacsin and occurred independent of the formation of abnormal bundles of intermediate filaments, a key feature of ARSACS. Although Rab1b was associated with both Golgi and ER in both Sacs+/+ and Sacs-/- cells, expression of the DNAJ domain or the Ubl domain of sacsin increased Rab1b association with ER and restored normal electrophoretic mobility. Finally, subcellular distribution of another membrane protein, neuroplastin, a key receptor for synapse formation and plasticity, also was impaired, pointing to a general problem in Rab-dependent membrane trafficking in the absence of sacsin.

cell biology↗

bioRxiv2023

Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

Protein misfolding and mislocalization are common themes in neurodegenerative disorders, including the motor neuron disease, amyotrophic lateral sclerosis (ALS). Maintaining proteostasis is a crosscutting therapeutic target, including upregulation of heat shock proteins (HSP) to increase chaperoning capacity. Motor neurons have a high threshold for upregulating stress inducible HSPA1A, but constitutively express high levels of HSPA8. This study compared expression of these HSPs in cultured motor neurons expressing three variants linked to familial ALS: TDP-43G348C, FUSR521G or SOD1G93A. All variants were poor inducers of Hspa1a, and reduced levels of Hspa8 mRNA and protein, indicating multiple compromises in chaperoning capacity. To promote HSP expression, cultures were treated with the putative HSP co-inducer, arimoclomol, class I histone deacetylase (HDAC) inhibitors to promote active chromatin for transcription, and the combination. Treatments had variable, often different effects on expression of Hspa1a and Hspa8, depending on the ALS variant expressed, mRNA distribution (somata and dendrites), and biomarker of toxicity measured (histone acetylation, maintaining nuclear TDP-43 and the nBAF chromatin remodeling complex component Brg1, mitochondrial transport, FUS aggregation). Overall, HDAC inhibition alone was effective on more measures than arimoclomol. In the TDP-43 model, arimoclomol failed to induce HSPA1A or preserve Hspa8 mRNA, despite preserving nuclear TDP-43 and Brg1, indicating neuroprotective properties other than HSP induction. The data speak to the complexity of drug mechanisms against multiple biomarkers of ALS pathogenesis, as well as to the importance of HSPA8 for neuronal proteostasis in both somata and dendrites.

neuroscience↗

bioRxiv2021

The J domain of sacsin disrupts intermediate filament assembly

Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS), is caused by loss of function mutations in the SACS gene, which encodes sacsin, a giant protein of 520 kDa. A key feature of the absence of sacsin in cells is the formation of abnormal bundles of intermediate filaments (IF) including neurofilaments (NF) in neurons and vimentin IF in fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs-/- neurons, however, its mechanism is still unclear. Here, we focused on the role of SacsJ in NF assembly. We report that the SacsJ domain directly interacts with NF proteins in vitro to disassemble NFL filaments, and to inhibit their initial assembly, in the absence of Hsp70. We generated a cell-penetrating peptide derived from this domain, SacsJ-myc-TAT, which was efficient in disassembling both vimentin IF and NF in cultured fibroblasts and Sacs+/+ motor neurons as well as NF bundles in cultured Sacs-/- motor neurons. Whereas a normal NF network was restored in Sacs-/- neurons treated with the SacsJ peptide, there was some loss of IF networks in Sacs+/+ fibroblasts or neurons. These results suggest that SacsJ is a key regulator of NF and IF networks in cells, with implications for its therapeutic use.

cell biology↗

bioRxiv2020

Functional characterization of Neurofilament Light b splicing and misbalance in zebrafish

Neurofilaments (NFs), a major cytoskeletal component of motor neurons, play a key role in their differentiation, establishment and maintenance of their morphology and mechanical strength. The de novo assembly of these neuronal intermediate filaments requires the presence of the neurofilament light subunit, NEFL, which expression is reduced in motor neurons in Amyotrophic Lateral Sclerosis (ALS). This study used zebrafish as a model to characterize the NEFL homologue neflb, which encodes two different isoforms via splicing of the primary transcript (neflbE4 and neflbE3). In vivo imaging showed that neflb is crucial for proper neuronal development, and that disrupting the balance between its two isoforms specifically affects NF assembly and motor axon growth, with resulting motor deficits. This equilibrium is also disrupted upon partial depletion of TDP-43, a RNA binding protein that is mislocalized into cytoplasmic inclusions in ALS. The study supports interaction of NEFL expression and splicing with TDP-43 in a common pathway, both biologically and pathogenetically.

neuroscience↗