Protecting Against Cytoplasmic Protein Aggregates With Cytoplasmic PML Variants

Cytoplasmic protein aggregation is a defining feature of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, and certain forms of motor neuron disease. Recent evidence indicates that promyelocytic leukemia protein (PML) and engineered PML-derived variants can act as versatile aggregate-remodeling factors. In particular, cytoplasmically redirected PML variants recognize pathological cytoplasmic inclusions and promote their clearance. Here, we describe a protocol to generate and validate two engineered cytoplasmic PML variants: full-length mPML, which is redirected to the cytoplasm by disruption of its nuclear localization sequence, and the truncated mPML^ΔRBC variant, which lacks the RING, B-box, and coiled-coil domain but retains aggregate-reducing activity. The protocol integrates fluorescence-based imaging, bimolecular fluorescence complementation, detergent-soluble/insoluble fractionation, and validation in primary rat cortical neurons. This workflow provides a practical platform for assessing cytoplasmic aggregate burden and for comparing the aggregate-remodeling activities of PML-derived constructs. It can also be adapted to other disease-associated aggregation-prone proteins, including TDP-43, SOD1, FUS, tau, polyGA, and polyQ-expanded proteins. Key features • Describes the generation and validation of two complementary cytoplasmic PML variants: full-length mPML and truncated mPML^ΔRBC. • Provides fluorescence-based and BiFC-based assays to visualize pathological cytoplasmic protein assemblies. • Includes detergent-soluble/insoluble fractionation for biochemical assessment of aggregate burden. • Establishes a primary rat cortical neuron workflow to evaluate the effect of mPML on TDP-43-CTF aggregates. • Can be adapted to other aggregation-prone proteins and additional PML-derived aggregate-remodeling candidates.