Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells.

USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or α-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated ß-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect ß-catenin, demonstrating USP14 but not UCHL5 has a specific effect on ß-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides ß-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.

Aß1-42 triggers the generation of a retrograde signaling complex from sentinel mRNAs in axons.

Neurons frequently encounter neurodegenerative signals first in their periphery. For example, exposure of axons to oligomeric Aß1-42 is sufficient to induce changes in the neuronal cell body that ultimately lead to degeneration. Currently, it is unclear how the information about the neurodegenerative insult is transmitted to the soma. Here, we find that the translation of pre-localized but normally silenced sentinel mRNAs in axons is induced within minutes of Aß1-42 addition in a Ca2+-dependent manner. This immediate protein synthesis following Aß1-42 exposure generates a retrograde signaling complex including vimentin. Inhibition of the immediate protein synthesis, knock-down of axonal vimentin synthesis, or inhibition of dynein-dependent transport to the soma prevented the normal cell body response to Aß1-42 These results establish that CNS axons react to neurodegenerative insults via the local translation of sentinel mRNAs encoding components of a retrograde signaling complex that transmit the information about the event to the neuronal soma.

Axonally synthesized ATF4 transmits a neurodegenerative signal across brain regions.

In Alzheimer's disease (AD) brain, exposure of axons to Aß causes pathogenic changes that spread retrogradely by unknown mechanisms, affecting the entire neuron. We found that locally applied Aß1-42 initiates axonal synthesis of a defined set of proteins including the transcription factor ATF4. Inhibition of local translation and retrograde transport or knockdown of axonal Atf4 mRNA abolished Aß-induced ATF4 transcriptional activity and cell loss. Aß1-42 injection into the dentate gyrus (DG) of mice caused loss of forebrain neurons whose axons project to the DG. Protein synthesis and Atf4 mRNA were upregulated in these axons, and coinjection of Atf4 siRNA into the DG reduced the effects of Aß1-42 in the forebrain. ATF4 protein and transcripts were found with greater frequency in axons in the brain of AD patients. These results reveal an active role for intra-axonal translation in neurodegeneration and identify ATF4 as a mediator for the spread of AD pathology.

The stability of myocilin olfactomedin domain variants provides new insight into glaucoma as a protein misfolding disorder.

Myocilin variants, localized to the olfactomedin (OLF) domain, are linked to early-onset, inherited forms of open-angle glaucoma. Disease-causing myocilin variants accumulate within trabecular meshwork cells instead of being secreted to the trabecular extracellular matrix of the eye. We hypothesize that, like in other diseases of protein misfolding, aggregation and downstream pathogenesis originate from the compromised thermal stability of mutant myocilins. In an expansion of our pilot study of four mutants, we compare 21 additional purified OLF variants by using a fluorescence stability assay and investigate the secondary structure of the most stable variants by circular dichroism. Variants with lower melting temperatures are correlated with earlier glaucoma diagnoses. The chemical chaperone trimethylamine N-oxide is capable of restoring the stability of most, but not all, variants to wild-type (WT) levels. Interestingly, three reported OLF disease variants, A427T, G246R, and A445V, exhibited properties indistinguishable from those of WT OLF, but an increased apparent aggregation propensity in vitro relative to that of WT OLF suggests that biophysical factors other than thermal stability, such as kinetics and unfolding pathways, may also be involved in myocilin glaucoma pathogenesis. Similarly, no changes from WT OLF stability and secondary structure were detected for three annotated single-nucleotide polymorphism variants. Our work provides the first quantitative demonstration of compromised stability among many identified OLF variants and places myocilin glaucoma in the context of other diseases of protein misfolding.