Deubiquitinating Enzyme UCH-L1 Regulates Neuronal Ca²⁺ Signaling

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Ubiquitination is a key posttranslational modification regulating protein fate.
• Deubiquitinating enzymes (DUBs) reverse ubiquitination, counterbalancing E3 ligase activity.
• DUBs play critical roles in numerous cellular processes, including protein trafficking and degradation.

Purpose of the Study:

• To investigate the effect of the broad-spectrum DUB inhibitor PR-619 on calcium (Ca2+) entry through voltage-gated Ca2+ channels (VGCCs) in cultured embryonic cortical neurons.
• To identify the specific DUB responsible for PR-619's inhibitory action on neuronal Ca2+ influx.
• To elucidate the role of UCH-L1 in neuronal calcium homeostasis.

Main Methods:

• Fura-2-based Ca2+ imaging to measure cytosolic Ca2+ changes upon depolarization.
• Pharmacological inhibition of DUBs using PR-619, UCH-L1 inhibitors (IMP1710, GK13S), USP19 inhibitor (ADC141), and UCH-L3 inhibitor (TCID).
• Assessment of Ca2+ storage capacities and CaV1.2 protein levels.

Main Results:

• PR-619 significantly reduced Ca2+ influx, primarily through dihydropyridine-sensitive (L-type) VGCCs.
• The inhibitory effect of PR-619 was sensitive to dynamin and lysosomal pathway inhibitors.
• Selective inhibition of UCH-L1 (using IMP1710 and GK13S) mimicked PR-619's effect, reducing Ca2+ uptake and CaV1.2 protein levels, and impairing neuronal Ca2+ storage.
• Inhibition of USP19 or UCH-L3 did not affect Ca2+ influx.

Conclusions:

• UCH-L1, a major brain DUB, plays a significant role in regulating neuronal Ca2+ uptake and storage.
• UCH-L1 inhibition impacts neuronal Ca2+ homeostasis, affecting CaV1.2 protein levels and Ca2+ storage.
• These findings highlight UCH-L1 as a crucial regulator of neuronal calcium dynamics with potential physiological implications.