Neuronal Proteasome Research Articles

Dynamic control of 26S proteasome function at CNS synapses (478.3)

The selective strengthening and weakening of synapses in response to past experience and activity is known as synaptic plasticity and is fundamental to all brain processes such as the ability to learn, form memories and modify behavior. In contrast, impairments in synaptic plasticity are considered to contribute to neuropsychiatric disorders and neurodegenerative diseases. Synaptic plasticity is largely mediated by both morphological and functional modifications to synapses. Local protein synthesis and protein degradation are known to contribute to these modifications. It is now clear that regulated proteolysis via the ubiquitin proteasome system (UPS) plays a major role in the growth and development, maintenance and remodeling of synaptic connections in the brain. We and others have previously found a novel form of regulation for the 26S proteasome in neurons involving the plasticity kinase Ca2+/calmodulin‐dependent protein kinases II alpha (CaMKIIα). CaMKIIα phosphorylates the ATPase 19S regulatory particle (RP) subunit, Rpt6, at serine 120 (S120) in an activity‐dependent fashion to modulate the activity and control the distribution of proteasomes in neurons (Djakovic et al., 2009; 2012; Bingol et al., 2010). We find that Rpt6 phosphorylation and proteasome function regulates synaptic strength and activity‐dependent generation of dendritic spines (Djakovic et al., 2012; Hamilton et al., 2012). I will discuss our more recent work and the functional relevance of proteasome phosphorylation in neurons using newly generated Rpt6 Ser120Ala and Ser120Asp (phospho‐dead and phospho‐mimetic, respectively) genetically modified mice.

Increased protein hydrophobicity in response to aging and Alzheimer disease

Increased levels of misfolded and damaged proteins occur in response to brain aging and Alzheimer disease (AD), which presumably increase the amount of aggregation-prone proteins via elevations in hydrophobicity. The proteasome is an intracellular protease that degrades oxidized and ubiquitinated proteins, and its function is known to be impaired in response to both aging and AD. In this study we sought to determine the potential for increased levels of protein hydrophobicity occurring in response to aging and AD, to identify the contribution of proteasome inhibition to increased protein hydrophobicity, and last to identify the contribution of ubiquitinated and oxidized proteins to the pool of hydrophobic proteins. In our studies we identified that aging and AD brain exhibited increases in protein hydrophobicity as detected using Bis ANS, with dietary restriction (DR) significantly decreasing age-related increases in protein hydrophobicity. Affinity chromatography purification of hydrophobic proteins from aging and AD brains identified increased levels of oxidized and ubiquitinated proteins in the pool of hydrophobic proteins. Pharmacological inhibition of the proteasome in neurons, but not astrocytes, resulted in an increase in protein hydrophobicity. Taken together, these data indicate that there is a relationship between increased protein oxidation and protein ubiquitination and elevations in protein hydrophobicity within the aging and the AD brain, which may be mediated in part by impaired proteasome activity in neurons. Our studies also suggest a potential role for decreased oxidized and hydrophobic proteins in mediating the beneficial effects of DR.

Regulation of the Proteasome by Neuronal Activity and Calcium/Calmodulin-dependent Protein Kinase II

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-D-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation.

Study on the activities' alteration of the proteasome in neurons of cortex and its relation with delayed neuron death after reperfusion following ischemia

To investigate the activities' alteration of the proteasome in neurons of cortex and its relation with delayed neuron death after reperfusion following ischemia. 20 minutes transient global ischemia rat model was used. Following different reperfusion period, all the 50 rats were divided into 5 groups, sham-operation group, 0.5 hour recovery group, 4 hours recovery group, 24 hours recovery group and 72 hours recovery group, 10 rats each group. Suc-llvy-amc was used as substrate for measuring proteasome's activities. Delayed neuron death after reperfusion following ischemia was observed under light microscope by HE staining. Proteasome's distribution was observed under laser scanning confocal microscope after immuno-histo-chemical staining. The proteasome activity of sham group was 54 602 +/- 1602, and that of 0.5 h reperfusion following ischemia was 42,036 +/- 1465 (compared with sham group, P < 0.01). Although the proteasome activity temporarily recovered to 47,536 +/- 2532 (P < 0.05) after 4 h reperfusion, it still decreased to 45,450 +/- 649 (P < 0.01) after 24 h reperfusion, and to 43,108 +/- 995 (P < 0.01) after 72 h reperfusion. HE staining showed that parts of neurons in the cortex died after 72 hours reperfusion. Under laser scanning microscope, we could observe that after 24 hours reperfusion, proteasome in both nucleus and cytoplasma significantly decreased; after 72 hours reperfusion, proteasome almost disappeared totally in nucleus and only a small part of proteasome still existed in cytoplasm. Decreasing of the proteasome's activities is an important factor for delayed neuron death after reperfusion following ischemia.