Synaptic impairment is known to be a prerequisite to the cognitive deficiencies exhibited in Alzheimer’s Disease (AD), so investigating the mechanisms which dysregulate the integrity of synaptic scaffolding components may provide insight into the genesis of AD.
The Rps23rg1 (ribosomal protein S23 retroposed) gene was recently identified as an anti β-amyloid (Aβ) component – relevant to the pathological formation of Aβ-enriched plaques in AD. The transmembrane protein, RPS23RG1, concurrently inhibits Aβ generation and tau hyperphosphorylation through interactions with adenylyl cyclases, enhancing cAMP/PKA to inhibit GSK activity. Given that the RPS23RG family is conserved in both mouse and human, the use of custom genetic mouse models has provided elucidation into the pathogenesis of AD that could improve human disease studies and uncover new therapeutic modalities.
The pathology of human AD and AD mouse was first evaluated in order to effectively address the therapeutic relevance of the model and identify areas of homology. The RPS23RG1 family is conserved in both mouse and human, with one functionally expressed gene in human (RPS23RG1) and two in mouse (Rps23rg1 and Rps23rg2) identified thus far. The scaffolding protein PSD-95 plays an important role in the organization of neuro-signaling factors. Dysregulation of PSD-95 and the related scaffolding protein PSD-93 impairs synaptic function, with subsequent effects on learning and memory. Previous studies have reported decreased PSD-95 and PSD-93 levels in both human AD brain and AD mouse models, which correlates with increasing pathological severity and dementia. Additional study has suggested that PSD-95 levels and functionality may fluctuate depending on the stage of the disease and pathological severity during AD progression. RPS23RG1 mRNA expression was found to be dramatically decreased in postmortem AD brain compared to controls; RPS23RG1 protein levels were shown to correlate positively with PSD-93 and PSD-95 protein levels in AD samples. Interestingly, researchers found that RPS23RG1, PSD-93, and PSD-95 levels declined at 7 months of age in AD mice – when disease-associate phenotypes begin to manifest. These key findings led to the working model that suggests RPS23RG1 reduction can promote PSD-93/95 destabilization and turnover, triggering cognitive impairment with the onset of AD.
The ubiquitin-proteasome system (UPS) regulates the protein environment within neurons, the compositional landscape within PSDs, and turnover of PSD-95. Ubiquitin-associated pathology is commonly observed in AD, suggesting that UPS-dependent dysregulation of PSD components may contribute to AD pathogenesis. The E3 ubiquitin ligase MDM2 (murine double minute 2) mediates poly-ubiquitination and degradation of PSD-95 – suggesting its role in the pathologic decrease of PSD-95 seen in AD. The following results provide a new mechanism underlying PSD destabilization and turnover through the ubiquitin-proteasome pathway.
Researchers generated a Rps23rg1 knockout (KO) mouse line: using a five-nucleotide deletion within the Rps23rg1 protein coding sequence to create a frame shift and early truncation of RPS23RG11. The homozygous Rps23rg1 KO mice exhibited no reproductive or viability issues, and, notably, did not differ in brain physiology compared to WT mouse brain sections. Behavioral tests indicate that Rps23rg1 mediates a variety of cognition-related behaviors and that its loss severely impairs learning and memory. To determine synaptic function mediated by AMPA and NMDA receptors, whole-cell mEPSC (miniature excitatory postsynaptic current) recordings revealed attenuated amplitudes for both AMPA and NMDA. Additionally, paired-pulse facilitation did not vary significantly between Rps23rg1 KO and WT mice, indicating that mEPSC frequency attenuation is likely caused by a reduction in functional synapses; this was further validated with immunocytochemistry. These results implicate that loss of Rps23rg1 is associated with reductions in surface glutamate receptors - impairing postsynaptic glutamatergic neurotransmission and synaptic function. Additionally, deletion of the Rps23rg1 gene resulted in marked reductions in PSD-93 and PSD-95 protein levels. However, the loss of Rps23rg1 had no effect on the mRNA expression of PSD-95 or PSD-93. Collectively, these findings suggest that dysregulated PSD scaffolding components may contribute to synaptic dysfunction and that Rps23rg1 influences posttranslational PSD-93/95 stability or turnover.
Endogenous PSD-93 and PSD-95 co-precipitated with an RPS23RG1 antibody in WT but not Rps23rg1 KO mouse brain lysates – with similar co-precipitation results for recombinant human RPS23RG1. Furthermore, it was found that RPS23RG1 overexpression attenuated MDM2 interactions with PSD-93/95 in a dose-dependent manner. Significantly, overexpression of full-length human RPS23RG1 distinctly reversed PSD-93 and PSD-95 poly-ubiquitination induced by MDM2 overexpression. Overall, results suggest that RPS23RG1 and MDM2 compete for PSD-93 and PSD-95 interactions. This was confirmed in-vivo as Rps23rg1 deletion enhanced MDM2 co-immunoprecipitation with PSD-93/95 in total brain lysates and synaptosomes. These results, taken together with the role of RPS23RG1 in inhibiting Aβ generation, provides the first look into how RPS23RG1 reduction in AD could simultaneously enhance Aβ proteotoxicity and drive synaptic degeneration – contributing greatly to AD pathogenesis.
The described research presents a model for RPS23RG1-mediated synaptic stabilization through interactions between the RPS23RG1 intracellular domain (ICD) and PSD-93/95 – displacing MDM2 from binding and poly-ubiquitination of PSD-93/95. Given the reported correlation of PSD-93/95 levels with AD progression, it was a significant finding that restoring both PSD-93/95 levels, but not either individually, can rescue synaptic and cognitive impairments in Rps23rg1 KO mice. A conserved “TTLAH” motif was identified to be present in both human (aa163-167) and mouse (aa130-134) RPS23RG1 as an essential region for interactions between RPS23RG1 and PSD-93/95; this unique sequence within the ICD was used to design a competing peptide to displace PSD-destabilizing interactions with MDM2. Results demonstrate that the human RPS23RG1 ICD-TAT peptide can penetrate the BBB and target PSD-93/95 – increasing PSD-93/95 protein levels, attenuating MDM2 interactions with PSD-93/95, and reducing poly-ubiquitination of PSD-93/95 in Rps23rg1 KO mice. In addition to stabilizing PSD-93/95 components with an RPS23RG1-derived ICD peptide, reconstituting depleted PSD components may serve as a complementary strategy to reverse neuronal impairment in late AD.
Numerous neurodegenerative disorders beside AD have presented defects in synaptic stability, homeostasis and PSD composition. Patients with Huntington’s disease (HD) present decreased PSD-95 levels, much like AD. Parkinson’s disease (PD) is known to present decreased spine density. Synaptic loss has additionally been observed in mouse models of amyotrophic lateral sclerosis. However, the way in which the described mechanisms could affect synaptic formation and function in various neurodegenerative disorders remains unclear. Given the therapeutic potential of enhancing RPS23RG1-associated PSD-93/PSD-95 stability in AD models, it is imperative to assess its potential to reverse synaptic deficits in these disorders.
Cyagen offers custom mouse, mouse embryo, and rat model generation services to enable your next discovery – the Rps23rg1 KO mouse used in this study is just one example. Since our founding in 2005, we have successfully delivered over 39,600 custom animal models to researchers worldwide, who have cited us in over 2,800 publications. All of our animal model projects are backed by 100% money-back guarantee, the best in the industry. With recent advances in our gene engineering toolbox and new proprietary methods developed by Cyagen, the efficiency of generating a KO/cKO/KI rat has been brought to a level similar to a mouse model.
Rats are physiologically, morphologically, and genetically closer to humans than mice, which in theory makes rats ideal models of neurodegenerative diseases. Its larger body and brain size facilitate administration of drugs, multiple samplings, in vivo electrophysiology, as well as neurosurgical and neuroimaging procedures. They have finer and more accurate motor coordination than mice, display a more complex social behavior, and are less stressed in tasks (such as the water maze). Furthermore, rats contain 6 isoforms of tau, like humans, and there is good homology between rat and human APOE amino acid sequences. Several established mouse models, such as transgene of Tau mutants and humanized APOE mouse, have been used widely in AD related research while their counterparts in rats have not been fully developed or explored. Rats are indicated to be highly suitable animal models for human tauopathies research, including study of the pathogenic mechanism of tau spreading. Cyagen has developed more than 900 custom rat models so far, and we can certainly assist you with your biomedical research – contact us today for a free gene targeting strategy design from our experts.