A novel H129-based anterograde monosynaptic tracer exhibits features of strong labeling intensity, high tracing efficiency, and reduced retrograde labeling.

BACKGROUND: Viral tracers are important tools for mapping brain connectomes. The feature of predominant anterograde transneuronal transmission offers herpes simplex virus-1 (HSV-1) strain H129 (HSV1-H129) as a promising candidate to be developed as anterograde viral tracers. In our earlier studies, we developed H129-derived anterograde polysynaptic tracers and TK deficient (H129-dTK) monosynaptic tracers. However, their broad application is limited by some intrinsic drawbacks of the H129-dTK tracers, such as low labeling intensity due to TK deficiency and potential retrograde labeling caused by axon terminal invasion. The glycoprotein K (gK) of HSV-1 plays important roles in virus entry, egress, and virus-induced cell fusion. Its deficiency severely disables virus egress and spread, while only slightly limits viral genome replication and expression of viral proteins. Therefore, we created a novel H129-derived anterograde monosynaptic tracer (H129-dgK) by targeting gK, which overcomes the limitations of H129-dTK. METHODS: Using our established platform and pipeline for developing viral tracers, we generated a novel tracer by deleting the gK gene from the H129-G4. The gK-deleted virus (H129-dgK-G4) was reconstituted and propagated in the Vero cell expressing wildtype H129 gK (gKwt) or the mutant gK (gKmut, A40V, C82S, M223I, L224V, V309M), respectively. Then the obtained viral tracers of gKmut pseudotyped and gKwt coated H129-dgK-G4 were tested in vitro and in vivo to characterize their tracing properties. RESULTS: H129-dgK-G4 expresses high levels of fluorescent proteins, eliminating the requirement of immunostaining for imaging detection. Compared to the TK deficient monosynaptic tracer H129-dTK-G4, H129-dgK-G4 labeled neurons with 1.76-fold stronger fluorescence intensity, and visualized 2.00-fold more postsynaptic neurons in the downstream brain regions. gKmut pseudotyping leads to a 77% decrease in retrograde labeling by reducing axon terminal invasion, and thus dramatically improves the anterograde-specific tracing of H129-dgK-G4. In addition, assisted by the AAV helper trans-complementarily expressing gKwt, H129-dgK-G4 allows for mapping monosynaptic connections and quantifying the circuit connectivity difference in the Alzheimer's disease and control mouse brains. CONCLUSIONS: gKmut pseudotyped H129-dgK-G4, a novel anterograde monosynaptic tracer, overcomes the limitations of H129-dTK tracers, and demonstrates desirable features of strong labeling intensity, high tracing efficiency, and improved anterograde specificity.

The Involvement of NR2B and tau Protein in MG132-Induced CREB Dephosphorylation.

Transcription factor cAMP response element-binding protein (CREB) plays a critical role in memory formation. Ubiquitin-proteasome system-dependent protein degradation affects the upstream signaling pathways which regulate CREB activity. However, the molecular mechanisms of proteasome inhibition on reductive CREB activity are still unclear. The current study demonstrated that MG132-inhibited proteasome activity resulted in a dose dependence of CREB dephosphorylation at Ser133 as well as decreased phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit NR2B (Tyr1472) and its tyrosine protein kinase Fyn (Tyr416). These dephosphorylations are probably caused by disturbance of expression and post-translational modifications of tau protein since tau siRNA decreased the activity of Fyn, NR2B, and CREB. To further confirm this perspective, HEK293 cells stably expressing human tau441 protein were treated with MG132 and dephosphorylations of CREB and NR2B were observed. The current research provides an alternative pathway, tau/Fyn/NR2B signaling, regulating CREB activity.