Stimulation of the cuneiform nucleus enables training and boosts recovery after spinal cord injury.

Severe spinal cord injuries result in permanent paraparesis in spite of the frequent sparing of small portions of white matter. Spared fibre tracts are often incapable of maintaining and modulating the activity of lower spinal motor centres. Effects of rehabilitative training thus remain limited. Here, we activated spared descending brainstem fibres by electrical deep brain stimulation of the cuneiform nucleus of the mesencephalic locomotor region, the main control centre for locomotion in the brainstem, in adult female Lewis rats. We show that deep brain stimulation of the cuneiform nucleus enhances the weak remaining motor drive in highly paraparetic rats with severe, incomplete spinal cord injuries and enables high-intensity locomotor training. Stimulation of the cuneiform nucleus during rehabilitative aquatraining after subchronic (n = 8 stimulated versus n = 7 unstimulated versus n = 7 untrained rats) and chronic (n = 14 stimulated versus n = 9 unstimulated versus n = 9 untrained rats) spinal cord injury re-established substantial locomotion and improved long-term recovery of motor function. We additionally identified a safety window of stimulation parameters ensuring context-specific locomotor control in intact rats (n = 18) and illustrate the importance of timing of treatment initiation after spinal cord injury (n = 14). This study highlights stimulation of the cuneiform nucleus as a highly promising therapeutic strategy to enhance motor recovery after subchronic and chronic incomplete spinal cord injury with direct clinical applicability.

Lysine acetyltransferase Tip60 acetylates the APP adaptor Fe65 to increase its transcriptional activity.

Proteolytic processing of the amyloid precursor protein (APP) releases the APP intracellular domain (AICD) from the membrane. Bound to the APP adaptor protein Fe65 and the lysine acetyltransferase (KAT) Tip60, AICD translocates to the nucleus. Here, the complex forms spherical condensates at sites of endogenous target genes, termed AFT spots (AICD-Fe65-Tip60). We show that loss of Tip60 KAT activity prevents autoacetylation, reduces binding of Fe65 and abolishes Fe65-mediated stabilization of Tip60. Autoacetylation is a prerequisite for AFT spot formation, with KAT-deficient Tip60 retained together with Fe65 in speckles. We identify lysine residues 204 and 701 of Fe65 as acetylation targets of Tip60. We do not detect acetylation of AICD. Mutation of Fe65 K204 and K701 to glutamine, mimicking acetylation-induced charge neutralization, increases the transcriptional activity of Fe65 whereas Tip60 inhibition reduces it. The lysine deacetylase (KDAC) class III Sirt1 deacetylates Fe65 and pharmacological modulation of Sirt1 activity regulates Fe65 transcriptional activity. A second acetylation/deacetylation cycle, conducted by CBP and class I/II KDACs at different lysine residues, regulates stability of Fe65. This is the first report describing a role for acetylation in the regulation of Fe65 transcriptional activity, with Tip60 being the only KAT tested that supports AFT spot formation.

Visualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation.

BACKGROUND: The amyloid precursor protein (APP) intracellular domain (AICD) is released from full-length APP upon sequential cleavage by either α- or ß-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein complexes (AFT complexes) that function in transcriptional regulation. OBJECTIVE: To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells. METHODS: We used cotransfection of bimolecular fluorescence complementation (BiFC) fusion constructs of APP and Tip60 for analysis of subcellular localization by confocal microscopy and quantification by flow cytometry (FC). RESULTS: Our novel BiFC-constructs show a nuclear localization of AFT complexes that is identical to conventional fluorescence-tagged constructs. Production of the BiFC signal is dependent on the adaptor protein Fe65 resulting in fluorescence complementation only after Fe65-mediated nuclear translocation of AICD and interaction with Tip60. We applied the AFT-BiFC system to show that the Swedish APP familial Alzheimer's disease mutation increases AFT complex formation, consistent with the notion that AICD mediated nuclear signaling mainly occurs following APP processing through the amyloidogenic ß-secretase pathway. Next, we studied the impact of posttranslational modifications of AICD on AFT complex formation. Mutation of tyrosine 682 in the YENPTY motif of AICD to phenylalanine prevents phosphorylation resulting in increased nuclear AFT-BiFC signals. This is consistent with the negative impact of tyrosine phosphorylation on Fe65 binding to AICD. Finally, we studied the effect of oxidative stress. Our data shows that oxidative stress, at a level that also causes cell death, leads to a reduction in AFT-BiFC signals. CONCLUSION: We established a new method for visualization and FC quantification of the interaction between AICD, Fe65 and Tip60 in the nucleus based on BiFC. It enables flow cytometric analysis of AICD nuclear signaling and is characterized by scalability and low background fluorescence.