Detection, visualization and quantification of protein complexes in human Alzheimer's disease brains using proximity ligation assay.

Examination of healthy and diseased human brain is essential to translational neuroscience. Protein-protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the in-situ proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in the human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer's disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that phosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies.

Recurrent TRAK1::RAF1 Fusions in pediatric low-grade gliomas.

Fusions involving CRAF (RAF1) are infrequent oncogenic drivers in pediatric low-grade gliomas, rarely identified in tumors bearing features of pilocytic astrocytoma, and involving a limited number of known fusion partners. We describe recurrent TRAK1::RAF1 fusions, previously unreported in brain tumors, in three pediatric patients with low-grade glial-glioneuronal tumors. We present the associated clinical, histopathologic and molecular features. Patients were all female, aged 8 years, 15 months, and 10 months at diagnosis. All tumors were located in the cerebral hemispheres and predominantly cortical, with leptomeningeal involvement in 2/3 patients. Similar to previously described activating RAF1 fusions, the breakpoints in RAF1 all occurred 5' of the kinase domain, while the breakpoints in the 3' partner preserved the N-terminal kinesin-interacting domain and coiled-coil motifs of TRAK1. Two of the three cases demonstrated methylation profiles (v12.5) compatible with desmoplastic infantile ganglioglioma (DIG)/desmoplastic infantile astrocytoma (DIA) and have remained clinically stable and without disease progression/recurrence after resection. The remaining tumor was non-classifiable; with focal recurrence 14 months after initial resection; the patient remains symptom free and without further recurrence/progression (5 months post re-resection and 19 months from initial diagnosis). Our report expands the landscape of oncogenic RAF1 fusions in pediatric gliomas, which will help to further refine tumor classification and guide management of patients with these alterations.