P1.16. PROTEOMIC ALTERATIONS AFTER SPINALIZATION: PERSPECTIVES FROM MOTONEURONS, DORSAL ROOT GANGLIA, AND EPENDYMA
Olga Gajewska-Woźniak1,2, Anna Głowacka2, Agata Pytyś1, Tomasz Wójtowicz1, Kasia Radwanska1, Małgorzata Skup2
1 Laboratory of Molecular Basis of Behavior, Nencki Institute of Experimental Biology
2 Group of Restorative Neurobiology, Nencki Institute of Experimental Biology
INTRODUCTION: Motoneurons (MNs) innervating soleus (SOL), a slow-twitch postural muscle, and tibialis anterior (TA), a fast-twitch phasic muscle exhibit distinct physiological and molecular features, influencing their response to spinal cord injury (SCI). Although type-specific MN responses to SCI have been reported, direct comparative proteomic data are lacking.
AIM(S): We aim to investigate proteomic differences between SOL and TA MNs in healthy rats and after spinal cord transection (SCT). We also profiled dorsal root ganglia (DRG), providing sensory input, and ependymal cells (EC) lining the central canal, supporting spinal cord homeostasis.
METHOD(S): Adult male Wistar rats (n=19) received retrograde tracers into SOL and TA. Rats were sacrificed at 2, or 6 weeks after thoracic SCT; healthy rats served as controls. Labeled SOL/ TA MNs and EC were isolated by laser microdissection (Leica).
Proteins were extracted, digested, and labeled with tandem mass tags (TMT). Peptides were separated on an UltiMate 3000 nano-LC system coupled to Q Exactive HF-X mass spectrometer. Data were processed in MaxQuant (v1.6.17.0) using Andromeda search engine against the Rattus norvegicus UniProt database.
RESULTS: We identified ~1200 proteins in MNs, 1500 in EC, and 3000- 5000 in DRGs. In controls, SOL MNs exhibited higher than TA MNs expression of proteins linked to cellular homeostasis and sustained activity: PICALM (autophagy and endocytosis), CST3 (neuroprotection), LNPK (endoplasmic reticulum structure), HAGH (metabolic detoxification), and CKAP4 (cytoskeleton-ER interaction).
After SCT, MN subtypes showed distinct proteomic changes. In SOL MNs, 35 proteins changed at 2 weeks and 15 at 6 weeks, while TA MNs showed fewer changes (11 and 4, respectively). EC changes were more prominent at 2 weeks, whereas DRG sensory neuron proteomes showed greater alterations at 6 weeks.
CONCLUSIONS: Our results show both baseline and injury-induced differences between MN subtypes, emphasizing the need for precision approaches in SCI therapies.
FINANCIAL SUPPORT: Supported by MINIATURA G01783 (O G-W.), OPUS G2789 (M S)