Failures in protein homeostasis are linked to Parkinson's disease (PD) and other neurodegenerative diseases. Lewy bodies, proteinaceous inclusions rich in phosphorylated alpha-synuclein are a hallmark of PD. Glial cell line-derived neurotrophic factor (GDNF) can eliminate Lewy body-like inclusions in mouse dopamine neurons. This study explores whether GDNF has protective effects against alpha-synuclein protofibril toxicity under proteasome inhibition by lactacystin, both in vitro and in vivo. GDNF did not shield midbrain dopamine neurons from lactacystin-induced neurodegeneration, but still prevented phosphorylated alpha-synuclein accumulation. In vivo experiment with control or GDNF-expressing viral vectors assessed alpha-synuclein pathology spread in the nigrostriatal pathway and lactacystin damage in the midbrain. GDNF overexpression reduced phosphorylated alpha-synuclein inclusions. Lactacystin-treated mice showed motor asymmetry and decreased spontaneous activity, exacerbated without AAV-GDNF pre-treatment. However, GDNF's neuroprotective effect could not be confirmed in vivo, due to side-effects from overexpression in the midbrain. Importantly, these findings show that GDNF continues to eliminate alpha-synuclein aggregation despite lactacystin-induced proteasome inhibition. Activating neurotrophic signaling pathways may protect against alpha-synuclein pathology in PD, even with impaired protein degradation mechanisms.