Development Of A Small A-Synuclein-Knockdown Peptide As A Potential Pd Therapy

Object:Convincing evidence supports the premise that toxic a-synuclein oligomers promote the pathogenesis of PD. And reducing a-synuclein levels may represent an effective and specific therapy for PD. However, a clinically applicable a-synuclein reducing therapeutic strategy remains lacking. Here we report the development of a plasma membrane-permeable a-synuclein knockdown peptide (Tat-psyn-degron) observe whether it could be via directing the endogenous a-synuclein for proteasomal degradation to reduce a-synuclein levels in neurons and hence, protect dopaminergic neurons against parkinsonian toxin-induced neuronal damage both in primary neuronal cultures, and in a mouse parkinsonian toxicity model of PD.Methods:1.Using a method that we recently developed to rapidly and reversibly decrease the levels of endogenous proteins by directing them for proteasomal degradation and the amino acids 36-45 of β-synuclein (βsyn) can specifically bind to a-synuclein with high affinity, we develop the proposed a-synuclein targeting peptide (Tat-βsyn-degron) and control peptide (Tat-βsyn). Bath application of synthetic Tat-βsyn-degron peptide(5μM-50μM) and Tat-psyn peptide (50μM) for 24hrs, the levels of a-synuclein were examined by the immunoblot; then to explore if the synthetic Tat-βsyn-degron peptide could degrade a-synuclein and it’s mechanism in the presence of the proteasome inhibitor MG132;2. In vitro model of PD, MPP+treatment (20 μM; 48hrs) in rat primary cultures the ventral midbrain. Then bath application of Tat-βsyn-degron peptide (25 μM; 48hrs) and Tat-βsyn peptide (25 μM; 48hrs), the expression of a-synuclein and TH were tested by immunoblot;3. In vivo model of PD, the C57BL/6(20-25g) received intraperitoneal (i.p.) injection of 30mg/kg parkinsonian toxin MPTP hydrochloride once a day for 5 days to induce dopaminergic neuron death in the substantia nigra, while the control mice received equal volumes of saline injection.6 umol/kg Tat-psyn-degron peptide or control Tat-βsyn peptide was i.p. injected into the MPTP-treated mice every 12hrs from the first day of MPTP injection until 7 days after the last injection of MPTP. All groups of mice then underwent a rotarode test to examine the role of the synthetic Tat-βsyn-degron peptide on Parkinson’s disease behavior; then the mice were sacrificed to examine the levels of a-synuclein and TH in striatum and substantia nigra by immunoblot and immunohistochemistry.Results:1.Tat-psyn-degron peptide dose-and time-dependently efficiently knocks down a-synuclein via directing the endogenous a-synuclein for proteasomal degradation.2. Tat-βsyn-degron peptide efficiently knocks down α-synuclein and protects against parkinsonian toxin induced neuronal damage in rat ventral midbrain cultures. However Tat-βsyn may potentially exert a certain degree of neuroprotection through competitively inhibiting the oligomerization of a-synuclein;3. Tat-βsyn-degron peptide-mediated knockdown of a-synuclein protects against parkinsonian toxin MPTP-induced dopaminergic neuronal damage and behavioral deficits in mice, but not Tat-βsyn peptide.Conclusion:1.The synthetic Tat-βsyn-degron peptide could efficiently knocks down a-synuclein via directing the endogenous α-synuclein for proteasomal degradation; 2.The synthetic Tat-βsyn-degron peptide-mediated knockdown of α-synuclein protects against parkinsonian toxin induced dopaminergic neuronal damage in model of PD.