Granzyme B-inhibitor serpina3n induces neuroprotection in vitro and in vivo.
BACKGROUND
Multiple sclerosis (MS) is an autoimmune inflammatory and neurodegenerative disease of the central nervous system (CNS). It is widely accepted that inflammatory cells play major roles in the pathogenesis of MS, possibly through the use of serine protease granzyme B (GrB) secreted from the granules of cytotoxic T cells. We have previously identified GrB as a mediator of axonal injury and neuronal death. In this study, our goal was to evaluate the effect of GrB inhibition in the human system in vitro, and in vivo in EAE using the newly isolated GrB-inhibitor serpina3n.


METHODS
We used a well-established in vitro model of neuroinflammation characterized by a co-culture system between human fetal neurons and lymphocytes. In vivo, we induced EAE in 10- to 12-week-old female C57/BL6 mice and treated them intravenously with serpina3n.


RESULTS
In the in vitro co-culture system, pre-treatment of lymphocytes with serpina3n prevented neuronal killing and cleavage of the cytoskeletal protein alpha-tubulin, a known substrate for GrB. Moreover, in EAE, 50 μg serpina3n substantially reduced the severity of the disease. This dose was administered intravenously twice at days 7 and 20 post EAE induction. serpina3n treatment reduced axonal and neuronal injury compared to the vehicle-treated control group and maintained the integrity of myelin. Interestingly, serpina3n treatment did not seem to reduce the infiltration of immune cells (CD4(+) and CD8(+) T cells) into the CNS.


CONCLUSIONS
Our data suggest further studies on serpina3n as a potentially novel therapeutic strategy for the treatment of inflammatory-mediated neurodegenerative diseases such as MS.
Department of Medicine, Division of Neurology, University of Alberta, 4C Kaye Edmonton Clinic, Edmonton, Alberta, T6G 1Z1, Canada.
Yohannes
Y
Haile
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Katia
K
Carmine-Simmen
Department of Anesthesiology and Pain Medicine, University of Alberta, Edmonton, Canada.
Camille
C
Olechowski
Department of Anesthesiology and Pain Medicine, University of Alberta, Edmonton, Canada.|Department of Pharmacology, University of Alberta, Edmonton, Canada.|Centre for Neuroscience, University of Alberta, Edmonton, Canada.
Bradley
B
Kerr
Department of Biochemistry, University of Alberta, Edmonton, Canada.
R Chris
RC
Bleackley
Department of Medicine, Division of Neurology, University of Alberta, 4C Kaye Edmonton Clinic, Edmonton, Alberta, T6G 1Z1, Canada. giuliani@ualberta.ca.
Fabrizio
F
Giuliani
D000209:Acute-Phase Proteins / Q000627:therapeutic use*; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000139:chemically induced / Q000517:prevention & control*; D005260:Female; D005333:Fetus; D005620:Freund's Adjuvant / Q000633:toxicity; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009186:Myelin Sheath / Q000378:metabolism / Q000473:pathology; D063308:Myelin-Oligodendrocyte Glycoprotein / Q000633:toxicity; D016900:Neurofilament Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects; D018696:Neuroprotective Agents / Q000627:therapeutic use*; D010446:Peptide Fragments / Q000633:toxicity; D015843:Serpins / Q000627:therapeutic use*; D013601:T-Lymphocytes / Q000187:drug effects; D014404:Tubulin / Q000378:metabolism
D000209:Acute-Phase Proteins; D015703:Antigens, CD; D063308:Myelin-Oligodendrocyte Glycoprotein; D016900:Neurofilament Proteins; D018696:Neuroprotective Agents; D010446:Peptide Fragments; C516624:Serpina3n protein, mouse; D015843:Serpins; D014404:Tubulin; C445081:myelin oligodendrocyte glycoprotein (35-55); C051774:neurofilament protein H; D005620:Freund's Adjuvant