A brain-targeted, modified neurosin (kallikrein-6) reduces α-synuclein accumulation in a mouse model of multiple system atrophy

Spencer et al. Molecular Neurodegeneration (2015) 10:48 DOI 10.1186/s13024-015-0043-6 RESEARCH ARTICLE Open Access A brain-targeted, modified neurosin (kallikrein-6) reduces α-synuclein accumulation in a mouse model of multiple system atrophy Brian Spencer1*†, Elvira Valera1†, Edward Rockenstein1, Margarita Trejo-Morales1, Anthony Adame1 and Eliezer Masliah1,2 Abstract Background: Multiple system atrophy (MSA) is a progressive, neurodegenerative disease characterized by parkinsonism, resistance to dopamine therapy, ataxia, autonomic dysfunction, and pathological accumulation of α-synuclein (α-syn) in oligodendrocytes. Neurosin (kallikrein-6) is a serine protease capable of cleaving α-syn in the CNS, and we have previously shown that lentiviral (LV) vector delivery of neurosin into the brain of a mouse model of dementia with Lewy body/ Parkinson’s disease reduces the accumulation of α-syn and improves neuronal synaptic integrity. Results: In this study, we investigated the ability of a modified, systemically delivered neurosin to reduce the levels of α-syn in oligodendrocytes and reduce the cell-to-cell spread of α-syn to glial cells in a mouse model of MSA (MBP-α-syn). We engineered a viral vector that expresses a neurosin genetically modified for increased half-life (R80Q mutation) that also contains a brain-targeting sequence (apoB) for delivery into the CNS. Peripheral administration of the LV-neurosin-apoB to the MBP-α-syn tg model resulted in accumulation of neurosin-apoB in the CNS, reduced accumulation of α-syn in oligodendrocytes and astrocytes, improved myelin sheath formation in the corpus callosum and behavioral improvements. Conclusion: Thus, the modified, brain-targeted neurosin may warrant further investigation as potential therapy for MSA. Introduction The synucleinopathies are a heterogeneous group of neurodegenerative disorders that affect 5 million people worldwide and includes Parkinson’s disease (PD), dementia with Lewy bodies (DLB), neurodegeneration with brain iron accumulation, pure autonomic failure (PAF) and multiple system atrophy (MSA) (Reviewed in [1]). Multiple system atrophy is a rapidly progressive, neurological condition characterized by parkinsonism resistant to dopamine therapy, ataxia, autonomic dysfunction, and pathological accumulation of α-synuclein (α-syn) [2–4]. This disorder differs from other synucleinopathies in that α-syn accumulates not only within * Correspondence: † Equal contributors 1 Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA Full list of author information is available at the end of the article neurons and astrocytes, but also within oligodendrocytes in the form of glial cytoplasmic inclusions [5]. This intracellular accumulation of toxic α-syn species leads to degeneration of oligodendroglial cells, loss of trophic support to neurons and subsequent neurodegeneration. The mechanisms through which α-syn leads to neurotoxicity are not completely clear, however recent evidence supports a role for oligomerization [6, 7]. Increasing evidence supports the notion that α-syn, which is primarily generated by neurons, can be toxic once released to the extracellular environment [6, 8, 9]. Extracellular aggregated α-syn can then propagate to other neurons and glial cells in a prion-like fashion [10, 11]. Although it had been previously suggested that the sole source of oligodendroglial α-syn was through endocytosis, a recent report showed α-syn mRNA in MSA oligodendrocytes suggesting that the origin of oligodendroglial α-syn might be both of endogenous nature and the result of © 2015 Spencer et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Spencer et al. Molecular Neurodegeneration (2015) 10:48 propagation from neurons and/or other oligodendroglial cells [12]. Furthermore, propagation and accumulation of α-syn within astrocytes could lead to activation of these cells and subsequent neuroinflammation [13–15]. Therefore, the development of therapeutic interventions for MSA and related neurodegenerative disorders has been focused simultaneously on reducing α-syn accumulation, increasing α-syn clearance and preventing α-syn propagation. Neurosin, (human kallikrein 6, KLK6, Zyme, Protease M), is a serine protease capable of cleaving α-syn [16– 19]. This enzyme is found to be expressed throughout the body in many tissues [20] including the CNS in the choroid plexus and in oligodendrocytes and astroglial cells [21] of healthy individuals [20], as well as neurons and microglia of the hippocampus of Alzheimer’s disease patients [16, 22]. Down-regulation of neurosin is associated with accumulation of α-syn in patients with DLB/PD [23–25] as well in animal models of DLB/PD [25], whereas over-expression of neurosin in the brain via lentiviral (LV) vector reduces the accumulation of α-syn and improves neuronal synaptic integrity in an αsyn tg mouse model of DLB/PD [25]. Neurosin is expressed as a catalytically inactive prepro protein and is activated through autocatalytic proteolysis. Upon expression and secretion, auto-activation occurs first via cleavage of the pre-pro neurosin at Q19 followed by cleavage at K21, which produces the mature neurosin enzyme. The mature neurosin can then autoproteolytically inactivate itself with cleavage at R80. This amino acid, when altered to Glutamine (Q), prevents the auto-inactivation generating a longer-acting enzyme [26]. In vitro proteolytic reactions with neurosin show that autocatalytic cleavage can begin as early as 10 min after incubation, and full cleavage to the inactive form occurs by 240 min incubation [26]. Since we have previously shown that stereotaxic injection of LV-neurosin into the hippocampus reduced the local accumulation of neuronal α-syn in a mouse model of DLB [25], for this study we sought to determine if gene therapy with this vector would also reduce the neurodegenerative process in a mouse model of MSA [27, 28]. However, given that the synucleinopathy in MSA is more disseminated throughout the CNS than PD, a more systemic approach is needed. For this reason, we engineered the recombinant neurosin for systemic delivery with the R80Q mutation to allow for longer half-life in order that the active form of the enzyme would reach the BBB, transcytose the endothelial cells, and reach the site of α-syn accumulation before it gets degraded. In addition, we fused the 38 amino acid LDL-R binding d (...truncated)