Volume 21, Issue 1, Pages 31-41 (January 2013) Lentivirus Mediated Delivery of Neurosin Promotes Clearance of Wild-type α-Synuclein and Reduces the Pathology in an α-Synuclein Model of LBD  Brian Spencer, Sarah Michael, Jay Shen, Kori Kosberg, Edward Rockenstein, Christina Patrick, Anthony Adame, Eliezer Masliah  Molecular Therapy  Volume 21, Issue 1, Pages 31-41 (January 2013) DOI: 10.1038/mt.2012.66 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Neurosin levels are reduced in postmortem brains of human or mouse Lewy body/Parkinson's disease. Temporal cortex sections of (a) control and dementia with Lewy bodies (DLB) brains or (d) non-tg control littermates and platelet-derived growth factor-β (PDGF-β)-α-syn transgenic (tg) mouse were immunostained with and antibody againt neurosin and reacted with diaminobenzidine or α-synuclein and detected with tyramide red. (b, e) Image analysis of the levels of neurosin immunoreactivity in the temporal cortex performed with NIH Image J show a reduction in the levels of neurosin immunostaining in the DLB cases and in the α-synuclein (α-syn) tg mouse. Representative image of the immunoblot analysis in human and tg mouse brain tissues (g, i). Total protein was extracted from temporal cortical from (g) human postmortem brains from control and DLB patients or (i) α-syn tg or non-tg mouse brains fractioned by centrifugation and examined by western blot for the levels of neurosin immunoreactivity. (h, j) Image analysis of the mature form of neurosin plotted as signal to actin levels, shows a reduction in the mature form of neurosin in DLB cases and α-syn tg mouse. Bar: 25 µm. *Statistical significance compared to control patients (P < 0.01, unpaired, Student's t-test). For each assay n = 6 controls and n = 8 DLB cases were used. For the mice n = 6 non-tg and n = 6 -α-syn tg mice were used (9 months old). Molecular Therapy 2013 21, 31-41DOI: (10.1038/mt.2012.66) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Neurosin degrades monomeric and oligomeric α-synuclein (α-syn) in vitro. (a) Recombinant pro-neurosin or recombinant neprilysin (Nep) at various concentrations was incubated with monomeric (m) recombinant wild-type α-synuclein (1 µmol/l) for 18 hours at 37 °C and then analyzed by western blot (anti-α-syn; BD Bioscience). (c) Oligomeric (oli) α-synuclein generated as described in Materials and Methods section was incubated with various concentrations of recombinant pro-neurosin or rNep for 18 hours at 37 °C and then analyzed by western blot (anti-α-syn; BD). Molecular weight marker is indicated on the left side of the blot and sizes of monomeric, oligomeric, and neurosin digestion products are indicated on the right side of the blot. (b, d) Image analysis of the resulting monomeric or oligomeric bands was plotted (n = 2). Molecular Therapy 2013 21, 31-41DOI: (10.1038/mt.2012.66) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Neurosin degradation of α-synuclein (α-syn) is protective for neuronal cultures. (a) Recombinant monomeric or oligomeric α-syn (1 µmol/l) was incubated with recombinant pro-neurosin (100 ng) for 18 hours at 37 °C and then analyzed by western blot (anti-α-syn; Millipore). Molecular weight marker is indicated on the left side of the blot and sizes of monomeric, oligomeric, and neurosin digestion products are indicated on the right side of the blot. Monomeric or oligomeric α-syn alone or following degradation by neurosin were incubated with B103 neuronal cells for 24 hours and then analyzed by confocal microscopy. (b) Cells were double immunolabeled with antibodies against α-syn (red) and the neuronal protein MAP2 (green) and imaged with the laser scanning confocal microscope. White arrows depict extracellular α-syn aggregates while yellow arrow heads indicate intracellular α-syn. (c) Neurite length for MAP2 immunosreactive processes was measured and plotted against vehicle-treated control neurons. Bar: 10 µm. *Statistical significance (P < 0.05, n = 3 per group, one-way ANOVA, post-hoc Dunnet's) compared to vehicle-treated cells. Molecular Therapy 2013 21, 31-41DOI: (10.1038/mt.2012.66) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Lentiviral vector driven expression of neurosin reduced the accumulation of α-synuclein (α-syn) in a neuronal cell line. The lentiviral vector expresses the pre-pro neurosin. B103 neuronal cells were infected with (a) an empty lentiviral vector (LV) control, (b) LV-α-syn wt, or (c) A53T mutant alone or in combination with LV-Neurosin or LV-siNeurosin (LV-siNeuro) double immunolabeled for neurosin (blue) and -α-syn (red) analyzed with the confocal laser scanning microscope. (d) Analysis of the levels of α-syn immunoreactivity from digital images acquired by confocal microscopy. Levels of α-syn immunoreactivity were reduced in cells treated with LV-Neurosin in neuronal cells expressing wild type but not in the A53T cells. Bar: 10 µm. *Statistical significance from LV-Control (P < 0.05, one-way ANOVA, post-hoc Dunnet's), #Statistical significance from LV-Neurosin (P < 0.05, one-way ANOVA, post-hoc Fisher). Molecular Therapy 2013 21, 31-41DOI: (10.1038/mt.2012.66) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Immunohistochemical analysis showing that delivery of lentiviral vector (LV)-Neurosin reduces the accumulation of wt α-synuclein (α-syn) in a mouse model of dementia with Lewy bodies (DLB). (a) Non-tg, (b) α-syn wild-type transgenic (tg), and (c) A53T-mutant tg mice were injected with LV-Control, LV-Neurosin, or LV-siNeurosin and after 3 months of the injection, sections were immunostained with an antibody against α-syn and analyzed with digital bright field microscopy. (d) Higher magnification images of selected areas of the cortex of LV-Control-, LV-Neurosin-, or LV-siNeurosin-treated α-syn tg mice. (e) Semiquantitative analysis of levels of α-syn immunostaining expressed as optical density. Levels of α-syn immunoreactivity were reduced in α-syn wild-type tg mice treated with LV-Neurosin but not in the A53T tg mice. Bar: for a, b, and c is 200 µm, for d is 40 µm. *Statistical significance (P <0.05, one-way ANOVA, post-hoc Dunnet's) compared to LV-Control-treated mice. #Statistical significance (P < 0.05, one-way ANOVA, post-hoc Tukey–Krammer) compared to LV-Neurosin-treated mice. N = 6 mice per group, 9 months of age. Molecular Therapy 2013 21, 31-41DOI: (10.1038/mt.2012.66) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Immunoblot analyses of the levels of α-synuclein (α-syn) mice treated with lentiviral vector (LV)-Neurosin. The neocortex and hippocampus around the injection site were dissected (~50 mg of tissue), homogenized and analyzed by western blot 3 months after injection of LV-Control, LV-Neurosin, or LV-siNeurosin in (a) non-tg and α-syn [(b) wild type and (c) A53T mutant] transgenic (tg) mice. Blots were probed with the anti-α-syn BD Bioscience antibody. Image analysis for the α-syn signal was plotted against the actin signal. Levels of α-syn immunoreactivity were reduced in α-syn wild-type tg mice treated with LV-Neurosin but not in the A53T tg mice. *Statistical significance (P < 0.05, one-way ANOVA, post-hoc Dunnet's) compared to LV-Control-treated mice. #Statistical significance (P < 0.05, one-way ANOVA, post-hoc Tukey–Krammer) compared to LV-Neurosin-treated mice. N = 6 mice per group, 9 months of age. Molecular Therapy 2013 21, 31-41DOI: (10.1038/mt.2012.66) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Effects of lentiviral vector (LV)-Neurosin delivery on levels of the dendritic marker MAP2. (a) Non-tg, (b) α-syn wild-type transgenic (tg), and (c) A53T-mutant tg mice were injected with LV-Control, LV-Neurosin, or LV-siNeurosin and after 3 months of the injection, mice were immunostained for the dendritic marker, MAP2, and analyzed with laser scanning confocal microscopy. Percent area of the neuropil in the (d) temporal cortex and (e) hippocampus occupied by MAP2 immunolabeled dendrites showing that treatment with LV-Neurosin rescued the loss of MAP2 dendrites in the temporal cortex and hippocampus of the α-synuclein (α-syn) wild-type tg but not the A53T-mutant tg mice. Bar: 10 µm. *Statistical significance (P < 0.05, one-way ANOVA, post-hoc Dunnet's) compared to non-tg LV-Control-treated mice. #Statistical significance (P < 0.05, one-way ANOVA, post-hoc Tuckey–Krammer) compared to LV-Control-treated tg mice. N = 6 mice per group, 9 months of age. Molecular Therapy 2013 21, 31-41DOI: (10.1038/mt.2012.66) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Lentiviral vector (LV)-Neurosin reverses neuropathological changes in α-synuclein (α-syn) transgenic (tg) mice. α-Syn tg mice injected with LV-Control, LV-Neurosin, or LV-siNeurosin were analyzed 3 months after injection for (a, b) Synaptophysin (synaptic marker), (c, d) NeuN (neuronal marker) and (e, f) GFAP (astroglial marker). (b) Percent area of hippocampus occupied by synaptophysin immunolabeled presynaptic terminals imaged by laser scanning confocal microscopy. (d) Stereological analysis using the disector method to estimate NeuN immunolabled neuronal counts in the hippocampus (CA2-3) neurons. (f) Optical density of levels of GFAP immunostained astroglia in the hippocampus. Bar: 20 µm. *Statistical significance (P <0.05, one-way ANOVA, post-hoc Dunnet's) compared to non-tg LV-Control-treated mice. #Statistical significance (P < 0.05, one-way ANOVA, post-hoc Tuckey–Krammer) compared to LV-Control-treated tg mice. N = 6 mice per group, 9 months of age. Molecular Therapy 2013 21, 31-41DOI: (10.1038/mt.2012.66) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions