Volume 16, Issue 4, Pages 649-656 (April 2008) Intraventricular Enzyme Replacement Improves Disease Phenotypes in a Mouse Model of Late Infantile Neuronal Ceroid Lipofuscinosis  Michael Chang, Jonathan D Cooper, David E Sleat, Seng H Cheng, James C Dodge, Marco A Passini, Peter Lobel, Beverly L Davidson  Molecular Therapy  Volume 16, Issue 4, Pages 649-656 (April 2008) DOI: 10.1038/mt.2008.9 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 Progressive reactive astrocytosis in the CLN2–/– mouse. Coronal brain sections from 35-, 63-, 99-, and 148-days-old CLN2 –/– mice were immunostained for glial fibrillary acidic protein (GFAP). (a–d) Images from primary motor cortex, (e) hippocampus, (f) striatum. (g) Thresholding analysis via Image J software indicates the fraction of total area that is positive for GFAP staining in primary motor cortex (n= 3 mice per timepoint, three sections per mouse). Data expressed as mean ± SEM. Scale bars for (a–d) is 100 μm, while for (e and f) it is 200 μm. Molecular Therapy 2008 16, 649-656DOI: (10.1038/mt.2008.9) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 Cerebellar and brainstem pathology. Sections through (a–f) cerebellum and (g and h) brainstem of 150-days-old CLN2 –/– mice (b–d,f,h) or age-matched CLN2+/– control littermates (a,e,g). (a and b) Immunostain for glial fibrillary acidic protein (GFAP) reveals gliosis in granular layer (GL) and Bergmann gliosis in molecular layer (ML, arrow) of CLN2 –/– mice (b) but not CLN2+/– controls (a). (c and d) Co-immunostain for GFAP (red) and the Purkinje cell marker calbindin (green). (e and f) Immunostain for Neu-N in cerebellum. (g and h) Nissl-stained coronal sections through the brainstem at the level of the lateral medullary reticular formation (arrows). Scale bars for panels (a and b) is 200 μm, while for panels (c and d) it is 100 μm, and for panels (e–h) is 500 μm. Molecular Therapy 2008 16, 649-656DOI: (10.1038/mt.2008.9) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 Neuropathology in human late infantile neuronal ceroid lipofuscinosis (LINCL). (aand b) Immunohistochemical staining for the astrocytic marker glial fibrillary acidic protein (GFAP) in human brain sections from the primary motor cortex of (a) control and (b) LINCL brains. In (b), arrowheads denote hypertrophied astrocytes and GFAP+ processes encircling persisting distended neurons (denoted by *). Arrows denote microglia clusters. (c and d) Representative hematoxylin-stained sections from the cerebellar vermis from (c) control and (d) LINCL brains. Purkinje neurons in control tissue (arrows, c), are diminished in LINCL cerebellar sections (d, arrowhead) and if present are distended. (e–g) Multichannel confocal microscopy to assess autofluorescence and GFAP immunoreactivity in LINCL brain. (e) Blue 405 nm; (f) red 546 nm; (g) GFAP immunoreactivity, 488 nm; (h) merged image. Autofluorescence is seen within GFAP-positive astrocytes (arrowheads) and neurons (arrow). (i and j) GFAP-stained sections from (i) control and (j) LINCL cerebellum. Arrowheads in (j) depict hypertrophied astrocytes in the granular layer (GL) and molecular layer (ML); *denotes Bergmann gliosis in the molecular layer. (k and l) Bielschowsky silverstain of axons in cerebellar white matter tract in sections from (k) control and (l) LINCL brain. Scale bar for panels a and b is 50 μm; for panels c and d is 500 μm (C–D); for panels e–h is 10 μm; for panels k–l 100 μm. Molecular Therapy 2008 16, 649-656DOI: (10.1038/mt.2008.9) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 Tripeptidyl peptidase 1 (TPP1) enzyme distribution following intraventricular infusion. (a) Nissl-stained serial sections through cerebral cortex. Entrance of cannula into ventricular space (arrow). (b–d) Immunofluorescent staining for TPP1 in periventricular parenchyma including the (b) hippocampus (*), (c) corpus callosum, and (e) meninges. At these camera settings, autofluorescence from endogenous storage material in the late infantile neuronal ceroid lipofuscinosis (LINCL) brain is not detectable. (e) Representative Western blot for TPP1 in brain homogenate from enzyme-treated [enzyme replacement therapy (ERT)] and mock-treated (mock) CLN2 –/– mice. PE denotes purified TPP1 proenzyme. Only the mature enzyme is detectable in ERT-treated mice indicating uptake and autoactivation in vivo. (f) In vitro fluorometric TPP1 enzyme activity assay with brain homogenates from serial bilateral coronal sections of enzyme-treated CLN2 –/–, mock-treated CLN2 –/–, and CLN2+/– mice. Data presented as mean ± SEM. Scale bars for panels b and d is 500 μm, while for panel cit is 100 μm. Molecular Therapy 2008 16, 649-656DOI: (10.1038/mt.2008.9) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 5 Effect of enzyme replacement on tremor phenotype and gliosis of motor cortex. (a) Mean tremor amplitude, expressed in millivolts, in enzyme-treated CLN2 –/–, mock-treated CLN2 –/–, and CLN2+/– mice from 60 days of age to 105 days of age. (b–d) Images from motor cortex of (b) CLN2+/–, (c) mock-treated CLN2 –/–, and (d) enzyme-treated CLN2 –/– mice immunostained for glial fibrillary acidic protein (GFAP). (e and f) Thresholding analysis via Image J software for (e) positive GFAP staining and (f) autofluorescence in primary motor cortex. Enzyme-treated group was compared to the mock-treated group at each time point and statistical significance was determined by student's t-test. P ≤ 0.05 are indicated by *. All data are expressed as mean ± SEM. Scale bars for panels b–d is 100 μm. ERT, enzyme replacement therapy. Molecular Therapy 2008 16, 649-656DOI: (10.1038/mt.2008.9) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 6 Effect of enzyme replacement on survival of neurons in deep cerebellar nuclei (DCNs). (a–c) Sagittal cerebellar sections immunostained for Neu-N from (a) CLN2+/–, (b) enzyme-treated CLN2 –/–, and (c) mock-treated CLN2 –/– mice. (d) Scatter plot of results from a blinded neuropathological grading of DCNs (1 represents few remaining intact neurons; 2 represents intermediate degree of neuron loss; 3 represents no discernable neuron loss). Scale bars for panels a–c is 500 μm. ERT, enzyme replacement therapy. Molecular Therapy 2008 16, 649-656DOI: (10.1038/mt.2008.9) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions