Amyloidosis, Synucleinopathy, and Prion Encephalopathy in a Neuropathic Lysosomal Storage Disease: The CNS-Biomarker Potential of Peripheral Blood

<div><p>Mucopolysaccharidosis (MPS) IIIB is a devastating neuropathic lysosomal storage disease with complex pathology. This study identifies molecular signatures in peripheral blood that may be relevant to MPS IIIB pathogenesis using a mouse model. Genome-wide gene expression microarrays on pooled RNAs showed dysregulation of 2,802 transcripts in blood from MPS IIIB mice, reflecting pathological complexity of MPS IIIB, encompassing virtually all previously reported and as yet unexplored disease aspects. Importantly, many of the dysregulated genes are reported to be tissue-specific. Further analyses of multiple genes linked to major pathways of neurodegeneration demonstrated a strong brain-blood correlation in amyloidosis and synucleinopathy in MPS IIIB. We also detected prion protein (Prnp) deposition in the CNS and Prnp dysregulation in the blood in MPS IIIB mice, suggesting the involvement of Prnp aggregation in neuropathology. Systemic delivery of trans-BBB-neurotropic rAAV9-hNAGLU vector mediated not only efficient restoration of functional <i>α-N</i>-acetylglucosaminidase and clearance of lysosomal storage pathology in the central nervous system (CNS) and periphery, but also the correction of impaired neurodegenerative molecular pathways in the brain and blood. Our data suggest that molecular changes in blood may reflect pathological status in the CNS and provide a useful tool for identifying potential CNS-specific biomarkers for MPS IIIB and possibly other neurological diseases.</p></div

Functional and disease association of blood transcriptional profile in MPS IIIB mice.

<p>Genome-wide gene expression microarrays were performed on pooled blood and brain RNA samples from 6 mo-old MPS IIIB mice and their wt littermates (n = 6/group, M:F = 1∶1). Genes of transcripts were identified by Ingenuity Pathway Analysis (IPA).</p>*<p>Genes associated with specific tissues according to enrichment analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID); <b>FC>2:</b> fold change >2;</p>**<p>% of known genes.</p

Dysregulation of neurodegeneration associated genes in 6-month-old MPS IIIB mice.

<p>*Gene expression microarray data were expressed as fold changes in MPS IIIB mice, relative to wt.</p><p>+increase;</p><p>−decrease.</p

Dysregulation of pathways involved in amyloidosis in the brain and blood in MPS IIIB mice and their response to rAAV9-hNAGLU gene delivery.

<p><b>a.</b> Total RNA from the brain (cortex), peripheral blood (PB) and/or representative control somatic tissues of 2 mo and/or 6 mo old wt, MPS IIIB mice, and MPS IIIB mice treated with an IV injection of rAAV9-CMV-hNAGLU (1×10¹³ vg/kg), were assayed by qRT-PCR (n = 9–12/group). Data: relative expression vs. wt. <b>b & c.</b> Brain tissue sections (4 µm) from 6 mo-old mice (n = 7) were assayed by IHC for Necab3. Necab3-positive calls/signals were stained brown (<b>b</b>) (red arrows). Necab3 staining intensity was quantitated using ImageJ (<b>c</b>). <b>d.</b> Whole cell proteins of brain cortical tissues from 6 mo-old mice were assayed by ELISA for pApp levels. <b>+/+:</b> wt mice; <b>−/−:</b> non-treated MPS IIIB mice; <b>AAV9:</b> rAAV9-treated MPS IIIB mice; CTX: cerebral cortex; DG: dentate gyrus of hippocampus. <b>*:</b> P<0.05 vs. wt; <b>#:</b> P<0.05 vs. AAV9-treated; <b>+:</b> P>0.05 vs. wt; <b>$:</b> P>0.05 vs.AAV9-treated. Scale bar: 50 µm.</p

Gene dysregulation involved in synucleinopathy in the brain and blood in MPS IIIB mice and their response to rAAV9-hNAGLU gene delivery.

<p><b>a & b</b>: Total RNA from the brain (cortex), peripheral blood (PB) and control somatic tissues of 6 mo old mice were assayed for Snca (<b>a</b>) and Park2 (<b>b</b>) by qRT-PCR (n = 9–12/group). Data: relative expression vs. wt. Brain tissue sections (4 µm) from 6 mo-old mice (n = 7) were assayed by IHC for Snca (<b>c</b>) and Park2 (<b>d</b>), positive cells/signals were stained brown (d). Snca staining intensity was quantitated using ImageJ (<b>c</b>). <b>+/+:</b> wt mice; <b>−/−:</b> non-treated MPS IIIB mice; <b>AAV9:</b> rAAV9-treated MPS IIIB mice; <b>CA3:</b> hippocampus CA3 region; <b>DG:</b> dentate gyrus; <b>Py:</b> pyramidal cell layer of hippocampus; <b>red arrows:</b> Park2-positive neuronal cell bodies; <b>black arrows:</b> Park2-positive neuronal processes. <b>*:</b> P<0.05 vs. wt; <b>#:</b> P<0.05 vs. AAV9-treated; <b>+:</b> P>0.05 vs. wt; <b>$:</b> P>0.05 vs.AAV9-treated. Scale bar: 50 µm.</p

Dysregulation in prion protein in the brain and blood in MPS IIIB mice and their response to rAAV9-hNAGLU gene delivery.

<p>Total RNA from the brain (cortex), peripheral blood (PB) and control somatic tissues of 6 mo old mice were assayed for Prnp by qRT-PCR (n = 9–12/group) (<b>a</b>). Data: relative expression vs. wt. Brain tissue sections (4 µm) from 6 mo-old mice (n = 7) were assayed for Prnp by immunohistochemistry (<b>b, c</b>). Prnp-positive signals were stained brown (<b>b</b>). Prnp staining intensity in PL was quantitated using ImageJ (<b>c</b>). <b>+/+:</b> wt mice; <b>−/−:</b> non-treated MPS IIIB mice; <b>AAV9:</b> rAAV9-treated MPS IIIB mice; <b>#1:</b> mouse with low Prnp IHC intensity; <b>#2:</b> mouse with high Prnp IHC intensity; <b>DG:</b> dentate gyrus of hippocampus. <b>PL:</b> polymoph layer of DG; <b>*:</b> P<0.05 vs. wt; <b>#:</b> P<0.05 vs. AAV9-treated; <b>+:</b> P>0.05 vs. wt; <b>$:</b> P>0.05 vs.AAV9-treated. Scale bar: 50 µm.</p

rAAV9-mediated restoration of NAGLU activity, correction of GAG storage and astrocytosis, and functional benefits MPS IIIB mice.

<p>4–6wk old MPS IIIB mice were treated with an IV injection of rAAV9-CMV-hNAGLU vector (1×10¹³ vg/kg). Mice were tested for behavior in Morris water maze at 5–5.5 mo old (n = 13) (<b>a</b>). Longevity studies are ongoing (n = 11) (<b>b</b>).Tissue analyses were performed at 6 mo of age. <b>c.</b> tissue NAGLU activity (no detectable NAGLU activity in tissues from non-treated MPS IIIB mice). <b>d.</b> Immunofluorescence (IF) for hNAGLU. Red fluorescence: hNAGLU-positive cells/signals. CTX: cerebral cortex; TH: thalamus; <b>BS:</b> brain stem; <b>Live:</b> liver; <b>Hrt:</b> heart. <b>Green arrows:</b> hNAGLU-positive neurons; <b>Yellow arrows:</b> hNAGLU positive vasculatures. <b>e.</b> tissue GAG contents; <b>f.</b> IF staining for LAMP-1. Red fluorescence: LAMP-1-positive calls/signals. g. IF staining for GFAP. Green fluorescence: GFAP-positive cells/signals. <b>*:</b> P<0.05 vs. WT; <b>#:</b> P<0.05 vs. AAV9-treated; <b>+:</b> P>0.05 vs. WT; ^∧: P>0.05 vs +/+ and AAV9-treated. Scale bar: 50 µm.</p