Heterologous HSPC Transplantation Rescues Neuroinflammation and Ameliorates Peripheral Manifestations in the Mouse Model of Lysosomal Transmembrane Enzyme Deficiency, MPS IIIC

Abstract

Mucopolysaccharidosis III type C (MPS IIIC) is an untreatable neuropathic lysosomal storage disease caused by a genetic deficiency of the lysosomal N-acetyltransferase, HGSNAT, catalyzing a transmembrane acetylation of heparan sulfate. HGSNAT is a transmembrane enzyme incapable of free diffusion between the cells or their cross-correction, which limits development of therapies based on enzyme replacement and gene correction. Since our previous work identified neuroinflammation as a hallmark of the CNS pathology in MPS IIIC, we tested whether it can be corrected by replacement of activated brain microglia with neuroprotective macrophages/microglia derived from a heterologous HSPC transplant. Eight-week-old MPS IIIC (Hgsnat^P304L) mice were transplanted with HSPC from congenic wild type mice after myeloablation with Busulfan and studied using behavior test battery, starting from the age of 6 months. At the age of ~8 months, mice were sacrificed to study pathological changes in the brain, heparan sulfate storage, and other biomarkers of the disease. We found that the treatment corrected several behavior deficits including hyperactivity and reduction in socialization, but not memory decline. It also improved several features of CNS pathology such as microastroglyosis, expression of pro-inflammatory cytokine IL-1β, and accumulation of misfolded amyloid aggregates in cortical neurons. At the periphery, the treatment delayed development of terminal urinary retention, potentially increasing longevity, and reduced blood levels of heparan sulfate. However, we did not observe correction of lysosomal storage phenotype in neurons and heparan sulfate brain levels. Together, our results demonstrate that neuroinflammation in a neurological lysosomal storage disease, caused by defects in a transmembrane enzyme, can be effectively ameliorated by replacement of microglia bearing the genetic defect with cells from a normal healthy donor. They also suggest that heterologous HSPC transplant, if used together with other methods, such as chaperone therapy or substrate reduction therapy, may constitute an effective combination therapy for MPS IIIC and other disorders with a similar etiology.

Keywords: HSPC; Sanfilippo disease; allogenic HSPC transplantation; heparan sulfate; microglia; mucopolysaccharidosis; neuroinflammation.

Figure 1

Transplantation of WT HSPC rescues several behavior abnormalities in Hgsnat^P304L mice. (A) Experimental design. (B–E) WT, untreated, and transplanted Hgsnat^P304L mice were analyzed at 6 months of age for presence of hyperactivity (increased total distance traveled) and reduced anxiety (increased number of entries to the center of arena) in the OF test (B), short-term memory deficit (reduced new object exploration time and discrimination index) using the NOR test (C), defects in associative learning (reduced freezing time) using the CFC test (D), and a reduced sociability (absence of interest in a stranger mouse) using the TCS test (E). Individual results, means and SD of experiments performed with 10–12 male and female mice per genotype, per treatment, are shown. (A–D) Mouse groups were compared with untreated Hgsnat^P304L mice and p values calculated using one-way ANOVA with Dunnett post hoc test. (E) p values were calculated using two-way ANOVA with Tukey post hoc test.

Figure 2

Transplantation of WT HSPC rescues urinary retention in Hgsnat^P304L mice but does not alter organomegaly. (A,B) Development of urinary retention in untransplanted and transplanted Hgsnat^P304L and WT mice. (A) Urinary retention score (0, none; 1, mild; 2, moderate; 3, severe; 4, very severe) was assessed for untransplanted and transplanted Hgsnat^P304L male and female mice, and their WT counterparts (n = 16 for each group). The significance of differences between untreated and treated groups was determined by two-way ANOVA (p < 0.0001). By the age of 34 weeks, all untreated Hgsnat^P304L mice but none of transplanted Hgsnat^P304L mice had severe urinary retention requiring euthanasia. (B) Volume of urine in the bladder of mice at sacrifice. The amount of urine in the bladders of transplanted Hgsnat^P304L mice is not significantly different from that in WT mice. (C–E) Wet organ weight of mice at sacrifice (34–36 weeks). Enlargement of liver (C), kidney (D), and spleen (E) compared to age-matched WT controls, consistent with the lysosomal storage and inflammatory cell infiltration, is detected in both untreated and transplanted Hgsnat^P304L mice. Graphs show individual data, means and SD. p values were calculated using ANOVA with Tukey post hoc test.

Figure 3

Transplanted Hgsnat^P304L mice show normalized HS levels in plasma and partial rescue of HGSNAT deficiency and increased activity of lysosomal β-hexosaminidase in bone marrow and peripheral tissues, but not in the CNS. (A,B) HGSNAT activity (A) and total lysosomal β-hexosaminidase activity (B) in the tissues of 8-month-old WT, Hgsnat^P304L and transplanted Hgsnat^P304L mice. (C,D) Levels of disaccharides produced by enzymatic digestion of HS, ΔDiHS-OS (C), and ΔDiHS-NS (D) measured by tandem mass spectrometry in blood serum, urine, and tissues of WT, Hgsnat^P304L and transplanted Hgsnat^P304L mice. All graphs show individual data, means and SD of experiments performed using tissues from five male and female mice per genotype per treatment except for the bone marrow, where results for three samples, each containing pooled tissues of two mice, are shown. p values were calculated by one-way ANOVA with Tukey post hoc test. Only p values < 0.05 are shown.

Figure 4

Systemic and brain inflammation is reduced in Hgsnat^P304L mice transplanted with WT HSPC. To estimate inflammatory activation of spleen macrophages, frequency of CX3CR1 labeling on CD11b-positive cells (A) and CD63 (B) or CD68 (C) labeling on CX3CR1/CD11b-positive cells was evaluated in populations of dissociated splenocytes from WT, Hgsnat^P304L, and Hgsnat^P304L mice transplanted with WT HSPC (Hgsnat^P304L–BM). To estimate the level of neuroinflammation, frequency for CD63 (D) and CD68 (E) was evaluated on CD45/CD11b/CX3CR1-positive cells in populations of dissociated brain cells of WT (grey traces), Hgsnat^P304L (blue traces), and Hgsnat^P304L–BM (red traces) mice. Data are shown as % of CD63+ or CD68+ cells among CD45+/CD11b+/CX3CR1+ cells (graphs on the left) or as total numbers of CD63+ or CD68+ cells per mg of brain tissue (graphs on the right). (F,G) Expression levels of pro-inflammatory cytokine IL-1β (F) were reduced in treated compared to untreated Hgsnat^P304L mice while the expression levels of an anti-inflammatory cytokine, TGF-β1 (G), was unchanged. Graphs show individual data, means and SD for 5 mice per group (A–E) and 3 mice per group (F,G). p values were calculated using one-way ANOVA with Dunn post hoc test. Only p values < 0.05 are shown.

Figure 5

Reduction in neuroinflammation is mediated by infiltration of WT donor-derived CX3CR1/CD45.1-positive cells. (A) Frequency for CD45.1 and CD45.2 was evaluated on CD45/CD11b/CX3CR1-positive dissociated brain cells of WT, untransplanted Hgsnat^P304L, and transplanted Hgsnat^P304L–BM mice. (B,C) MFI for CD63 (B) and CD68 (C) was evaluated on CD45/CD11b/CX3CR1-positive dissociated brain cells of WT, Hgsnat^P304L, and transplanted Hgsnat^P304L–BM mice. Graphs show individual data, means and SD for five mice per group. p values were calculated using one-way ANOVA with Dunn post hoc test.

Figure 6

Hgsnat^P304L mice transplanted with WT HSPC reveal recovery of astrogliosis and amelioration of levels of CNS pathology biomarkers in the somatosensory cortex and hippocampus. (A,B) Levels of activated CD68-positive microglia are reduced in the somatosensory cortices (A) and hippocampi (B) of 8-month-old transplanted mice compared to untransplanted Hgsnat^P304L mice. Levels of GFAP-positive astrocytes are reduced in the hippocampus, but not in the cortex. Panels show representative images of the somatosensory cortex (layers 4–5) and the CA1 region of the hippocampus labeled for GFAP (green) and CD68 (red). DAPI was used as a nuclear counterstain. Bar graphs show quantification of CD68- and GFAP-positive areas with ImageJ 1.54i software. (C) Reduction in thioflavin S-positive β-amyloid aggregates in the cortical neurons of transplanted Hgsnat^P304L mice. Panels show representative images of brain cortex (layers 4–5) labeled for β-amyloid (red) and misfolded proteins (Thioflavin-S, green). Graphs show quantification of β-amyloid and Thioflavin-S staining with ImageJ software. (D) Levels of misfolded SCMAS aggregates in cortical neurons are reduced in transplanted Hgsnat^P304L mice. Panels show representative images of somatosensory cortex (layers 4–5), labeled for SCMAS (red) and neuronal marker NeuN (green). DAPI (blue) was used as a nuclear counterstain. The bar graph shows quantification of SCMAS staining with ImageJ software. (E) Reduction in granular autofluorescent ceroid material in cortical neurons. Panels display representative images of brain cortex showing autofluorescent ceroid inclusions in the neurons (green). The graph shows quantification of autofluorescence with ImageJ software. (F) Levels of G_M2 ganglioside in cortical neurons are not reduced by transplantation. Panels show representative images of somatosensory cortex (layers 4–5) immunolabeled for G_M2 ganglioside (green). The bar graphs show quantification of G_M2 staining with ImageJ software. In all panels, scale bars equal 25 µm. All graphs show individual results, means and SD from experiments conducted with five mice (three panels per mouse) per genotype per treatment. p values were calculated using Nested one-way ANOVA test with Tukey post hoc test. Only p values < 0.05 are shown.