Suppression of agrin-22 production and synaptic dysfunction in Cln1 (-/-) mice

Abstract

Objective: Oxidative stress in the brain is highly prevalent in many neurodegenerative disorders including lysosomal storage disorders, in which neurodegeneration is a devastating manifestation. Despite intense studies, a precise mechanism linking oxidative stress to neuropathology in specific neurodegenerative diseases remains largely unclear.

Methods: Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease caused by mutations in the ceroid lipofuscinosis neuronal-1 (CLN1) gene encoding palmitoyl-protein thioesterase-1. Previously, we reported that in the brain of Cln1 (-/-) mice, which mimic INCL, and in postmortem brain tissues from INCL patients, increased oxidative stress is readily detectable. We used molecular, biochemical, immunohistological, and electrophysiological analyses of brain tissues of Cln1 (-/-) mice to study the role(s) of oxidative stress in mediating neuropathology.

Results: Our results show that in Cln1 (-/-) mice oxidative stress in the brain via upregulation of the transcription factor, CCAAT/enhancer-binding protein-δ, stimulated expression of serpina1, which is an inhibitor of a serine protease, neurotrypsin. Moreover, in the Cln1 (-/-) mice, suppression of neurotrypsin activity by serpina1 inhibited the cleavage of agrin (a large proteoglycan), which substantially reduced the production of agrin-22, essential for synaptic homeostasis. Direct whole-cell recordings at the nerve terminals of Cln1 (-/-) mice showed inhibition of Ca(2+) currents attesting to synaptic dysfunction. Treatment of these mice with a thioesterase-mimetic small molecule, N-tert (Butyl) hydroxylamine (NtBuHA), increased agrin-22 levels.

Interpretation: Our findings provide insight into a novel pathway linking oxidative stress with synaptic pathology in Cln1 (-/-) mice and suggest that NtBuHA, which increased agrin-22 levels, may ameliorate synaptic dysfunction in this devastating neurodegenerative disease.

Figure 1

Dendritic filopodia abnormality in the hippocampus of Cln1 ^−/− mice. Representative Golgi‐Cox‐stained neuron images of dendrites from WT mouse hippocampus (left panels) and their Cln1 ^−/− littermates (right panels) at 1 month (A), 3 months (C) and 6 months (E) of age. The majority of the spine‐type affected was stubby spines (A, C, and E). The reductions of total spine in the hippocampi of Cln1 ^−/− mice occur in a time‐dependent manner. The numbers of total spines are 9% less in 1‐month old Cln1 ^−/− mouse hippocampus compared to those of their WT littermates (B). The total spine levels decline further in 3‐month‐old Cln1 ^−/− mice (D). There is a significant reduction in dendritic filopodia levels in the hippocampi of 6‐month‐old Cln1 ^−/− mice (35% of WT; P < 0.01, one way ANOVA) (F). The results are presented as the mean spine counts from five randomly selected hippocampal neurons in each mouse brain. A 30 μm long dendrite was used as a counting standard, and five dendrites were counted separately in each neuron. WT, wild‐type; ANOVA, analysis of variance.

Figure 2

Exocytosis, endocytosis and Ca²⁺ currents at the calyces of Held. Exocytosis of synaptic vesicles and RRP in the calyx of Held from both WT and Cln1 ^−/− mice were measured by monitoring the whole‐cell membrane capacitance (C _m) (A and B). Compared to its WT littermate, Cln1 ^−/− mice had 25% reduction in exocytosis (upper panel), a small, but insignificant decrease in the RRP size (middle panel), and significant inhibition of vesicle mobilization of releasable vesicles (C). The depolarization‐evoked Ca²⁺ currents were also detected. (D) The peak amplitude (ICa) was reduced from 1.7 nA (WT) to 1.2 nA (Cln1 ^−/−); while the charge of Ca²⁺ influx was reduced from 24 pC (WT) to 20 pC (Cln1 ^−/−) for a single 20 msec pulse, and from 190 pC (WT) to 144 pC (Cln1 ^−/−) for a train of 10 pulses (E). Compared to the WT mice (F), Cln1 ^−/− littermates showed substantial reduction in rapid endocytosis (221 fF/sec vs. 136 fF/sec) (F and G) and in slow endocytosis (52 fF/sec vs. 37 fF/sec) (H and I). RRP, readily releasable vesicle pool; WT, wild‐type; ICa, calcium current. *means that the results are significantly different between wt and Ppt1 ^−/−, at the significance level: P < 0.05 (two‐tailed student t‐test).

Figure 3

Neurotrypsin‐mediated agrin‐22 production is disrupted in Cln1 ^−/− mouse brain. Western blots analysis of intact and cleaved agrin (agrin‐22) proteins in wild‐type (WT) and Cln1 ^−/− mouse brains (A). The lower panel showed that the mean densitometry of each agin‐22 from WT and Cln1 ^−/− mouse brains. Compared with WT mice the levels of agrin‐22 in the brain of Cln1 ^−/− littermates were markedly reduced (P < 0.05, Excel student t‐test) (A). Neurotrypsin‐mRNA levels measured by qRT‐PCR showed no significant difference between WT mice and their Cln1 ^−/− littermates (1 and 2 for 1‐month‐old WT and Cln1 ^−/− mice, respectively; 3 and 4 for 3‐month‐old WT and Cln1 ^−/− mice, respectively; 5 and 6 for 6‐month old WT and Cln1 ^−/− mice, respectively) (B). Western blot analysis showed that neurotrypsin protein levels between Cln1 ^−/− and their WT littermates were virtually no difference (C). Enzymatic activities of neurotrypsin in brain tissues of 6‐month old Cln1 ^−/− mice was significantly lower compared with those of their WT littermates (D). Quantitative RT‐PCR and Western blot analyses showed increased levels of serpina1‐mRNA (E; 1 and 2 for 1‐month‐old WT and Cln1 ^−/− mice; 3 and 4 for 3‐month‐old WT and Cln1 ^−/− mice; 5 and 6 for 6‐month‐old WT and Cln1 ^−/− mice, P < 0.05, Excel student t‐test) and protein (F) from cortical tissues of Cln1 ^−/− mice compared with those of their WT littermates. Serpina1 overexpression in WT mouse glial cells significantly reduced the level of agrin‐22 (G). To further confirm the inhibition of serpina1 to neurotrypsin activity, serpina1 knockdown in Cln1 ^−/− glial cells showed that agrin‐22 protein level was increased (H).

Figure 4

Agrin and Serpina1 expression in brain tissues and cultured astroglia. Serpina1‐mRNA levels in cultured neurons and astroglia (A and B) (P < 0.01; Excel student t‐test); (C) Western blot analysis from cultured neurons from wild‐type (WT) and Cln1 ^−/− mice. β‐actin was used as loading controls. (D) Western blot analysis cultured astroglia from WT and Cln1 ^−/− mice. (E) Cytochemical localization of serpina1 in cultured neuron from WT and Cln1 ^−/− mice. Cultured neurons were stained with anti‐serpina1 and beta‐3‐tublin (neuronal marker) antibodies, respectively; (F) Cultured astroglia stained with anti‐serpina1, and anti‐GFAP (astrocyte marker), respectively. (G) We performed experiments to cytochemically detect agrin and neurotrypsin in cultured astroglia and the results showed that cultured astroglia from WT mice (upper panel) and their Cln1 ^−/− littermates (lower panel) were stained positive for agrin as well as neurotrypsin.

Figure 5

Colocalization of neurotrypsin with serpina1 in astrocyte‐neuron cocultures. (A) Pull‐down assay using serpina1 antibody showed that there was much more neurotrypsin in immunoprecipitates of synaptosome preparations from Cln1 ^−/− mouse brains compared with those of its wild‐type (WT) littermates; (B) Western blot analysis of the immunoprecipitates with neurotrypsin antibody showed serpina1 is pulled down in synaptosomal preparations from WT mice and their Cln1 ^−/− littermates; (C) Astroglia and neurons were cocultured and stained with anti‐serpina1 and anti‐synaptophysin (a synaptosome marker); (D) Cocultured astrocyte with neurons from WT and Cln1 ^−/− mice were stained with anti‐neurotrypsin and synaptophysin.

Figure 6

Oxidative stress via C/EBP‐δ upregulates serpina1 expression: effect of anti‐oxidants. The levels of mRNA (A) and protein (B) of oxidative‐stress sensitive transcription factor, C/EBP‐δ, were significantly higher in the WT glia cells treated with H₂O₂ compared with those of untreated WT glia cells. H₂O₂ treatment markedly increased levels of both serpina1‐mRNA (C) and serpina1‐protein (D and E) in cultured WT glia cells. Compared to the untreated and scramble siRNA‐treated controls, C/EBP‐δ‐mRNA knockdown significantly reduced the levels of sperpina1‐mRNA (F): 1, untreated control; 2, H₂O₂‐treated; 3, scramble siRNA‐treated; 4, siRNA‐ + H₂O₂‐treated; 5, C/EBP‐δ‐siRNA‐treated; 6, C/EBP‐δ‐siRNA‐ + H₂O₂‐treated; and serpina1‐protein (G) in cultured Cln1 ^−/−astroglia that were treated with H₂O₂ (400 μmol/L for 1 h), (n = 3, P < 0.05). Administration of either NtBuHA or RSV to Cln1 ^−/− mice for 3 months significantly lower levels of C/EBP‐δ ‐mRNA (H) and C/EBP‐δ‐protein (I) compared with untreated Cln1 ^−/− mice (P < 0.05, Excel student t‐test). Levels of serpina1‐mRNA (J) and serpina1‐protein (K) were reduced in the anti‐oxidant treated mice compared with those of untreated Cln1 ^−/− mice. C/EBP‐δ, CCAAT enhancer binding protein; WT, wild‐type; RSV, resveratrol.

Figure 7

Loss of hippocampal interneurons during early development of Cln1 ^−/− mice. Representative images of hippocampal sections from WT mice (upper panels) and those from their Cln1 ^−/− littermates (lower panels) stained for GABAergic interneuron markers at 1 month (A and C) and 6 months of age (B and D). Markers examined were CCK, CoupTFII, SOM, and PV. Pooled group data for cell density counts of the labeled interneurons in WT and Cln1 ^−/− are provided in (C) and (D) for mice aged 1 and 6 months, respectively; (C) a total of 24 sections per marker from 3 different WT and Cln1 ^−/− mice were processed and counted parallel; (D) a total of 8–12 sections per marker from a WT mouse and those from a Cln1 ^−/− littermate were examined. Excel student t‐test was used for statistical analysis of the data, P < 0.01. WT, wild‐type; CCK, cholecystokinin; CoupTFII, COUP transcription factor 2; SOM, somatostatin; PV, parvalbumin.