Evidence for Aberrant Astrocyte Hemichannel Activity in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL)

Abstract

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3 that leads to vision loss, progressive cognitive and motor decline, and premature death. Morphological evidence of astrocyte activation occurs early in the disease process and coincides with regions where neuronal loss eventually ensues. However, the consequences of CLN3 mutation on astrocyte function remain relatively ill-defined. Astrocytes play a critical role in CNS homeostasis, in part, by their ability to regulate the extracellular milieu via the formation of extensive syncytial networks coupled by gap junction (GJ) channels. In contrast, unopposed hemichannels (HCs) have been implicated in CNS pathology by allowing the non-discriminant passage of molecules between the intracellular and extracellular milieus. Here we examined acute brain slices from CLN3 mutant mice (CLN3Δex7/8) to determine whether CLN3 loss alters the balance of GJ and HC activity. CLN3Δex7/8 mice displayed transient increases in astrocyte HC opening at postnatal day 30 in numerous brain regions, compared to wild type (WT) animals; however, HC activity steadily decreased at postnatal days 60 and 90 in CLN3Δex7/8 astrocytes to reach levels lower than WT cells. This suggested a progressive decline in astrocyte function, which was supported by significant reductions in glutamine synthetase, GLAST, and connexin expression in CLN3Δex7/8 mice compared to WT animals. Based on the early increase in astrocyte HC activity, CLN3Δex7/8 mice were treated with the novel carbenoxolone derivative INI-0602 to inhibit HCs. Administration of INI-0602 for a one month period significantly reduced lysosomal ceroid inclusions in the brains of CLN3Δex7/8 mice compared to WT animals, which coincided with significant increases in astrocyte GJ communication and normalization of astrocyte resting membrane potential to WT levels. Collectively, these findings suggest that alterations in astrocyte communication may impact the progression of JNCL and could offer a potential therapeutic target.

Efficacy of phosphodiesterase‐4 inhibitors in juvenile Batten disease (CLN3)

“…PF‐06266047 also led to significant reductions in microglial activation (see Fig 4C, D). GFAP immunoreactivity was significantly enhanced in the S1BF and TH of Cln3 Δex7/8 mice as compared to WT controls, in agreement with previous observations from us and others 6, 7. Roflumilast significantly reduced GFAP immunoreactivity in both brain regions of Cln3 Δex7/8 mice (Fig 5A, B).…”

“…Additionally, Cln3 Δex7/8 astrocytes show reduced expression of the glutamate transporter GLAST and glutamine synthetase, indicating altered glutamate recycling capacity, which supports the neurotransmitter imbalance associated with JNCL 6, 9, 10, 11. We have also reported that Cln3 Δex7/8 microglia are primed toward a proinflammatory phenotype when exposed to danger signals present in the Cln3 Δex7/8 brain 8.…”

“…In JNCL mouse models, reactive glia are apparent within 1 to 3 months of age and coincide with areas of eventual neuron loss, which is significantly delayed in comparison (ie, 12–18 months) 6, 7. Current evidence supports non–cell‐autonomous influences of diseased glia in JNCL pathogenesis in addition to intrinsic neuronal deficits.…”

“…For example, Cln3 Δex7/8 microglia are primed to produce exaggerated levels of several inflammatory mediators in response to danger signals encountered in the JNCL brain 8. Likewise, molecules critical for astrocyte glutamate regulation are dramatically reduced in Cln3 Δex7/8 mice, including glutamate/aspartate transporter (GLAST) and glutamine synthetase 6. These findings support the neurotransmitter imbalance described in CLN3 mouse models, namely excessive glutamate and reduced γ‐aminobutyric acid (GABA), which has been implicated in neurotoxicity 9, 10, 11…”

“…PDE4 inhibitors have shown beneficial effects in a wide range of neurodegenerative disorders, by limiting neuronal apoptosis and neuroinflammation as well as augmenting glutamate transporter expression 14, 18, 19, 20. Based on our previous findings demonstrating reductions in GLAST expression in Cln3 Δex7/8 astrocytes,6 exaggerated proinflammatory mediator production in Cln3 Δex7/8 microglia,8 and intrinsic neuron pathology as shown by other groups,7, 21 we surmised that PDE4 inhibitors would represent an excellent means to target this triad of cellular dysfunction.…”