Sodium oligomannate alters gut microbiota, reduces cerebral amyloidosis and reactive microglia in a sex-specific manner.

It has recently become well-established that there is a connection between Alzheimer's disease pathology and gut microbiome dysbiosis. We have previously demonstrated that antibiotic-mediated gut microbiota perturbations lead to attenuation of Aß deposition, phosphorylated tau accumulation, and disease-associated glial cell phenotypes in a sex-dependent manner. In this regard, we were intrigued by the finding that a marine-derived oligosaccharide, GV-971, was reported to alter gut microbiota and reduce Aß amyloidosis in the 5XFAD mouse model that were treated at a point when Aß burden was near plateau levels. Utilizing comparable methodologies, but with distinct technical and temporal features, we now report on the impact of GV-971 on gut microbiota, Aß amyloidosis and microglial phenotypes in the APPPS1-21 model, studies performed at the University of Chicago, and independently in the 5X FAD model, studies performed at Washington University, St. Louis.Methods To comprehensively characterize the effects of GV-971 on the microbiota-microglia-amyloid axis, we conducted two separate investigations at independent institutions. There was no coordination of the experimental design or execution between the two laboratories. Indeed, the two laboratories were not aware of each other's experiments until the studies were completed. Male and female APPPS1-21 mice were treated daily with 40, 80, or 160 mg/kg of GV-971 from 8, when Aß burden was detectable upto 12 weeks of age when Aß burden was near maximal levels. In parallel, and to corroborate existing published studies and further investigate sex-related differences, male and female 5XFAD mice were treated daily with 100 mg/kg of GV-971 from 7 to 9 months of age when Aß burden was near peak levels. Subsequently, the two laboratories independently assessed amyloid-ß deposition, metagenomic, and neuroinflammatory profiles. Finally, studies were initiated at the University of Chicago to evaluate the metabolites in cecal tissue from vehicle and GV-971-treated 5XFAD mice.Results These studies showed that independent of the procedural differences (dosage, timing and duration of treatment) between the two laboratories, cerebral amyloidosis was reduced primarily in male mice, independent of strain. We also observed sex-specific microbiota differences following GV-971 treatment. Interestingly, GV-971 significantly altered multiple overlapping bacterial species at both institutions. Moreover, we discovered that GV-971 significantly impacted microbiome metabolism, particularly by elevating amino acid production and influencing the tryptophan pathway. The metagenomics and metabolomics changes correspond with notable reductions in peripheral pro-inflammatory cytokine and chemokine profiles. Furthermore, GV-971 treatment dampened astrocyte and microglia activation, significantly decreasing plaque-associated reactive microglia while concurrently increasing homeostatic microglia only in male mice. Bulk RNAseq analysis unveiled sex-specific changes in cerebral cortex transcriptome profiles, but most importantly, the transcriptome changes in the GV-971-treated male group revealed the involvement of microglia and inflammatory responses.Conclusions In conclusion, these studies demonstrate the connection between the gut microbiome, neuroinflammation, and Alzheimer's disease pathology while highlighting the potential therapeutic effect of GV-971. GV-971 targets the microbiota-microglia-amyloid axis, leading to the lowering of plaque pathology and neuroinflammatory signatures in a sex-dependent manner when given at the onset of Aß deposition or when given after Aß deposition is already at higher levels.

Amelioration of Tau and ApoE4-linked glial lipid accumulation and neurodegeneration with an LXR agonist.

Apolipoprotein E (APOE) is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). APOE4 increases and APOE2 decreases risk relative to APOE3. In the P301S mouse model of tauopathy, ApoE4 increases tau pathology and neurodegeneration when compared with ApoE3 or the absence of ApoE. However, the role of ApoE isoforms and lipid metabolism in contributing to tau-mediated degeneration is unknown. We demonstrate that in P301S tau mice, ApoE4 strongly promotes glial lipid accumulation and perturbations in cholesterol metabolism and lysosomal function. Increasing lipid efflux in glia via an LXR agonist or Abca1 overexpression strongly attenuates tau pathology and neurodegeneration in P301S/ApoE4 mice. We also demonstrate reductions in reactive astrocytes and microglia, as well as changes in cholesterol biosynthesis and metabolism in glia of tauopathy mice in response to LXR activation. These data suggest that promoting efflux of glial lipids may serve as a therapeutic approach to ameliorate tau and ApoE4-linked neurodegeneration.

Parenchymal border macrophages regulate tau pathology and tau-mediated neurodegeneration.

Parenchymal border macrophages (PBMs) reside close to the central nervous system parenchyma and regulate CSF flow dynamics. We recently demonstrated that PBMs provide a clearance pathway for amyloid-ß peptide, which accumulates in the brain in Alzheimer's disease (AD). Given the emerging role for PBMs in AD, we explored how tau pathology affects the CSF flow and the PBM populations in the PS19 mouse model of tau pathology. We demonstrated a reduction of CSF flow, and an increase in an MHCII+PBM subpopulation in PS19 mice compared with WT littermates. Consequently, we asked whether PBM dysfunction could exacerbate tau pathology and tau-mediated neurodegeneration. Pharmacological depletion of PBMs in PS19 mice led to an increase in tau pathology and tau-dependent neurodegeneration, which was independent of gliosis or aquaporin-4 depolarization, essential for the CSF-ISF exchange. Together, our results identify PBMs as novel cellular regulators of tau pathology and tau-mediated neurodegeneration.

Astrocytic APOE4 removal confers cerebrovascular protection despite increased cerebral amyloid angiopathy.

BACKGROUND: Alzheimer Disease (AD) and cerebral amyloid angiopathy (CAA) are both characterized by amyloid-ß (Aß) accumulation in the brain, although Aß deposits mostly in the brain parenchyma in AD and in the cerebrovasculature in CAA. The presence of CAA can exacerbate clinical outcomes of AD patients by promoting spontaneous intracerebral hemorrhage and ischemia leading to CAA-associated cognitive decline. Genetically, AD and CAA share the ε4 allele of the apolipoprotein E (APOE) gene as the strongest genetic risk factor. Although tremendous efforts have focused on uncovering the role of APOE4 on parenchymal plaque pathogenesis in AD, mechanistic studies investigating the role of APOE4 on CAA are still lacking. Here, we addressed whether abolishing APOE4 generated by astrocytes, the major producers of APOE, is sufficient to ameliorate CAA and CAA-associated vessel damage. METHODS: We generated transgenic mice that deposited both CAA and plaques in which APOE4 expression can be selectively suppressed in astrocytes. At 2-months-of-age, a timepoint preceding CAA and plaque formation, APOE4 was removed from astrocytes of 5XFAD APOE4 knock-in mice. Mice were assessed at 10-months-of-age for Aß plaque and CAA pathology, gliosis, and vascular integrity. RESULTS: Reducing the levels of APOE4 in astrocytes shifted the deposition of fibrillar Aß from the brain parenchyma to the cerebrovasculature. However, despite increased CAA, astrocytic APOE4 removal reduced overall Aß-mediated gliosis and also led to increased cerebrovascular integrity and function in vessels containing CAA. CONCLUSION: In a mouse model of CAA, the reduction of  APOE4 derived specifically from astrocytes, despite increased fibrillar Aß deposition in the vasculature, is sufficient to reduce Aß-mediated gliosis and cerebrovascular dysfunction.

Staphylococcus aureus ATP Synthase Promotes Biofilm Persistence by Influencing Innate Immunity.

Staphylococcus aureus is a major cause of prosthetic joint infection (PJI), which is characterized by biofilm formation. S. aureus biofilm skews the host immune response toward an anti-inflammatory profile by the increased recruitment of myeloid-derived suppressor cells (MDSCs) that attenuate macrophage proinflammatory activity, leading to chronic infection. A screen of the Nebraska Transposon Mutant Library identified several hits in the ATP synthase operon that elicited a heightened inflammatory response in macrophages and MDSCs, including atpA, which encodes the alpha subunit of ATP synthase. An atpA transposon mutant (ΔatpA) had altered growth kinetics under both planktonic and biofilm conditions, along with a diffuse biofilm architecture that was permissive for leukocyte infiltration, as observed by confocal laser scanning microscopy. Coculture of MDSCs and macrophages with ΔatpA biofilm elicited significant increases in the proinflammatory cytokines interleukin 12p70 (IL-12p70), tumor necrosis factor alpha (TNF-α), and IL-6. This was attributed to increased leukocyte survival resulting from less toxin and protease production by ΔatpA biofilm as determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The enhanced inflammatory response elicited by ΔatpA biofilm was cell lysis-dependent since it was negated by polyanethole sodium sulfanate treatment or deletion of the major autolysin, Atl. In a mouse model of PJI, ΔatpA-infected mice had decreased MDSCs concomitant with increased monocyte/macrophage infiltrates and proinflammatory cytokine production, which resulted in biofilm clearance. These studies identify S. aureus ATP synthase as an important factor in influencing the immune response during biofilm-associated infection and bacterial persistence.IMPORTANCE Medical device-associated biofilm infections are a therapeutic challenge based on their antibiotic tolerance and ability to evade immune-mediated clearance. The virulence determinants responsible for bacterial biofilm to induce a maladaptive immune response remain largely unknown. This study identified a critical role for S. aureus ATP synthase in influencing the host immune response to biofilm infection. An S. aureus ATP synthase alpha subunit mutant (ΔatpA) elicited heightened proinflammatory cytokine production by leukocytes in vitro and in vivo, which coincided with improved biofilm clearance in a mouse model of prosthetic joint infection. The ability of S. aureus ΔatpA to augment host proinflammatory responses was cell lysis-dependent, as inhibition of bacterial lysis by polyanethole sodium sulfanate or a ΔatpAΔatl biofilm did not elicit heightened cytokine production. These studies reveal a critical role for AtpA in shaping the host immune response to S. aureus biofilm.

Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogramme the host immune response during persistent infection.

Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI), resulting in considerable disability and prolonged treatment. It is known that host leukocyte IL-10 production is required for S. aureus biofilm persistence in PJI. An S. aureus bursa aurealis Tn library consisting of 1,952 non-essential genes was screened for mutants that failed to induce IL-10 in myeloid-derived suppressor cells (MDSCs), which identified a critical role for bacterial lactic acid biosynthesis. We generated an S. aureus ddh/ldh1/ldh2 triple Tn mutant that cannot produce D- or L-lactate. Co-culture of MDSCs or macrophages with ddh/ldh1/ldh2 mutant biofilm produced substantially less IL-10 compared with wild-type S. aureus, which was also observed in a mouse model of PJI and led to reduced biofilm burden. Using MDSCs recovered from the mouse PJI model and in vitro leukocyte-biofilm co-cultures, we show that bacterial-derived lactate inhibits histone deacetylase 11, causing unchecked HDAC6 activity and increased histone 3 acetylation at the Il-10 promoter, resulting in enhanced Il-10 transcription in MDSCs and macrophages. Finally, we show that synovial fluid of patients with PJI contains elevated amounts of D-lactate and IL-10 compared with control subjects, and bacterial lactate increases IL-10 production by human monocyte-derived macrophages.

Astrocytes in juvenile neuronal ceroid lipofuscinosis (CLN3) display metabolic and calcium signaling abnormalities.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by autosomal recessive mutations in ceroid lipofuscinosis 3 (CLN3). Children with JNCL experience progressive visual, cognitive, and motor deterioration with a decreased life expectancy (late teens-early 20s). Neuronal loss is thought to occur, in part, via glutamate excitotoxicity; however, little is known about astrocyte glutamate regulation in JNCL. Spontaneous Ca2+ oscillations were reduced in murine Cln3Δex7/8 astrocytes, which were also observed following glutamate or cytokine exposure. Astrocyte glutamate transport is an energy-demanding process and disruptions in metabolic pathways could influence glutamate homeostasis in Cln3Δex7/8 astrocytes. Indeed, basal mitochondrial respiration and ATP production were significantly reduced in Cln3Δex7/8 astrocytes. These changes were not attributable to reduced mitochondria, since mitochondrial DNA levels were similar between wild type and Cln3Δex7/8 astrocytes. Interestingly, despite these functional deficits in Cln3Δex7/8 astrocytes, glutamate transporter expression and glutamate uptake were not dramatically affected. Concurrent with impaired astrocyte metabolism and Ca2+ signaling, murine Cln3Δex7/8 neurons were hyper-responsive to glutamate, as reflected by heightened and prolonged Ca2+ signals. These findings identify intrinsic metabolic and Ca2+ signaling defects in Cln3Δex7/8 astrocytes that may contribute to neuronal dysfunction in CLN3 disease. This article is part of the Special Issue "Lysosomal Storage Disorders".

Caspase 1 activity influences juvenile Batten disease (CLN3) pathogenesis.

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is an autosomal recessive lysosomal storage disease caused by loss-of-function mutations in CLN3. Symptoms appear between 5 and 10 years of age, beginning with blindness and seizures, followed by progressive cognitive and motor decline, and premature death. Glial activation and impaired neuronal activity are early signs of pathology in the Cln3Δex7/8 mouse model of JNCL, whereas neuron death occurs much later in the disease process. We previously reported that Cln3Δex7/8 microglia are primed toward a pro-inflammatory phenotype typified by exaggerated caspase 1 inflammasome activation and here we extend those findings to demonstrate heightened caspase activity in the Cln3Δex7/8 mouse brain. Based on the ability of caspase 1 to cleave a large number of substrates that have been implicated in JNCL pathology, we examined the functional implications of caspase 1 inflammasome activity by crossing Cln3Δex7/8 and caspase 1-deficient mice to create Cln3Δex7/8 /Casp-1-/- animals. Caspase 1 deletion influenced motor behavior deficits and astrocyte activation in the context of CLN3 mutation, since both were significantly reversed in Cln3Δex7/8 /Casp-1-/- mice, with phenotypes approaching that of wild-type animals. We also report a progressive age-dependent reduction in whisker length in Cln3Δex7/8 mice that was partially caspase 1-dependent. However, not all CLN3 phenotypes were reversed following caspase 1 deletion, since no significant differences in lysosomal accumulation or microglial activation were observed between Cln3Δex7/8 and Cln3Δex7/8 /Casp-1-/- mice. Although the molecular targets of aberrant caspase 1 activity in the context of CLN3 mutation remain to be identified, our studies suggest that caspase 1 may represent a potential therapeutic target to mitigate some attributes of CLN3 disease. This article is part of the Special Issue "Lysosomal Storage Disorders".

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

OBJECTIVE: Juvenile neuronal ceroid lipofuscinosis (JNCL), or juvenile Batten disease, is a pediatric lysosomal storage disease caused by autosomal recessive mutations in CLN3, typified by blindness, seizures, progressive cognitive and motor decline, and premature death. Currently, there is no treatment for JNCL that slows disease progression, which highlights the need to explore novel strategies to extend the survival and quality of life of afflicted children. Cyclic adenosine monophosphate (cAMP) is a second messenger with pleiotropic effects, including regulating neuroinflammation and neuronal survival. Here we investigated whether 3 phosphodiesterase-4 (PDE4) inhibitors (rolipram, roflumilast, and PF-06266047) could mitigate behavioral deficits and cell-specific pathology in the Cln3Δex7/8 mouse model of JNCL. METHODS: In a randomized, blinded study, wild-type (WT) and Cln3Δex7/8 mice received PDE4 inhibitors daily beginning at 1 or 3 months of age and continuing for 6 to 9 months, with motor deficits assessed by accelerating rotarod testing. The effect of PDE4 inhibitors on cAMP levels, astrocyte and microglial activation (glial fibrillary acidic protein and CD68, respectively), lysosomal pathology (lysosomal-associated membrane protein 1), and astrocyte glutamate transporter expression (glutamate/aspartate transporter) were also examined in WT and Cln3Δex7/8 animals. RESULTS: cAMP levels were significantly reduced in the Cln3Δex7/8 brain, and were restored by PF-06266047. PDE4 inhibitors significantly improved motor function in Cln3Δex7/8 mice, attenuated glial activation and lysosomal pathology, and restored glutamate transporter expression to levels observed in WT animals, with no evidence of toxicity as revealed by blood chemistry analysis. INTERPRETATION: These studies reveal neuroprotective effects for PDE4 inhibitors in Cln3Δex7/8 mice and support their therapeutic potential in JNCL patients. Ann Neurol 2016;80:909-923.

Self-Complementary AAV9 Gene Delivery Partially Corrects Pathology Associated with Juvenile Neuronal Ceroid Lipofuscinosis (CLN3).

UNLABELLED: Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal lysosomal storage disease caused by autosomal-recessive mutations in CLN3 for which no treatment exists. Symptoms appear between 5 and 10 years of age, beginning with blindness and seizures, followed by progressive cognitive and motor decline and premature death (late teens to 20s). We explored a gene delivery approach for JNCL by generating two self-complementary adeno-associated virus 9 (scAAV9) constructs to address CLN3 dosage effects using the methyl-CpG-binding protein 2 (MeCP2) and ß-actin promoters to drive low versus high transgene expression, respectively. This approach was based on the expectation that low CLN3 levels are required for cellular homeostasis due to minimal CLN3 expression postnatally, although this had not yet been demonstrated in vivo One-month-old Cln3(Δex7/8) mice received one systemic (intravenous) injection of scAAV9/MeCP2-hCLN3 or scAAV9/ß-actin-hCLN3, with green fluorescent protein (GFP)-expressing viruses as controls. A promoter-dosage effect was observed in all brain regions examined, in which hCLN3 levels were elevated 3- to 8-fold in Cln3(Δex7/8) mice receiving scAAV9/ß-actin-hCLN3 versus scAAV9/MeCP2-hCLN3. However, a disconnect occurred between CLN3 levels and disease improvement, because only the scAAV9 construct driving low CLN3 expression (scAAV9/MeCP2-hCLN3) corrected motor deficits and attenuated microglial and astrocyte activation and lysosomal pathology. This may have resulted from preferential promoter usage because transgene expression after intravenous scAAV9/MeCP2-GFP injection was primarily detected in NeuN(+) neurons, whereas scAAV9/ß-actin-GFP drove transgene expression in GFAP(+) astrocytes. This is the first demonstration of a systemic delivery route to restore CLN3 in vivo using scAAV9 and highlights the importance of promoter selection for disease modification in juvenile animals. SIGNIFICANCE STATEMENT: Juvenile neuronal ceroid lipofuscinosis (JNCL) is a fatal lysosomal storage disease caused by CLN3 mutations. We explored a gene delivery approach using two self-complementary adeno-associated virus 9 (scAAV9) constructs to address CLN3 dosage effects using the methyl-CpG-binding protein 2 (MeCP2) and ß-actin promoters. hCLN3 levels were elevated 3- to 8-fold in Cln3(Δex7/8) mice receiving scAAV9/ß-actin-hCLN3 versus scAAV9/MeCP2-hCLN3 after a single systemic injection. However, only scAAV9/MeCP2-hCLN3 corrected motor deficits and attenuated glial activation and lysosomal pathology. This may reflect preferential promoter usage because transgene expression with scAAV9/MeCP2-green fluorescent protein (GFP) was primarily in neurons, whereas scAAV9/ß-actin-GFP drove transgene expression in astrocytes. This is the first demonstration of systemic delivery for CLN3 using scAAV9 and highlights the importance of promoter selection for disease modification in juvenile animals.

Neuroinflammatory paradigms in lysosomal storage diseases.

Lysosomal storage diseases (LSDs) include approximately 70 distinct disorders that collectively account for 14% of all inherited metabolic diseases. LSDs are caused by mutations in various enzymes/proteins that disrupt lysosomal function, which impairs macromolecule degradation following endosome-lysosome and phagosome-lysosome fusion and autophagy, ultimately disrupting cellular homeostasis. LSDs are pathologically typified by lysosomal inclusions composed of a heterogeneous mixture of various proteins and lipids that can be found throughout the body. However, in many cases the CNS is dramatically affected, which may result from heightened neuronal vulnerability based on their post-mitotic state. Besides intrinsic neuronal defects, another emerging factor common to many LSDs is neuroinflammation, which may negatively impact neuronal survival and contribute to neurodegeneration. Microglial and astrocyte activation is a hallmark of many LSDs that affect the CNS, which often precedes and predicts regions where eventual neuron loss will occur. However, the timing, intensity, and duration of neuroinflammation may ultimately dictate the impact on CNS homeostasis. For example, a transient inflammatory response following CNS insult/injury can be neuroprotective, as glial cells attempt to remove the insult and provide trophic support to neurons. However, chronic inflammation, as seen in several LSDs, can promote neurodegeneration by creating a neurotoxic environment due to elevated levels of cytokines, chemokines, and pro-apoptotic molecules. Although neuroinflammation has been reported in several LSDs, the cellular basis and mechanisms responsible for eliciting neuroinflammatory pathways are just beginning to be defined. This review highlights the role of neuroinflammation in select LSDs and its potential contribution to neuron loss.

AAV2/1 CD74 Gene Transfer Reduces ß-amyloidosis and Improves Learning and Memory in a Mouse Model of Alzheimer's Disease.

Modulation of the amyloid-ß (Aß) trafficking pathway heralds a new therapeutic frontier for Alzheimer's disease (AD). As CD74 binds to the amyloid-ß precursor protein (APP) and can suppresses Aß processing, we investigated whether recombinant adeno-associated virus (AAV) delivery of CD74 could reduce Aß production and affect disease outcomes. This idea was tested in a mouse AD model. Cotransduction of AAV-tetracycline-controlled transactivator (tTA) and AAV-tet-response element (TRE)-CD74 resulted in CD74 expression, reduced Aß production in mouse neurons containing the human APP with familial AD-linked mutations. Stereotaxic injection of AAV-TRE-GFP or CD74 into the hippocampi of an AD mouse, defined as a TgCRND8 × calmodulin-dependent protein kinase II derived promoter-tTA double-transgenic, reduced Aß loads and pyramidal neuronal Aß accumulation in the hippocampus. Immunofluorescent studies showed that APP colocalization with Lamp1 was increased in CD74-expressing neurons. Moreover, Morris water maze tasks demonstrated that mice treated with AAV-TRE-CD74 showed improved learning and memory compared to AAV-TRE-GFP control animals. These results support the idea that CD74-induced alteration of Aß processing could improve AD-associated memory deficits as shown in mouse models of human disease.

Hemichannels in neurodegenerative diseases: is there a link to pathology?

Although originally considered a structural component of gap junctions, connexin hemichannels (HCs) are now recognized as functional entities capable of influencing metabolic gradients within the CNS, allowing direct communication between the intra- and extracellular milieus. Besides connexins, HCs can also be formed by pannexins, which are not capable of gap junction assembly. Both positive and negative effects have been attributed to HC activity in the context of neurodegenerative diseases. For example, HCs can exert neuroprotective effects by promoting the uptake of neurotoxic molecules, whereas chronic HC opening can disrupt molecular gradients leading to cellular dysfunction and death. The latter scenario has been suggested for multiple neurodegenerative disorders, including Alzheimer's disease (AD) and more recently, lysosomal storage disorders, which are the focus of this perspective. Currently available evidence suggests a complex role for HCs in neurodegenerative disorders, which sets the stage for future studies to determine whether targeting HC action may improve disease outcomes.

Evidence for aberrant astrocyte hemichannel activity in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL).

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.