👤 Tammy Kielian

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 Ca Show less

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 Cln3 Show less

CLN3-Batten disease is a rare, autosomal recessive disorder involving seizures, visual, motor and cognitive decline, and premature death. The Cln3Δex7/8 mouse model recapitulates several phenotypic characteristics of the most common 1.02kb disease-associated deletion. Identification of reproducible biomarker(s) to facilitate longitudinal monitoring of disease progression and provide readouts for therapeutic response has remained elusive. One factor that has complicated the identification of suitable biomarkers in this mouse model has been that variations in animal husbandry appear to significantly influence readouts. In the current study, we cross-compared a number of biological parameters in blood from Cln3Δex7/8 mice and control, non-disease mice on the same genetic background from multiple animal facilities in an attempt to better define a surrogate marker of CLN3-Batten disease. Interestingly, we found that significant differences between Batten and non-disease mice found at one site were generally not maintained across different facilities. Our results suggest that colony variation in the Cln3Δex7/8 mouse model of CLN3-Batten disease can influence potential biomarkers of the disease. Show less

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a fatal lysosomal storage disease caused by autosomal recessive mutations in CLN3. JNCL is typified by progressive neurodegeneration that has been suggested to occur from excessive excitatory and impaired inhibitory synaptic input; however, no studies to date have directly evaluated neuronal function. To examine changes in neuronal activity with advancing disease, electrophysiological recordings were performed in the CA1 hippocampus (HPC) and visual cortex (VC) of acute brain slices from Cln3 Show less

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 Cln3 In a randomized, blinded study, wild-type (WT) and Cln3 cAMP levels were significantly reduced in the Cln3 These studies reveal neuroprotective effects for PDE4 inhibitors in Cln3 Show less

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 β-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/β-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/β-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/β-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. 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 β-actin promoters. hCLN3 levels were elevated 3- to 8-fold in Cln3(Δex7/8) mice receiving scAAV9/β-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/β-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. Show less

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. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3. Regions of microglial activation precede and predict areas of neuronal loss in JNCL; however, the functional role of activated microglia remains to be defined. The inflammasome is a key molecular pathway for activating pro-IL-1β in microglia, and IL-1β is elevated in the brains of JNCL patients and can induce neuronal cell death. Here, we utilized primary microglia isolated from CLN3(Δex7/8) mutant and wild-type (WT) mice to examine the impact of CLN3 mutation on microglial activation and inflammasome function. Treatment with neuronal lysates and ceramide, a lipid intermediate elevated in the JNCL brain, led to inflammasome activation and IL-1β release in CLN3(Δex7/8) microglia but not WT cells, as well as increased expression of additional pro-inflammatory mediators. Similar effects were observed following either TNF-α or IL-1β treatment, suggesting that CLN3(Δex7/8) microglia exist in primed state and hyper-respond to several inflammatory stimuli compared to WT cells. CLN3(Δex7/8) microglia displayed constitutive caspase-1 activity that when blocked led to increased glutamate release that coincided with hemichannel opening. Conditioned medium from activated CLN3(Δex7/8) or WT microglia induced significant cell death in CLN3(Δex7/8) but not WT neurons, demonstrating that intrinsically diseased CLN3(Δex7/8) neurons are less equipped to withstand cytotoxic insults generated by activated microglia. Collectively, aberrant microglial activation may contribute to the pathological chain of events leading to neurodegeneration during later stages of JNCL. Show less