[Electroacupuncture at "Baihui"（GV20） and "Yongquan"（KI1） improves learning-memory ability of APP/PS1 double transgenic mice by regulating endosomal-lysosomal system].

OBJECTIVE: To explore the mechanism of electroacupuncture（EA） in improving learning-memory ability in Alzheimer's disease （AD） mice from the perspective of endosomal-lysosomal system. METHODS: Male APP/PS1 transgenic mice were randomly divided into model group and EA group （n=10 in each group） and 10 male C57BL/6 wild mice were taken as the normal group. EA （1 Hz/50 Hz, 1 mA） was applied at bilateral "Yongquan"（KI1） and acupuncture was applied at "Baihui" （GV20） for 15 min. The mice of the model and normal groups were subjected to restriction with the same method as those of the EA group for 15 min. The treatment was conducted once every other day for 6 weeks. The spatial learning-memory ability （shown by escape latency of place navigation test and the time of crossing the target platform and total swimming distance in the target quadrant in 1 min of spatial probe test ） was detected by Morris water maze test. The immunoactivity of senile plaques （SP） in the hippocampus tissue was detected by immunohistochemistry. The ultrastructural characters of hippocampal neurons were observed by transmission electron microscope, and the expression levels of Ras-related protein 5 （Rab5）, Ras-related protein 7 （Rab7） and cathepsin D （CTSD） in the hippocampus were detected by Western blot, separately. RESULTS: Compared with the normal group, the escape latency, SP immunoactivity, and protein expression levels of Rab5, Rab7 and CTSD were significantly increased （P<0.05, P<0.01）, while the number of crossing the original platform and the total swimming distance in the platform quadrant were considerably reduced （P<0.05） in the model group. In contrast to the model group, the EA group had a marked decrease in the escape latency, SP immunoactivity, and protein expression levels of Rab5, Rab7 and CTSD （P<0.05, P<0.01）, and a striking increase in the number of crossing the original platform and the swimming distance in the platform quadrant （P<0.05）. Results of transmission electron microscope showed an accumulation of endosome, lysosome, and endolysosomes in the hippocampal neurons in the model group, which was evidently milder in the EA group. CONCLUSION: EA of GV20 and KI1 can improve the learning-memory ability of AD mice, which may be related to its function in reducing hippocampal Aß deposition and down-regulating endosomal-lysosomal system activity.

Chemical and genetic rescue of in vivo progranulin-deficient lysosomal and autophagic defects.

In 2006, GRN mutations were first linked to frontotemporal dementia (FTD), the leading cause of non-Alzheimer dementias. While much research has been dedicated to understanding the genetic causes of the disease, our understanding of the mechanistic impacts of GRN deficiency has only recently begun to take shape. With no known cure or treatment available for GRN-related FTD, there is a growing need to rapidly advance genetic and/or small-molecule therapeutics for this disease. This issue is complicated by the fact that, while lysosomal dysfunction seems to be a key driver of pathology, the mechanisms linking a loss of GRN to a pathogenic state remain unclear. In our attempt to address these key issues, we have turned to the nematode, Caenorhabditis elegans, to model, study, and find potential therapies for GRN-deficient FTD. First, we show that the loss of the nematode GRN ortholog, pgrn-1, results in several behavioral and molecular defects, including lysosomal dysfunction and defects in autophagic flux. Our investigations implicate the sphingolipid metabolic pathway in the regulation of many of the in vivo defects associated with pgrn-1 loss. Finally, we utilized these nematodes as an in vivo tool for high-throughput drug screening and identified two small molecules with potential therapeutic applications against GRN/pgrn-1 deficiency. These compounds reverse the biochemical, cellular, and functional phenotypes of GRN deficiency. Together, our results open avenues for mechanistic and therapeutic research into the outcomes of GRN-related neurodegeneration, both genetic and molecular.

Diosgenyl Saponin Inducing Endoplasmic Reticulum Stress and Mitochondria-Mediated Apoptotic Pathways in Liver Cancer Cells.

Diosgenin and diosgenyl saponins as the major bioactive compounds isolated from dietary fenugreek seeds, yam roots, etc. possessed strong antitumor effects. To understand their detailed antitumor mechanisms, a fluorophore-appended derivative of diosgenin [Glc/CNHphth-diosgenin (GND)] was synthesized, starting from diosgenin and glucosamine hydrochloride in overall yields of 7-12% over 7-10 steps. Co-localization of GND with organelle-specific stains, transmission electron microscopy, and relative protein analyses demonstrated that GND crossed the plasma membrane through organic anion-transporting polypeptide 1B1 and distributed in the endoplasmic reticulum (ER), lysosome, and mitochondria. In this process, GND induced ER swelling, mitochondrial damage, and autophagosome and upregulating IRE-1α to induce autophagy and apoptosis. Furthermore, autophagy inhibitor chloroquine delayed the appearance of cleaved poly(ADP-ribose) polymerase and inhibited cleaved caspase 8, which indicated that GND induced autophagy to activate caspase-8-dependent apoptosis. These observations suggested that diosgenyl saponin was a potent anticancer agent that elicited ER stress and mitochondria-mediated apoptotic pathways in liver cancer.

Investigating the functional link between TMEM165 and SPCA1.

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.

YY1 cooperates with TFEB to regulate autophagy and lysosomal biogenesis in melanoma.

Autophagy is a self-proteolytic process that degrades intracellular material to maintain cellular homeostasis. Transcription factor EB (TFEB) is the master activator that regulates the transcription of genes involved in autophagy and lysosomal biogenesis. However, the cotranscriptional factors of TFEB are rarely identified. Here, we found that Yin Yang 1 (YY1) regulated autophagy and lysosome biogenesis in melanoma cells. YY1 cooperates with TFEB to regulate autophagy through controlling the transcription of autophagy and lysosome biogenesis related genes. Moreover, suppression of YY1 enhanced the antitumor efficiency of vemurafenib both in vitro and in vivo. Collectively, these studies identify YY1 as a novel cotranscription factor of TFEB in regulating autophagy and lysosomal functions and suggest YY1 could be a therapeutic target in cancer treatment.

Paricalcitol accelerates BACE1 lysosomal degradation and inhibits calpain-1 dependent neuronal loss in APP/PS1 transgenic mice.

BACKGROUND: Recent studies have revealed that vitamin D deficiency may increase the risk of Alzheimer's disease, and vitamin D supplementation may be effective strategy to ameliorate the neurodegenerative process in Alzheimer's disease patients. Paricalcitol (PAL), a low-calcemic vitamin D receptor agonist, is clinically used to treat secondary hyperparathyroidism. However, the potential application of PAL for treating neurodegenerative disorders remains unexplored. METHODS: The APP/PS1 mice were intraperitoneally injected with PAL or vehicle every other day for 15 weeks. The ß-amyloid (Aß) production was confirmed using immunostaining and enzyme linked immunosorbent assay. The underlying mechanism was verified by western blot and immunostaining in vivo and in vitro. FINDINGS: Long-term PAL treatment clearly reduced ß-amyloid (Aß) generation and neuronal loss in APP/PS1 transgenic mouse brains. PAL stimulated the expression of low-density lipoprotein receptor-related protein 1 (LRP1) possibly through inhibiting sterol regulatory element binding protein-2 (SREBP2); PAL also promoted LRP1-mediated ß-site APP cleavage enzyme 1 (BACE1) transport to late endosomes, thus increasing the lysosomal degradation of BACE1. Furthermore, PAL diminished 8-hydroxyguanosine (8-OHdG) generation in neuronal mitochondria via enhancing base excision repair (BER), resulting in the attenuation of calpain-1-mediated neuronal loss. INTERPRETATION: The present data demonstrate that PAL can reduce Aß generation through accelerating BACE1 lysosomal degradation and can inhibit neuronal loss through suppressing mitochondrial 8-OHdG generation. Hence, PAL might be a promising agent for treating Alzheimer's disease. FUND: This study was financially supported by the Natural Science Foundation of China (U1608282).

Impaired TFEB-mediated lysosomal biogenesis promotes the development of pancreatitis in mice and is associated with human pancreatitis.

Impaired macroautophagy/autophagy has been implicated in experimental and human pancreatitis. However, the transcriptional control governing the autophagy-lysosomal process in pancreatitis is largely unknown. We investigated the role and mechanisms of TFEB (transcription factor EB), a master regulator of lysosomal biogenesis, in the pathogenesis of experimental pancreatitis. We analyzed autophagic flux, TFEB nuclear translocation, lysosomal biogenesis, inflammation and fibrosis in GFP-LC3 transgenic mice, acinar cell-specific tfeb knockout (KO) and tfeb and tfe3 double-knockout (DKO) mice as well as human pancreatitis samples. We found that cerulein activated MTOR (mechanistic target of rapamycin kinase) and increased the levels of phosphorylated TFEB as well as pancreatic proteasome activities that led to rapid TFEB degradation. As a result, cerulein decreased the number of lysosomes resulting in insufficient autophagy in mouse pancreas. Pharmacological inhibition of MTOR or proteasome partially rescued cerulein-induced TFEB degradation and pancreatic damage. Furthermore, genetic deletion of tfeb specifically in mouse pancreatic acinar cells increased pancreatic edema, necrotic cell death, infiltration of inflammatory cells and fibrosis in pancreas after cerulein treatment. tfeb and tfe3 DKO mice also developed spontaneous pancreatitis with increased pancreatic trypsin activities, edema and infiltration of inflammatory cells. Finally, decreased TFEB nuclear staining was associated with human pancreatitis. In conclusion, our results indicate a critical role of impaired TFEB-mediated lysosomal biogenesis in promoting the pathogenesis of pancreatitis. Abbreviations: AC: acinar cell; AMY: amylase; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATP6V1B2: ATPase, H+ transporting, lysosomal V1 subunit B2; ATP6V1D: ATPase, H+ transporting, lysosomal V1 subunit D; ATP6V1H: ATPase, H+ transporting, lysosomal V1 subunit H; AV: autophagic vacuole; CDE: choline-deficient, ethionine-supplemented; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EM: electron microscopy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H & E: hematoxylin and eosin; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: normal donor; NEU: neutrophil; PPARGC1A/PGC1α: peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha; RIPA: radio-immunoprecipitation; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TM: tamoxifen; WT: wild-type; ZG: zymogen granule.

Identification of apilimod as a first-in-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma.

We identified apilimod as an antiproliferative compound by high-throughput screening of clinical-stage drugs. Apilimod exhibits exquisite specificity for phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) lipid kinase and has selective cytotoxic activity in B-cell non-Hodgkin lymphoma (B-NHL) compared with normal cells. Apilimod displays nanomolar activity in vitro, and in vivo studies demonstrate single-agent efficacy as well as synergy with approved B-NHL drugs. Using biochemical and knockdown approaches, and discovery of a kinase domain mutation conferring resistance, we demonstrate that apilimod-mediated cytotoxicity is driven by PIKfyve inhibition. Furthermore, a critical role for lysosome dysfunction as a major factor contributing to apilimod's cytotoxicity is supported by a genome-wide CRISPR screen. In the screen, TFEB (master transcriptional regulator of lysosomal biogenesis) and endosomal/lysosomal genes CLCN7, OSTM1, and SNX10 were identified as important determinants of apilimod sensitivity. These findings thus suggest that disruption of lysosomal homeostasis with apilimod represents a novel approach to treat B-NHL.

Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder caused by GLA mutations, resulting in α-galactosidase (α-Gal) deficiency and accumulation of lysosomal substrates. Migalastat, an oral pharmacological chaperone being developed as an alternative to intravenous enzyme replacement therapy (ERT), stabilises specific mutant (amenable) forms of α-Gal to facilitate normal lysosomal trafficking. METHODS: The main objective of the 18-month, randomised, active-controlled ATTRACT study was to assess the effects of migalastat on renal function in patients with Fabry disease previously treated with ERT. Effects on heart, disease substrate, patient-reported outcomes (PROs) and safety were also assessed. RESULTS: Fifty-seven adults (56% female) receiving ERT (88% had multiorgan disease) were randomised (1.5:1), based on a preliminary cell-based assay of responsiveness to migalastat, to receive 18 months open-label migalastat or remain on ERT. Four patients had non-amenable mutant forms of α-Gal based on the validated cell-based assay conducted after treatment initiation and were excluded from primary efficacy analyses only. Migalastat and ERT had similar effects on renal function. Left ventricular mass index decreased significantly with migalastat treatment (-6.6 g/m2 (-11.0 to -2.2)); there was no significant change with ERT. Predefined renal, cardiac or cerebrovascular events occurred in 29% and 44% of patients in the migalastat and ERT groups, respectively. Plasma globotriaosylsphingosine remained low and stable following the switch from ERT to migalastat. PROs were comparable between groups. Migalastat was generally safe and well tolerated. CONCLUSIONS: Migalastat offers promise as a first-in-class oral monotherapy alternative treatment to intravenous ERT for patients with Fabry disease and amenable mutations. TRIAL REGISTRATION NUMBER: NCT00925301; Pre-results.

Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons.

Autophagy is an important cell recycling program responsible for the clearance of damaged or long-lived proteins and organelles. Pharmacological modulators of this pathway have been extensively utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics. To address this, we cultured primary rat cortical neurons from E18 embryos and used the Seahorse XF96 analyzer and a targeted metabolomics approach to measure the effects of bafilomycin A1 and chloroquine on bioenergetics and metabolism. We found that both bafilomycin and chloroquine could significantly increase the autophagosome marker LC3-II and inhibit key parameters of mitochondrial function, and increase mtDNA damage. Furthermore, we observed significant alterations in TCA cycle intermediates, particularly those downstream of citrate synthase and those linked to glutaminolysis. Taken together, these data demonstrate a significant impact of bafilomycin and chloroquine on cellular bioenergetics and metabolism consistent with decreased mitochondrial quality associated with inhibition of autophagy.

Electroacupuncture protects against ischemic stroke by reducing autophagosome formation and inhibiting autophagy through the mTORC1-ULK1 complex-Beclin1 pathway.

In a previous study by our group, we demonstrated that electroacupuncture (EA) activates the class I phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. There is considerable evidence that the downstream mammalian target of rapamycin complex 1 (mTORC1) plays an important role in autophagy following ischemic stroke. The aim of the present study was to determine whether EA exerts a neuroprotective effect through mTORC1-mediated autophagy following ischemia/reperfusion injury. Our results revealed that EA at the LI11 and ST36 acupoints attenuated motor dysfunction, improved neurological deficit outcomes and decreased the infarct volumes. The number of autophagosomes, autolysosomes and lysosomes was decreased following treatment with EA. Simultaneously, the levels of the autophagosome membrane maker, microtubule-associated protein 1 light chain 3 beta (LC3B)Ⅱ/Ⅰ, Unc-51-like kinase 1 (ULK1), autophagy related gene 13 Atg13) and Beclin1 (ser14) were decreased, whereas mTORC1 expression was increased in the peri-infarct cortex. These results suggest that EA protects against ischemic stroke through the inhibition of autophagosome formation and autophagy, which is mediated through the mTORC1-ULK complex-Beclin1 pathway.

Clearance of Apoptotic Cells and Pyrenocytes.

Apoptotic cells are engulfed and digested by macrophages to maintain homeostasis in animals. If dead cells are not engulfed swiftly, they undergo secondary necrosis and release intracellular components that activate the immune system. Apoptotic cells are efficiently cleared due to phosphatidylserine (PtdSer) exposed on the cell surface that acts as an "eat me" signal. PtdSer is exposed through the activation of phospholipid scramblase and the inactivation of phospholipid flippase, which are both caspase-mediated events. Macrophages express a variety of molecules to recognize PtdSer, and use a sophisticated mechanism to engulf apoptotic cells. In red blood cells, the nucleus is lost when it is extruded as a pyrenocyte during definitive erythropoiesis. These pyrenocytes (nuclei surrounded by plasma membrane) also expose PtdSer on their surface and are efficiently engulfed by macrophages in a PtdSer-dependent manner. Macrophages transfer the engulfed apoptotic cell or pyrenocyte into lysosomes, where the components of the dead cell or pyrenocyte are degraded. If lysosomes cannot digest the DNA from apoptotic cells or pyrenocytes, the undigested DNA accumulates in the lysosome and activates macrophages to produce type I interferon (IFN) via a STING-dependent pathway; in embryos, this causes severe anemia. Here, we discuss how macrophages clear apoptotic cells and pyrenocytes.

Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px)-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective stage of T. brucei. The respective knockout of the cytosolic px I-II in the procyclic insect form resulted in cells that were fully viable in Trolox-free medium.

Candidate molecules for chemical chaperone therapy of GM1-gangliosidosis.

A growing body of evidence suggests that misfolding of a mutant protein followed by its aggregation or premature degradation in the endoplasmic reticulum is one of the main mechanisms that underlie inherited neurodegenerative diseases, including lysosomal storage diseases. Chemical or pharmacological chaperones are small molecules that bind to and stabilize mutant lysosomal enzyme proteins in the endoplasmic reticulum. A number of chaperone compounds for lysosomal hydrolases have been identified in the last decade. They have gained attention because they can be orally administrated, and also because they can penetrate the blood-brain barrier. In this article, we describe two chaperone candidates for the treatment of GM1-gangliosidosis. We also discuss the future direction of this strategy targeting other lysosomal storage diseases as well as protein misfolding diseases in general.

Systemic delivery of therapeutic small interfering RNA using a pH-triggered amphiphilic poly-L-lysine nanocarrier to suppress prostate cancer growth in mice.

Prostate cancer is associated with high mortality and new therapeutic strategies are necessary for improved patient outcome. The utilisation of potent, sequence-specific small interfering RNA (siRNA) to facilitate down-regulation of complementary mRNA sequences in vitro and in vivo has stimulated the development of siRNA-based cancer therapies. However, the lack of an effective siRNA delivery system significantly retards clinical application. Amphiphilic polycations with 'stealth' capacity have previously been synthesised by PEGylation of poly-l-lysine-cholic acid (PLL-CA). The benzoic imine linker between PEG and PLL-CA was designed to be stable at physiological pH but cleavable at lower pHs, consistent with the extracellular environment of tumours and the interior of endosomes/lysosomes. The selective hydrolysis of the PEG linker at these targeted sites should provide enhanced cellular uptake and endosomal escape while simultaneously ensuring prolonged blood circulation times. In this study, physicochemical profiling demonstrated nano-complex formation between the PLL derivatives and siRNA (200-280 nm in diameter). At physiological pH only a slight cationic surface charge (<20 mV) was detected, due to the masking effect of the PEG. In contrast, significantly higher positive charges (∼20 to 30 mV and >40 mV) were detected upon hydrolysis of the PEG linker at acidic pHs (pH=6.8 and 5.5, respectively). The PEGylated complexes were stable in serum without significant aggregation or decomplexation of siRNA for up to 48 h. At the cellular level, PEG-PLLs were comparable with the commercial carrier INTERFRin, in terms of cellular uptake, endosomal escape and in vitro reporter gene knockdown. In vivo, utilising a mouse model grafted with prostate carcinoma, significant tumour suppression was achieved using PEGylated complexes without marked toxicity or undesirable immunological response, this was accompanied by a simultaneous reduction in target mRNA levels. In summary, the advantages of these vectors include: the in vitro and in vivo silencing efficiency, and the low toxicity and immunogenicity.

Lysosomal exocytosis is impaired in mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is an autosomal recessive disease characterized by severe neurological impairment, ophthalmologic defects, and gastric dysfunction. MLIV cells have a deficiency in the late endosomal/lysosomal (LEL) pathway that results in the buildup of lysosomal inclusions. Using a Xenopus oocyte expression system, we previously showed that mucolipin-1 (MLN1), the protein encoded by the MCOLN1 gene is a Ca2+ -permeable non-selective cation channel that is transiently modulated by elevations in intracellular Ca2+. We further showed that MLN1 is translocated to the plasma membrane during lysosomal exocytosis. In this study we show that lysosomal exocytosis is impaired in fibroblasts from MLIV patients, indicating that MLN1 plays an active role in this process. Further, we show that transfection with wild type MLN1 cDNA rescues exocytosis, suggesting the possibility of treatments based on the restoration of this crucial cellular function.

Thalamocortical neuron loss and localized astrocytosis in the Cln3Deltaex7/8 knock-in mouse model of Batten disease.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is the result of mutations in the Cln3 gene. The Cln3 knock-in mouse (Cln3Deltaex7/8) reproduces the most common Cln3 mutation and we have now characterized the CNS of these mice at 12 months of age. With the exception of the thalamus, Cln3Deltaex7/8 homozygotes displayed no significant regional atrophy, but a range of changes in individual laminar thickness that resulted in variable cortical thinning across subfields. Stereological analysis revealed a pronounced loss of neurons within individual laminae of somatosensory cortex of affected mice and the novel finding of a loss of sensory relay thalamic neurons. These affected mice also exhibited profound astrocytic reactions that were most pronounced in the neocortex and thalamus, but diminished in other brain regions. These data provide the first direct evidence for neurodegenerative and reactive changes in the thalamocortical system in JNCL and emphasize the localized nature of these events.