Generation of a humanized Aß expressing mouse demonstrating aspects of Alzheimer's disease-like pathology.

The majority of Alzheimer's disease (AD) cases are late-onset and occur sporadically, however most mouse models of the disease harbor pathogenic mutations, rendering them better representations of familial autosomal-dominant forms of the disease. Here, we generated knock-in mice that express wildtype human Aß under control of the mouse App locus. Remarkably, changing 3 amino acids in the mouse Aß sequence to its wild-type human counterpart leads to age-dependent impairments in cognition and synaptic plasticity, brain volumetric changes, inflammatory alterations, the appearance of Periodic Acid-Schiff (PAS) granules and changes in gene expression. In addition, when exon 14 encoding the Aß sequence was flanked by loxP sites we show that Cre-mediated excision of exon 14 ablates hAß expression, rescues cognition and reduces the formation of PAS granules.

p-Tau immunotherapy reduces soluble and insoluble tau in aged 3xTg-AD mice.

Alzheimer's disease (AD) is a proteinopathy characterized by the accumulation of ß-amyloid (Aß) and tau. To date, clinical trials indicate that Aß immunotherapy does not improve cognition. Consequently, it is critical to modulate other aspects of AD pathology. As such, tau represents an excellent target, as its accumulation better correlates with cognitive impairment. To determine the effectiveness of targeting pathological tau, with Aß pathology present, we administered a single injection of AT8, or control antibody, into the hippocampus of aged 3xTg-AD mice. Extensive data indicates that phosphorylated Ser(202) and Thr(205) sites of tau (corresponding to the AT8 epitope) represent a pathologically relevant target for AD. We report that immunization with AT8 reduced somatodendritic tau load, p-tau immunoreactivity, and silver stained positive neurons, without affecting Aß pathology. We also discovered that tau pathology soon reemerges post-injection, possibly due to persistent Aß pathology. These studies provide evidence that targeting p-tau may represent an effective treatment strategy: potentially in conjunction with Aß immunotherapy.

Presenilin-null cells have altered two-pore calcium channel expression and lysosomal calcium: implications for lysosomal function.

Presenilins are necessary for calcium homeostasis and also for efficient proteolysis through the autophagy/lysosome system. Presenilin regulates both endoplasmic reticulum calcium stores and autophagic proteolysis in a γ-secretase independent fashion. The endo-lysosome system can also act as a calcium store, with calcium efflux channels being recently identified as two-pore channels 1 and 2. Here we investigated lysosomal calcium content and the channels that mediate calcium release from these acidic stores in presenilin knockout cells. We report that presenilin loss leads to a lower total lysosomal calcium store despite the buildup of lysosomes found in these cells. Additionally, we find alterations in two-pore calcium channel protein expression, with loss of presenilin preventing the formation of a high molecular weight species of TPC1 and TPC2. Finally, we find that treatments that disturb lysosomal calcium release lead to a reduction in autophagy function yet lysosomal inhibitors do not alter two-pore calcium channel expression. These data indicate that alterations in lysosomal calcium in the absence of presenilins might be leading to disruptions in autophagy.

A highly toxic cellular prion protein induces a novel, nonapoptotic form of neuronal death.

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.

Lysosome dysfunction triggers Atg7-dependent neural apoptosis.

Macroautophagy (autophagy) is a process wherein bulk cytosolic proteins and damaged organelles are sequestered and degraded via the lysosome. Alterations in autophagy-associated proteins have been shown to cause neural tube closure defects, neurodegeneration, and tumor formation. Normal lysosome function is critical for autophagy completion and when altered may lead to an accumulation of autophagic vacuoles (AVs) and caspase activation. The tumor suppressor p53 is highly expressed in neural precursor cells (NPCs) and has an important role in the regulation of both autophagy and apoptosis. We hypothesized that altered lysosome function would lead to NPC death via an interaction between autophagy- and apoptosis-associated proteins. To test our hypothesis, we utilized FGF2-expanded NPCs and the neural stem cell line, C17.2, in combination with the lysosomotropic agent chloroquine (CQ) and the vacuolar ATPase inhibitor bafilomycin A1 (Baf A1). Both CQ and Baf A1 caused concentration- and time-dependent AV accumulation, p53 phosphorylation, increased damage regulator autophagy modulator levels, caspase-3 activation, and cell death. Short hairpin RNA knockdown of Atg7, but not Beclin1, expression significantly inhibited CQ- and Baf A1-induced cell death, indicating that Atg7 is an upstream mediator of lysosome dysfunction-induced cell death. Cell death and/or caspase-3 activation was also attenuated by protein synthesis inhibition, p53 deficiency, or Bax deficiency, indicating involvement of the intrinsic apoptotic death pathway. In contrast to lysosome dysfunction, starvation-induced AV accumulation was inhibited by either Atg7 or Beclin1 knockdown, and Atg7 knockdown had no effect on starvation-induced death. These findings indicate that Atg7- and Beclin1-induced autophagy plays a cytoprotective role during starvation but that Atg7 has a unique pro-apoptotic function in response to lysosome dysfunction.

The proapoptotic BH3-only, Bcl-2 family member, Puma is critical for acute ethanol-induced neuronal apoptosis.

Synaptogenesis in humans occurs in the last trimester of gestation and in the first few years of life, whereas it occurs in the postnatal period in rodents. A single exposure of neonatal rodents to ethanol during this period evokes extensive neuronal apoptosis. Previous studies indicate that ethanol triggers the intrinsic apoptotic pathway in neurons, and that this requires the multi-BH domain, proapoptotic Bcl-2 family member Bax. To define the upstream regulators of this apoptotic pathway, we examined the possible roles of p53 and a subclass of proapoptotic Bcl-2 family members (i.e. the BH3 domain-only proteins) in neonatal wild-type and gene-targeted mice that lack these cell death inducers. Acute ethanol exposure produced greater caspase-3 activation and neuronal apoptosis in wild-type mice than in saline-treated littermate controls. Loss of p53-upregulated mediator of apoptosis (Puma) resulted in marked protection from ethanol-induced caspase-3 activation and apoptosis. Although Puma expression has been reported to be regulated by p53, p53-deficient mice exhibited a similar extent of ethanol-induced caspase-3 activation and neuronal apoptosis as wild-type mice. Mice deficient in other proapoptotic BH3-only proteins, including Noxa, Bim, or Hrk, showed no significant protection from ethanol-induced neuronal apoptosis. Collectively, these studies indicate a p53-independent, Bax- and Puma-dependent mechanism of neuronal apoptosis and identify Puma as a possible molecular target for inhibiting the effects of intrauterine ethanol exposure in humans.

Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity.

α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

Differential methylation hybridization array of endometrial cancers reveals two novel cancer-specific methylation markers.

PURPOSE: To identify novel endometrial cancer-specific methylation markers and to determine their association with clinicopathologic variables and survival outcomes. EXPERIMENTAL DESIGN: Differential methylation hybridization analysis (DMH) was done for 20 endometrioid endometrial cancers using normal endometrial DNA as a reference control. Combined bisulfite restriction analysis (COBRA) was used to verify methylation of sequences identified by DMH. Bisulfite sequencing was undertaken to further define CpG island methylation and to confirm the reliability of the COBRA. The methylation status of newly identified markers and the MLH1 promoter was evaluated by COBRA in a large series of endometrioid (n=361) and non-endometrioid uterine cancers (n=23). RESULTS: DMH and COBRA identified two CpG islands methylated in tumors but not in normal DNAs: SESN3 (PY2B4) and TITF1 (SC77F6/154). Bisulfite sequencing showed dense methylation of the CpG islands and confirmed the COBRA assays. SESN3 and TITF1 were methylated in 20% and 70% of endometrioid tumors, respectively. MLH1 methylation was seen in 28% of the tumors. TITF1 and SESN3 methylation was highly associated with MLH1 methylation (P<0.0001). SESN3 and TITF1 were methylated in endometrioid and non-endometrioid tumors, whereas MLH1 methylation was restricted to endometrioid tumors. Methylation at these markers was not associated with survival outcomes. CONCLUSIONS: The 5' CpG islands for SESN3 and TITF1 are novel cancer-specific methylation markers. Methylation at these loci is strongly associated with aberrant MLH1 methylation in endometrial cancers. SESN3, TITF1 and MLH1 methylation did not predict overall survival or disease-free survival in this large cohort of patients with endometrioid endometrial cancer.

Altered regulation of phosphatidylinositol 3-kinase signaling in cathepsin D-deficient brain.

Cathepsin D (CD) is an essential lysosomal protease and mice lacking this enzyme exhibit neuropathology similar to that observed in brains of patients with neuronal ceroid lipofuscinosces (NCL/Batten disease), a group of autosomal recessive pediatric neurodegenerative diseases. CD-deficient (CD-/-) brains exhibit a dramatic induction of autophagic stress as defined by the aberrant accumulation of autophagosomes, which is concomitant with markers of apoptosis. However, the signaling abnormalities which lead to CD deficiency-induced neurodegeneration are poorly defined. Since phosphatidylinositol-3 kinase (PI3-K) is known to regulate both apoptosis and autophagy, PI3-K-mediated signaling events were assessed in CD-/- brain at P14 and P25-26. Compared to WT littermate controls, CD-/- cortical neurons exhibited a widespread decrease in phosphorylation of Akt (inactivation) and GSK3beta (disinhibition) at P25-26, while levels of total Akt and GSK3beta remained unchanged. This P25-26-specific decrease in phosphorylation of Akt and GSK-3beta in CD-/- brain coincided temporally with markers of apoptosis but followed the induction of autophagic stress observed at both P14 and P25-26. In addition, levels and/or activation of mTOR and Beclin were not affected by CD deficiency, suggesting that the accumulation of autophagosomes is not due to an increased synthesis of autophagosomes but rather from an inhibition of autophagosome recycling, due most likely to a compromise in lysosome function. Together these observations indicate a pronounced decrease in pro-survival PI3-K signaling in CD-/- brain that may contribute to autophagic stress-induced and apoptotic neuropathology.

Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers.

Endometrial cancer is the most common gynecologic malignancy in the United States and the most frequent extracolonic tumor in hereditary nonpolyposis colorectal cancer (HNPCC). HNPCC patients have inherited defects in DNA mismatch repair and the microsatellite instability (MSI) tumor phenotype. Sporadic endometrial cancers also exhibit MSI, usually associated with methylation of the MLH1 promoter. Germ-line MSH6 mutations, which are rare in HNPCC, have been reported in several families with multiple members affected with endometrial carcinoma. We reasoned that MSH6 mutation might account for loss of mismatch repair in MSI-positive endometrial cancers in which the cause of MSI is unknown. We therefore investigated MSI and MLH1 promoter methylation in 441 endometrial cancer patients unselected for age or personal and family history of cancers. MSI and MLH1 promoter methylation status were associated with age of onset and tumor histology. One hundred cases (23% of the entire series) were evaluated for MSH6 defects. Inactivating germ-line MSH6 mutations were identified in seven women with MSI-positive, MLH1 promoter unmethylated cancers. Most of the MSI in these cases was seen with mononucleotide repeat markers. The MSH6 mutation carriers were significantly younger than the rest of the population (mean age 54.8 versus 64.6, P = 0.04). Somatic mutations were seen in 17 tumors, all of which had MSI. Our data suggest that inherited defects in MSH6 in women with endometrial cancer are relatively common. The minimum estimate of the prevalence of inherited MSH6 mutation in endometrial cancer is 1.6% (7 of 441), comparable with the predicted prevalence for patients with colorectal cancer.