Ken C. Walls

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

Abstractα-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, including Parkinsons 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.

Acute neonatal glucocorticoid exposure produces selective and rapid cerebellar neural progenitor cell apoptotic death

There has been a growing controversy regarding the continued use of glucocorticoid therapy to treat respiratory dysfunction associated with prematurity, as mounting clinical evidence has shown neonatal exposure produces permanent neuromotor and cognitive deficits. Here we report that, during a selective neonatal window of vulnerability, a single glucocorticoid injection in the mouse produces rapid and selective apoptotic cell death of the proliferating neural progenitor cells in the cerebellar external granule layer and permanent reductions in neuronal cell counts of their progeny, the cerebellar internal granule layer neurons. Our estimates suggest that this mouse window of vulnerability would correspond in the human to a period extending from approximately 20 weeks gestation to 6.5 weeks after birth. This death pathway is critically regulated by the proapoptotic Bcl-2 family member Puma and is independent of p53 expression. These rodent data indicate that there exists a previously unknown window of vulnerability during which a single glucocorticoid exposure at clinically relevant doses can produce neural progenitor cell apoptosis and permanent cerebellar pathology that may be responsible for some of the iatrogenically induced neurodevelopmental abnormalities seen in children exposed to this drug. This vulnerability may be related to the physiological role of glucocorticoids in regulating programmed cell death in the mammalian cerebellum.

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.

Cytoplasmic p53 and Activated Bax Regulate p53-dependent, Transcription-independent Neural Precursor Cell Apoptosis

The prodeath effects of p53 are typically mediated via its transcriptional upregulation of proapoptotic Bcl-2 family members, including PUMA, Noxa, and/or Bax. We previously reported that staurosporine (STS), a broad-spectrum kinase inhibitor and prototypical apoptosis-inducing agent, produced p53-dependent, Bax-dependent, neural precursor cell (NPC) apoptosis, but that this effect occurred independently of new gene transcription and PUMA expression. To further characterize the mechanism by which p53 regulates NPC death, we used primary cerebellar NPCs derived from wild-type, p53-deficient, and Bax-deficient neonatal mice and the mouse cerebellar neural stem cell line, C17.2. We found that STS rapidly increased p53 cytoplasmic immunoreactivity in neuritic-like processes in C17.2 cells, which preceded Bax activation and caspase-3 cleavage. Confocal microscopy analysis of STS-treated cells revealed partial colocalization of p53 with the mitochondrial marker pyruvate dehydrogenase as well as with conformationally altered “activated” Bax, suggesting an interaction between these proapoptotic molecules in triggering apoptotic death. Nucleophosmin (NPM), a CRM1-dependent nuclear chaperone, also exhibited partial colocalization with both activated Bax and p53 following STS treatment. These observations suggest that cytoplasmic p53 can trigger transcription-independent NPC apoptosis through its potential interaction with NPM and activated Bax.

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.

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

Alzheimers disease (AD) is a proteinopathy characterized by the accumulation of β-amyloid (Aβ) and tau. To date, clinical trials indicate that Aβ 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 Aβ 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 Aβ pathology. We also discovered that tau pathology soon reemerges post-injection, possibly due to persistent Aβ pathology. These studies provide evidence that targeting p-tau may represent an effective treatment strategy: potentially in conjunction with Aβ immunotherapy.

Swedish Alzheimer mutation induces mitochondrial dysfunction mediated by HSP60 mislocalization of amyloid precursor protein (APP) and beta-amyloid.

Background: Alzheimer is associated with mitochondrial dysfunction, yet the mechanism leading to APP and beta-amyloid accumulation is unknown. Results: Beta-amyloid and γ-secretase components accumulate in mitochondria via HSP60-mediated interactions. Conclusion: HSP60 mediates accumulation of APP and beta-amyloid in the mitochondria of Alzheimer transgenic and human brains. Significance: This study identifies a molecular player that translocates beta-amyloid and APP to mitochondria, contributing to its dysfunction. Alzheimer disease (AD) is a complex disorder that involves numerous cellular and subcellular alterations including impairments in mitochondrial homeostasis. To better understand the role of mitochondrial dysfunction in the pathogenesis of AD, we analyzed brains from clinically well-characterized human subjects and from the 3xTg-AD mouse model of AD. We find Aβ and critical components of the γ-secretase complex, presenilin-1, -2, and nicastrin, accumulate in the mitochondria. We used a proteomics approach to identify binding partners and show that heat shock protein 60 (HSP60), a molecular chaperone localized to mitochondria and the plasma membrane, specifically associates with APP. We next generated stable neural cell lines expressing human wild-type or Swedish APP, and provide corroborating in vitro evidence that HSP60 mediates translocation of APP to the mitochondria. Viral-mediated shRNA knockdown of HSP60 attenuates APP and Aβ mislocalization to the mitochondria. Our findings identify a novel interaction between APP and HSP60, which accounts for its translocation to the mitochondria.

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.

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.

bcl-2/Adenovirus E1B 19-kd interacting protein 3 (BNIP3) regulates hypoxia-induced neural precursor cell death.

Perinatal hypoxia-ischemia may result in long-term neurological deficits. In addition to producing neuron death, HI causes death of neural precursor cells (NPCs) in the developing brain. To characterize the molecular pathways that regulate hypoxia-induced death of NPCs, we treated a mouse neural stem cell line (C17.2 cells) and fibroblastic growth factor II-expanded primary NPCs derived from wild-type or gene-disrupted mice, with oxygen glucose deprivation or the hypoxia mimetics desferrioxamine or cobalt chloride. Neural precursor cells undergoing hypoxia exhibited time- and concentration-dependent caspase-3 activation and cell death, which was significantly reduced by treatment with a broad caspase inhibitor or protein synthesis inhibition. Bax/Bak-deficient NPCs were protected from desferrioxamine-induced death and exhibited minimal caspase-3 activation. Oxygen glucose deprivation or hypoxia-mimetic exposure also resulted in increased hypoxia-inducible factor &agr; and bcl-2/adenovirus E1B 19-kd interacting protein 3 (BNIP3) expression. BNIP3 shRNA treatment failed to affect hypoxia-induced caspase-3 activation but inhibited cell death and nuclear translocation of apoptosis-inducing factor, indicating that BNIP3 is an important regulator of caspase-independent NPC death after hypoxia. These studies demonstrate that hypoxia activates both caspase-dependent and -independent NPC death pathways that are critically regulated by multiple Bcl-2 family members.