Social orienting in gaze leading: a mechanism for shared attention

Here, we report a novel social orienting response that occurs after viewing averted gaze. We show, in three experiments, that when a person looks from one location to an object, attention then shifts towards the face of an individual who has subsequently followed the person''s gaze to that same object. That is, contrary to ‘gaze following’, attention instead orients in the opposite direction to observed gaze and towards the gazing face. The magnitude of attentional orienting towards a face that ‘follows’ the participant''s gaze is also associated with self-reported autism-like traits. We propose that this gaze leading phenomenon implies the existence of a mechanism in the human social cognitive system for detecting when one''s gaze has been followed, in order to establish ‘shared attention’ and maintain the ongoing interaction.     

Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis

Autophagy-related gene (Atg) 5 is a gene product required for the formation of autophagosomes. Here, we report that Atg5, in addition to the promotion of autophagy, enhances susceptibility towards apoptotic stimuli. Enforced expression of Atg5-sensitized tumour cells to anticancer drug treatment both in vitro and in vivo. In contrast, silencing the Atg5 gene with short interfering RNA (siRNA) resulted in partial resistance to chemotherapy. Apoptosis was associated with calpain-mediated Atg5 cleavage, resulting in an amino-terminal cleavage product with a relative molecular mass of 24,000 (Mr 24K). Atg5 cleavage was observed independent of the cell type and the apoptotic stimulus, suggesting that calpain activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the anti-apoptotic molecule Bcl-xL and triggered cytochrome c release and caspase activation. Taken together, calpain-mediated Atg5 cleavage provokes apoptotic cell death, therefore, represents a molecular link between autophagy and apoptosis--a finding with potential importance for clinical anticancer therapies.     

Rat tau proteome consists of six tau isoforms: implication for animal models of human tauopathies

Human brain encompasses six tau isoforms, containing either three (3R) or four (4R) repeat domains, all of which participate in the pathogenesis of human tauopathies. To investigate the role of tau protein in the disease, transgenic rat models have been created. However, unlike humans, it has been suggested that rat brain expresses only three 4R tau isoforms. Because of the significance of the number of tau isoforms for faithful reproducibility of neurofibrillary pathology in transgenic rat models, we reopened this issue. Surprisingly, our results showed that adult rat brain contains six tau isoforms like humans. Protein expression of 4R tau isoforms was ninefold higher than 3R isoforms. Furthermore, the protein levels of tau isoforms with none, one or two N-terminal inserts were 30%, 35%, and 35% of total tau, respectively. Moreover, amount and ratio of tau isoforms were developmentally regulated. The levels of 4R tau isoforms progressively increased from early postnatal period until adulthood, whereas the expression of 3R tau isoforms reached maximum at P10 and then gradually declined. Our results show that rat brain encompasses full tau proteome similar to humans. These findings support the use of rat as an animal model in human tauopathies research.     

Calpain-mediated cleavage of the cyclin-dependent kinase-5 activator p39 to p29.

The activity of cyclin-dependent kinase-5 (Cdk5) is tightly regulated by binding of its neuronal activators p35 and p39. Upon neurotoxic insults, p35 is cleaved to p25 by the Ca(2+)-dependent protease calpain. p25 is accumulated in ischemic brains and in brains of patients with Alzheimer's disease. p25 deregulates Cdk5 activity by causing prolonged activation and mislocalization of Cdk5. It is unknown whether p39, which is expressed throughout the adult rat brain, is cleaved by calpain, and whether this contributes to deregulation of Cdk5. Here, we show that calpain cleaved p39 in vitro, resulting in generation of a C-terminal p29 fragment. In vivo, p29 was generated in ischemic brain concomitant with increased calpain activity. In fresh brain lysates, generation of p29 was Ca(2+)-dependent, and calpain inhibitors abolished p29 production. The Ca(2+) ionophore ionomycin and the excitotoxin glutamate induced production of p29 in cultures of cortical neurons in a calpain-dependent manner. Like p25, p29 was more stable than p39 and caused redistribution of Cdk5 in cortical neurons. Our data suggest that neurotoxic insults lead to calpain-mediated conversion of p39 to p29, which might contribute to deregulation of Cdk5.     

Inability of tau to properly regulate neuronal microtubule dynamics: a loss-of-function mechanism by which tau might mediate neuronal cell death

Interest in the microtubule-associated protein tau stems from its critical roles in neural development and maintenance, as well as its role in Alzheimer's, FTDP-17 and related neurodegenerative diseases. Under normal circumstances, tau performs its functions by binding to microtubules and powerfully regulating their stability and growing and shortening dynamics. On the other hand, genetic analyses have established a clear cause-and-effect relationship between tau dysfunction/mis-regulation and neuronal cell death and dementia in FTDP-17, but the molecular basis of tau's destructive action(s) remains poorly understood. One attractive model suggests that the intracellular accumulation of abnormal tau aggregates causes cell death, i.e., a gain-of-toxic function model. Here, we describe the evidence and arguments for an alternative loss-of-function model in which tau-mediated neuronal cell death is caused by the inability of affected cells to properly regulate their microtubule dynamic due to mis-regulation by tau. In support of this model, our recent data demonstrate that missense FTDP-17 mutations that alter amino acid residues near tau's microtubule binding region strikingly modify the ability of tau to modulate microtubule dynamics. Additional recent data from our labs support the notion that the same dysfunction occurs in the FTDP-17 regulatory mutations that alter tau RNA splicing patterns. Our model posits that the dynamics of microtubules in neuronal cells must be tightly regulated to enable them to carry out their diverse functions, and that microtubules that are either over-stabilized or under-stabilized, that is, outside an acceptable window of dynamic activity, lead to neurodegeneration. An especially attractive aspect of this model is that it readily accommodates both the structural and regulatory classes of FTDP-17 mutations.     

mTOR regulates tau phosphorylation and degradation: implications for Alzheimer's disease and other tauopathies

Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer's disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3β and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology, while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence that reducing mTOR signaling increases lifespan and healthspan, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide preclinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies.     

Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis

Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.     

Inhibition of tau polymerization by its carboxy-terminal caspase cleavage fragment

Abnormal aggregation of the microtubule-associated protein, tau, occurs in many neurodegenerative diseases, making it important to understand the mechanisms of tau polymerization. Previous work has indicated that the C-terminal region of tau inhibits polymerization in vitro, and a growing body of evidence implicates caspase cleavage of tau at Asp 421 in the C-terminus as an important inducer of tau polymerization in Alzheimer's disease. In the present study, we provide evidence that the C-terminal peptide fragment produced by caspase cleavage inhibits tau polymerization, suggesting that caspase cleavage of tau enhances its polymerization by removing the inhibitory control element. Moreover, we provide evidence that the peptide assumes an alpha-helical configuration and inhibits tau assembly by interacting with residues 321-375 in the microtubule binding repeat region. These findings indicate that formation of the fibrillar pathologies during the course of Alzheimer's disease may be driven or sustained by apoptotic events leading to caspase activation.     

Modulation of microtubule dynamics by tau in living cells: implications for development and neurodegeneration

The neural microtubule-associated protein tau binds to and stabilizes microtubules. Because of alternative mRNA splicing, tau is expressed with either 3 or 4 C-terminal repeats. Two observations indicate that differences between these tau isoforms are functionally important. First, the pattern of tau isoform expression is tightly regulated during development. Second, mutation-induced changes in tau RNA splicing cause neuronal cell death and dementia simply by altering the isoform expression ratio. To investigate whether 3- and 4-repeat tau differentially regulate microtubule behavior in cells, we microinjected physiological levels of these two isoforms into EGFP-tubulin-expressing cultured MCF7 cells and measured the effects on the dynamic instability behavior of individual microtubules by time-lapse microscopy. Both isoforms suppressed microtubule dynamics, though to different extents. Specifically, 4-repeat tau reduced the rate and extent of both growing and shortening events. In contrast, 3-repeat tau stabilized most dynamic parameters about threefold less potently than 4-repeat tau and had only a minimal ability to suppress shortening events. These differences provide a mechanistic rationale for the developmental shift in tau isoform expression and are consistent with a loss-of-function model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal cell death and dementia.     

Calpain-mediated proteolytic cleavage of troponin I induced by hypoxia or metabolic inhibition in cultured neonatal cardiomyocytes

While ischemic damage to myofibrillar proteins is thought to be responsible in part for depressed cardiac function, the relation between myofilament protein breakdown and chronic hypoxia has not been defined. We previously characterized a chemical hypoxia model of neonatal cardiomyocytes mediated by 1 mM azide that exhibits features of calpain activation (Mol Cell Biochem 178:141-149, 1998). We here show that both hypoxia and azide-mediated metabolic inhibition induced heme oxygenase-1 expression, and caused cell death associated with lipid peroxidation. While blocking calcium influx or inhibiting calpain activity efficiently attenuated hypoxia-induced cell injury, it failed to prevent cell injury caused by adenoviral overexpression of the tumor suppressor protein p53. Inhibitors of caspases, on the other hand, suppressed cell injury caused by p53 overexpression. Hypoxia caused selective cleavage of troponin I (TnI), which could be suppressed by either nifedipine or calpeptin. Other myofilament proteins such as troponin T, myosin heavy chain, and actin appeared to remain largely intact. p53-mediated cell injury exhibited proteolysis of the caspase protein substrate lamin B without appreciable breakdown of TnI. We suggest that calpain-induced TnI breakdown may constitute a unique biochemical marker associated with chronically hypoxic cardiomyocytes.     

MINCR: A long non-coding RNA shared between cancer and neurodegeneration

The multitasking nature of lncRNAs allows them to play a central role in both physiological and pathological conditions. Often the same lncRNA can participate in different diseases. Specifically, the MYC-induced Long non-Coding RNA MINCR is upregulated in various cancer types, while downregulated in Amyotrophic Lateral Sclerosis patients. Therefore, this work aims to investigate MINCR potential mechanisms of action and its implications in cancer and neurodegeneration in relation to its expression levels in SH-SY5Y cells through RNA-sequencing approach. Our results show that MINCR overexpression causes massive alterations in cancer-related genes, leading to disruption in many fundamental processes, such as cell cycle and growth factor signaling. On the contrary, MINCR downregulation influences a small number of genes involved in different neurodegenerative disorders, mostly concerning RNA metabolism and inflammation. Thus, understanding the cause and functional consequences of MINCR deregulation gives important insights on potential pathogenetic mechanisms both in cancer and in neurodegeneration.     

The complex relationship between soluble and insoluble tau in tauopathies revealed by efficient dephosphorylation and specific antibodies

Phosphorylated tau is deposited as insoluble inclusion bodies in the tauopathies. We have used a new efficient method to dephosphorylate tau extracted from control and tauopathy brain. In some tauopathies, including Alzheimer's disease and progressive supranuclear palsy, the pattern of insoluble tau isoforms reflected that of soluble tau. In contrast, in corticobasal degeneration, Pick's disease, and some forms of fronto-temporal dementia, specific tau isoforms were selectively sequestered into insoluble inclusion-forming tau. Therefore the overall expression of individual tau isoforms does not predict which tau isoforms are deposited in all tauopathies and different mechanisms must operate that result in the deposition of specific tau isoforms.     

Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration

The signaling adapter p62 plays a coordinating role in mediating phosphorylation and ubiquitin-dependent trafficking of interacting proteins. However, there is little known about the physiologic role of this protein in brain. Here, we report age-dependent constitutive activation of glycogen synthase kinase 3β, protein kinase B, mitogen-activated protein kinase, and c- Jun -N-terminal kinase in adult p62^−/− mice resulting in hyperphosphorylated tau, neurofibrillary tangles, and neurodegeneration. Biochemical fractionation of p62^−/− brain led to recovery of aggregated K63-ubiquitinated tau. Loss of p62 was manifested by increased anxiety, depression, loss of working memory, and reduced serum brain-derived neurotrophic factor levels. Our findings reveal a novel role for p62 as a chaperone that regulates tau solubility thereby preventing tau aggregation. This study provides a clear demonstration of an Alzheimer-like phenotype in a mouse model in the absence of expression of human genes carrying mutations in amyloid-beta protein precursor, presenilin, or tau. Thus, these findings provide new insight into manifestation of sporadic Alzheimer disease and the impact of obesity.     

Soluble oligomers from a non-disease related protein mimic Abeta-induced tau hyperphosphorylation and neurodegeneration

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.     

Imaging multiple phases of neurodegeneration: a novel approach to assessing cell death in vivo

Nerve cell death is the key event in all neurodegenerative disorders, with apoptosis and necrosis being central to both acute and chronic degenerative processes. However, until now, it has not been possible to study these dynamically and in real time. In this study, we use spectrally distinct, well-recognised fluorescent cell death markers to enable the temporal resolution and quantification of the early and late phases of apoptosis and necrosis of single nerve cells in different disease models. The tracking of single-cell death profiles in the same living eye over hours, days, weeks and months is a significant advancement on currently available techniques. We identified a numerical preponderance of late-phase versus early-phase apoptotic cells in chronic models, reinforcing the commonalities between cellular mechanisms in different disease models. We showed that MK801 effectively inhibited both apoptosis and necrosis, but our findings support the use of our technique to investigate more specific anti-apoptotic and anti-necrotic strategies with well-defined targets, with potentially greater clinical application. The optical properties of the eye provide compelling opportunities for the quantitative monitoring of disease mechanisms and dynamics in experimental neurodegeneration. Our findings also help to directly observe retinal nerve cell death in patients as an adjunct to refining diagnosis, tracking disease status and assessing therapeutic intervention.     

Aberrant Cdk5 activation by p25 triggers pathological events leading to neurodegeneration and neurofibrillary tangles

Cyclin-dependent kinase 5 (Cdk5) and its regulatory subunit p35 are integral players in the proper development of the mammalian central nervous system. Proteolytic cleavage of p35 generates p25, leading to aberrant Cdk5 activation. The accumulation of p25 is implicated in several neurodegenerative diseases. In primary neurons, p25 causes apoptosis and tau hyperphosphorylation. Current mouse models expressing p25, however, fail to rigorously recapitulate these phenotypes in vivo. Here, we generated inducible transgenic mouse lines overexpressing p25 in the postnatal forebrain. Induction of p25 preferentially directed Cdk5 to pathological substrates. These animals exhibited neuronal loss in the cortex and hippocampus, accompanied by forebrain atrophy, astrogliosis, and caspase-3 activation. Endogenous tau was hyperphosphorylated at many epitopes, aggregated tau accumulated, and neurofibrillary pathology developed progressively in these animals. Our cumulative findings provide compelling evidence that in vivo deregulation of Cdk5 by p25 plays a causative role in neurodegeneration and the development of neurofibrillary pathology.