Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations.

Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.

Neurofilaments: neurobiological foundations for biomarker applications.

Interest in neurofilaments has risen sharply in recent years with recognition of their potential as biomarkers of brain injury or neurodegeneration in CSF and blood. This is in the context of a growing appreciation for the complexity of the neurobiology of neurofilaments, new recognition of specialized roles for neurofilaments in synapses and a developing understanding of mechanisms responsible for their turnover. Here we will review the neurobiology of neurofilament proteins, describing current understanding of their structure and function, including recently discovered evidence for their roles in synapses. We will explore emerging understanding of the mechanisms of neurofilament degradation and clearance and review new methods for future elucidation of the kinetics of their turnover in humans. Primary roles of neurofilaments in the pathogenesis of human diseases will be described. With this background, we then will review critically evidence supporting use of neurofilament concentration measures as biomarkers of neuronal injury or degeneration. Finally, we will reflect on major challenges for studies of the neurobiology of intermediate filaments with specific attention to identifying what needs to be learned for more precise use and confident interpretation of neurofilament measures as biomarkers of neurodegeneration.

Lysosomal Dysfunction in Down Syndrome Is APP-Dependent and Mediated by APP-ßCTF (C99).

Lysosomal failure underlies pathogenesis of numerous congenital neurodegenerative disorders and is an early and progressive feature of Alzheimer's disease (AD) pathogenesis. Here, we report that lysosomal dysfunction in Down ayndrome (trisomy 21), a neurodevelopmental disorder and form of early onset AD, requires the extra gene copy of amyloid precursor protein (APP) and is specifically mediated by the ß cleaved carboxy terminal fragment of APP (APP-ßCTF, C99). In primary fibroblasts from individuals with DS, lysosomal degradation of autophagic and endocytic substrates is selectively impaired, causing them to accumulate in enlarged autolysosomes/lysosomes. Direct measurements of lysosomal pH uncovered a significant elevation (0.6 units) as a basis for slowed LC3 turnover and the inactivation of cathepsin D and other lysosomal hydrolases known to be unstable or less active when lysosomal pH is persistently elevated. Normalizing lysosome pH by delivering acidic nanoparticles to lysosomes ameliorated lysosomal deficits, whereas RNA sequencing analysis excluded a transcriptional contribution to hydrolase declines. Cortical neurons cultured from the Ts2 mouse model of DS exhibited lysosomal deficits similar to those in DS cells. Lowering APP expression with siRNA or BACE1 inhibition reversed cathepsin deficits in both fibroblasts and neurons. Deleting one Bace1 allele from adult Ts2 mice had similar rescue effects in vivo The modest elevation of endogenous APP-ßCTF needed to disrupt lysosomal function in DS is relevant to sporadic AD where APP-ßCTF, but not APP, is also elevated. Our results extend evidence that impaired lysosomal acidification drives progressive lysosomal failure in multiple forms of AD.SIGNIFICANCE STATEMENT Down syndrome (trisomy 21) (DS) is a neurodevelopmental disorder invariably leading to early-onset Alzheimer's disease (AD). We showed in cells from DS individuals and neurons of DS models that one extra copy of a normal amyloid precursor protein (APP) gene impairs lysosomal acidification, thereby depressing lysosomal hydrolytic activities and turnover of autophagic and endocytic substrates, processes vital to neuronal survival. These deficits, which were reversible by correcting lysosomal pH, are mediated by elevated levels of endogenous ß-cleaved carboxy-terminal fragment of APP (APP-ßCTF). Notably, similar endosomal-lysosomal pathobiology emerges early in sporadic AD, where neuronal APP-ßCTF is also elevated, underscoring its importance as a therapeutic target and underscoring the functional and pathogenic interrelationships between the endosomal-lysosomal pathway and genes causing AD.

Lysosome trafficking and signaling in health and neurodegenerative diseases.

Lysosomes, single-membrane organelles defined by a uniquely strong acidic lumenal pH and high content of acid hydrolases, are the shared degradative compartments of the endocytic and autophagic pathways. These pathways, and especially lysosomes, are points of particular vulnerability in many neurodegenerative diseases. Beyond the role of lysosomes in substrate degradation, new findings have ascribed to lysosomes the leading role in sensing and responding to cellular nutrients, growth factors and cellular stress. This review aims to integrate recent concepts of basic lysosome biology and pathobiology as a basis for understanding neurodegenerative disease pathogenesis. Here, we discuss the newly recognized signaling functions of lysosomes and specific aspects of lysosome biology in neurons while re-visiting the classical defining criteria for lysosomes and the importance of preserving strict definitions. Our discussion emphasizes dynein-mediated axonal transport of maturing degradative organelles, with further consideration of their roles in synaptic function. We finally examine how distinctive underlying disturbances of lysosomes in various neurodegenerative diseases result in unique patterns of auto/endolysosomal mistrafficking. The rapidly emerging understanding of lysosomal trafficking and disruptions in lysosome signaling is providing valuable clues to new targets for disease-modifying therapies.

Cyclodextrin has conflicting actions on autophagy flux in vivo in brains of normal and Alzheimer model mice.

2-hydroxypropyl-ß-cyclodextrin (CYCLO), a modifier of cholesterol efflux from cellular membrane and endo-lysosomal compartments, reduces lysosomal lipid accumulations and has therapeutic effects in animal models of Niemann-Pick disease type C and several other neurodegenerative states. Here, we investigated CYCLO effects on autophagy in wild-type mice and TgCRND8 mice-an Alzheimer's Disease (AD) model exhibiting ß-amyloidosis, neuronal autophagy deficits leading to protein and lipid accumulation within greatly enlarged autolysosomes. A 14-day intracerebroventricular administration of CYCLO to 8-month-old TgCRND8 mice that exhibit moderately advanced neuropathology markedly diminished the sizes of enlarged autolysosomes and lowered their content of GM2 ganglioside and Aß-immunoreactivity without detectably altering amyloid precursor protein processing or extracellular Aß/ß-amyloid burden. We identified two major actions of CYCLO on autophagy underlying amelioration of lysosomal pathology. First, CYCLO stimulated lysosomal proteolytic activity by increasing cathepsin D activity, levels of cathepsins B and D and two proteins known to interact with cathepsin D, NPC1 and ABCA1. Second, CYCLO impeded autophagosome-lysosome fusion as evidenced by the accumulation of LC3, SQSTM1/p62, and ubiquitinated substrates in an expanded population of autophagosomes in the absence of greater autophagy induction. By slowing substrate delivery to lysosomes, autophagosome maturational delay, as further confirmed by our in vitro studies, may relieve lysosomal stress due to accumulated substrates. These findings provide in vivo evidence for lysosomal enhancing properties of CYCLO, but caution that prolonged interference with cellular membrane fusion/autophagosome maturation could have unfavorable consequences, which might require careful optimization of dosage and dosing schedules.

Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and ß-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote ß-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies ß-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-ß, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.-Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

Calpastatin inhibits motor neuron death and increases survival of hSOD1(G93A) mice.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1(G93A) transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1(G93A) mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1(G93A) /CAST mice compared to littermate hSOD1(G93A) mice is delayed, which accounts for their longer time of survival. We also find that neuronal over-expression of CAST in hSOD1(G93A) transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model. CAST (encoding calpastatin) inhibits hyperactivated calpain to prevent motor neuron disease operating through a cascade of events as indicated in the schematic, with relevance to amyotrophic lateral sclerosis (ALS). We propose that over-expression of CAST in motor neurons of hSOD1(G93A) mice inhibits activation of CDK5, breakdown of cytoskeletal proteins (NFs, MAP2 and Tau) and regulatory molecules (Cam Kinase IV, Calcineurin A), and disease-causing proteins (TDP-43, α-Synuclein and Huntingtin) to prevent neuronal loss and delay neurological deficits. In our experiments, CAST could also inhibit cleavage of Bid, Bax, AIF to prevent mitochondrial, ER and lysosome-mediated cell death mechanisms. Similarly, CAST over-expression in neurons attenuated pathological effects of TDP-43, α-synuclein and Huntingtin. These results suggest a potential value of specific small molecule inhibitors of calpains in delaying the development of ALS. Read the Editorial Highlight for this article on page 140.

Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice.

Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD.

Specific calpain inhibition by calpastatin prevents tauopathy and neurodegeneration and restores normal lifespan in tau P301L mice.

Tau pathogenicity in Alzheimer's disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5. Calpain overactivation in brains of these mice is accelerated as a result of the marked depletion of the endogenous calpain inhibitor, calpastatin. When levels of this inhibitor are restored in neurons of JNPL3 mice by overexpressing calpastatin, tauopathy is prevented, including calpain-mediated breakdown of cytoskeletal proteins, cdk5 activation, tau hyperphosphorylation, formation of potentially neurotoxic tau fragments by either calpain or caspase-3, and tau oligomerization. Calpastatin overexpression also prevents loss of motor axons, delays disease onset, and extends survival of JNPL3 mice by 3 months to within the range of normal lifespan. Our findings support the therapeutic promise of highly specific calpain inhibition in the treatment of tauopathies and other neurodegenerative states.

Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits.

Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimer's disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin B inhibition on lysosomal proteases suggests that enhancing lysosomal proteolysis improves the overall environment of the lysosome and its clearance functions, which may be possibly relevant to a broader range of lysosomal disorders beyond Alzheimer's disease.

Single-walled carbon nanotubes alleviate autophagic/lysosomal defects in primary glia from a mouse model of Alzheimer's disease.

Defective autophagy in Alzheimer's disease (AD) promotes disease progression in diverse ways. Here, we demonstrate impaired autophagy flux in primary glial cells derived from CRND8 mice that overexpress mutant amyloid precursor protein (APP). Functionalized single-walled carbon nanotubes (SWNT) restored normal autophagy by reversing abnormal activation of mTOR signaling and deficits in lysosomal proteolysis, thereby facilitating elimination of autophagic substrates. These findings suggest SWNT as a novel neuroprotective approach to AD therapy.

2014 Report on the Milestones for the US National Plan to Address Alzheimer's Disease.

With increasing numbers of people with Alzheimer's and other dementias across the globe, many countries have developed national plans to deal with the resulting challenges. In the United States, the National Alzheimer's Project Act, signed into law in 2011, required the creation of such a plan with annual updates thereafter. Pursuant to this, the US Department of Health and Human Services (HHS) released the National Plan to Address Alzheimer's Disease in 2012, including an ambitious research goal of preventing and effectively treating Alzheimer's disease by 2025. To guide investments, activities, and the measurement of progress toward achieving this 2025 goal, in its first annual plan update (2013) HHS also incorporated into the plan a set of short, medium and long-term milestones. HHS further committed to updating these milestones on an ongoing basis to account for progress and setbacks, and emerging opportunities and obstacles. To assist HHS as it updates these milestones, the Alzheimer's Association convened a National Plan Milestone Workgroup consisting of scientific experts representing all areas of Alzheimer's and dementia research. The workgroup evaluated each milestone and made recommendations to ensure that they collectively constitute an adequate work plan for reaching the goal of preventing and effectively treating Alzheimer's by 2025. This report presents these Workgroup recommendations.

mTOR inhibition in Q175 Huntington's disease model mice facilitates neuronal autophagy and mutant huntingtin clearance.

Huntington's disease (HD) is caused by expansion of the polyglutamine stretch in huntingtin protein (HTT) resulting in hallmark aggresomes/inclusion bodies (IBs) composed of mutant huntingtin protein (mHTT) and its fragments. Stimulating autophagy to enhance mHTT clearance is considered a potential therapeutic strategy for HD. Our recent evaluation of the autophagic-lysosomal pathway (ALP) in human HD brain reveals upregulated lysosomal biogenesis and relatively normal autophagy flux in early Vonsattel grade brains, but impaired autolysosome clearance in late grade brains, suggesting that autophagy stimulation could have therapeutic benefits as an earlier clinical intervention. Here, we tested this hypothesis by crossing the Q175 HD knock-in model with our autophagy reporter mouse TRGL ( T hy-1- R FP- G FP- L C3) to investigate in vivo neuronal ALP dynamics. In the Q175 and/or TRGL/Q175 mice, mHTT was detected in autophagic vacuoles and also exhibited high level colocalization with autophagy receptors p62/SQSTM1 and ubiquitin in the IBs. Compared to the robust lysosomal pathology in late-stage human HD striatum, ALP alterations in Q175 models are also late-onset but milder that included a lowered phospho-p70S6K level, lysosome depletion and autolysosome elevation including more poorly acidified autolysosomes and larger-sized lipofuscin granules, reflecting impaired autophagic flux. Administration of a mTOR inhibitor to 6-mo-old TRGL/Q175 normalized lysosome number, ameliorated aggresome pathology while reducing mHTT-, p62- and ubiquitin-immunoreactivities, suggesting beneficial potential of autophagy modulation at early stages of disease progression.

Pathobiology of the autophagy-lysosomal pathway in the Huntington's disease brain.

Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntington's disease (HD). Although lowering mHTT by stimulating autophagy has been considered a possible therapeutic strategy, the role and competence of autophagy-lysosomal pathway (ALP) during HD progression in the human disease remains largely unknown. Here, we used multiplex confocal and ultrastructural immunocytochemical analyses of ALP functional markers in relation to mHTT aggresome pathology in striatum and the less affected cortex of HD brains staged from HD2 to HD4 by Vonsattel neuropathological criteria compared to controls. Immunolabeling revealed the localization of HTT/mHTT in ALP vesicular compartments labeled by autophagy-related adaptor proteins p62/SQSTM1 and ubiquitin, and cathepsin D (CTSD) as well as HTT-positive inclusions. Although comparatively normal at HD2, neurons at later HD stages exhibited progressive enlargement and clustering of CTSD-immunoreactive autolysosomes/lysosomes and, ultrastructurally, autophagic vacuole/lipofuscin granules accumulated progressively, more prominently in striatum than cortex. These changes were accompanied by rises in levels of HTT/mHTT and p62/SQSTM1, particularly their fragments, in striatum but not in the cortex, and by increases of LAMP1 and LAMP2 RNA and LAMP1 protein. Importantly, no blockage in autophagosome formation and autophagosome-lysosome fusion was detected, thus pinpointing autophagy substrate clearance deficits as a basis for autophagic flux declines. The findings collectively suggest that upregulated lysosomal biogenesis and preserved proteolysis maintain autophagic clearance in early-stage HD, but failure at advanced stages contributes to progressive HTT build-up and potential neurotoxicity. These findings support the prospect that ALP stimulation applied at early disease stages, when clearance machinery is fully competent, may have therapeutic benefits in HD patients.

Peripherin is a subunit of peripheral nerve neurofilaments: implications for differential vulnerability of CNS and peripheral nervous system axons.

Peripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament triplet proteins [neurofilament light (NFL), medium (NFM), and heavy (NFH) chain] but has an unknown function. The earlier peak expression of peripherin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that peripherin and neurofilament triplets form separate filament systems. However, here, we demonstrate that, despite a postnatal decline in expression, peripherin is as abundant as the triplet in the adult PNS and exists in a relatively fixed stoichiometry with these subunits. Peripherin exhibits a distribution pattern identical to those of triplet proteins in sciatic axons and colocalizes with NFL on single neurofilaments by immunogold electron microscopy. Peripherin also coassembles into a single network of filaments containing NFL, NFM, and NFH with and without α-internexin in quadruple- or quintuple-transfected SW13vim(-) cells. Genetically deleting NFL in mice dramatically reduces peripherin content in sciatic axons. Moreover, peripherin mutations has been shown to disrupt the neurofilament network in transfected SW13vim(-) cells. These data show that peripherin and the neurofilament proteins are functionally interdependent. The results strongly support the view that, rather than forming an independent structure, peripherin is a subunit of neurofilaments in the adult PNS. Our findings provide a basis for its close relationship with neurofilaments in PNS diseases associated with neurofilament accumulation.

G9a-mediated histone methylation regulates ethanol-induced neurodegeneration in the neonatal mouse brain.

Rodent exposure to binge-like ethanol during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces neuronal cell loss. However, the molecular mechanisms underlying these neuronal losses are still poorly understood. Here, we tested the possibility of histone methylation mediated by G9a (lysine dimethyltransferase) in regulating neuronal apoptosis in P7 mice exposed to ethanol. G9a protein expression, which is higher during embryogenesis and synaptogenic period compared to adult brain, is entirely confined to the cell nuclei in the developing brain. We found that ethanol treatment at P7, which induces apoptotic neurodegeneration in neonatal mice, enhanced G9a activity followed by increased histone H3 lysine 9 (H3K9me2) and 27 (H3K27me2) dimethylation. In addition, it appears that increased dimethylation of H3K9 makes it susceptible to proteolytic degradation by caspase-3 in conditions in which ethanol induces neurodegeneration. Further, pharmacological inhibition of G9a activity prior to ethanol treatment at P7 normalized H3K9me2, H3K27me2 and total H3 proteins to basal levels and prevented neurodegeneration in neonatal mice. Together, these data demonstrate that G9a mediated histone H3K9 and K27 dimethylation critically regulates ethanol-induced neurodegeneration in the developing brain. Furthermore, these findings reveal a novel link between G9a and neurodegeneration in the developing brain exposed to postnatal ethanol and may have a role in fetal alcohol spectrum disorders.

Autophagy failure in Alzheimer's disease and the role of defective lysosomal acidification.

Autophagy is a lysosomal degradative process which recycles cellular waste and eliminates potentially toxic damaged organelles and protein aggregates. The important cytoprotective functions of autophagy are demonstrated by the diverse pathogenic consequences that may stem from autophagy dysregulation in a growing number of neurodegenerative disorders. In many of the diseases associated with autophagy anomalies, it is the final stage of autophagy-lysosomal degradation that is disrupted. In several disorders, including Alzheimer's disease (AD), defective lysosomal acidification contributes to this proteolytic failure. The complex regulation of lysosomal pH makes this process vulnerable to disruption by many factors, and reliable lysosomal pH measurements have become increasingly important in investigations of disease mechanisms. Although various reagents for pH quantification have been developed over several decades, they are not all equally well suited for measuring the pH of lysosomes. Here, we evaluate the most commonly used pH probes for sensitivity and localisation, and identify LysoSensor yellow/blue-dextran, among currently used probes, as having the optimal profile of properties for measuring lysosomal pH. In addition, we review evidence that lysosomal acidification is defective in AD and extend our original findings, of elevated lysosomal pH in presenilin 1 (PS1)-deficient blastocysts and neurons, to additional cell models of PS1 and PS1/2 deficiency, to fibroblasts from AD patients with PS1 mutations, and to neurons in the PS/APP mouse model of AD.

Lysosome and calcium dysregulation in Alzheimer's disease: partners in crime.

Early-onset FAD (familial Alzheimer's disease) is caused by mutations of PS1 (presenilin 1), PS2 (presenilin 2) and APP (amyloid precursor protein). Beyond the effects of PS1 mutations on proteolytic functions of the γ-secretase complex, mutant or deficient PS1 disrupts lysosomal function and Ca2+ homoeostasis, both of which are considered strong pathogenic factors in FAD. Loss of PS1 function compromises assembly and proton-pumping activity of the vacuolar-ATPase on lysosomes, leading to defective lysosomal acidification and marked impairment of autophagy. Additional dysregulation of cellular Ca2+ by mutant PS1 in FAD has been ascribed to altered ion channels in the endoplasmic reticulum; however, rich stores of Ca2+ in lysosomes are also abnormally released in PS1-deficient cells secondary to the lysosomal acidification defect. The resultant rise in cytosolic Ca2+ activates Ca2+-dependent enzymes, contributing substantially to calpain overactivation that is a final common pathway leading to neurofibrillary degeneration in all forms of AD (Alzheimer's disease). In the present review, we discuss the close inter-relationships among deficits of lysosomal function, autophagy and Ca2+ homoeostasis as a pathogenic process in PS1-related FAD and their relevance to sporadic AD.

Alzheimer's-related endosome dysfunction in Down syndrome is Abeta-independent but requires APP and is reversed by BACE-1 inhibition.

An additional copy of the beta-amyloid precursor protein (APP) gene causes early-onset Alzheimer's disease (AD) in trisomy 21 (DS). Endosome dysfunction develops very early in DS and AD and has been implicated in the mechanism of neurodegeneration. Here, we show that morphological and functional endocytic abnormalities in fibroblasts from individuals with DS are reversed by lowering the expression of APP or beta-APP-cleaving enzyme 1 (BACE-1) using short hairpin RNA constructs. By contrast, endosomal pathology can be induced in normal disomic (2N) fibroblasts by overexpressing APP or the C-terminal APP fragment generated by BACE-1 (betaCTF), all of which elevate the levels of betaCTFs. Expression of a mutant form of APP that cannot undergo beta-cleavage had no effect on endosomes. Pharmacological inhibition of APP gamma-secretase, which markedly reduced Abeta production but raised betaCTF levels, also induced AD-like endosome dysfunction in 2N fibroblasts and worsened this pathology in DS fibroblasts. These findings strongly implicate APP and the betaCTF of APP, and exclude Abeta and the alphaCTF, as the cause of endocytic pathway dysfunction in DS and AD, underscoring the potential multifaceted value of BACE-1 inhibition in AD therapeutics.

Posttranscriptional regulation of neurofilament proteins and tau in health and disease.

Neurofilament and tau proteins are neuron-specific cytoskeletal proteins that are enriched in axons, regulated by many of the same protein kinases, interact physically, and are the principal constituents of neurofibrillary lesions in major adult-onset dementias. Both proteins share functions related to the modulation of stability and functions of the microtubule network in axons, axonal transport and scaffolding of organelles, long-term synaptic potentiation, and learning and memory. Expression of these proteins is regulated not only at the transcriptional level but also through posttranscriptional control of pre-mRNA splicing, mRNA stability, transport, localization, local translation and degradation. Current evidence suggests that posttranscriptional determinants of their levels are usually regulated by RNA-binding proteins and microRNAs primarily through 3'-untranslated regions of neurofilament and tau mRNAs. Dysregulations of neurofilament and tau expression caused by mutations or pathologies of RNA-binding proteins such as TDP43, FUS and microRNAs are increasingly recognized in association with varied neurological disorders. In this review, we summarize the current understanding of posttranscriptional control of neurofilament and tau by examining the posttranscriptional regulation of neurofilament and tau by RNA-binding proteins and microRNAs implicated in health and diseases.