Corrinne M. Peterhoff

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 Alzheimers 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.

Endocytic Pathway Abnormalities Precede Amyloid β Deposition in Sporadic Alzheimer’s Disease and Down Syndrome: Differential Effects of APOE Genotype and Presenilin Mutations

Endocytosis is critical to the function and fate of molecules important to Alzheimers disease (AD) etiology, including the beta protein precursor (betaPP), amyloid beta (Abeta) peptide, and apolipoprotein E (ApoE). Early endosomes, a major site of Abeta peptide generation, are markedly enlarged within neurons in the Alzheimer brain, suggesting altered endocytic pathway (EP) activity. Here, we show that neuronal EP activation is a specific and very early response in AD. To evaluate endocytic activation, we used markers of internalization (rab5, rabaptin 5) and recycling (rab4), and found that enlargement of rab5-positive early endosomes in the AD brain was associated with elevated levels of rab4 immunoreactive protein and translocation of rabaptin 5 to endosomes, implying that both endocytic uptake and recycling are activated. These abnormalities were evident in pyramidal neurons of the neocortex at preclinical stages of disease when Alzheimer-like neuropathology, such as Abeta deposition, was restricted to the entorhinal region. In Down syndrome, early endosomes were significantly enlarged in some pyramidal neurons as early as 28 weeks of gestation, decades before classical AD neuropathology develops. Markers of EP activity were only minimally influenced by normal aging and other neurodegenerative diseases studied. Inheritance of the epsilon4 allele of APOE, however, accentuated early endosome enlargement at preclinical stages of AD. By contrast, endosomes were normal in size at advanced stages of familial AD caused by mutations of presenilin 1 or 2, indicating that altered endocytosis is not a consequence of Abeta deposition. These results identify EP activation as the earliest known intraneuronal change to occur in sporadic AD, the most common form of AD. Given the important role of the EP in Abeta peptide generation and ApoE function, early endosomal abnormalities provide a mechanistic link between EP alterations, genetic susceptibility factors, and Abeta generation and suggest differences that may be involved in Abeta generation and beta amyloidogenesis in subtypes of AD.

Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits

Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimers disease brain contributes to Alzheimers disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-β peptide/amyloid and lysosomal system pathology in the Alzheimers disease mouse model TgCRND8 similar to that previously described in Alzheimers disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-β peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimers disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimers disease.

Aβ localization in abnormal endosomes: association with earliest Aβ elevations in AD and Down syndrome

Early endosomes are a major site of amyloid precursor protein (APP) processing and a convergence point for molecules of pathologic relevance to Alzheimers disease (AD). Neuronal endosome enlargement, reflecting altered endocytic function, is a disease-specific response that develops years before the earliest stage of AD and Down syndrome (DS). We examined how endocytic dysfunction is related to Abeta accumulation and distribution in early stage AD and DS. We found by ELISA and immunocytochemistry that the appearance of enlarged endosomes coincided with an initial rise in soluble Abeta40 and Abeta42 peptides, which preceded amyloid deposition. Double-immunofluorescence using numerous Abeta antibodies showed that intracellular Abeta localized principally to rab5-positive endosomes in neurons from AD brains and was prominent in enlarged endosomes. Abeta was not detectable in neurons from normal controls and was diminished after amyloid deposition in neuropathologically confirmed AD. These studies support growing evidence that endosomal pathology contributes significantly to Abeta overproduction and accumulation in sporadic AD and in AD associated with DS and may signify earlier disease-relevant disturbances of the signaling functions of endosomes.

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

An additional copy of the β-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 β-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 (βCTF), all of which elevate the levels of βCTFs. Expression of a mutant form of APP that cannot undergo β-cleavage had no effect on endosomes. Pharmacological inhibition of APP γ-secretase, which markedly reduced Aβ production but raised βCTF levels, also induced AD-like endosome dysfunction in 2N fibroblasts and worsened this pathology in DS fibroblasts. These findings strongly implicate APP and the βCTF of APP, and exclude Aβ and the αCTF, as the cause of endocytic pathway dysfunction in DS and AD, underscoring the potential multifaceted value of BACE-1 inhibition in AD therapeutics.

Axonal Transport Rates In Vivo Are Unaffected by Tau Deletion or Overexpression in Mice

Elevated tau expression has been proposed as a possible basis for impaired axonal transport in Alzheimers disease. To address this hypothesis, we analyzed the movement of pulse radiolabeled proteins in vivo along retinal ganglion cell (RGC) axons of mice that lack tau or overexpress human tau isoforms. Here, we show that the global axonal transport rates of slow and fast transport cargoes in axons are not significantly impaired when tau expression is eliminated or increased. In addition, markers of slow transport (neurofilament light subunit) and fast transport (snap25) do not accumulate in retinas and are distributed normally along optic axons in mice that lack or overexpress tau. Finally, ultrastructural analyses revealed no abnormal accumulations of vesicular organelles or neurofilaments in RGC perikarya or axons in mice overexpressing or lacking tau. These results suggest that tau is not essential for axonal transport and that transport rates in vivo are not significantly affected by substantial fluctuations in tau expression.

Calpain mediates calcium-induced activation of the Erk1,2 MAPK pathway and cytoskeletal phosphorylation in neurons relevance to Alzheimer's disease

Aberrant phosphorylation of the neuronal cytoskeleton is an early pathological event in Alzheimers disease (AD), but the underlying mechanisms are unclear. Here, we demonstrate in the brains of AD patients that neurofilament hyperphosphorylation in neocortical pyramidal neurons is accompanied by activation of both Erk1,2 and calpain. Using immunochemistry, Western blot analysis, and kinase activity measurements, we show in primary hippocampal and cerebellar granule (CG) neurons that calcium influx activates calpain and Erk1,2 and increases neurofilament phosphorylation on carboxy terminal polypeptide sites known to be modulated by Erk1,2 and to be altered in AD. Blocking Erk1,2 activity either with antisense oligonucleotides to Erk1,2 mRNA sequences or by specifically inhibiting its upstream activating kinase MEK1,2 markedly reduced neurofilament phosphorylation. Calpeptin, a cell-permeable calpain inhibitor, blocked both Erk1,2 activation and neurofilament hyperphosphorylation at concentrations that inhibit calpain-mediated cleavage of brain spectrin. By contrast, inhibiting Erk1,2 with U-0126, a specific inhibitor of Mek1,2, had no appreciable effect on ionomycin-induced calpain activation. These findings demonstrate that, under conditions of calcium injury in neurons, calpains are upstream activators of Erk1,2 signaling and are likely to mediate in part the hyperphosphorylation of neurofilaments and tau seen at early stages of AD as well as the neuron survival-related functions of the MAP kinase pathway.

Down Syndrome Fibroblast Model of Alzheimer-Related Endosome Pathology: Accelerated Endocytosis Promotes Late Endocytic Defects

Endocytic dysfunction is an early pathological change in Alzheimers disease (AD) and Downs syndrome (DS). Using primary fibroblasts from DS individuals, we explored the interactions among endocytic compartments that are altered in AD and assessed their functional consequences in AD pathogenesis. We found that, like neurons in both AD and DS brains, DS fibroblasts exhibit increased endocytic uptake, fusion, and recycling, and trafficking of lysosomal hydrolases to rab5-positive early endosomes. Moreover, late endosomes identified using antibodies to rab7 and lysobisphosphatidic acid increased in number and appeared as enlarged, perinuclear vacuoles, resembling those in neurons of both AD and DS brains. In control fibroblasts, similar enlargement of rab5-, rab7-, and lysobisphosphatidic acid-positive endosomes was induced when endocytosis and endosomal fusion were increased by expression of either a rab5 or an active rab5 mutant, suggesting that persistent endocytic activation results in late endocytic dysfunction. Conversely, expression of a rab5 mutant that inhibits endocytic uptake reversed early and late endosomal abnormalities in DS fibroblasts. Our results indicate that DS fibroblasts recapitulate the neuronal endocytic dysfunction of AD and DS, suggesting that increased trafficking from early endosomes can account, in part, for downstream endocytic perturbations that occur in neurons in both AD and DS brains.

Proteomic identification of sorting nexin 6 as a negative regulator of BACE1-mediated APP processing

The β‐site APP cleaving enzyme‐1 (BACE1) mediates the first cleavage of the β‐amyloid precursor protein (APP) to yield the amyloid β‐peptide (Aβ), a key pathogenic agent in Alzheimers disease (AD). Using a proteomic approach based on in‐cell chemical cross‐linking and tandem affinity purification (TAP), we herein identify sorting nexin 6 (SNX6) as a BACE1‐associated protein. SNX6, a PX domain protein, is a putative component of retromer, a multiprotein cargo complex that mediates the retrograde trafficking of the cation‐independent mannose‐6‐phosphate receptor (CI‐MPR) and sortilin. RNA interference suppression of SNX6 increased BACE1‐dependent secretion of soluble APP (sAPPβ) and cell‐associated fragments (C99), resulting in increased Aβ secretion. Furthermore, SNX6 reduction led to elevated steady‐state BACE1 levels as well as increased retrograde transport of BACE1 in the endocytic pathway, suggesting that SNX6 modulates the retrograde trafficking and basal levels of BACE1, thereby regulating BACE1‐mediated APP processing and Aβ biogenesis. Our study identifies a novel cellular pathway by which SNX6 negatively modulates BACE1‐mediated cleavage of APP.—Okada, H., Zhang, W., Peterhoff, C., Hwang, J. C., Nixon, R. A., Ryu, S. H., Kim, T.‐W. Proteomic identification of sorting nexin 6 as a negative regulator of BACE1‐mediated APP processing. FASEB J. 24, 2783–2794 (2010). www.fasebj.org

Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis

The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.