Luigi Puglielli

Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid β-peptide

The pathogenic event common to all forms of Alzheimers disease is the abnormal accumulation of the amyloid β-peptide (Aβ). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Aβ. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Aβ production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Aβ through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Aβ generation, indicating their potential for use in the treatment of Alzheimers disease.

Alzheimer disease β-amyloid activity mimics cholesterol oxidase

The abnormal accumulation of amyloid beta-peptide (Abeta) in the form of senile (or amyloid) plaques is one of the main characteristics of Alzheimer disease (AD). Both cholesterol and Cu2+ have been implicated in AD pathogenesis and plaque formation. Abeta binds Cu2+ with very high affinity, forming a redox-active complex that catalyzes H2O2 production from O2 and cholesterol. Here we show that Abeta:Cu2+ complexes oxidize cholesterol selectively at the C-3 hydroxyl group, catalytically producing 4-cholesten-3-one and therefore mimicking the activity of cholesterol oxidase, which is implicated in cardiovascular disease. Abeta toxicity in neuronal cultures correlated with this activity, which was inhibited by Cu2+ chelators including clioquinol. Cell death induced by staurosporine or H2O2 did not elevate 4-cholesten-3-one levels. Brain tissue from AD subjects had 98% more 4-cholesten-3-one than tissue from age-matched control subjects. We observed a similar increase in the brains of Tg2576 transgenic mice compared with nontransgenic littermates; the increase was inhibited by in vivo treatment with clioquinol, which suggests that brain Abeta accumulation elevates 4-cholesten-3-one levels in AD. Cu2+-mediated oxidation of cholesterol may be a pathogenic mechanism common to atherosclerosis and AD.

An aging pathway controls the TrkA to p75NTR receptor switch and amyloid β‐peptide generation

Aging of the brain is characterized by marked changes in the expression levels of the neurotrophin receptors, TrkA and p75NTR. An expression pattern in which TrkA predominates in younger animals switches to one in which p75NTR predominates in older animals. This TrkA‐to‐p75NTR switch is accompanied by activation of the second messenger ceramide, stabilization of β‐site amyloid precursor protein‐cleaving enzyme‐1 (BACE1), and increased production of amyloid β‐peptide (Aβ). Here, we show that the insulin‐like growth factor‐1 receptor (IGF1‐R), the common regulator of lifespan and age‐related events in many different organisms, is responsible for the TrkA‐to‐p75NTR switch in both human neuroblastoma cell lines and primary neurons from mouse brain. The signaling pathway that controls the level of TrkA and p75NTR downstream of the IGF1‐R requires IRS2, PIP3/Akt, and is under the control of PTEN and p44, the short isoform of p53. We also show that hyperactivation of IGF1‐R signaling in p44 transgenic animals, which show an accelerated form of aging, is characterized by early TrkA‐to‐p75NTR switch and increased production of Aβ in the brain.

A reversible form of lysine acetylation in the ER and Golgi lumen controls the molecular stabilization of BACE1

The lipid second messenger ceramide regulates the rate of beta cleavage of the Alzheimers disease APP (amyloid precursor protein) by affecting the molecular stability of the beta secretase BACE1 (beta-site APP cleaving enzyme 1). Such an event is stimulated in the brain by the normal process of aging, and is under the control of the general aging programme mediated by the insulin-like growth factor 1 receptor. In the present study we report that BACE1 is acetylated on seven lysine residues of the N-terminal portion of the nascent protein. This process involves lysine acetylation in the lumen of the ER (endoplasmic reticulum) and is followed by deacetylation in the lumen of the Golgi apparatus, once the protein is fully mature. We also show that specific enzymatic activities acetylate (in the ER) and deacetylate (in the Golgi apparatus) the lysine residues. This process requires carrier-mediated translocation of acetyl-CoA into the ER lumen and is stimulated by ceramide. Site-directed mutagenesis indicates that lysine acetylation is necessary for nascent BACE1 to leave the ER and move ahead in the secretory pathway, and for the molecular stabilization of the protein.

PCSK9 is required for the disposal of non‐acetylated intermediates of the nascent membrane protein BACE1

We have recently identified a new form of post‐translational regulation of BACE1 (β‐site amyloid precursor protein (APP)‐cleaving enzyme 1), a membrane protein that acts as the rate‐limiting enzyme in the generation of the Alzheimer disease amyloid β‐peptide (Aβ). Specifically, BACE1 is transiently acetylated on seven lysine residues in the lumen of the endoplasmic reticulum/endoplasmic reticulum–Golgi intermediate compartment (ER/ERGIC). The acetylated intermediates of the nascent protein are able to reach the Golgi apparatus, whereas the non‐acetylated ones are retained and degraded in a post‐ER compartment. Here, we report that the serine protease PCSK9 (proprotein convertase subtilisin kexin type 9) contributes to the disposal of non‐acetylated BACE1. Both overexpression and small interfering RNA‐mediated downregulation of PCSK9 affected the levels of BACE1. The downregulation of PCSK9 affected the levels of the loss‐of‐acetylation mutants (BACE1Ala and BACE1Arg) but not those of the gain‐of‐acetylation mutant (BACE1Gln). In addition, Pcsk9−/− mice showed increased levels of BACE1 and Aβ in the brain. Finally, we found that nascent low‐density lipoprotein receptor, a known substrate of PCSK9, is also acetylated.

Two Endoplasmic Reticulum (ER)/ER Golgi Intermediate Compartment-based Lysine Acetyltransferases Post-translationally Regulate BACE1 Levels

We have recently identified a novel form of post-translational regulation of BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), a membrane protein that acts as the rate-limiting enzyme in the generation of the Alzheimer disease amyloid β-peptide. Specifically, nascent BACE1 is transiently acetylated in seven lysine residues clustered in a highly disordered region of the protein that faces the lumen of the endoplasmic reticulum (ER)/ER Golgi intermediate compartment (ER/ERGIC). The acetylation protects the nascent protein from degradation by PCSK9/NARC-1 in the ERGIC and allows it to reach the Golgi apparatus. Here we report the identification of two ER/ERGIC-based acetyltransferases, ATase1 and ATase2. Both proteins display acetyl-CoA:lysine acetyltransferase activity, can interact with and acetylate BACE1, and display an ER/ERGIC localization with the catalytic site facing the lumen of the organelle. Both ATase1 and ATase2 regulate the steady-state levels of BACE1 and the rate of amyloid β-peptide generation. Finally, their transcripts are up-regulated by ceramide treatment. In conclusion, our studies have identified two new enzymes that may be involved in the pathogenesis of late-onset Alzheimer disease. The biochemical characterization of the above events could lead to the identification of novel pharmacological strategies for the prevention of this form of dementia.

Effects of Simvastatin on Cerebrospinal Fluid Biomarkers and Cognition in Middle-Aged Adults at Risk for Alzheimer's Disease

BACKGROUND Statins reduce amyloid-beta (Abeta) levels in the brain and cerebrospinal fluid (CSF) in animals and may thereby favorably alter the pathobiology of AD. It is unclear if statins modify Abeta metabolism or improve cognition in asymptomatic middle-aged adults at increased risk for AD. METHODS In a 4-month randomized, double-blind, controlled study, we evaluated the effects of simvastatin 40 mg daily vs. placebo on CSF Abeta42 levels and cognition in 57 asymptomatic middle-aged adult children of persons with AD. RESULTS Compared to placebo, individuals randomized to simvastatin for 4 months had similar changes in CSF Abeta42 (p=0.344) and total tau levels (p=0.226), yet greater improvements in some measures of verbal fluency (p=0.024) and working memory (p=0.015). APOE4 genotype, gender, and vascular risk factors were associated with CSF biomarker levels, but did not modify treatment effects. CONCLUSION In asymptomatic middle-aged adults at increased risk for AD, simvastatin use improved selected measures of cognitive function without significantly changing CSF Abeta42 or total tau levels. Further studies are needed to clarify the impact of higher dose and/or longer duration statin therapy on not only Abeta metabolism, but also other preclinical processes related to the development of AD.

Impairment of the Golgi GDP-L-fucose transport and unresponsiveness to fucose replacement therapy in LAD II patients

Leukocyte adhesion deficiency type II is an autosomal recessive syndrome characterized by generalized reduction of l-fucose in glycoconjugates; the specific molecular defect is still undefined. The most important clinical symptoms include severe growth and mental retardation and severe immunodeficiency. Patients from two ethnic groups have been reported, i.e. Arab and Turkish. We have observed that GDP-l-fucose transport into Golgi vesicles was specifically impaired in an Arab patient, with a significant reduction of the Vmax but no significant differences in the Km from control and parents. GDP-l-fucose transport showed simple saturation kinetics in all samples. Transport of UDP-galactose, UDP-N-acetylglucosamine, and CMP-sialic acid was comparable into vesicles from the Arab patient, parents, and control. These kinetic parameters probably account for the failure to obtain any clinical and biochemical response to fucose therapy in Arab patients. This contrasts both with the distinctive kinetic properties of GDP-l-fucose transport and with the success of fucose therapy, which have been recently reported in one patient of Turkish origin. Accordingly, the biochemical properties of GDP-l-fucose transport into the Golgi are consistent with different variants of leukocyte adhesion deficiency type II that are probably the result of different molecular defects.

CSF T-Tau/Aβ42 predicts white matter microstructure in healthy adults at risk for Alzheimer's disease.

Cerebrospinal fluid (CSF) biomarkers T-Tau and Aβ42 are linked with Alzheimer’s disease (AD), yet little is known about the relationship between CSF biomarkers and structural brain alteration in healthy adults. In this study we examined the extent to which AD biomarkers measured in CSF predict brain microstructure indexed by diffusion tensor imaging (DTI) and volume indexed by T1-weighted imaging. Forty-three middle-aged adults with parental family history of AD received baseline lumbar puncture and MRI approximately 3.5 years later. Voxel-wise image analysis methods were used to test whether baseline CSF Aβ42, total tau (T-Tau), phosphorylated tau (P-Tau) and neurofilament light protein predicted brain microstructure as indexed by DTI and gray matter volume indexed by T1-weighted imaging. T-Tau and T-Tau/Aβ42 were widely correlated with indices of brain microstructure (mean, axial, and radial diffusivity), notably in white matter regions adjacent to gray matter structures affected in the earliest stages of AD. None of the CSF biomarkers were related to gray matter volume. Elevated P-Tau and P-Tau/Aβ42 levels were associated with lower recognition performance on the Rey Auditory Verbal Learning Test. Overall, the results suggest that CSF biomarkers are related to brain microstructure in healthy adults with elevated risk of developing AD. Furthermore, the results clearly suggest that early pathological changes in AD can be detected with DTI and occur not only in cortex, but also in white matter.

Altered longevity-assurance activity of p53:p44 in the mouse causes memory loss, neurodegeneration and premature death

The longevity‐assurance activity of the tumor suppressor p53 depends on the levels of Δ40p53 (p44), a short and naturally occurring isoform of the p53 gene. As such, increased dosage of p44 in the mouse leads to accelerated aging and short lifespan. Here we show that mice homozygous for a transgene encoding p44 (p44+/+) display cognitive decline and synaptic impairment early in life. The synaptic deficits are attributed to hyperactivation of insulin‐like growth factor 1 receptor (IGF‐1R) signaling and altered metabolism of the microtubule‐binding protein tau. In fact, they were rescued by either Igf1r or Mapt haploinsufficiency. When expressing a human or a ‘humanized’ form of the amyloid precursor protein (APP), p44+/+ animals developed a selective degeneration of memory‐forming and ‐retrieving areas of the brain, and died prematurely. Mechanistically, the neurodegeneration was caused by both paraptosis‐ and autophagy‐like cell deaths. These results indicate that altered longevity‐assurance activity of p53:p44 causes memory loss and neurodegeneration by affecting IGF‐1R signaling. Importantly, Igf1r haploinsufficiency was also able to correct the synaptic deficits of APP695/swe mice, a model of Alzheimer’s disease.