Fabrizio Piazza

Ubiquitin regulates GGA3-mediated degradation of BACE1

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.

Anti–amyloid β autoantibodies in cerebral amyloid angiopathy–related inflammation: Implications for amyloid-modifying therapies

Cerebral amyloid angiopathy–related inflammation (CAA‐ri) is characterized by vasogenic edema and multiple cortical/subcortical microbleeds, sharing several aspects with the recently defined amyloid‐related imaging abnormalities (ARIA) reported in Alzheimers disease (AD) passive immunization therapies. Herein, we investigated the role of anti–amyloid β (Aβ) autoantibodies in the acute and remission phases of CAA‐ri.

Aβ42 production in brain capillary endothelial cells after oxygen and glucose deprivation

Although the diverse triggers of AD are still under debate, the hypothesis of the contribution of cerebrovascular deficiencies has emerged in recent years. Cerebrovascular dysfunction may precede cognitive decline and onset of neurodegeneration. Indeed, the toxic Aβ(42) aggregates constituting senile plaques, one of AD hallmarks, is often detected as amorphous material or fine fibrils in the brain capillary of AD patients. Aβ(42) causing cerebral microangiopathy might originate either from the circulating blood, the vessel wall itself or the brain parenchyma. In the present investigation we show, for the first time, that in rat brain capillary endothelial cells (RBE4), in vitro oxygen glucose deprivation treatment elicits 250% of Aβ(42) peptide production increase through a mechanism that involves the hypoxia inducible factor-1-mediated β-secretase (BACE1) up-regulation. Furthermore, we observed a time dependent increase of amyloid protein precursor (AβPP) gene and protein expression, confirming previous reports which established the existence of AβPP in the cerebrovascular domain. Our experimental evidences point out that ischemic events may directly contribute in brain capillary endothelial cells to the enhancement of the amyloidogenic metabolism, leading to intracellular deposition of Aβ(42). This events may contribute to the impairment of Aβ brain clearance and AD related blood brain barrier dysfunctions.

Validation of Clinicoradiological Criteria for the Diagnosis of Cerebral Amyloid Angiopathy-Related Inflammation.

IMPORTANCE Cerebral amyloid angiopathy-related inflammation (CAA-ri) is an important diagnosis to reach in clinical practice because many patients with the disease respond to immunosuppressive therapy. Reliable noninvasive diagnostic criteria for CAA-ri would allow some patients to avoid the risk of brain biopsy. OBJECTIVE To test the sensitivity and specificity of clinical and neuroimaging-based criteria for CAA-ri. DESIGN, SETTING, AND PARTICIPANTS We modified the previously proposed clinicoradiological criteria and retrospectively analyzed clinical medical records and magnetic resonance imaging fluid-attenuated inversion recovery and gradient-echo scans obtained from individuals with CAA-ri and noninflammatory CAA. At 2 referral centers between October 1, 1995, and May 31, 2013, and between January 1, 2009, and December 31, 2011, participants included 17 individuals with pathologically confirmed CAA-ri and 37 control group members with pathologically confirmed noninflammatory CAA. The control group was further divided into those with past lobar intracerebral hemorrhage (ICH) (n = 21) and those with cerebral microbleeds only and no history of ICH (n = 16). The dates of our analysis were September 1, 2012, to August 31, 2015. MAIN OUTCOMES AND MEASURES The sensitivity and specificity of prespecified criteria for probable CAA-ri (requiring asymmetric white matter hyperintensities extending to the subcortical white matter) and possible CAA-ri (not requiring the white matter hyperintensities to be asymmetric). RESULTS The 17 patients in the CAA-ri group were a mean (SD) of 68 (8) years and 8 (47%) were women. In the CAA-ri group, 14 of 17 (82%) met the criteria for both probable and possible CAA-ri. In the control group having noninflammatory CAA with lobar ICH, 1 of 21 (5%) met the criteria for possible CAA-ri, and none met the criteria for probable CAA-ri. In the control group having noninflammatory CAA with no ICH, 11 of 16 (69%) met the criteria for possible CAA-ri, and 1 of 16 (6%) met the criteria for probable CAA-ri. These findings yielded a sensitivity and specificity of 82% and 97%, respectively, for the probable criteria and a sensitivity and specificity of 82% and 68%, respectively, for the possible criteria. CONCLUSIONS AND RELEVANCE Our data suggest that a reliable diagnosis of CAA-ri can be reached from basic clinical and magnetic resonance imaging information alone, with good sensitivity and excellent specificity.

BACE1 protein endocytosis and trafficking are differentially regulated by ubiquitination at lysine 501 and the Di-leucine motif in the carboxyl terminus.

Background: BACE1 inhibition is a primary drug target for Alzheimer disease. Results: BACE1 endocytosis, trafficking, and degradation are differentially regulated by the di-leucine motif and ubiquitination at Lys-501 in the BACE1 carboxyl terminus. Conclusion: BACE1 ubiquitination regulates BACE1 degradation but not its endocytosis. Significance: Therapies able to increase BACE1 ubiquitination may represent a potential treatment for Alzheimer disease. β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal (495DDISLL500) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules.

Prodromal Alzheimer's disease presenting as cerebral amyloid angiopathy-related inflammation with spontaneous amyloid-related imaging abnormalities and high cerebrospinal fluid anti-Aβ autoantibodies.

Cerebral amyloid angiopathy-related inflammation (CAA-ri), a rare form of vasculitis associated with amyloid-β (Aβ) deposition in vessel walls, has been proposed as a spontaneous human model of the amyloid-related imaging abnormalities (ARIA) occurring after anti-Aβ immunotherapy for the treatment of Alzheimers disease (AD). We describe a case of a patient with biopsy-proven CAA-ri and prodromal AD, confirmed by means of neuropsychological examination after 20 months follow-up, presenting with ARIA and high levels of cerebrospinal fluid anti-Aβ autoantibodies. This case further supports the analogies between the inflammatory response driven by anti-Aβ immunotherapy and that spontaneously occurring in CAA-ri.

Anti-Aβ Autoantibodies in Amyloid Related Imaging Abnormalities (ARIA): Candidate Biomarker for Immunotherapy in Alzheimer's Disease and Cerebral Amyloid Angiopathy.

Amyloid-related imaging abnormalities (ARIA) represent the major severe side effect of amyloid-beta (Aβ) immunotherapy for Alzheimer’s disease (AD). Early biomarkers of ARIA represent an important challenge to ensure safe and beneficial effects of immunotherapies, given that different promising clinical trials in prodromal and subjects at risk for AD are underway. The recent demonstration that cerebrospinal fluid (CSF) anti-Aβ autoantibodies play a key role in the development of the ARIA-like events characterizing cerebral amyloid angiopathy-related inflammation generated great interest in the field of immunotherapy. Herein, we critically review the growing body of evidence supporting the monitoring of CSF anti-Aβ autoantibody as a promising candidate biomarker for ARIA in clinical trials.

Natural Amyloid-Beta Oligomers Acutely Impair the Formation of a Contextual Fear Memory in Mice

Memory loss is one of the hallmark symptoms of Alzheimers disease (AD). It has been proposed that soluble amyloid-beta (Abeta) oligomers acutely impair neuronal function and thereby memory. We here report that natural Abeta oligomers acutely impair contextual fear memory in mice. A natural Abeta oligomer solution containing Abeta monomers, dimers, trimers, and tetramers was derived from the conditioned medium of 7PA2 cells, a cell line that expresses human amyloid precursor protein containing the Val717Phe familial AD mutation. As a control we used 7PA2 conditioned medium from which Abeta oligomers were removed through immunodepletion. Separate groups of mice were injected with Abeta and control solutions through a cannula into the lateral brain ventricle, and subjected to fear conditioning using two tone-shock pairings. One day after fear conditioning, mice were tested for contextual fear memory and tone fear memory in separate retrieval trials. Three experiments were performed. For experiment 1, mice were injected three times: 1 hour before and 3 hours after fear conditioning, and 1 hour before context retrieval. For experiments 2 and 3, mice were injected a single time at 1 hour and 2 hours before fear conditioning respectively. In all three experiments there was no effect on tone fear memory. Injection of Abeta 1 hour before fear conditioning, but not 2 hours before fear conditioning, impaired the formation of a contextual fear memory. In future studies, the acute effect of natural Abeta oligomers on contextual fear memory can be used to identify potential mechanisms and treatments of AD associated memory loss.

Cerebrospinal fluid levels of BAFF and APRIL in untreated multiple sclerosis

OBJECTIVE AND SUBJECTS To examine in vivo levels of BAFF (B-cell activating factor of the tumor necrosis factor family) and APRIL (a proliferation-inducing ligand) in both the cerebrospinal fluid (CSF) and serum of 30 naïve MS patients and 79 subjects affected by acute or chronic inflammatory or non-inflammatory neurological diseases. DESIGN Case-control study. RESULTS No difference among groups was evidenced in serum BAFF or APRIL levels. By contrast, CSF levels of BAFF in MS (mean 144.3 pg/ml+/-141.2), although not significantly different from those observed in NIND (164.2 pg/ml+/-92.0), acute peripheral OIND (243.1 pg/ml+/-139.0) or chronic OIND (240.2 pg/ml+/-122.5), were significantly higher in acute central OIND patients (1274.0 pg/ml+/-803.8; p<0.001 vs. all groups). Similarly, CSF APRIL levels in MS (1541.0 pg/ml+/-1071.0), NIND (2629.0 pg/ml+/-1669.0), acute peripheral OIND (2834.0 pg/ml+/-1118.) or chronic OIND (2764.0 pg/ml+/-659.7) were not significantly different, while they were significantly higher in acute central OIND (6218.0 pg/ml+/-3790.0; p<0.001 vs. MS and NIND; and p<0.05 vs. acute peripheral OIND). CONCLUSIONS Our results strongly suggest that further investigation is warranted to elucidate the role of BAFF and APRIL in MS and that serum levels of BAFF and APRIL do not reflect CSF levels.

Amyloid-Related Imaging Abnormalities (ARIA) in Immunotherapy Trials for Alzheimer’s Disease: Need for Prognostic Biomarkers?

At the 8th International Conference on Clinical Trials in Alzheimers Disease held November 5-7, 2015 in Barcelona, Spain, promising data were presented on two candidate Alzheimers disease immunotherapeutic agents, gantenerumab and aducanumab. Trial results demonstrated that the implementation of cerebrospinal fluid and Aβ-PET biomarkers improves trial enrichment and outcome, which has led to a change in targeting strategy as clinical trials would be conducted with earlier, even presymptomatic, stages of the disease. Promising findings of outcomes, as measured by Aβ-PET and cerebrospinal fluid tau and P-tau, were, nevertheless, associated with antibody dose-dependent increased risk of severe adverse effects, specifically amyloid-related imaging abnormalities (ARIA). Aducanumab was associated with concomitant time-, dose-, and APOE-related incidence of ARIA in more than one-half of the patients within the high-dose arm. The future challenge will thus be to find biomarkers more favorably balanced between effective dosing of antibody to remove Aβ versus dosing to limit deleterious side effects. Interest was shown by Roche and Biogen, which promoted high-dose phase 3 trials. However, this generated some concerns related to a reasonable expected further increase in the incidence of severe side effects. What has been learned is challenging primary industry strategies for following-up and monitoring safety and effectiveness of anti-Aβ antibodies in clinical trials. Here, we debate the issue of what is an acceptable balance of treatment side effects, i.e., therapeutic-induced ARIA, versus the positive prospects. Indeed, implementation of biomarkers for ARIA might increase value and reduce waste in the design of immunotherapy trials of Alzheimers disease.