Retromer binds the FANSHY sorting motif in SorLA to regulate amyloid precursor protein sorting and processing

sorLA is a sorting receptor for amyloid precursor protein (APP) genetically linked to Alzheimer's disease (AD). Retromer, an adaptor complex in the endosome-to-Golgi retrieval pathway, has been implicated in APP transport because retromer deficiency leads to aberrant APP sorting and processing and levels of retromer proteins are altered in AD. Here we report that sorLA and retromer functionally interact in neurons to control trafficking and amyloidogenic processing of APP. We have identified a sequence (FANSHY) in the cytoplasmic domain of sorLA that is recognized by the VPS26 subunit of the retromer complex. Accordingly, we characterized the interaction between the retromer complex and sorLA and determined the role of retromer on sorLA-dependent sorting and processing of APP. Mutations in the VPS26 binding site resulted in receptor redistribution to the endosomal network, similar to the situation seen in cells with VPS26 knockdown. The sorLA mutant retained APP-binding activity but, as opposed to the wild-type receptor, misdirected APP into a distinct non-Golgi compartment, resulting in increased amyloid processing. In conclusion, our data provide a molecular link between reduced retromer expression and increased amyloidogenesis as seen in patients with sporadic AD.     

The Use of Pharmacological Retromer Chaperones in Alzheimer’s Disease and other Endosomal-related Disorders

The retromer is an evolutionary conserved multiprotein complex involved in the sorting and retrograde trafficking of cargo from endosomal compartments to the Golgi network and to the cell surface. The neuronal retromer traffics the amyloid precursor protein away from the endosomes, a site where amyloid precursor protein is enzymatically cleaved into pathogenic fragments in Alzheimer’s disease. In recent years, deficiencies in retromer-mediated transport have been implicated in several neurological and non-neurological diseases, including Parkinson’s disease, suggesting that improving the efficacy of the retromer trafficking pathway would result in decreased pathology. We recently identified a new family of small molecules that appear to stabilize the interaction between members of the retromer complex and enhance its function in neurons: the retromer pharmacological chaperones. Here we discuss the role of these molecules in the improvement of retromer trafficking and endosomal dysfunction, as well as their potential as therapeutics for neurological and non-neurological disorders.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0321-y) contains supplementary material, which is available to authorized users.Key Words: Retromer, Alzheimer’s disease, neurodegeneration, pharmacological chaperones     

Model-guided microarray implicates the retromer complex in Alzheimer's disease

Although, in principle, gene expression profiling is well suited to isolate pathogenic molecules associated with Alzheimer's disease (AD), techniques such as microarray present unique analytic challenges when applied to disorders of the brain. Here, we addressed these challenges by first constructing a spatiotemporal model, predicting a priori how a molecule underlying AD should behave anatomically and over time. Then, guided by the model, we generated gene expression profiles of the entorhinal cortex and the dentate gyrus, harvested from the brains of AD cases and controls covering a broad age span. Among many expression differences, the retromer trafficking molecule VPS35 best conformed to the spatiotemporal model of AD. Western blotting confirmed the abnormality, establishing that VPS35 levels are reduced in brain regions selectively vulnerable to AD. VPS35 is the core molecule of the retromer trafficking complex and further analysis revealed that VPS26, another member of the complex, is also downregulated in AD. Cell culture studies, using small interfering RNAs or expression vectors, showed that VPS35 regulates Abeta peptide levels, establishing the relevance of the retromer complex to AD. Reviewing our findings in the context of recent studies suggests how downregulation of the retromer complex in AD can regulate local levels of Abeta peptide.     

Role of the VPS35 D620N mutation in Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder involving the loss of dopaminergic neurons in the brain. Following the discovery of the PD-causing D620N mutation in the VPS35 (Vacuolar sorting protein 35) gene, dysfunction in the subcellular retromer complex has been strongly implicated in pathogenesis of PD. Although the function and dysfunction of the retromer has been a focus of study for some time, the role of this complex in the development of PD is not fully understood. Investigating cellular alterations that occur when the retromer is rendered dysfunctional, such as when the D620N disease-causing mutation is introduced into various model systems, shows that endosomal processing defects are major contributors to the disease phenotype. Altered trafficking of retromer cargo molecules, reduced cellular survival and altered processing of alpha-synuclein have all been observed in the presence of the D620N mutation. In addition, interactions between the retromer and the protein products of other familial Parkinsonism-related genes, has made the retromer a prime target of research in PD. This review gives an overview of the changes in retromer function, identified thus far, that may contribute to the neurodegeneration observed in PD.     

Causative and susceptibility genes for Alzheimer's disease: a review

Alzheimer's disease (AD) is the most common type of dementia in the elderly population. Three genes have been identified as responsible for the rare early-onset familial form of the disease: the amyloid precursor protein (APP) gene, the presenilin 1 (PSEN1) gene and the presenilin 2 (PSEN2) gene. Mutations in these genes, however, account for less than 5% of the total number of AD cases. The remaining 95% of AD patients are mostly sporadic late-onset cases, with a complex aetiology due to interactions between environmental conditions and genetic features of the individual. In this paper, we review the most important genes supposed to be involved in the pathogenesis of AD, known as susceptibility genes, in an attempt to provide a comprehensive picture of what is known about the genetic mechanisms underlying the onset and progression of AD. Hypotheses about the role of each gene in the pathogenic pathway are discussed, taking into account the functions and molecular features, if known, of the coded protein. A major susceptibility gene, the apolipoprotein E (APOE) gene, found to be associated with sporadic late-onset AD cases and the only one, whose role in AD has been confirmed in numerous studies, will be included in a specific chapter. As the results reported by association studies are conflicting, we conclude that a better understanding of the complex aetiology that underlies AD may be achieved likely through a multidisciplinary approach that combines clinical and neurophysiological characterization of AD subtypes and in vivo functional brain imaging studies with molecular investigations of genetic components.     

Molecular genetics of Alzheimer’s disease

Alzheimer disease (AD) is the most common cause of dementia. In the past decade, many advances in the understanding of the etiology of AD have been reported. Familial early onset AD is a heterogeneous disorder that can be caused by mutations in at least three different genes. Current studies are focused on identifying genetic risk factors for late onset AD. In this article, the authors will review the progress in understanding the pathogenic implications of the genes mutated in familial early onset AD and the mapping studies to identify additional genes involved in late-onset AD.     

Retromer disruption promotes amyloidogenic APP processing

Retromer deficiency has been implicated in sporadic AD and animals deficient in retromer components exhibit pronounced neurodegeneration. Because retromer performs retrograde transport from the endosome to the Golgi apparatus and neuronal Aβ is found in late endosomal compartments, we speculated that retromer malfunction might enhance amyloidogenic APP processing by promoting interactions between APP and secretase enzymes in late endosomes. We have evaluated changes in amyloid precursor protein (APP) processing and trafficking as a result of disrupted retromer activity by knockdown of Vps35, a vacuolar sorting protein that is an essential component of the retromer complex. Knocking down retromer activity produced no change in the quantity or cellular distribution of total cellular APP and had no affect on internalization of cell-surface APP. Retromer deficiency did, however, increase the ratio of secreted Aβ42:Aβ40 in HEK-293 cells over-expressing APP695, due primarily to a decrease in Aβ40 secretion. Recent studies suggest that the retromer-trafficked protein, Wntless, is secreted at the synapse in exosome vesicles and that these same vesicles contain Aβ. We therefore hypothesized that retromer deficiency may be associated with altered exosomal secretion of APP and/or secretase fragments. Holo-APP, Presenilin and APP C-terminal fragments were detected in exosomal vesicles secreted from HEK-293 cells. Levels of total APP C-terminal fragments were significantly increased in exosomes secreted by retromer deficient cells. These data suggest that reduced retromer activity can mimic the effects of familial AD Presenilin mutations on APP processing and promote export of amyloidogenic APP derivatives.     

Clinical and histologic findings in autosomal centronuclear myopathy

Centronuclear myopathy (CNM) is a congenital myopathy characterized by chains of centrally located nuclei in a large number of muscle fibers. Clinically, an early-onset form was reported in several autosomal-recessive (AR) families and many sporadic patients, whereas a late-onset form was found in most autosomal-dominant (AD) families. The boundary between these two forms remains unclear, and the molecular basis of autosomal CNM is still unresolved. To better define the clinical and morphologic characteristics of autosomal CNM, the authors analyzed a series of 29 patients from 12 families. Two subgroups were identified in three AD families: two families had a relatively late onset of disease and a slow progression of diffuse weakness, whereas the third family, who had a similar clinical course, also presented a unique diffuse muscle hypertrophy. Two presumed AR families and seven sporadic patients were analyzed together, and three subgroups were identified: 1) an early-onset form with ophthalmoparesis; 2) an early-onset form without ophthalmoparesis; and 3) a late-onset form without ophthalmoparesis. Overall, 23 muscle biopsies were reviewed; a majority of patients had >20% central nuclei, fiber type 1 predominance, and a radial distribution of sarcoplasmic strands on oxidative stains. A marked endomysial fibrosis was observed in three sporadic patients with a relatively severe clinical course. The classification reported in this study will be useful for the diagnosis and the follow-up evaluation of patients with autosomal CNM and for the research into the molecular defects underlying the condition.     

Apolipoprotein E4 as a target for developing new therapeutics for Alzheimer’s disease

The identification of factors that influence the onset or progression of the sporadic form of Alzheimer’s disease (AD) is a key step toward understanding its mechanism(s) and developing successful rational therapies. The apoE genotype has been identified as a powerful risk factor for AD that may account for as much as 50% of the sporadic form of the disease. As the major risk factor for late-onset AD, apolipoprotein E4 (apoE4) should be considered a good target for AD drug discovery. However, despite knowing for over a decade that apoE4 is detrimental to the disease process, we still remain uncertain about the molecular mechanisms subserving the risk-factor activity of apoE4. This, coupled with the fact that we know relatively little about the function(s) of apoE in brain, has presented a barrier to developing apoE-based therapeutics for AD. Progress has been made in understanding the neurobiology of apoE; a number of potentially overlapping functions have been ascribed, which include lipid transport, neuronal repair, dendritic growth, maintenance of synaptic plasticity, and anti-inflammatory activities. Until the gaps are filled in our understanding of the pathogenic function(s) of apoE4, therapeutic strategies targeting this protein will lag behind the development of other AD therapies. Putative pathological functions, or risk-factor activities, of apoE4 include its role in β-amyloid deposition, neurofibrillary tangle formation, synaptic loss, lipid dysfunction, neuroinflammation, and oxidative stress.     

Pathophysiology of Alzheimer's disease

Tremendous progress has been made in understanding the processes of the Alzheimer's disease (AD) cascade, laying the groundwork for improvements in diagnosis and treatment. Advancement has been made in understanding the genetic basis of AD, with identification of causative genes for early-onset familial AD, and the role of the polymorphism of the APOE gene in the late-onset form of the disease. Understanding cerebral degeneration and accumulation of beta-amyloid has generated hopes for discovery of disease-modifying treatments. Progress is needed in understanding the mechanisms that link beta-amyloid accumulation and neuronal death. The next 5 years will be crucial in this respect.     

White matter low attenuation on computed tomography in Alzheimer''s disease and vascular dementia – diagnostic and pathogenetic aspects

Demented patients with early-onset Alzheimer's disease (AD) (n = 17), late-onset Alzheimer's disease (n = 30) and vascular dementia (VD) (n = 20) were studied with computed tomography of the brain. Semiquantitative evaluation of white matter low attenuation (WMLA) and central and cortical atrophy was performed without knowledge of the clinical diagnosis. In early onset AD there was almost complete absence of WMLA and central atrophy compared with the other groups, which showed moderate to severe changes. This suggests that early-onset AD should be separated from the late-onset form. The increased systolic blood pressure found in the WMLA group supports the opinion that WMLA has a vascular origin. The high percentage of WMLA in VD and late-onset AD indicates that subcortical factors have to be considered in the pathogenesis of these disorders.     

Early diagnosis and the clinical genetics of Alzheimer’s disease

Alzheimer’s disease (AD) has a significant genetic background manifested as autosomal dominant inheritance in some early-onset families and as familial risk in late-onset cases. Three genes responsible for early-onset autosomal dominant AD have been identified, and one gene, apolipoprotein E, has been confirmed as a susceptibility gene for late-onset forms of the disorder. These findings raise the possibility of genetic testing, either for early diagnosis or prediction. For early-onset autosomal dominant AD genetic testing will have a limited but useful role in confirming diagnosis in established cases and in predictive counselling for relatives; a situation analogous to that for Huntington’s disease. For late-onset AD significant problems remain to be overcome before the advances in molecular genetics have a direct clinical application Received: 24 August 1997 Accepted: 23 July 1988     

Alzheimer's disease: from pathogenesis to disease-modifying approaches

The two major neuropathologic hallmarks of AD are extracellular amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles (NFTs). A number of additional pathogenic mechanisms, possibly overlapping with Aβ plaques and NFTs formation, have been described, including inflammation, oxidative damage, iron dysregulation, and alterations in cholesterol metabolism. In this review, all of these mechanisms will be discussed and treatments that are under development to interfere with these pathogenic steps will be presented. A primary goal of work in this area is identification of novel compounds that can block the course of the disease in early phases. For this reason they are currently termed "disease modifying" drugs. These drugs are designed to modify pathological steps leading to AD, thus acting on the evolution and progression of the disease. Some of these molecules are undergoing clinical testing whereas others are in preclinical phases of development. Several approaches have been considered, including mainly Aβ deposition interference by anti- Aβ aggregation agents, vaccination, γ-secretase inhibition or selective Aβ 42-lowering agents (SALAs), tau deposition interference by methyl thioninium chloride (MTC), and methods for reduction of inflammation and oxidative damage.     

An update on cellular and molecular determinants of Parkinson's disease with emphasis on the role of the retromer complex

Parkinson's disease (PD) is a highly prevalent neurodegenerative condition. The disease involves the progressive degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Among late‐onset, familial forms of Parkinson are cases with mutations in the PARK17 locus encoding the vacuolar protein sorting 35 (Vps35), a subunit of the retromer complex. The retromer complex is composed of a heterotrimeric protein core (Vps26‐Vps35‐Vps29). The best‐known role of retromer is the retrieval of cargoes from endosomes to the Golgi complex or the plasma membrane. However, recent literature indicates that retromer performs roles associated with lysosomal and mitochondrial functions and degradative pathways such as autophagy. A common point mutation affecting the retromer subunit Vps35 is D620N, which has been linked to the alterations in the aforementioned cellular processes as well as with neurodegeneration. Here, we review the main aspects of the malfunction of the retromer complex and its implications for PD pathology. Besides, we highlight several controversies still awaiting clarification.     

SORLA/SORL1, a neuronal sorting receptor implicated in Alzheimer's disease

The proteolytic breakdown of the amyloid precursor protein (APP) to neurotoxic amyloid-beta peptides in the brain has been recognized as a major pathological pathway in Alzheimer's disease (AD). Yet, the factors that control the processing of APP and their potential contribution to the common sporadic form of AD remain poorly understood. Here, we review recent findings from studies in patients and in animal models that led to the identification of a unique sorting receptor for APP in neurons, designated SORLA/SORL1, that emerges as a key player in amyloidogenic processing and as major genetic risk factor for AD.     

Revisiting Alzheimer's Disease from a New Perspective: Can "Risk Factors" Play a Key Role?

Alzheimer's disease (AD) has been intensively studied for decades, but why has its common pathological cause remained so enigmatic? Our studies have suggested that plaques and tangles occur spontaneously during aging as a result of a natural decline of energy metabolism and Ca2+ signaling, but not necessarily due to conventional pathogens. This view would lead to an unexpected outcome; that is, natural aging plays a more important role in neurodegeneration than is currently recognized. Does this model over-simplify the disease origin? We know that AD-type neurodegeneration typically occurs at the end stages of life when not only do plaques and tangles appear, but also many other bodily changes as well (bone loss and skin wrinkling, etc). Neurodegeneration differs from the latter changes mainly by "social" consequences, not by "physiological" origin. If neurodegeneration is a natural event, then why do only some people, but not others, develop AD? Obviously, additional factors are required for neurodegeneration to develop into AD. By comparing current models and ruling out other possibilities, we think that several known "risk factors" most likely play a critical role in the late-onset sporadic AD. These risk factors can exert their effects either by providing the conditions for ailing neurons to die (extended longevity and sedentary lifestyle), or by enhancing the individual's "vulnerability" to natural neurodegeneration (low synapse reserve). In this context, late-onset sporadic AD would be similar to many other age-related conditions where perhaps no single pathogen can be held exclusively responsible for most cases; rather, many risk factors are important to allow the initial defect to turn into clinical diseases. Accordingly, these factors should be the primary targets for AD prevention. Yet, some other AD cases, especially the early-onset ones, may be complicated by the concomitant involvement of other diseases in the brain.     

Genetic heterogeneity of Alzheimer's disease: complexity and advances

Most of what we know about the pathological process of Alzheimer's disease (AD) results from research on the amyloid cascade hypothesis. This hypothesis in turn is derived largely from the characterization of rare disease-causing mutations in three genes, which code for the amyloid precursor protein (APP), presenilin 1 (PS-1) and presenilin 2 (PS-2) and account for most cases of early-onset autosomal dominant familial AD. These genetic findings also suggested that better understanding of the genetic components of AD, even in the late-onset sporadic forms of the disease, might help to identify central pathways of the AD process and lead to the rapid development of active molecules. Twin studies have reinforced the rationale of this approach, for they indicate that more than 50% of the late-onset AD risk may be attributable to genetic factors. The 1993 discovery that the apolipoprotein E4 (ApoE4) allele is genetically associated with increased risk in both sporadic and familial late-onset Alzheimer's disease strongly supports the validity of this genetic approach. Further progress based on this major finding has nonetheless been disappointing and raises questions about it. First, despite intensive researches, the exact role of APOE in the pathophysiological process still remains unknown. Second, the APOE gene is the only gene so far recognized as a consistent genetic determinant of sporadic forms of AD, even though numerous studies have looked for such genes; these disappointing results suggest persistent methodological limitations. However, recent methodologies allowing new strategies may allow important breakthrough.     

Intracellular amyloid precursor protein sorting and amyloid-β secretion are regulated by Src-mediated phosphorylation of Mint2

Mint adaptor proteins bind to the membrane-bound amyloid precursor protein (APP) and affect the production of pathogenic amyloid-β (Aβ) peptides related to Alzheimer's disease (AD). Previous studies have shown that loss of each of the three Mint proteins delays the age-dependent production of amyloid plaques in transgenic mouse models of AD. However, the cellular and molecular mechanisms underlying Mints effect on amyloid production are unclear. Because Aβ generation involves the internalization of membrane-bound APP via endosomes and Mints bind directly to the endocytic motif of APP, we proposed that Mints are involved in APP intracellular trafficking, which in turn, affects Aβ generation. Here, we show that APP endocytosis was attenuated in Mint knock-out neurons, revealing a role for Mints in APP trafficking. We also show that the endocytic APP sorting processes are regulated by Src-mediated phosphorylation of Mint2 and that internalized APP is differentially sorted between autophagic and recycling trafficking pathways. A Mint2 phosphomimetic mutant favored endocytosis of APP along the autophagic sorting pathway leading to increased intracellular Aβ accumulation. Conversely, the Mint2 phospho-resistant mutant increased APP localization to the recycling pathway and back to the cell surface thereby enhancing Aβ42 secretion. These results demonstrate that Src-mediated phosphorylation of Mint2 regulates the APP endocytic sorting pathway, providing a mechanism for regulating Aβ secretion.     

Decreased expression of hippocampal cholinergic neurostimulating peptide precursor protein mRNA in the hippocampus in Alzheimer disease

Hippocampal cholinergic neurostimulating peptide (HCNP) is involved in the phenotype development of the septo-hippocampal system. HCNP precursor protein (HCNP-pp) is known to interact with other molecules including phosphatidylethanolamine and Raf-1 kinase, and is also known as phosphatidylethanolamine-binding protein and raf kinase-inhibitory protein. To assess whether HCNP-pp is involved in the pathogenesis of Alzheimer disease (AD), the expression levels of its mRNA in the hippocampus of autopsy brains from patients with dementia (including AD and ischemic vascular dementia) were compared with those of non-demented control subjects. The in situ hybridization analysis revealed that the expression of HCNP-pp mRNA in patients with clinically late-onset AD was decreased in the hippocampal CA1 field, but not in the CA3 field or the dentate gyrus. The early-onset AD patients showed a wide range of expression levels in the hippocampal sub-regions. Northern blot analysis of HCNP-pp mRNA in brain tissue supported these observations. Since HCNP is known to stimulate the enzymatic activity of choline acetyltransferase in neurons, its low expression in the CAI field of AD patients may explain the downregulation of cholinergic neurons seen in these patients and may thus contribute to the pathogenic processes underlying AD.