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.     

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.     

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     

Retromers in Alzheimer's disease

Amyloid-β peptide (Aβ), the key pathogenic agent in Alzheimer's disease (AD), is released after sequential proteolytic cleavage of the transmembrane amyloid precursor protein (APP). β-Site APP-cleaving enzyme 1 (BACE1) cleaves APP in early endosomes, and the cause of increased BACE cleavage of APP in AD is not fully resolved yet. It has been proposed that perturbed intracellular trafficking of APP, which leads to prolonged residence time in early endosomes, influences Aβ production and hence the risk for AD. Retromers are a family of proteins that mediate the retrieval of transmembrane proteins from the endosomes to the trans-Golgi network. Misregulation of retromers or retromer-associated proteins influences endosomal localization of APP/BACE1. Here we review the role of retromers in the amyloidogenic processing of APP and their pathogenic role in AD.     

Recombinant adenovirus is an appropriate vector for endocytotic protein trafficking studies in cultured neurons.

Endocytosis of full-length beta-amyloid precursor protein (APP) from the plasma membrane contributes to beta-amyloid peptide (Abeta) secretion, and, hence, potentially contributes to the molecular pathogenesis of Alzheimer's disease. We recently have demonstrated that central neuronal APP is endocytosed in a common vesicular compartment with recycling synaptic vesicle integral membrane proteins, but is then sorted away from synaptic vesicles for retrograde transport to the neuronal soma. For this report, we explore whether recombinant adenovirus can be used to modulate APP expression in cultured central neurons to study APP processing by the endocytotic pathway in these cells. Using a replication-deficient recombinant adenovirus that expresses a lacZ reporter (Ad5/CMV-lacZ), we demonstrate high efficiency of transfection (30-35%) at low viral titer (10-20 MOI), with no significant neuronal toxicity or cytoarchitectural change. In addition, we demonstrate that infection with the control virus does not result in re-direction of endogenous neuronal APP from usual endocytotic pathways. We have prepared, using the same genomic background as the control virus, an adenoviral vector that expresses the neuronal isoform of human APP (Ad5/CMV-APP). Infection with Ad5/CMV-APP at 10-20 MOI results in significantly increased immunoreactivity for endocytosed APP with preservation of usual endocytotic trafficking. These results demonstrate that recombinant adenovirus at low titer is an appropriate and effective vector for protein trafficking/processing studies in cultured central neurons.     

Dopamine Transporter Localization in Medial Forebrain Bundle Axons Indicates Its Long-Range Transport Primarily by Membrane Diffusion with a Limited Contribution of Vesicular Traffic on Retromer-Positive Compartments

Dopamine transporter (DAT) controls dopamine neurotransmission by clearing synaptically released dopamine. However, trafficking itineraries of DAT, which determine its cell-surface concentration near synapses, are poorly characterized. It is especially unknown how DAT is transported between spatially distant midbrain somatodendritic and striatal axonal compartments. To examine this “long-range” trafficking, the localization and membrane diffusion of HA-epitope tagged DAT in the medial forebrain bundle (MFB) of a knock-in mouse (both sexes) were analyzed using confocal, super-resolution and EM in intact brain and acute brain slices. HA-DAT was abundant in the plasma membrane of MFB axons, similar to the striatum, although the intracellular fraction of HA-DAT in MFB was more substantial. Intracellular HA-DAT colocalized with VPS35, a subunit of the retromer complex mediating recycling from endosomes, in a subset of axons. Late endosomes, lysosomes, and endoplasmic reticulum were abundant in the soma but minimally present in MFB axons, suggesting that biosynthesis and lysosomal degradation of DAT are confined to soma. Together, the data suggest that membrane diffusion is the main mode of long-range DAT transport through MFB, although the contribution of vesicular traffic can be significant in a population of MFB axons. Based on HA-DAT diffusion rates, plasma membrane DAT in MFB axons turns over with a halftime of ∼20 d, which explains the extremely slow turnover of DAT protein in the brain. Unexpectedly, the mean diameter of DAT-labeled MFB axons was observed to be twice larger than reported for striatum. The implications of this finding for dopamine neuron physiology are discussed.SIGNIFICANCE STATEMENT The dopamine transporter (DAT) is a key regulator of dopamine neurotransmission and a target of abused psychostimulants. In the present study, we examined, for the first time, mechanisms of the long-range traffic of DAT in intact brain and acute brain slices from the knock-in mouse expressing epitope-tagged DAT. Using a combination of confocal, super-resolution and EM, we defined DAT localization and its membrane diffusion parameters in medial forebrain bundle axonal tracts connecting midbrain somatodendritic and striatal axonal compartments of dopaminergic neurons. In contrast to the widely accepted model of long-range axonal transport, our studies suggest that DAT traffics between midbrain and striatum, mainly by lateral diffusion in the plasma membrane with only a limited contribution of vesicular transport in recycling endosomes.     

Loss of Christianson Syndrome Na+/H+ Exchanger 6 (NHE6) Causes Abnormal Endosome Maturation and Trafficking Underlying Lysosome Dysfunction in Neurons

Loss-of-function mutations in endosomal Na⁺/H⁺ exchanger 6 (NHE6) cause the X-linked neurologic disorder Christianson syndrome. Patients exhibit symptoms associated with both neurodevelopmental and neurodegenerative abnormalities. While loss of NHE6 has been shown to overacidify the endosome lumen, and is associated with endolysosome neuropathology, NHE6-mediated mechanisms in endosome trafficking and lysosome function have been understudied. Here, we show that NHE6-null mouse neurons demonstrate worsening lysosome function with time in culture, likely as a result of defective endosome trafficking. NHE6-null neurons exhibit overall reduced lysosomal proteolysis despite overacidification of the endosome and lysosome lumen. Akin to Nhx1 mutants in Saccharomyces cerevisiae, we observe decreased endosome-lysosome fusion in NHE6-null neurons. Also, we find premature activation of pH-dependent cathepsin D (CatD) in endosomes. While active CatD is increased in endosomes, CatD activation and CatD protein levels are reduced in the lysosome. Protein levels of another mannose 6-phosphate receptor (M6PR)-dependent enzyme, β-N-acetylglucosaminidase, were also decreased in lysosomes of NHE6-null neurons. M6PRs accumulate in late endosomes, suggesting defective M6PR recycling and retromer function in NHE6-null neurons. Finally, coincident with decreased endosome-lysosome fusion, using total internal reflection fluorescence, we also find a prominent increase in fusion between endosomal multivesicular bodies and the plasma membrane, indicating enhanced exosome secretion from NHE6-null neurons. In summary, in addition to overacidification of endosomes and lysosomes, loss of NHE6 leads to defects in endosome maturation and trafficking, including enhanced exosome release, contributing to lysosome deficiency and potentially leading to neurodegenerative disease.SIGNIFICANCE STATEMENT Loss-of-function mutations in the endosomal Na⁺/H⁺ exchanger 6 (NHE6) cause Christianson syndrome, an X-linked neurologic disorder. Loss of NHE6 has been shown to overacidify endosomes; however, endosome trafficking mechanisms have been understudied, and the mechanisms leading to neurodegeneration are largely unknown. In NHE6-null mouse neurons in vitro, we find worsening lysosome function with days in culture. Notably, pH-dependent lysosome enzymes, such as cathepsin D, have reduced activity in lysosomes yet increased, precocious activity in endosomes in NHE6-null neurons. Further, endosomes show reduced fusion to lysosomes, and increased fusion to the plasma membrane with increased exosome release. This study identifies new mechanisms involving defective endosome maturation and trafficking that impair lysosome function in Christianson syndrome, likely contributing to neurodegeneration.     

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.     

Loss of glial fibrillary acidic protein results in decreased glutamate transport and inhibition of PKA-induced EAAT2 cell surface trafficking

Loss of the astrocyte-specific intermediate filament protein, glial fibrillary acidic protein (GFAP) results in an increased susceptibility to ischemic insult, enhanced hippocampal LTP, and decreased cerebellar long-term depression (LTD). Because glutamate receptor activation plays a key role in cell death and cellular plasticity responses, we wanted to determine if alterations in glial glutamate transport could contribute to the GFAP null phenotype. To address functional changes in glutamate transport, we measured glutamate uptake in cortical, cerebellar, and hippocampal synaptosomal preparations from age-matched adult wild type and GFAP null mice and demonstrated a 25-30% reduction in the V(max) for d-aspartate uptake in the cortex and hippocampus of GFAP null animals. Western blot analysis of cortical synaptosomal fractions from wild type and GFAP null animals demonstrated that loss of GFAP results in decreases in both astrocytic (EAAT1) and neuronal (EAAT3) glutamate transporter subtypes. Immunohistochemical analysis demonstrated a region-specific modification of neuronal glutamate transporter, EAAT3 trafficking in the GFAP null phenotype. Analysis of primary cortical astrocyte cultures prepared from GFAP null and wild type mice demonstrated that loss of GFAP results in an inability to traffic the glial glutamate transporter, EAAT2, to the surface of the cell following protein kinase A (PKA) stimulation by dibutyryl cAMP. Taken together, these results suggest that the intermediate filament protein, GFAP plays a key role in modulating astrocytic and neuronal glutamate transporter trafficking and function.     

Lipoprotein receptors in Alzheimer's disease

Lipoprotein receptors have important roles in pathological processes that lead to Alzheimer's disease (AD). Previously, they were believed to act mainly by modulating the neuronal metabolism of cholesterol and apolipoprotein E, major risk factors for spontaneous AD. However, recent findings point towards an unexpected new function for lipoprotein receptors in regulation of intracellular transport and processing of the amyloid precursor protein (APP) to give amyloid-beta peptide, the principal component of senile plaques. Here, we will discuss how lipoprotein receptors might modulate distinct steps in neuronal trafficking of APP, and how an intricate balance between opposing receptor activities might be a crucial determinant of APP processing, and of onset and progression of neurodegeneration.     

β-amyloid precursor protein fragments and lysosomal dense bodies are found in rat brain neurons after ventricular infusion of leupeptin

Infusion of the serine and thiol protease inhibitor, leupeptin, is known to cause a reduction of fast axoplasmic transport, and accumulation of lysosomal dense bodies in neuronal perikarya. We have found these dense bodies in hippocampal and cerebellar neurons contain ubiquitin conjugated proteins. We now demonstrate that these accumulated neuronal lysosomes are labeled by antisera to the cytoplasmic, transmembrane and extracellular domains of β-amyloid precursor protein (APP) and also that lysosomal APP is fragmented. This in vivo model confirms that neurons can process APP via a lysosomal pathway and that neuronal lysosomes in vivo contain both N-terminal and potentially amyloidogenic C-terminal fragments of APP. We also show that increased APP immunoreactivity after leupeptin treatment is seen first in neurons and later in astrocytes. On recovery from infusion, APP N-terminal immunoreactivity diminishes whilst C-terminal reactivity remains in neurons. These findings are consistent with production in whole brain of potentially amyloidogenic fragments of APP within neuronal lysosomes in perikarya and dendrites implying that neurons may play a role in forming the β-amyloid of plaques.     

Amyloid precursor protein (APP) regulates synaptic structure and function

The amyloid precursor protein (APP) plays a critical role in Alzheimer's disease (AD) pathogenesis. APP is proteolytically cleaved by β- and γ-secretases to generate the amyloid β-protein (Aβ), the core protein component of senile plaques in AD. It is also cleaved by α-secretase to release the large soluble APP (sAPP) luminal domain that has been shown to exhibit trophic properties. Increasing evidence points to the development of synaptic deficits and dendritic spine loss prior to deposition of amyloid in transgenic mouse models that overexpress APP and Aβ peptides. The consequence of loss of APP, however, is unsettled. In this study, we investigated whether APP itself plays a role in regulating synaptic structure and function using an APP knock-out (APP-/-) mouse model. We examined dendritic spines in primary cultures of hippocampal neurons and CA1 neurons of hippocampus from APP-/- mice. In the cultured neurons, there was a significant decrease (~35%) in spine density in neurons derived from APP-/- mice compared to littermate control neurons that were partially restored with sAPPα-conditioned medium. In APP-/- mice in vivo, spine numbers were also significantly reduced but by a smaller magnitude (~15%). Furthermore, apical dendritic length and dendritic arborization were markedly diminished in hippocampal neurons. These abnormalities in neuronal morphology were accompanied by reduction in long-term potentiation. Strikingly, all these changes in vivo were only seen in mice that were 12-15months in age but not in younger animals. We propose that APP, specifically sAPP, is necessary for the maintenance of dendritic integrity in the hippocampus in an age-associated manner. Finally, these age-related changes may contribute to AD pathology independent of Aβ-mediated synaptic toxicity.     

Upregulation of amyloid precursor protein and its mRNA in an experimental model of paediatric head injury.

Amyloid precursor protein (APP), a membrane spanning glycoprotein which plays an important role in neuronal growth and synaptic plasticity, is increased after traumatic brain injury (TBI) and has been used as a sensitive marker of neuronal damage in an adult sheep head impact model. We hypothesised that APP expression would similarly be increased in lambs, suggesting that in the immature injured brain APP is also upregulated as an acute phase response to trauma. Ten anaesthetised and ventilated 4-5 week old Merino lambs sustained a left temporal head impact from a humane stunner. At 2 h after impact, there was widespread and intense neuronal cell body APP immunoreactivity which was more widely distributed than axonal APP. APP messenger RNA (mRNA) expression was also markedly increased with a distribution similar to that of APP antigen. These results demonstrate that APP antigen and mRNA are sensitive early indicators of TBI in paediatric cases.     

Electrophysiological and cerebrovascular effects of the alpha-secretase-derived form of amyloid precursor protein in young and middle-aged rats

Cleavage of the beta-amyloid precursor protein (APP) by alpha-secretase releases a secreted form of APP (sAPP) from cells. sAPP is released from neurons in an activity-dependent manner and is believed to play roles in synaptic plasticity and neuroprotection. We determined whether sAPP modulates electrophysiological and cerebrovascular processes in vivo. The effects of recombinant sAPP, applied by intracerebroventricular infusion, on hippocampal and cortical electroencephalographic (EEG) activity and hippocampal blood flow in young adult and middle-aged Long-Evans rats were measured. sAPP increased the power spectrum density of low frequency EEG bands in the hippocampus and cortex of middle-aged rats without affecting hippocampal blood flow. The neurophysiological effects of sAPP were observed in middle-aged, but not in young rats. The results of this study indicate that hippocampal and cortical electrophysiological processes are sensitive to sAPP, whereas the cerebral vasculature may not be regulated by sAPP. The age-dependent change in the sensitivity of neuronal activity to sAPP suggests the possibility of an important role for this APP product in brain functioning in mid life.     

Dendrite-selective redistribution of the chemokine receptor CXCR4 following agonist stimulation

The chemokine SDF-1 is a secreted protein that plays a critical role in several aspects of neuron development through interaction with its unique receptor CXCR4. A key mechanism that controls neuron responsiveness to extracellular signals during neuronal growth is receptor endocytosis. Since we previously reported that SDF-1 regulates axon development without affecting the other neurites, we asked whether this could correlate with a compartment-selective trafficking of CXCR4. We thus studied CXCR4 behavior upon SDF-1 exposure in rat hippocampus slices and in transfected neuron cultures. A massive agonist-induced redistribution of CXCR4 in endosomes was observed in dendrites whereas no modification was evidenced in axons. Our data suggest that CXCR4 trafficking may play a role in mediating selective effects of SDF-1 on distinct neuronal membrane subdomains.     

A macromolecular complex involving the amyloid precursor protein (APP) and the cytosolic adapter FE65 is a negative regulator of axon branching

Several studies suggest a role for the amyloid precursor protein (APP) in neurite outgrowth and synaptogenesis, but the downstream interactions that mediate the function of APP during neuron development are unknown. By introducing interaction-deficient FE65 into cultured hippocampal neurons using adenovirus, we show that a complex including APP, FE65 and an additional protein is involved in neurite outgrowth at early stages of neuronal development. Both FE65 that is unable to interact with APP (PID2 mutants) or a WW mutant increased axon branching. Although the FE65 mutants did not affect total neurite output, both mutants decreased axon segment length, consistent with an overall slowing of axonal growth cones. FE65 mutants did not alter the localization of either APP or FE65 in axonal growth cones, suggesting that the effects on neurite outgrowth are achieved by alterations in local complex formation within the axonal growth cone.     

Perinatal iron deficiency alters apical dendritic growth in hippocampal CA1 pyramidal neurons

Iron deficiency early in life is associated with cognitive disturbances that persist beyond the period of iron deficiency. Within cognitive processing circuitry, the hippocampus is particularly susceptible to insults during the perinatal period. During the hippocampal growth spurt, which is predominantly postnatal in rodents, iron transport proteins and their messenger RNA stabilizing proteins are upregulated, suggesting an increased demand for iron import during this developmental period. Rat pups deprived of iron during the perinatal period show a 30-40% decrease in hippocampal metabolic activity during postnatal hippocampal development. We hypothesized that this reduced hippocampal neuronal metabolism impedes developmental processes such as neurite outgrowth. The goals of the current study were to investigate the effects of perinatal iron deficiency on apical dendritic segment growth in the postnatal day (P) 15 hippocampus and to determine if structural abnormalities persist into adulthood (P65) following iron treatment. Qualitative and quantitative immunohistochemical analyses of dendritic structure and growth using microtubule-associated protein-2 as an index showed that iron-deficient P15 pups have truncated apical dendritic morphology in CA1 and a persistence of an immature apical dendritic pattern at P65. These results demonstrate that perinatal iron deficiency disrupts developmental processes in the hippocampal subarea CA1 and that these changes persist despite iron repletion. These structural abnormalities may contribute to the learning and memory deficits that occur during and following early iron deficiency.     

Schwann cell‐specific deletion of the endosomal PI 3‐kinase Vps34 leads to delayed radial sorting of axons,arrested myelination,and abnormal ErbB2‐ErbB3 tyrosine kinase signaling

The PI 3‐kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3‐phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot‐Marie‐Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34‐Schwann cell knockout (Vps34^SCKO) mice show severe hypomyelination in peripheral nerves. Vps34^–/– Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34^–/– Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34^SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34^SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34^–/– Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34^SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic through endosomes. In Vps34^SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to neuregulin 1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34^SCKO mice.