Critical Roles of Embryonic Born Dorsal Dentate Granule Neurons for Activity-Dependent Increases in BDNF, Adult Hippocampal Neurogenesis, and Antianxiety-like Behaviors.

BACKGROUND: Dentate gyrus (DG), a "gate" that controls information flow into the hippocampus, plays important roles in regulating both cognitive (e.g., spatial learning and memory) and mood behaviors. Deficits in DG neurons contribute to the pathogenesis of not only neurological, but also psychiatric, disorders, such as anxiety disorder. Whereas DG's function in spatial learning and memory has been extensively investigated, its role in regulating anxiety remains elusive. METHODS: Using c-Fos to mark DG neuron activation, we identified a group of embryonic born dorsal DG (dDG) neurons, which were activated by anxiogenic stimuli and specifically express osteocalcin (Ocn)-Cre. We further investigated their functions in regulating anxiety and the underlying mechanisms by using a combination of chemogenetic, electrophysiological, and RNA-sequencing methods. RESULTS: The Ocn-Cre+ dDG neurons were highly active in response to anxiogenic environment but had lower excitability and fewer presynaptic inputs than those of Ocn-Cre- or adult born dDG neurons. Activating Ocn-Cre+ dDG neurons suppressed anxiety-like behaviors and increased adult DG neurogenesis, whereas ablating or chronically inhibiting Ocn-Cre+ dDG neurons exacerbated anxiety-like behaviors, impaired adult DG neurogenesis, and abolished activity (e.g., voluntary wheel running)-induced anxiolytic effect and adult DG neurogenesis. RNA-sequencing screening for factors induced by activation of Ocn-Cre+ dDG neurons identified BDNF, which was required for Ocn-Cre+ dDG neurons mediated antianxiety-like behaviors and adult DG neurogenesis. CONCLUSIONS: These results demonstrate critical functions of Ocn-Cre+ dDG neurons in suppressing anxiety-like behaviors but promoting adult DG neurogenesis, and both functions are likely through activation of BDNF.

Ganglioside GD3 regulates dendritic growth in newborn neurons in adult mouse hippocampus via modulation of mitochondrial dynamics.

Ganglioside GD3, a major ganglioside species in neural stem cells, plays a crucial role in maintenance of the self-renewal capacity of these cells. However, its bioactivity in postnatally differentiated neurons in the neurogenic regions of adult brains has not been elucidated. Here, we describe for the first time that deletion of GD3 not only impairs neurotrophin-induced stem cell proliferation, but also alters the dendritic structure as well as the number of synapses of nascent neurons in the dentate gyrus of adult brain. When examining the behavioral phenotypes, GD3 synthase-knockout (GD3S-KO) mice displayed impairment in hippocampus-dependent memory function. To further gain insight into its cellular function, we examined GD3-binding partners from mouse brain extract using a GD3-specific monoclonal antibody, R24, followed by LC-MS/MS analysis and identified a mitochondrial fission protein, the dynamin-related protein-1 (Drp1), as a novel GD3-binding protein. Biochemical and imaging analyses revealed mitochondrial fragmentation in GD3-depleted dentate gyrus neurons, suggesting that GD3 is essential for the mitochondrial Drp1 turnover that is required for efficient mitochondrial fission. These results suggest that GD3 is required for proper dendritic and spine maturation of newborn neurons in adult brain through the regulation of mitochondrial dynamics.

Neuron-Derived Estrogen Is Critical for Astrocyte Activation and Neuroprotection of the Ischemic Brain.

17ß-Estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but the functions of neuron-derived E2 in the ischemic brain are unclear. Here, we used a forebrain neuron-specific aromatase KO (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain and determine its roles after global cerebral ischemia. We demonstrated that ovariectomized female FBN-ARO-KO mice exhibited significantly attenuated astrocyte activation, astrocytic aromatization, and decreased hippocampal E2 levels compared with FLOX mice. Furthermore, FBN-ARO-KO mice had exacerbated neuronal damage and worse cognitive dysfunction after global cerebral ischemia. Similar results were observed in intact male mice. RNA-seq analysis revealed alterations in pathways and genes associated with astrocyte activation, neuroinflammation, and oxidative stress in FBN-ARO-KO mice. The compromised astrocyte activation in FBN-ARO-KO mice was associated with robust downregulation of the astrocyte-derived neurotrophic factors, BDNF and IGF-1, as well as the astrocytic glutamate transporter, GLT-1. Νeuronal FGF2, which acts in a paracrine manner to suppress astrocyte activation, was increased in FBN-ARO-KO neurons. Interestingly, blocking FGF2 signaling by central injection of FGFR3-neutralizing antibody was able to reverse the diminishment in neuroprotective astrocyte reactivity, and attenuate neuronal damage in FBN-ARO-KO mice. Moreover, in vivo E2 replacement suppressed FGF2 signaling and rescued the compromised reactive astrogliosis and cognitive deficits. Collectively, our data provide novel genetic evidence for a beneficial role of neuron-derived E2 in astrocyte activation, neuroprotection, and cognitive preservation following ischemic injury to the brain.SIGNIFICANCE STATEMENT Following cerebral ischemia, astrocytes become highly reactive and can exert neuroprotection through the release of neurotrophic factors and clearance of neurotoxic glutamate. The current study advances our understanding of this process by demonstrating that neuron-derived 17ß-estradiol (E2) is neuroprotective and critical for induction of reactive astrocytes and their ability to produce astrocyte-derived neurotrophic factors, BDNF and IGF-1, and the glutamate transporter, GLT-1 after ischemic brain damage. These beneficial effects of neuron-derived E2 appear to be due, at least in part, to suppression of neuronal FGF2 signaling, which is a known suppressor of astrocyte activation. These findings suggest that neuron-derived E2 is neuroprotective after ischemic brain injury via a mechanism that involves suppression of neuronal FGF2 signaling, thereby facilitating astrocyte activation.

Enhanced Susceptibility to Chemoconvulsant-Induced Seizures in Ganglioside GM3 Synthase Knockout Mice.

Ganglioside GM3 synthase (α-2,3-sialyltransferase, ST3GAL5, GM3S) is a key enzyme involved in the biosynthesis of gangliosides. ST3GAL5 deficiency causes an absence of GM3 and all downstream biosynthetic derivatives. The affected individuals manifest deafness, severe irritability, intractable seizures, and profound intellectual disability. To investigate whether deficiency of GM3 is involved in seizure susceptibility, we induced seizures with different chemoconvulsants in ST3GAL5 knockout mice. We report here that ST3GAL5 knockout mice are hyperactive and more susceptible to seizures induced by chemoconvulsants, including kainate and pilocarpine, compared with normal controls. In the hippocampal dentate gyrus, loss of GM3 aggravates seizure-induced aberrant neurogenesis. These data indicate that GM3 and gangliosides derived from GM3 may serve as important regulators of epilepsy and may play an important role in aberrant neurogenesis associated with seizures.

Ependymal Vps35 Promotes Ependymal Cell Differentiation and Survival, Suppresses Microglial Activation, and Prevents Neonatal Hydrocephalus.

Hydrocephalus is a pathologic condition associated with various brain diseases, including Alzheimer's disease (AD). Dysfunctional ependymal cells (EpCs) are believed to contribute to the development of hydrocephalus. It is thus of interest to investigate EpCs' development and function. Here, we report that vacuolar protein sorting-associated protein 35 (VPS35) is critical for EpC differentiation, ciliogenesis, and survival, and thus preventing neonatal hydrocephalus. VPS35 is abundantly expressed in EpCs. Mice with conditional knock-out (cKO) of Vps35 in embryonic (Vps35GFAP-Cre and Vps35Emx1-Cre) or postnatal (Vps35Foxj1-CreER) EpC progenitors exhibit enlarged lateral ventricles (LVs) and hydrocephalus-like pathology. Further studies reveal marked reductions in EpCs and their cilia in both Vps35GFAP-Cre and Vps35Foxj1-CreER mutant mice. The reduced EpCs appear to be due to impairments in EpC differentiation and survival. Additionally, both Vps35GFAP-Cre and Vps35Foxj1-CreER neonatal pups exhibit increased cell proliferation and death largely in a region close to LV-EpCs. Many microglia close to the mutant LV-EpC region become activated. Depletion of the microglia by PLX3397, an antagonist of colony-stimulating factor 1 receptor (CSF1R), restores LV-EpCs and diminishes the pathology of neonatal hydrocephalus in Vps35Foxj1-CreER mice. Taken together, these observations suggest unrecognized functions of Vps35 in EpC differentiation, ciliogenesis, and survival in neonatal LV, and reveal pathologic roles of locally activated microglia in EpC homeostasis and hydrocephalus development.SIGNIFICANCE STATEMENT This study reports critical functions of vacuolar protein sorting-associated protein 35 (VPS35) not only in promoting ependymal cell (EpC) differentiation, ciliogenesis, and survival, but also in preventing local microglial activation. The dysfunctional EpCs and activated microglia are likely to induce hydrocephalus.

Coupling of terminal differentiation deficit with neurodegenerative pathology in Vps35-deficient pyramidal neurons.

Vps35 (vacuolar protein sorting 35) is a key component of retromer that regulates transmembrane protein trafficking. Dysfunctional Vps35 is a risk factor for neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Vps35 is highly expressed in developing pyramidal neurons, and its physiological role in developing neurons remains to be explored. Here, we provide evidence that Vps35 in embryonic neurons is necessary for axonal and dendritic terminal differentiation. Loss of Vps35 in embryonic neurons results in not only terminal differentiation deficits, but also neurodegenerative pathology, such as cortical brain atrophy and reactive glial responses. The atrophy of neocortex appears to be in association with increases in neuronal death, autophagosome proteins (LC3-II and P62), and neurodegeneration associated proteins (TDP43 and ubiquitin-conjugated proteins). Further studies reveal an increase of retromer cargo protein, sortilin1 (Sort1), in lysosomes of Vps35-KO neurons, and lysosomal dysfunction. Suppression of Sort1 diminishes Vps35-KO-induced dendritic defects. Expression of lysosomal Sort1 recapitulates Vps35-KO-induced phenotypes. Together, these results demonstrate embryonic neuronal Vps35's function in terminal axonal and dendritic differentiation, reveal an association of terminal differentiation deficit with neurodegenerative pathology, and uncover an important lysosomal contribution to both events.

Microglial VPS35 deficiency regulates microglial polarization and decreases ischemic stroke-induced damage in the cortex.

BACKGROUND: Vacuolar sorting protein 35 (VPS35), a critical component of retromer, is essential for selective endosome-to-Golgi retrieval of membrane proteins. It is highly expressed in microglial cells, in addition to neurons. We have previously demonstrated microglial VPS35's functions in preventing hippocampal, but not cortical, microglial activation, and in promoting adult hippocampal neurogenesis. However, microglial VPS35's role in the cortex in response to ischemic stroke remains largely unclear. METHODS: We used mice with VPS35 cKO (conditional knockout) in microglial cells and examined and compared their responses to ischemic stroke with control mice. The brain damage, cell death, changes in glial cells and gene expression, and sensorimotor deficits were assessed by a combination of immunohistochemical and immunofluorescence staining, RT-PCR, Western blot, and neurological functional behavior tests. RESULTS: We found that microglial VPS35 loss results in an increase of anti-inflammatory microglia in mouse cortex after ischemic stroke. The ischemic stroke-induced brain injury phenotypes, including brain damage, neuronal death, and sensorimotor deficits, were all attenuated by microglial VPS35-deficiency. Further analysis of protein expression changes revealed a reduction in CX3CR1 (CX3C chemokine receptor 1) in microglial VPS35-deficient cortex after ischemic stroke, implicating CX3CR1 as a potential cargo of VPS35 in this event. CONCLUSION: Together, these results reveal an unrecognized function of microglial VPS35 in enhancing ischemic brain injury-induced inflammatory microglia, but suppressing the injury-induced anti-inflammatory microglia. Consequently, microglial VPS35 cKO mice exhibit attenuation of ischemic brain injury response.

pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo.

ß-site APP-cleaving enzyme 1 (BACE1) initiates amyloid precursor protein (APP) cleavage and ß-amyloid (Aß) production, a critical step in the pathogenesis of Alzheimer's disease (AD). It is thus of considerable interest to investigate how BACE1 activity is regulated. BACE1 has its maximal activity at acidic pH and GFP variant-pHluorin-displays pH dependence. In light of these observations, we generated three tandem fluorescence-tagged BACE1 fusion proteins, named pHluorin-BACE1-mCherry, BACE1-mCherry-pHluorin and BACE1-mCherry-EGFP. Comparing the fluorescence characteristics of these proteins in response to intracellular pH changes induced by chloroquine or bafilomycin A1, we found that pHluorin-BACE1-mCherry is a better pH sensor for BACE1 because its fluorescence intensity responds to pH changes more dramatically and more quickly. Additionally, we found that (pro)renin receptor (PRR), a subunit of the v-ATPase complex, which is critical for maintaining vesicular pH, regulates pHluorin's fluorescence and BACE1 activity in pHluorin-BACE1-mCherry expressing cells. Finally, we found that the expression of Swedish mutant APP (APPswe) suppresses pHluorin fluorescence in pHluorin-BACE1-mCherry expressing cells in culture and in vivo, implicating APPswe not only as a substrate but also as an activator of BACE1. Taken together, these results suggest that the pHluorin-BACE1-mCherry fusion protein may serve as a useful tool for visualizing active/inactive BACE1 in culture and in vivo.

YAP promotes osteogenesis and suppresses adipogenic differentiation by regulating ß-catenin signaling.

YAP (yes-associated protein) is a transcriptional factor that is negatively regulated by Hippo pathway, a conserved pathway for the development and size control of multiple organs. The exact function of YAP in bone homeostasis remains controversial. Here we provide evidence for YAP's function in promoting osteogenesis, suppressing adipogenesis, and thus maintaining bone homeostasis. YAP is selectively expressed in osteoblast (OB)-lineage cells. Conditionally knocking out Yap in the OB lineage in mice reduces cell proliferation and OB differentiation and increases adipocyte formation, resulting in a trabecular bone loss. Mechanistically, YAP interacts with ß-catenin and is necessary for maintenance of nuclear ß-catenin level and Wnt/ß-catenin signaling. Expression of ß-catenin in YAP-deficient BMSCs (bone marrow stromal cells) diminishes the osteogenesis deficit. These results thus identify YAP-ß-catenin as an important pathway for osteogenesis during adult bone remodeling and uncover a mechanism underlying YAP regulation of bone homeostasis.

Neogenin, a regulator of adult hippocampal neurogenesis, prevents depressive-like behavior.

Adult neurogenesis in hippocampal dentate gyrus (DG) is a complex, but precisely controlled process. Dysregulation of this event contributes to multiple neurological disorders, including major depression. Thus, it is of considerable interest to investigate how adult hippocampal neurogenesis is regulated. Here, we present evidence for neogenin, a multifunctional transmembrane receptor, to regulate adult mouse hippocampal neurogenesis. Loss of neogenin in adult neural stem cells (NSCs) or neural progenitor cells (NPCs) impaired NSCs/NPCs proliferation and neurogenesis, whereas increased their astrocytic differentiation. Mechanistic studies revealed a role for neogenin to positively regulate Gli1, a crucial downstream transcriptional factor of sonic hedgehog, and expression of Gli1 into neogenin depleted NSCs/NPCs restores their proliferation. Further morphological and functional studies showed additional abnormities, including reduced dendritic branches and spines, and impaired glutamatergic neuro-transmission, in neogenin-depleted new-born DG neurons; and mice with depletion of neogenin in NSCs/NPCs exhibited depressive-like behavior. These results thus demonstrate unrecognized functions of neogenin in adult hippocampal NSCs/NPCs-promoting NSCs/NPCs proliferation and neurogenesis and preventing astrogliogenesis and depressive-like behavior, and suggest neogenin regulation of Gli1 signaling as a possible underlying mechanism.

Astrocytic Lrp4 (Low-Density Lipoprotein Receptor-Related Protein 4) Contributes to Ischemia-Induced Brain Injury by Regulating ATP Release and Adenosine-A2AR (Adenosine A2A Receptor) Signaling.

BACKGROUND AND PURPOSE: Lrp4 (low-density lipoprotein receptor-related protein 4) is predominantly expressed in astrocytes, where it regulates glutamatergic neurotransmission by suppressing ATP release. Here, we investigated Lrp4's function in ischemia/stroke-induced brain injury response, which includes glutamate-induced neuronal death and reactive astrogliosis. METHODS: The brain-specific Lrp4 conditional knockout mice (Lrp4GFAP-Cre), astrocytic-specific Lrp4 conditional knockout mice (Lrp4GFAP-creER), and their control mice (Lrp4f/f) were subjected to photothrombotic ischemia and the transient middle cerebral artery occlusion. After ischemia/stroke, mice or their brain samples were subjected to behavior tests, brain histology, immunofluorescence staining, Western blot, and quantitative real-time polymerase chain reaction. In addition, primary astrocytes and neurons were cocultured with or without oxygen and glucose deprivation and in the presence or absence of the antagonist for adenosine-A2AR (adenosine A2A receptor) or ATP-P2X7R (P2X purinoceptor 7) signaling. Gliotransmitters, such as glutamate, d-serine, ATP, and adenosine, in the condition medium of cultured astrocytes were also measured. RESULTS: Lrp4, largely expressed in astrocytes, was increased in response to ischemia/stroke. Both Lrp4GFAP-Cre and Lrp4GFAP-creER mice showed less brain injury, including reduced neuronal death, and impaired reactive astrogliosis. Mechanistically, Lrp4 conditional knockout in astrocytes increased ATP release and the production of ATP derivative, adenosine, which were further elevated by oxygen and glucose deprivation. Pharmacological inhibition of ATP-P2X7R or adenosine-A2AR signaling diminished Lrp4GFAP-creER's protective effect. CONCLUSIONS: The astrocytic Lrp4 plays an important role in ischemic brain injury response. Lrp4 deficiency in astrocytes seems to be protective in response to ischemic brain injury, likely because of the increased ATP release and adenosine-A2AR signaling.

Vps35-deficiency impairs SLC4A11 trafficking and promotes corneal dystrophy.

Vps35 (vacuolar protein sorting 35) is a major component of retromer that selectively promotes endosome-to-Golgi retrieval of transmembrane proteins. Dysfunction of retromer is a risk factor for the pathogenesis of Parkinson's disease (PD) and Alzheimer's disease (AD). However, Vps35/retromer's function in the eye or the contribution of Vps35-deficiency to eye degenerative disorders remains to be explored. Here we provide evidence for a critical role of Vps35 in mouse corneal dystrophy. Vps35 is expressed in mouse and human cornea. Mouse cornea from Vps35 heterozygotes (Vps35+/-) show features of dystrophy, such as loss of both endothelial and epithelial cell densities, disorganizations of endothelial, stroma, and epithelial cells, excrescences in the Descemet membrane, and corneal edema. Additionally, corneal epithelial cell proliferation was reduced in Vps35-deficient mice. Intriguingly, cell surface targeting of SLC4A11, a membrane transport protein (OH- /H+ /NH3 /H2O) of corneal endothelium, whose mutations have been identified in patients with corneal dystrophy, was impaired in Vps35-deficient cells and cornea. Taken together, these results suggest that SLC4A11 appears to be a Vps35/retromer cargo, and Vps35-regulation of SLC4A11 trafficking may underlie Vps35/retromer regulation of corneal dystrophy.

Retromer in Osteoblasts Interacts With Protein Phosphatase 1 Regulator Subunit 14C, Terminates Parathyroid Hormone's Signaling, and Promotes Its Catabolic Response.

Parathyroid hormone (PTH) plays critical, but distinct, roles in bone remodeling, including bone formation (anabolic response) and resorption (catabolic response). Although its signaling and function have been extensively investigated, it just began to be understood how distinct functions are induced by PTH activating a common receptor, the PTH type 1 receptor (PTH1R), and how PTH1R signaling is terminated. Here, we provide evidence for vacuolar protein sorting 35 (VPS35), a major component of retromer, in regulating PTH1R trafficking, turning off PTH signaling, and promoting its catabolic function. VPS35 is expressed in osteoblast (OB)-lineage cells. VPS35-deficiency in OBs impaired PTH(1-34)-promoted PTH1R translocation to the trans-Golgi network, enhanced PTH(1-34)-driven signaling, and reduced PTH(1-34)'s catabolic response in culture and in mice. Further mechanical studies revealed that VPS35 interacts with not only PTH1R, but also protein phosphatase 1 regulatory subunit 14C (PPP1R14C), an inhibitory subunit of PP1 phosphatase. PPP1R14C also interacts with PTH1R, which is necessary for the increased endosomal PTH1R signaling and decreased PTH(1-34)'s catabolic response in VPS35-deficient OB-lineage cells. Taken together, these results suggest that VPS35 deregulates PTH1R-signaling likely by its interaction with PTH1R and PPP1R14C. This event is critical for the control of PTH(1-34)-signaling dynamics, which may underlie PTH-induced catabolic response and adequate bone remodeling.

Neogenin Promotes BMP2 Activation of YAP and Smad1 and Enhances Astrocytic Differentiation in Developing Mouse Neocortex.

UNLABELLED: Neogenin, a DCC (deleted in colorectal cancer) family receptor, is highly expressed in neural stem cells (NSCs). However, its function in NSCs remains to be explored. Here we provide in vitro and in vivo evidence for neogenin's function in NSCs to promote neocortical astrogliogenesis, but not self-renewal or neural differentiation. Mechanistically, neogenin in neocortical NSCs was required for BMP2 activation of YAP (yes associated protein). The active/nuclear YAP stabilized phospho-Smad1/5/8 and was necessary for BMP2 induction of astrocytic differentiation. Deletion of yap in mouse neocortical NSCs caused a similar deficit in neocortical astrogliogenesis as that in neogenin mutant mice. Expression of YAP in neogenin mutant NSCs diminished the astrocytic differentiation deficit in response to BMP2. Together, these results reveal an unrecognized function of neogenin in increasing neocortical astrogliogenesis, and identify a pathway of BMP2-neogenin-YAP-Smad1 for astrocytic differentiation in developing mouse neocortex. SIGNIFICANCE STATEMENT: Astrocytes, a major type of glial cells in the brain, play important roles in modulating synaptic transmission and information processing, and maintaining CNS homeostasis. The abnormal astrocytic differentiation during development contributes to dysfunctions of synaptic plasticity and neuropsychological disorders. Here we provide evidence for neogenin's function in regulation of the neocortical astrocyte differentiation during mouse brain development. We also provide evidence for the necessity of neogenin in BMP2/Smad1-induced astrocyte differentiation through YAP. Thus, our findings identify an unrecognized function of neogenin in mouse neocortical astrocyte differentiation, and suggest a signaling pathway, BMP2-neogenin-YAP-Smad1, underlying astrogliogenesis in developing mouse neocortex.

Analysis of Expression Pattern and Genetic Deletion of Netrin5 in the Developing Mouse.

Boundary cap cells (BCC) are a transient, neural-crest-derived population found at the motor exit point (MEP) and dorsal root entry zone (DREZ) of the embryonic spinal cord. These cells contribute to the central/peripheral nervous system (CNS/PNS) boundary, and in their absence neurons and glia from the CNS migrate into the PNS. We found Netrin5 (Ntn5), a previously unstudied member of the netrin gene family, to be robustly expressed in BCC. We generated Ntn5 knockout mice and examined neurodevelopmental and BCC-related phenotypes. No abnormalities in cranial nerve guidance, dorsal root organization, or sensory projections were found. However, Ntn5 mutant embryos did have ectopic motor neurons (MNs) that migrated out of the ventral horn and into the motor roots. Previous studies have implicated semaphorin6A (Sema6A) in BCC signaling to plexinA2 (PlxnA2)/neuropilin2 (Nrp2) in MNs in restricting MN cell bodies to the ventral horn, particularly in the caudal spinal cord. In Ntn5 mutants, ectopic MNs are likely to be a different population, as more ectopias were found rostrally. Furthermore, ectopic MNs in Ntn5 mutants were not immunoreactive for NRP2. The netrin receptor deleted in colorectal cancer (DCC) is a potential receptor for NTN5 in MNs, as similar ectopic neurons were found in Dcc mutant mice, but not in mice deficient for other netrin receptors. Thus, Ntn5 is a novel netrin family member that is expressed in BCC, functioning to prevent MN migration out of the CNS.

VPS35-deficiency results in an impaired AMPA receptor trafficking and decreased dendritic spine maturation.

BACKGROUND: Vacuolar protein sorting 35 (VPS35), a key component of retromer, plays an important role in endosome-to-Golgi retrieval of membrane proteins. Dysfunction of VPS35/retromer is a risk factor for neurodegenerative disorders, including AD (Alzheimer's disease) and PD (Parkinson's disease). However, exactly how VPS35-deficiency contributes to AD or PD pathogenesis remains poorly understood. RESULTS: We found that VPS35-deficiency impaired dendritic spine maturation and decreased glutamatergic transmission. AMPA receptors, GluA1 and GluA2, are significantly reduced in purified synaptosomal and PSD fractions from VPS35-deficient brain. The surface levels of AMPA receptors are also decreased in VPS35-deficient neurons. Additionally, VPS35 interacted with AMPA-type receptors, GluA1 and GluA2. Overexpression of GluA2, but not GluA1, could partially restore the spine maturation deficit in VPS35-deficient neurons. CONCLUSIONS: These results provide evidence for VPS35's function in promoting spine maturation, which is likely through increasing AMPA receptor targeting to the postsynaptic membrane. Perturbation of such a VPS35/retromer function may contribute to the impaired glutamatergic transmission and pathogenesis of neurodegenerative disorders, such as AD and PD.

VPS35 Deficiency or Mutation Causes Dopaminergic Neuronal Loss by Impairing Mitochondrial Fusion and Function.

Vacuolar protein sorting-35 (VPS35) is a retromer component for endosomal trafficking. Mutations of VPS35 have been linked to familial Parkinson's disease (PD). Here, we show that specific deletion of the VPS35 gene in dopamine (DA) neurons resulted in PD-like deficits, including loss of DA neurons and accumulation of α-synuclein. Intriguingly, mitochondria became fragmented and dysfunctional in VPS35-deficient DA neurons, phenotypes that could be restored by expressing VPS35 wild-type, but not PD-linked mutant. Concomitantly, VPS35 deficiency or mutation increased mitochondrial E3 ubiquitin ligase 1 (MUL1) and, thus, led to mitofusin 2 (MFN2) degradation and mitochondrial fragmentation. Suppression of MUL1 expression ameliorated MFN2 reduction and DA neuron loss but not α-synuclein accumulation. These results provide a cellular mechanism for VPS35 dysfunction in mitochondrial impairment and PD pathogenesis.

VPS35 in Dopamine Neurons Is Required for Endosome-to-Golgi Retrieval of Lamp2a, a Receptor of Chaperone-Mediated Autophagy That Is Critical for α-Synuclein Degradation and Prevention of Pathogenesis of Parkinson's Disease.

Vacuolar protein sorting-35 (VPS35) is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with autosomal dominant PD. However, it remains poorly understood if and how VPS35 deficiency or mutation contributes to PD pathogenesis. Here we provide evidence that links VPS35 deficiency to PD-like neuropathology. VPS35 was expressed in mouse dopamine (DA) neurons in substantia nigra pars compacta (SNpc) and STR (striatum)--regions that are PD vulnerable. VPS35-deficient mice exhibited PD-relevant deficits including accumulation of α-synuclein in SNpc-DA neurons, loss of DA transmitter and DA neurons in SNpc and STR, and impairment of locomotor behavior. Further mechanical studies showed that VPS35-deficient DA neurons or DA neurons expressing PD-linked VPS35 mutant (D620N) had impaired endosome-to-Golgi retrieval of lysosome-associated membrane glycoprotein 2a (Lamp2a) and accelerated Lamp2a degradation. Expression of Lamp2a in VPS35-deficient DA neurons reduced α-synuclein, supporting the view for Lamp2a as a receptor of chaperone-mediated autophagy to be critical for α-synuclein degradation. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis. Significance statement: VPS35 is a key component of the retromer complex that is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with PD. However, if and how VPS35 deficiency or mutation contributes to PD pathogenesis remains unclear. We demonstrated that VPS35 deficiency or mutation (D620N) in mice leads to α-synuclein accumulation and aggregation in the substantia nigra, accompanied with DA neurodegeneration. VPS35-deficient DA neurons exhibit impaired endosome-to-Golgi retrieval of Lamp2a, which may contribute to the reduced α-synuclein degradation through chaperone-mediated autophagy. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis, and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis.

Vps35 haploinsufficiency results in degenerative-like deficit in mouse retinal ganglion neurons and impairment of optic nerve injury-induced gliosis.

VPS35 (vacuolar protein sorting 35) is a major component of retromer that selectively promotes endosome-to-Golgi retrieval of transmembrane proteins. Dysfunction of retromer is a risk factor for the pathogenesis of Parkinson's disease (PD) and Alzheimer's disease (AD), both neuro-degeneration disorders. However, VPS35/retromer's function in retina or the contribution of Vps35-deficiency to retinal neuro-degenerative disorders has not been investigated. Here we provide evidence for a role of VPS35 in mouse retinal ganglion cell (RGC) survival and regeneration. VPS35 is selectively expressed in developing mouse RGCs. RGCs from young adult Vps35 heterozygotes (Vps35+/m) show degenerative-like features, such as dystrophic dendrites, reduced axon fibers, and increased TUNEL labeled RGCs. Additionally, gliosis in the optic nerve is transiently elevated in neonatal, but reduced in aged Vps35+/m mice. Optic nerve injury-induced gliosis is also attenuated in Vps35+/m mice. These results suggest that Vps35 is necessary for mouse RGC survival and regeneration, and Vps35-deficiency may contribute to the pathogenesis of retinal ganglion neuro-degeneration, a critical pathology leading to the blindness of many retinal degenerative disorders.

Vps35 loss promotes hyperresorptive osteoclastogenesis and osteoporosis via sustained RANKL signaling.

Receptor activator of NF-κB (RANK) plays a critical role in osteoclastogenesis, an essential process for the initiation of bone remodeling to maintain healthy bone mass and structure. Although the signaling and function of RANK have been investigated extensively, much less is known about the negative regulatory mechanisms of its signaling. We demonstrate in this paper that RANK trafficking, signaling, and function are regulated by VPS35, a major component of the retromer essential for selective endosome to Golgi retrieval of membrane proteins. VPS35 loss of function altered RANK ligand (RANKL)-induced RANK distribution, enhanced RANKL sensitivity, sustained RANKL signaling, and increased hyperresorptive osteoclast (OC) formation. Hemizygous deletion of the Vps35 gene in mice promoted hyperresorptive osteoclastogenesis, decreased bone formation, and caused a subsequent osteoporotic deficit, including decreased trabecular bone volumes and reduced trabecular thickness and density in long bones. These results indicate that VPS35 critically deregulates RANK signaling, thus restraining increased formation of hyperresorptive OCs and preventing osteoporotic deficits.