Parkin mediates the ubiquitination of VPS35 and modulates retromer-dependent endosomal sorting.

Mutations in a number of genes cause familial forms of Parkinson's disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) and parkin genes. In this study, we identify a novel functional interaction between parkin and VPS35. We demonstrate that parkin interacts with and robustly ubiquitinates VPS35 in human neural cells. Familial parkin mutations are impaired in their ability to ubiquitinate VPS35. Parkin mediates the attachment of an atypical poly-ubiquitin chain to VPS35 with three lysine residues identified within the C-terminal region of VPS35 that are covalently modified by ubiquitin. Notably, parkin-mediated VPS35 ubiquitination does not promote the proteasomal degradation of VPS35. Furthermore, parkin does not influence the steady-state levels or turnover of VPS35 in neural cells and VPS35 levels are normal in the brains of parkin knockout mice. These data suggest that ubiquitination of VPS35 by parkin may instead serve a non-degradative cellular function potentially by regulating retromer-dependent sorting. Accordingly, we find that components of the retromer-associated WASH complex are markedly decreased in the brain of parkin knockout mice, suggesting that parkin may modulate WASH complex-dependent retromer sorting. Parkin gene silencing in primary cortical neurons selectively disrupts the vesicular sorting of the autophagy receptor ATG9A, a WASH-dependent retromer cargo. Parkin is not required for dopaminergic neurodegeneration induced by the expression of PD-linked D620N VPS35 in mice, consistent with VPS35 being located downstream of parkin function. Our data reveal a novel functional interaction of parkin with VPS35 that may be important for retromer-mediated endosomal sorting and PD.

Membrane activity detection in cultured cells using phase-sensitive plasmonics.

Despite the existence of various neural recording and mapping techniques, there is an open territory for the emergence of novel techniques. The current neural imaging and recording techniques suffer from invasiveness, a time-consuming labeling process, poor spatial/ temporal resolution, and noisy signals. Among others, neuroplasmonics is a label-free and nontoxic recording technique with no issue of photo-bleaching or signal-averaging. We introduced an integrated plasmonic-ellipsometry platform for membrane activity detection with cost-effective and high-quality grating extracted from commercial DVDs. With ellipsometry technique, one can measure both amplitude (intensity) and phase difference of reflected light simultaneously with high signal to noise ratio close to surface plasmon resonances, which leads to the enhancement of sensitivity in plasmonic techniques. We cultured three different types of cells (primary hippocampal neurons, neuroblastoma SH-SY5Y cells, and human embryonic kidney 293 (HEK293) cells) on the grating surface. By introducing KCl solution as a chemical stimulus, we can differentiate the neural activity of distinct cell types and observe the signaling event in a label-free, optical recording platform. This method has potential applications in recording neural signal activity without labeling and stimulation artifacts.

Functional interaction of Parkinson's disease-associated LRRK2 with members of the dynamin GTPase superfamily.

Mutations in LRRK2 cause autosomal dominant Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase domains, and putative protein-protein interaction domains. Familial PD mutations alter the GTPase and kinase activity of LRRK2 in vitro. LRRK2 is suggested to regulate a number of cellular pathways although the underlying mechanisms are poorly understood. To explore such mechanisms, it has proved informative to identify LRRK2-interacting proteins, some of which serve as LRRK2 kinase substrates. Here, we identify common interactions of LRRK2 with members of the dynamin GTPase superfamily. LRRK2 interacts with dynamin 1-3 that mediate membrane scission in clathrin-mediated endocytosis and with dynamin-related proteins that mediate mitochondrial fission (Drp1) and fusion (mitofusins and OPA1). LRRK2 partially co-localizes with endosomal dynamin-1 or with mitofusins and OPA1 at mitochondrial membranes. The subcellular distribution and oligomeric complexes of dynamin GTPases are not altered by modulating LRRK2 in mouse brain, whereas mature OPA1 levels are reduced in G2019S PD brains. LRRK2 enhances mitofusin-1 GTP binding, whereas dynamin-1 and OPA1 serve as modest substrates of LRRK2-mediated phosphorylation in vitro. While dynamin GTPase orthologs are not required for LRRK2-induced toxicity in yeast, LRRK2 functionally interacts with dynamin-1 and mitofusin-1 in cultured neurons. LRRK2 attenuates neurite shortening induced by dynamin-1 by reducing its levels, whereas LRRK2 rescues impaired neurite outgrowth induced by mitofusin-1 potentially by reversing excessive mitochondrial fusion. Our study elucidates novel functional interactions of LRRK2 with dynamin-superfamily GTPases that implicate LRRK2 in the regulation of membrane dynamics important for endocytosis and mitochondrial morphology.

Parkinson's disease-linked mutations in VPS35 induce dopaminergic neurodegeneration.

Mutations in the vacuolar protein sorting 35 homolog (VPS35) gene at the PARK17 locus, encoding a key component of the retromer complex, were recently identified as a new cause of late-onset, autosomal dominant Parkinson's disease (PD). Here we explore the pathogenic consequences of PD-associated mutations in VPS35 using a number of model systems. VPS35 exhibits a broad neuronal distribution throughout the rodent brain, including within the nigrostriatal dopaminergic pathway. In the human brain, VPS35 protein levels and distribution are similar in tissues from control and PD subjects, and VPS35 is not associated with Lewy body pathology. The common D620N missense mutation in VPS35 does not compromise its protein stability or localization to endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, sortilin, SorLA and cation-independent mannose 6-phosphate receptor, in rodent primary neurons or patient-derived human fibroblasts. In yeast we show that PD-linked VPS35 mutations are functional and can normally complement VPS35 null phenotypes suggesting that they do not result in a loss-of-function. In rat primary cortical cultures the overexpression of human VPS35 induces neuronal cell death and increases neuronal vulnerability to PD-relevant cellular stress. In a novel viral-mediated gene transfer rat model, the expression of D620N VPS35 induces the marked degeneration of substantia nigra dopaminergic neurons and axonal pathology, a cardinal pathological hallmark of PD. Collectively, these studies establish that dominant VPS35 mutations lead to neurodegeneration in PD consistent with a gain-of-function mechanism, and support a key role for VPS35 in the development of PD.

A Parkinson's disease gene regulatory network identifies the signaling protein RGS2 as a modulator of LRRK2 activity and neuronal toxicity.

Mutations in LRRK2 are one of the primary genetic causes of Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, and familial PD mutations affect both enzymatic activities. However, the signaling mechanisms regulating LRRK2 and the pathogenic effects of familial mutations remain unknown. Identifying the signaling proteins that regulate LRRK2 function and toxicity remains a critical goal for the development of effective therapeutic strategies. In this study, we apply systems biology tools to human PD brain and blood transcriptomes to reverse-engineer a LRRK2-centered gene regulatory network. This network identifies several putative master regulators of LRRK2 function. In particular, the signaling gene RGS2, which encodes for a GTPase-activating protein (GAP), is a key regulatory hub connecting the familial PD-associated genes DJ-1 and PINK1 with LRRK2 in the network. RGS2 expression levels are reduced in the striata of LRRK2 and sporadic PD patients. We identify RGS2 as a novel interacting partner of LRRK2 in vivo. RGS2 regulates both the GTPase and kinase activities of LRRK2. We show in mammalian neurons that RGS2 regulates LRRK2 function in the control of neuronal process length. RGS2 is also protective against neuronal toxicity of the most prevalent mutation in LRRK2, G2019S. We find that RGS2 regulates LRRK2 function and neuronal toxicity through its effects on kinase activity and independently of GTPase activity, which reveals a novel mode of action for GAP proteins. This work identifies RGS2 as a promising target for interfering with neurodegeneration due to LRRK2 mutations in PD patients.

α-Synuclein-induced dopaminergic neurodegeneration in a rat model of Parkinson's disease occurs independent of ATP13A2 (PARK9).

Mutations in the ATP13A2 (PARK9) gene cause early-onset, autosomal recessive Parkinson's disease (PD) and Kufor-Rakeb syndrome. ATP13A2 mRNA is spliced into three distinct isoforms encoding a P5-type ATPase involved in regulating heavy metal transport across vesicular membranes. Here, we demonstrate that three ATP13A2 mRNA isoforms are expressed in the normal human brain and are modestly increased in the cingulate cortex of PD cases. ATP13A2 can mediate protection toward a number of stressors in mammalian cells and can protect against α-synuclein-induced toxicity in cellular and invertebrate models of PD. Using a primary cortical neuronal model combined with lentiviral-mediated gene transfer, we demonstrate that human ATP13A2 isoforms 1 and 2 display selective neuroprotective effects toward toxicity induced by manganese and hydrogen peroxide exposure through an ATPase-independent mechanism. The familial PD mutations, F182L and G504R, abolish the neuroprotective effects of ATP13A2 consistent with a loss-of-function mechanism. We further demonstrate that the AAV-mediated overexpression of human ATP13A2 is not sufficient to attenuate dopaminergic neurodegeneration, neuropathology, and striatal dopamine and motoric deficits induced by human α-synuclein expression in a rat model of PD. Intriguingly, the delivery of an ATPase-deficient form of ATP13A2 (D513N) to the substantia nigra is sufficient to induce dopaminergic neuronal degeneration and motor deficits in rats, potentially suggesting a dominant-negative mechanism of action. Collectively, our data demonstrate a distinct lack of ATP13A2-mediated protection against α-synuclein-induced neurotoxicity in the rat nigrostriatal dopaminergic pathway, and limited neuroprotective capacity overall, and raise doubts about the potential of ATP13A2 as a therapeutic target for PD.

GTPase activity regulates kinase activity and cellular phenotypes of Parkinson's disease-associated LRRK2.

Mutations in the LRRK2 gene cause autosomal dominant Parkinson's disease. LRRK2 encodes a multi-domain protein containing a Ras-of-complex (Roc) GTPase domain, a C-terminal of Roc domain and a protein kinase domain. LRRK2 can function as a GTPase and protein kinase, although the interplay between these two enzymatic domains is poorly understood. Although guanine nucleotide binding is critically required for the kinase activity of LRRK2, the contribution of GTP hydrolysis is not known. In general, the molecular determinants regulating GTPase activity and how the GTPase domain contributes to the properties of LRRK2 remain to be clarified. Here, we identify a number of synthetic missense mutations in the GTPase domain that functionally modulate GTP binding and GTP hydrolysis and we employ these mutants to comprehensively explore the contribution of GTPase activity to the kinase activity and cellular phenotypes of LRRK2. Our data demonstrate that guanine nucleotide binding and, to a lesser extent, GTP hydrolysis are required for maintaining normal kinase activity and both activities contribute to the GTP-dependent activation of LRRK2 kinase activity. Guanine nucleotide binding but not GTP hydrolysis regulates the dimerization, structure and stability of LRRK2. Furthermore, GTP hydrolysis regulates the LRRK2-dependent inhibition of neurite outgrowth in primary cortical neurons but is unable to robustly modulate the effects of the familial G2019S mutation. Our study elucidates the role of GTPase activity in regulating kinase activity and cellular phenotypes of LRRK2 and has important implications for the validation of the GTPase domain as a molecular target for attenuating LRRK2-mediated neurodegeneration.

GTPase activity and neuronal toxicity of Parkinson's disease-associated LRRK2 is regulated by ArfGAP1.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 encodes a large multi-domain protein with GTPase and kinase activity. Initial data indicates that an intact functional GTPase domain is critically required for LRRK2 kinase activity. PD-associated mutations in LRRK2, including the most common G2019S variant, have variable effects on enzymatic activity but commonly alter neuronal process morphology. The mechanisms underlying the intrinsic and extrinsic regulation of LRRK2 GTPase and kinase activity, and the pathogenic effects of familial mutations, are incompletely understood. Here, we identify a novel functional interaction between LRRK2 and ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1). LRRK2 and ArfGAP1 interact in vitro in mammalian cells and in vivo in brain, and co-localize in the cytoplasm and at Golgi membranes. PD-associated and functional mutations that alter the GTPase activity of LRRK2 modulate the interaction with ArfGAP1. The GTP hydrolysis activity of LRRK2 is markedly enhanced by ArfGAP1 supporting a role for ArfGAP1 as a GTPase-activating protein for LRRK2. Unexpectedly, ArfGAP1 promotes the kinase activity of LRRK2 suggesting a potential role for GTP hydrolysis in kinase activation. Furthermore, LRRK2 robustly and directly phosphorylates ArfGAP1 in vitro. Silencing of ArfGAP1 expression in primary cortical neurons rescues the neurite shortening phenotype induced by G2019S LRRK2 overexpression, whereas the co-expression of ArfGAP1 and LRRK2 synergistically promotes neurite shortening in a manner dependent upon LRRK2 GTPase activity. Neurite shortening induced by ArfGAP1 overexpression is also attenuated by silencing of LRRK2. Our data reveal a novel role for ArfGAP1 in regulating the GTPase activity and neuronal toxicity of LRRK2; reciprocally, LRRK2 phosphorylates ArfGAP1 and is required for ArfGAP1 neuronal toxicity. ArfGAP1 may represent a promising target for interfering with LRRK2-dependent neurodegeneration in familial and sporadic PD.

Conditional expression of Parkinson's disease-related R1441C LRRK2 in midbrain dopaminergic neurons of mice causes nuclear abnormalities without neurodegeneration.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). The clinical and neurochemical features of LRRK2-linked PD are similar to idiopathic disease although neuropathology is somewhat heterogeneous. Dominant mutations in LRRK2 precipitate neurodegeneration through a toxic gain-of-function mechanism which can be modeled in transgenic mice overexpressing human LRRK2 variants. A number of LRRK2 transgenic mouse models have been developed that display abnormalities in dopaminergic neurotransmission and alterations in tau metabolism yet without consistently inducing dopaminergic neurodegeneration. To directly explore the impact of mutant LRRK2 on the nigrostriatal dopaminergic pathway, we developed conditional transgenic mice that selectively express human R1441C LRRK2 in dopaminergic neurons from the endogenous murine ROSA26 promoter. The expression of R1441C LRRK2 does not induce the degeneration of substantia nigra dopaminergic neurons or striatal dopamine deficits in mice up to 2years of age, and fails to precipitate abnormal protein inclusions containing alpha-synuclein, tau, ubiquitin or autophagy markers (LC3 and p62). Furthermore, mice expressing R1441C LRRK2 exhibit normal motor activity and olfactory function with increasing age. Intriguingly, the expression of R1441C LRRK2 induces age-dependent abnormalities of the nuclear envelope in nigral dopaminergic neurons including reduced nuclear circularity and increased invaginations of the nuclear envelope. In addition, R1441C LRRK2 mice display increased neurite complexity of cultured midbrain dopaminergic neurons. Collectively, these novel R1441C LRRK2 conditional transgenic mice reveal altered dopaminergic neuronal morphology with advancing age, and provide a useful tool for exploring the pathogenic mechanisms underlying the R1441C LRRK2 mutation in PD.

PARK9-associated ATP13A2 localizes to intracellular acidic vesicles and regulates cation homeostasis and neuronal integrity.

Mutations in the ATP13A2 gene (PARK9, OMIM 610513) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome and early-onset parkinsonism. ATP13A2 is an uncharacterized protein belonging to the P(5)-type ATPase subfamily that is predicted to regulate the membrane transport of cations. The physiological function of ATP13A2 in the mammalian brain is poorly understood. Here, we demonstrate that ATP13A2 is localized to intracellular acidic vesicular compartments in cultured neurons. In the human brain, ATP13A2 is localized to pyramidal neurons within the cerebral cortex and dopaminergic neurons of the substantia nigra. ATP13A2 protein levels are increased in nigral dopaminergic and cortical pyramidal neurons of Parkinson's disease brains compared with normal control brains. ATP13A2 levels are increased in cortical neurons bearing Lewy bodies (LBs) compared with neurons without LBs. Using short hairpin RNA-mediated silencing or overexpression to explore the function of ATP13A2, we find that modulating the expression of ATP13A2 reduces the neurite outgrowth of cultured midbrain dopaminergic neurons. We also find that silencing of ATP13A2 expression in cortical neurons alters the kinetics of intracellular pH in response to cadmium exposure. Furthermore, modulation of ATP13A2 expression leads to reduced intracellular calcium levels in cortical neurons. Finally, we demonstrate that silencing of ATP13A2 expression induces mitochondrial fragmentation in neurons. Oppositely, overexpression of ATP13A2 delays cadmium-induced mitochondrial fragmentation in neurons consistent with a neuroprotective effect. Collectively, this study reveals a number of intriguing neuronal phenotypes due to the loss- or gain-of-function of ATP13A2 that support a role for this protein in regulating intracellular cation homeostasis and neuronal integrity.

Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1.

Mutations in the parkin gene cause early-onset, autosomal recessive Parkinson's disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non-degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly-ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.

Comprehensive analysis of PM20D1 QTL in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a complex disorder caused by a combination of genetic and non-genetic risk factors. In addition, an increasing evidence suggests that epigenetic mechanisms also accompany AD. Genetic and epigenetic factors are not independent, but multiple loci show genetic-epigenetic interactions, the so-called quantitative trait loci (QTLs). Recently, we identified the first QTL association with AD, namely Peptidase M20 Domain Containing 1 (PM20D1). We observed that PM20D1 DNA methylation, RNA expression, and genetic background are correlated and, in turn, associated with AD. We provided mechanistic insights for these correlations and had shown that by genetically increasing and decreasing PM20D1 levels, AD-related pathologies were decreased and accelerated, respectively. However, since the PM20D1 QTL region encompasses also other genes, namely Nuclear Casein Kinase and Cyclin Dependent Kinase Substrate 1 (NUCKS1); RAB7, member RAS oncogene family-like 1 (RAB7L1); and Solute Carrier Family 41 Member 1 (SLC41A1), we investigated whether these genes might also contribute to the described AD association. RESULTS: Here, we report a comprehensive analysis of these QTL genes using a repertoire of in silico methods as well as in vivo and in vitro experimental approaches. First, we analyzed publicly available databases to pinpoint the major QTL correlations. Then, we validated these correlations using a well-characterized set of samples and locus-specific approaches-i.e., Sanger sequencing for the genotype, cloning/sequencing and pyrosequencing for the DNA methylation, and allele-specific and real-time PCR for the RNA expression. Finally, we defined the functional relevance of the observed alterations in the context of AD in vitro. Using this approach, we show that only PM20D1 DNA methylation and expression are significantly correlated with the AD-risk associated background. We find that the expression of SLC41A1 and PM20D1-but not NUCKS1 and RAB7L1-is increased in mouse models and human samples of AD, respectively. However, SLC41A1 and PM20D1 are differentially regulated by AD-related stressors, with only PM20D1 being upregulated by amyloid-ß and reactive oxygen species, and with only PM20D1 being neuroprotective when overexpressed in cell and primary cultures. CONCLUSIONS: Our findings reinforce PM20D1 as the most likely gene responsible of the previously reported PM20D1 QTL association with AD.

Modulation of receptor cycling by neuron-enriched endosomal protein of 21 kD.

Although correct cycling of neuronal membrane proteins is essential for neurite outgrowth and synaptic plasticity, neuron-specific proteins of the implicated endosomes have not been characterized. Here we show that a previously cloned, developmentally regulated, neuronal protein of unknown function binds to syntaxin 13. We propose to name this protein neuron-enriched endosomal protein of 21 kD (NEEP21), because it is colocalized with transferrin receptors, internalized transferrin (Tf), and Rab4. In PC12 cells, NEEP21 overexpression accelerates Tf internalization and recycling, whereas its down-regulation strongly delays Tf recycling. In primary neurons, NEEP21 is localized to the somatodendritic compartment, and, upon N-methyl-d-aspartate (NMDA) stimulation, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunit GluR2 is internalized into NEEP21-positive endosomes. NEEP21 down-regulation retards recycling of GluR1 to the cell surface after NMDA stimulation of hippocampal neurons. In summary, NEEP21 is a neuronal protein that is localized to the early endosomal pathway and is necessary for correct receptor recycling in neurons.

Subunit-specific surface mobility of differentially labeled AMPA receptor subunits.

Lateral mobility of AMPA-type glutamate receptors as well as their trafficking between plasma membrane and intracellular compartments are major mechanisms for the regulation of synaptic plasticity. Here we applied a recently established labeling technique in combination with lentiviral expression in hippocampal neurons to label individual ACP-tagged AMPA receptor subunits specifically at the surface of neurons. We show that this technique allows the differential labeling of two receptor subunits on the same cell. Moreover, these subunits are integrated into heteromeric receptors together with endogenous subunits, and these labeled receptors are targeted to active synapses. Sequential labeling experiments indicate that there is basal surface insertion of GluR1, GluR2 and GluR3, and that this insertion is strongly increased following potassium depolarization. Moreover, we found that ACP-labeled GluR3 shows the highest surface mobility among GluR1, GluR2, and GluR3. In double-infected neurons the diffusion coefficient of labeled GluR2 at the surface of living neurons is significantly higher in GluR2/GluR3-infected neurons compared to GluR1/GluR2-infected neurons suggesting a higher mobility of GluR2/3 receptors compared to GluR1/2 receptors. These results indicate that surface mobility is regulated by different subunit compositions of AMPA receptors.

Reactivation of recall-induced neurons contributes to remote fear memory attenuation.

Whether fear attenuation is mediated by inhibition of the original memory trace of fear with a new memory trace of safety or by updating of the original fear trace toward safety has been a long-standing question in neuroscience and psychology alike. In particular, which of the two scenarios underlies the attenuation of remote (month-old) fear memories is completely unknown, despite the impetus to better understand this process against the backdrop of enduring traumata. We found-chemogenetically and in an engram-specific manner-that effective remote fear attenuation is accompanied by the reactivation of memory recall-induced neurons in the dentate gyrus and that the continued activity of these neurons is critical for fear reduction. This suggests that the original memory trace of fear actively contributes to remote fear attenuation.

PM20D1 is a quantitative trait locus associated with Alzheimer's disease.

The chances to develop Alzheimer's disease (AD) result from a combination of genetic and non-genetic risk factors 1 , the latter likely being mediated by epigenetic mechanisms 2 . In the past, genome-wide association studies (GWAS) have identified an important number of risk loci associated with AD pathology 3 , but a causal relationship remains difficult to establish. In contrast, locus-specific or epigenome-wide association studies (EWAS) have revealed site-specific epigenetic alterations, which provide mechanistic insights for a particular risk gene but often lack the statistical power of GWAS 4 . Here, combining both approaches, we report a previously unidentified association of the peptidase M20-domain-containing protein 1 (PM20D1) with AD. We find that PM20D1 is a methylation and expression quantitative trait locus coupled to an AD-risk associated haplotype, which displays enhancer-like characteristics and contacts the PM20D1 promoter via a haplotype-dependent, CCCTC-binding-factor-mediated chromatin loop. Furthermore, PM20D1 is increased following AD-related neurotoxic insults at symptomatic stages in the APP/PS1 mouse model of AD and in human patients with AD who are carriers of the non-risk haplotype. In line, genetically increasing or decreasing the expression of PM20D1 reduces and aggravates AD-related pathologies, respectively. These findings suggest that in a particular genetic background, PM20D1 contributes to neuroprotection against AD.

Phosphorylation of 4E-BP1 in the mammalian brain is not altered by LRRK2 expression or pathogenic mutations.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of autosomal dominant familial Parkinson's disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase enzymatic domains. Disease-associated mutations in LRRK2 variably influence enzymatic activity with the common G2019S variant leading to enhanced kinase activity. Mutant LRRK2 induces neuronal toxicity through a kinase-dependent mechanism suggesting that kinase activity is important for mediating the pathogenic effects of LRRK2 mutations. A number of LRRK2 kinase substrates have been identified in vitro but whether they represent authentic physiological substrates in mammalian cells or tissues is not yet clear. The eukaryotic initiation factor 4E (eIF4E)-binding protein, 4E-BP1, was recently identified as a potential substrate of LRRK2 kinase activity in vitro and in Drosophila with phosphorylation occurring at Thr37 and Thr46. Here, we explore a potential interaction of LRRK2 and 4E-BP1 in mammalian cells and brain. We find that LRRK2 can weakly phosphorylate 4E-BP1 in vitro but LRRK2 overexpression is not able to alter endogenous 4E-BP1 phosphorylation in mammalian cells. In mammalian neurons LRRK2 and 4E-BP1 display minimal co-localization, whereas the subcellular distribution, protein complex formation and covalent post-translational modification of endogenous 4E-BP1 are not altered in the brains of LRRK2 knockout or mutant LRRK2 transgenic mice. In the brain, the phosphorylation of 4E-BP1 at Thr37 and Thr46 does not change in LRRK2 knockout or mutant LRRK2 transgenic mice, nor is 4E-BP1 phosphorylation altered in idiopathic or G2019S mutant PD brains. Collectively, our results suggest that 4E-BP1 is neither a major nor robust physiological substrate of LRRK2 in mammalian cells or brain.

Common pathogenic effects of missense mutations in the P-type ATPase ATP13A2 (PARK9) associated with early-onset parkinsonism.

Mutations in the ATP13A2 gene (PARK9) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS), a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s) by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and heterozygous missense mutations in ATP13A2 associated with early-onset forms of parkinsonism.

Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.

Phosphorylation of glutamate receptor interacting protein 1 regulates surface expression of glutamate receptors.

The number of synaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling. Here we show that an about 85-kDa protein kinase phosphorylates GRIP1 on serine 917. This kinase is present in NEEP21 immunocomplexes and is activated in okadaic acid-treated neurons. Pulldown assays and atomic force microscopy indicate that phosphorylated GRIP shows reduced binding to NEEP21. AMPA or N-methyl-D-aspartate stimulation of hippocampal neurons induces delayed phosphorylation of the same serine 917. A wild type carboxy-terminal GRIP1 fragment expressed in hippocampal neurons interferes with GluR2 surface expression. On the contrary, a S917D mutant fragment does not interfere with GluR2 surface expression. Likewise, coexpression of GluR2 together with full-length wild type GRIP1 enhances GluR2 surface expression in fibroblasts, whereas full-length GRIP1-S917D had no effect. This indicates that this serine residue is implicated in AMPAR cycling. Our results identify an important regulatory mechanism in the trafficking of AMPAR subunits between internal compartments and the plasma membrane.