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Abstract: Mutations in leucine-rich repeat kinase 2 (LRRK2) have been causally linked to neuronal cell death in Parkinson''s disease. LRRK2 expression has also been detected in B lymphocytes and macrophages, suggesting a role in immune responses. In the present study, we demonstrate that LRRK2 is expressed in primary microglial cells isolated from brains of adult mice. Moreover, lipopolysaccharide (LPS)-activated microglial cells from mice overexpressing the Parkinson''s disease-linked LRRK2(R1441G) mutation exhibit increased expression and secretion of proinflammatory cytokines compared with wild-type control microglia. Expression of the LPS receptor Toll-like receptor 4 (TLR4) and downstream signaling proteins did not differ between LRRK2(R1441G) transgenic microglia and wild-type controls. Consistently, conditioned medium from LPS-stimulated LRRK2(R1441G) transgenic microglia induced significant cell death when added to neuronal cultures. These findings indicate that enhanced neuroinflammation may contribute to neurodegeneration in Parkinson''s disease patients carrying LRRK2 mutations. [Copyright &y& Elsevier]

Abstract: Autosomal dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset Parkinson''s disease. However, the regulators/effectors contributing to the (patho−)physiological functions of LRRK2 remain poorly defined. Here we show that human LRRK2 co-purifies/co-immunoprecipitates with elongation factor 1-alpha (EF1A). Co-incubation of recombinant LRRK2 and EF1A significantly reduces the kinase activity of LRRK2, whereas its GTPase activity remains unchanged. In addition to its canonical role in mRNA translation, EF1A maintains stability of the microtubule cytoskeleton. In the present study, EF1A promotes microtubule assembly in an in vitro tubulin polymerization assay which is impaired by co-incubation with LRRK2 at sub-stoichiometric concentrations. These findings suggest that the interaction between LRRK2 and EF1A may reciprocally modulate their physiological function. [Copyright &y& Elsevier]

Cerebrospinal fluid (CSF) proteins may provide important information about the pathomechanisms present in multiple sclerosis (MS). Although diagnostic criteria for early MS are available, there is still a need for biomarkers, predicting disease subtype and progression to improve individually tailored treatment. Using the two-dimensional difference gel electrophoresis (2-D-DIGE) technology for comparative analysis, we compared CSF samples from patients with MS of the relapse-remitting type (RRMS, n = 12) and from patients with clinically isolated syndrome (CIS, n = 12) suggestive of a first demyelinating attack with neurologically normal controls. Protein spots that showed more than two-fold difference between patients and controls were selected for further analysis with MALDI-TOF mass spectrometry. Immunoblot analysis was performed to confirm the validity of individual candidate proteins. In RRMS, we identified 1 up-regulated and 10 down-regulated proteins. In CIS, 2 up-regulated and 11 down-regulated proteins were identified. One of these proteins (Apolipoprotein A1) was confirmed by immunoblot. Though the pathophysiological role of these proteins still remains to be elucidated in detail and further validation is needed, these findings may have a relevant impact on the identification of disease-specific markers. [ABSTRACT FROM AUTHOR]

AIMS--Immunohistochemical techniques were used to investigate c-Fos and c-Jun proto-oncogene expression in the cornea, lens, and retina after ultraviolet irradiation of the rat eye. METHODS--Eyes of anaesthetised rats were exposed to 1.5 J/cm2 of ultraviolet radiation (280-380 nm). Animals were perfused 1, 6, or 24 hours after irradiation and tissue sections were incubated with specific antiserum to c-Fos and c-Jun, respectively. RESULTS--Non-irradiated contralateral eyes displayed no c-Fos and c-Jun immunoreactivity. One and 6 hours after ultraviolet exposure numerous c-Fos and c-Jun immunopositive nuclei were observed mainly in the epithelial cell layers of the cornea and the lens epithelium. Scattered labelled nuclei were detectable in the retinal ganglion cell layer and the inner nuclear layer. Twenty four hours after irradiation c-Fos and c-Jun protein expression returned to near control levels. Histological signs of ultraviolet damage (for example, chromatin condensation, nuclear fragmentation) were first recognisable in the corneal epithelium 6 hours after irradiation and became more apparent at later times. CONCLUSION--Thus, the rapid and sustained activation of c-Fos and c-Jun expression in the eye after single ultraviolet exposure may represent the molecular mechanism underlying ultraviolet induced photodamage and initiation of cell death. Furthermore, topical application of a c-fos antisense oligodeoxynucleotide to the ultraviolet exposed rat eye inhibited the increase in c-Fos expression in the cornea, suggesting therapeutic activity of antisense drugs in corneal malignant and infectious diseases. [ABSTRACT FROM PUBLISHER]

Induction of c- protooncogene expression following exposure of mammalian skin to UV irradiation suggests an involvement in UV-induced alterations of epidermal cell proliferation and viability. In the present study we have investigated whether topically administered c- antisense oligodeoxynucleotides (ODNs) inhibit c- activation in the UV-exposed rat skin and thereby modulate the delayed increase in cellular proliferative activity. The accumulation of c- immunolabeled nuclei in the epidermis was almost completely blocked 18h postirradiation by topical treatment with the c- antisense ODN. The co-expression of c-Jun was not affected and a random sequence control ODN was ineffective. Epicutaneous application of fluorescein-labeled ODNs revealed penetration into the underlying epidermis. The appearance of nuclear immunoreactivity for proliferating cell nuclear antigen (PCNA) 18 h after UV exposure was significantly suppressed in the epidermis treated with c- antisense ODNs. PCNA is involved in both DNA repair synthesis and DNA replication, and the expression of PCNA mRNA is increased after W irradiation. Thus, it may be speculated that UV-induced c-Fos transcription factor may be linked to repair of photadamaged DNA and/or cell cycle progression by -activating PCNA gene expression. [ABSTRACT FROM PUBLISHER]

Converging evidence suggests that the Parkinson’s disease-linked leucine-rich repeat kinase 2 (LRRK2) modulates cellular function by regulating actin dynamics. In the present study we investigate the role of LRRK2 in functional synaptic terminals of adult LRRK2-knockout and LRRK2(R1441G)-transgenic mice as well as in primary fibroblasts of LRRK2(G2019S) mutation carriers. We show that lack of LRRK2 decreases and overexpression of mutant LRRK2 age-dependently increases the effect of the actin depolymerizing agent Latrunculin A (LatA) on the synaptic cytoskeleton. Similarly, endogenous mutant LRRK2 increases sensitivity to LatA in primary fibroblasts. Under basal conditions however, these fibroblasts show an increase in F-actin bundles and a decrease in filopodial length which can be rescued by LatA treatment. Our data suggest that LRRK2 alters actin dynamics and F-actin structure both in brain neurons and skin fibroblasts. We hypothesize that increased F-actin bundling represents a compensatory mechanism to protect F-actin from the depolymerizing effect of mutant LRRK2 under basal conditions. Our data further indicate that LRRK2-dependent changes in the cytoskeleton might have functional consequences on postsynaptic NMDA receptor localization. [ABSTRACT FROM AUTHOR]

Abstract: Aggregation of α-synuclein may contribute to neuropathology in Parkinson''s disease patients and in transgenic animal models. Natively unfolded α-synuclein binds to various proteins and conformational changes due to α-synuclein misfolding may alter physiological interactions. In the present study, we used protein arrays spotted with 5000 recombinant human proteins for a large scale interaction analysis of monomeric versus oligomeric α-synuclein. Monomeric α-synuclein bound to arrayed cAMP regulated phosphoprotein 19 and binding appears to be disrupted by α-synuclein oligomerization. Incubation with recombinant α-synuclein oligomers lead to the identification of several GTPase activating proteins and Cdc42 effector proteins as binding partners. Protein database searches revealed a Cdc42/Rac interactive binding domain in some interactors. To demonstrate in vivo relevance, we analyzed brainstem protein extracts from α-synuclein(A30P) transgenic mice. Pull-down assays using beads conjugated with a Cdc42/Rac interactive binding domain lead to an enrichment of endogenous α-synuclein oligomers. Cdc42 effector proteins were also co-immunoprecipitated with α-synuclein from brainstem lysates and were colocalized with α-synuclein aggregates in brain sections by double immunostaining. By two-dimensional gel electrophoretic analysis of synaptosomal fractions from transgenic mouse brains we detected additional isoforms of septin 6, a downstream target of Cdc42 effector proteins. Small GTPases have recently been identified in a genetic modifier screen to suppress α-synuclein toxicity in yeast. Our data indicate that components of small GTPase signal transduction pathways may be directly targeted by α-synuclein oligomers which potentially leads to signaling deficits and neurodegeneration. [Copyright &y& Elsevier]

Background: Recombinant adeno-associated virus (AAV) is the most widely used vector for gene therapy in clinical trials. To increase transduction efficiency and specificity, novel engineered AAV variants with modified capsid sequences are evaluated in human cell cultures and non-human primates., Methods: We tested two novel AAV capsid variants, AAV2-NNPTPSR and AAV9-NVVRSSS, in human cortical neurons, which were directly converted from human induced pluripotent stem cells and cocultured with rat primary astrocytes., Results: AAV2-NNPTPSR variant efficiently transduced both induced human cortical glutamatergic neurons and induced human cortical GABAergic interneurons. By contrast, AAV9-NVVRSSS variant transduced both induced human cortical neurons and cocultured rat primary astrocytes. High viral titers (1E+5 viral genomes per cell) caused a significant decrease in viability of induced human cortical neurons. Low viral titers (1E+4 viral genomes per cell) led to a significant increase in the neuronal activity marker c-Fos in transduced human neurons following treatment with a potassium channel blocker., Conclusions: We identified two engineered AAV capsid variants that efficiently transduce induced human cortical neurons. The threefold higher percentage of c-Fos positive, transduced human neurons may indicate functional alterations induced by viral transduction and/or transgene expression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bi-transgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in β-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. The utility of this model is enhanced by the ability to form tumor-derived allografts. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics., (© 2021. The Author(s).)

Although the molecular underpinnings of schizophrenia (SZ) are still incompletely understood, deficits in synaptic activity and neuronal connectivity have been identified as core pathomechanisms of SZ and other neuropsychiatric disorders. In this study, we generated induced pluripotent stem cell (iPSC) lines from skin fibroblasts from healthy donors and patients diagnosed with idiopathic SZ. We differentiated the human iPSC into cortical neurons both as adherent monolayers and as three-dimensional spheroids. RNA sequencing revealed little overlap in differentially expressed genes between 2D and 3D neuron cultures from SZ iPSC compared with controls. Notably, mRNA transcripts encoding dipeptidyl peptidase-like protein 6 (DPP6), an accessory subunit of Kv4.2 voltage-gated potassium channels, were massively increased in cortical neurons from SZ iPSC in the 2D and 3D model. Consistently, multielectrode array recordings and calcium imaging showed significantly decreased neuronal activity both in 2D and in 3D cultures from SZ neurons. To show a causal relationship, we treated iPSC-derived neurons in 2D cultures with lentiviral DPP6 shRNA vectors and the Kv4.2 channel blocker AmmTx3, respectively. Both treatments successfully reversed neuronal hypoexcitability and hypoactivity in cortical neurons from SZ iPSC. Our data highlight a contribution of DPP6 and Kv4.2 to the deficit in neurotransmission in an iPSC model for SZ, which may be of therapeutic relevance for a subset of SZ patients.

Both rare, high risk, loss-of-function mutations and common, low risk, genetic variants in the CUL3 gene are strongly associated with neuropsychiatric disorders. Network analyses of neuropsychiatric risk genes have shown high CUL3 expression in the prenatal human brain and an enrichment in neural precursor cells (NPCs) and cortical neurons. The role of CUL3 in human neurodevelopment however, is poorly understood. In the present study, we used CRISPR/Cas9 nickase to knockout CUL3 in human induced pluripotent stem cells (iPSCs). iPSCs were subsequently differentiated into cortical glutamatergic neurons using two different protocols and tested for structural/functional alterations. Immunocytochemical analysis and transcriptomic profiling revealed that pluripotency of heterozygous CUL3 knockout (KO) iPSCs remained unchanged compared to isogenic control iPSCs. Following small molecule-mediated differentiation into cortical glutamatergic neurons however, we detected a significant delay in transition from proliferating radial glia cells/NPCs to postmitotic neurons in CUL3 KO cultures. Notably, direct neural conversion of CUL3 KO iPSCs by lentiviral expression of Neurogenin-2 massively attenuated the neurodevelopmental delay. However, both optogenetic and electrical stimulation of induced neurons revealed decreased excitability in Cullin-3 deficient cultures, while basal synaptic transmission remained unchanged. Analysis of target gene expression pointed to alterations in FGF signaling in CUL3 KO NPCs, which is required for NPC proliferation and self-renewal, while RhoA and Notch signaling appeared unaffected. Our data provide first evidence for a major role of Cullin-3 in neuronal differentiation, and for neurodevelopmental deficits underlying neuropsychiatric disorders associated with CUL3 mutations., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. nonsensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies., Competing Interests: The authors declare no competing interest.

The human KCTD13 gene is located within the 16p11.2 locus and copy number variants of this locus are associated with a high risk for neuropsychiatric diseases including autism spectrum disorder and schizophrenia. Studies in zebrafish point to a role of KCTD13 in proliferation of neural precursor cells which may contribute to macrocephaly in 16p11.2 deletion carriers. KCTD13 is highly expressed in the fetal human brain and in mouse cortical neurons, but its contribution to the development and function of mammalian neurons is not completely understood. In the present study, we deleted the KCTD13 gene in human-induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nickase. Following neural differentiation of KCTD13 deficient and isogenic control iPSC lines, we detected a moderate but significant inhibition of DNA synthesis and proliferation in KCTD13 deficient human neural precursor cells. KCTD13 deficient cortical neurons derived from iPSCs showed decreased neurite formation and reduced spontaneous network activity. RNA-sequencing and pathway analysis pointed to a role for ERBB signaling in these phenotypic changes. Consistently, activating and inhibiting ERBB kinases rescued and aggravated, respectively, impaired neurite formation. In contrast to findings in non-neuronal human HeLa cells, we did not detect an accumulation of the putative KCTD13/Cullin-3 substrate RhoA, and treatment with inhibitors of RhoA signaling did not rescue decreased neurite formation in human KCTD13 knockout neurons. Taken together, our data provide insight into the role of KCTD13 in neurodevelopmental disorders, and point to ERBB signaling as a potential target for neuropsychiatric disorders associated with KCTD13 deficiency.

Mutations in leucine-rich repeat kinase 2 (LRRK2) age-dependently cause Parkinson's disease and are associated with several inflammatory diseases. So far, the potential role of LRRK2 expression in glial cells as mediators of neuroinflammation and the influence of aging have not been investigated in viral vector-based LRRK2 animal models. In this study, we compared the effect of striatal injection of high-capacity adenoviral vectors expressing either a kinase-overactive LRRK2 with the familial G2019S mutation or a kinase-inactive LRRK2 variant in young and old C57BL/6J mice. The intrinsic adenovirus tropism guided preferentially glial transduction, and the vector design led to stable expression for at least 6 months. In histopathological analysis, young mice expressing either LRRK2 variant presented with transient vacuolization of striatal white fiber tracts accompanied by accumulation of microglial cells and astrogliosis, but inflammation resolved without permanent damage. Old mice had a stronger and prolonged inflammatory reaction and experienced permanent damage in form of partial neuron loss after 3 months exclusively in case of LRRK2_G2019S expression. The autophagic receptor p62 accumulated in cells with high levels of either LRRK2 variant, even more so in old mice. We conclude that the aging mouse brain is more susceptible to LRRK2-associated pathology, and in this model, glial LRRK2 expression significantly contributed to neuroinflammation, ultimately causing neurodegeneration., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide and characterized by the loss of dopaminergic neurons in the patients' midbrains. Both the presence of the protein α-synuclein in intracellular protein aggregates in surviving neurons and the genetic linking of the α-synuclein encoding gene point towards a major role of α-synuclein in PD etiology. The exact pathogenic mechanisms of PD development are not entirely described to date, neither is the specific role of α-synuclein in this context. Previous studies indicate that one aspect of α-synuclein-related cellular toxicity might be direct proteasome impairment. The 20/26S proteasomal machinery is an important instrument of intracellular protein degradation. Thus, direct proteasome impairment by α-synuclein might explain or at least contribute to the formation of intracellular protein aggregates. Therefore this study investigates direct proteasomal impairment by α-synuclein both in vitro using recombinant α-synuclein and isolated proteasomes as well as in living cells. Our experiments demonstrate that the impairment of proteasome activity by α-synuclein is highly dependent upon the cellular background and origin. We show that recombinant α-synuclein oligomers and fibrils scarcely affect 20S proteasome function in vitro, neither does transient α-synuclein expression in U2OS ps 2042 (Ubi(G76V)-GFP) cells. However, stable expression of both wild-type and mutant α-synuclein in dopaminergic SH-SY5Y and PC12 cells results in a prominent impairment of the chymotrypsin-like 20S/26S proteasomal protein cleavage. Thus, our results support the idea that α-synuclein in a specific cellular environment, potentially present in dopaminergic cells, cannot be processed by the proteasome and thus contributes to a selective vulnerability of dopaminergic cells to α-synuclein pathology.

Background and Objective: Reduced progranulin levels are a hallmark of frontotemporal dementia (FTD) caused by loss-of-function (LoF) mutations in the progranulin gene (GRN). However, alterations of central nervous progranulin expression also occur in neurodegenerative disorders unrelated to GRN mutations, such as Alzheimer's disease. We hypothesised that central nervous progranulin levels are also reduced in GRN-negative FTD., Methods: Progranulin levels were determined in both cerebrospinal fluid (CSF) and serum in 75 subjects (37 FTD patients and 38 controls). All FTD patients were assessed by whole-exome sequencing for GRN mutations, yielding a target cohort of 34 patients without pathogenic mutations in GRN (GRN-negative cohort) and 3 GRN mutation carriers (2 LoF variants and 1 novel missense variant)., Results: Not only the GRN mutation carriers but also the GRN-negative patients showed decreased CSF levels of progranulin (serum levels in GRN-negative patients were normal). The decreased CSF progranulin levels were unrelated to patients' increased CSF levels of total tau, possibly indicating different destructive neuronal processes within FTD neurodegeneration. The patient with the novel GRN missense variant (c.1117C>T, p.P373S) showed substantially decreased CSF levels of progranulin, comparable to the 2 patients with GRN LoF mutations, suggesting a pathogenic effect of this missense variant., Conclusions: Our results indicate that central nervous progranulin reduction is not restricted to the relatively rare cases of FTD caused by GRN LoF mutations, but also contributes to the more common GRN-negative forms of FTD. Central nervous progranulin reduction might reflect a partially distinct pathogenic mechanism underlying FTD neurodegeneration and is not directly linked to tau alterations., (© 2016 S. Karger AG, Basel.)

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are strongly associated with familial Parkinson's disease (PD). High expression levels in immune cells suggest a role of LRRK2 in regulating the immune system. In this study, we investigated the effect of the LRRK2 (G2019S) mutation in monocytes, using a human stem cell-derived model expressing LRRK2 at endogenous levels. We discovered alterations in the differentiation pattern of LRRK2 mutant, compared to non-mutant isogenic controls, leading to accelerated monocyte production and a reduction in the non-classical CD14+CD16+ monocyte subpopulation in the LRRK2 mutant cells. LPS-treatment of the iPSC-derived monocytes significantly increased the release of pro-inflammatory cytokines, demonstrating a functional response without revealing any significant differences between the genotypes. Assessment of the migrational capacity of the differentiated monocytes revealed moderate deficits in LRRK2 mutant cells, compared to their respective controls. Our findings indicate a pivotal role of LRRK2 in hematopoietic fate decision, endorsing the involvement of the immune system in the development of PD., Competing Interests: Anna Speidel, Sandra Felk and Frank Gillardon are employed by Boehringer Ingelheim Pharma GmbH & Co. KG. This does neither constitute conflicts of interest regarding the present study nor does it alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Extracellular alpha-synuclein (αsyn) oligomers, associated to exosomes or free, play an important role in the pathogenesis of Parkinson's disease (PD). Increasing evidence suggests that these extracellular moieties activate microglia leading to enhanced neuronal damage. Despite extensive efforts on studying neuroinflammation in PD, little is known about the impact of age on microglial activation and phagocytosis, especially of extracellular αsyn oligomers. Here, we show that microglia isolated from adult mice, in contrast to microglia from young mice, display phagocytosis deficits of free and exosome-associated αsyn oligomers combined with enhanced TNFα secretion. In addition, we describe a dysregulation of monocyte subpopulations with age in mice and humans. Accordingly, human monocytes from elderly donors also show reduced phagocytic activity of extracellular αsyn. These findings suggest that these age-related alterations may contribute to an increased susceptibility to pathogens or abnormally folded proteins with age in neurodegenerative diseases.

Altered progranulin levels play a major role in neurodegenerative diseases, like Alzheimer's dementia (AD), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), even in the absence of GRN mutations. Increasing progranulin levels could hereby provide a novel treatment strategy. However, knowledge on progranulin regulation in neurodegenerative diseases remains limited. We here demonstrate that cerebrospinal fluid progranulin levels do not correlate with its serum levels in AD, FTD and ALS, indicating a differential regulation of its central and peripheral levels in neurodegeneration. Blood progranulin levels thus do not reliably predict central nervous progranulin levels and their response to future progranulin-increasing therapeutics.

Mutations within the LRRK2 gene have been identified in Parkinson's disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.

Increasing evidence suggests that Parkinson's disease (PD)-linked Leucine-rich repeat kinase 2 (LRRK2) has a role in peripheral and brain-resident immune cells. Furthermore, dysregulation of the anti-inflammatory, neurotrophic protein progranulin (PGRN) has been demonstrated in several chronic neurodegenerative diseases. Here we show that PGRN levels are significantly reduced in conditioned medium of LRRK2(R1441G) mutant mouse fibroblasts, leukocytes, and microglia, whereas levels of proinflammatory factors, like interleukin-1β and keratinocyte-derived chemokine, were significantly increased. Decreased PGRN levels were also detected in supernatants of cultured human fibroblasts isolated from presymptomatic LRRK2(G2019S) mutation carriers, while mitochondrial function was unaffected. Furthermore, medium levels of matrix metalloprotease (MMP) 2 increased, whereas MMP 9 decreased in LRRK2(R1441G) mutant microglia. Increased proteolytic cleavage of the MMP substrates ICAM-5 and α-synuclein in synaptoneurosomes from LRRK2(R1441G) mutant mouse brain indicates increased net synaptic MMP activity. PGRN levels were decreased in the cerebrospinal fluid of presymptomatic LRRK2 mutant mice, whereas PGRN levels were increased in aged symptomatic mutant mice. Notably, PGRN levels were also increased in the cerebrospinal fluid of PD patients carrying LRRK2 mutations, but not in idiopathic PD patients and in healthy control donors. Our data suggest that proinflammatory activity of peripheral and brain-resident immune cells may particularly contribute to the early stages of Parkinson's disease caused by LRRK2 mutations., (Copyright © 2014 Wiley Periodicals, Inc.)

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause familial as well as sporadic Parkinson's disease (PD) that is characterized by an age-dependent degeneration of dopaminergic neurons. LRRK2 is strongly expressed in neural stem cells (NSCs), but still the exact molecular function of LRRK2 in these cells remains unknown. By performing a systemic analysis of the gene expression profile of LRRK2-deficient NSCs, we found that the expression of several PD-associated genes, such as oxidation and reduction in mitochondria, are deregulated on LRRK2 absence. Our data, indeed, indicate that LRRK2 regulates the level of cellular oxidative stress and thereby influences the survival of NSCs. Furthermore, the lack of LRRK2 leads to an up-regulation of neuronal differentiation-inducing processes, including the Let-7a pathway. On the other hand, the constitutive mutant of LRRK2(R1441G), known to cause PD, leads to down-regulation of the same pathway. In agreement with the function of Let-7a during neuronal differentiation, LRRK2-deficient NSCs differentiate faster than wild-type cells, while LRRK2(R1441G)-expressing NSCs show impaired neuronal differentiation. These results might help better characterize the molecular mechanisms underlying the role of LRRK2 in NSCs and would further improve potential cell-replacement strategies as well as drug discovery approaches.

Recent studies indicate that the Parkinson's disease-linked leucine-rich repeat kinase 2 (LRRK2) modulates cytoskeletal functions by regulating actin and tubulin dynamics, thereby affecting neurite outgrowth. By interactome analysis we demonstrate that the binding of LRRK2 to tubulins is significantly enhanced by pharmacological LRRK2 inhibition in cells. Co-incubation of LRRK2 with microtubules increased the LRRK2 GTPase activity in a cell-free assay. Destabilization of microtubules causes a rapid decrease in cellular LRRK2(S935) phosphorylation indicating a decreased LRRK2 kinase activity. Moreover, both human LRRK2(G2019S) fibroblasts and mouse LRRK2(R1441G) fibroblasts exhibit alterations in cell migration in culture. Treatment of mouse fibroblasts with the selective LRRK2 inhibitor LRRK2-IN1 reduces cell motility. These findings suggest that LRRK2 and microtubules mutually interact both in non-neuronal cells and in neurons, which might contribute to our understanding of its pathogenic effects in Parkinson's disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. LRRK2 kinase activity is required for toxicity in neuronal cell cultures suggesting that selective kinase inhibitors may prevent neurodegeneration in patients. Directly monitoring LRRK2 activity in cells would be advantageous for the development of small molecule LRRK2 inhibitors. Here, we demonstrate that a monoclonal anti-LRRK2 antibody directed against the activation segment binds less efficiently to native LRRK2 protein in the presence of ATP-competitive LRRK2 inhibitors. Since kinase inhibitors prevent autophosphorylation and refolding of the activation segment, we hypothesize that the antibody preferentially binds to the active conformation of LRRK2 under native conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Adult neurogenesis, the formation of new neurons in the mammalian forebrain, is one important mechanism maintaining lifelong neuronal plasticity. The generation and maturation of adult neural stem and progenitor cells is impaired in models of neurodegenerative diseases, in particular Parkinson's disease (PD). Monogenetic forms of PD were identified and associated with several genes including the leucine-rich-repeat kinase 2 (LRRK2). Some of the underlying mechanisms in neurodegenerative diseases are closely linked to neuronal plasticity, and induce changes in adult neurogenesis, neuritic maintenance, synaptic transmission, and neural connectivity. We investigated adult neurogenesis and neuritic development of newly formed neurons in the hippocampal dentate gyrus of LRRK2 knockout mice. Proliferation and survival of newly generated cells were unchanged. However, the expression profile of maturation markers in surviving newly generated cells was altered. While immature neuronal phenotypes were significantly increased, the mature neuronal phenotype of surviving cells remained unchanged. Importantly, the absolute number of immature doublecortin positive neuroblasts was significantly increased in the hippocampus of LRRK2 knockout mice. These neuroblasts presented extended dendritic length with a more complex arborization. Furthermore, LRRK2 deletion resulted in an increased volume of the axonal mossy fiber bundle projecting from dentate granule cells to CA3 pyramidal neurons. Our findings suggest that LRRK2 influences neurogenesis and particularly neuronal morphogenesis. As neurogenesis and the pre-/post- synaptic compartments are significantly altered in PD, our data advance LRRK2 as a potent candidate in addressing neuroregenerative processes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment., Methodology/principal Findings: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture., Conclusion/significance: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.

Background: Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson's disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro., Methodology/principal Findings: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7., Conclusions/significance: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson's disease pathogenesis.

Background: Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease, however, the underlying pathogenic mechanisms are poorly understood. Several in vitro studies have shown that the most frequent mutation, LRRK2(G2019S), increases kinase activity and impairs neuronal survival. LRRK2 has been linked to the mitogen-activated protein kinase kinase kinase family and the receptor-interacting protein kinases based on sequence similarity within the kinase domain and in vitro substrate phosphorylation., Methodology/principal Findings: We used an unbiased proteomic approach to identify the kinase signaling pathways wherein LRRK2 may be active. By incubation of protein microarrays containing 260 signal transduction proteins we detected four arrayed Ste20 serine/threonine kinase family members (TAOK3, STK3, STK24, STK25) as novel LRRK2 substrates and LRRK2 interacting proteins, respectively. Moreover, we found that protein kinase C (PKC) zeta binds and phosphorylates LRRK2 both in vitro and in vivo., Conclusions/significance: Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations.

Mutations within the LRRK2 (leucine-rich repeat kinase 2) gene predispose humans to develop late-onset Parkinson's disease (PD). The most prevalent of these mutations, G2019S, has been shown to increase LRRK2 kinase activity. Therefore, the discovery of small molecule inhibitors of LRRK2(G2019S) through high-throughput screening (HTS) may provide a novel therapeutic strategy for treating PD. Current biochemical assays monitoring the activity of LRRK2(G2019S) either are radioactive or use short peptidic substrates. Here we describe the development and optimization of a novel HTS AlphaScreen assay for measuring the catalytic activity of full-length LRRK2(G2019S) using its putative physiological protein substrate moesin. The high sensitivity of this optimized 384-well assay allowed the use of enzyme concentrations as low as 0.75nM. The estimated apparent K(m) value for adenosine triphosphate (6 microM) using the glutathione S-transferase-moesin substrate was much lower than the one previously reported using LRRKtide, a synthetic peptide derived from moesin. Testing of nonselective kinase inhibitors (staurosporine, H-1152, and Y-27632) generated potencies consistent with published data. Finally, robotic validation of the assay yielded an average Z' factor of 0.80. Overall, these results indicate that the present HTS AlphaScreen assay might provide a more relevant biochemical approach for the discovery of novel LRRK2(G2019S) inhibitors., (2010 Elsevier Inc. All rights reserved.)

Point mutations in the mitochondrial genome are associated with a variety of metabolic disorders. The myopathy, encephalopathy, lactic acidosis, stroke-like episodes syndrome (MELAS), is most frequently associated with an A to G transition at position 3243 of the mitochondrial tRNA(Leu(UUR)) gene, and is characterized by biochemical and structural alterations of mitochondria. In the present study, we analyzed proteomic changes in an immortalized B-cell line harbouring the MELAS A3243G mutation by two-dimensional difference gel electrophoresis and immunoblot analysis. Although the cell line contained only 10% mutated mitochondrial genomes, we detected significant alterations in numerous proteins associated with the actin cytoskeleton and in nuclear-encoded subunits of mitochondrial respiratory chain complexes. Notably, mitochondrial Lon protease and Hsp60 were deregulated in MELAS cells, indicating an effect on the mitochondrial protein quality control system. By immunofluorescence microscopy, we detected mitochondrial Lon protease accumulation and changes in actin-binding proteins preferentially in MELAS cells containing numerous mitochondria with mutated genomes. Enzymatic assays revealed that Lon protease activity is increased in MELAS cell lysates. Although Lon protease has been shown to degrade misfolded proteins and to stabilize respiratory chain complexes within mitochondria, our MELAS cell line exhibited a higher sensitivity to mitochondrial stress. These findings provide novel insights into the cellular response to dysfunctional mitochondria containing mutated genomes., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The E46K is a point mutation in alpha-synuclein (alpha-syn) that causes familial Parkinsonism with Lewy body dementia. We have now generated a cell model of Parkinsonism/Parkinson's disease (PD) and demonstrated cell toxicity after expression of E46K in the differentiated PC12 cells. E46K alpha-syn inhibited proteasome activity and induced mitochondrial depolarization in the cell model. Baicalein has been reported to inhibit fibrillation of wild type alpha-syn in vitro, and to protect neurons against several chemical-induced models of PD. We now report that baicalein significantly attenuated E46K-induced mitochondrial depolarization and proteasome inhibition, and protected cells against E46K-induced toxicity in a cell model of PD. Baicalein also reduced E46K fibrilization in vitro, with a concentration-dependent decrease in beta sheet conformation, though it increased some oligomeric species, and decreased formation of E46K alpha-syn-induced aggregates and rescued toxicity in N2A cells. Taken together, these data indicate that mitochondrial dysfunction, proteasome inhibition and specific aspects of abnormal E46K aggregation accompany E46K alpha-syn-induced cell toxicity, and baicalein can protect as well as altering aggregation properties. Baicalein has potential as a tool to understand the relation between different aggregation species and toxicity, and might be a candidate compound for further validation by using in vivo alpha-syn genetic PD models.

CNS regeneration is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. Inhibition of ROCK has been previously shown to promote regeneration in retinal ganglion cells (RGC) whereas Cdk5 inhibition mainly promoted survival. Therefore, we have evaluated the effects of combined treatment with inhibitors of ROCK and Cdk5. We show that in vitro, the co-application of the Cdk5 inhibitor, Indolinone A, and the ROCK inhibitor, Y-27632, potentiated the survival-promoting effect of either substance alone. However, neurite outgrowth in vitro was promoted only by the presence of Y-27632, not by Indolinone A alone. In the ex vivo explant and the in vivo optic nerve crush model the combination of both inhibitors significantly increased neurite outgrowth at small distances, but this effect leveled off for longer neurites. In summary, the combined treatment with the Cdk5 inhibitor Indolinone A and the ROCK inhibitor Y-27632 results in a strong additive effect on neuronal survival, but is not able to increase the regenerative response beyond the effect of the ROCK inhibitor.

Autosomal dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset Parkinson's disease. The most prevalent LRRK2(G2019S) mutation has repeatedly been shown to enhance kinase activity and neurotoxicity, however, the molecular mechanisms leading to neurodegeneration remain poorly defined. Here we show that recombinant human LRRK2 preferentially phosphorylates tubulin-beta purified from bovine brain and that phosphorylation is three-fold enhanced by the LRRK2(G2019S) mutation. By tandem mass spectrometry, Thr107 was identified as phosphorylation site which is highly conserved between tubulin-beta family members and also between tubulin-beta genes of different species. LRRK2 was co-immunoprecipitated with tubulin-beta both from wild-type mouse brain and from LRRK2 over-expressing, non-neuronal human embryonic kidney 293 cells. However, an effect of LRRK2 on tubulin phosphorylation and assembly was only detectable in mouse brain samples. In vitro co-incubation of bovine brain tubulins with LRRK2 increased microtubule stability in the presence of microtubule-associated proteins which may explain the reduction in neurite length in LRRK2-deficient neurons in culture. These findings suggest that LRRK2(G2019S)-induced neurodegeneration in Parkinsonian brains may be partly mediated by increased phosphorylation of tubulin-beta and constraining of microtubule dynamics.