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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.)

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., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

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 © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

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.

Aggregation of alpha-synuclein may contribute to neuropathology in Parkinson's disease patients and in transgenic animal models. Natively unfolded alpha-synuclein binds to various proteins and conformational changes due to alpha-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 alpha-synuclein. Monomeric alpha-synuclein bound to arrayed cAMP regulated phosphoprotein 19 and binding appears to be disrupted by alpha-synuclein oligomerization. Incubation with recombinant alpha-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 alpha-synuclein(A30P) transgenic mice. Pull-down assays using beads conjugated with a Cdc42/Rac interactive binding domain lead to an enrichment of endogenous alpha-synuclein oligomers. Cdc42 effector proteins were also co-immunoprecipitated with alpha-synuclein from brainstem lysates and were colocalized with alpha-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 alpha-synuclein toxicity in yeast. Our data indicate that components of small GTPase signal transduction pathways may be directly targeted by alpha-synuclein oligomers which potentially leads to signaling deficits and neurodegeneration.

The temporospatial expression pattern of the nuclear DNA repair enzyme redox factor-1 (ref-1), the p53-activated gene (pag) 608 and the effector caspase-3 was examined by in situ hybridization histochemistry in gerbils subjected to two 10-min episodes of unilateral common carotid artery occlusion, separated by 5h. Gene responses were correlated with the metabolic state, as revealed by regional adenosine 5'-triphosphate bioluminescent imaging, and with the degree of histological damage, as assessed by haematoxylin-eosin staining and terminal deoxynucleotidyl transferase-mediated-dUTP nick end labeling (TUNEL), in order to evaluate the role of these genes in the maturation of injury. Focal infarcts developed in the dorsolateral cerebral cortex at the bregma level and the nucleus caudate-putamen within four days after repeated unilateral ischemia, as indicated by a secondary adenosine 5'-triphosphate loss after initial adenosine 5'-triphosphate recovery and by histomorphological signs of pannecrosis. The more caudal cortex at hippocampal levels and the hippocampus (CA1>CA3 area), however, exhibited selective neuronal injury without adenosine 5'-triphosphate depletion. TUNEL+ cells appeared starting 5h after repeated unilateral ischemia. TUNEL+ cells reached maximum levels in the caudate-putamen at 12-24h, but much later in the cortex and hippocampus at two days after ischemia. Remarkably few TUNEL+ cells were noticed in the thalamus, where adenosine 5'-triphosphate state did not recover after reperfusion. Following repeated unilateral ischemia, a transient elevation of ref-1 mRNA was detected after 5h in the cerebral cortex and hippocampal CA1 area. Ref-1 mRNA levels decreased within 12-24h, before the onset of tissue damage. Subsequently, pag608 and caspase-3 mRNA levels increased, closely in parallel with the appearance of DNA fragmented cells, but slightly prior to the deterioration of adenosine 5'-triphosphate state. In the caudate-putamen, pag608 and caspase-3 mRNAs reached maximum levels already 12-24h after repeated common carotid artery occlusion, when DNA fragmentation was most prominent, and declined thereafter. In the cortex and hippocampal CA1-3 areas, where DNA damage appeared more slowly, pag608 and caspase-3 mRNAs were induced starting 24h after ischemia, and remained elevated even after two to four days. The levels of pag608 and caspase-3 mRNAs were similar at rostral and caudal levels of the cortex, as well as in the hippocampal CA1 and CA3 area, although the degree of injury differed considerably between these structures. Notably, pag608 and caspase-3 mRNAs were not elevated in the thalamus after repeated unilateral ischemia. The present report shows a close temporal association between the induction of ref-1, pag608 and caspase-3 mRNAs, the manifestation of cell injury and the secondary adenosine 5'-triphosphate depletion in infarcting brain areas, suggesting (i) that de novo responses of these genes may be involved in the maturation of cell injury and (ii) that apoptotic programs and the secondary deterioration of cerebral energy state may interfere with each other after ischemia.

An essential role for caspases in programmed neuronal cell death has been demonstrated in various in vitro studies, and synthetic caspase inhibitors have recently been shown to prevent neuronal cell loss in animal models of focal cerebral ischemia and traumatic brain injury, respectively. The therapeutic utility of caspase inhibitors, however, will depend on preservation of both structural and functional integrity of neurons under stressful conditions. The present study demonstrates that expression and proteolytic activity of caspase-3 is up-regulated in the rat hippocampus after transient forebrain ischemia. Continuous i.c.v. infusion of the caspase inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone significantly attenuated caspase-3-like enzymatic activity, and blocked delayed cell loss of hippocampal CA1 neurons after ischemia. Administration of N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, however, did not prevent impairment of induction of long-term potentiation in post-ischemic CA1 cells, suggesting that caspase inhibition alone does not preserve neuronal functional plasticity.

Administration of inhibitors of neuronal nitric oxide synthase or deletion of the encoding gene in rodents provided evidence that neuronal nitric oxide synthase activity may contribute to neuronal cell death following global and focal cerebral ischemia. In the present study, we investigated by in situ hybridization the expression of an endogenous inhibitor of neuronal nitric oxide synthase activity, designated protein inhibitor of neuronal nitric oxide synthase and homologous to cytoplasmic dynein light chain, in the post-ischemic rat brain. Following global ischemia induced by cardiac arrest, messenger RNA expression of protein inhibitor of neuronal nitric oxide synthase was rapidly induced in pyramidal neurons of the hippocampal CA3 region and granule cell of the dentate gyrus which are resistant to ischemic damage. In vulnerable CA1 pyramidal neurons however, protein inhibitor of neuronal nitric oxide synthase expression remained at basal level after global ischemia and was associated with an increase in nicotinamide adenine dinucleotide phosphate-diaphorase activity and subsequent DNA fragmentation indicating ischemia-mediated neuronal cell death. Following focal cerebral ischemia induced by permanent occlusion of the middle cerebral artery, transcripts of protein inhibitor of neuronal nitric oxide synthase progressively accumulated in cortical neurons bordering the infarct area. After transient middle cerebral artery occlusion however, messenger RNA levels of protein inhibitor of neuronal nitric oxide synthase increased in the reperfused neocortex. Our findings indicate that cerebral ischemia leads to an increase in neuronal expression of protein inhibitor of neuronal nitric oxide synthase in brain regions where sustained or "uncoupled" nitric oxide synthase activity may be detrimental to neurons. Lack of post-ischemic induction of protein inhibitor of neuronal nitric oxide synthase in CA1 pyramidal neurons may result in high nitric oxide synthase activity after global ischemia and could contribute to delayed neuronal cell death.

Contact hypersensitivity responsiveness to dinitrofluorobenzene is depressed in mice that are sensitized through skin sites exposed to ultraviolet (UV) radiation. Local impairment of contact hypersensitivity by UV has been associated with a reduction in antigen-presenting cell activity within UV-irradiated skin sites marked by a decrease in the density of Ia-positive epidermal Langerhans cells. Our recent studies have demonstrated that neurogenic mediators (e.g. calcitonin gene-related peptide (CGRP) and nitric oxide (NO) contribute to cutaneous inflammation following exposure of rats to high-dose UV radiation. Since CGRP and NO inhibit antigen presentation by dendritic cells in vitro, we have investigated the possible involvement of CGRP and NO in local immunosuppression in UV-irradiated rodents. Hindpaw skin of Sprague-Dawley rats and back skin of UV-susceptible C57BL/6 mice was exposed to acute UV radiation (2.0 J/cm2 and 0.5 J/cm2, respectively). Alterations in cutaneous CGRP content were analyzed by a specific radioimmunoassay (RIA). In separate experiments, the CGRP receptor antagonist CGRP-(8-37) (10-5 M) and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (2 X 10-5 M) were topically applied to UV-exposed skin before induction of contact hypersensitivity with dinitrofluorobenzene. Finally, we examined the effects of UV irradiation and epicutaneous application of CGRP on Ia-positive Langerhans cells by immunohistochemical analysis of epidermal sheets. It was found that UV exposure lead to a decrease in skin CGRP levels starting already 2 h after irradiation and reaching a minimum (less than 40% of non-irradiated control skin) at 6-12 h. Contact hypersensitivity reactions were significantly suppressed by UV radiation in rat skin (by 51%) and murine skin (by 80%). Topical administration of both CGRP-(8-37) and L-NAME before sensitization restored the capacity to respond to haptens applied to UV-exposed skin. Both UV exposure and topical CGRP reduced the density of Ia-positive epidermal cells. Our data indicate that CGRP may be released from sensory neurons following cutaneous UV irradiation and that CGRP and NO contribute of UV-induced local immunosuppression. Moreover, topical administration of CGRP or its antagonist may be able to modulate epidermal Langerhans cell activity in vivo.

Evidence from our previous work suggests that neurogenic mediators contribute to the inflammation following ultraviolet (UV) irradiation of the skin. We have investigated whether calcitonin gene-related peptide (CGRP), substance P and nitric oxide (NO) participate in the cutaneous inflammatory reaction of the rat hind paw and ear to UV irradiation. Skin blood flow was measured by laser Doppler technique. Oedema was quantified using a spring loaded micrometer to measure ear thickness. UV irradiation of the rat skin lead to a long lasting increase in skin blood flow. This increase was dose dependently attenuated by the CGRP receptor antagonist CGRP-(8-37) (0.15 nmol in 25 microliters to 6.0 nmol in 25 microliters, s.c.) up to 51% with a maximum of effectiveness at 24 h post irradiation. The inhibitor of NO synthase NG-nitro-L-arginine methyl ester hydrochloride (L-NAME, 25 nmol in 25 microliters, s.c.) attenuated skin blood flow by 38%. Concurrent injections s.c. of CGRP-(8-37) (1.5 nmol in 12.5 microliters) and L-NAME (25 nmol in 12.5 microliters) demonstrated an augmentive effect in attenuating skin blood flow. The tachykinin NK1 receptor antagonist CP-96,345 (6.0 nmol in 25 microliters, s.c.) attenuated skin blood flow by 27%. NG-Nitro-D-arginine methyl ester hydrochloride (D-NAME) and CP-96.344 showed no effects on skin blood flow after UV irradiation. CGRP-(8-37) (0.6 nmol in 10 microliters) i.d. and L-NAME (10 nmol in 10 microliters) i.d. had no effect of oedema formation after UV irradiation. Furthermore, post UV irradiation enhanced CGRP- and NO synthase-immunoreactivity in nerve fibres in the exposed skin area were visible. Taken these findings together we suggest the involvement of the neuropeptides CGRP and substance P and of neuronal NO on the vasodilatory component of the UV-induced inflammatory reaction of the rat skin. CGRP contributing to UV-induced vasodilation acts in an endothelial NO-independent manner.