Absence of tissue transglutaminase reduces amyloid-beta pathology in APP23 mice.

AIMS: Alzheimer's disease (AD) is characterised by amyloid-beta (Aß) aggregates in the brain. Targeting Aß aggregates is a major approach for AD therapies, although attempts have had little to no success so far. A novel treatment option is to focus on blocking the actual formation of Aß multimers. The enzyme tissue transglutaminase (TG2) is abundantly expressed in the human brain and plays a key role in post-translational modifications in Aß resulting in covalently cross-linked, stable and neurotoxic Aß oligomers. In vivo absence of TG2 in the APP23 mouse model may provide evidence that TG2 plays a key role in development and/or progression of Aß-related pathology. METHODS: Here, we compared the effects on Aß pathology in the presence or absence of TG2 using 12-month-old wild type, APP23 and a crossbreed of the TG2-/- mouse model and APP23 mice (APP23/TG2-/-). RESULTS: Using immunohistochemistry, we found that the number of Aß deposits was significantly reduced in the absence of TG2 compared with age-matched APP23 mice. To pinpoint possible TG2-associated mechanisms involved in this observation, we analysed soluble brain Aß1-40 , Aß1-42 and/or Aß40/42 ratio, and mRNA levels of human APP and TG2 family members present in brain of the various mouse models. In addition, using immunohistochemistry, both beta-pleated sheet formation in Aß deposits and the presence of reactive astrocytes associated with Aß deposits were analysed. CONCLUSIONS: We found that absence of TG2 reduces the formation of Aß pathology in the APP23 mouse model, suggesting that TG2 may be a suitable therapeutic target for reducing Aß deposition in AD.

Increased transcription of transglutaminase 1 mediates neuronal death in in vitro models of neuronal stress and Aß1-42-mediated toxicity.

Alzheimer's disease (AD) is the most common cause of dementia. At the pre-symptomatic phase of the disease, the processing of the amyloid precursor protein (APP) produces toxic peptides, called amyloid-ß 1-42 (Aß 1-42). The downstream effects of Aß 1-42 production are not completely uncovered. Here, we report the involvement of transglutaminase 1 (TG1) in in vitro AD models of neuronal toxicity. TG1 was increased at late stages of the disease in the hippocampus of a mouse model of AD and in primary cortical neurons undergoing stress. Silencing of TGM1 gene was sufficient to prevent Aß-mediated neuronal death. Conversely, its overexpression enhanced cell death. TGM1 upregulation was mediated at the transcriptional level by an activator protein 1 (AP1) binding site that when mutated halted TGM1 promoter activation. These results indicate that TG1 acts downstream of Aß-toxicity, and that its stress-dependent increase makes it suitable for pharmacological intervention.

Tissue Transglutaminase contributes to myelin phagocytosis in interleukin-4-treated human monocyte-derived macrophages.

Macrophages exert either a detrimental or beneficial role in Multiple Sclerosis (MS) pathology, depending on their inflammatory environment. Tissue Transglutaminase (TG2), a calcium-dependent cross-linking enzyme, has been described as a novel marker for anti-inflammatory, interleukin-4 (IL-4) polarized macrophages (M(IL-4)), which represent a subpopulation of macrophages with phagocytic abilities. Since TG2 is expressed in macrophages in active human MS lesions, we questioned whether TG2 drives the differentiation of M(IL-4) into an anti-inflammatory phenotype and whether it plays a role in the phagocytosis of myelin by these cells. In macrophage-differentiated THP-1 monocytes, TG2 was increased upon IL-4 treatment. Reducing TG2 expression impairs the differentiation of M(IL-4) macrophages into an anti-inflammatory phenotype and drives them into a pro-inflammatory state. In addition, reduced TG2 expression resulted in increased presence of myelin basic protein in macrophages upon myelin exposure of M(IL-4) macrophages. Moreover, the elevated presence of an early endosome marker and equal expression of a lysosome marker compared to control macrophages, suggest that TG2 plays a role in phagosome maturation in M(IL-4) macrophages These data suggest that tuning macrophages into TG2 producing anti-inflammatory cells by IL-4 treatment may benefit effective myelin phagocytosis in e.g. demyelinating MS lesions and open avenues for successful regeneration.

Interaction between tissue transglutaminase and amyloid-beta: Protein-protein binding versus enzymatic crosslinking.

Self-interaction, chaperone binding and posttranslational modification of amyloid-beta (Aß) is essential in the initiation and propagation of Aß aggregation. Aggregation results in insoluble Aß deposits characteristic of Alzheimer's disease (AD) brain lesions, i.e. senile plaques and cerebral amyloid angiopathy. Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes posttranslational modifications including the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds (molecular crosslinks), and colocalizes with Aß deposits in AD. Two independent groups recently found that apart from the induction of Aß oligomerization, the blood-derived transglutaminase member FXIIIa forms stable protein-protein complexes with Aß independent of the transamidation reaction. Here, we investigated whether also tTG forms rigid protein complexes with Aß in the absence of catalytic activation. We found that both Aß1-40 and Aß1-42 are substrates for tTG-catalyzed crosslinking. In addition, in the absence of calcium or the presence of a peptidergic inhibitor of tTG, stable tTG-Aß1-40 complexes were found. Interestingly, the stable complexes between tTG and Aß1-40, were only found at 'physiological' concentrations of Aß1-40. Together, our data suggest that depending on the Aß species at hand, and on the concentration of Aß, rigid protein-complexes are formed between tTG and Aß1-40 without the involvement of the crosslinking reaction.

A Qualitative Study on Experiences and Perspectives of Members of a Dutch Medical Research Ethics Committee.

The aim of this research was to gain insight into the experiences and perspectives of individual members of a Medical Research Ethics Committee (MREC) regarding their individual roles and possible tensions within and between these roles. We conducted a qualitative interview study among members of a large MREC, supplemented by a focus group meeting. Respondents distinguish five roles: protector, facilitator, educator, advisor and assessor. Central to the role of protector is securing valid informed consent and a proper risk-benefit analysis. The role of facilitator implies that respondents want to think along with and assist researchers in order to help medical science progress. As educators, the respondents want to raise ethical and methodological awareness of researchers. The role of advisor implies that respondents bring in their own expertise. The role of assessor points to contributing to the overall evaluation of the research proposal. Various tensions were identified within and between roles. Within the role of protector, a tension is experienced between paternalism and autonomy. Between the role of protector and facilitator tensions occur when the value of a study is questioned while risks and burdens for the subjects are negligible. Within the role of assessor, a tension is felt between the implicit nature of judgments and the need for more explicit formulations. Awareness of various roles and responsibilities may prevent one-sided views on MREC work, not only by members themselves, but also by researchers. Tensions within and between the roles require reflection by MREC members.

Tissue transglutaminase in astrocytes is enhanced by inflammatory mediators and is involved in the formation of fibronectin fibril-like structures.

BACKGROUND: During multiple sclerosis (MS) lesion formation, inflammatory mediators are produced by microglial cells and invading leukocytes. Subsequently, hypertrophic astrocytes fill the lesion and produce extracellular matrix (ECM) proteins that together form the astroglial scar. This is beneficial because it seals off the site of central nervous system (CNS) damage. However, astroglial scarring also forms an obstacle that inhibits remyelination of brain lesions. This is possibly an important cause for incomplete remyelination of the CNS in early stage MS patients and for failure of remyelination when the disease progresses. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme that can cross-link proteins, appears in astrocytes in inflammatory MS lesions and may contribute to the rearrangement of ECM protein deposition and aggregation. METHODS: The effect of different inflammatory mediators on TG2 and fibronectin, an ECM protein, protein levels was examined in primary rat microglia and astrocytes by western blotting. Also, TG2 activity was analyzed in primary rat astrocytes by a TG activity assay. To determine the role of TG2 in the deposition and cross-linking of fibronectin, a TG2 inhibitor and TG2 knockdown astrocytes were used. RESULTS: Our data show that under inflammatory conditions in vitro, TG2 production is enhanced in astrocytes and microglia. We observed that in particular, astrocytes produce fibronectin that can be cross-linked and aggregated by exogenous TG2. Moreover, inflammatory stimulus-induced endogenously produced TG2 is involved in the appearance of morphological fibril-like fibronectin deposits but does not lead to cross-linked fibronectin aggregates. CONCLUSIONS: Our in vitro observations suggest that during MS lesion formation, when inflammatory mediators are produced, astrocyte-derived TG2 may contribute to ECM rearrangement, and subsequent astroglial scarring.

Monocyte-derived tissue transglutaminase in multiple sclerosis patients: reflecting an anti-inflammatory status and function of the cells?

BACKGROUND: Leukocyte infiltration into the central nervous system is an important feature of multiple sclerosis (MS) pathology. Among the infiltrating cells, monocytes comprise the largest population and are considered to play a dual role in the course of the disease. The enzyme tissue transglutaminase (TG2), produced by monocytes, plays a central role in monocyte adhesion/migration in animal models of MS. In the present study, we questioned whether TG2 expression is altered in monocytes from MS patients compared to healthy control (HC) subjects. Moreover, we determined the inflammatory status of these TG2-expressing monocytes, what inflammatory factor regulates TG2 expression, and whether TG2 can functionally contribute to their adhesion/migration processes. METHODS: Primary human monocytes from MS patients and HC subjects were collected, RNA isolated and subjected to qPCR analysis. Human THP-1 monocytes were lentivirally transduced with TG2 siRNA or control and treated with various cytokines. Subsequently, mRNA levels of inflammatory factors, adhesion properties, and activity of RhoA were analyzed in interleukin (IL)-4-treated monocytes. RESULTS: TG2 mRNA levels are significantly increased in monocytes derived from MS patients compared to HC subjects. In addition, correlation analyses indicated that TG2-expressing cells display a more anti-inflammatory, migratory profile in MS patients. Using THP-1 monocytes, we observed that IL-4 is a major trigger of TG2 expression in these cells. Furthermore, knockdown of TG2 expression leads to a pro-inflammatory profile and reduced adhesion/migration properties of IL-4-treated monocytes. CONCLUSIONS: TG2-expressing monocytes in MS patients have a more anti-inflammatory profile. Furthermore, TG2 mediates IL-4-induced anti-inflammatory status in THP-1 monocytes, adhesion, and cytoskeletal rearrangement in vitro. We thus propose that IL-4 upregulates TG2 expression in monocytes of MS patients, driving them into an anti-inflammatory status.

Strategies towards in vivo imaging of active transglutaminase type 2 using positron emission tomography.

Transglutaminase type 2 (TG2) is increasingly linked to the pathogenesis of several diseases, such as celiac disease, cancer, and fibrotic and neurodegenerative diseases. In parallel with becoming an attractive target for therapy, interest in the development of compounds for in vivo imaging of TG2 is rising. Such imaging biomarkers might assist in clarifying the role of TG2 in pathology and in monitoring TG2 inhibition in vivo and thus assist in drug development. In this review, the latest results together with various strategies in TG2 PET tracer development are discussed, including radiolabelling of irreversible and reversible active-site inhibitors, as well as allosteric inhibitors, acyl-donor and acyl-acceptor substrates, and anti-TG2 monoclonal antibodies.

Is monocyte- and macrophage-derived tissue transglutaminase involved in inflammatory processes?

Monocytes and macrophages are key players in inflammatory processes following an infection or tissue damage. Monocytes adhere and extravasate into the inflamed tissue, differentiate into macrophages, and produce inflammatory mediators to combat the pathogens. In addition, they take up dead cells and debris and, therefore, take part in the resolution of inflammation. The multifunctional enzyme tissue Transglutaminase (TG2, tTG) is known to participate in most of those monocyte- and macrophage-mediated processes. Moreover, TG2 expression and activity can be regulated by inflammatory mediators. In the present review, we selectively elaborate on the expression, regulation, and contribution of TG2 derived from monocytes and macrophages to inflammatory processes mediated by those cells. In addition, we discuss the role of TG2 in certain pathological conditions, in which inflammation and monocytes and/or macrophages are prominently present, including atherosclerosis, sepsis, and multiple sclerosis. Based on the studies and considerations reported in this review, we conclude that monocyte- and macrophage-derived TG2 is clearly involved in various processes contributing to inflammation. However, TG2's potential as a therapeutic target to counteract the possible detrimental effects or stimulate the potential beneficial effects on monocyte and macrophage responses during inflammation should be carefully considered. Alternatively, as TG2-related parameters can be used as a marker of disease, e.g., in celiac disease, or of disease-stage, e.g., in cancer, we put forward that this could be subject of research for monocyte- or macrophage-derived TG2 in inflammatory diseases.

Monocyte behaviour and tissue transglutaminase expression during experimental autoimmune encephalomyelitis in transgenic CX3CR1gfp/gfp mice.

Leukocyte infiltration into the central nervous system (CNS) is a key pathological feature in multiple sclerosis (MS) and the MS animal model experimental autoimmune encephalomyelitis (EAE). Recently, preventing leukocyte influx into the CNS of MS patients is the main target of MS therapies and insight into cell behaviour in the circulation is needed for further elucidation of such therapies. In this study, we aimed at in vivo visualization of monocytes in a time-dependent manner during EAE. Using intravital two-photon microscopy (IVM), we imaged CX3CR1gfp/gfp mice during EAE, visualizing CX3CR1-GFP+ monocytes and their dynamics in the spinal cord vasculature. Our observations showed that intraluminal crawling of CX3CR1-GFP+ monocytes increased even before the clinical onset of EAE due to immunization of the animals. Furthermore, intraluminal crawling remained elevated during ongoing clinical disease. Besides, the displacement of these cells was larger during the peak of EAE compared to the control animals. In addition, we showed that the enzyme tissue transglutaminase (TG2), which is present in CNS-infiltrated cells in MS patients, is likewise found in CX3CR1-GFP+ monocytes in the spinal cord lesions and at the luminal side of the vasculature during EAE. It might thereby contribute to adhesion and crawling of monocytes, facilitating extravasation into the CNS. Thus, we put forward that interference with monocyte adhesion, by e.g. inhibition of TG2, should be applied at a very early stage of EAE and possibly MS, to effectively combat subsequent pathology.

Tissue transglutaminase-catalysed cross-linking induces Apolipoprotein E multimers inhibiting Apolipoprotein E's protective effects towards amyloid-beta-induced toxicity.

Cerebral amyloid angiopathy (CAA) is a pathological hallmark of Alzheimer's disease (AD) and characterized by deposition of amyloid-ß (Aß) protein and smooth muscle cell (SMC) death in cerebral vessel walls. Apolipoprotein E (ApoE) is of importance in both Aß accumulation and Aß-mediated toxicity towards SMCs in the cerebral vessel wall, although its exact role in CAA pathogenesis remains unclear. Tissue transglutaminase (tTG) is an enzyme capable of inducing both protein complexes and altered protein bioactivity via post-translational cross-linking. In CAA, tTG and its catalytic activity are associated with deposited Aß. Furthermore, several apolipoproteins are known substrates of tTG. We therefore investigated whether ApoE is a substrate for tTG and if this affects ApoE's bioactivity. We found strong binding of different ApoE isoforms with tTG and demonstrated tTG-catalysed ApoE multimers. In post-mortem human AD cases, ApoE colocalized with in situ active tTG in CAA. Moreover, human brain SMCs treated with Aß demonstrated enhanced secretion of both ApoE and tTG, and of TG cross-links in the extracellular matrix. Interestingly, tTG-catalysed cross-linked ApoE failed to protect SMCs against Aß-mediated cytotoxicity. Together, our data demonstrate a novel tTG-driven post-translational modification of ApoE that might play an important role in CAA. Cerebral amyloid angiopathy (CAA) is a pathological hallmark of Alzheimer's disease (AD) and characterized by amyloid-ß (Aß) protein deposition and cerebral smooth muscle cell (SMC) death. We found that, in contrast to normal vessels, in CAA apolipoprotein E (ApoE) is cross-linked by tissue transglutaminase (tTG) resulting in stable ApoE complexes. These complexes no longer protect cerebral SMC from Aß-mediated toxicity. Our findings demonstrate a novel mechanism explaining the Aß-mediated cerebral SMC cell death characteristic of CAA in AD cases.

Tissue Transglutaminase contributes to experimental multiple sclerosis pathogenesis and clinical outcome by promoting macrophage migration.

Multiple sclerosis is a serious neurological disorder, resulting in e.g., sensory, motor and cognitive deficits. A critical pathological aspect of multiple sclerosis (MS) is the influx of immunomodulatory cells into the central nervous system (CNS). Identification of key players that regulate cellular trafficking into the CNS may lead to the development of more selective treatment to halt this process. The multifunctional enzyme tissue Transglutaminase (TG2) can participate in various inflammation-related processes, and is known to be expressed in the CNS. In the present study, we question whether TG2 activity contributes to the pathogenesis of experimental MS, and could be a novel therapeutic target. In human post-mortem material, we showed the appearance of TG2 immunoreactivity in leukocytes in MS lesions, and particular in macrophages in rat chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE), an experimental MS model. Clinical deficits as observed in mouse EAE were reduced in TG2 knock-out mice compared to littermate wild-type mice, supporting a role of TG2 in EAE pathogenesis. To establish if the enzyme TG2 represents an attractive therapeutic target, cr-EAE rats were treated with TG2 activity inhibitors during ongoing disease. Reduction of TG2 activity in cr-EAE animals dramatically attenuated clinical deficits and demyelination. The mechanism underlying these beneficial effects pointed toward a reduction in macrophage migration into the CNS due to attenuated cytoskeletal flexibility and RhoA GTPase activity. Moreover, iNOS and TNFα levels were selectively reduced in the CNS of cr-EAE rats treated with a TG2 activity inhibitor, whereas other relevant inflammatory mediators were not affected in CNS or spleen by reducing TG2 activity. We conclude that modulating TG2 activity opens new avenues for therapeutic intervention in MS which does not affect peripheral levels of inflammatory mediators.

Antioxidative defense mechanisms controlled by Nrf2: state-of-the-art and clinical perspectives in neurodegenerative diseases.

Activation of microglial cells and impaired mitochondrial function are common pathological characteristics of many neurological diseases and contribute to increased generation of reactive oxygen species (ROS). It is nowadays accepted that oxidative damage and mitochondrial dysfunction are key hallmarks of classical neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease and Huntington's disease. To counteract the detrimental effects of ROS and restore the delicate redox balance in the central nervous system (CNS), cells are equipped with an endogenous antioxidant defense mechanism consisting of several antioxidant enzymes. The production of many antioxidant enzymes is regulated at the transcriptional level by the transcription factor nuclear factor E2-related factor 2 (Nrf2). Although evidence is accumulating that activation of the Nrf2 pathway represents a promising therapeutic approach to restore the CNS redox balance by reducing ROS-mediated neuronal damage in experimental models of neurodegenerative disorders, only a few Nrf2-activating compounds have been tested in a clinical setting. We here provide a comprehensive synopsis on the role of ROS in common neurodegenerative disorders and discuss the therapeutic potential of the Nrf2 pathway.

Hippocampal proliferation is increased in presymptomatic Parkinson's disease and due to microglia.

Besides dopamine-deficiency related motor symptoms, nonmotor symptoms, including cognitive changes occur in Parkinson's disease (PD) patients, that may relate to accumulation of α-synuclein in the hippocampus (HC). This brain region also contains stem cells that can proliferate. This is a well-regulated process that can, for example, be altered by neurodegenerative conditions. In contrast to proliferation in the substantia nigra and subventricular zone, little is known about the HC in PD. In addition, glial cells contribute to neurodegenerative processes and may proliferate in response to PD pathology. In the present study, we questioned whether microglial cells proliferate in the HC of established PD patients versus control subjects or incidental Lewy body disease (iLBD) cases as a prodromal state of PD. To this end, proliferation was assessed using the immunocytochemical marker minichromosome maintenance protein 2 (MCM2). Colocalization with Iba1 was performed to determine microglial proliferation. MCM2-positive cells were present in the HC of controls and were significantly increased in the presymptomatic iLBD cases, but not in established PD patients. Microglia represented the majority of the proliferating cells in the HC. This suggests an early microglial response to developing PD pathology in the HC and further indicates that neuroinflammatory processes play an important role in the development of PD pathology.

Transglutaminase 1 and its regulator tazarotene-induced gene 3 localize to neuronal tau inclusions in tauopathies.

Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and Pick's disease (PiD) are commonly known as tauopathies. Neurodegeneration observed in these diseases is linked to neuronal fibrillary hyperphosphorylated tau protein inclusions. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular cross-links. Both transglutaminase 1 (TG1) and transglutaminase 2 (TG2) are abundantly expressed in the brain and are associated with fibrillary hyperphosphorylated tau protein inclusions in neurons of AD, so-called neurofibrillary tangles (NFTs). However, other data obtained by our group suggested that tau pathology in the brain may be primarily related to TG1 and not to TG2 activity. To obtain more information on this issue, we set out to investigate the association of TG1, TG2, and TG-catalysed cross-links with fibrillary hyperphosphorylated tau inclusions in tauopathies other than AD, using immunohistochemistry. We found strong TG1 and TG-catalysed cross-link staining in neuronal tau inclusions characteristic of PSP, FTDP-17 with mutations in the tau gene (FTDP-17T), and PiD brain, whereas, in contrast to AD, TG2 was only rarely observed in these inclusions. Furthermore, using a biochemical approach, we demonstrated that tau is a substrate for TG1-mediated cross-linking. Interestingly, we found co-localization of the TG1 activator, tazarotene-induced gene 3 (TIG3), in the neuronal tau inclusions of PSP, FTDP-17T, and PiD, but not in NFTs of AD cases, indicating that these tau-containing protein aggregates are not identical. We conclude that TG1-catalysed cross-linking, regulated by TIG3, might play an important role in the formation of neuronal tau inclusions in PSP, FTDP-17T, and PiD brain.

Thinking about the action potential: the nerve signal as a window to the physical principles guiding neuronal excitability.

Ever since the work of Edgar Adrian, the neuronal action potential has been considered as an electric signal, modeled and interpreted using concepts and theories lent from electronic engineering. Accordingly, the electric action potential, as the prime manifestation of neuronal excitability, serving processing and reliable "long distance" communication of the information contained in the signal, was defined as a non-linear, self-propagating, regenerative, wave of electrical activity that travels along the surface of nerve cells. Thus, in the ground-breaking theory and mathematical model of Hodgkin and Huxley (HH), linking Nernst's treatment of the electrochemistry of semi-permeable membranes to the physical laws of electricity and Kelvin's cable theory, the electrical characteristics of the action potential are presented as the result of the depolarization-induced, voltage- and time-dependent opening and closure of ion channels in the membrane allowing the passive flow of charge, particularly in the form of Na+ and K+ -ions, into and out of the neuronal cytoplasm along the respective electrochemical ion gradient. In the model, which treats the membrane as a capacitor and ion channels as resistors, these changes in ionic conductance across the membrane cause a sudden and transient alteration of the transmembrane potential, i.e., the action potential, which is then carried forward and spreads over long(er) distances by means of both active and passive conduction dependent on local current flow by diffusion of Na+ ion in the neuronal cytoplasm. However, although highly successful in predicting and explaining many of the electric characteristics of the action potential, the HH model, nevertheless cannot accommodate the various non-electrical physical manifestations (mechanical, thermal and optical changes) that accompany action potential propagation, and for which there is ample experimental evidence. As such, the electrical conception of neuronal excitability appears to be incomplete and alternatives, aiming to improve, extend or even replace it, have been sought for. Commonly misunderstood as to their basic premises and the physical principles they are built on, and mistakenly perceived as a threat to the generally acknowledged explanatory power of the "classical" HH framework, these attempts to present a more complete picture of neuronal physiology, have met with fierce opposition from mainstream neuroscience and, as a consequence, currently remain underdeveloped and insufficiently tested. Here we present our perspective that this may be an unfortunate state of affairs as these different biophysics-informed approaches to incorporate also non-electrical signs of the action potential into the modeling and explanation of the nerve signal, in our view, are well suited to foster a new, more complete and better integrated understanding of the (multi)physical nature of neuronal excitability and signal transport and, hence, of neuronal function. In doing so, we will emphasize attempts to derive the different physical manifestations of the action potential from one common, macroscopic thermodynamics-based, framework treating the multiphysics of the nerve signal as the inevitable result of the collective material, i.e., physico-chemical, properties of the lipid bilayer neuronal membrane (in particular, the axolemma) and/or the so-called ectoplasm or membrane skeleton consisting of cytoskeletal protein polymers, in particular, actin fibrils. Potential consequences for our view of action potential physiology and role in neuronal function are identified and discussed.

Insulin modulates cocaine-sensitive monoamine transporter function and impulsive behavior.

Because insulin acutely enhances the function of dopamine transporters, the tyrosine kinase receptors activated by this hormone may modulate transporter-dependent neurochemical and behavioral effects of psychoactive drugs. In this respect, we examined the effects of insulin on exocytotic monoamine release and the efficacy of the monoamine transporter blocker cocaine in rat nucleus accumbens. Whereas insulin reduced electrically evoked exocytotic [(3)H]dopamine release in nucleus accumbens slices, the hormone potentiated the release-enhancing effect of cocaine thereon. The phosphatidylinositol 3-kinase inhibitor LY294002 abolished these effects, indicating the involvement of insulin receptors. Similar insulin effects were observed on the release of [(3)H]norepinephrine in nucleus accumbens slices, but not on that of [(3)H]serotonin, and were also apparent in medial prefrontal cortex slices. As might then be expected, insulin also potentiated the dopamine and norepinephrine release-enhancing effects of the selective monoamine uptake inhibitors GBR12909 and desmethylimipramine, respectively. In subsequent behavioral experiments, we investigated the role of insulin in motor impulsivity that depends on monoamine neurotransmission in the nucleus accumbens. Intracranial administration of insulin in the nucleus accumbens alone reduced premature responses in the five-choice serial reaction time task and enhanced the stimulatory effect of peripheral cocaine administration on impulsivity, resembling the observed neurochemical effects of the hormone. In contrast, cocaine-induced locomotor activity remained unchanged by intra-accumbal insulin application. These data reveal that insulin presynaptically regulates cocaine-sensitive monoamine transporter function in the nucleus accumbens and, as a consequence, impulsivity. Therefore, insulin signaling proteins may represent targets for the treatment of inhibitory control deficits such as addictive behaviors.

Increase in endoplasmic reticulum-associated tissue transglutaminase and enzymatic activation in a cellular model of Parkinson's disease.

Parkinson's disease (PD) is characterized by accumulation of α-synuclein aggregates and degeneration of melanized, catecholaminergic neurons. The tissue transglutaminase (tTG) enzyme catalyzes molecular protein cross-linking. In PD, tTG levels are increased and cross-linking has been identified as an important factor in α-synuclein aggregation. In our quest to link tTGs distribution in the human brain to the hallmarks of PD pathology, we recently reported that catecholaminergic neurons in PD disease-affected brain areas display typical endoplasmic reticulum (ER) granules showing tTG immunoreactivity. In the present study, we set out to elucidate the nature of the interaction between tTG and the ER in PD pathogenesis, using retinoic-acid differentiated SH-SY5Y cells exposed to the PD-mimetic 1-methyl-4-phenylpyridinium (MPP(+)). Alike our observations in PD brain, MPP(+)-treated cells displayed typical TG-positive granules, that were also induced by other PD mimetics and by ER-stress inducing toxins. Additional immunocytochemical and biochemical investigation revealed that tTG is indeed associated to the ER, in particular at the cytoplasmic face of the ER. Upon MPP(+) exposure, additional recruitment of tTG toward the ER was found. In addition, we observed that MPP(+)-induced tTG activity results in transamidation of ER membrane proteins, like calnexin. Our data provide strong evidence for a, so far unrecognized, localization of tTG at the ER, at least in catecholaminergic neurons, and suggests that in PD activation of tTG may have a direct impact on ER function, in particular via post-translational modification of ER membrane proteins.

Apolipoprotein E and LRP1 Increase Early in Parkinson's Disease Pathogenesis.

Parkinson's disease (PD) is characterized by α-synuclein-containing Lewy bodies (LBs) and loss of melanized neurons in the substantia nigra (SN). Recently, a link between apolipoprotein E (ApoE) expression, α-synuclein aggregation, and neurodegeneration was suggested. Here, we report on ApoE expression appearing in melanized neurons of the SN and in LBs in both PD and incidental LB disease cases. Interestingly, increased expression of the low-density lipoprotein receptor-related protein 1 (the receptor for ApoE) was also observed in incidental LB disease and PD. Our data suggest that alterations in lipoprotein homeostasis/signaling in melanized neurons of the SN are an early event during PD pathogenesis.

Tissue transglutaminase: a novel therapeutic target in cerebral amyloid angiopathy.

Accumulation of amyloid-ß (Aß) in brain vessel walls, known as cerebral amyloid angiopathy (CAA), plays a key role in Alzheimer's disease pathogenesis. CAA might result from impaired transport of Aß out of the brain. Although the mechanisms underlying reduced Aß transport are largely unknown, thickening of basement membrane extracellular matrix (ECM) is likely involved. Tissue transglutaminase (tTG) is an enzyme capable of modulating the ECM by covalently cross-linking ECM proteins. Recently, our group found that tTG and its cross-linking activity are associated with CAA pathology, suggesting a role for tTG in ECM modulation in CAA. Therefore, inhibition of tTG activity might be a promising novel therapeutic target to counteract CAA.