Ana Luísa Carvalho

Role of the brain‐derived neurotrophic factor at glutamatergic synapses

The neurotrophin brain‐derived neurotrophic factor (BDNF) plays an important role in the activity‐dependent regulation of synaptic structure and function, particularly of the glutamatergic synapses. BDNF may be released in the mature form, which activates preferentially TrkB receptors, or as proBDNF, which is coupled to the stimulation of the p75NTR. In the mature form BDNF induces rapid effects on glutamate release, and may induce short‐ and long‐term effects on the postsynaptic response to the neurotransmitter. BDNF may affect glutamate receptor activity by inducing the phosphorylation of the receptor subunits, which may also affect the interaction with intracellular proteins and, consequently, their recycling and localization to defined postsynaptic sites. Stimulation of the local protein synthesis and transcription activity account for the delayed effects of BDNF on glutamatergic synaptic strength. Several evidences show impaired synaptic plasticity of glutamatergic synapses in diseases where compromised BDNF function has been observed, such as Huntingtons disease, depression, anxiety, and the BDNF polymorphism Val66Met, suggesting that upregulating BDNF‐activated pathways may be therapeutically relevant. This review focuses on recent advances in the understanding of the regulation of the glutamatergic synapse by BDNF, and its implications in synaptic plasticity.

Bdnf regulates the expression and traffic of NMDA receptors in cultured hippocampal neurons

The neurotrophin BDNF regulates the activity-dependent modifications of synaptic strength in the CNS. Physiological and biochemical evidences implicate the NMDA glutamate receptor as one of the targets for BDNF modulation. In the present study, we investigated the effect of BDNF on the expression and plasma membrane abundance of NMDA receptor subunits in cultured hippocampal neurons. Acute stimulation of hippocampal neurons with BDNF differentially upregulated the protein levels of the NR1, NR2A and NR2B NMDA receptor subunits, by a mechanism sensitive to transcription and translation inhibitors. Accordingly, BDNF also increased the mRNA levels for NR1, NR2A and NR2B subunits. The neurotrophin NT3 also upregulated the protein levels of NR2A and NR2B subunits, but was without effect on the NR1 subunit. The amount of NR1, NR2A and NR2B proteins associated with the plasma membrane of hippocampal neurons was differentially increased by BDNF stimulation for 30 min or 24 h. The rapid upregulation of plasma membrane-associated NMDA receptor subunits was correlated with an increase in NMDA receptor activity. The results indicate that BDNF increases the abundance of NMDA receptors and their delivery to the plasma membrane, thereby upregulating receptor activity in cultured hippocampal neurons.

Photoacoustic Measurements of Porphyrin Triplet-State Quantum Yields and Singlet-Oxygen Efficiencies

Photoacoustic calorimetry was used to measure the quantum yields of singlet molecular oxygen production by the triplet states of tetraphenylporphyrin (TPP), ZnTPP and CuTPP in toluene, yielding values of 0.67 0.14, 0.68 0.19 and 0.03 0.01, respectively. We show that a novel dichlorophenyl derivative of ZnTPP is capable of singlet-oxygen production with a 0.90 0.07 quantum yield. The synthesis and characterisation of a new photostable chlorin with high absorptivity in the red that is capable of singlet-oxygen production with 0.54 0.06 quantum yield is described. Our results suggest that chlorinated chlorins may be interesting new sensitisers for photodynamic therapy.

In vivo and in vitro experiments show that betaxolol is a retinal neuroprotective agent

The aim of the study was to determine whether betaxolol is a neuroprotective agent and can therefore slow down the changes seen in the retina following ischaemia/reperfusion. Ischaemia was induced in one rat eye by raising the intraocular pressure for 45 min. Three days later electroretinograms were recorded from both eyes and the retinas were examined immunohistochemically for the localisation of calretinin and choline acetyltransferase (ChAT) immunoreactivities. The effect of glutamate agonists, hypoxia or experimental ischaemia was examined on the GABA immunoreactivity, lactate dehydrogenase (LDH) and internal calcium levels ([Ca2+]i) of the isolated rabbit retina, rat cortical cultures and chick retinal cell cultures respectively. Betaxolol was tested to see whether it can attenuate the influence of the glutamate agonists, hypoxia or experimental ischaemia. Ischaemia for 45 min causes a change in the nature of the normal calretinin immunoreactivity, an obliteration of the ChAT immunoreactivity and a drastic reduction in the b-wave of the electroretinogram after 3 days of reperfusion. When betaxolol was injected i.p. into the rats before ischaemia and on the days of reperfusion the changes to the calretinin and ChAT immunoreactivities were reduced and the reduction of the b-wave was prevented. Rabbit retinas incubated in vitro in physiological solution lacking oxygen/glucose or containing the glutamate agonists kainate or NMDA caused a change in the nature of the GABA immunoreactivity. Inclusion of betaxolol partially prevented the changes caused by NMDA and lack of oxygen/glucose. Rat cortical cultures exposed to glutamate or hypoxia/reoxygenation resulted in a release of LDH. The release of the enzyme was almost completely attenuated when betaxolol was included in the culture medium. Kainate increased the [Ca2+]i in chick retinal cultures, as measured with Indo-1. In a medium with sodium, this kainate-induced elevation of [Ca2+]i was significantly reduced by betaxolol. The combined data show that betaxolol is a neuroprotective agent and attenuates the effects on the retina induced by raising the intraocular pressure to simulate an ischaemic insult as may occur in glaucoma.

Regulation of AMPA receptors and synaptic plasticity.

Neuronal activity controls the strength of excitatory synapses by mechanisms that include changes in the postsynaptic responses mediated by AMPA receptors. These receptors account for most fast responses at excitatory synapses of the CNS, and their activity is regulated by various signaling pathways which control the electrophysiological properties of AMPA receptors and their interaction with numerous intracellular regulatory proteins. AMPA receptor phosphorylation/dephosphorylation and interaction with other proteins control their recycling and localization to defined postsynaptic sites, thereby regulating the strength of the synapse. This review focuses on recent advances in the understanding of the molecular mechanisms of regulation of AMPA receptors, and the implications in synaptic plasticity.

Polyglutamine diseases: the special case of ataxin-3 and Machado-Joseph disease.

Polyglutamine (polyQ) diseases are a group of nine neurodegenerative disorders caused by an unstable CAG expansion in the codifying region of their respective associated genes. However, each polyQ disease displays a different symptomatic and pathoanatomic profile and the proteins involved share no homology outside the polyQ tract. This suggests that the other regions of the proteins and the cellular functions they mediate are important in defining disease progression and specificity. Machado-Joseph disease (MJD), the most common form of spinocerebellar ataxia worldwide, is a progressive and ultimately fatal neurodegenerative disorder caused by polyQ expansion in ataxin-3 (atx3), a conserved and ubiquitous protein known to bind polyubiquitin chains and to function as a deubiquitinating enzyme. Atx3 has been linked to protein homeostasis maintenance, transcription, cytoskeleton regulation and myogenesis, but its precise biologic function remains a mystery, limiting the understanding of the mechanisms by which the mutated protein leads to the selective neuronal death profile observed in MJD patients. A number of recent evidence support the idea that the toxic entities behind neuronal demise may be either the dysfunctional expanded atx3 or the soluble amyloid-like oligomers formed by self-assembly of the aggregation-prone mutated protein. Expanded atx3 pathogenicity is likely the result of a series of events implicating both atx3 dysfunction and aggregation, possibly involving both full-length atx3 and polyQ-containing fragments that may act as seeds for protein aggregation. A deeper understanding of polyQ protein biology, the way the expansion alters their features, and the consequences of these changes for cell functioning and survival are sure to be of critical importance for developing future treatment of polyQ diseases.

Leptin regulates AMPA receptor trafficking via PTEN inhibition

The hormone leptin can cross the blood–brain barrier and influences numerous brain functions (Harvey, 2007). Indeed, recent studies have demonstrated that leptin regulates activity-dependent synaptic plasticity in the CA1 region of the hippocampus (Shanley et al., 2001; Li et al., 2002; Durakoglugil et al., 2005; Moult et al., 2009). It is well documented that trafficking of AMPA receptors is pivotal for hippocampal synaptic plasticity (Collingridge et al., 2004), but there is limited knowledge of how hormonal systems like leptin influence this process. In this study we have examined how leptin influences AMPA receptor trafficking and in turn how this impacts on excitatory synaptic function. Here we show that leptin preferentially increases the cell surface expression of GluR1 and the synaptic density of GluR2-lacking AMPA receptors in adult hippocampal slices. The leptin-induced increase in surface GluR1 required NMDA receptor activation and was associated with an increase in cytoplasmic PtdIns(3,4,5)P3 levels. In addition, leptin enhanced phosphorylation of the lipid phosphatase PTEN which inhibits PTEN function and elevates PtdIns(3,4,5)P3 levels. Moreover, inhibition of PTEN mimicked and occluded the effects of leptin on GluR1 trafficking and excitatory synaptic strength. These data indicate that leptin, via a novel pathway involving PTEN inhibition, promotes GluR1 trafficking to hippocampal synapses. This process has important implications for the role of leptin in hippocampal synaptic function in health and disease.

Regulation of AMPA receptors by phosphorylation.

The AMPA receptors for glutamate are oligomeric structures that mediate fast excitatory responses in the central nervous system. Phosphorylation of AMPA receptors is an important mechanism for short-term modulation of their function, and is thought to play an important role in synaptic plasticity in different brain regions. Recent studies have shown that phosphorylation of AMPA receptors by cAMP-dependent protein kinase (PKA) and Ca2+ - and calmodulin-dependent protein kinase II (CaMKII) potentiates their activity, but phosphorylation of the receptor subunits may also affect their interaction with intracellular proteins, and their expression at the plasma membrane. Phosphorylation of AMPA receptor subunits has also been investigated in relation to processes of synaptic plasticity. This review focuses on recent advances in understanding the molecular mechanisms of regulation of AMPA receptors, and their implications in synaptic plasticity.

Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study, we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors (LV), to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD. LV encoding mutant or wild-type human ataxin-3 was injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue. In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; alpha-synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and alpha-synuclein. This study demonstrates the use of LV encoding mutant ataxin-3 to produce a model of MJD and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapies.

Ischemic insults induce necroptotic cell death in hippocampal neurons through the up-regulation of endogenous RIP3

Global cerebral ischemia induces selective acute neuronal injury of the CA1 region of the hippocampus. The type of cell death that ensues may include different programmed cell death mechanisms namely apoptosis and necroptosis, a recently described type of programmed necrosis. We investigated whether necroptosis contributes to hippocampal neuronal death following oxygen-glucose deprivation (OGD), an in vitro model of global ischemia. We observed that OGD induced a death receptor (DR)-dependent component of necroptotic cell death in primary cultures of hippocampal neurons. Additionally, we found that this ischemic challenge upregulated the receptor-interacting protein kinase 3 (RIP3) mRNA and protein levels, with a concomitant increase of the RIP1 protein. Together, these two related proteins form the necrosome, the complex responsible for induction of necroptotic cell death. Interestingly, we found that caspase-8 mRNA, a known negative regulator of necroptosis, was transiently decreased following OGD. Importantly, we observed that the OGD-induced increase in the RIP3 protein was paralleled in an in vivo model of transient global cerebral ischemia, specifically in the CA1 area of the hippocampus. Moreover, we show that the induction of endogenous RIP3 protein levels influenced neuronal toxicity since we found that RIP3 knock-down (KD) abrogated the component of OGD-induced necrotic neuronal death while RIP3 overexpression exacerbated neuronal death following OGD. Overexpression of RIP1 also had deleterious effects following the OGD challenge. Taken together, our results highlight that cerebral ischemia activates transcriptional changes that lead to an increase in the endogenous RIP3 protein level which might contribute to the formation of the necrosome complex and to the subsequent component of necroptotic neuronal death that follows ischemic injury.