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Papers by Luís Pereira Almeida

Molecular Therapy - Nucleic Acids, 2013

The present work aimed at the development and application of a lipid-based nanocarrier for target... more The present work aimed at the development and application of a lipid-based nanocarrier for targeted delivery of nucleic acids to glioblastoma (GBM). For this purpose, chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells while showing no affinity for non-neoplastic cells, was covalently coupled to liposomes encapsulating antisense oligonucleotides (asOs) or small interfering RNAs (siRNAs). The resulting targeted nanoparticles, designated CTX-coupled stable nucleic acid lipid particles (SNALPs), exhibited excellent features for in vivo application, namely small size (<180 nm) and neutral surface charge. Cellular association and internalization studies revealed that attachment of CTX onto the liposomal surface enhanced particle internalization into glioma cells, whereas no significant internalization was observed in noncancer cells. Moreover, nanoparticle-mediated miR-21 silencing in U87 human GBM and GL261 mouse glioma cells resulted in increased levels of the tumor suppressors PTEN and PDCD4, caspase 3/7 activation and decreased tumor cell proliferation. Preliminary in vivo studies revealed that CTX enhances particle internalization into established intracranial tumors. Overall, our results indicate that the developed targeted nanoparticles represent a valuable tool for targeted nucleic acid delivery to cancer cells. Combined with a drug-based therapy, nanoparticle-mediated miR-21 silencing constitutes a promising multimodal therapeutic approach towards GBM.

Neurobiology of Disease, 2001

Neurodegenerative diseases represent promising targets for gene therapy approaches provided effec... more Neurodegenerative diseases represent promising targets for gene therapy approaches provided effective transfer vectors. In the present study, we evaluated the effectiveness of LacZ-expressing lentiviral vectors with two different internal promoters, the mouse phosphoglycerate kinase 1 (PGK) and cytomegalovirus (CMV), to infect striatal cells. The intrastriatal injection of lenti-␤-Gal vectors lead to 207, 400 ؎ 11,500 and 303,100 ؎ 4,300 infected cells in adult rats, respectively. Importantly, the ␤-galactosidase activity was higher in striatal extracts from PGK-LacZ-injected animals as compared to CMV-LacZ animals. The efficacy of the system was further examined with a potential therapeutic gene for the treatment of Huntington's disease, the human ciliary neurotrophic factor (CNTF). PGK-LacZ-or PGK-CNTF-expressing viruses were stereotaxically injected into the striatum of rats, 3 weeks later the animals were unilaterally lesioned with 180 nmol of quinolinic acid (QA). Control animals displayed 148 ؎ 43 apomorphine-induced rotations ipsilateral to the lesion 5 days postlesion as compared to 26 ؎ 22 turns/45 min in the CNTF-treated group. The extent of the striatal damage was significantly diminished in the CNTF-treated rats as indicated by the 52 ؎ 9.7% decrease of the lesion volume and the sparing of DARPP-32, ChAT and NADPH-d neuronal populations. These results further establish that lentiviruses may represent an efficient gene delivery system for the screening of therapeutic molecules in Huntington's disease.

The Journal of Gene Medicine, 2007

BackgroundRNA interference provides a powerful technology for specific gene silencing. Therapeuti... more BackgroundRNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation, protection, and extra‐ and intracellular delivery of these nucleic acids. Here, we evaluated the potential of transferrin (Tf)‐associated liposomes for siRNA complexation and gene silencing.MethodsCationic liposomes composed of DOTAP : Cholesterol associated with or without transferrin (Tf) were complexed with siRNA at different lipid/siRNA charge ratios. Complexation and protection of siRNA from enzymatic degradation was assessed with the PicoGreen intercalation assay and gel electrophoresis. Cellular internalization of these siRNA Tf‐lipoplexes was detected by confocal microscopy. Luciferase assay, immunoblot and fluorescence‐activated cell sorting (FACS) analysis were used to evaluate reporter gene silencing in Huh‐7 hepatocarcinoma and U‐373 glioma cells. c‐Jun knockdown in HT‐...

Journal of Controlled Release, 2008

Although RNAi-based gene silencing holds a great potential for treatment of neurological disorder... more Although RNAi-based gene silencing holds a great potential for treatment of neurological disorders, its application to the CNS has been restricted by low levels of tissue distribution and cellular uptake. In this work we report that cationic lipid-based vectors can enhance siRNA delivery to neurons both in vitro and in vivo. DOTAP:Chol liposomes associated with transferrin (Tf) and complexed with siRNAs (Tf-lipoplexes) were delivered to primary cultures of luciferase-expressing cortical neurons. Confocal microscopy studies revealed efficient cellular uptake of Cy3-labelled siRNAs after Tf-lipoplex delivery, which was reduced but not completely inhibited by blocking the Tf-receptor with excess Tf. Gene silencing was also evaluated after delivery of anti-luciferase or anti-c-Jun siRNAs. Our results demonstrate that Tf-lipoplexes achieve up to 50% luciferase and c-Jun knockdown, 48 h after transfection, without significant cytotoxicity. Similar results were observed in vivo, where a 40% reduction of luciferase activity was found in the striatum of luciferase mice. In addition, fluorescence microscopy studies showed extensive local distribution and internalization of Tflipoplex-associated Cy3-siRNAs without tissue toxicity. Overall, our results demonstrate that Tf-lipoplexes can mediate efficient gene silencing in neuronal cells, both in vitro an in vivo, which may prove useful in therapeutic approaches to neuronal protection and repair.

Gene Therapy, 2005

The development of efficient systems for in vivo gene transfer to the central nervous system (CNS... more The development of efficient systems for in vivo gene transfer to the central nervous system (CNS) may provide a useful therapeutic strategy for the alleviation of several neurological disorders. In this study, we evaluated the feasibility of nonviral gene therapy to the CNS mediated by cationic liposomes. We present evidence of the successful delivery and expression of both a reporter and a therapeutic gene in the rodent brain, as evaluated by immunohistochemical assays. Our results indicate that transferrin-associated cationic liposome/DNA complexes (Tf-lipoplexes) allow a significant enhancement of transfection activity as compared to plain complexes, and that 8/1 (+/À) Tf-lipoplexes constitute the best formulation to mediate in vivo gene transfer. We demonstrated that Tf-lipoplex-mediated nerve growth factor transgene expression attenuates the morphological damages of the kainic acid-induced lesion as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) vital staining. These findings suggest the usefulness of these lipid-based vectors in mediating the delivery of therapeutic genes to the CNS.

European Journal of Neuroscience, 2001

Ciliary neurotrophic factor prevents behavioural de®cits and striatal degeneration in rat and pri... more Ciliary neurotrophic factor prevents behavioural de®cits and striatal degeneration in rat and primate models of Huntington's disease. Interleukin-6, another member of the cytokine family, and the chimeric molecule (IL6/IL6R) in which interleukin-6 and its soluble receptor are fused, have been shown to exert trophic action on various neuronal populations in the central nervous system. Therefore, we investigated the neuroprotective effect of these two molecules in the quinolinic acid model of Huntington's disease. LacZ-, interleukin-6-and IL6/IL6R-expressing lentiviral vectors were stereotaxically injected into the striatum of Wistar rats. Three weeks later the animals were lesioned through the intrastriatal injection of 180 nmol of quinolinic acid. The extent of the striatal damage was signi®cantly diminished in the rats that had been treated with interleukin-6 or IL6/IL6R. The neuroprotective effect was, however, more pronounced with the IL6/IL6R chimera than with interleukin-6 as indicated by the volume of the lesions (38.6 T 10% in the IL6/IL6R group, 63.3 T 3.6% in the IL-6 group and 84.3 T 2.9% in the control group). Quantitative analysis of striatal interneurons further demonstrated that the IL6/IL6R chimera is more neuroprotective than IL-6 on ChAT-and NADPH-d-immunoreactive neurons. These results suggest that the IL6/IL6R chimera is a potential treatment for Huntington's disease.

Human Gene Therapy, 2002

The ability to regulate gene expression constitutes a prerequisite for the development of gene th... more The ability to regulate gene expression constitutes a prerequisite for the development of gene therapy strategies aimed at the treatment of neurologic disorders. In the present work, we used tetracycline (Tet)-regulated lentiviral vectors to investigate the dose-dependent neuroprotective effect of human ciliary neurotrophic factor (CNTF) in the quinolinic acid (QA) model of Huntington's disease (HD). The Tet system was split in two lentiviruses, the first one containing the CNTF or green fluorescent protein (GFP) cDNAs under the control of the Tet-response element (TRE) and a second vector encoding the transactivator (tTA). Preliminary coinfection study demonstrated that 63.8% 6 2.0% of infected cells contain at least two viral copies. Adult rats were then injected with CNTF-and GFP-expressing viral vectors followed 3 weeks later by an intrastriatal administration of QA. A significant reduction of apomorphine-induced rotations was observed in the CNTFon group. In contrast, GFP-treated animals or CNTF-off rats displayed an ipsilateral turning behavior in response to apomorphine. A selective sparing of DARPP-32-, choline acetyltransferase (ChAT)-, and NADPHd-positive neurons was observed in the striatum of CNTF-on rats compared to GFP animals and CNTF-off group. Enzyme-linked immunosorbent assay (ELISA) performed on striatal samples of rats sacrificed at the same time point indicated that this neuroprotective effect was associated with the production of 15.5 6 4.7 ng CNTF per milligram of protein whereas the residual CNTF expression in the off state (0.54 6 0.02 ng/mg of protein) was not sufficient to protect against QA toxicity. These results establish the proof of principle of neurotrophic factor dosing for neurodegenerative diseases and demonstrate the feasibility of lentiviral-mediated tetracycline-regulated gene transfer in the brain.

Human Molecular Genetics, 2019

Machado–Joseph disease or spinocerebellar ataxia type 3 is an inherited neurodegenerative disease... more Machado–Joseph disease or spinocerebellar ataxia type 3 is an inherited neurodegenerative disease associated with an abnormal glutamine over-repetition within the ataxin-3 protein. This mutant ataxin-3 protein affects several cellular pathways, leading to neuroinflammation and neuronal death in specific brain regions resulting in severe clinical manifestations. Presently, there is no therapy able to modify the disease progression. Nevertheless, anti-inflammatory pharmacological intervention has been associated with positive outcomes in other neurodegenerative diseases. Thus, the present work aimed at investigating whether ibuprofen treatment would alleviate Machado–Joseph disease.We found that ibuprofen-treated mouse models presented a significant reduction in the neuroinflammation markers, namely Il1b and TNFa mRNA and IKB-α protein phosphorylation levels. Moreover, these mice exhibited neuronal preservation, cerebellar atrophy reduction, smaller mutant ataxin-3 inclusions and moto...

Pharmaceutics, 2021

A major bottleneck in the successful development of central nervous system (CNS) drugs is the dis... more A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit der...

Communications biology, May 19, 2022

Cell membrane-based biovesicles (BVs) are important candidate drug delivery vehicles and comprise... more Cell membrane-based biovesicles (BVs) are important candidate drug delivery vehicles and comprise extracellular vesicles, virus-like particles, and lentiviral vectors. Here, we introduce a non-enzymatic assembly of purified BVs, supercharged proteins, and plasmid DNA called pDNA-scBVs. This multicomponent vehicle results from the interaction of negative sugar moieties on BVs and supercharged proteins that contain positively charged amino acids on their surface to enhance their affinity for pDNA. pDNA-scBVs were demonstrated to mediate floxed reporter activation in culture by delivering a Cre transgene. We introduced pDNA-scBVs containing both a CRE-encoding plasmid and a BV-packaged floxed reporter into the brains of Ai9 mice. Successful delivery of both payloads by pDNA-scBVs was confirmed with reporter signal in the striatal brain region. Overall, we developed a more efficient method to load isolated BVs with cargo that functionally modified recipient cells. Augmenting the natural properties of BVs opens avenues for adoptive extracellular interventions using therapeutic loaded cargo.

Neuropathology and Applied Neurobiology, 2021

Aims: Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), is the most common a... more Aims: Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), is the most common autosomal dominantly-inherited ataxia worldwide and is characterised by the accumulation of mutant ataxin-3 (mutATXN3) in different brain regions, leading to neurodegeneration. Currently, there are no available treatments able to block disease progression. In this study, we investigated whether carbamazepine (CBZ) would activate autophagy and mitigate MJD pathology. Methods: The autophagy-enhancing activity of CBZ and its effects on clearance of mutATXN3 were evaluated using in vitro and in vivo models of MJD. To investigate the optimal treatment regimen, a daily or intermittent CBZ administration was applied to MJD transgenic mice expressing a truncated human ATXN3 with 69 glutamine repeats. Motor behaviour tests and immunohistology was performed to access the alleviation of MJD-associated motor deficits and neuropathology. A retrospective study was conducted to evaluate the CBZ effect in MJD patients. Results: We found that CBZ promoted the activation of autophagy and the degradation of mutATXN3 in MJD models upon short or intermittent, but not daily prolonged, treatment regimens. CBZ up-regulated autophagy through activation of AMPK, which was dependent on the myo-inositol levels. In addition, intermittent CBZ treatment improved motor performance, as well as prevented neuropathology in MJD transgenic mice. However, in patients, no evident differences in SARA scale were found, which was not unexpected given the small number of patients included in the study. Conclusions: Our data support the autophagy-enhancing activity of CBZ in the brain and suggest this pharmacological approach as a promising therapy for MJD and other polyglutamine disorders.

Journal of Translational Medicine, 2020

Background Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is the most... more Background Machado–Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is the most common of the dominantly inherited ataxias worldwide and is characterized by mutant ataxin-3 aggregation and neuronal degeneration. There is no treatment available to block or delay disease progression. In this work we investigated whether trehalose, a natural occurring disaccharide widely used in food and cosmetic industry, would rescue biochemical, behavioral and neuropathological features of an in vitro and of a severe MJD transgenic mouse model. Methods Two MJD animal models, a lentiviral based and a transgenic model, were orally treated with 2% trehalose solution for a period of 4 and 30 weeks, respectively. Motor behavior (rotarod, grip strength and footprint patterns) was evaluated at different time points and neuropathological features were evaluated upon in-life phase termination. Results Trehalose-treated MJD mice equilibrated for a longer time in the rotarod apparatus and exh...

The Journal of Neuroscience, 2002

XXI program). We thank Xiao-Jiang Li, Lesley Jones, Paul Greengard, and Hugh C. Hemmings for gene... more XXI program). We thank Xiao-Jiang Li, Lesley Jones, Paul Greengard, and Hugh C. Hemmings for generous gifts of antibodies and Jillian K. Cooper for the huntingtin expressing vectors. We also thank Albert Spicher, Yvan Arsenijevic, Fabienne Pidoux, Maria Rey, and Christel Sadeghi for contributions to this study and Etienne Régulier for critical reading of this manuscript.

Advances in experimental medicine and biology, 2018

Polyglutamine diseases are hereditary degenerative disorders of the nervous system that have rema... more Polyglutamine diseases are hereditary degenerative disorders of the nervous system that have remained, to this date, untreatable. Promisingly, investigation into their molecular etiology and the development of increasingly perfected tools have contributed to the design of novel strategies with therapeutic potential. Encouraging studies have explored gene therapy as a means to counteract cell demise and loss in this context. The current chapter addresses the two main focuses of research in the area: the characteristics of the systems used to deliver nucleic acids to cells and the molecular and cellular actions of the therapeutic agents. Vectors used in gene therapy have to satisfyingly reach the tissues and cell types of interest, while eliciting the lowest toxicity possible. Both viral and non-viral systems have been developed for the delivery of nucleic acids to the central nervous system, each with its respective advantages and shortcomings. Since each polyglutamine disease is cau...

Trends in Endocrinology & Metabolism, 2017

Ataxin-2 is a polyglutamine protein implicated in several biological processes such as RNA metabo... more Ataxin-2 is a polyglutamine protein implicated in several biological processes such as RNA metabolism and cytoskeleton reorganization. Ataxin-2 is highly expressed in various tissues including the hypothalamus, a brain region that controls food intake and energy balance. Ataxin-2 expression is influenced by nutritional status. Emerging studies discussed here now show that ataxin-2 deficiency correlates with insulin resistance and dyslipidemia, an action mediated via the mTOR pathway, suggesting that ataxin-2 might play key roles in metabolic homeostasis including body weight regulation, insulin sensitivity, and cellular stress responses. In this review we also discuss the relevance of ataxin-2 in the hypothalamic regulation of energy balance, and its potential as a therapeutic target in metabolic disorders such as obesity.

Brain, 2014

Machado-Joseph disease is a neurodegenerative disease without effective treatment. Patients with ... more Machado-Joseph disease is a neurodegenerative disease without effective treatment. Patients with Machado-Joseph disease exhibit significant motor impairments such as gait ataxia, associated with multiple neuropathological changes including mutant ATXN3 inclusions, marked neuronal loss and atrophy of the cerebellum. Thus, an effective treatment of symptomatic patients with Machado-Joseph disease may require cell replacement, which we investigated in this study. For this purpose, we injected cerebellar neural stem cells into the cerebellum of adult Machado-Joseph disease transgenic mice and assessed the effect on the neuropathology, neuroinflammation mediators and neurotrophic factor levels and motor coordination. We found that upon transplantation into the cerebellum of adult Machado-Joseph disease mice, cerebellar neural stem cells differentiate into neurons, astrocytes and oligodendrocytes. Importantly, cerebellar neural stem cell transplantation mediated a significant and robust alleviation of the motor behaviour impairments, which correlated with preservation from Machado-Joseph disease-associated neuropathology, namely reduction of Purkinje cell loss, reduction of cellular layer shrinkage and mutant ATXN3 aggregates. Additionally, a significant reduction of neuroinflammation and an increase of neurotrophic factors levels was observed, indicating that transplantation of cerebellar neural stem cells also triggers important neuroprotective effects. Thus, cerebellar neural stem cells have the potential to be used as a cell replacement and neuroprotective approach for Machado-Joseph disease therapy.

Additional file 1: Supplementary Table 1. Additional pathological data summary of analyzed postmo... more Additional file 1: Supplementary Table 1. Additional pathological data summary of analyzed postmortem tissue from patients

Polyglutamine (polyQ) diseases are a group of 9 rare neurodegenerative disorders caused by an abn... more Polyglutamine (polyQ) diseases are a group of 9 rare neurodegenerative disorders caused by an abnormal expansion of the CAG trinucleotide in the codifying regions of the respective disease-associated gene. The trinucleotide abnormal expansion leads to the translation of a protein containing an overexpanded tract of glutamines. PolyQ mutant proteins undergo a gain of toxic function disrupting normal cellular pathways leading to neuronal death and, consequently, leading to selective neurodegeneration of specific brain regions. Spinocerebellar ataxia (SCA) 2 and SCA3 (also known as Machado-Joseph disease) are two different polyQ diseases in which the ataxin-2 and ataxin-3 proteins, respectively, bear abnormally long polyQ tracts. Until now, there is no treatment for these fatal diseases or therapies that could delay the normal pathologic progression. Stress granules (SGs) are important structures formed in response to cellular stress, having an important role in mRNA triage. A core com...

Molecular Therapy - Nucleic Acids, 2013

Neurobiology of Disease, 2001

The Journal of Gene Medicine, 2007

Journal of Controlled Release, 2008

Gene Therapy, 2005

European Journal of Neuroscience, 2001

Human Gene Therapy, 2002

Human Molecular Genetics, 2019

Pharmaceutics, 2021

Communications biology, May 19, 2022

Neuropathology and Applied Neurobiology, 2021

Journal of Translational Medicine, 2020

The Journal of Neuroscience, 2002

Advances in experimental medicine and biology, 2018

Trends in Endocrinology & Metabolism, 2017

Brain, 2014

Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative ... more Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease.

Machado–Joseph disease, also known as spinocerebellar ataxia type 3, is the most common of the do... more Machado–Joseph disease, also known as spinocerebellar ataxia type 3, is the most common of the dominantly inherited ataxias worldwide and is characterized by mutant ataxin-3 misfolding, intracellular accumulation of aggregates and neuronal degener-ation. Here we investigated the implication of autophagy, the major pathway for organelle and protein turnover, in the accumulation of mutant ataxin-3 aggregates and neurodegeneration found in Machado–Joseph disease and we assessed whether specific stimulation of this pathway could mitigate the disease. Using tissue from patients with Machado–Joseph disease, transgenic mice and a lentiviral-based rat model, we found an abnormal expression of endogenous autophagic markers, accumulation of autophagosomes and decreased levels of beclin-1, a crucial protein in the early nucleation step of autophagy. Lentiviral vector-mediated overexpression of beclin-1 led to stimulation of autophagic flux, mutant ataxin-3 clearance and overall neuroprotective effects in neuronal cultures and in a lentiviral-based rat model of Machado–Joseph disease. These data demonstrate that autophagy is a key degradation pathway, with beclin-1 playing a significant role in alleviating Machado–Joseph disease pathogenesis. Abbreviations: Atg = autophagic related protein; DARPP-32 = dopamine-and-cyclic AMP-regulated phosphoprotein of 32 kDa; p62 = sequestosome 1/p62 protein

Machado–Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-... more Machado–Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinu-cleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado–Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado–Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado–Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado–Joseph disease. Abbreviations: AAV = adeno-associated virus; DARPP-32 = dopamine-and cyclic AMP-regulated neuronal phosphoprotein; GFP = green fluorescent protein