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Target-derived molecules are essential for the maintenance of neuron survival. In the present work, we introduce the electric organ of Torpedo marmorata as a tool for the study of trophic interactions in a polyinnervate system. This electric organ maintains a large number of cholinergic terminals on the postsynaptic cell surface. We have observed that a soluble extract derived from the electric organ induces the maturation of Xenopus oocytes injected with presynaptic plasma membranes (PSPM), indicating that a trophic system may exist. Moreover, we have detected a p75(NGFR) related protein in PSPM by Western blot analysis. These results suggest the presence of a neurotrophin-related system maintaining the polyinnervate electric organ. Furthermore, molecular experiments showed that the brain-derived neurotrophic factor (BDNF) is the neurotrophin operating in our model. Using degenerate oligonucleotides which comprise a conserved fragment of all neurotrophins, we have only amplified by polymerase chain reaction a BDNF fragment. In a similar way, we have amplified and cloned a fragment of the TrkB/C high affinity BDNF receptor. The fact that degenerate oligonucleotides only amplify BDNF allows us to conclude that the polyinnervation is maintained by this neurotrophin either alone or in combination with other trophic factors., This work was supported by grants from DGCYT.

A cDNA (alpha T6) encoding an alpha-tubulin from Torpedo marmorata (Tm) was isolated and sequenced. The deduced 451-amino-acid (aa) sequence codes for an alpha-tubulin of 50,161 Da. The aa sequence of alpha T6 of Tm showed a 70-99.6% identity to the other alpha-tubulins previously described. Moreover, the alpha T6 aa sequence was 95-99.6% identical to neural-specific tubulins of mouse, rat, human and siberian salmon. The corresponding mRNA is highly represented in the giant motoneurons of the electric lobe. All neuronal populations of the Tm brain exhibit variable levels of alpha T6 expression, with the highest levels in the long-axon-projecting neurons. These results suggest that this alpha-tubulin isoform may play an important role in the maintenance and/or remodeling of the neuronal cytoskeleton.

Botulinum neurotoxin type A blocks acetylcholine release from the peripheral nervous system. We have previously described a putative botulinum neurotoxin type A receptor of presynaptic plasma membranes from Torpedo. The electric organ of Torpedo, which is largely enriched in cholinergic nerve endings, is homologous to the neuromuscular junction, allowing us to isolate large scale of presynaptic components. In order to characterize this protein we have raised a polyclonal antibody (a-P140) against this receptor. The antiserum a-P140 recognizes a 140,000 mol. wt band in non-reducing conditions and an 80,000 band in reducing conditions. The immunohistochemistry assay reveals the P140 protein on the ventral face of the electrocytes where the nerve terminals are localized. Moreover, a-P140 antiserum recognizes the P140-BoNT/A complex after binding and cross-linking experiments. In addition, we have immunoprecipitated an in vitro translated product which is closely coincident in mol. wt to the 80,000 band of the receptor.

In order to examine the role of cysteine (Cys)-rich domains in the accumulation of maize (Zea mays L.) γ-zein within the endoplasmic-reticulum-derived protein bodies, we studied the localization of γ-zein and of two truncated forms of γ-zein in Xenopus laevis oocytes. The two derivatives were constructed from a DNA encoding the γ-zein: one by deletion of the Pro-X linker region (21 amino acids) and the other by deletion of the Cys-rich domain (94 amino acids). In-vitro-synthesized transcripts were injected into oocytes and the distribution of the translation products was then analyzed. The entire γ-zein and both truncated forms of the γ-zein had accumulated efficiently in microsomes and no traces of secretion were observed. We suggest that neither C-terminal Cys-rich nor Pro-X domains are essential for γ-zein retention in oocyte vesicles. Therefore, structural features derived from disulphide bonds are not necessary for γ-zein targeting on the endoplasmic reticulum., This research was supported by grants from the Comisidn Interministerial de Ciencia y Tecnologia (CICYT), Bio-90 (to P.P) and BIO 92/186 (to M.D.L.). M.I.G. and J.M.C. are recipient of a postgraduate fellowship from the Formaci6n del Personal Investigador (FPI).

Two artificial models for acetylcholine secretion have been used in order to study if they are sensitive to the action of Botulinum toxin type A. We have used the electric organ of Torpedo as the source to obtain subcellular components of the cholinergie nerve terminals. Giant proteoliposomes which have incorporated presynaptic plasma membrane proteins are able to translocate acetylcholine in a calcium dependent manner, in the presence of A23187 calcium ionophore. This transport is sensitive to BoNT/A. In addition the fluidity of such artificial membrane is also sensitive to BoNT/A, suggesting that BoNT/A may inhibit ACh release by making synaptic membranes more rigid. The second model utilized is Xenopus oocytes injected with cholinergie synaptic vesicles and presynaptic plasma membrane. These injected cells are able to release ACh under potassium depolarization or by A23187 ionophore. BoNT/A is capable to inhibit such depolarization-induced release.

Human Dopamine- and cAMP-regulated phosphoprotein of molecular weight 32kDa (DARPP-32, also known as PPP1R1B) gene codes for different transcripts that are mainly translated into two DARPP-32 protein isoforms, full length (fl)-DARPP-32 and truncated (t)-DARPP. The t-DARPP lacks the first 36 residues at the N-terminal, which alters its function. In the central nervous system, fl-DARPP-32 is highly expressed in GABAergic striatal medium spiny neurons (MSNs), where it integrates dopaminergic and glutamatergic input signaling. However, no information about human DARPP-32 isoform expression during MSNs maturation is available. In this study, our aim is to determine the expression of the two DARPP-32 isoforms in human fetal and adult striatal samples. We show that DARPP-32 isoform expression is differentially regulated during human striatal development, with the t-DARPP isoform being virtually absent from whole ganglionic eminence (WGE) and highly induced in the adult striatum (in both caudate and putamen). We next compared the four most common anti-DARPP-32 antibodies used in human specimens, to study their recognition of the two isoforms in fetal and adult human striatal samples by western blot and immunohistochemistry. The four antibodies specifically identify the fl-DARPP-32 in both fetal and adult samples, while t-DARPP form was only detected in adult striatal samples. In addition, the lack of t-DARPP recognition in human adult striatum by the antibody generated against the full-length domain produces in turn different efficacy by immunohistochemical analysis. In conclusion, our results show that expression of human DARPP-32 protein isoforms depends on the striatal neurodevelopmental stage with t-DARPP being specific for the human adult striatum.

A growing body of evidence indicates that the fundamental molecular mechanism of exocytosis in the secretory pathway may be structurally similar in all eukaryotic cells. The synaptosomal-associated protein of 25 kDa (SNAP-25) is a plasma membrane protein involved in regulated exocytosis in neurons. In order to compare exocytotic components in neurons and endocrine cells, we have analyzed the expression of SNAP-25 in the rat anterior pituitary. Western blotting analysis documented the presence of SNAP-25 in anterior pituitary homogenates and cultured anterior pituitary cells. In addition to SNAP-25, other neuronal proteins involved in exocytosis (syntaxin, VAMP/synaptobrevin and Rab3A) were also detected in the anterior pituitary. The specific expression of SNAP-25 mRNA in anterior pituitary cells was also corroborated by Northern analysis. SNAP-25 immunoreactivity was located at the plasma membrane of endocrine anterior pituitary cells. Characteristically, patches of fine punctate deposits exhibited intense SNAP-25 immunoreactivity. Double-labeling immunocytochemistry revealed that SNAP-25 was mainly associated with gonadotroph cell populations. Furthermore, we demonstrate that in the anterior pituitary, SNAP-25 is selectively cleaved by clostridial neurotoxins. In conclusion, our results establish the presence of SNAP-25 in secretory anterior pituitary cells and suggest a potential role of this protein in the secretion of adenohypophysial hormone.

The first derivation of human embryonic stem cells (hESCs) (Thomson et al., 1998) and the more recently development of human induced pluripotent stem cells (iPSCs) (Park et al., 2008; Takahashi et al., 2007; Takahashi & Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007) have marked the beginning of a new era in biomedical research. These two types of human pluripotent stem cells (hPSCs) are characterized by an unlimited capacity to selfrenew while retaining their potential to differentiate into almost all cell types of the body (Odorico et al., 2001; Reubinoff et al., 2000; Silva & Smith, 2008). These remarkable properties turn hPSCs into one of the most interesting cell types for toxicology and drug discovery, tissue engineering and regenerative medicine (Battey, 2007; Mountford, 2008). In fact, work with hPSCs has already provided new and exciting developments that may eventually lead to the creation of novel cell-based therapies for the treatment of a wide range of human diseases including Parkinson’s and other neurodegenerative diseases, diabetes, cardiac and vascular diseases (Kiskinis & Eggan, 2010; Ronaghi et al., 2010). However, a major challenge for the widespread application of hPSCs is the development of efficient protocols for cryopreservation...

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson’s disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients’ genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type., AS-D was partially supported by a pre-doctoral fellowship from MEC. Additional support was provided by grants from MICINN (BFU2009- 13277, PLE2009-0144 and ACI2010-1117 to AR; RyC-2008- 02772 and BFU2010-21823 to AC; SAF2008-04360, to JA; SAF2009-07774 and PLE2009-0089, to JMC), FIS (PI10/00849 to MV; RD06/0010/0006 to JMC), NIH/NIA AG031782/ AG038072 (to AMC) and Fondazione Guido Berlucchi (to AC). The Cell Therapy Program was supported by the CMRB (Promt-0901 to JMC). The work was also part of a CIBERNED Cooperative Project (to JA, JL-B, MV, ET and AR).

Neurodegenerative disorders affecting the central nervous system, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s chorea (HD) and amyotrophic lateral sclerosis are characterized by the loss of selected neuronal populations. Another striking feature shared by these diseases is the deposition of proteinaceous inclusion bodies in the brain, which may be intracytoplasmatic or intranuclear, or even extracellular. However, the density and prevalence of aggregates are not always directly related to neurodegeneration. Although some of these diseases are the result of mutations in known proteins, with HD a clear example, the expression and location of the affected protein do not explain the selective neurodegeneration. Therefore, other intrinsic mechanisms, characteristic of each neuronal population, might be involved in the neurodegenerative process. In this review we focus on several proposed mechanisms such as excitotoxicity, mitochondrial dysfunction and altered expression of trophic factors, which could account for the pathogenesis of HD.

In the present study we have evaluated changes in nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophin 3 (NT-3) mRNA expression induced by different glutamate receptor agonists injected into the neostriatum. Up-regulation of NGF expression was observed at 24 h after intrastriatal quinolinate injection, an N -methyl- d -aspartate receptor agonist, and this increase was maintained up to 7 days after lesion. NGF up-regulation was also apparent in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) treatment from 6 to 16 h postinjection. Instead, BDNF was up-regulated only at 6 h after kainate or AMPA excitotoxicity. Interestingly, NT-3 mRNA was down-regulated from 10 to 16 h following AMPA lesion, while 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid injection enhanced NT-3 mRNA levels at 10 h. Our results show a specific neurotrophin response induced by stimulation of each glutamate receptor. These activity-dependent changes might be involved in neuronal plasticity processes and may underlie the differential vulnerability of striatal neurons observed in neurodegenerative disorders.