Number of Nanoparticles per Cell through a Spectrophotometric Method - A key parameter to Assess Nanoparticle-based Cellular Assays

Engineered nanoparticles (eNPs) for biological and biomedical applications are produced from functionalised nanoparticles (NPs) after undergoing multiple handling steps, giving rise to an inevitable loss of NPs. Herein we present a practical method to quantify nanoparticles (NPs) number per volume in an aqueous suspension using standard spectrophotometers and minute amounts of the suspensions (up to 1 μL). This method allows, for the first time, to analyse cellular uptake by reporting NPs number added per cell, as opposed to current methods which are related to solid content (w/V) of NPs. In analogy to the parameter used in viral infective assays (multiplicity of infection), we propose to name this novel parameter as multiplicity of nanofection.JJDM thanks Spanish Ministerio de Economía y Competitividad for a Ramon y Cajal Fellowship and for supporting this work partially by Grant CTQ2012-34778. This research was partially supported by Marie Curie Career Integration Grants within the 7th European Community Framework Programme (FP7-PEOPLE-2011-CIG-Project Number 294142 and FP7-PEOPLE-2012-CIG-Project Number 322276) to RMSM and JJDM, respectively. This research was partially supported by the Consejería de Economía, Innovación y Ciencia de la Junta de Andalucía (BIO-1778) to JJDM. RMSM and JDUB thank CEI Biotic Granada for funding P_BS_54 and mP_BS_37 projects. JDUB thanks Spanish Ministerio de Economía y Competitividad for a Torres Quevedo fellowship (PTQ-13-06046)

Expression of activity-dependent neuroprotective protein in the brain of adult rats

Activity-dependent neuroprotective protein (ADNP) is a VIP-regulated gene, which is essential for brain development. A synthetic peptide (NAP) derived from the ADNP sequence is highly neuroprotective, therefore it has been hypothesised that ADNP has a similar role. ADNP contains classical transcription factor motifs and nuclear localisation domains, but it has also been reported to be secreted and to co-localise with microtubules, indicating that ADNP may have multiple functions. We investigated the pattern of ADNP expression by immunohistology in normal rat brain, in order to generate a framework for future studies examining changes in ADNP expression in response to noxious stimuli or in models of disease. We found widespread ADNP-like immunoreactivity in neurons throughout the rat brain, with the highest expression in the cerebellum, and strong expression in the thalamus, mesencephalon, pons and medulla oblongata. ADNPlike immunoreactivity was mainly observed in the cytoplasm of neurons, and fibre tracts were often strongly positive as well. In addition, positive neuronal nuclei were occasionally observed. ADNP-like immunoreactivity was lost in degenerating ‘dark’ neurons, the morphologically unaltered adjacent cells. Occasional astrocyte and microglial cells were also positive. We suggest that the widespread expression of ADNP may correlate with the wide-ranging protective effects of NAP, and that the cytoplasmic and axonal localisation of ADNP-like immunoreactivity suggests additional, nontranscriptional functions of ADN

FolR1: a novel cell surface marker for isolating midbrain dopamine neural progenitors and nascent dopamine neurons

Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson’s disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1), a GPI-anchored cell surface molecule, specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a, a bona-fide mesDA lineage marker, during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development, as well as in ESC-derived mesDA lineage. FolR1+ neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3, whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons