Mice in translational neuroscience: What R we doing?

Animal models play a pivotal role in translational neuroscience but recurrent problems in data collection, an-alyses, and interpretation, lack of biomarkers, and a tendency to over-reliance on mice have marred neuroscience progress, leading to one of the highest attrition rates in drug translation. Global initiatives to improve repro-ducibility and model selection are being implemented. Notwithstanding, mice are still the preferred animal species to model human brain disorders even when the translation has been shown to be limited. Non-human primates are better positioned to provide relevant translational information because of their higher brain complexity and homology to humans. Among others, lack of resources and formal training, strict legislation, and ethical issues may impede broad access to large animals. We propose that instead of increasingly restrictive legislation, more resources for training, education, husbandry, and data sharing are urgently needed. The cre-ation of multidisciplinary teams, in which veterinarians need to play a key role, would be critical to improve translational efficiency. Furthermore, it is not usually acknowledged by researchers and regulators the value of comparative studies in lower species, that are instrumental in toxicology, target identification, and mechanistic studies. Overall, we highlight here the need for a conceptual shift in neuroscience research and policies to reach the patients

Looking Backward to Move Forward: a Meta-Analysis of Stem Cell Therapy in Amyotrophic Lateral Sclerosis

Transplantation of several types of stem cells (SC) for the treatment of amyotrophic lateral sclerosis (ALS) has been evaluated in numerous Phase I/II clinical trials with inconclusive results. Here, we conducted a meta-analysis to systematically assess the outcome of SC therapy trials which report the evolution of each patient before and after cell administration. In this way, we aimed to determine the effect of the SC intervention despite individual heterogeneity in disease progression. We identified 670 references by electronic search and 90 full-text studies were evaluated according to the eligibility criteria. Eleven studies were included comprising 220 cell-treated patients who received mesenchymal (M) SC (n=152), neural (N) SC (n=57), or mononuclear cells (MNC: CD34, CD117, and CD133 positive cells) (n=11). Our analyses indicate that whereas intrathecal injection of mesenchymal stromal cells appears to have a transient positive effect on clinical progression, as measured by the ALS functional rating score, there was a worsening of respiratory function measured by forced vital capacity after all interventions. Based on current evidence, we conclude that optimal cell product and route of administration need to be determined in properly controlled preclinical models before further advancing into ALS patients. In addition, in-depth understanding of disease mechanisms in subsets of patients will help tailoring SC therapy to specific targets and increase the likelihood of improving outcomesR.S.P. is the recipient of a Talentia Senior grant from the Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades of the Andalusian Government. L.M. is partly supported by the AGING Project for Department of Excellence at the Department of Translational Medicine (DIMET), Università del Piemonte Orientale, Novara, Italy

Selective COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of Parkinson's disease

Several lines of evidence point to a significant role of neuroinflammation in Parkinson's disease (PD) and other neurodegenerative disorders. In the present study we examined the protective effect of celecoxib, a selective inhibitor of the inducible form of cyclooxygenase (COX-2), on dopamine (DA) cell loss in a rat model of PD. We used the intrastriatal administration of 6-hydroxydopamine (6-OHDA) that induces a retrograde neuronal damage and death, which progresses over weeks. Animals were randomized to receive celecoxib (20 mg/kg/day) or vehicle starting 1 hour before the intrastriatal administration of 6-OHDA. Evaluation was performed in vivo using micro PET and selective radiotracers for DA terminals and microglia. Post mortem analysis included stereological quantification of tyrosine hydroxylase, astrocytes and microglia. 12 days after the 6-OHDA lesion there were no differences in DA cell or fiber loss between groups, although the microglial cell density and activation was markedly reduced in animals receiving celecoxib (p < 0.01). COX-2 inhibition did not reduce the typical astroglial response in the striatum at any stage. Between 12 and 21 days, there was a significant progression of DA cell loss in the vehicle group (from 40 to 65%) that was prevented by celecoxib. Therefore, inhibition of COX-2 by celecoxib appears to be able, either directly or through inhibition of microglia activation to prevent or slow down DA cell degeneration

The Role of the Subthalamic Nucleus in L-DOPA Induced Dyskinesia in 6-Hydroxydopamine Lesioned Rats

14 p.L-DOPA is the most effective treatment for Parkinson's disease (PD), but prolonged use leads to disabling motor complications including dyskinesia. Strong evidence supports a role of the subthalamic nucleus (STN) in the pathophysiology of PD whereas its role in dyskinesia is a matter of controversy. Here, we investigated the involvement of STN in dyskinesia, using single-unit extracellular recording, behavioural and molecular approaches in hemi-parkinsonian rats rendered dyskinetic by chronic L-DOPA administration. Our results show that chronic L-DOPA treatment does not modify the abnormal STN activity induced by the 6-hydroxydopamine lesion of the nigrostriatal pathway in this model. Likewise, we observed a loss of STN responsiveness to a single L-DOPA dose both in lesioned and sham animals that received daily L-DOPA treatment. We did not find any correlation between the abnormal involuntary movement (AIM) scores and the electrophysiological parameters of STN neurons recorded 24 h or 20–120 min after the last L-DOPA injection, except for the axial subscores. Nonetheless, unilateral chemical ablation of the STN with ibotenic acid resulted in a reduction in global AIM scores and peak-severity of dyskinesia. In addition, STN lesion decreased the anti-dyskinetogenic effect of buspirone in a reciprocal manner. Striatal protein expression was altered in dyskinetic animals with increases in ΔFosB, phosphoDARPP-32, dopamine receptor (DR) D3 and DRD2/DRD1 ratio. The STN lesion attenuated the striatal molecular changes and normalized the DRD2/DRD1 ratio. Taken together, our results show that the STN plays a role, if modest, in the physiopathology of dyskinesias.This study was supported by grants from the Spanish Ministry of Science SAF 2009-08664 (LU), the department of Industry of the Basque Government S-PE10UN24 (LU and RSP) and Kutxa Obra social (RSP). AA and AS have a fellowship from the University of the Basque Country. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of manuscript

Mutations in LRRK2 impair NF-κB pathway in iPSC-derived neurons

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both familial and idiopathic forms of Parkinson's disease (PD). Neuroinflammation is a key event in neurodegeneration and aging, and there is mounting evidence of LRRK2 involvement in inflammatory pathways. In a previous study, we described an alteration of the inflammatory response in dermal fibroblasts from PD patients expressing the G2019S and R1441G mutations in LRRK2. Methods: Taking advantage of cellular reprogramming, we generated induced pluripotent stem cell (iPSC) lines and neurons thereafter, harboring LRRK2G2019S and LRRK2R1441G mutations. We used gene silencing and functional reporter assays to characterize the effect of the mutations. We examined the temporal profile of TNF alpha-induced changes in proteins of the NF-kappa B pathway and optimized western blot analysis to capture alpha-synuclein dynamics. The effects of the mutations and interventions were analyzed by two-way ANOVA tests with respect to corresponding controls. Results: LRRK2 silencing decreased alpha-synuclein protein levels in mutated neurons and modified NF-kappa B transcriptional targets, such as PTGS2 (COX-2) and TNFAIP3 (A20). We next tested whether NF-kappa B and alpha-synuclein pathways converged and found that TNF alpha modulated alpha-synuclein levels, although we could not detect an effect of LRRK2 mutations, partly because of the individual variability. Nevertheless, we confirmed NF-kappa B dysregulation in mutated neurons, as shown by a protracted recovery of I kappa B alpha and a clear impairment in p65 nuclear translocation in the LRRK2 mutants. Conclusions: Altogether, our results show that LRRK2 mutations affect alpha-synuclein regulation and impair NF-kappa B canonical signaling in iPSC-derived neurons. TNFa modulated alpha-synuclein proteostasis but was not modified by the LRRK2 mutations in this paradigm. These results strengthen the link between LRRK2 and the innate immunity system underscoring the involvement of inflammatory pathways in the neurodegenerative process in PDThis study is funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO), Fondo de Investigaciones Sanitarias PI15/00486, the European Commission FP7 Health -278871, and the Joint Program in Neurodegenerative Diseases AC 14/0041 (DAMNDPATHS) to RSP

Small fiber neuropathy and phosphorylated alpha-synuclein in the skin of E46K- SNCA mutation carriers

Background and objective: In 2004 we described the E46K mutation in alpha-synuclein gene (E46K-SNCA), a rare point mutation causing an aggressive Lewy body disease with early prominent non-motor features and small fiber denervation of myocardium. Considering the potential interest of the skin as a target for the development of biomarkers in Parkinson's Disease (PD), in this work we aimed to evaluate structural and functional integrity of small autonomic nerve fibers and phosphorylated alpha-synuclein (p-synuclein) deposition in the skin of E46K- SNCA carriers as compared to those observed in parkin gene mutation (PARK2) carriers and healthy controls. Patients and methods: We studied 7 E46K-SNCA carriers (3 dementia with Lewy bodies, 2 pure autonomic failure, 1 PD and 1 asymptomatic), 2 PARK2 carriers and 2 healthy controls to quantify intraepidermal nerve fiber density and p-synuclein deposition with cervical skin punch biopsies (immunohistochemistry against anti PGP9.5/UCHL-1, TH and p-synuclein) and sudomotor function with electrochemical skin conductance (ESC) (SudoScan). Results: All E46K-SNCA carriers had moderate to severe p-synuclein deposits and small fiber neurodegeneration in different epidermal and dermal structures including nerve fascicles and glands, especially in carriers with Pure Autonomic Failure, while p-synuclein aggregates where absent in healthy controls and in one of two PARK2 carriers. The severity of the latter skin abnormalities in E46K-SNCA were correlated with sudomotor dysfunction (lower ESC) in hands (p = 0.035). Interpretation: These results together with our previous findings support the relevance of E46K-SNCA mutation as a suitable model to study small fiber neuropathy in Lewy body diseases

Successful development and clinical translation of a novel anterior lamellar artificial cornea

We thank the Andalusian Public Foundation Progress and Health, through the Andalusian Initiative for Advanced Therapies, for assuming the roles and responsibilities of sponsoring this clinical trial. We thank Dr. Manuel de la Rosa and Dr. Salvador Arias Santiago for providing insight and expertise that assisted the research.The datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus (GEO) public repository, ref. GSE86584 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86584Blindness due to corneal diseases is a common pathology affecting up to 23 million individuals worldwide. The tissue‐engineered anterior human cornea, which is currently being tested in a Phase I/II clinical trial to treat severe corneal trophic ulcers with preliminary good feasibility and safety results. This bioartificial cornea is based on a nanostructured fibrin–agarose biomaterial containing human allogeneic stromal keratocytes and cornea epithelial cells, mimicking the human native anterior cornea in terms of optical, mechanical, and biological behavior. This product is manufactured as a clinical‐grade tissue engineering product, fulfilling European requirements and regulations. The clinical translation process included several phases: an initial in vitro and in vivo preclinical research plan, including preclinical advice from the Spanish Medicines Agency followed by additional preclinical development, the adaptation of the biofabrication protocols to a good manufacturing practice manufacturing process, including all quality controls required, and the design of an advanced therapy clinical trial. The experimental development and successful translation of advanced therapy medicinal products for clinical application has to overcome many obstacles, especially when undertaken by academia or SMEs. We expect that our experience and research strategy may help future researchers to efficiently transfer their preclinical results into the clinical settings.This study was supported by the Spanish National Plan for Scientific and Technical Research and Innovation (I + D + I) from the Spanish Ministry of Economy and Competitiveness (Carlos III Institute of Health), grants FIS PI14/0955 and FIS PI17/0391 (both cofinanced by ERDF‐FEDER, European Union); by the Spanish Ministry of Health, Social Policy and Equity, grant EC10‐285; and by preclinical research funds from the Regional Ministry of Health through the Andalusian Initiative for Advanced Therapies

Retrieval of germinal zone neural stem cells from the cerebrospinal fluid of premature infants with intraventricular hemorrhage

Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology