Multiomic Analysis of Neurons with Divergent Projection Patterns Identifies Novel Regulators of Axon Pathfinding.

Axon pathfinding is a key step in neural circuits formation. However, the transcriptional mechanisms regulating its progression remain poorly understood. The binary decision of crossing or avoiding the midline taken by some neuronal axons during development represents a robust model to investigate the mechanisms that control the selection of axonal trajectories. Here, to identify novel regulators of axon guidance, this work compares the transcriptome and chromatin occupancy profiles of two neuronal subpopulations, ipsilateral (iRGC) and contralateral retinal ganglion cells (cRGC), with similar functions but divergent axon trajectories. These analyses retrieved a number of genes encoding for proteins not previously implicated in axon pathfinding. In vivo functional experiments confirm the implication of some of these candidates in axonal navigation. Among the candidate genes, γ-synuclein is identified as essential for inducing midline crossing. Footprint and luciferase assays demonstrate that this small-sized protein is regulated by the transcription factor (TF) Pou4f1 in cRGCs. It is also shown that Lhx2/9 are specifically expressed in iRGCs and control a program that partially overlaps with that regulated by Zic2, previously described as essential for iRGC specification. Overall, the analyses identify dozens of new molecules potentially involved in axon guidance and reveal the regulatory logic behind the selection of axonal trajectories.

Altered Levels and Isoforms of Tau and Nuclear Membrane Invaginations in Huntington's Disease.

Since the early reports of neurofibrillary Tau pathology in brains of some Huntington's disease (HD) patients, mounting evidence of multiple alterations of Tau in HD brain tissue has emerged in recent years. Such Tau alterations range from increased total levels, imbalance of isoforms generated by alternative splicing (increased 4R-/3R-Tau ratio) or by post-translational modifications such as hyperphosphorylation or truncation. Besides, the detection in HD brains of a new Tau histopathological hallmark known as Tau nuclear rods (TNRs) or Tau-positive nuclear indentations (TNIs) led to propose HD as a secondary Tauopathy. After their discovery in HD brains, TNIs have also been reported in hippocampal neurons of early Braak stage AD cases and in frontal and temporal cortical neurons of FTD-MAPT cases due to the intronic IVS10+16 mutation in the Tau gene (MAPT) which results in an increased 4R-/3R-Tau ratio similar to that observed in HD. TNIs are likely pathogenic for contributing to the disturbed nucleocytoplasmic transport observed in HD. A key question is whether correction of any of the mentioned Tau alterations might have positive therapeutic implications for HD. The beneficial effect of decreasing Tau expression in HD mouse models clearly implicates Tau in HD pathogenesis. Such beneficial effect might be exerted by diminishing the excess total levels of Tau or specifically by diminishing the excess 4R-Tau, as well as any of their downstream effects. In any case, since gene silencing drugs are under development to attenuate both Huntingtin (HTT) expression for HD and MAPT expression for FTD-MAPT, it is conceivable that the combined therapy in HD patients might be more effective than HTT silencing alone.

The peripheral eye: A neurogenic area with potential to treat retinal pathologies?

Numerous degenerative diseases affecting visual function, including glaucoma and retinitis pigmentosa, are produced by the loss of different types of retinal cells. Cell replacement therapy has emerged as a promising strategy for treating these and other retinal diseases. The retinal margin or ciliary body (CB) of mammals has been proposed as a potential source of cells to be used in degenerative conditions affecting the retina because it has been reported it might hold neurogenic potential beyond embryonic development. However, many aspects of the origin and biology of the CB are unknown and more recent experiments have challenged the capacity of CB cells to generate different types of retinal neurons. Here we review the most recent findings about the development of the marginal zone of the retina in different vertebrates and some of the mechanisms underlying the proliferative and neurogenic capacity of this fascinating region of the vertebrates eye. In addition, we performed experiments to isolate CB cells from the mouse retina, generated neurospheres and observed that they can be expanded with a proliferative ratio similar to neural stem cells. When induced to differentiate, cells derived from the CB neurospheres start to express early neural markers but, unlike embryonic stem cells, they are not able to fully differentiate in vitro or generate retinal organoids. Together with previous reports on the neurogenic capacity of CB cells, also reviewed here, our results contribute to the current knowledge about the potentiality of this peripheral region of the eye as a therapeutic source of functional retinal neurons in degenerative diseases.

Bi-directional genetic modulation of GSK-3ß exacerbates hippocampal neuropathology in experimental status epilepticus.

Glycogen synthase kinase-3 (GSK-3) is ubiquitously expressed throughout the brain and involved in vital molecular pathways such as cell survival and synaptic reorganization and has emerged as a potential drug target for brain diseases. A causal role for GSK-3, in particular the brain-enriched GSK-3ß isoform, has been demonstrated in neurodegenerative diseases such as Alzheimer's and Huntington's, and in psychiatric diseases. Recent studies have also linked GSK-3 dysregulation to neuropathological outcomes in epilepsy. To date, however, there has been no genetic evidence for the involvement of GSK-3 in seizure-induced pathology. Status epilepticus (prolonged, damaging seizure) was induced via a microinjection of kainic acid into the amygdala of mice. Studies were conducted using two transgenic mouse lines: a neuron-specific GSK-3ß overexpression and a neuron-specific dominant-negative GSK-3ß (GSK-3ß-DN) expression in order to determine the effects of increased or decreased GSK-3ß activity, respectively, on seizures and attendant pathological changes in the hippocampus. GSK-3 inhibitors were also employed to support the genetic approach. Status epilepticus resulted in a spatiotemporal regulation of GSK-3 expression and activity in the hippocampus, with decreased GSK-3 activity evident in non-damaged hippocampal areas. Consistent with this, overexpression of GSK-3ß exacerbated status epilepticus-induced neurodegeneration in mice. Surprisingly, decreasing GSK-3 activity, either via overexpression of GSK-3ß-DN or through the use of specific GSK-3 inhibitors, also exacerbated hippocampal damage and increased seizure severity during status epilepticus. In conclusion, our results demonstrate that the brain has limited tolerance for modulation of GSK-3 activity in the setting of epileptic brain injury. These findings caution against targeting GSK-3 as a treatment strategy for epilepsy or other neurologic disorders where neuronal hyperexcitability is an underlying pathomechanism.

Tau-positive nuclear indentations in P301S tauopathy mice.

Increased incidence of neuronal nuclear indentations is a well-known feature of the striatum of Huntington's disease (HD) brains and, in Alzheimer's disease (AD), neuronal nuclear indentations have recently been reported to correlate with neurotoxicity caused by improper cytoskeletal/nucleoskeletal coupling. Initial detection of rod-shaped tau immunostaining in nuclei of cortical and striatal neurons of HD brains and in hippocampal neurons of early Braak stage AD led us to coin the term "tau nuclear rods (TNRs)." Although TNRs traverse nuclear space, they in fact occupy narrow cytoplasmic extensions that fill indentations of the nuclear envelope and we will here refer to this histological hallmark as Tau-immunopositive nuclear indentations (TNIs). We reasoned that TNI formation is likely secondary to tau alterations as TNI detection in HD correlates with an increase in total tau, particularly of the isoforms with four tubulin binding repeats (4R-tau). Here we analyze transgenic mice that overexpress human 4R-tau with a frontotemporal lobar degeneration-tau point mutation (P301S mice) to explore whether tau alteration is sufficient for TNI formation. Immunohistochemistry with various tau antibodies, immunoelectron microscopy and double tau-immunofluorescence/DAPI-nuclear counterstaining confirmed that excess 4R-tau in P301S mice is sufficient for the detection of abundant TNIs that fill nuclear indentations. Interestingly, this does not correlate with an increase in the number of nuclear indentations, thus suggesting that excess total tau or an isoform imbalance in favor of 4R-tau facilitates tau detection inside preexisting nuclear indentations but does not induce formation of the latter. In summary, here we demonstrate that tau alteration is sufficient for TNI detection and our results suggest that the neuropathological finding of TNIs becomes a possible indicator of increased total tau and/or increased 4R/3R-tau ratio in the affected neurons apart from being an efficient way to monitor pathology-associated nuclear indentations.

Faulty splicing and cytoskeleton abnormalities in Huntington's disease.

Huntington's disease (HD) is caused by a CAG-repeat encoding a polyglutamine (polyQ) tract in the huntingtin protein. There is plenty of evidence of polyQ-driven toxicity. However, CAG repeat RNA-driven alteration of splicing has recently been proposed in analogy to CUG-repeat diseases. Here we review the reported alteration of the CAG-repeat associated splicing factor SRSF6 in brains of HD patients and mouse models and how this correlates with altered splicing of, at least, two microtubule-associated proteins in HD, namely MAPT (tau) and MAP2. Regarding tau, altered splicing of exon 10 has been reported, along with increased levels and 4R/3R-tau ratio and detection of tau in a new nuclear rod-shaped histopathological hallmark termed tau nuclear rod (TNR) or tau nuclear indentation (TNI). These findings, together with an attenuation of HD phenotype in R6/1 mice with tau deficiency and subsequent studies showing increased phosphorylation in mouse models and increased levels in CSF of patients, has led to proposing HD as a tauopathy. Regarding MAP2, an increase in its juvenile form and a decrease in total MAP2 together with redistribution from dendrites to soma is observed in HD patients, which may contribute to the dendritic atrophy in HD. Furthermore, MAP2 positive structures filling nuclear indentations have occasionally been found and co-localized with tau. Therefore, altered MAP function with imbalance in tau/MAP2 content could contribute to HD striatal atrophy and dysfunction. Besides, TNIs might be indicative of such MAP abnormalities. TNIs are also found in early pathology Alzheimer's disease and in tauopathy mice over-expressing mutant 4R-tau. This indicates that tau alteration is sufficient for TNI detection, which becomes a marker of increased total tau and/or altered 4R/3R-tau ratio and reporter of pathology-associated nuclear indentations. Altogether, these recent studies suggest that correcting the SRSF6-driven missplicing and/or microtubule-associated imbalance might be of therapeutic value in HD.

Impaired PLP-dependent metabolism in brain samples from Huntington disease patients and transgenic R6/1 mice.

Oxidative stress has been described as important to Huntington disease (HD) progression. In a previous HD study, we identified several carbonylated proteins, including pyridoxal kinase and antiquitin, both of which are involved in the metabolism of pyridoxal 5´-phosphate (PLP), the active form of vitamin B6. In the present study, pyridoxal kinase levels were quantified and showed to be decreased both in HD patients and a R6/1 mouse model, compared to control samples. A metabolomic analysis was used to analyze metabolites in brain samples of HD patients and R6/1 mice, compared to control samples using mass spectrometry. This technique allowed detection of increased concentrations of pyridoxal, the substrate of pyridoxal kinase. In addition, PLP, the product of the reaction, was decreased in striatum from R6/1 mice. Furthermore, glutamate and cystathionine, both substrates of PLP-dependent enzymes were increased in HD. This reinforces the hypothesis that PLP synthesis is impaired, and could explain some alterations observed in the disease. Together, these results identify PLP as a potential therapeutic agent.

Decreased glycogen synthase kinase-3 levels and activity contribute to Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by brain atrophy particularly in striatum leading to personality changes, chorea and dementia. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase in the crossroad of many signaling pathways that is highly pleiotropic as it phosphorylates more than hundred substrates including structural, metabolic, and signaling proteins. Increased GSK-3 activity is believed to contribute to the pathogenesis of neurodegenerative diseases like Alzheimer's disease and GSK-3 inhibitors have been postulated as therapeutic agents for neurodegeneration. Regarding HD, GSK-3 inhibitors have shown beneficial effects in cell and invertebrate animal models but no evident efficacy in mouse models. Intriguingly, those studies were performed without interrogating GSK-3 level and activity in HD brain. Here we aim to explore the level and also the enzymatic activity of GSK-3 in the striatum and other less affected brain regions of HD patients and of the R6/1 mouse model to then elucidate the possible contribution of its alteration to HD pathogenesis by genetic manipulation in mice. We report a dramatic decrease in GSK-3 levels and activity in striatum and cortex of HD patients with similar results in the mouse model. Correction of the GSK-3 deficit in HD mice, by combining with transgenic mice with conditional GSK-3 expression, resulted in amelioration of their brain atrophy and behavioral motor and learning deficits. Thus, our results demonstrate that decreased brain GSK-3 contributes to HD neurological phenotype and open new therapeutic opportunities based on increasing GSK-3 activity or attenuating the harmful consequences of its decrease.

Huntington's disease is a four-repeat tauopathy with tau nuclear rods.

An imbalance of tau isoforms containing either three or four microtubule-binding repeats causes frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) in families with intronic mutations in the MAPT gene. Here we report equivalent imbalances at the mRNA and protein levels and increased total tau levels in the brains of subjects with Huntington's disease (HD) together with rod-like tau deposits along neuronal nuclei. These tau nuclear rods show an ordered filamentous ultrastructure and can be found filling the neuronal nuclear indentations previously reported in HD brains. Finally, alterations in serine/arginine-rich splicing factor-6 coincide with tau missplicing, and a role of tau in HD pathogenesis is evidenced by the attenuation of motor abnormalities of mutant HTT transgenic mice in tau knockout backgrounds.