Fate of Cajal–Retzius Neurons in the Postnatal Mouse Neocortex

Cajal–Retzius (CR) neurons play a critical role in cortical neuronal migration, but their exact fate after the completion of neocortical lamination remains a mystery. Histological evidence has been unable to unequivocally determine whether these cells die or undergo a phenotypic transformation to become resident interneurons of Layer 1 in the adult neocortex. To determine their ultimate fate, we performed chronic in vivo two-photon imaging of identified CR neurons during postnatal development in mice that express the green fluorescent protein (GFP) under the control of the early B-cell factor 2 (Ebf2) promoter. We find that, after birth, virtually all CR neurons in mouse neocortex express Ebf2. Although postnatal CR neurons undergo dramatic morphological transformations, they do not migrate to deeper layers. Instead, their gradual disappearance from the cortex is due to apoptotic death during the second postnatal week. A small fraction of CR neurons present at birth survive into adulthood. We conclude that, in addition to orchestrating cortical layering, a subset of CR neurons must play other roles beyond the third postnatal week

The E3 ubiquitin ligase IDOL regulates synaptic ApoER2 levels and is important for plasticity and learning.

Neuronal ApoE receptors are linked to learning and memory, but the pathways governing their abundance, and the mechanisms by which they affect the function of neural circuits are incompletely understood. Here we demonstrate that the E3 ubiquitin ligase IDOL determines synaptic ApoER2 protein levels in response to neuronal activation and regulates dendritic spine morphogenesis and plasticity. IDOL-dependent changes in ApoER2 abundance modulate dendritic filopodia initiation and synapse maturation. Loss of IDOL in neurons results in constitutive overexpression of ApoER2 and is associated with impaired activity-dependent structural remodeling of spines and defective LTP in primary neuron cultures and hippocampal slices. IDOL-deficient mice show profound impairment in experience-dependent reorganization of synaptic circuits in the barrel cortex, as well as diminished spatial and associative learning. These results identify control of lipoprotein receptor abundance by IDOL as a post-transcriptional mechanism underlying the structural and functional plasticity of synapses and neural circuits

Glutamate Induces the Elongation of Early Dendritic Protrusions via mGluRs in Wild Type Mice, but Not in Fragile X Mice

Fragile X syndrome (FXS), the most common inherited from of autism and mental impairment, is caused by transcriptional silencing of the Fmr1 gene, resulting in the loss of the RNA-binding protein FMRP. Dendritic spines of cortical pyramidal neurons in affected individuals are abnormally immature and in Fmr1 knockout (KO) mice they are also abnormally unstable. This could result in defects in synaptogenesis, because spine dynamics are critical for synapse formation. We have previously shown that the earliest dendritic protrusions, which are highly dynamic and might serve an exploratory role to reach out for axons, elongate in response to glutamate. Here, we tested the hypothesis that this process is mediated by metabotropic glutamate receptors (mGluRs) and that it is defective in Fmr1 KO mice. Using time-lapse imaging with two-photon microscopy in acute brain slices from early postnatal mice, we find that early dendritic protrusions in layer 2/3 neurons become longer in response to application of glutamate or DHPG, a Group 1 mGluR agonist. Blockade of mGluR5 signaling, which reverses some adult phenotypes of KO mice, prevented the glutamate-mediated elongation of early protrusions. In contrast, dendritic protrusions from KO mice failed to respond to glutamate. Thus, absence of FMRP may impair the ability of cortical pyramidal neurons to respond to glutamate released from nearby pre-synaptic terminals, which may be a critical step to initiate synaptogenesis and stabilize spines

Development and Validation of a Canine-Specific Profiling Array to Examine Expression of Pro-Apoptotic and Pro-Survival Genes in Retinal Degenerative Diseases

We developed an expression profiling array to examine pro-apoptotic and pro-survival genes in dog retinal degeneration models. Gene-specific canine TaqMan assays were developed and included in a custom real-time quantitative reverse transcription-PCR (qRT-PCR) array. Of the 96 selected genes, 93 belonged to known relevant pro-apoptotic and pro-survival pathways, and/or were positive controls expressed in retina, while three were housekeeping genes. Ingenuity Pathway Analysis (IPA) showed that the selected genes belonged to expected biological functions (cell death, cell-mediated immune response, cellular development, function, and maintenance) and pathways (death receptor signaling, apoptosis, TNFR1 signaling, and induction of apoptosis by HIV1). Validation of the profiling array was performed with RNA extracted from cultured MDCK cells in the presence or absence of treatment with 10 μM staurosporin for 5 or 10 h. The vast majority of the genes showed positive amplifications, and a number of them also had fold change (FC) differences \u3e ±3 between control and staurosporin-treated cells. To conclude, we established a profiling array that will be used to identify differentially expressed genes associated with photoreceptor death or survival in canine models of retinal degenerative diseases with mutations in genes that cause human inherited blindness with comparable phenotypes

Three-Dimensional Analysis of Spiny Dendrites Using Straightening and Unrolling Transforms

Current understanding of the synaptic organization of the brain depends to a large extent on knowledge about the synaptic inputs to the neurons. Indeed, the dendritic surfaces of pyramidal cells (the most common neuron in the cerebral cortex) are covered by thin protrusions named dendritic spines. These represent the targets of most excitatory synapses in the cerebral cortex and therefore, dendritic spines prove critical in learning, memory and cognition. This paper presents a new method that facilitates the analysis of the 3D structure of spine insertions in dendrites, providing insight on spine distribution patterns. This method is based both on the implementation of straightening and unrolling transformations to move the analysis process to a planar, unfolded arrangement, and on the design of DISPINE, an interactive environment that supports the visual analysis of 3D patterns

Proteins That Promote Filopodia Stability, but Not Number, Lead to More Axonal-Dendritic Contacts

Dendritic filopodia are dynamic protrusions that are thought to play an active role in synaptogenesis and serve as precursors to spine synapses. However, this hypothesis is largely based on a temporal correlation between filopodia formation and synaptogenesis. We investigated the role of filopodia in synapse formation by contrasting the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. We used a filopodia inducing motif that is found in GAP-43, as a molecular tool, and found this palmitoylated motif enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, RNAi knockdown of NLG-1 reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, increased the rate at which filopodia transformed into spines by stabilization of the initial contact with axons. Taken together, these results suggest that increased filopodia stability and not density, may be the rate-limiting step for synapse formation

Impaired perceptual learning in a mouse model of Fragile X syndrome is mediated by parvalbumin neuron dysfunction and is reversible.

To uncover the circuit-level alterations that underlie atypical sensory processing associated with autism, we adopted a symptom-to-circuit approach in the Fmr1-knockout (Fmr1-/-) mouse model of Fragile X syndrome. Using a go/no-go task and in vivo two-photon calcium imaging, we find that impaired visual discrimination in Fmr1-/- mice correlates with marked deficits in orientation tuning of principal neurons and with a decrease in the activity of parvalbumin interneurons in primary visual cortex. Restoring visually evoked activity in parvalbumin cells in Fmr1-/- mice with a chemogenetic strategy using designer receptors exclusively activated by designer drugs was sufficient to rescue their behavioral performance. Strikingly, human subjects with Fragile X syndrome exhibit impairments in visual discrimination similar to those in Fmr1-/- mice. These results suggest that manipulating inhibition may help sensory processing in Fragile X syndrome

Delayed Treatment with Systemic (S)-Roscovitine Provides Neuroprotection and Inhibits In Vivo CDK5 Activity Increase in Animal Stroke Models

Although quite challenging, neuroprotective therapies in ischemic stroke remain an interesting strategy to counter mechanisms of ischemic injury and reduce brain tissue damage. Among potential neuroprotective drug, cyclin-dependent kinases (CDK) inhibitors represent interesting therapeutic candidates. Increasing evidence indisputably links cell cycle CDKs and CDK5 to the pathogenesis of stroke. Although recent studies have demonstrated promising neuroprotective efficacies of pharmacological CDK inhibitors in related animal models, none of them were however clinically relevant to human treatment.In the present study, we report that systemic delivery of (S)-roscovitine, a well known inhibitor of mitotic CDKs and CDK5, was neuroprotective in a dose-dependent manner in two models of focal ischemia, as recommended by STAIR guidelines. We show that (S)-roscovitine was able to cross the blood brain barrier. (S)-roscovitine significant in vivo positive effect remained when the compound was systemically administered 2 hrs after the insult. Moreover, we validate one of (S)-roscovitine in vivo target after ischemia. Cerebral increase of CDK5/p25 activity was observed 3 hrs after the insult and prevented by systemic (S)-roscovitine administration. Our results show therefore that roscovitine protects in vivo neurons possibly through CDK5 dependent mechanisms.Altogether, our data bring new evidences for the further development of pharmacological CDK inhibitors in stroke therapy

XIAP Protection of Photoreceptors in Animal Models of Retinitis Pigmentosa

BACKGROUND: Retinitis pigmentosa (RP) is a blinding genetic disorder that is caused by the death of photoreceptors in the outer nuclear layer of the retina. To date, 39 different genetic loci have been associated with the disease, and 28 mutated genes have been identified. Despite the complexity of the underlying genetic basis for RP, the final common pathway is photoreceptor cell death via apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, P23H and S334ter rhodopsin transgenic rat models of RP were used to test the neuroprotective effects of anti-apoptotic gene therapy. Adeno-associated viruses (AAV) carrying the X-linked inhibitor of apoptosis (XIAP) or green fluorescent protein (GFP) were delivered subretinally into the eye of transgenic rat pups. Histological and functional measures were used to assess neuroprotection. XIAP is known to block apoptosis by inhibiting the action of caspases-3, -7 and -9. The results show that XIAP gene therapy provides long-term neuroprotection of photoreceptors at both structural and functional levels. CONCLUSIONS/SIGNIFICANCE: Our gene therapy strategy targets the apoptotic cascade, which is the final common pathway in all forms of retinitis pigmentosa. This strategy holds great promise for the treatment of RP, as it allows for the broad protection of photoreceptors, regardless of the initial disease causing mutation

A Partial Structural and Functional Rescue of a Retinitis Pigmentosa Model with Compacted DNA Nanoparticles

Previously we have shown that compacted DNA nanoparticles can drive high levels of transgene expression after subretinal injection in the mouse eye. Here we delivered compacted DNA nanoparticles containing a therapeutic gene to the retinas of a mouse model of retinitis pigmentosa. Nanoparticles containing the wild-type retinal degeneration slow (Rds) gene were injected into the subretinal space of rds+/− mice on postnatal day 5. Gene expression was sustained for up to four months at levels up to four times higher than in controls injected with saline or naked DNA. The nanoparticles were taken up into virtually all photoreceptors and mediated significant structural and biochemical rescue of the disease without histological or functional evidence of toxicity. Electroretinogram recordings showed that nanoparticle-mediated gene transfer restored cone function to a near-normal level in contrast to transfer of naked plasmid DNA. Rod function was also improved. These findings demonstrate that compacted DNA nanoparticles represent a viable option for development of gene-based interventions for ocular diseases and obviate major barriers commonly encountered with non-viral based therapies