personalized-needles-for-microinjections-in-the-rodent-brain

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

University of ferrara

HIV-1 associated neurocognitive disorders (HAND) are a major complication of HIV-1 infection. The mechanism(s) underlying HAND are not completely understood but, based on in vitro studies, the HIV-1 Tat protein may play an important role. In this study, the effect of prolonged exposure to endogenously produced Tat in the brain was investigated using a tat-transgenic (TT) mouse model constitutively expressing the HIV-1 tat gene. We found that stimulus-evoked glutamate exocytosis in the hippocampus and cortex was significantly increased in TT as compared with wild-type control (CC) mice, while GABA exocytosis was unchanged in the hippocampus and decreased in the cortex. This suggests that Tat generates a latent hyper-excitability state, which favors the detrimental effects of neurotoxic and/or excitotoxic agents. To challenge this idea, TT mice were tested for susceptibility to kainate-induced seizures and neurodegeneration, and found to exhibit significantly greater responses to the convulsant agent than CC mice. These results support the concept that constitutive expression of tat in the brain generates a latent excitatory state, which may increase the negative effects of damaging insults. These events may play a key role in the development of HAND.

Increased excitability in tat-transgenic mice: role of tat in HIV-related neurological disorders.

The pharmacological treatment of mesial temporal lobe epilepsy (mTLE), the most common epileptic syndrome in adults, is still unsatisfactory, as one-third of the patients are or become refractory to antiepileptic agents. Refractoriness may depend upon drug-induced alterations, but the disease per se may also undergo a progressive evolution that affects the sensitivity to drugs. mTLE has been shown to be associated with a dysfunction of the inhibitory signaling mediated by GABAA receptors. In particular, the repetitive activation of GABAA receptors produces a use-dependent decrease (rundown) of the evoked currents (I GABA), which is markedly enhanced in the hippocampus and cortex of drug-resistant mTLE patients. This phenomenon has been also observed in the pilocarpine model, where the increased I GABA rundown is observed in the hippocampus at the time of the first spontaneous seizure, then extends to the cortex and remains constant in the chronic phase of the disease. Here, we examined the sensitivity of I GABA to pharmacological modulation. We focused on the antiepileptic agent levetiracetam (LEV) and on the neurotrophin brain-derived neurotrophic factor (BDNF), which were previously reported to attenuate mTLE-induced increased rundown in the chronic human tissue. In the pilocarpine model, BDNF displayed a paramount effect, decreasing rundown in the hippocampus at the time of the first seizure, as well as in the hippocampus and cortex in the chronic period. In contrast, LEV did not affect rundown in the hippocampus, but attenuated it in the cortex. Interestingly, this effect of LEV was also observed on the still unaltered rundown observed in the cortex at the time of the first spontaneous seizure. These data suggest that the sensitivity of GABAA receptors to pharmacological interventions undergoes changes during the natural history of mTLE, implicating that the site of seizure initiation and the timing of treatment may highly affect the therapeutic outcome.

Changes in the sensitivity of GABAA current rundown to drug treatments in a model of temporal lobe epilepsy.

The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity.

Previously we reported a new series of highly defective herpes simplex virus type 1 (HSV-1) vectors that were functionally devoid of all viral immediately early (IE) genes, resulting in virtual absence of viral gene expression. Nevertheless, a reporter gene cassette inserted into the vector flanked by boundary elements from the viral latency locus showed high, persistent reporter gene activity in non-neuronal cells while an independent expression cassette inserted into a deleted ICP4 locus remained almost silent. In contrast to non-neuronal cells, we show here that the ICP4 locus cassette permitted robust reporter gene expression in a diversity of neurons following stereotactic injection of different rat brain regions; transgene expression in the hippocampus lasted up to 6 months and was essentially restricted to neurons. No evidence of neuronal cell toxicity or induction of inflammatory cell infiltrates was observed. An independent reporter gene cassette located in an intergenic region remained silent, indicating that the transgene promoter and/or insertion site are critical for sustained expression. These findings suggest the suitability of this vector for therapeutic intervention into diseases of the central nervous system that require the expression of large and/or multiple therapeutic transgenes.

Engineered HSV vector achieves safe long-term transgene expression in the central nervous system.

The ability of herpes simplex virus (HSV) to establish lifelong latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently, we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes.

Article	Year
Increased excitability in tat-transgenic mice: role of tat in HIV-related neurological disorders. Neurobiology of disease\| PubMed ID: 23454193	2013
Changes in the sensitivity of GABAA current rundown to drug treatments in a model of temporal lobe epilepsy. Frontiers in cellular neuroscience\| PubMed ID: 23874269	2013
Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 25775541	2015
Engineered HSV vector achieves safe long-term transgene expression in the central nervous system. Scientific reports\| PubMed ID: 28473703	2017
Deletion of the Virion Host Shut-off Gene Enhances Neuronal-Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes. Molecular therapy. Methods & clinical development\| PubMed ID: 28702475	2017

Gianluca Verlengia

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