Early and reversible neuropathology induced by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum

The ability to overexpress full-length huntingtin or large fragments represents an important challenge to mimic Huntington's pathology and reproduce all stages of the disease in a time frame compatible with rodent life span. In the present study, tetracycline-regulated lentiviral vectors leading to high expression levels were used to accelerate the pathological process. Rats were simultaneously injected with vectors coding for the transactivator and wild type (WT) or mutated huntingtin (TRE-853-19Q/82Q) in the left and right striatum, respectively, and analyzed in the ‘on' and ‘off' conditions. Overexpression of TRE-853-19Q protein or residual expression of TRE-853-82Q in ‘off' condition did not cause any significant neuronal pathology. Overexpressed TRE-853-82Q protein led to proteolytic release of N-terminal htt fragments, nuclear aggregation, and a striatal dysfunction as revealed by decrease of DARPP-32 staining but absence of NeuN down-regulation. The differential effect on the DARPP-32/NeuN neuronal staining was observed as early as 1 month after injection and maintained at 3 months. In contrast, expression of a shorter htt form (htt171-82Q) did not require processing prior formation of nuclear aggregates and caused decrease of both DARPP-32 and NeuN neuronal markers at one month post-injection suggesting that polyQ pathology may be dependent on protein context. Finally, the reversibility of the pathology was assessed. Huntingtin expression was turn ‘on' for 1 month and then shut ‘off' for 2 months. Recovery of DARPP-32 immunoreactivity and clearance of huntingtin aggregates were observed in animals treated with doxycycline. These results suggest that a tetracycline-regulated system may be particularly attractive to model Huntington's disease and induce early and reversible striatal neuropathology in viv

Fetal Spinal Cord Tissue in Mini-Guidance Channels Promotes Longitudinal Axonal Growth after Grafting into Hemisected Adult Rat Spinal Cords

Solid fetal spinal cord (FSC) tissue, seeded into semipermeable mini-guidance channels, was tested for the ability to promote axonal growth across the gap created by a midthoracic (T8) hemisection in adult rats. Fetal thoracic spinal cords, at embryonic days 13 to 15, were harvested and gently aspirated into mini-guidance channels (1.25 mm in diameter and 3.0 mm in length). Care was taken to maintain the rostro-caudal orientation of the FSC. In control rats, the FSC-channel congraft struct was exposed to 5 freeze/thaw cycles to produce non-viable grafts before implantation into the hemisected cord. All cases revealed intact tissue cables of various diameters spanning the rostro-caudal extent of the lesion cavity, with integration of host-graft tissues at both interfaces. Immunofluorescence results indicated that numerous neurofilament-positive axons were present within the FSC tissue cable. Double-labeling of a subpopulation of these axons with calcitonin generelated peptide indicated their peripheral nervous system (PNS) origin. Descending serotonergic and noradrenergic axons were found in the proximity of the rostral host-graft interface, but were not observed to grow into the FSC-graft. Anterograde tracing of propriospinal axons with Phaseolus vulgaris-leucoagglutinin demonstrated that axons had regenerated into the FSC-graft and had traveled longitudinally to the distal end of the channel. Few axons were observed to cross the distal host-graft interface to enter the host spinal cord. Cross-sectional analysis at the midpoint of the tissue cable stained with toluidine blue demonstrated a significant increase (P<0.01) in myelinated axons in viable FSC grafts (1455±663, mean±S.E.M.; n=6) versus freeze-thaw control grafts (155±50; n=5). In addition to the myelinated axons, many unmyelinated axons were observed in the tissue cable at the electron microscopic level. Areas resembling the PNS with typical Schwann cells, as well as those resembling the central nervous system with neurons and central neuropil, were also seen. In freeze-thaw control grafts, neither viable neurons nor central neuropil were observed. Retrograde tracing with Fast Blue and Diamidino Yellow demonstrated that neurons within the FSC graft extended axons into the host spinal cord at least for 2 mm from both the rostral and caudal host-graft interfaces. We conclude that viable FSC grafts within semipermeable guidance channels may serve both as a permissive bridge for longitudinally directed axonal growth and a potential relay for conveying information across a lesion site in the adult rat spinal cord

AAV-mediated expression of wild-type and ALS-linked mutant VAPB selectively triggers death of motoneurons through a Ca2+-dependent ER-associated pathway

A dominant mutation in the gene coding for the vesicle-associated membrane protein-associated protein B (VAPB) was associated with amyotrophic lateral sclerosis, a fatal paralytic disorder characterized by the selective loss of motoneurons in the brain and spinal cord. Adeno-associated viral vectors that we show to transduce up to 90% of motoneurons in vitro were used to model VAPB-associated neurodegenerative process. We observed that Adeno-associated viral-mediated over-expression of both wild-type and mutated form of human VAPB selectively induces death of primary motoneurons, albeit with different kinetics. We provide evidence that ER stress and impaired homeostatic regulation of calcium (Ca2+) are implicated in the death process. Finally, we found that completion of the motoneuron death program triggered by the over-expression of wild-type and mutant VAPB implicates calpains, caspase 12 and 3. Our viral-based in vitro model, which recapitulates the selective vulnerability of motoneurons to the presence of mutant VAPB and also to VAPB gene dosage effect, identifies aberrant Ca2+ signals and ER-derived death pathways as important events in the motoneuron degenerative process

Over-expression of alpha-synuclein in human neural progenitors leads to specific changes in fate and differentiation

Missense mutations and extra copies of the α-Synuclein gene result in Parkinson disease (PD). Human stem and progenitor cells can be expanded from embryonic tissues and provide a source of non-transformed neural cells to explore the effects of these pathogenic mutations specifically in human nervous tissue. We over-expressed the wild type, A53T and A30P forms of α-synuclein in expanded populations of progenitors derived from the human fetal cortex. The protein localized in the nucleus and around microvesicles. Only the A53T form was acutely toxic, suggesting a unique vulnerability of these progenitors to this mutation. Interestingly, constitutive over-expression of wild-type α-synuclein progressively impaired the innate ability of progenitors to switch toward gliogenesis at later passages. To explore the effect of α-synuclein on neuronal subtypes selectively affected in PD, such as dopaminergic neurons, α-synuclein and its mutations were also over-expressed in terminally differentiating neuroectodermal cultures derived from human embryonic stem cells (hESC). Alpha-synuclein induced acute cytotoxicity and reduced the number of neurons expressing either tyrosine hydroxylase or gamma-aminobutyric acid over time. Consistent with the selective vulnerability of ventral midbrain dopaminergic neurons, α-synuclein cytotoxicity appeared most pronounced following FGF8/SHH specification and was decreased by inhibition of dopamine synthesis. Together, these data show that α-synuclein over-expressed in human neural embryonic cells results in patterns of degeneration that in some cases match features of Parkinson Disease. Thus, neural cells derived from hESC provide a useful model system to understand the development of α-synuclein-related pathologies and allow therapeutic drug screenin

Polo-like kinase 2 regulates selective autophagic α-synuclein clearance and suppresses its toxicity in vivo

An increase in α-synuclein levels due to gene duplications/triplications or impaired degradation is sufficient to trigger its aggregation and cause familial Parkinson disease (PD). Therefore, lowering α-synuclein levels represents a viable therapeutic strategy for the treatment of PD and related synucleinopathies. Here, we report that Polo-like kinase 2 (PLK2), an enzyme up-regulated in synucleinopathy-diseased brains, interacts with, phosphorylates and enhances α-synuclein autophagic degradation in a kinase activity-dependent manner. PLK2-mediated degradation of α-synuclein requires both phosphorylation at S129 and PLK2/α-synuclein complex formation. In a rat genetic model of PD, PLK2 overexpression reduces intraneuronal human α-synuclein accumulation, suppresses dopaminergic neurodegeneration, and reverses hemiparkinsonian motor impairments induced by α-synuclein overexpression. This PLK2-mediated neuroprotective effect is also dependent on PLK2 activity and α-synuclein phosphorylation. Collectively, our findings demonstrate that PLK2 is a previously undescribed regulator of α-synuclein turnover and that modulating its kinase activity could be a viable target for the treatment of synucleinopathies

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

BACKGROUND: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. RESULTS: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (&lt; 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. CONCLUSION: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain

Phosphorylation does not prompt, nor prevent, the formation of α-synuclein toxic species in a rat model of Parkinson's disease

Phosphorylation is involved in numerous neurodegenerative diseases. In particular, alpha-synuclein is extensively phosphorylated in aggregates in patients suffering from synucleinopathies. However, the share of this modification in the events that lead to the conversion of alpha-synuclein to aggregated toxic species needed to be clarified. The rat model that we developed through rAAV2/6-mediated expression of alpha-synuclein demonstrates a correlation between neurodegeneration and formation of small filamentous alpha-synuclein aggregates. A mutation preventing phosphorylation (S129A) significantly increases alpha-synuclein toxicity and leads to enhanced formation of beta-sheet-rich, proteinase K-resistant aggregates, increased affinity for intracellular membranes, a disarrayed network of neurofilaments and enhanced alpha-synuclein nuclear localization. The expression of a mutation mimicking phosphorylation (S129D) does not lead to dopaminergic cell loss. Nevertheless, fewer but larger aggregates are formed, and signals of apoptosis are also activated in rats expressing the phosphorylation-mimicking form of alpha-synuclein. These observations strongly suggest that phosphorylation does not play an active role in the accumulation of cytotoxic pre-inclusion aggregates. Unexpectedly, the study also demonstrates that constitutive expression of phosphorylation-mimicking forms of alpha-synuclein does not protect from neurodegeneration. The role of phosphorylation at Serine 129 in the early phase of Parkinson's disease is examined, which brings new perspective to therapeutic approaches focusing on the modulation of kinases/phosphatases activity to control alpha-synuclein toxicit

Perineuronal net digestion with chondroitinase restores memory in mice with tau pathology.

Alzheimer's disease is the most prevalent tauopathy and cause of dementia. We investigate the hypothesis that reactivation of plasticity can restore function in the presence of neuronal damage resulting from tauopathy. We investigated two models with tau hyperphosphorylation, aggregation and neurodegeneration: a transgenic mouse model in which the mutant P301S tau is expressed in neurons (Tg P301S), and a model in which an adeno-associated virus expressing P301S tau (AAV-P301S) was injected in the perirhinal cortex, a region critical for object recognition (OR) memory. Both models show profound loss of OR memory despite only 15% neuronal loss in the Tg P301S and 26% in AAV-P301S-injected mice. Recordings from perirhinal cortex slices of 3month-old P301S transgenic mice showed a diminution in synaptic transmission following temporal stimulation. Chondroitinase ABC (ChABC) can reactivate plasticity and affect memory through actions on perineuronal nets. ChABC was injected into the perirhinal cortex and animals were tested for OR memory 1week later, demonstrating restoration of OR memory to normal levels. Synaptic transmission indicated by fEPSP amplitude was restored to control levels following ChABC treatment. ChABC did not affect the progression of neurodegenerative tauopathy. These findings suggest that increasing plasticity by manipulation of perineuronal nets offers a novel therapeutic approach to the treatment of memory loss in neurodegenerative disorders.This work was supported by the European Union Framework 7 Project Plasticise (S.Y., M.C., M.G.S., J.W.F., P.R., P.A., B.L.S.), the Wellcome Trust (L.M.S., T.J.B.), the Alzheimer's Research UK (M.G.S.), and the UK Medical Research Council (L.M.S., T.J.B.). J.W.F.'s work is supported by the ERC-ECMneuro, the Christopher and Dana Reeve Foundation and the NIHR Cambridge biomedical research center.This article was originally published in Experimental Neurology (S Yang, M Cacqueve, LM Saksida, TJ Bussey, BL Schneider, P Aebischer, R Melani, T Pizzorusso, JW Fawcett, MG Spillantini, Experimental Neurology 2015, 265, 48-58