Pascal Barneoud

Direct intracerebral gene transfer of an adenoviral vector expressing tyrosine hydroxylase in a rat model of Parkinson's disease

DIRECT intracerebral gene transfer to neural cells has been demonstrated with recombinant adenovirus encoding β-galactosidase. To explore the potential of recombinant adenovirus for the therapy of neurological disease we constructed a recombinant adenovirus encoding tyrosine hydroxylase, and optimized intracerebral injection to express the gene in the striatum of unilaterally denervated rats. These animals have dopamine depletion in their lesioned striatum, causing a rotation asymmetry induced by apomorphine. One, and two weeks after intracerebral injection this sensorimotor asymmetry was decreased by the adenovirus encoding tyrosine hydroxylase, and not by a control adenovirus encoding β-galactosidase. Histological analysis showed that tyrosine hydroxylase was preferentially expressed in astrocytes.

Nicotinic acetylcholine subunit mRNA expression in dopaminergic neurons of the rat substantia nigra and ventral tegmental area.

THE molecular composition of the nicotinic acetylcholine receptors (nAChRs) located on dopaminergic neurons and modulating their activity is unclear. Using the reverse transcriptase-polymerase chain reaction we have analyzed the mRNA for nAChR subunits expressed in the substantia nigra (SN) and ventral tegmental area (VTA) following unilateral 6-hydroxy-dopamine lesion of the dopaminergic system. In contrast to the unlesioned hemisphere, no signal was found in the lesioned hemisphere for α3, α5, alpha;6, and β4 subunits in the SN nor for α2, α3, α5, α6, α7 and β4 subunits in the VTA, indicating the expression of these subunits in dopaminergic neurons. mRNA for α4, β2 and β3 subunits (and α7 in the SN) were still detected after lesion, suggesting that they are expressed in GABAergic neurons and interneurons of these brain areas. These results demonstrate the selective localisation of a number of nAChR subunit mRNA within dopaminergic neurons, strongly suggesting that a heterogenous population of nAChRs play a role in modulating dopaminergic neuronal activity.

Evaluation of simple and complex sensorimotor behaviours in rats with a partial lesion of the dopaminergic nigrostriatal system

We have examined the behavioural consequences of a partial unilateral dopaminergic denervation of the rat striatum. This partial lesion was obtained by an intrastriatal 6‐hydroxy‐dopamine injection (6‐OHDA, 20 or 10 μg divided between two injection sites) and was compared with a unilateral complete lesion resulting from an injection of 6‐OHDA (2 × 6 μg) into the medial forebrain bundle. Quantification of striatal dopamine (DA) and its metabolites, and the immunohistochemical evaluation of the nigrostriatal DA system confirmed the complete and partial lesions. Animals with complete striatal denervation displayed both apomorphine‐ and amphetamine‐induced rotations whereas the partial denervation elicited amphetamine‐induced rotations only. However, the rates of amphetamine‐induced rotation were not correlated with the size of the lesion. In contrast, the paw‐reaching impairments were significantly correlated with the striatal dopaminergic depletion. When evaluated in the staircase test, animals with partial denervation were impaired exclusively for the paw contralateral to the side of the lesion. This motor deficit (50–75%) included all components of the skilled paw use (i.e. attempt, motor coordination and success) and was observed at least 12 weeks after the lesion. However, these animals were able to perform normal stepping adjustments with the impaired paw, indicating that the partial lesion induced a coordination deficit of the paw rather than a deficit of movement initiation. After a complete lesion, stepping adjustments of the contralateral paw were dramatically impaired (by 80%), an akinesia which almost certainly accounted for the great deficit in skilled paw use. The paw‐reaching impairments resulting from the partial striatal denervation are proposed as a model of the early symptoms of Parkinsons disease and may be useful for the development of restorative therapies.

Quantitative motor assessment in Fals mice: a longitudinal study

WE have evaluated the G1H line of transgenic mice overexpressing a familial ALS mutation of SOD1 (Gly93→Ala) in tasks assessing different aspects of motor function to determine how early these deficits could be detected and their order of appearance. The earliest deficits were observed in tests of muscle strength and coordination as early as 8 weeks of age and their development appeared to be biphasic, whereas spontaneous activity was not impaired until 15 weeks of age. These studies show that, in addition to the previously demonstrated histological and electromyographic deficits, this transgenic mouse also presents changes in motor function reminiscent of the human disease, reinforcing and extending its validity as an animal model of familial amyotrophic lateral sclerosis (FALS) and allowing the investigation of novel drug treatment for ALS.

Effects of mild traumatic brain injury on immunoreactivity for the inducible transcription factors c-Fos, c-Jun, JunB, and Krox-24 in cerebral regions associated with conditioned fear responding.

We have previously demonstrated that mild traumatic brain injury (TBI) of the right parietal cortex results in a relatively selective deficit in conditioned fear responding. However, this behavioural deficit is very consistent and unrelated to the extent of the cortical necrotic lesion. We were therefore interested in determining if other brain regions might show a consistent response to mild TBI, and therefore, more reliably relate to the behavioural change. Increased expression of inducible transcription factors (ITFs) has been used to study which brain regions respond to a variety of events. In the present study, we examined the expression patterns of immunoreactivity (IR) for four ITFs (c-Fos, c-Jun, JunB, and Krox-24) at 3 h after mild fluid percussion TBI. Changes in ITF expression were only observed ipsilateral to the side of TBI. The clearest changes were observed in brain regions known to be involved in conditioned fear responding, such as the amygdala complex and hippocampal formation and several cortical regions. In contrast, no changes in IR for any of the ITFs were observed in the striatum, nucleus accumbens, nucleus basalis magnocellularis, septum or periacqueductal grey. Unlike the extent of visible damage to the cortex at the site of impact, the overexpression of ITFs showed a notable consistency between animals subjected to TBI. This consistency in regions known to be involved in conditioned fear responding (i.e., amygdala complex and hippocampal formation) lead us to suggest that it is these changes, rather than the more variable cortical necrotic lesion, that is responsible for the behavioural deficits we observe following mild TBI. Importantly, our results demonstrate that like the hippocampus, the amygdala is a sub-cortical structure particularly sensitive to the effects of mild brain trauma and underline the fact that cerebral regions distant from the location of the fluid impact can be affected.

Protection of axotomized ganglion cells by salicylic acid.

Neuronal survival is influenced by the redox environment, and it has been shown that antioxidants protect developing neurons from the effects of axotomy. Here, we show that the intraocular injection of salicylic acid (SA) reduces the number of dying axotomized ganglion cells in the chick embryo. The antioxidant properties of SA are probably responsible for its protective effects, whose U-shaped dose-dependency matches that of several other antioxidants. We conclude that SA protects axotomized neurons by maintaining the redox status near an optimal set-point.

Systemic immune‐checkpoint blockade with anti‐PD1 antibodies does not alter cerebral amyloid‐β burden in several amyloid transgenic mouse models

Chronic inflammation represents a central component in the pathogenesis of Alzheimers disease (AD). Recent work suggests that breaking immune tolerance by Programmed cell Death‐1 (PD1) checkpoint inhibition produces an IFN‐γ‐dependent systemic immune response, with infiltration of the brain by peripheral myeloid cells and neuropathological as well as functional improvements even in mice with advanced amyloid pathology (Baruch et al., ( ): Nature Medicine, 22:135–137). Immune checkpoint inhibition was therefore suggested as potential treatment for neurodegenerative disorders when activation of the immune system is appropriate. Because a xenogeneic rat antibody (mAb) was used in the study, whether the effect was specific to PD1 target engagement was uncertain. In the present study we examined whether PD1 immunotherapy can lower amyloid‐β pathology in a range of different amyloid transgenic models performed at three pharmaceutical companies with the exact same anti‐PD1 isotype and two mouse chimeric variants. Although PD1 immunotherapy stimulated systemic activation of the peripheral immune system, monocyte‐derived macrophage infiltration into the brain was not detected, and progression of brain amyloid pathology was not altered. Similar negative results of the effect of PD1 immunotherapy on amyloid brain pathology were obtained in two additional models in two separate institutions. These results show that inhibition of PD1 checkpoint signaling by itself is not sufficient to reduce amyloid pathology and that additional factors might have contributed to previously published results (Baruch et al., ( ): Nature Medicine, 22:135–137). Until such factors are elucidated, animal model data do not support further evaluation of PD1 checkpoint inhibition as a therapeutic modality for Alzheimers disease.

NF-L in cerebrospinal fluid and serum is a biomarker of neuronal damage in an inducible mouse model of neurodegeneration

Accumulation of neurofilaments (NFs), the major constituents of the neuronal cytoskeleton, is a distinctive feature of neurological diseases and several studies have shown that soluble NFs can be detected in the cerebrospinal fluid (CSF) of patients with neurological diseases, such as multiple sclerosis and frontotemporal dementia. Here we have used an inducible transgenic mouse model of neurodegeneration, CamKII-TetOp25 mice, to evaluate whether NF-L levels in CSF or blood can be used as a biochemical biomarker of neurodegeneration. Induction of p25 transgene brain expression led to increase in CSF and serum NF-L levels that correlated with ongoing neurodegeneration. Switching off p25 prevented further increases in both CSF and serum NF-L levels and concomitantly stopped the progression of neurodegeneration. The levels of CSF NF-L detected in p25 mice are about 4-fold higher than the CSF levels detected in patients with chronic neurodegenerative diseases, such as symptomatic FTD (bvFTD). In addition, our data indicate that the NF-L detected in CSF is most likely a cleaved form of NF-L. These results suggest that CSF and serum NF-L are of interest to be further explored as potential translational dynamic biomarkers of neurodegeneration or as pharmacodynamics biomarkers at least in preclinical animal studies.

Nicotinic Receptor Subunit mRNA Expression in Dopaminergic Neurons of the Rat Brain

icotinic agonists have been shown to increase dopamine release both in vivo and in vitro. In particular, modulation of dopaminergic neurotransmission in the mesolimbic system is thought to be responsible for the addictive properties of nicotine. However, the composition of the native nicotinic acetylcholine receptors (nAChRs) involved remains unclear. In situ hybridization studies have shown the presence of most of the known neuronal nAChR subunit mRNA (α3–6 and β2–3) in the substantia nigra (SN) and ventral tegmental area (VTA). In addition, studies with β2 subunit knockout mice suggest that β2-containing nAChRs play an important role in nicotine-evoked dopamine release, and pharmacological analyses indicate that at least two different receptor subtypes, one containing an α3/β2 subunit interface, may be involved. However, only the α6 subunit has been recently unambiguously localized on dopaminergic neurons. In order to determine the nAChR subunits specifically expressed in dopaminergic neurons, we have used the RT-PCR technique to analyze nAChR mRNA expression in rat SN and VTA following total unilateral lesion of the dopaminergic system induced by injection of 6-hydroxydopamine (6-OHDA).

SAR110894, a potent histamine H3-receptor antagonist, displays disease-modifying activity in a transgenic mouse model of tauopathy

Tau hyperphosphorylation and neurofibrillary tangles are histopathologic hallmarks of tauopathies. Histamine H3‐receptor antagonists have been proposed to reduce tau hyperphosphorylation in preclinical models.