Elsa Diguet

Human ESC-Derived Dopamine Neurons Show Similar Preclinical Efficacy and Potency to Fetal Neurons when Grafted in a Rat Model of Parkinson’s Disease

Summary Considerable progress has been made in generating fully functional and transplantable dopamine neurons from human embryonic stem cells (hESCs). Before these cells can be used for cell replacement therapy in Parkinson’s disease (PD), it is important to verify their functional properties and efficacy in animal models. Here we provide a comprehensive preclinical assessment of hESC-derived midbrain dopamine neurons in a rat model of PD. We show long-term survival and functionality using clinically relevant MRI and PET imaging techniques and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal dopamine neurons. Furthermore, we show that hESC-derived dopamine neurons can project sufficiently long distances for use in humans, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures. This provides strong preclinical support for clinical translation of hESC-derived dopamine neurons using approaches similar to those established with fetal cells for the treatment of Parkinson’s disease.

A simple method to measure stride length as an index of nigrostriatal dysfunction in mice.

Reduced stride length characterizes Parkinsonian gait. We aimed to demonstrate that it could be measured simply and reliably in mice by pawprints and used as an index of basal ganglia dysfunction. In C57BL/6 mice, stride length measurements proved to be consistent across measurements and experimenters. It was slightly lower in the hindlimbs and was correlated to femur size and animal velocity. Dopamine depletion by reserpine and striatal dopamine receptor blockade by haloperidol resulted in reduced mean stride length in four limbs. Significant forelimb/hindlimb difference was also observed both in mice with 3-nitropropionic acid (3-NP) induced striatal lesions and in those with MPTP-induced nigral cell loss. Reduction of hindlimb stride length was correlated significantly with the magnitude of cell loss, either in the substantia nigra or in the lateral mid-striatum. Stride length is, therefore, a simple method to obtain an index of motor disorders due to basal ganglia dysfunction in mice.

Deleterious effects of minocycline in animal models of Parkinson's disease and Huntington's disease.

Minocycline has been shown to exert anti‐inflammatory effects underlying its putative neuroprotective properties in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of Parkinsons disease and in the R6/2 mouse model of Huntingtons disease (HD). However, contradictory results have recently been reported. We report deleterious effects of minocycline in two phenotypic (toxic) models of Parkinsons disease and HD in monkey and mouse. Of seven MPTP‐intoxicated female cynomolgus monkeys (0.2 mg/kg, i.v. until day 15), three received minocycline (200 mg b.i.d.). While placebo‐MPTP‐treated animals displayed mild parkinsonism at day 15, the minocycline/MPTP‐treated animals tended to be more affected (P = 0.057) and showed a greater loss of putaminal dopaminergic nerve endings (P < 0.0001). In the 3‐nitropropionic acid (3‐NP) mouse model of HD, minocycline (45 mg/kg i.p.) was administered 30 min before each i.p. injection of 3‐NP (b.i.d., cumulated dose, 360 mg/kg in 5 days). Mice receiving minocycline exhibited a worsening of the mean motor score with a slower recovery slope, more impaired general activity and significantly deteriorated performances on the rotarod, pole test and beam‐traversing tasks. The histopathological outcome demonstrated that minocycline‐treated mice presented significantly more severe neuronal cell loss in the dorsal striatum. The effect of minocycline vs. 3‐NP was also investigated on hippocampal and cortical cell cultures. minocycline blocked 3‐NP‐induced neurotoxicity at certain doses (1 mm cortical neurons) but not at higher doses (10 mm). Thus, minocycline may have variable and even deleterious effects in different species and models according to the mode of administration and dose.

SUBACUTE SYSTEMIC 3-NITROPROPIONIC ACID INTOXICATION INDUCES A DISTINCT MOTOR DISORDER IN ADULT C57Bl/6 MICE: BEHAVIOURAL AND HISTOPATHOLOGICAL CHARACTERISATION

Data on motor behavioural disorders induced by systemic 3-nitropropionic acid, an irreversible inhibitor of mitochondrial succinate dehydrogenase and their histopathological correlates in mice, are sparse. We thus further characterised the subacute 3-nitropropionic-acid-induced motor disorder and its time course in C57Bl/6 mice using standard behavioural tests, histopathological correlates and in vivo magnetic resonance imaging. Firstly, we studied two intoxication paradigms (340 and 560 mg 3-nitropropionic acid/kg, 7 days) compared to controls. The low-dose regimen induced only slight motor changes (reduced hindlimb stride length and rearing). The high-dose regimen induced significant (P<0.05) behavioural and sensorimotor integration deficits (pole test, rotarod, stride length, open-field spontaneous activity) but with 37.5% lethality at week one. The clinical motor disorder consisted of hindlimb clasping and dystonia, truncal dystonia, bradykinesia and impaired postural control. Histopathologically, there were discrete lesions of the dorsolateral striatum in 62.5% of mice together with a 32% reduction (P<0.0001) of the striatal volume, reduced caldbindin-D28K immunoreactivity in the lateral striatum, and met-enkephalin and substance P in the striatal output pathways. There was also a significant (P<0.05) 30-40% dopaminergic cell loss within the substantia nigra pars compacta. Secondly, we validated a semi-quantitative behavioural scale to describe the time course of the motor deficits and to predict the occurrence of striatal damage. We sought to determine whether it could also be disclosed in vivo by magnetic resonance imaging. The scale correlated with the striatal volume reduction (r(2)=0.57) and striatal cell loss (r(2)=0.87) but not with the loss of striatal dopaminergic terminals (dopamine transporter binding). Increased T2-signal intensity within the striatal lesion correlated with the cell loss (r(2)=0.66). We conclude that systemic administration of 3-nitropropionic acid in C57Bl/6 mice induces a distinct motor disorder and dose-dependent striatonigral damage, which are potentially useful to model human diseases of the basal ganglia.

MOTOR BEHAVIOUR DEFICITS AND THEIR HISTOPATHOLOGICAL AND FUNCTIONAL CORRELATES IN THE NIGROSTRIATAL SYSTEM OF DOPAMINE TRANSPORTER KNOCKOUT MICE

Chronic dysregulation of dopamine homeostasis has been shown to induce behavioural impairment in dopamine transporter knockout mutant mice arising from the dysfunction of the mesolimbic and hypothalamo-infundibular system. Here, we assessed whether there are also any motor consequences of a chronic and constitutive hyperdopaminergia in the nigrostriatal system in dopamine transporter knockout mutant mice. For this, we analysed motor performances using tests assessing balance, coordinated motor skills (rotarod, pole test), stride lengths and locomotor activity. Dopamine transporter knockout mutant mice were markedly hyperactive in the open field with central compartment avoidance, as previously shown. However, sensorimotor integration was also found to be altered in dopamine transporter knockout mutant mice which displayed a reduced fore- and hind-limb mean stride length, impaired motor coordination on the pole test and reduced rearings in the open field. Moreover, dopamine transporter knockout mutant mice showed a slower task acquisition on the rotarod. Six-week-old dopamine transporter knockout wild type mice having the same femur size as adult dopamine transporter knockout mutant mice ruled out a possible size-effect bias. Whilst there was no significant difference in the striatal volume, we found a slight but significant reduction in neuronal density in the striatum but not in the nucleus accumbens of dopamine transporter knockout mutant mice. There was a reduced binding in the striatum and nucleus accumbens of dopamine(1) receptors ([(3)H]SCH 23390) and dopamine(2) receptors ([(3)H]YM-09151-2). There was no significant difference in the number of dopaminergic neurons in the substantia nigra between dopamine transporter knockout mutant mice and dopamine transporter knockout wild type mice. These results suggest an impaired functioning of the nigrostriatal system in dopamine transporter knockout mutant hyperdopaminergic mice, as illustrated by motor and sensorimotor integration deficits, despite their apparent hyperactivity. These dysfunctions may arise from combined striatal cell loss and/or functional changes of dopaminergic neurotransmission.

Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II

In neurodegenerative disorders associated with primary or secondary mitochondrial defects such as Huntingtons disease (HD), cells of the striatum are particularly vulnerable to cell death, although the mechanisms by which this cell death is induced are unclear. Dopamine, found in high concentrations in the striatum, may play a role in striatal cell death. We show that in primary striatal cultures, dopamine increases the toxicity of an N-terminal fragment of mutated huntingtin (Htt-171-82Q). Mitochondrial complex II protein (mCII) levels are reduced in HD striatum, indicating that this protein may be important for dopamine-mediated striatal cell death. We found that dopamine enhances the toxicity of the selective mCII inhibitor, 3-nitropropionic acid. We also demonstrated that dopamine doses that are insufficient to produce cell loss regulate mCII expression at the mRNA, protein and catalytic activity level. We also show that dopamine-induced down-regulation of mCII levels can be blocked by several dopamine D2 receptor antagonists. Sustained overexpression of mCII subunits using lentiviral vectors abrogated the effects of dopamine, both by high dopamine concentrations alone and neuronal death induced by low dopamine concentrations together with Htt-171-82Q. This novel pathway links dopamine signaling and regulation of mCII activity and could play a key role in oxidative energy metabolism and explain the vulnerability of the striatum in neurodegenerative diseases.

A role of mitochondrial complex II defects in genetic models of Huntington's disease expressing N-terminal fragments of mutant huntingtin

Huntingtons disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of a CAG repeat encoding a polyglutamine tract in the huntingtin (Htt) protein. The mutation leads to neuronal death through mechanisms which are still unknown. One hypothesis is that mitochondrial defects may play a key role. In support of this, the activity of mitochondrial complex II (C-II) is preferentially reduced in the striatum of HD patients. Here, we studied C-II expression in different genetic models of HD expressing N-terminal fragments of mutant Htt (mHtt). Western blot analysis showed that the expression of the 30 kDa Iron–Sulfur (Ip) subunit of C-II was significantly reduced in the striatum of the R6/1 transgenic mice, while the levels of the FAD containing catalytic 70 kDa subunit (Fp) were not significantly changed. Blue native gel analysis showed that the assembly of C-II in mitochondria was altered early in N171-82Q transgenic mice. Early loco-regional reduction in C-II activity and Ip protein expression was also demonstrated in a rat model of HD using intrastriatal injection of lentiviral vectors encoding mHtt. Infection of the rat striatum with a lentiviral vector coding the C-II Ip or Fp subunits induced a significant overexpression of these proteins that led to significant neuroprotection of striatal neurons against mHtt neurotoxicity. These results obtained in vivo support the hypothesis that structural and functional alterations of C-II induced by mHtt may play a critical role in the degeneration of striatal neurons in HD and that mitochondrial-targeted therapies may be useful in its treatment.

MPTP potentiates 3-nitropropionic acid-induced striatal damage in mice: reference to striatonigral degeneration.

Striatonigral degeneration (SND) is a parkinsonian disorder due to the combined degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and striatal output neurons. The aims of this study were to explore (1) the behavioral and histopathological consequences of combined MPTP plus 3-nitropropionic acid (3-NP) intoxication in C57/Bl6 mice and (2) its ability to reproduce the neuropathological hallmarks of SND. 3-NP was administered i.p. every 12 h (total dose=450 mg/kg in 9 days) and MPTP i.p. at 10 mg/(kg day) (total dose=90 mg/kg in 9 days). Four groups of mice (n=10) were compared: control, 3-NP alone, MPTP alone, MPTP + 3-NP. Mice intoxicated with 3-NP and MPTP + 3-NP developed motor symptoms, including hindlimb dystonia and clasping, truncal dystonia and impaired balance adjustments. The severity of motor disorder was worse and lasted longer in MPTP + 3-NP-treated mice compared to 3-NP alone, MPTP alone and controls. 3-NP and MPTP + 3-NP-treated mice also displayed altered gait patterns, impaired motor performance on the pole test, rotarod and traversing a beam tasks and activity parameters. Several of these sensorimotor deficits were also more severe and lasted longer in MPTP + 3-NP-treated mice. Histology demonstrated increased neuronal loss along with astrocytic activation (glial fibrillary acid protein, GFAP) and a higher incidence of circumscribed striatal lateral lesions in MPTP + 3-NP-treated mice compared to 3-NP. Neuronal loss and astrocytic activation were increased in the lateral part of the striatum in 3-NP-intoxicated mice while observed both in the medial and lateral part in MPTP + 3-NP-intoxicated mice. There was also a significant loss of SNc dopaminergic neurons and striatal terminals, similar to that in MPTP-treated mice. Altogether, these results suggest that MPTP potentiates striatal damage and behavioral impairments induced by 3-NP intoxication in mice and constitutes a useful model of the motor disorder and its histopathological correlates in SND.

Minocycline is not beneficial in a phenotypic mouse model of Huntington's disease

Smith and colleagues evaluate minocycline and doxycycline in the R6/2 mouse model of Huntington’s disease (HD) and conclude that these drugs are not effective. The findings are in sharp contrast with ours. Given the differences between the studies and the sweeping conclusions Smith and colleagues draw about the appropriateness of considering clinical trials of minocycline for HD, we feel compelled to address certain issues. In our study, daily intraperitoneal minocycline administration improved Rotarod performance, increased survival, inhibited caspase activation, and inhibited nitric oxide synthase. Minocycline, at the dose tested, did not affect mouse weight (a sign of lack of toxicity). Higher minocycline doses in R6/2 mice are toxic. We have repeated the original protocol of intraperitoneal minocycline delivery in a larger cohort of mice (n 20), reproducing our previously published data (unpublished data). Minocycline, in addition to HD, is effective in models of stroke, brain trauma, amyotrophic lateral sclerosis, Parkinson’s disease, and multiple sclerosis. A key to the effectiveness of minocycline is its mechanisms of action, targeting the core pathophysiology of these disorders. Smith and colleagues delivered minocycline and doxycycline with the drinking water. Two minocycline doses were evaluated, 1 and 10mg/ml. Whereas 1 mg/ml was neither effective nor toxic, 10mg/ml had toxic effects and was reduced to 5mg/ml during the trial. This is a major flaw that hinders interpretation of the results. It has been our experience that R6/2 mice may not recover from toxicity of compounds administered at excessive doses that are subsequently lowered. The only information that can be concluded from this cohort is that 10mg/ml minocycline is toxic. Given that mice were adversely affected by this dosing, valid conclusions as to the lack of neuroprotection cannot be drawn. Furthermore, the informativeness of this study is rather limited because no data were provided on the length of survival, the most powerful and relevant end point for neuroprotection. A brain drug concentration of 5.8 M is reported for mice receiving 2mg/ml minocycline. The authors state that this steady state brain concentration is higher than what was achieved in our study injecting 5mg/kg/day IP and substantiate it by their measurement of brain minocycline levels after a single intraperitoneal injection. Plasma elimination halflife of minocycline is 12 to 18 hours. Plasma steady-state is reached within four to five half-lives. Hence, single-dose kinetics yields expectedly a plasma concentration that underestimates the levels after repetitive dosing. The evaluation performed in this publication on the minocycline brain concentration after intraperitoneal administration therefore bears no relevance to determining whether their doses and ours are equivalent. The experimental design raises concerns that their results may have been compromised because of problems with stability of minocycline. Minocycline was kept in solution for 7 days. Although the authors provide high-performance liquid chromotography analysis of drug stability, we have compared drug efficacy of daily and once a week preparations and found little or no improvement in the phenotype of R6/2 mice in doses made weekly (unpublished data). Therefore, the HPLC analysis is questionable in the detection of structural alterations to the drugs that might render it ineffective. The results of this study bear little relevance to our original study. The route of administration (oral vs intraperitoneal) the manner the drug was prepared (weekly vs daily), and the outcome measurements are different (no survival component reported, the gold standard in a therapeutic trial). If this group was attempting to validate our study, it should have followed our testing protocol. These types of experiments are difficult and many variables can cause negative results. We agree with Smith and colleagues that taking a drug into human clinical trials bears great responsibility. However, not advancing a safe drug into a trial for a universally lethal disease with no current treatment based on negative but inconclusive/flawed studies would, in our opinion, not be responsible.

Dopamine transporter knock-out mice are hypersensitive to 3-nitropropionic acid-induced striatal damage.

Evidence suggests that dopamine is involved in the modulation of striatal excitotoxic processes. To further investigate this issue, we studied the effects of systemic ‘low‐dose’ (total dose, 340 mg/kg in 7 days) 3‐nitropropionic acid (3‐NP) intoxication in dopamine transporter knock‐out mice (DAT–/–) compared to wildtype (DAT+/+) mice. Systemic ‘low‐dose’ 3‐NP induced a significant impairment in a rotarod task only in DAT–/– mice. Histopathology also demonstrated a significant reduction of the striatal volume (−7%, P < 0.05), neuronal density (−12.5%, P < 0.001) and absolute number estimates of striatal neurons (−11.5%, P < 0.001) in DAT–/– compared to DAT+/+ mice, with increased glial activation, independent of the degree of succinate dehydrogenase inhibition. These findings strengthen the hypothesis for dopamine modulation of excitotoxicity within the nigrostriatal system.