Validation of memory assessment in the Starmaze task: Data from 14 month-old APPPS1 mice and controls.

This article describes navigation data of 14 month-old APPPS1 and C57Bl6 in the Starmaze task. These data were acquired as positive controls of memory deficit in a model of the familial form of Alzheimers's disease (see Schmitt et al., Flexibility as a marker of early cognitive decline in humanized Apolipoprotein E ε4 (ApoE4) mice, Neurobiol Aging, 2021). They were acquired in a reduced version of the Starmaze environment and accompanied by a number of acquisitions in different control groups at 6 and 14 months to assess the robustness of the procedure and its associated memory scores. These data illustrate the extraction of a variety of navigation scores (including search strategy, spatial learning and memory) and provide a reference of navigation data in the Starmaze task for healthy 6-month-old controls, normal aging and a model of pathological memory deficit.

Modifications of physical and functional integrity of the blood-brain barrier in an inducible mouse model of neurodegeneration.

The inducible p25 overexpression mouse model recapitulate many hallmark features of Alzheimer's disase including progressive neuronal loss, elevated Aß, tau pathology, cognitive dysfunction, and impaired synaptic plasticity. We chose p25 mice to evaluate the physical and functional integrity of the blood-brain barrier (BBB) in a context of Tau pathology (pTau) and severe neurodegeneration, at an early (3 weeks ON) and a late (6 weeks ON) stage of the pathology. Using in situ brain perfusion and confocal imaging, we found that the brain vascular surface area and the physical integrity of the BBB were unaltered in p25 mice. However, there was a significant 14% decrease in cerebrovascular volume in 6 weeks ON mice, possibly explained by a significant 27% increase of collagen IV in the basement membrane of brain capillaries. The function of the BBB transporters GLUT1 and LAT1 was evaluated by measuring brain uptake of d-glucose and phenylalanine, respectively. In 6 weeks ON p25 mice, d-glucose brain uptake was significantly reduced by about 17% compared with WT, without any change in the levels of GLUT1 protein or mRNA in brain capillaries. The brain uptake of phenylalanine was not significantly reduced in p25 mice compared with WT. Lack of BBB integrity, impaired BBB d-glucose transport have been observed in several mouse models of AD. In contrast, reduced cerebrovascular volume and an increased basement membrane thickness may be more specifically associated with pTau in mouse models of neurodegeneration.

Flexibility as a marker of early cognitive decline in humanized apolipoprotein E ε4 (ApoE4) mice.

To test the hypothesis that ApoE4 may be involved in cognitive deficits associated with aging, we investigated the impact of APOE4 status and aging on the flexibility and memory components of spatial learning in mice. Young adult (6 months) and middle-aged (14 months) ApoE4, ApoE3 and C57BL/6 male mice were tested for flexibility in an aquatic Y-maze, and for spatio-temporal memory acquisition in the Starmaze. Our results revealed a flexibility deficit of the 6-month-old ApoE4 mice compared to controls. However, this deficit was not associated with spatio-temporal memory deficit at the same age. Importantly, the ApoE4 flexibility deficit did not increase with age, nor turn into memory deficit, or was able to predict individual variations of memory performance at 14 months. By contrast, control ApoE3 mice showed a decline of flexibility at 14 months resulting in performance similar to that of ApoE4. Overall, our results suggest that ApoE4 could be associated with an acceleration of the flexibility decrease otherwise observed in normal aging.

Dopaminergic neurodegeneration induced by Parkinson's disease-linked G2019S LRRK2 is dependent on kinase and GTPase activity.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of late-onset, autosomal-dominant familial Parkinson's disease (PD). LRRK2 functions as both a kinase and GTPase, and PD-linked mutations are known to influence both enzymatic activities. While PD-linked LRRK2 mutations can commonly induce neuronal damage in culture models, the mechanisms underlying these pathogenic effects remain uncertain. Rodent models containing familial LRRK2 mutations often lack robust PD-like neurodegenerative phenotypes. Here, we develop a robust preclinical model of PD in adult rats induced by the brain delivery of recombinant adenoviral vectors with neuronal-specific expression of human LRRK2 harboring the most common G2019S mutation. In this model, G2019S LRRK2 induces the robust degeneration of substantia nigra dopaminergic neurons, a pathological hallmark of PD. Introduction of a stable kinase-inactive mutation or administration of the selective kinase inhibitor, PF-360, attenuates neurodegeneration induced by G2019S LRRK2. Neuroprotection provided by pharmacological kinase inhibition is mediated by an unusual mechanism involving the robust destabilization of human LRRK2 protein in the brain relative to endogenous LRRK2. Our study further demonstrates that G2019S LRRK2-induced dopaminergic neurodegeneration critically requires normal GTPase activity, as hypothesis-testing mutations that increase GTP hydrolysis or impair GTP-binding activity provide neuroprotection although via distinct mechanisms. Taken together, our data demonstrate that G2019S LRRK2 induces neurodegeneration in vivo via a mechanism that is dependent on kinase and GTPase activity. Our study provides a robust rodent preclinical model of LRRK2-linked PD and nominates kinase inhibition and modulation of GTPase activity as promising disease-modifying therapeutic targets.

Gene delivery of a modified antibody to Aß reduces progression of murine Alzheimer's disease.

Antibody therapies for Alzheimer's Disease (AD) hold promise but have been limited by the inability of these proteins to migrate efficiently across the blood brain barrier (BBB). Central nervous system (CNS) gene transfer by vectors like adeno-associated virus (AAV) overcome this barrier by allowing the bodies' own cells to produce the therapeutic protein, but previous studies using this method to target amyloid-ß have shown success only with truncated single chain antibodies (Abs) lacking an Fc domain. The Fc region mediates effector function and enhances antigen clearance from the brain by neonatal Fc receptor (FcRn)-mediated reverse transcytosis and is therefore desirable to include for such treatments. Here, we show that single chain Abs fused to an Fc domain retaining FcRn binding, but lacking Fc gamma receptor (FcγR) binding, termed a silent scFv-IgG, can be expressed and released into the CNS following gene transfer with AAV. While expression of canonical IgG in the brain led to signs of neurotoxicity, this modified Ab was efficiently secreted from neuronal cells and retained target specificity. Steady state levels in the brain exceeded peak levels obtained by intravenous injection of IgG. AAV-mediated expression of this scFv-IgG reduced cortical and hippocampal plaque load in a transgenic mouse model of progressive ß-amyloid plaque accumulation. These findings suggest that CNS gene delivery of a silent anti-Aß scFv-IgG was well-tolerated, durably expressed and functional in a relevant disease model, demonstrating the potential of this modality for the treatment of Alzheimer's disease.

Data on synthesis, ADME and pharmacological properties and early safety pharmacology evaluation of a series of novel NURR1/NOT agonist potentially useful for the treatment of Parkinson's disease.

This article describes the chemical synthesis, ADME and pharmacological properties and early safety pharmacology evaluation of a series of novel Nurr1/NOT agonist. It is meant as a support to an article recently published in Bioorganic and Medicinal chemistry Letters and entitled "Development of a novel NURR1/NOT agonist from hit to lead and candidate for the potential treatment of Parkinson's disease" [1] and presenting the discovery, scope and potential of these new ligands of these nuclear receptors.

Development of a novel NURR1/NOT agonist from hit to lead and candidate for the potential treatment of Parkinson's disease.

In the course of a programme aimed at identifying Nurr1/NOT agonists for potential treatment of Parkinson's disease, a few hits from high throughput screening were identified and characterized. A combined optimization pointed to a very narrow and stringent structure activity relationship. A comprehensive program of optimization led to a potent and safe candidate drug displaying neuroprotective and anti-inflammatory activity in several in vitro and in vivo models.

SAR228810: an antibody for protofibrillar amyloid ß peptide designed to reduce the risk of amyloid-related imaging abnormalities (ARIA).

BACKGROUND: Anti-amyloid ß (Aß) immunotherapy represents a major area of drug development for Alzheimer's disease (AD). However, Aß peptide adopts multiple conformations and the pathological forms to be specifically targeted have not been identified. Aß immunotherapy-related vasogenic edema has also been severely dose limiting for antibodies with effector functions binding vascular amyloid such as bapineuzumab. These two factors might have contributed to the limited efficacy demonstrated so far in clinical studies. METHODS: To address these limitations, we have engineered SAR228810, a humanized monoclonal antibody (mAb) with limited Fc effector functions that binds specifically to soluble protofibrillar and fibrillar forms of Aß peptide and we tested it together with its murine precursor SAR255952 in vitro and in vivo. RESULTS: Unlike gantenerumab and BAN2401, SAR228810 and SAR255952 do not bind to Aß monomers, low molecular weight Aß oligomers or, in human brain sections, to Aß diffuse deposits which are not specific of AD pathology. Both antibodies prevent Aß42 oligomer neurotoxicity in primary neuronal cultures. In vivo, SAR255952, a mouse aglycosylated IgG1, dose-dependently prevented brain amyloid plaque formation and plaque-related inflammation with a minimal active dose of 3 mg/kg/week by the intraperitoneal route. No increase in plasma Aß levels was observed with SAR255952 treatment, in line with its lack of affinity for monomeric Aß. The effects of SAR255952 translated into synaptic functional improvement in ex-vivo hippocampal slices. Brain penetration and decoration of cerebral amyloid plaques was documented in live animals and postmortem. SAR255952 (up to 50 mg/kg/week intravenously) did not increase brain microhemorrhages and/or microscopic changes in meningeal and cerebral arteries in old APPSL mice while 3D6, the murine version of bapineuzumab, did. In immunotolerized mice, the clinical candidate SAR228810 demonstrated the same level of efficacy as the murine SAR255952. CONCLUSION: Based on the improved efficacy/safety profile in non-clinical models of SAR228810, a first-in-man single and multiple dose administration clinical study has been initiated in AD patients.

High brain distribution of a new central nervous system drug candidate despite its P-glycoprotein-mediated efflux at the mouse blood-brain barrier.

Efficacy of drugs aimed at treating central nervous system (CNS) disorders rely partly on their ability to cross the cerebral endothelium, also called the blood-brain barrier (BBB), which constitutes the main interface modulating exchanges of compounds between the brain and blood. In this work, we used both, conventional pharmacokinetics (PK) approach and in situ brain perfusion technique to study the blood and brain PK of PKRinh, an inhibitor of the double-stranded RNA-dependent protein kinase (PKR) activation, in mice. PKRinh showed a supra dose-proportional blood exposure that was not observed in the brain, and a brain to blood AUC ratio of unbound drug smaller than 1 at all tested doses. These data suggested the implication of an active efflux at the BBB. Using in situ brain perfusion technique, we showed that PKRinh has a very high brain uptake clearance which saturates with increasing concentrations. Fitting the data to a Michaelis-Menten equation revealed that PKRinh transport through the BBB is composed of a passive unsaturable flux and an active saturable protein-mediated efflux with a km of ≅ 3 µM. We were able to show that the ATP-binding cassette (ABC) transporter P-gp (Abcb1), but not Bcrp (Abcg2), was involved in the brain to blood efflux of PKRinh. At the circulating PKRinh concentrations of this study, the P-gp was not saturated, in accordance with the linear brain PKRinh PK. Finally, PKRinh had high brain uptake clearance (14 µl/g/s) despite it is a good P-gp substrate (P-gp Efflux ratio ≅ 3.6), and reached similar values than the cerebral blood flow reference, diazepam, in P-gp saturation conditions. With its very unique brain transport properties, PKRinh improves our knowledge about P-gp-mediated efflux across the BBB for the development of new CNS directed drugs.

Systemic immune-checkpoint blockade with anti-PD1 antibodies does not alter cerebral amyloid-ß burden in several amyloid transgenic mouse models.

Chronic inflammation represents a central component in the pathogenesis of Alzheimer's 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-ß 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 Alzheimer's 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.

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

INTRODUCTION: Tau hyperphosphorylation and neurofibrillary tangles are histopathologic hallmarks of tauopathies. Histamine H3-receptor antagonists have been proposed to reduce tau hyperphosphorylation in preclinical models. METHODS: We evaluated the ability of SAR110894, a selective histamine H3-receptor antagonist, to inhibit tau pathology and prevent cognitive deficits in a tau transgenic mouse model (THY-Tau22). RESULTS: SAR110894 treatment for 6 months (but not 2 weeks) in THY-Tau22 mice decreased both tau hyperphosphorylation at pSer396-pSer404 (AD2 signal) in the hippocampus and the number of AT8 (pSer199/202-Thr205) positive cells in the cortex and decreased the formation of neurofibrillary tangles in the cortex, hippocampus, and amygdala. Macrophage inflammatory protein 1-alpha messenger RNA expression was decreased in the hippocampus. SAR110894 also prevented episodic memory deficits, and this effect was still detected after treatment washout. DISCUSSION: Long-term SAR110894 treatment could have potential disease modifying activity in neurodegenerative tauopathies.

A Nurr1 agonist causes neuroprotection in a Parkinson's disease lesion model primed with the toll-like receptor 3 dsRNA inflammatory stimulant poly(I:C).

Dopaminergic neurons in the substantia nigra pars compacta (SNpc) are characterized by the expression of genes required for dopamine synthesis, handling and reuptake and the expression of these genes is largely controlled by nuclear receptor related 1 (Nurr1). Nurr1 is also expressed in astrocytes and microglia where it functions to mitigate the release of proinflammatory cytokines and neurotoxic factors. Given that Parkinson's disease (PD) pathogenesis has been linked to both loss of Nurr1 expression in the SNpc and inflammation, increasing levels of Nurr1 maybe a promising therapeutic strategy. In this study a novel Nurr1 agonist, SA00025, was tested for both its efficiency to induce the transcription of dopaminergic target genes in vivo and prevent dopaminergic neuron degeneration in an inflammation exacerbated 6-OHDA-lesion model of PD. SA00025 (30mg/kg p.o.) entered the brain and modulated the expression of the dopaminergic phenotype genes TH, VMAT, DAT, AADC and the GDNF receptor gene c-Ret in the SN of naive rats. Daily gavage treatment with SA00025 (30mg/kg) for 32 days also induced partial neuroprotection of dopaminergic neurons and fibers in rats administered a priming injection of polyinosinic-polycytidylic acid (poly(I:C) and subsequent injection of 6-OHDA. The neuroprotective effects of SA00025 in this dopamine neuron degeneration model were associated with changes in microglial morphology indicative of a resting state and a decrease in microglial specific IBA-1 staining intensity in the SNpc. Astrocyte specific GFAP staining intensity and IL-6 levels were also reduced. We conclude that Nurr1 agonist treatment causes neuroprotective and anti-inflammatory effects in an inflammation exacerbated 6-OHDA lesion model of PD.