Debby Van Dam

Age-dependent cognitive decline in the APP23 model precedes amyloid deposition.

Heterozygous APP23 mice, expressing human amyloid‐precursor protein with the Swedish double mutation and control littermates, were subjected to behavioral and neuromotor tasks at the age of 6–8 weeks, 3 and 6 months. A hidden‐platform Morris‐type water maze showed an age‐dependent decline of spatial memory capacities in the APP23 model. From the age of 3 months onwards, the APP23 mice displayed major learning and memory deficits as demonstrated by severely impaired learning curves during acquisition and impaired probe trial performance. In addition to the cognitive deficit, APP23 mice displayed disturbed activity patterns. Overnight cage‐activity recording showed hyperactivity in the transgenics for the three age groups tested. However, a short 2‐h recording during dusk phase demonstrated lower activity levels in 6‐month‐old APP23 mice as compared to controls. Moreover, at this age, APP23 mice differed from control littermates in exploration and activity levels in the open‐field paradigm. These findings are reminiscent of disturbances in circadian rhythms and activity observed in Alzheimer patients. Determination of plaque‐associated human amyloid‐β1–42 peptides in brain revealed a fivefold increase in heterozygous APP23 mice at 6 months as compared to younger transgenics. This increase coincided with the first appearance of plaques in hippocampus and neocortex. Spatial memory deficits preceded plaque formation and increase in plaque‐associated amyloid‐β1–42 peptides, but probe trial performance did correlate negatively with soluble amyloid‐β brain concentration in 3‐month‐old APP23 mutants. Detectable plaque formation is not the (only) causal factor contributing to memory defects in the APP23 model.

Decreased expression of the GABAA receptor in fragile X syndrome.

After our initial discovery of under expression of the GABA(A) receptor delta subunit in a genome wide screening for differentially expressed mRNAs in brain of fragile X mice, a validated model for fragile X mental retardation syndrome, we analyzed expression of the 17 remaining subunits of the GABA(A) receptor using real-time PCR. We confirmed nearly 50% under expression of the delta subunit and found a significant 35%-50% reduction in expression of 7 additional subunit mRNAs, namely alpha(1), alpha(3), and alpha(4), beta(1) and beta(2) and gamma(1) and gamma(2), in fragile X mice compared to wild-type littermates. In concordance with previous results, under expression was found in cortex, but not in cerebellum. Moreover, decreased expression of specific GABA(A) receptor subunits in fragile X syndrome seems to be an evolutionary conserved hallmark since in the fragile X fly (Drosophila melanogaster) model we also found almost 50% under expression of all 3 subunits which make up the invertebrate GABA receptor, namely Grd, Rdl and Lcch3. In addition, we demonstrated a direct correlation between the amount of dFmrp and the expression of the GABA receptor subunits Rdl and Grd. Our results add evidence to previous observations of an altered GABAergic system in fragile X syndrome. Because GABA(A) receptors are the major inhibitory receptors in brain, involved in anxiety, depression, insomnia, learning and memory and epilepsy, processes also disturbed in fragile X patients, the well described GABA(A) receptor pharmacology might open new powerful opportunities for treatment of the behavioral and epileptic phenotype associated with fragile X syndrome.

Spatial learning, contextual fear conditioning and conditioned emotional response in Fmr1 knockout mice

Fmr1 knockout mice are an animal model for fragile X syndrome, the most common form of heritable mental retardation in humans. Fmr1 knockout mice exhibit macro-orchidism and cognitive and behavioural deficits reminiscent of the human phenotype. In the present study additional behavioural and cognitive testing was performed. Knockouts and control littermates were subjected to a spatial learning test using a plus-shaped water maze. Animals had to learn the position of a hidden escape platform during training trials. The position of this platform was changed during subsequent reversal trials. Previously reported deficits in reversal learning were replicated, but we also observed significant differences during the acquisition trials. A plus-shaped water maze experiment with daily changing platform positions failed to provide clear evidence for a working memory impairment, putatively underlying the spatial learning deficits. Two different test settings were used to examine the reported deficit of Fmr1 knockout mice in fear conditioning. Conditioned fear responses were observed in a contextual fear test, and the ability to acquire an emotional response was tested by means of response suppression in a conditioned emotional response procedure. Neither protocol revealed significant differences between controls and knockouts.

Adeno-associated Virus Gene Therapy With Cholesterol 24-Hydroxylase Reduces the Amyloid Pathology Before or After the Onset of Amyloid Plaques in Mouse Models of Alzheimer's Disease

The development of Alzheimers disease (AD) is closely connected with cholesterol metabolism. Cholesterol increases the production and deposition of amyloid-beta (Abeta) peptides that result in the formation of amyloid plaques, a hallmark of the pathology. In the brain, cholesterol is synthesized in situ but cannot be degraded nor cross the blood-brain barrier. The major exportable form of brain cholesterol is 24S-hydroxycholesterol, an oxysterol generated by the neuronal cholesterol 24-hydroxylase encoded by the CYP46A1 gene. We report that the injection of adeno-associated vector (AAV) encoding CYP46A1 in the cortex and hippocampus of APP23 mice before the onset of amyloid deposits markedly reduces Abeta peptides, amyloid deposits and trimeric oligomers at 12 months of age. The Morris water maze (MWM) procedure also demonstrated improvement of spatial memory at 6 months, before the onset of amyloid deposits. AAV5-wtCYP46A1 vector injection in the cortex and hippocampus of amyloid precursor protein/presenilin 1 (APP/PS) mice after the onset of amyloid deposits also reduced markedly the number of amyloid plaques in the hippocampus, and to a less extent in the cortex, 3 months after the injection. Our data demonstrate that neuronal overexpression of CYP46A1 before or after the onset of amyloid plaques significantly reduces Abeta pathology in mouse models of AD.

Cognitive decline, neuromotor and behavioural disturbances in a mouse model for fragile-X-associated tremor/ataxia syndrome (FXTAS)

Carriers of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene are spared the major neurodevelopmental symptomatology of fragile X syndrome patients carrying a full mutation (>200 repeats). In a proportion of premutation carriers, the repeat expansion is associated with a specific neurological profile involving intention tremor, ataxia, intellectual decline compatible with dementia syndrome, Parkinsonism and autonomic dysfunction at older age, commonly referred to as fragile-X-associated tremor/ataxia syndrome (FXTAS). Typical CNS changes include hyperintense signals on T2 weighted magnetic resonance images and the presence of ubiquitin-positive intranuclear neuronal inclusions. A knock-in mouse model with a (CGG)98 repeat in the premutation range has been generated and shown to exhibit elevated Fmr1 mRNA levels and ubiquitin-positive intranuclear neuronal inclusions, suggesting it may be a valid model for the human disease. Given the specific clinical profile of FXTAS patients, the expanded CGG repeat model was assessed for cognitive, behavioural and neuromotor performance at different ages (20, 52 and 72 weeks). The Morris water maze task exposed age-dependent decline of visual-spatial memory. Open field recordings revealed decreased exploration of the centre of the arena in the oldest group of expanded CGG repeat mice, potentially reflecting increased anxiety. Neuromotor tasks primarily showed decline of performance on the accelerating rotarod with age in the premutation carriers but not in control littermates. The age-dependent cognitive decline and neuromotor disturbances may be related to the progressive cognitive and behavioural difficulties observed in FXTAS patients.

Animal models in the drug discovery pipeline for Alzheimer's disease

With increasing feasibility of predicting conversion of mild cognitive impairment to dementia based on biomarker profiling, the urgent need for efficacious disease‐modifying compounds has become even more critical. Despite intensive research, underlying pathophysiological mechanisms remain insufficiently documented for purposeful target discovery. Translational research based on valid animal models may aid in alleviating some of the unmet needs in the current Alzheimers disease pharmaceutical market, which includes disease‐modification, increased efficacy and safety, reduction of the number of treatment unresponsive patients and patient compliance. The development and phenotyping of animal models is indeed essential in Alzheimers disease‐related research as valid models enable the appraisal of early pathological processes – which are often not accessible in patients, and subsequent target discovery and evaluation. This review paper summarizes and critically evaluates currently available animal models, and discusses their value to the Alzheimer drug discovery pipeline. Models dealt with include spontaneous models in various species, including senescence‐accelerated mice, chemical and lesion‐induced rodent models, and genetically modified models developed in Drosophila melanogaster, Caenorhabditis elegans, Danio rerio and rodents. Although highly valid animal models exist, none of the currently available models recapitulates all aspects of human Alzheimers disease, and one should always be aware of the potential dangers of uncritical extrapolating from model organisms to a human condition that takes decades to develop and mainly involves higher cognitive functions.

Pharmacological treatment of fragile X syndrome with GABAergic drugs in a knockout mouse model.

Molecular and electrophysiological studies have provided evidence for a general downregulation of the GABAergic system in the Fmr1 knockout mouse. GABA(A) receptors are the main inhibitory receptors in the brain and the GABA(A) receptor was proposed as a novel target for treatment of the fragile X syndrome, the most frequent form of intellectual disability. This study examined the functionality of the GABA(A) receptor in rotarod and elevated plus maze tests with fragile X mice treated with GABA(A) receptor agonists, the benzodiazepine diazepam and the neuroactive steroid alphaxalone. In addition, the effect of GABA(A) receptor activation on the audiogenic seizure activity was determined. We proved that the GABA(A) receptor is still sensitive to GABAergic drugs as the sedative effect of diazepam resulted in a decreased latency time on the rotarod and alphaxalone had a clear anxiolytic effect in the elevated plus maze, decreasing the frequency of entries, the total time spent and the path length in the closed arms. We also observed that treatment with ganaxolone could rescue audiogenic seizures in Fmr1 knockout mice. These findings support the hypothesis that the GABA(A) receptor is a potential therapeutic target for fragile X syndrome.

Cellular ageing, increased mortality and FTLD-TDP-associated neuropathology in progranulin knockout mice†

Loss‐of‐function mutations in progranulin (GRN) are associated with frontotemporal lobar degeneration with intraneuronal ubiquitinated protein accumulations composed primarily of hyperphosphorylated TDP‐43 (FTLD‐TDP). The mechanism by which GRN deficiency causes TDP‐43 pathology or neurodegeneration remains elusive. To explore the role of GRN in vivo, we established Grn knockout mice using a targeted genomic recombination approach and Cre‐LoxP technology. Constitutive Grn homozygous knockout (Grn−/−) mice were born in an expected Mendelian pattern of inheritance and showed no phenotypic alterations compared to heterozygous (Grn+/−) or wild‐type (Wt) littermates until 10 months of age. From then, Grn−/− mice showed reduced survival accompanied by significantly increased gliosis and ubiquitin‐positive accumulations in the cortex, hippocampus, and subcortical regions. Although phosphorylated TDP‐43 could not be detected in the ubiquitinated inclusions, elevated levels of hyperphosphorylated full‐length TDP‐43 were recovered from detergent‐insoluble brain fractions of Grn−/− mice. Phosphorylated TDP‐43 increased with age and was primarily extracted from the nuclear fraction. Grn−/− mice also showed degenerative liver changes and cathepsin D‐positive foamy histiocytes within sinusoids, suggesting widespread defects in lysosomal turnover. An increase in insulin‐like growth factor (IGF)‐1 was observed in Grn−/− brains, and increased IGF‐1 signalling has been associated with decreased longevity. Our data suggest that progranulin deficiency in mice leads to reduced survival in adulthood and increased cellular ageing accompanied by hyperphosphorylation of TDP‐43, and recapitulates key aspects of FTLD‐TDP neuropathology. Copyright

Model organisms: Drug discovery in dementia: the role of rodent models

Key PointsAlzheimers disease (AD), including both early-onset familial and late-onset senile dementia of the Alzheimer type, accounts for the major proportion of dementia cases and neurodegenerative diseases.AD is characterized by progressive cognitive, functional and behavioural impairment, evolving into a dramatic loss of most cortical and subcortical functions, and ultimately death. The major pathological hallmarks include amyloid plaques, neurofibrillary tangles and neuronal cell loss.Valid animal models for AD could help to further unravel underlying degenerative processes and discover therapeutic strategies to alleviate and/or prevent this devastating condition. We describe the major rodent models of AD and evaluate their effectiveness for drug discovery research.For a therapeutic intervention to slow down or halt disease progression — that is, to be disease-modifying — it must interfere with a central pathophysiological pathway.Rodent models have acquired a strong position in the evaluation of the preventive and disease-modifying efficacy of potential therapeutics because they rapidly develop symptoms and/or pathology, allow the assessment of large groups of subjects, improve accessibility to early-stage CNS changes and enable time-linked observations.Non-cognitive symptoms represent a major source of physical and psychological caregiver burden often motivating institutionalization of the patient. Animal models mimicking these symptoms are indispensable tools for evaluating new psychopharmacological strategiesWe empahsize the need for validation of all new models and thorough standardization of procedures, good knowledge of strains, compounds and paradigm characteristics, and skilled personnel.The implementation of a multidisciplinary approach combining valid animal models with new technologies improving biomarker profiling and early diagnosis of dementia subtypes, as well as prediction of patient-specific treatment outcome, will create new paths for improved treatment and prevention of AD.Animal models contribute to advancing our understanding of the pathophysiology of complex disorders and the preclinical assessment of therapeutics for these diseases. Van Dam and De Deyn review the major rodent models of Alzheimers disease and evaluate their usefulness for drug discovery researchAbstractRecent advances in the understanding of the pathophysiological mechanisms underlying Alzheimers disease have pointed to novel strategies for drug development. Animal models have contributed considerably to these advances, and will have a key role in the evaluation of therapeutics that could have the potential not just to alleviate the dementia associated with Alzheimers disease, but to modify the disease process. Here, we summarize and critically evaluate current rodent models of dementia, and discuss their role in drug discovery and development.Recent advances in the understanding of the pathophysiological mechanisms underlying Alzheimers disease have pointed to novel strategies for drug development. Animal models have contributed considerably to these advances, and will have a key role in the evaluation of therapeutics that could have the potential not just to alleviate the dementia associated with Alzheimers disease, but to modify the disease process. Here, we summarize and critically evaluate current rodent models of dementia, and discuss their role in drug discovery and development.

Altered circadian locomotor activity in APP23 mice: a model for BPSD disturbances.

Over the past decade, clinical Alzheimers disease research has been challenged with an increased interest in noncognitive symptomatology, commonly referred to as behavioural and psychological signs and symptoms of dementia (BPSD). In accordance, major attention is being paid to behavioural alterations in the phenotyping of transgenic mouse models. Besides an age‐dependent decline of cognitive functions, the APP23 model was previously shown to exhibit cage activity disturbances, reminiscent of diurnal rhythm disturbances in Alzheimer patients. To further scrutinize these observations, circadian patterns of horizontal locomotor activity were assessed in 3‐, 6‐ and 12‐month‐old APP23 mice and wild‐type littermates in a test paradigm continuously recording cage activity over a period ranging from 1 to 3 days. At the age of 3 months, APP23 profiles resembled the wild‐type pattern to a large extent, although minor differences were already noticeable. Six‐month‐old APP23 mice displayed an altered activity profile with a first indication of increased activity during the second half of the active phase, reminiscent of sundowning behaviour in Alzheimer patients. This bimodal overnight activity pattern became even more evident at the age of 12 months. The APP23 model was therefore shown to display an age‐dependent development of cage activity disturbances and sundowning‐like behaviour. A comparison is made with actigraphic recordings of human Alzheimer patients exhibiting sundowning behaviour. This first report of diurnal rhythm disturbances and sundowning‐like phenomena in a transgenic mouse model greatly adds to the validity of the APP23 model.