Neuronal expression of F-box and leucine-rich-repeat protein 2 decreases over Braak stages in the brains of Alzheimer's disease patients.

BACKGROUND: The dysfunction of protein degradation through the ubiquitin-proteasome system is now widely accepted as one of the causes of Alzheimer's disease (AD), the pathological hallmarks of which are abnormal protein accumulation such as senile plaques and neurofibrillary tangles in the brain. OBJECTIVE: To examine the expression of F-box and leucine-rich-repeat protein 2 (FBL2), a member of the ubiquitin-protein ligase complex expected to be involved in the ubiquitin-proteasome system. METHODS AND RESULTS: We investigated the expression profile of FBL2 in the brains of AD patients by quantitative PCR and immunohistochemical analysis. In healthy subjects, the FBL2 mRNA level was very high in the brain when compared to other tissues. FBL2 immunoreactivities were detected in somata and dendrites in the neurons, but not detected in astrocytes or microglia. The FBL2 mRNA level decreased progressively in the brains of AD patients over Braak stages; this was more prominent in the temporal cortex (known to be a vulnerable region) than in the frontal cortex. Interestingly, the decrease was more severe in AD patients carrying the apolipoprotein E4 allele. The FBL2 IR also decreased over Braak stages, and was hardly detected at Braak stage 5 in both NeuN-positive and EAAC1-positive glutamatergic neurons. CONCLUSION: These results suggest that the involvement of the reduction of FBL2 level is related to AD progression.

Characterization of HTT inclusion size, location, and timing in the zQ175 mouse model of Huntington's disease: an in vivo high-content imaging study.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.

Dendritic structural degeneration is functionally linked to cellular hyperexcitability in a mouse model of Alzheimer's disease.

Dendritic structure critically determines the electrical properties of neurons and, thereby, defines the fundamental process of input-to-output conversion. The diversity of dendritic architectures enables neurons to fulfill their specialized circuit functions during cognitive processes. It is known that this dendritic integrity is impaired in patients with Alzheimer's disease and in relevant mouse models. It is unknown, however, whether this structural degeneration translates into aberrant neuronal function. Here we use in vivo whole-cell patch-clamp recordings, high-resolution STED imaging, and computational modeling of CA1 pyramidal neurons in a mouse model of Alzheimer's disease to show that structural degeneration and neuronal hyperexcitability are crucially linked. Our results demonstrate that a structure-dependent amplification of synaptic input to action potential output conversion might constitute a novel cellular pathomechanism underlying network dysfunction with potential relevance for other neurodegenerative diseases with abnormal changes of dendritic morphology.

Small-animal PET imaging of amyloid-beta plaques with [11C]PiB and its multi-modal validation in an APP/PS1 mouse model of Alzheimer's disease.

In vivo imaging and quantification of amyloid-ß plaque (Aß) burden in small-animal models of Alzheimer's disease (AD) is a valuable tool for translational research such as developing specific imaging markers and monitoring new therapy approaches. Methodological constraints such as image resolution of positron emission tomography (PET) and lack of suitable AD models have limited the feasibility of PET in mice. In this study, we evaluated a feasible protocol for PET imaging of Aß in mouse brain with [(11)C]PiB and specific activities commonly used in human studies. In vivo mouse brain MRI for anatomical reference was acquired with a clinical 1.5 T system. A recently characterized APP/PS1 mouse was employed to measure Aß at different disease stages in homozygous and hemizygous animals. We performed multi-modal cross-validations for the PET results with ex vivo and in vitro methodologies, including regional brain biodistribution, multi-label digital autoradiography, protein quantification with ELISA, fluorescence microscopy, semi-automated histological quantification and radioligand binding assays. Specific [(11)C]PiB uptake in individual brain regions with Aß deposition was demonstrated and validated in all animals of the study cohort including homozygous AD animals as young as nine months. Corresponding to the extent of Aß pathology, old homozygous AD animals (21 months) showed the highest uptake followed by old hemizygous (23 months) and young homozygous mice (9 months). In all AD age groups the cerebellum was shown to be suitable as an intracerebral reference region. PET results were cross-validated and consistent with all applied ex vivo and in vitro methodologies. The results confirm that the experimental setup for non-invasive [(11)C]PiB imaging of Aß in the APP/PS1 mice provides a feasible, reproducible and robust protocol for small-animal Aß imaging. It allows longitudinal imaging studies with follow-up periods of approximately one and a half years and provides a foundation for translational Alzheimer neuroimaging in transgenic mice.

Early-onset and robust amyloid pathology in a new homozygous mouse model of Alzheimer's disease.

BACKGROUND: Transgenic mice expressing mutated amyloid precursor protein (APP) and presenilin (PS)-1 or -2 have been successfully used to model cerebral beta-amyloidosis, one of the characteristic hallmarks of Alzheimer's disease (AD) pathology. However, the use of many transgenic lines is limited by premature death, low breeding efficiencies and late onset and high inter-animal variability of the pathology, creating a need for improved animal models. Here we describe the detailed characterization of a new homozygous double-transgenic mouse line that addresses most of these issues. METHODOLOGY/PRINCIPAL FINDINGS: The transgenic mouse line (ARTE10) was generated by co-integration of two transgenes carrying the K670N/M671L mutated amyloid precursor protein (APP(swe)) and the M146V mutated presenilin 1 (PS1) both under control of a neuron-specific promoter. Mice, hemi- as well as homozygous for both transgenes, are viable and fertile with good breeding capabilities and a low rate of premature death. They develop robust AD-like cerebral beta-amyloid plaque pathology with glial inflammation, signs of neuritic dystrophy and cerebral amyloid angiopathy. Using our novel image analysis algorithm for semi-automatic quantification of plaque burden, we demonstrate an early onset and progressive plaque deposition starting at 3 months of age in homozygous mice with low inter-animal variability and 100%-penetrance of the phenotype. The plaques are readily detected in vivo by PiB, the standard human PET tracer for AD. In addition, ARTE10 mice display early loss of synaptic markers and age-related cognitive deficits. By applying a gamma-secretase inhibitor we show a dose dependent reduction of soluble amyloid beta levels in the brain. CONCLUSIONS: ARTE10 mice develop a cerebral beta-amyloidosis closely resembling the beta-amyloid-related aspects of human AD neuropathology. Unifying several advantages of previous transgenic models, this line particularly qualifies for the use in target validation and for evaluating potential diagnostic or therapeutic agents targeting the amyloid pathology of AD.

The novel cytosolic RING finger protein dactylidin is up-regulated in brains of patients with Alzheimer's disease.

Alzheimer's disease (AD) is characterized by a progressive degeneration of neurons along with deposition of amyloid plaques and the formation of neurofibrillary tangles. Neurodegeneration in AD follows both a spatial pattern of selective vulnerability and temporal staging of affected neurons. In order to address transcriptional changes associated with this selective vulnerability, we used subtractive hybridization of transcripts derived from human frontal cortex, which degenerates in late stages of AD, against transcripts of the inferior temporal cortex, which is affected both heavily and early in the course of AD. Moreover, we compared these to brain sections obtained from age-matched control subjects. We isolated a differentially expressed novel gene encoding a polypeptide that contained an amino-terminal C3HC4 RING finger domain, called dactylidin. It is ubiquitously expressed in all tissues examined and in situ hybridization of mouse brain sections revealed specific expression in neurons. Further, heterologous expression studies revealed a cytoplasmic localization of dactylidin and as all known cytoplasmic RING finger proteins function as ubiquitin protein ligases, an E3-like ligase function of dactylidin is probable. However, the up-regulation of dactylidin in highly vulnerable brain tissues of AD patients was confirmed by a quantitative PCR approach, suggesting that dactylidin may function early in the progression of neurodegenerative diseases.

Cholesterol 25-hydroxylase on chromosome 10q is a susceptibility gene for sporadic Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia. It is characterized by beta-amyloid (A beta) plaques, neurofibrillary tangles and the degeneration of specifically vulnerable brain neurons. We observed high expression of the cholesterol 25-hydroxylase (CH25H) gene in specifically vulnerable brain regions of AD patients. CH25H maps to a region within 10q23 that has been previously linked to sporadic AD. Sequencing of the 5' region of CH25H revealed three common haplotypes, CH25Hchi2, CH25Hchi3 and CH25Hchi4; CSF levels of the cholesterol precursor lathosterol were higher in carriers of the CH25Hchi4 haplotype. In 1,282 patients with AD and 1,312 healthy control subjects from five independent populations, a common variation in the vicinity of CH25H was significantly associated with the risk for sporadic AD (p = 0.006). Quantitative neuropathology of brains from elderly non-demented subjects showed brain A beta deposits in carriers of CH25Hchi4 and CH25Hchi3 haplotypes, whereas no A beta deposits were present in CH25Hchi2 carriers. Together, these results are compatible with a role of CH25Hchi4 as a putative susceptibility factor for sporadic AD; they may explain part of the linkage of chromosome 10 markers with sporadic AD, and they suggest the possibility that CH25H polymorphisms are associated with different rates of brain A beta deposition.