Ángela González-Martínez

Plasma β-amyloid peptides in canine aging and cognitive dysfunction as a model of Alzheimer's disease

Aging dogs naturally demonstrate cognitive impairment and neuropathology that model early Alzheimers disease (AD). In particular, there is evidence that canine cognitive dysfunction syndrome (CDS) in aged dogs is accompanied by cortical deposition of Aβ peptides and neurodegeneration. Plasma Aβ levels have been examined in humans as putative biomarkers for AD, but to date, no similar studies have been conducted for canine dementia. The aim of the present study was to assess plasma Aβ1-42 and Aβ1-40 levels in a blind study using pet dogs that were either successfully aging or exhibiting CDS. The severity of cognitive impairment was assessed using an owner-based questionnaire. On average, young dogs presented significantly higher plasma levels of Aβ1-42 and Aβ1-40 than aged, cognitively unimpaired dogs. Notably, among aged dogs, the levels of Aβ1-42 and the Aβ42/40 ratio were significantly higher in those showing mild cognitive impairment than in either cognitively unimpaired or severely affected dogs. These results suggest that increased plasma Aβ1-42 levels and Aβ42/40 ratio could be a biomarker for canine cognitive dysfunction, which is considered an excellent natural model of early AD.

Effect of age and severity of cognitive dysfunction on two simple tasks in pet dogs

Dogs exhibit age-dependent losses in learning and memory as well as a progressive accumulation of neuropathology that parallels that observed in normal human aging and early Alzheimers disease. These deficits have been extensively studied using a number of standard cognitive tasks in the laboratory; however, appropriate tools for their assessment in veterinary clinics are still lacking. The aim of this study was to evaluate the effect of age and the severity of cognitive dysfunction syndrome (CDS) on two simple tests conducted in a clinical setting. A food searching (FS) task and a problem-solving (PS) task were administered to young (1-4 years, n=9), middle-aged (5-8 years, n=10), cognitively unimpaired aged (≥9 years, n=31), and cognitively impaired aged (≥ 9 years, n=37) dogs. Cognitive status was classified using an owner-based questionnaire, and in the impaired group, dogs were categorized as having either mild or severe CDS. During the FS task, younger dogs (<9 years) were able to locate the food more quickly and with more success than the aged groups (≥9 years). Dogs with severe CDS exhibited poorer performance than those with mild CDS or their healthy counterparts. In the PS task, younger dogs performed better than the aged dogs in obtaining food, but there were no differences related to CDS severity. The FS task might help to better characterize cognitively affected dogs in the clinical setting than the PS task. These and similar tasks require further investigations in the field.

Effect of age and severity of cognitive dysfunction on spontaneous activity in pet dogs – Part 2: Social responsiveness

Changes in social interactions with owners and other dogs are frequently observed in dogs with cognitive dysfunction syndrome (CDS). The aim of this work was to assess the effect of age and severity of CDS on social responsiveness. This is the second part of a 2-part report on spontaneous activity in pet dogs. A human interaction test and a mirror test were administered at baseline and 6 months later to assess social responses to humans and conspecifics, respectively, to four groups of privately-owned dogs: young (n=9), middle-aged (n=9), cognitively unimpaired aged (n=31), and cognitively impaired aged (n=36). The severity of cognitive impairment was considered in the last group and dogs were categorised as having either mild or severe CDS. The influence of the person and the mirror on locomotion and exploratory behaviour was also studied. Dogs were recorded in a testing room and the video recordings were subsequently analysed. Young dogs displayed more interactions involving physical contact with a person. Young and middle-aged dogs showed more vocalisations in response to social isolation. In contrast, aged animals spent more time in front of the mirror. Changes in social responsiveness associated with severe CDS included decreased response to social isolation and human interaction and increased time in front of the mirror, suggesting a deficit in habituation. Testing of spontaneous activity might help to characterise CDS in aged dogs, a condition increasingly diagnosed in veterinary clinics and a potentially useful natural model of Alzheimers disease in humans.

Effect of age and severity of cognitive dysfunction on spontaneous activity in pet dogs - Part 1: Locomotor and exploratory behaviour

Age-related cognitive dysfunction syndrome (CDS) has been reported in dogs and it is considered a natural model for Alzheimers disease in humans. Changes in spontaneous activity (including locomotor and exploratory behaviour) and social responsiveness have been related to the age and cognitive status of kennel-reared Beagle dogs. The aim of this study was to assess the influence of age and severity of CDS on locomotor and exploratory behaviour of privately owned dogs. This is the first part of a two-part report on spontaneous activity in pet dogs. An open-field (OF) test and a curiosity test were administered at baseline and 6 months later to young (1-4 years, n=9), middle-aged (5-8 years, n=9), cognitively unimpaired aged (≥ 9 years, n=31), and cognitively impaired aged ( ≥ 9 years, n=36) animals. Classification of cognitive status was carried out using an owner-based observational questionnaire, and in the cognitively impaired group, the dogs were categorised as having either mild or severe cognitive impairment. Dogs were recorded during sessions in the testing room and the video-recordings were subsequently analysed. The severity of CDS (but not age) influenced locomotion and exploratory behaviour so that the more severe the impairment, the higher the locomotor activity and frequency of corner-directed (aimless) behaviours, and the lower the frequency of door-aimed activities. Curiosity directed toward novel stimuli exhibited an age-dependent decline although severely affected animals displayed more sniffing episodes directed towards the objects. OF activity did not change after 6 months. Testing aged pet dogs for spontaneous behaviour might help to better characterise cognitively affected individuals.

Expression of p75(NTR), a marker for basal forebrain cholinergic neurons, in young and aged dogs with or without cognitive dysfunction syndrome.

The canine cognitive dysfunction syndrome (CDS) has been identified as a natural model for Alzheimers disease (AD). We have used unbiased stereology to estimate the total number of basal forebrain cholinergic neurons expressing the nerve growth factor p75(NTR) receptor in young, aged cognitively-unimpaired (CU) and aged cognitively-impaired (CI) dogs. Aged-CI dogs showed a ∼20% decrement (p = 0.009) in p75(NTR) neurons compared to both the young and the aged-CU animals. These results suggest that the basal forebrain cholinergic system is affected in dogs with CDS and provide additional support for the use this canine syndrome as a model for AD research.

Neprilysin Is Poorly Expressed in the Prefrontal Cortex of Aged Dogs with Cognitive Dysfunction Syndrome

Neprilysin (NEP) is the principal amyloid β (Aβ) degrading peptidase; this activity may protect against Alzheimers disease (AD), the most important age-related neurodegenerative process. The aim of this work was to analyze NEP mRNA expression in the frontal cortex of dogs with and without canine cognitive dysfunction syndrome (CDS), which is considered a natural model for AD. Expression of canine cerebral NEP mRNA was assessed by RT-PCR followed by qPCR in young, aged-cognitively unimpaired (CU), and aged-cognitively impaired (CI) dogs. On average, aged-CI dogs showed 80% (P < 0.01) lower expression levels of NEP mRNA than their aged-CU counterparts. Furthermore, the standard deviation of the qPCR measurements was more than 6 times higher in the cognitively healthy animals (young and aged-CU) than in the aged-CI group. Another interesting find is the determination of a positive correlation between NEP expression and the number of cholinergic neurons in basal telencephalon, indicating a probable connection between both events in these types of neurodegeneration processes. These results suggest that high expression levels of NEP might be a protective factor for canine CDS and, most likely, for other Aβ-associated neurodegenerative diseases, such as AD.

Immunization of dogs with C-terminal sequences of beta-amyloid protein is safe and depletes the protein from the blood

Background: Immunization with Ab has been proposed as a promising therapeutic way in the fight against Alzheimer disease. Methods: We have carried out a pilot immunization assay against Ab peptide in four groups of dogs (n 1⁄4 3) that were vaccinated with 7 biweekly injections containing 400 mg of Ab x-42 conjugated to either hemocyanin (groups A and B) or albumin (groups C and D) and mixed with Th1-type adjuvant (groups B and D), or Th2-type adjuvant (groups A and C). Antibody titers were determined by direct ELISA assay. Levels of Ab 1-42 and Ab 1-40 in plasma were measured with high sensitive proprietary ELISA assay kits. All animals received clinical examination, including blood cell and chemistry analyses, at the beginning and the end of the experiment and were diagnosed to be healthy. Results: After the third immunization, dogs of groups C and D reached high antibody titers, which remained practically unchanged up to the time of the seventh immunization. At this time point, plasma levels of both Ab 1-42 and Ab 1-40 had dropped 100 % and 62 %, respectively, with regard to pre-immune levels. Four months after the last immunization the antibody titers in these dogs had fallen down to the preimmunization levels again. At this time point, the plasma levels of Ab 1-42 and Ab 1-40 showed a tendency to increase, but they were still significantly lower than in the pre-immune samples.Dogs in groups A and B did not respond to immunization and did not show significant changes in the levels of plasma Ab 1-42 and Ab 1-40 across the experiment. Conclusions: Vaccines designed from the C-terminal sequence of Ab are safe for the animals and efficiently eliminate plasmatic Ab in the dog, which represents a natural model for testing Ab modulating therapies against Alzheimer disease.

Early diagnosis and preventive treatment of Alzheimer's with isoform-specific Aβ antibodies

Background: Until now attempts to measure Ab peptides in blood have given inconclusive and discouraging results; mostly for a variety of technical reasons that certainly compromise, but in our opinion do not invalidate the working hypothesis. Due to their biochemical nature, Ab1-40 and Ab1-42 peptides will be found free in the plasma (FP), bound to the plasma components and bound to the blood cells (comprehensively described here as the bamyloid pool in blood (bAPB)). Consequently, a complete quantification of Ab in blood should consider the levels of Ab1-40 and Ab1-42 total in plasma (TP) and bound to cells (CB), which obviously require the use of Ab C-terminal specific antibodies. In addition, contrasting to the preferred N-terminal targeted strategy, immunization against the C-terminus of Ab, which has been suggested to be mainly hidden in the fibrillar aggregates within senile plaques and vascular deposits, might be advantageous to remove Ab soluble monomers and oligomers without undesirable side effects. Methods: We carried out a pilot study including 16 healthy controls, 8 mild cognitive impairment patients and 16 Alzheimer disease patients to explore if quantification of the bAPB by ELISA with Ab C-terminus specific antibodies could provide useful and reliable biomarkers for early stages of Alzheimer disease. Simultaneously, the same C-terminus fragments used to generate these antibodies were used for the active immunization of aged dogs, considered as a very useful animal model for AD research. Results: Main results in the diagnostic study were that several directly markers, including TP and CB Ab140, FP and CB Ab1-42 and other calculated markers as the total bAPB reached a sensitivity and specificity over 80 % to discriminate between MCI patients and healthy controls. For the other side the active immunization of dogs with C-terminus fragment of Ab40 and Ab42 was completely safe, immunogenic and produced a complete wash up of the Ab peptides from the blood. Conclusions: Thus, these results suggest that C-terminal fragments of Ab peptides can be used to generate specific antibodies for the diagnosis of early stages of AD and for preventive therapeutic strategies. Validation of these results in a large sample population is in progress.