Amyloid-ß targeting immunisation in aged non-human primate (Microcebus murinus).

Non-human primates have an important translational value given their close phylogenetic relationship to humans. Studies in these animals remain essential for evaluating efficacy and safety of new therapeutic approaches, particularly in aging primates that display Alzheimer's disease (AD) -like pathology. With the objective to improve amyloid-ß (Aß) targeting immunotherapy, we investigated the safety and efficacy of an active immunisation with an Aß derivative, K6Aß1-30-NH2, in old non-human primates. Thirty-two aged (4-10 year-old) mouse lemurs were enrolled in the study, and received up to four subcutaneous injections of the vaccine in alum adjuvant or adjuvant alone. Even though antibody titres to Aß were not high, pathological examination of the mouse lemur brains showed a significant reduction in intraneuronal Aß that was associated with reduced microgliosis, and the vaccination did not lead to microhemorrhages. Moreover, a subtle cognitive improvement was observed in the vaccinated primates, which was probably linked to Aß clearance. This Aß derivative vaccine appeared to be safe as a prophylactic measure based on the brain analyses and because it did not appear to have detrimental effects on the general health of these old animals.

The three-panel runway maze adapted to Microcebus murinus reveals age-related differences in memory and perseverance performances.

Microcebus murinus, a mouse lemur primate appears to be a valuable model for cerebral aging study and for Alzheimer's disease model since they can develop beta-amyloid plaques with age. Although the biological and biochemical analyses of cerebral aging are well documented, the cognitive abilities of this primate have not been thoroughly characterized. In this study, we adapted a spatial working memory procedure described in rodents, the sequential choice task in the three-panel runway, to mouse lemurs. We analyzed the age-related differences in a procedural memory task in the absence or presence of visual cues. Sixty percent of young adult and 48% of aged lemurs completed the exploratory, choice habituation and testing phases at the beginning of the procedure. Young adult lemurs showed a higher level of perseverative errors compared with aged animals, particularly in the presence of visual stimuli. Over trials, old animals made more reference errors compared to young ones that improved quickly their performances under random level. No significant improvement was observed in young adults and old ones over sessions. This study showed that behavioural performances of M. murinus assessed on the sequential choice task in the three-panel runway markedly differ from the previously reported abilities of rodents. The behavioural response of young adult lemurs was influenced by novelty-related anxiety that contributed to their performance in terms of perseverative errors. Conversely, aged lemurs showed less perseverative errors, a rapid habituation to the three-panel runway maze, but made more memory errors. Overall, these findings demonstrate the feasibility to use the three-panel runway task in assessing memory performance, particularly in aged mouse lemurs.

Linking cognition to age and amyloid-ß burden in the brain of a nonhuman primate (Microcebus murinus).

The gray mouse lemur (Microcebus murinus) is a valuable model in research on age-related proteopathies. This nonhuman primate, comparable to humans, naturally develops tau and amyloid-ß proteopathies during aging. Whether these are linked to cognitive alterations is unknown. Here, standardized cognitive testing in pairwise discrimination and reversal learning in a sample of 37 aged (>5 years) subjects was combined with tau and amyloid-ß histochemistry in individuals that died naturally. Correlation analyses in successfully tested subjects (n = 22) revealed a significant relation between object discrimination learning and age, strongly influenced by outliers, suggesting pathological cases. Where neuroimmunohistochemistry was possible, as subjects deceased, the naturally developed cortical amyloid-ß burden was significantly linked to pretraining success (intraneuronal accumulations) and discrimination learning (extracellular deposits), showing that cognitive (pairwise discrimination) performance in old age predicts the natural accumulation of amyloid-ß at death. This is the first description of a direct relation between the cortical amyloid-ß burden and cognition in a nonhuman primate.

Exogenous LRRK2G2019S induces parkinsonian-like pathology in a nonhuman primate.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among the elderly. To understand its pathogenesis and to test therapies, animal models that faithfully reproduce key pathological PD hallmarks are needed. As a prelude to developing a model of PD, we tested the tropism, efficacy, biodistribution, and transcriptional effect of canine adenovirus type 2 (CAV-2) vectors in the brain of Microcebus murinus, a nonhuman primate that naturally develops neurodegenerative lesions. We show that introducing helper-dependent (HD) CAV-2 vectors results in long-term, neuron-specific expression at the injection site and in afferent nuclei. Although HD CAV-2 vector injection induced a modest transcriptional response, no significant adaptive immune response was generated. We then generated and tested HD CAV-2 vectors expressing leucine-rich repeat kinase 2 (LRRK2) and LRRK2 carrying a G2019S mutation (LRRK2G2019S), which is linked to sporadic and familial autosomal dominant forms of PD. We show that HD-LRRK2G2019S expression induced parkinsonian-like motor symptoms and histological features in less than 4 months.

Amyloid beta immunization worsens iron deposits in the choroid plexus and cerebral microbleeds.

Anti-amyloid beta (Aß) immunotherapy provides potential benefits in Alzheimer's disease patients. Nevertheless, strategies based on Aß1-42 peptide induced encephalomyelitis and possible microhemorrhages. These outcomes were not expected from studies performed in rodents. It is critical to determine if other animal models better predict side effects of immunotherapies. Mouse lemur primates can develop amyloidosis with aging. Here we used old lemurs to study immunotherapy based on Aß1-42 or Aß-derivative (K6Aß1-30). We followed anti-Aß40 immunoglobulin G and M responses and Aß levels in plasma. In vivo magnetic resonance imaging and histology were used to evaluate amyloidosis, neuroinflammation, vasogenic edema, microhemorrhages, and brain iron deposits. The animals responded mainly to the Aß1-42 immunogen. This treatment induced immune response and increased Aß levels in plasma and also microhemorrhages and iron deposits in the choroid plexus. A complementary study of untreated lemurs showed iron accumulation in the choroid plexus with normal aging. Worsening of iron accumulation is thus a potential side effect of Aß-immunization at prodromal stages of Alzheimer's disease, and should be monitored in clinical trials.

Antibody response and plasma Abeta1-40 levels in young Microcebus murinus primates immunized with Abeta1-42 and its derivatives.

We have been developing Abeta derivative vaccines with the objective to improve the safety of Abeta targeting immunotherapy. Our Abeta homologs are designed to have less direct toxicity and to produce a modified immune response compared to Abeta. In extensive mouse studies, all our vaccines have improved cognition in transgenic mice while eliciting different immune responses and reducing brain amyloid burden to a variable degree. While we are continuing to characterize these vaccines in mice, in preparation for studies in old primates and for human trials we assessed their effect in young lemur primates (n=25) that with age develop Abeta plaques and tau aggregates as seen in Alzheimer's disease. In the primates, all the peptides administered with alum adjuvant elicited a moderate to robust anti-Abeta IgM response. Abeta1-42, K6Abeta1-30 and K6Abeta1-30[E(18)E(19)] resulted in a high anti-Abeta IgG response, whereas Abeta1-30[E(18)E(19)] produced a weaker more variable IgG titer. Notably, 22 weeks after the 3rd immunization, IgM and IgG levels in derivative-vaccinated primates were similar to preimmune values whereas Abeta1-42 treated primates maintained a moderate IgG titer. The increase in antibodies that recognized Abeta1-40 often correlated with increase in Abeta1-40 in plasma, which suggests that the antibodies were binding to Abeta in vivo. Interestingly, significant transient weight gain was observed (K6Abeta1-30-, Abeta1-30[E(18)E(19)]- and Abeta1-42-treated) or a trend in the same direction (K6Abeta1-30[E(18)E(19)]-treated, adjuvant controls) following the injections. Based on these findings, we have chosen K6Abeta1-30 for immunizations in old primates as the antibody response to this vaccine was less variable compared to other Abeta derivatives. Our present findings indicate that most of our Abeta derivatives elicit a substantial antibody response in primates, and importantly this effect is reversible which enhances the safety profile of our approach.